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Sample records for bead based immunoassay

  1. Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Y; Hara, C A; Knize, M G; Hwang, M H; Venkatesteswaran, K S; Wheeler, E K; Bell, P M; Renzi, R F; Fruetel, J A; Bailey, C G

    2008-05-01

    As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

  2. Magnetic bead-based reverse colorimetric immunoassay strategy for sensing biomolecules.

    Science.gov (United States)

    Gao, Zhuangqiang; Xu, Mingdi; Hou, Li; Chen, Guonan; Tang, Dianping

    2013-07-16

    A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostate-specific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic bead-based peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanoparticles as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a colorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional colorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RCIA exhibited a wide dynamic range of 0.05-20 ng mL(-1) toward PSA with a detection limit of 0.03 ng mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method.

  3. Single-bead arrays for fluorescence-based immunoassays on capillary-driven microfluidic chips

    Science.gov (United States)

    Temiz, Yuksel; Lim, Michel; Delamarche, Emmanuel

    2016-03-01

    We report a concept for the simple fabrication of easy-to-use chips for immunoassays in the context of point-of-care diagnostics. The chip concept comprises mainly three features: (1) the efficient integration of reagents using beads functionalized with receptors, (2) the generation of capillary-driven liquid flows without using external pumps, and (3) a high-sensitivity detection of analytes using fluorescence microscopy. We fabricated prototype chips using dry etching of Si wafers. 4.5-μm-diameter beads were integrated into hexagonal arrays by sedimentation and removing the excess using a stream of water. We studied the effect of different parameters and showed that array occupancies from 30% to 50% can be achieved by pipetting a 250 nL droplet of 1% bead solution and allowing the beads sediment for 3 min. Chips with integrated beads were sealed using a 50-μm-thick dry-film resist laminated at 45 °C. Liquids pipetted to loading pads were autonomously pulled by capillary pumps at a rate of 0.35 nL s-1 for about 30 min. We studied ligand-receptor interactions and binding kinetics using time-lapse fluorescence microscopy and demonstrated a 5 pM limit of detection (LOD) for an anti-biotin immunoassay. As a clinically-relevant example, we implemented an immunoassay to detect prostate specific antigen (PSA) and showed an LOD of 108 fM (i.e. 3.6 pg mL-1). While a specific implementation is provided here for the detection of PSA, we believe that combining capillary-driven microfluidics with arrays of single beads and fluorescence readout to be very flexible and sufficiently sensitive for the detection of other clinically-relevant analytes.

  4. Magnetic-bead-based sub-femtomolar immunoassay using resonant Raman scattering signals of ZnS nanoparticles.

    Science.gov (United States)

    Ding, Yadan; Cong, Tie; Chu, Xueying; Jia, Yan; Hong, Xia; Liu, Yichun

    2016-07-01

    Highly sensitive, specific, and selective immunoassays are of great significance for not only clinical diagnostics but also food safety, environmental monitoring, and so on. Enzyme-linked immunosorbent assays and fluorescence-based and electrochemical immunoassays are important intensively investigated immunoassay techniques. However, they might suffer from low sensitivity or false-positive results. In this work, a simple, reliable, and ultrasensitive magnetic-bead-based immunoassay was performed using biofunctionalized ZnS semiconductor nanocrystals as resonant Raman probes. The resonant Raman scattering of ZnS nanocrystals displays evenly spaced multi-phonon resonant Raman lines with narrow bandwidths and has strong resistance to environmental variation due to the nature of the electron-phonon interaction, thus rendering reliable signal readout in the immunoassays. The superparamagnetic Fe3O4 nanoparticles facilitated greatly the separation, purification, and concentration processes. It is beneficial for both reducing the labor intensity and amplifying the detection signals. The immobilization of antibodies on the surface of magnetic beads, the preparation of resonant Raman probes, and the immunological recognition between the antibody and analyte all occurred in the liquid phase, which minimized the diffusion barriers and boundary layer constraints. All these factors contributed to the ultralow detection limit of human IgG, which was determined to be about 0.5 fM (∼0.08 pg/ml). It is nearly the highest sensitivity obtained for IgG detection. This work shall facilitate the design of nanoplatforms for ultrasensitive detections of proteins, DNAs, bacteria, explosives, and so on. Graphical abstract An ultrasensitive magnetic-bead-based immunoassay was performed using multi-phonon resonant Raman lines of ZnS nanoparticles as detection signals. PMID:27173389

  5. Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels.

    Science.gov (United States)

    Liu, Lifen; Wu, Simin; Jing, Fengxiang; Zhou, Hongbo; Jia, Chunping; Li, Gang; Cong, Hui; Jin, Qinghui; Zhao, Jianlong

    2016-06-15

    In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody-antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers-carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)-in 10 μl of human serum, with a wide linear dynamic range (1.03-111 ng/mL for CEA and CYFRA21-1; 9.26-1000 ng/ml for NSE) and a low detection limit (CEA: 0.19 ng/ml; CYFRA21-1: 0.97 ng/ml; NSE: 0.37 ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins. PMID:26852198

  6. Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk.

    Science.gov (United States)

    Xu, Jing; Yin, Weiwei; Zhang, Yuanyang; Yi, Jian; Meng, Meng; Wang, Yabin; Xue, Huyin; Zhang, Taichang; Xi, Rimo

    2012-10-15

    In this research, magnetic beads-based enzyme immunoassays were investigated for rapid analysis of chloramphenicol (CAP) in milk. To improve sensitivity of CAP determination, two kinds of immunomagnetic separation methods were designed and compared. Magnetic polystyrene microspheres were conjugated with anti-CAP antibody (Method I) or goat-anti-mouse IgG (Method II). The whole determination could be finished in 1.25 h. Both methods showed high sensitivity to CAP in buffer, and obtained an IC(50) value of 0.05 ng mL(-1) for Method I and 0.4 ng mL(-1) for Method II. The methods showed high specificity, only showing a little cross-reaction towards CAP succinate. The two methods were applied to detect CAP in milk. The recovery rates were 80-106% and the coefficients of variation (CVs) were 4.7-15%. The immunomagnetic assay showed promising potential in rapid screening field for CAP analysis. Between the two methods, Method I is more sensitive, and Method II is more suitable for producing a general assay by changing a primary antibody for another analyte. PMID:23442720

  7. Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.

    Science.gov (United States)

    Shoma, S; Verkaik, N J; de Vogel, C P; Hermans, P W M; van Selm, S; Mitchell, T J; van Roosmalen, M; Hossain, S; Rahman, M; Endtz, H Ph; van Wamel, W J B; van Belkum, A

    2011-04-01

    Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner. PMID:21086008

  8. Flow injection chemiluminescence immunoassay based on resin beads, enzymatic amplification and a novel monoclonal antibody for determination of Hg(2+).

    Science.gov (United States)

    Xu, Mingxia; Chen, Mengting; Dong, Tiantian; Zhao, Kang; Deng, Anping; Li, Jianguo

    2015-09-21

    In the present work, a simple and sensitive flow injection chemiluminescent competitive immunoassay was developed for the determination of mercury(II) ion (Hg(2+)) based on carboxylic resin beads, a novel specific monoclonal antibody (McAb) and HRP enzyme-amplification. Resin beads with carboxyl groups were creatively employed as supports for immobilizing more coating antigen through acylamide bonds. With a competitive-type assay mode, the Hg(2+) in solution competed with the immobilized coating antigen for the limited McAb. Then, the second antibody labeled with HRP was introduced, and an effectively increased CL was obtained, which was ascribed to the catalytic activity of HRP for the luminol-PIP-H2O2 reaction. With increasing concentration of Hg(2+), the CL of this system decreases because less HRP is present in the CL reaction. At optimal conditions, the CL signal displayed a good linear relation toward Hg(2+) in the range of 0.05-200 ng mL(-1) with a detection limit (3σ) of 0.015 ng mL(-1). The immunosensor possessed high specificity, acceptable accuracy and reproducibility, and was examined in real samples with favorable results. This immunoassay will have intriguing application prospects for the determination of other heavy metal ions and environmental contaminants.

  9. Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

    Science.gov (United States)

    Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

    2015-04-15

    In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples.

  10. Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

    Science.gov (United States)

    Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

    2016-06-21

    Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity. PMID:27185215

  11. Rapid bead-based immunoassay for measurement of mannose-binding lectin

    DEFF Research Database (Denmark)

    Bay, J T; Garred, P

    2009-01-01

    Mannose-binding lectin (MBL) is a serum protein, which functions as an opsonin and initiator of the lectin pathway of complement. The serum concentration of MBL shows great interindividual variation because of common polymorphisms in the MBL2 gene. Although several quantitative MBL immunoassays...

  12. Streptavidin Capture and Detection Using Individual Agarose Bead-based Microfluidic Immunoassay Devices

    Institute of Scientific and Technical Information of China (English)

    Xiaoguang Du

    2009-01-01

    @@ A single microwell on polycarbonate substratc was fabricated using hot embossing by silicon master.The silicon master (85 μm in top,100 μm in bottom,53 μm in height) and 0.25 mm-thick polycarbonate substrate were sandwiched between two glass plates in hot embossing system.The system was heated to 155-160℃ and pressed with a force of 300 psi for 10-30 s.The single microwell was stampted on polycarbonate substrate.Apply a~0.2 μL aliquot of agarose beads to the single microwell.

  13. Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

    Science.gov (United States)

    Lin, Yen-Heng; Peng, Po-Yu

    2015-04-15

    Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.

  14. Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus.

    Science.gov (United States)

    Li, Fang; Mei, Li; Li, Yaoming; Zhao, Kaihong; Chen, Huanchun; Wu, Peng; Hu, Yonggang; Cao, Shengbo

    2011-06-15

    A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.

  15. Integrated electrokinetic magnetic bead-based electrochemical immunoassay on microfluidic chips for reliable control of permitted levels of zearalenone in infant foods.

    Science.gov (United States)

    Hervás, Mirian; López, Miguel A; Escarpa, Alberto

    2011-05-21

    Microfluidic technology has now become a novel sensing platform where different analytical steps, biological recognition materials and suitable transducers can be cleverly integrated yielding a new sensor generation. A novel "lab-on-a-chip" strategy integrating an electrokinetic magnetic bead-based electrochemical immunoassay on a microfluidic chip for reliable control of permitted levels of zearalenone in infant foods is proposed. The strategy implies the creative use of the simple channel layout of the double-T microchip to perform sequentially the immunointeraction and enzymatic reaction by applying a program of electric fields suitably connected to the reservoirs for driving the fluidics at different chambers in order to perform the different reactions. Both zones are used with the aid of a magnetic field to avoid in a very simple and elegant way the non-specific adsorption. Immunological reaction is performed under a competitive enzyme-linked immunosorbent assay (ELISA) where the mycotoxin ZEA and an enzyme-labelled derivative compete for the binding sites of the specific monoclonal antibody immobilised onto protein G modified magnetic beads. Horseradish peroxidase (HRP), in the presence of hydrogen peroxide, catalyses the oxidation of hydroquinone (HQ) to benzoquinone (BQN), whose back electrochemical reduction was detected at +0.1 V. Controlled-electrokinetic fluidic handling optimized conditions are addressed for all analytical steps cited above, and allows performing the complete immunoassay for the target ZEA analyte in less than 15 minutes with unique analytical merits: competitive immunoassay currents showed a very well-defined concentration dependence with a good precision as well as a suitable limit of detection of 0.4 µg L(-1), well below the legislative requirements, and an extremely low systematic error of 2% from the analysis of a maize certified reference material revealing additionally an excellent accuracy. Also, the reliability of the

  16. A Bead-Based Multiplexed Immunoassay to Evaluate Breast Cancer Biomarkers for Early Detection in Pre-Diagnostic Serum

    Directory of Open Access Journals (Sweden)

    Carla H. van Gils

    2012-10-01

    Full Text Available This study investigates whether a set of ten potential breast cancer serum biomarkers and cancer antigens (osteopontin (OPN, haptoglobin, cancer antigen 15-3 (CA15-3, carcinoembryonic antigen (CEA, cancer antigen 125 (CA-125, prolactin, cancer antigen 19-9 (CA19-9, α-fetoprotein (AFP, leptin and migration inhibitory factor (MIF can predict early stage breast cancer in samples collected before clinical diagnosis (phase III samples. We performed a nested case-control study within the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition cohort. We examined to what extent the biomarker panel could discriminate between 68 women diagnosed with breast cancer up to three years after enrollment and 68 matched healthy controls (all 56-64 years at baseline. Using a quantitative bead-based multiplexed assay, we determined protein concentrations in serum samples collected at enrollment. Principal Component Analysis (PCA and Random Forest (RF analysis revealed that on the basis of all ten proteins, early cases could not be separated from controls. When we combined serum protein concentrations and subject characteristics related to breast cancer risk in the RF analysis, this did not result in classification accuracy scores that could correctly classify the samples (sensitivity: 50%, specificity: 50%. Our findings indicate that this panel of selected tumor markers cannot be used for diagnosis of early breast cancer.

  17. Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.

    Science.gov (United States)

    Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

    2014-05-20

    This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody.

  18. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

    Energy Technology Data Exchange (ETDEWEB)

    Hervas, Miriam; Lopez, Miguel Angel [Departamento Quimica Analitica, Universidad de Alcala, Ctra. Madrid-Barcelona, Km. 33600, E-28871 Alcala de Henares, Madrid (Spain); Escarpa, Alberto, E-mail: alberto.escarpa@uah.es [Departamento Quimica Analitica, Universidad de Alcala, Ctra. Madrid-Barcelona, Km. 33600, E-28871 Alcala de Henares, Madrid (Spain)

    2009-10-27

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 {mu}g L{sup -1} and EC{sub 50} 0.079 {mu}g L{sup -1} were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  19. Hydrogel nanoparticle based immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  20. Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

    Directory of Open Access Journals (Sweden)

    Dorota G. Pijanowska

    2013-06-01

    Full Text Available This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead–protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

  1. Immunoassays

    Science.gov (United States)

    Hsieh, Y.-H. Peggy

    Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

  2. Single bead-based electrochemical biosensor

    OpenAIRE

    LIU, CHANGCHUN; Schrlau, Michael G.; Bau, Haim H.

    2009-01-01

    A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor’s working electrode consists of an electrochemically-etched platinum wire, with a nominal diameter of 25 μm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bea...

  3. Viral RNA testing and automation on the bead-based CBNE detection microsystem.

    Energy Technology Data Exchange (ETDEWEB)

    Galambos, Paul C.; Bourdon, Christopher Jay; Farrell, Cara M.; Rossito, Paul (University of California at Davis); McClain, Jaime L.; Derzon, Mark Steven; Cullor, James Sterling (University of California at Davis); Rahimian, Kamayar

    2008-09-01

    We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and

  4. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Directory of Open Access Journals (Sweden)

    Angela Buys

    2014-02-01

    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  5. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

    Science.gov (United States)

    Buys, Angela; Macdonald, Raynard; Crafford, Jannie; Theron, Jacques

    2014-01-01

    Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required. PMID:24832497

  6. Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

    Science.gov (United States)

    Ahn, Junhyoung; Shin, Yong-Beom; Lee, JaeJong; Kim, Min-Gon

    2015-09-15

    The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).

  7. An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

    Science.gov (United States)

    Choi, Jin-Woo; Oh, Kwang W; Thomas, Jennifer H; Heineman, William R; Halsall, H Brian; Nevin, Joseph H; Helmicki, Arthur J; Henderson, H Thurman; Ahn, Chong H

    2002-02-01

    This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems. PMID:15100857

  8. Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase-thionine conjugate for ultrasensitive electrochemical immunoassay.

    Science.gov (United States)

    Lin, Youxiu; Zhou, Qian; Lin, Yuping; Tang, Dianping; Niessner, Reinhard; Knopp, Dietmar

    2015-08-18

    A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B1 (AFB1). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB1 antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB1-bovine serum albumin conjugate (AFB1-BSA) and invertase were utilized as the trace tag. Upon target AFB1 introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB1-BSA on the nanogold particles for the immobilized anti-AFB1 antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H2O2-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB1 in a dynamic working range from 3.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.7 pg mL(-1). The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB1

  9. Lab-on-a-Chip Magneto-Immunoassays: How to Ensure Contact between Superparamagnetic Beads and the Sensor Surface

    Directory of Open Access Journals (Sweden)

    Andreas Hütten

    2013-09-01

    Full Text Available Lab-on-a-chip immuno assays utilizing superparamagnetic beads as labels suffer from the fact that the majority of beads pass the sensing area without contacting the sensor surface. Different solutions, employing magnetic forces, ultrasonic standing waves, or hydrodynamic effects have been found over the past decades. The first category uses magnetic forces, created by on-chip conducting lines to attract beads towards the sensor surface. Modifications of the magnetic landscape allow for additional transport and separation of different bead species. The hydrodynamic approach uses changes in the channel geometry to enhance the capture volume. In acoustofluidics, ultrasonic standing waves force µm-sized particles onto a surface through radiation forces. As these approaches have their disadvantages, a new sensor concept that circumvents these problems is suggested. This concept is based on the granular giant magnetoresistance (GMR effect that can be found in gels containing magnetic nanoparticles. The proposed design could be realized in the shape of paper-based test strips printed with gel-based GMR sensors.

  10. In vivo behavior of hydrogel beads based on amidated pectins.

    Science.gov (United States)

    Munjeri, O; Collett, J H; Fell, J T; Sharma, H L; Smith, A M

    1998-01-01

    Radio-labeled hydrogel beads, based on amidated pectin, have been produced by adding droplets of an amidated pectin solution to calcium chloride. Incorporation of model drugs into the beads and measurement of the dissolution rate showed that the properties of the beads were unaffected by the incorporation of the radiolabel. The labeled beads were used to carry out an in vivo study of their behavior in the gastrointestinal tract using human volunteers. The volunteers were given the beads after an overnight fast and images were obtained at frequent intervals during transit through the upper gastrointestinal tract and the colon. The beads exhibited rapid gastric emptying and proceeded to pass through the small intestine individually before regrouping at the ileo-caecal junction. Once in the colon, the beads again proceeded as individuals and evidence of the degradation of the beads was observed.

  11. Detection of Avian Influenza Virus by Fluorescent DNA Barcode-based Immunoassay with Sensitivity Comparable to PCR

    DEFF Research Database (Denmark)

    Cao, Cuong; Dhumpa, Raghuram; Bang, Dang Duong;

    2010-01-01

    in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great......In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection...

  12. Polystyrene Based SPR Biosensor Chip for Use in Immunoassay

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Biosensors are widely used in immunoassay.The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor.This paper describes a polystyrene-based biosensor chip developed and used as part of a surface plasmon resonance (SPR) biosensor.The SPR biosensor has a much higher detecting sensitivity than enzyme-linked immunoserbent assay (ELISA).

  13. Bead Collage: An Arts-Based Research Method

    Science.gov (United States)

    Kay, Lisa

    2013-01-01

    In this paper, "bead collage," an arts-based research method that invites participants to reflect, communicate and construct their experience through the manipulation of beads and found objects is explained. Emphasizing the significance of one's personal biography and experiences as a researcher, I discuss how my background as an…

  14. Magnetic immunoassay platform based on the planar frequency mixing magnetic technique.

    Science.gov (United States)

    Kim, Chang-Beom; Lim, Eul-Gyoon; Shin, Sung Woong; Krause, Hans Joachim; Hong, Hyobong

    2016-09-15

    We represent the experimental results of our planar-frequency mixing magnetic detection (p-FMMD) technique to obtain 2D superparamagnetic images for magnetic immunoassay purpose. The imaging of magnetic beads is based on the nonlinear magnetic characteristics inherent in superparamagnetic materials. The p-FMMD records the sum-frequency components originating from both a high and a low frequency magnetic field incident on the magnetically nonlinear nanoparticles. In this study, we apply the p-FMMD technique to 2D scanning imaging of superparamagnetic iron oxide nanoparticles (SPIONs) in a microfluidic platform. Our p-FMMD system enables to acquire planar images of SPIONs filled in a microchannel as narrow as 30µm in width. The minimum detectable amount is ~1.0×10(8) beads of 100nm size. The system shows a spatial resolution enabling to distinguish between two distinct channels even 2mm apart from each other. Our p-FMMD system as a magnetic immunoassaying system has permitted the detection of amyloid beta 42 (Aβ42), a promising biomarker of Alzheimer's disease, at the minimum concentration of 23.8pg/ml. This may enable the identification of the Aβ42 levels for the early-stage of Alzheimer's disease with the assistance of the MPI using p-FMMD technique. The results show that the deployment of the p-FMMD can be an alternative to conventional biosensing analytical methods, and can be used as a fast and portable screening method. PMID:27135936

  15. Non-constrictive bead immobilization leading to decreased and uniform shear stress in microfluidic bead-based ELISA

    CERN Document Server

    Mitra, Kinshuk; Chidambaram, Preethi; Maharry, Aaron P; Xu, Ronald X; Tweedle, Michael F

    2014-01-01

    Microfluidic biosensors have been utilized for sensing a wide range of antigens using numerous configurations. Bead based microfluidic sensors have been a popular modality due to the plug and play nature of analyte choice and the favorable geometry of spherical sensor scaffolds. While constriction of beads against fluid flow remains a popular method to immobilize the sensor, it results in poor fluidic regimes and shear conditions around sensor beads that can affect sensor performance. We present an alternative means of sensor bead immobilization using poly-carbonate membrane. This system results in several orders of magnitude lower variance of flow radially around the sensor bead. Shear stress experienced by our non-constrictive immobilized bead was three orders of magnitude lower. We demonstrate ability to quantitatively sense EpCAM protein, a marker for cancer stem cells and operation under both far-red and green wavelengths with no auto-fluorescence.

  16. Digital microfluidic magnetic separation for particle-based immunoassays.

    Science.gov (United States)

    Ng, Alphonsus H C; Choi, Kihwan; Luoma, Robert P; Robinson, John M; Wheeler, Aaron R

    2012-10-16

    We introduce a new format for particle-based immunoassays relying on digital microfluidics (DMF) and magnetic forces to separate and resuspend antibody-coated paramagnetic particles. In DMF, fluids are electrostatically controlled as discrete droplets (picoliters to microliters) on an array of insulated electrodes. By applying appropriate sequences of potentials to these electrodes, multiple droplets can be manipulated simultaneously and various droplet operations can be achieved using the same device design. This flexibility makes DMF well-suited for applications that require complex, multistep protocols such as immunoassays. Here, we report the first particle-based immunoassay on DMF without the aid of oil carrier fluid to enable droplet movement (i.e., droplets are surrounded by air instead of oil). This new format allowed the realization of a novel on-chip particle separation and resuspension method capable of removing greater than 90% of unbound reagents in one step. Using this technique, we developed methods for noncompetitive and competitive immunoassays, using thyroid stimulating hormone (TSH) and 17β-estradiol (E2) as model analytes, respectively. We show that, compared to conventional methods, the new DMF approach reported here reduced reagent volumes and analysis time by 100-fold and 10-fold, respectively, while retaining a level of analytical performance required for clinical screening. Thus, we propose that the new technique has great potential for eventual use in a fast, low-waste, and inexpensive instrument for the quantitative analysis of proteins and small molecules in low sample volumes. PMID:23013543

  17. Sensitivity Enhancement of Bead-based Electrochemical Impedance Spectroscopy (BEIS) biosensor by electric field-focusing in microwells.

    Science.gov (United States)

    Shin, Kyeong-Sik; Ji, Jae Hoon; Hwang, Kyo Seon; Jun, Seong Chan; Kang, Ji Yoon

    2016-11-15

    This paper reports a novel electrochemical impedance spectroscopy (EIS) biosensors that uses magnetic beads trapped in a microwell array to improve the sensitivity of conventional bead-based EIS (BEIS) biosensors. Unloading the previously measured beads by removing the magnetic bar enables the BEIS sensor to be used repeatedly by reloading it with new beads. Despite its recyclability, the sensitivity of conventional BEIS biosensors is so low that it has not attracted much attentions from the biosensor industry. We significantly improved the sensitivity of the BEIS system by introducing of a microwell array that contains two electrodes (a working electrode and a counter electrode) to concentrate the electric field on the surfaces of the beads. We confirmed that the performance of the BEIS sensor in a microwell array using an immunoassay of prostate specific antigen (PSA) in PBS buffer and human plasma. The experimental results showed that a low concentration of PSA (a few tens or hundreds of fg/mL) were detectable as a ratio of the changes in the impedance of the PBS buffer or in human plasma. Therefore, our BEIS sensor with a microwell array could be a promising platform for low cost, high-performance biosensors for applications that require high sensitivity and recyclability.

  18. Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B.

    Science.gov (United States)

    Lai, Wenqiang; Wei, Qiaohua; Zhuang, Junyang; Lu, Minghua; Tang, Dianping

    2016-06-15

    We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resolution 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addition of hydrogen peroxide (H2O2), the equivalent iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concentration in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quantitative screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidation of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150 ng kg(-1) with a low detection limit (LOD) of 0.076 ng kg(-1). The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not observed. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit. PMID:26851583

  19. History of inductively coupled plasma mass spectrometry-based immunoassays

    International Nuclear Information System (INIS)

    The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA‐ICP‐MS. - Highlights: ► We discuss the fundamentals of elemental tagging for ICP‐MS applications. ► We propose a definition for the expressions “label” and “tag”. ► We highlight LA‐ICP‐MS‐based heteroelement detection. ► We give an historic overview on ICP-MS and LA‐ICP‐MS-based immunoassays. ► In a personal outlook, we discuss future improvements realistically attainable.

  20. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus

    Science.gov (United States)

    Acharya, Dhiraj; Bastola, Pradip; Le, Linda; Paul, Amber M.; Fernandez, Estefania; Diamond, Michael S.; Miao, Wujian; Bai, Fengwei

    2016-01-01

    Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research. PMID:27554037

  1. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus.

    Science.gov (United States)

    Acharya, Dhiraj; Bastola, Pradip; Le, Linda; Paul, Amber M; Fernandez, Estefania; Diamond, Michael S; Miao, Wujian; Bai, Fengwei

    2016-01-01

    Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research. PMID:27554037

  2. Bead-Based Microfluidic Sediment Analogues: Fabrication and Colloid Transport.

    Science.gov (United States)

    Guo, Yang; Huang, Jingwei; Xiao, Feng; Yin, Xiaolong; Chun, Jaehun; Um, Wooyong; Neeves, Keith B; Wu, Ning

    2016-09-13

    Mobile colloids can act as carriers for low-solubility contaminants in the environment. However, the dominant mechanism for this colloid-facilitated transport of chemicals is unclear. Therefore, we developed a bead-based microfluidic platform of sediment analogues and measured both single and population transport of model colloids. The porous medium is assembled through a bead-by-bead injection method. This approach has the versatility to build both electrostatically homogeneous and heterogeneous media at the pore scale. A T-junction at the exit also allowed for encapsulation and enumeration of colloids effluent at single particle resolution to give population dynamics. Tortuosity calculated from pore-scale trajectory analysis and its comparison with lattice Boltzmann simulations revealed that transport of colloids was influenced by the size exclusion effect. The porous media packed by positively and negatively charged beads into two layers showed distinctive colloidal particle retention and significant remobilization and re-adsorption of particles during water flushing. We demonstrated the potential of our method to fabricate porous media with surface heterogeneities at the pore scale. With both single and population dynamics measurement, our platform has the potential to connect pore-scale and macroscale colloid transport on a lab scale and to quantify the impact of grain surface heterogeneities that are natural in the subsurface environment. PMID:27548505

  3. Highly sensitive SERS-based immunoassay of aflatoxin B1 using silica-encapsulated hollow gold nanoparticles.

    Science.gov (United States)

    Ko, Juhui; Lee, Chankil; Choo, Jaebum

    2015-03-21

    Aflatoxin B1 (AFB1) is a well-known carcinogenic contaminant in foods. It is classified as an extremely hazardous compound because of its potential toxicity to the human nervous system. AFB1 has also been extensively used as a biochemical marker to evaluate the degree of food spoilage. In this study, a novel surface-enhanced Raman scattering (SERS)-based immunoassay platform using silica-encapsulated hollow gold nanoparticles (SEHGNs) and magnetic beads was developed for highly sensitive detection of AFB1. SEHGNs were used as highly stable SERS-encoding nano tags, and magnetic beads were used as supporting substrates for the high-density loading of immunocomplexes. Quantitative analysis of AFB1 was performed by monitoring the intensity change of the characteristic peaks of Raman reporter molecules. The limit of detection (LOD) of AFB1, determined by this SERS-based immunoassay, was determined to be 0.1 ng/mL. This method has some advantages over other analytical methods with respect to rapid analysis (less than 30 min), good selectivity, and reproducibility. The proposed method is expected to be a new analytical tool for the trace analysis of various mycotoxins.

  4. Highly sensitive SERS-based immunoassay of aflatoxin B1 using silica-encapsulated hollow gold nanoparticles.

    Science.gov (United States)

    Ko, Juhui; Lee, Chankil; Choo, Jaebum

    2015-03-21

    Aflatoxin B1 (AFB1) is a well-known carcinogenic contaminant in foods. It is classified as an extremely hazardous compound because of its potential toxicity to the human nervous system. AFB1 has also been extensively used as a biochemical marker to evaluate the degree of food spoilage. In this study, a novel surface-enhanced Raman scattering (SERS)-based immunoassay platform using silica-encapsulated hollow gold nanoparticles (SEHGNs) and magnetic beads was developed for highly sensitive detection of AFB1. SEHGNs were used as highly stable SERS-encoding nano tags, and magnetic beads were used as supporting substrates for the high-density loading of immunocomplexes. Quantitative analysis of AFB1 was performed by monitoring the intensity change of the characteristic peaks of Raman reporter molecules. The limit of detection (LOD) of AFB1, determined by this SERS-based immunoassay, was determined to be 0.1 ng/mL. This method has some advantages over other analytical methods with respect to rapid analysis (less than 30 min), good selectivity, and reproducibility. The proposed method is expected to be a new analytical tool for the trace analysis of various mycotoxins. PMID:25462866

  5. Sandwich immunoassay for alpha-fetoprotein in human sera using gold nanoparticle and magnetic bead labels along with resonance Rayleigh scattering readout

    International Nuclear Information System (INIS)

    We describe a sensitive sandwich immunoassay for alpha-fetoprotein (AFP). It is making use of gold nanoparticles (GNPs) and magnetic beads (MBs) as labels, and of resonance Rayleigh scattering for detection. Two antibodies were labeled with GNPs and MBs, respectively, and MB-antigen-GNP complexes were formed in the presence of antigens. The MB labels also serve as solid phase carriers that can be used to magnetically separate the immuno complex. The GNP labels are used as optical probes, and Rayleigh scattering was used to determine the concentration of free GNPs-antibody after separation of the MB-antigen-GNP complexes. The concentration of AFP is related to the intensity of light scattered by free GNPs in the 13.6 pM to 436 pM concentration range, and the limit of detection is 13.6 pM. The method was applied to the determination of AFP in sera of cancer patients, and the results agree well with those obtained by conventional ELISA. (author)

  6. An on-bacterium flow cytometric immunoassay for protein quantification.

    Science.gov (United States)

    Lan, Wen-Jun; Lan, Wei; Wang, Hai-Yan; Yan, Lei; Wang, Zhe-Li

    2013-09-01

    The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein. PMID:23739299

  7. Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

    OpenAIRE

    Debora Pomponi; Maria Livia Bernardi; Marina Liso; Paola Palazzo; Lisa Tuppo; Chiara Rafaiani; Mario Santoro; Alexis Labrada; Maria Antonietta Ciardiello; Adriano Mari; Enrico Scala

    2012-01-01

    BACKGROUND: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. OBJECTIVE AND METHODS: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Molecule-based micr...

  8. Development of surface-plasmon-resonance-based immunoassay for cephalexin

    Science.gov (United States)

    Dillon, Paul P.; Daly, Stephen J.; Browne, Johnathan; Manning, Bernadette M.; O'Kennedy, Richard; van Amerongen, Aart

    2003-03-01

    The public concern surrounding antibiotic contamination in food and food products has made it imperative to develop analytical methods for their detection. Polyclonal antibodies and protein-hapten conjugates to cephalexin were used in the development of a surface plasmon resonance (SPR)-based inhibition immunoassay to cephalexin. A conjugate consisting of cephalexin-bovine serum albumin (BSA) was immobilised on the dextran gel surface. Dissociation between the antibody and antigen was easily achieved with 10 mmol l-1 NaOH and was very reproducible. Standards of free hapten were prepared and premixed with antibody and, after a suitable incubation time, passed over the surface of the chip with the protein-hapten conjugate immobilised. The hapten in solution inhibited the binding of antibody to the surface resulting in higher response units of antibody bound at lower concentrations of free drug. Model inhibition immunoassays to cephalexin were developed in PBS and spiked milk samples. These assays had detection ranges between 4.88 to 2,500 ng ml-1 and 244 to 3,900 ng ml-1, respectively, with reproducible results.

  9. Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

    NARCIS (Netherlands)

    Peters, J.; Thomas, D.; Boers, E.A.M.; Rijk, de T.C.; Berthiller, F.; Haasnoot, W.; Nielen, M.W.F.

    2013-01-01

    A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins w

  10. Nanoparticle-based immunosensors and immunoassays for aflatoxins.

    Science.gov (United States)

    Wang, Xu; Niessner, Reinhard; Tang, Dianping; Knopp, Dietmar

    2016-03-17

    Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety.

  11. A capillary based chemiluminscent multi-target immunoassay.

    Science.gov (United States)

    Cao, Yuan-Cheng

    2015-05-01

    Renewed interest in capillary format immunoassays has lead to increasingly costly and complex approaches to preparation and readout. This study describes a simple multi-target method based on a capillary platform using horseradish peroxidase (HRP) labelled IgG to visualize an antibody antigen complex. When goat-anti-human IgG was employed as the probe and human IgG as target, the system allowed detection of target to less than 1 ng/mL using a standard detection approach. The capillaries were read visually or with a commercial grade CCD camera. Multi-target detection was demonstrated using a model system of rat-anti-mouse, goat-anti-human and mouse-anti-rat IgG. These probes were encoded to different locations in the capillary, providing a simple inexpensive approach to achieve multi-target assays. PMID:25731812

  12. Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection.

    Science.gov (United States)

    Guirgis, Bassem S S; Sá e Cunha, Cláudia; Gomes, Inês; Cavadas, Miguel; Silva, Isabel; Doria, Gonçalo; Blatch, Gregory L; Baptista, Pedro V; Pereira, Eulália; Azzazy, Hassan M E; Mota, Maria M; Prudêncio, Miguel; Franco, Ricardo

    2012-01-01

    The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

  13. First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

    Science.gov (United States)

    Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

    2007-02-21

    An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

  14. Dynamic light scattering (DLS)-based immunoassay for ultra-sensitive detection of tumor marker protein.

    Science.gov (United States)

    Li, Chao; Ma, Jiehua; Fan, Qiongxuan; Tao, Yaqin; Li, Genxi

    2016-06-14

    A novel dynamic light scattering (DLS)-based immunoassay that utilizes manganese dioxide nanosheet-modified gold nanoparticles (MnO2-GNPs) as an activatable nanoprobe has been developed to detect tumor markers down to femtomolar levels. PMID:27247980

  15. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Østerberg, Frederik Westergaard; Henriksen, Anders Dahl;

    2014-01-01

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the di......We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches...

  16. Bioelectrochemical Immunoassay of Polychlorinated Biphenyl

    International Nuclear Information System (INIS)

    A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP-PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP-PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP-PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 5 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring

  17. Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.

    Directory of Open Access Journals (Sweden)

    Debora Pomponi

    Full Text Available BACKGROUND: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. OBJECTIVE AND METHODS: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Molecule-based micro-bead Array system (ABA was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. RESULTS: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE to nAct d 1, nAct d 11, rAln g 1, nAmb a 1, nArt v 3, rBet v 1, rCor a 1, nCup a 1, nDer p 1, nDer s 1, rHev b 5, nOle e 1, rPar j 2, nPen m 1, rPhl p 1, and nPru p 3. Results obtained by ABA IgE testing were highly correlated to ISAC testing (r = 0.87, p<0.0001. No unspecific binding was recorded because of high total IgE values. CONCLUSION: The ABA assay represents a useful and flexible method for multiplex IgE detection using allergenic molecules. As also shown by our initial experiments with monoclonals and polyclonals, ABA is suitable for detecting other human and non-human immunoglobulins.

  18. Evaluation of enzyme immunoassay for hepatitis B virus DNA based on anti-double-stranded DNA.

    OpenAIRE

    F. Garcia(Helsinki U); Bernal, M.C.; Leyva, A.; Piedrola, G.; Maroto, M C

    1995-01-01

    We have evaluated a new enzyme immunoassay technology to detect the products of PCR-based amplification that may be applicable to routine testing of hepatitis B virus (HBV) DNA. Two hundred eight serum samples were studied: 73 were basal samples and 135 were sequential serum samples from patients with chronic hepatitis, some of whom were being treated with alpha interferon. We compared the new detection method (PCR-DNA enzyme immunoassay [DEIA]) with dot blot hybridization performed without p...

  19. A Compact Immunoassay Platform Based on a Multicapillary Glass Plate

    Directory of Open Access Journals (Sweden)

    Shuhua Xue

    2014-05-01

    Full Text Available A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP and a polydimethylsiloxane (PDMS slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays.

  20. Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

    Science.gov (United States)

    Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

    2016-04-01

    There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.

  1. Simultaneous Determination of the Main Peanut Allergens in Foods Using Disposable Amperometric Magnetic Beads-Based Immunosensing Platforms

    Directory of Open Access Journals (Sweden)

    Víctor Ruiz-Valdepeñas Montiel

    2016-06-01

    Full Text Available In this work, a novel magnetic beads (MBs-based immunosensing approach for the rapid and simultaneous determination of the main peanut allergenic proteins (Ara h 1 and Ara h 2 is reported. It involves the use of sandwich-type immunoassays using selective capture and detector antibodies and carboxylic acid-modified magnetic beads (HOOC-MBs. Amperometric detection at −0.20 V was performed using dual screen-printed carbon electrodes (SPdCEs and the H2O2/hydroquinone (HQ system. This methodology exhibits high sensitivity and selectivity for the target proteins providing detection limits of 18.0 and 0.07 ng/mL for Ara h 1 and Ara h 2, respectively, with an assay time of only 2 h. The usefulness of the approach was evaluated by detecting the endogenous content of both allergenic proteins in different food extracts as well as trace amounts of peanut allergen (0.0001% or 1.0 mg/kg in wheat flour spiked samples. The developed platform provides better Low detection limits (LODs in shorter assay times than those claimed for the allergen specific commercial ELISA kits using the same immunoreagents and quantitative information on individual food allergen levels. Moreover, the flexibility of the methodology makes it readily translate to the detection of other food-allergens.

  2. Magnetic bead detection using domain wall-based nanosensor

    International Nuclear Information System (INIS)

    We investigate the effect of a single magnetic bead (MB) on the domain wall (DW) pinning/depinning fields of a DW trapped at the corner of an L-shaped magnetic nanodevice. DW propagation across the device is investigated using magnetoresistance measurements. DW pinning/depinning fields are characterized in as-prepared devices and after placement of a 1 μm-sized MB (Dynabeads® MyOne™) at the corner. The effect of the MB on the DW dynamics is seen as an increase in the depinning field for specific orientations of the device with respect to the external magnetic field. The shift of the depinning field, ΔBdep = 4.5–27.0 mT, is highly stable and reproducible, being significantly above the stochastic deviation which is about 0.5 mT. The shift in the deppinning field is inversely proportional to the device width and larger for small negative angles between the device and the external magnetic field. Thus, we demonstrate that DW-based devices can be successfully used for detection of single micron size MB

  3. Design and Fabrication of a PDMS Microchip Based Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Guocheng; Wang, Wanjun; Wang, Jun; Lin, Yuehe

    2010-07-01

    In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close a 200µm wide micro channel with flow rate up to 20µl/min.

  4. A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs)

    International Nuclear Information System (INIS)

    A disposable immunomagnetic electrochemical sensor involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed and applied for the detection of polychlorinated biphenyls (PCBs). The sensor was based on a direct competitive immunoassay scheme in which antibody-coated magnetic beads were used as solid phase; then SPCEs were employed as transducers for the evaluation of the extent of the immunochemical reaction using an alkaline phosphatase (AP) labelled tracer. The α-naphthyl phosphate was used as enzymatic substrate and the α-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). A detection limit of 0.4 ng/mL for Aroclor 1248 PCB mixture was obtained. The performance of the sensor was successfully evaluated on marine sediment extracts and soil samples spiked with different Aroclor mixtures indicating that this new and sensitive technique offers great promise for decentralized environmental applications

  5. A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs)

    Energy Technology Data Exchange (ETDEWEB)

    Centi, S. [Universita degli Studi di Firenze, Dipartimento di Chimica, Polo Scientifico, via della Lastruccia 3, 50019 Sesto Fiorentino, Florence (Italy)]. E-mail: marco.mascini@unifi.it; Laschi, S. [Universita degli Studi di Firenze, Dipartimento di Chimica, Polo Scientifico, via della Lastruccia 3, 50019 Sesto Fiorentino, Florence (Italy); Franek, M. [Veterinary Research Institute, Hudcova 70, 62132 Brno (Czech Republic); Mascini, M. [Universita degli Studi di Firenze, Dipartimento di Chimica, Polo Scientifico, via della Lastruccia 3, 50019 Sesto Fiorentino, Florence (Italy)

    2005-05-04

    A disposable immunomagnetic electrochemical sensor involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed and applied for the detection of polychlorinated biphenyls (PCBs). The sensor was based on a direct competitive immunoassay scheme in which antibody-coated magnetic beads were used as solid phase; then SPCEs were employed as transducers for the evaluation of the extent of the immunochemical reaction using an alkaline phosphatase (AP) labelled tracer. The {alpha}-naphthyl phosphate was used as enzymatic substrate and the {alpha}-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). A detection limit of 0.4 ng/mL for Aroclor 1248 PCB mixture was obtained. The performance of the sensor was successfully evaluated on marine sediment extracts and soil samples spiked with different Aroclor mixtures indicating that this new and sensitive technique offers great promise for decentralized environmental applications.

  6. Highly sensitive immunoassay based on E. coli with autodisplayed Z-domain

    Energy Technology Data Exchange (ETDEWEB)

    Jose, Joachim [Institute of Pharmaceutical Chemistry, Heinrich Heine University, Duesseldorf (Germany); Park, Min [School of Materials and Sciences, College of Engineering, Yonsei University, 134 Shin-chon-dong, Seo-dae-mun-gu, Seoul 120-749 (Korea, Republic of); Pyun, Jae-Chul, E-mail: jcpyun@yonsei.ac.kr [School of Materials and Sciences, College of Engineering, Yonsei University, 134 Shin-chon-dong, Seo-dae-mun-gu, Seoul 120-749 (Korea, Republic of)

    2010-05-14

    The Z-domain of protein A has been known to bind specifically to the F{sub c} region of antibodies (IgGs). In this work, the Z-domain of protein A was expressed on the outer membrane of Escherichia coli by using 'Autodisplay' technology as a fusion protein of autotransport domain. The E. coli with autodisplayed Z-domain was applied to the sandwich-type immunoassay as a solid-support of detection-antibodies against a target analyte. For the feasibility demonstration of the E. coli based immunoassay, C-reactive protein (CRP) assay was carried out by using E. coli with autodisplayed Z-domain. The limit of detection (LOD) and binding capacity of the E. coli based immunoassay were estimated to be far more sensitive than the conventional ELISA. Such a far higher sensitivity of E. coli based immunoassay than conventional ELISA was explained by the orientation control of immobilized antibodies and the mobility of E. coli in assay matrix. From the test results of 45 rheumatoid arthritis (RA) patients' serum and 15 healthy samples, a cut-off value was established to have optimal sensitivity and selectivity values for RA. The CRP test result of each individual sample was compared with ELISA which is the reference method for RA diagnosis. From this work, the E. coli with Z-domain was proved to be feasible for the medical diagnosis based on sandwich-type immunoassay.

  7. Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles.

    Science.gov (United States)

    Castilho, M de Souza; Laube, T; Yamanaka, H; Alegret, S; Pividori, M I

    2011-07-15

    Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.

  8. Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides.

    Science.gov (United States)

    Kim, Hee-Joo; Ahn, Ki Chang; González-Techera, Andrés; González-Sapienza, Gualberto G; Gee, Shirley J; Hammock, Bruce D

    2009-03-01

    Noncompetitive immunoassays are advantageous over competitive assays for the detection of small molecular weight compounds. We recently demonstrated that phage peptide libraries can be an excellent source of immunoreagents that facilitate the development of sandwich-type noncompetitive immunoassays for the detection of small analytes, avoiding the technical challenges of producing anti-immunocomplex antibody. In this work we explore a new format that may help to optimize the performance of the phage anti-immunocomplex assay (PHAIA) technology. As a model system we used a polyclonal antibody to 3-phenoxybenzoic acid (3-PBA) and an anti-immunocomplex phage clone bearing the cyclic peptide CFNGKDWLYC. The assay setup with the biotinylated antibody immobilized onto streptavidin-coated magnetic beads significantly reduced the amount of coating antibody giving identical sensitivity (50% saturation of the signal (SC(50))=0.2-0.4ng/ml) to the best result obtained with direct coating of the antibody on ELISA plates. The bead-based assay tolerated up to 10 and 5% of methanol and urine matrix, respectively. This assay system accurately determined the level of spiked 3-PBA in different urine samples prepared by direct dilution or clean-up with solid-phase extraction after acidic hydrolysis with overall recovery of 80-120%.

  9. Enzyme immunoassay of pancreatic oncofetal antigen (POA) as a marker of pancreatic cancer.

    OpenAIRE

    Nishida, K; Sugiura, M; Yoshikawa, T; Kondo, M

    1985-01-01

    For the quantitative measurement of pancreatic oncofetal antigen (POA), an enzyme immunoassay for POA has been developed, and is based on the sandwich method using antibody-coupled glass beads and enzyme (peroxidase)-labelled antibody. Serum POA concentrations were increased significantly in patients with pancreatic cancer, but not in those with chronic pancreatitis or other miscellaneous diseases, or in normal subjects. It is concluded that the enzyme immunoassay could be used for the assay ...

  10. REVIEW: CHITOSAN BASED HYDROGEL POLYMERIC BEADS – AS DRUG DELIVERY SYSTEM

    Directory of Open Access Journals (Sweden)

    Manjusha Rani

    2010-11-01

    Full Text Available Chitosan obtained by alkaline deacetylation of chitin is a non-toxic, biocompatible, and biodegradable natural polymer. Chitosan-based hydrogel polymeric beads have been extensively studied as micro- or nano-particulate carriers in the pharmaceutical and medical fields, where they have shown promise for drug delivery as a result of their controlled and sustained release properties, as well as biocompatibility with tissue and cells. To introduce desired properties and enlarge the scope of the potential applications of chitosan, graft copolymerization with natural or synthetic polymers on it has been carried out, and also, various chitosan derivatives have been utilized to form beads. The desired kinetics, duration, and rate of drug release up to therapeutical level from polymeric beads are limited by specific conditions such as beads material and their composition, bead preparation method, amount of drug loading, drug solubility, and drug polymer interaction. The present review summarizes most of the available reports about compositional and structural effects of chitosan-based hydrogel polymeric beads on swelling, drug loading, and releasing properties. From the studies reviewed it is concluded that chitosan-based hydrogel polymeric beads are promising drug delivery systems.

  11. A bacteria-based bead for possible self-healing marine concrete applications

    Science.gov (United States)

    Palin, D.; Wiktor, V.; Jonkers, H. M.

    2016-08-01

    This work presents a bacteria-based bead for potential self-healing concrete applications in low-temperature marine environments. The bead consisting of calcium alginate encapsulated bacterial spores and mineral precursor compounds was assessed for: oxygen consumption, swelling, and its ability to form a biocomposite in a simulative marine concrete crack solution (SMCCS) at 8 °C. After six days immersion in the SMCCS the bacteria-based beads formed a calcite crust on their surface and calcite inclusions in their network, resulting in a calcite-alginate biocomposite. Beads swelled by 300% to a maximum diameter of 3 mm, while theoretical calculations estimate that 0.112 g of the beads were able to produce ˜1 mm3 of calcite after 14 days immersion; providing the bead with considerable crack healing potential. The bacteria-based bead shows great potential for the development of self-healing concrete in low-temperature marine environments, while the formation of a biocomposite healing material represents an exciting avenue for self-healing concrete research.

  12. Clinical validation of surface-enhanced Raman scattering-based immunoassays in the early diagnosis of rheumatoid arthritis.

    Science.gov (United States)

    Chon, Hyangah; Wang, Rui; Lee, Sangyeop; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol; Hong, Sung Hyun; Yoon, Young Ho; Lim, Dong Woo; deMello, Andrew J; Choo, Jaebum

    2015-11-01

    We assessed the clinical feasibility of conducting immunoassays based on surface-enhanced Raman scattering (SERS) in the early diagnosis of rheumatoid arthritis (RA). An autoantibody against citrullinated peptide (anti-CCP) was used as a biomarker, magnetic beads conjugated with CCP were used as substrates, and the SERS nanotags were comprised of anti-human IgG-conjugated hollow gold nanospheres (HGNs). We were able to determine the anti-CCP serum levels successfully by observing the distinctive Raman intensities corresponding to the SERS nanotags. At high concentrations of anti-CCP (>25 U/mL), the results obtained from the SERS assay confirmed those obtained via an ELISA-based assay. Nevertheless, quantitation via our SERS-based assay is significantly more accurate at low concentrations (25 U/mL) revealed a good correlation between the ELISA and SERS-based assays. However, in the anti-CCP-negative group (n = 43, <25 U/mL), the SERS-based assay was shown to be more reproducible. Accordingly, we suggest that SERS-based assays are novel and potentially useful tools in the early diagnosis of RA.

  13. Nitrocellulose membrane-based enzyme-linked immunoassay for dengue serotype-1 IgM detection

    International Nuclear Information System (INIS)

    To evaluate the sensitivity and specifity of a nitrocellulose membrane-based immunoassay for dengue IgM, with respect to capture enzyme immunoassay, for the diagnosis of dengue virus infection. 101 serum samples were processed and divided into 2 groups: 53 from dengue serotype 1 (DEN1) infected patients, and 48 from healthy subjects. Both groups were tested with a nitrocellulose membrane-based IgM capture enzyme immunoassay (NMB-EIA) and also with an ELISA as referential pattern. NMB-EIA testing detected IgM anti-DEN1 in 94,34% of samples from infected patients, and in 14,58% of control samples, whereas ELISA fails to report false positive or false negative results: NMB-EIA appears to be a good alternative for dengue infection diagnosis. (authors)

  14. Rapid polyelectrolyte-based membrane immunoassay for the herbicide butachlor.

    Science.gov (United States)

    Dzantiev, B B; Byzova, N A; Zherdev, A V; Hennion, M C

    2005-01-01

    Oppositely charged water-soluble polyelectrolytes were used in the developed membrane immunoenzyme assay for the herbicide butachlor. High-affinity and rapid binding between polyanion polymethacrylate and polycation poly(N-ethyl-4-vinylpyridinium) was applied to separate reacted and free immunoreactants. Competitive immunoassay format with peroxidase-labeled antigen was realized. The insoluble colored product of the peroxidase reaction was formed by bound labeled immune complexes and was reflectometrically detected. The assay combines short duration (15 min), high sensitivity (0.03 g/mL) and availability for out-of-laboratory testing. Different image processing algorithms were used to determine the herbicide content. Low variation coefficients of the measurements in the proposed quantitative assay, namely 4.8-9.0% for the range of antigen concentrations from 0.1 to 3.0 ng/mL, are evidence of the assay effectiveness. Possibility to control the butachlor content in mineral, artesian, and drinking water was demonstrated.

  15. A novel biosensor based on competitive SERS immunoassay and magnetic separation for accurate and sensitive detection of chloramphenicol.

    Science.gov (United States)

    Yang, Kang; Hu, Yongjun; Dong, Ning

    2016-06-15

    The accurate and sensitive detection of chloramphenicol (CAP) is particularly imperative to public health and safety. Here, we present a novel sensor for residual CAP detection based on competitive surface-enhanced Raman scattering (SERS) immunoassay and magnetic separation. In this nanosensor, functionalized Au nanoparticles (AuNPs) were labeled with the Raman reporter molecule (e.g. 4,4'-dipyridyl). With the addition of free CAP, a competitive immune reaction was initiated between free CAP and above AuNPs for conjugating with CAP antibody-modified magnetic nanoparticles (MNPs). Instead of the solid substrate, the antibody conjugated-magnetic beads were used as supporting materials and separation tools in the present sensor. With the aid of a magnet, the mixture was removed from the supernatant for concentration effects. This caused obvious change of SERS signal intensity obtained from supernatant. The SERS signals were collected from the supernatant directly, which made the SERS measurements more stable, repeatable and reliable. The proposed SERS-based magnetic immunosensor allows us to detect CAP in a fast, selective and sensitive (1.0 pg/mL) manner over a wide concentration range ( 1-1 × 10(4)pg/mL). In addition, these results demonstrate that this immunosensor holds great potential for the detection of antibiotics in real aquatic environment, which is crucial to our life. PMID:26866562

  16. Surface plasmon resonance-based immunoassay for procalcitonin.

    Science.gov (United States)

    Vashist, Sandeep Kumar; Schneider, E Marion; Barth, Eberhard; Luong, John H T

    2016-09-28

    A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This "single-step" PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4-324 ng mL(-1) of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL(-1) and 9.2 ng mL(-1), respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks. PMID:27619095

  17. PicoMolar level detection of protein biomarkers based on electronic sizing of bead aggregates: theoretical and experimental considerations.

    Science.gov (United States)

    Lin, Z; Cao, X; Xie, P; Liu, M; Javanmard, Mehdi

    2015-12-01

    We demonstrate a novel method for electronically detecting and quantifying protein biomarkers using microfluidic impedance cytometry. Our biosensor, which consists of gold electrodes micro-fabricated in a microchannel, detects the differences between bead aggregates of varying sizes in a micro-pore sandwiched between two micro channels. We perform a sandwich immunoassay, where the complementary antibody pairs are immobilized on two different bead types, and the presence of antigen results in bead aggregation, the amount of which depends on antigen quantity. When single beads or bead aggregates pass through the impedance sensor, differences in impedance change are detected. In this manuscript, we perform a comprehensive theoretical study on the limits imposed on sensitivity of this technique due to electronic noise and also mass transfer and reaction limits. We also experimentally characterize the performance of this technique by validating the technique on an IgG detection assay. A detection limit at the picoMolar level is demonstrated, thus comparable in sensitivity to a sandwich ELISA. PMID:26589228

  18. Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool.

    OpenAIRE

    Stibbs, H H

    1989-01-01

    A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed spe...

  19. Online measurement of bead geometry in GMAW-based additive manufacturing using passive vision

    International Nuclear Information System (INIS)

    Additive manufacturing based on gas metal arc welding is an advanced technique for depositing fully dense components with low cost. Despite this fact, techniques to achieve accurate control and automation of the process have not yet been perfectly developed. The online measurement of the deposited bead geometry is a key problem for reliable control. In this work a passive vision-sensing system, comprising two cameras and composite filtering techniques, was proposed for real-time detection of the bead height and width through deposition of thin walls. The nozzle to the top surface distance was monitored for eliminating accumulated height errors during the multi-layer deposition process. Various image processing algorithms were applied and discussed for extracting feature parameters. A calibration procedure was presented for the monitoring system. Validation experiments confirmed the effectiveness of the online measurement system for bead geometry in layered additive manufacturing. (paper)

  20. Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

    DEFF Research Database (Denmark)

    Hudson, Elton P.; Nikoshkov, Andrej; Uhlén, Mathias;

    2012-01-01

    gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close...... contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for...

  1. A Novel Magnetic Bead-based Biosensor Using Flip Chip Bonding Techniques

    Institute of Scientific and Technical Information of China (English)

    Bin Wang; Xiang Chen; Qinghui Jin; Jianlong Zhao; Yuansen Xu

    2006-01-01

    Based on flip-chip packaging, a novel approach towards integrated magnetic bio-separator was designed. The magnetic field and the force on the bead were simulated and analyzed, leading to the optimization of the fabrication parameters of the micro-magnetic unit. The planar coil as an electromagnet was fabricated through electroplating on a single seed layer.The PDMS microfluidic channel was bonded on the inverse side after Si etching. The results presented in this paper provide a novel design and fabrication to approach a microfluidic bio-separation system with magnetic beads.

  2. Accelerated surface-enhanced Raman spectroscopy (SERS)-based immunoassay on a gold-plated membrane.

    Science.gov (United States)

    Penn, Michelle A; Drake, David M; Driskell, Jeremy D

    2013-09-17

    A rapid and simple SERS-based immunoassay has been developed to overcome diffusion-limited binding kinetics that often impedes rapid analysis in conventional heterogeneous immunoassays. This paper describes the development of an antibody-modified membrane as a flow-through capture substrate for a nanoparticle-enabled SERS immunoassay to enhance antibody-antigen binding kinetics. A thin layer of gold is plated onto polycarbonate track-etched nanoporous membranes via electroless deposition. Capture antibody is immobilized onto the surface of a gold-plated membrane via thiolate coupling chemistry to serve as a capture substrate. A syringe is then used to actively transport the analyte and extrinsic Raman labels to the capture substrate. The fabrication of the gold-plated membrane is thoroughly investigated and established as a viable capture substrate for a SERS-based immunoassay in the absence of sample/SERS label flow. A syringe pump is used to systematically investigate the effect of flow rate on antibody-antigen binding kinetics and demonstrate that active transport to the capture membrane surface expedites antibody-antigen binding. Mouse IgG and goat anti-mouse IgG are selected as a model antigen-antibody system to establish proof of principle. It is demonstrated that the assay for mouse IgG is reduced from 24 h to 10 min and a 10-fold improvement in detection limit is achieved with the flow assay developed herein relative to the passive, i.e., no flow, assay. Moreover, mouse serum is directly analyzed and IgG level is determined using the flow assay. PMID:23972208

  3. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    Energy Technology Data Exchange (ETDEWEB)

    Rizzi, Giovanni, E-mail: giori@nanotech.dtu.dk; Østerberg, Frederik W.; Henriksen, Anders D.; Dufva, Martin; Hansen, Mikkel F., E-mail: mikkel.hansen@nanotech.dtu.dk

    2015-04-15

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface. The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover, we demonstrate that a single sensor bridge can be used to genotype a SNP. - Highlights: • We apply magnetoresistive sensors to study solid-surface hybridization kinetics of DNA. • We measure DNA melting profiles for perfectly matching DNA duplexes and for a single base mismatch. • We present a procedure to correct for temperature dependencies of the sensor output. • We reliably extract melting temperatures for the DNA hybrids. • We demonstrate direct measurement of differential binding signal for two probes on a single sensor.

  4. Double-antibody based immunoassay for the detection of β-casein in bovine milk samples.

    Science.gov (United States)

    Zhou, Y; Song, F; Li, Y S; Liu, J Q; Lu, S Y; Ren, H L; Liu, Z S; Zhang, Y Y; Yang, L; Li, Z H; Zhang, J H; Wang, X R

    2013-11-01

    The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1-10.0 μg mL(-1). The detection limit was 0.04 μg mL(-1). In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.

  5. Blu-ray optomagnetic measurement based competitive immunoassay for Salmonella detection.

    Science.gov (United States)

    Tian, Bo; Bejhed, Rebecca S; Svedlindh, Peter; Strömberg, Mattias

    2016-03-15

    A turn-on competitive immunoassay using a low-cost Blu-ray optomagnetic setup and two differently sized magnetic particles (micron-sized particles acting as capture particles and nano-sized particles acting as detection particles) is here presented. For Salmonella detection, a limit of detection of 8×10(4)CFU/mL is achieved within a total assay time of 3h. The combination of a competitive strategy and an optomagnetic setup not only enables a turn-on read-out format, but also results in a sensitivity limit about a factor of 20 times lower than of volumetric magnetic stray field detection device based immunoassays. The improvement of sensitivity is enabled by the formation of immuno-magnetic aggregates providing steric hindrance protecting the interior binding sites from interaction with the magnetic nanoparticle labels. The formation of immuno-magnetic aggregates is confirmed by fluorescence microscopy. The system exhibits no visible cross-reaction with other common pathogenic bacteria, even at concentrations as high as 10(7)CFU/mL. Furthermore, we present results when using the setup for a qualitative and homogeneous biplex immunoassay of Escherichia coli and Salmonella typhimurium.

  6. A flow cytometric assay technology based on quantum dots-encoded beads

    International Nuclear Information System (INIS)

    A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

  7. Preparation and Characterization of Alginate-Hyaluronic Acid-chitosan based Composite Gel Beads

    Institute of Scientific and Technical Information of China (English)

    HU Yan; ZHENG Mengzhu; DONG Xiaoying; ZHAO Dan; CHENG Han; XIAO Xincai

    2015-01-01

    The aim of this study was to fabricate composite gel beads based on natural polysaccharides. Hyaluronic acid (HA) and Chitosan (CS) were successfully admixed with Ca2+/alginate (SA) gel system to produce SA/HA/CS gel beads by dual crosslinking:the ionic gelation and the polyelectrolyte complexation. The preparation procedure was that the weight ratio of SA (2%, m/v) to HA (2%, m/v) was kept at 2:1, then the mixture was dripped into the Ca2+solution for ion-crosslinking, and finally polyelectrolyte crosslinked with 2%low molecular weight CS (LMW-CS) for 1.5 hours. The optimal formulation was achieved by adjusting the concentration and the weight ratio of SA, HA and LMW-CS. Due to the incorporation of HA and LMW-CS, the swelling ratio of the beads at pH 7.4 was increased up to 120, and the time for the maximum swelling degree was prolonged to 7.5 h. The swelling behavior was obviously improved compared to the pure SA/Ca2+system. The preliminary results clearly suggest that the SA/HA/CS gel beads may be a potential candidate for biomedical delivery vehicles.

  8. Development of surface plasmon resonance-based immunoassay for cephalexin

    NARCIS (Netherlands)

    Dillon, P.; Daly, S.; Browne, J.; Manning, B.; O'Kennedy, R.; Amerongen, van A.

    2003-01-01

    The public concern surrounding antibiotic contamination in food and food products has made it imperative to develop analytical methods for their detection. Polyclonal antibodies and protein-hapten conjugates to cephalexin were used in the development of a surface plasmon resonance (SPR)-based inhibi

  9. Optimizing a waveguide-based sandwich immunoassay for tumor biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    Mukundan, Harshini [Los Alamos National Laboratory; Swanson, Basil I [Los Alamos National Laboratory; Xie, Hongzhi [Los Alamos National Laboratory; Anderson, Aaron S [Los Alamos National Laboratory; Grace, W Kevin [Los Alamos National Laboratory; Shively, John E [NON LANL

    2008-01-01

    The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with the disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In this publication, we report improvements to this technology that will facilitate transition to a point-of-care diagnostic system and/or robust research tool.

  10. A 3D porous polymer monolith-based platform integrated in poly(dimethylsiloxane) microchips for immunoassay.

    Science.gov (United States)

    Kang, Qin-Shu; Shen, Xiao-Fan; Hu, Na-Na; Hu, Meng-Jia; Liao, Hui; Wang, Han-Zhong; He, Zhi-Ke; Huang, Wei-Hua

    2013-05-01

    In this work, we demonstrate the immunocapture and on-line fluorescence immunoassay of protein and virus based on porous polymer monoliths (PPM) in microfluidic devices. Poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)] monoliths were successfully synthesized in the polydimethylsiloxane (PDMS) microfluidic channels by in situ UV-initiated free radical polymerization. After surface modification, PPM provides a high-surface area and specific affinity 3D substrate for immunoassays. Combining with well controlled microfluidic devices, the direct immunoassay of IgG and sandwich immunoassay of inactivated H1N1 influenza virus using 5 μL sample has been accomplished, with detection limits of 4 ng mL(-1) and less than 10 pg mL(-1), respectively. The enhanced detection sensitivity is due to both high surface area of PPM and flow-through design. The detection time was obviously decreased mainly due to the shortened diffusion distance and improved convective mass transfer inside the monolith, which accelerates the reaction kinetics between antigen and antibody. This work provides a novel microfluidic immunoassay platform with high efficiency thereby enabling fast and sensitive immunoassay.

  11. Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody.

    Science.gov (United States)

    Zhang, Ruiqi; Hirakawa, Koji; Seto, Daisuke; Soh, Nobuaki; Nakano, Koji; Masadome, Takashi; Nagata, Kazumi; Sakamoto, Kazuhira; Imato, Toshihiko

    2005-12-15

    A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500ppb) under optimum conditions. The time required for analysis is less than 15min. PMID:18970310

  12. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

    Directory of Open Access Journals (Sweden)

    Michele D. Kattke

    2011-06-01

    Full Text Available In this study, a fluorescence resonance energy transfer (FRET-based quantum dot (QD immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.

  13. Development of a new passive sampler based on diffusive milligel beads for copper analysis in water

    Energy Technology Data Exchange (ETDEWEB)

    Perez, M.; Reynaud, S.; Lespes, G.; Potin-Gautier, M. [Université de Pau et des Pays de l’Adour/CNRS UMR IPREM 5254, Hélioparc, 2 av. du Président Angot, 64053 Pau (France); Mignard, E. [CNRS-Solvay-Université Bordeaux, UMR5258, Laboratoire du Futur, 178 Avenue du Dr. A. Schweitzer, 33608 Pessac Cedex (France); Chéry, P. [Bordeaux Science Agro, 1 cours du Général De Gaulle, Gradignan, 33175 (France); Schaumlöffel, D. [Université de Pau et des Pays de l’Adour/CNRS UMR IPREM 5254, Hélioparc, 2 av. du Président Angot, 64053 Pau (France); Grassl, B., E-mail: bruno.grassl@univ-pau.fr [Université de Pau et des Pays de l’Adour/CNRS UMR IPREM 5254, Hélioparc, 2 av. du Président Angot, 64053 Pau (France)

    2015-08-26

    A new passive sampler was designed and characterized for the determination of free copper ion (Cu{sup 2+}) concentration in aqueous solution. Each sampling device was composed of a set of about 30 diffusive milligel (DMG) beads. Milligel beads with incorporated cation exchange resin (Chelex) particles were synthetized using an adapted droplet-based millifluidic process. Beads were assumed to be prolate spheroids, with a diameter of 1.6 mm and an anisotropic factor of 1.4. The milligel was controlled in chemical composition of hydrogel (monomer, cross-linker, initiator and Chelex concentration) and characterized in pore size. Two types of sampling devices were developed containing 7.5% and 15% of Chelex, respectively, and 6 nm pore size. The kinetic curves obtained demonstrated the accumulation of copper in the DMG according to the process described in the literature as absorption (and/or adsorption) and release following the Fick's first law of diffusion. For their use in water monitoring, the typical physico-chemical characteristics of the samplers, i.e. the mass-transfer coefficient (k{sub 0}) and the sampler-water partition coefficient (K{sub sw}), were determined based on a static exposure design. In order to determine the copper concentration in the samplers after their exposure, a method using DMG bead digestion combined to Inductively Coupled Plasma – Atomic Emission Spectrometry (ICP-AES) analysis was developed and optimized. The DMG devices proved to be capable to absorb free copper ions from an aqueous solution, which could be accurately quantified with a mean recovery of 99% and a repeatability of 7% (mean relative uncertainty). - Highlights: • Controlled geometry of new passive sampler with ellipsoidal shape. • Original manufacturing process based on droplet-based millifluidic device. • Pore size characterization of the sampler. • Mass-transfer and sampler-water partitioning coefficients by static exposure experiments.

  14. Highly Sensitive Homogeneous Immunoassays Based on Construction of Silver Triangular Nanoplates-Quantum Dots FRET System

    Science.gov (United States)

    Zeng, Qinghui; Li, Qin; Ji, Wenyu; Bin, Xue; Song, Jie

    2016-05-01

    With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D-A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form.

  15. 340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

    Science.gov (United States)

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

    2016-09-19

    We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems. PMID:27661948

  16. Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

    Directory of Open Access Journals (Sweden)

    John McDevitt

    2012-11-01

    Full Text Available Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications.

  17. Assessment of colorimetric amplification methods in a paper-based immunoassay for diagnosis of malaria.

    Science.gov (United States)

    Lathwal, Shefali; Sikes, Hadley D

    2016-04-21

    Colorimetric detection methods that produce results readable by eye are important for diagnostic tests in resource-limited settings. In this work, we have compared three main types of colorimetric methods - enzymatic reactions, silver deposition catalyzed by gold nanoparticles, and polymerization-based amplification - in a paper-based immunoassay for detection of Plasmodium falciparum histidine-rich protein 2, a biomarker of malarial infection. We kept the binding events in the immunoassay constant in order to isolate the effect of the detection method on the outcome of the test. We have highlighted that the optimal readout time in a test can vary significantly - ranging from immediately after addition of a visualization agent to 25 minutes after addition of a visualization agent - depending on the colorimetric method being used, and accurate time keeping is essential to prevent false positives in methods where substantial color develops over time in negative tests. We have also shown that the choice of a colorimetric method impacts the calculated limit-of-detection, the ease of visual perception of the readout, and the total cost of the assay, and therefore directly impacts the feasibility and the ease-of-use of a test in field settings.

  18. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  19. Aggregation-induced emissive nanoparticles for fluorescence signaling in a low cost paper-based immunoassay.

    Science.gov (United States)

    Engels, Jan F; Roose, Jesse; Zhai, Demi Shuang; Yip, Ka Man; Lee, Mei Suet; Tang, Ben Zhong; Renneberg, Reinhard

    2016-07-01

    Low cost paper based immunoassays are receiving interest due to their fast performance and small amounts of biomolecules needed for developing an immunoassay complex. In this work aggregation-induced emissive (AIE) nanoparticles, obtained from a diastereoisomeric mixture of 1,2-di-(4-hydroxyphenyl)-1,2-diphenylethene (TPEDH) in a one-step top-down method, are characterized through Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Zeta potential. By measuring the Zeta potential before and after labeling the nanoparticles with antibodies we demonstrate that the colloidal system is stable in a wide pH-range. The AIE-active nanoparticles are deposited on chitosan and glutaraldehyde modified paper pads overcoming the common aggregation-caused quenching (ACQ) effect. Analyte concentrations from 1000ng and below are applied in a model immunocomplex using Goat anti-Rabbit IgG and Rabbit IgG. In the range of 7.81ng-250ng, linear trends with a high R(2) are observed, which leads to a strong increase of the blue fluorescence from the TPEDH nanoparticles. PMID:27037781

  20. Alginate and Algal-Based Beads for the Sorption of Metal Cations: Cu(II) and Pb(II).

    Science.gov (United States)

    Wang, Shengye; Vincent, Thierry; Faur, Catherine; Guibal, Eric

    2016-01-01

    Alginate and algal-biomass (Laminaria digitata) beads were prepared by homogeneous Ca ionotropic gelation. In addition, glutaraldehyde-crosslinked poly (ethyleneimine) (PEI) was incorporated into algal beads. The three sorbents were characterized by scanning electron microscopy (SEM) coupled with energy dispersive X-ray analysis (EDX): the sorption occurs in the whole mass of the sorbents. Sorption experiments were conducted to evaluate the impact of pH, sorption isotherms, and uptake kinetics. A special attention was paid to the effect of drying (air-drying vs. freeze-drying) on the mass transfer properties. For alginate, freeze drying is required for maintaining the porosity of the hydrogel, while for algal-based sorbents the swelling of the material minimizes the impact of the drying procedure. The maximum sorption capacities observed from experiments were 415, 296 and 218 mg Pb g(-1) and 112, 77 and 67 mg Cu g(-1) for alginate, algal and algal/PEI beads respectively. Though the sorption capacities of algal-beads decreased slightly (compared to alginate beads), the greener and cheaper one-pot synthesis of algal beads makes this sorbent more competitive for environmental applications. PEI in algal beads decreases the sorption properties in the case of the sorption of metal cations under selected experimental conditions. PMID:27598128

  1. Bead-based suspension array for simultaneous differential detection of five major swine viruses.

    Science.gov (United States)

    Chen, Ru; Yu, Xiao-Lu; Gao, Xiao-Bo; Xue, Cun-Yi; Song, Chang-Xu; Li, Yan; Cao, Yong-Chang

    2015-01-01

    A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen. PMID:25557628

  2. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus

    Science.gov (United States)

    Doerflinger, Sylvie Y.; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna

    2016-01-01

    ABSTRACT Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical

  3. Development of a bead-based multiplex genotyping method for diagnostic characterization of HPV infection.

    Directory of Open Access Journals (Sweden)

    Mee Young Chung

    Full Text Available The accurate genotyping of human papillomavirus (HPV is clinically important because the oncogenic potential of HPV is dependent on specific genotypes. Here, we described the development of a bead-based multiplex HPV genotyping (MPG method which is able to detect 20 types of HPV (15 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 5 low-risk HPV types 6, 11, 40, 55, 70 and evaluated its accuracy with sequencing. A total of 890 clinical samples were studied. Among these samples, 484 were HPV positive and 406 were HPV negative by consensus primer (PGMY09/11 directed PCR. The genotyping of 484 HPV positive samples was carried out by the bead-based MPG method. The accuracy was 93.5% (95% CI, 91.0-96.0, 80.1% (95% CI, 72.3-87.9 for single and multiple infections, respectively, while a complete type mismatch was observed only in one sample. The MPG method indiscriminately detected dysplasia of several cytological grades including 71.8% (95% CI, 61.5-82.3 of ASCUS (atypical squamous cells of undetermined significance and more specific for high grade lesions. For women with HSIL (high grade squamous intraepithelial lesion and SCC diagnosis, 32 women showed a PPV (positive predictive value of 77.3% (95% CI, 64.8-89.8. Among women >40 years of age, 22 women with histological cervical cancer lesions showed a PPV of 88% (95% CI, 75.3-100. Of the highest risk HPV types including HPV-16, 18 and 31 positive women of the same age groups, 34 women with histological cervical cancer lesions showed a PPV of 77.3% (95% CI, 65.0-89.6. Taken together, the bead-based MPG method could successfully detect high-grade lesions and high-risk HPV types with a high degree of accuracy in clinical samples.

  4. Luminex-Based Triplex Immunoassay for the Simultaneous Detection of Soy, Pea and Soluble Wheat proteins in Milk Powder

    NARCIS (Netherlands)

    Haasnoot, W.; Pre, du J.G.

    2007-01-01

    An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder.

  5. Updates in immunoassays: parasitology.

    Science.gov (United States)

    Josko, Deborah

    2012-01-01

    Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

  6. Reconfigurable and resettable arithmetic logic units based on magnetic beads and DNA

    Science.gov (United States)

    Zhang, Siqi; Wang, Kun; Huang, Congcong; Sun, Ting

    2015-12-01

    Based on the characteristics of magnetic beads and DNA, a simple and universal platform was developed for the integration of multiple logic gates to achieve resettable half adder and half subtractor functions. The signal reporter was composed of a split G-quadruplex DNAzyme and AuNP-surface immobilized molecular beacon molecule. The novel feature of the designed system is that the inputs (split G-quadruplexes) can interact with hairpin-modified Au NPs linked to magnetic particles. Another novel feature is that the logic operations can be reset by heating the output system and by using the magnetic separation of the computing modules. Moreover, the developed half adder and half subtractor are realized on a simple DNA/magnetic bead platform in an enzyme-free system and share a constant threshold setpoint. Due to the diversity and design flexibility of DNA, these investigations may provide a new method for the development of resettable DNA-based arithmetic operations.Based on the characteristics of magnetic beads and DNA, a simple and universal platform was developed for the integration of multiple logic gates to achieve resettable half adder and half subtractor functions. The signal reporter was composed of a split G-quadruplex DNAzyme and AuNP-surface immobilized molecular beacon molecule. The novel feature of the designed system is that the inputs (split G-quadruplexes) can interact with hairpin-modified Au NPs linked to magnetic particles. Another novel feature is that the logic operations can be reset by heating the output system and by using the magnetic separation of the computing modules. Moreover, the developed half adder and half subtractor are realized on a simple DNA/magnetic bead platform in an enzyme-free system and share a constant threshold setpoint. Due to the diversity and design flexibility of DNA, these investigations may provide a new method for the development of resettable DNA-based arithmetic operations. Electronic supplementary information

  7. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM;

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis...

  8. On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

    International Nuclear Information System (INIS)

    Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL−1 and 50 μg mL−1 of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities

  9. On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

    Energy Technology Data Exchange (ETDEWEB)

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier, E-mail: didier.gasparutto@cea.fr

    2014-02-17

    Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL{sup −1} and 50 μg mL{sup −1} of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair

  10. Peptidomic analysis of Chinese shrimp (Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDITOF MS

    Institute of Scientific and Technical Information of China (English)

    WANG Baojie; LIU Mei; JIANG Keyong; ZHANG Guofan; WANG Lei

    2013-01-01

    Peptides in shrimp hemolymph play an important role in the innate immune response.Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection.We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides.Shrimp serum and plasma were used as the source of samples for comparative analysis,and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis.To screen potential specific biomarkers in serum of immune-challenged shrimps,we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps.The spectra were analyzed using FlexAnalysis 3.0and ClinProTools 2.1 software.Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection.The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%.Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph,and will help to enable a better understanding of the innate immune response of shrimps.

  11. A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

    Directory of Open Access Journals (Sweden)

    Hali Bordelon

    Full Text Available Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3 to 5×10(8 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6, 14×10(6, and 8×10(6 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

  12. A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

    Science.gov (United States)

    Bordelon, Hali; Russ, Patricia K; Wright, David W; Haselton, Frederick R

    2013-01-01

    Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

  13. A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

    Directory of Open Access Journals (Sweden)

    Tsai Chueh-Jen

    2010-01-01

    Full Text Available Abstract There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α and nuclear factor-kappa B (NF-κB were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

  14. Specificity of immunoassays. Pt. 2

    International Nuclear Information System (INIS)

    Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay. (orig.)

  15. Electrochemical magnetic beads-based immunosensing platform for the determination of α-lactalbumin in milk.

    Science.gov (United States)

    Ruiz-Valdepeñas Montiel, Víctor; Campuzano, Susana; Torrente-Rodríguez, Rebeca M; Reviejo, A Julio; Pingarrón, José M

    2016-12-15

    Alpha-lactalbumin (α-LA) is one of the whey proteins in cows' milk that has been identified as allergenic. In this work, we present, for the first time, a very sensitive magnetic beads (MBs)-based immunosensor for the determination of α-LA. A sandwich configuration involving selective capture and horseradish peroxidase-labeled detector antibodies was implemented on carboxylic acid-modified magnetic beads, captured magnetically under the surface of a disposable screen-printed carbon electrode for amperometric detection using the hydroquinone (HQ)/H2O2 system. The α-LA immunosensor exhibited a wide linear range (37.0-5000pg/ml), a low limit of detection (LOD, 11.0pg/ml) and noteworthy selectivity against other non-target proteins. The MBs-based immunosensing platform was applied successfully for the determination of α-LA in several varieties of milk (raw and UHT cows' milk as well as human milk) and infant formulations. The results were corroborated with those obtained using a commercial ELISA method, thereby substantiating the analytical merits of this unique method. PMID:27451223

  16. Design, fabrication and test of a pneumatically controlled, renewable, microfluidic bead trapping device for sequential injection analysis applications

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Guocheng; Lu, Donglai; Fu, Zhifeng; Du, Dan; Ozanich, Richard M.; Wang, Wanjun; Lin, Yuehe

    2016-01-01

    This paper describes the design, fabrication, and testing of a pneumatically controlled,renewable, microfluidic device for conducting bead-based assays in an automated sequential injection analysis system. The device used a “brick wall”-like pillar array (pillar size: 20 μm length X 50 μm width X 45 μm height) with 5 μm gaps between the pillars serving as the micro filter. The flow channel where bead trapping occurred is 500 μm wide X 75 μm deep. An elastomeric membrane and an air chamber were located underneath the flow channel. By applying pressure to the air chamber, the membrane is deformed and pushed upward against the filter structure. This effectively traps beads larger than 5 μm and creates a “bed” or micro column of beads that can be perfused and washed with liquid samples and reagents. Upon completion of the assay process, the pressure is released and the beads are flushed out from underneath the filter structure to renew the device. Mouse IgG was used as a model analyte to test the feasibility of using the proposed device for immunoassay applications. Resulting microbeads from an on-chip fluorescent immunoassay were individually examined using flow cytometry. The results show that the fluorescence signal intensity distribution is fairly narrow indicating high chemical reaction uniformity among the beads population. Electrochemical onchip assay was also conducted. A detection limit of 0.1 ng/mL1 ppb was achieved and good device reliability and repeatability were demonstrated. The novel microfluidic-based beadstrapping device thus opens up a new pathway to design micro-bead based biosensor immunoassays for clinical and othervarious applications.

  17. Bifunctional 4MBA mediated recyclable SERS-based immunoassay induced by photocatalytic activity of TiO2 nanotube arrays.

    Science.gov (United States)

    Wang, Xiaolong; Zhou, Lu; Lai, Wei; Jiang, Tao; Zhou, Jun

    2016-09-14

    We first report here a novel recyclable surface-enhanced Raman scattering (SERS)-based immunoassay via the photocatalytic ability of anatase titania nanotube (TiO2-NT) arrays. In this immunoassay, an immune probe was realized by immobilizing anti-CA19-9 onto Ag@SiO2@Ag three core-shell nanoparticles (TCSNPs), which showed a much higher SERS activity than bare Ag NPs with an enhancement ratio of 1.75. Then, the vertically oriented TiO2-NT immune substrate was synthesized by ultra-fast anodic oxidation of flexible titanium foils and functionalised with 4-mercaptobenzoic acid (4MBA) molecules to link them with anti-CA19-9. The immunoassay using the above immune probe and the substrate exhibited a wide linear range from 1000 to 0.5 U mL(-1) and a low detection limit of 0.5 U mL(-1) for CA19-9 due to the excellent SERS performance of Ag@SiO2@Ag TCSNPs. More importantly, the linkage between TiO2-NTs and 4MBA was destroyed by catalyzing 4MBA into 4-sulfobenzoate upon UV irradiation in O2-saturated water. The target antigen and the immune probe were simultaneously removed leading to a recyclable immunoassay and a detection limit of 5 U mL(-1) was achieved after six cycles. The simplicity and versatility of this strategy may bridge the technology gap between academia and practical detection, which makes it promising for clinical SERS-based immunoassay. PMID:27523026

  18. Magnetic bead-based salivary peptidome profiling for periodontal-orthodontic treatment

    Directory of Open Access Journals (Sweden)

    Zhang Jieni

    2012-11-01

    Full Text Available Abstract Background Patients with periodontitis seek periodontal-orthodontic treatment to address certain functional and aesthetic problems. However, little is known of the effect of periodontitis on orthodontic treatment. Thus, we compared the differences in peptide mass fingerprints of orthodontic patients with and without periodontitis by MALDI-TOF MS using a magnetic bead-based peptidome analysis of saliva samples. In this way, we aimed to identify and explore a panel of differentially-expressed specific peptides. Results Saliva samples from 24 patients (eight orthodontic patients without periodontitis, eight with periodontitis and another eight with periodontitis but no orthodontic treatment were analyzed, and peptide mass fingerprints were created by scanning MS signals using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS combined with magnetic beads. Nine mass peaks showed significant differences. Orthodontic patients in the group without periodontal disease showed higher mass peaks for seven peptides of the nine, whereas the mass peaks for the other two peptides were higher in the periodontal-orthodontic patients. Besides, these differentially-expressed peptides were sequenced. Conclusions The elucidated candidate biomarkers indicated interactions between periodontal condition and orthodontic treatment and their contributions to the changes of saliva protein profiles. Our results provide novel insight into the altered salivary protein profile during periodontal-orthodontic treatment, and may lead to the development of a therapeutic monitoring strategy for periodontics and orthodontics.

  19. Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

    Science.gov (United States)

    Salminen, Teppo; Juntunen, Etvi; Khanna, Navin; Pettersson, Kim; Talha, Sheikh M

    2016-02-01

    There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.

  20. PRELIMINARY STUDY ON RETRO-REFLECTIVE COATED PAPER BASED ON MICRO-GLASS BEADS

    Institute of Scientific and Technical Information of China (English)

    Yulong Wang; Chuanshan Zhao; Tao Zhang

    2004-01-01

    High-reflectivity micro-glass bead, as a kind of retro-reflective material, is widely used in reflective fabric or film and other reflective coatings. But it is rarely used in coated paper. The retro-reflective theory of micro-bead is described. Also the effect of size of micro-bead, dosage of binder and different color layers on reflective properties of coated paper are discussed in this article. The results show that its retro-reflective efficiency is good, equally to reflective fabric or film when the micro-glass bead is used in coated paper.

  1. PRELIMINARY STUDY ON RETRO-REFLECTIVE COATED PAPER BASED ON MICRO-GLASS BEADS

    Institute of Scientific and Technical Information of China (English)

    YulongWang; ChuanshanZhao; TaoZhang

    2004-01-01

    High-reflectivity micro-glass bead, as a kind ofretro-reflective material, is widely used in reflectivefabric or film and other reflective coatings. But it israrely used in coated paper. The retro-reflectivetheory of micro-bead is described. Also the effect ofsize of micro-bead, dosage of binder and differentcolor layers on reflective properties of coated paperare discussed in this article. The results show that itsretro-reflective efficiency is good, equally toreflective fabric or film when the micro-glass bead isused in coated paper.

  2. Development of a Smartphone-based reading system for lateral flow immunoassay.

    Science.gov (United States)

    Lee, Sangdae; Kim, Giyoung; Moon, Jihea

    2014-11-01

    This study was conducted to develop and evaluate the performance of the Smartphone-based reading system for the lateral flow immunoassay (LFIA). Smartphone-based reading system consists of a Samsung Galaxy S2 Smartphone, Smartphone application, and a LFIA reader. LFIA reader is composed of the close-up lens with a focal length up to 30 mm, white LED light, lithium polymer battery, and main body. The Smartphone application for image acquisition and data analysis was developed on the Android platform. The standard curve was obtained by plotting the measured P(T)/P(c) or A(T)/A(c) ratio versus Salmonella standard concentration. The mean, standard deviation (SD), recovery, and relative standard deviation (RSD) were also calculated using additional experimental results. These data were compared with that obtained from the benchtop LFIA reader. The LOD in both systems was observed with 10(6) CFU/mL. The results show high accuracy and good reproducibility with a RSD less than 10% in the range of 10(6) to 10(9) CFU/mL. Due to the simple structure, good sensitivity, and high accuracy of the Smartphone-based reading system, this system can be substituted for the benchtop LFIA reader for point-of-care medical diagnostics. PMID:25958545

  3. Color-encoded microcarriers based on nano-silicon dioxide film for multiplexed immunoassays.

    Science.gov (United States)

    Li, Qiang; Zhang, Kaihuan; Wang, Tongzhou; Zhou, Xinying; Wang, Jia; Wang, Chen; Lin, Haixiao; Li, Xin; Lu, Ying; Huang, Guoliang

    2012-08-21

    Multiplexed analysis allows researchers to obtain high-density information with minimal assay time, sample volume and cost. Currently, microcarrier or particle-based approaches for multiplexed analysis involve complicated or expensive encoding and decoding processes. In this paper, a novel optical encoding technique based on nano-silicon dioxide film is presented. Microcarriers composed of thermally grown silicon dioxide (SiO(2)) film and monocrystalline silicon (Si) substrate were fabricated. The nano-silicon dioxide film exhibited unique surface color by low-coherence interference. Hence the colors can be used for encoding at least 100 microcarriers loaded with films of different thickness. We demonstrated that color-encoded microcarriers loaded with antigens could be used for multiplexed immunoassays to detect goat anti-human IgG, goat anti-mouse IgG and goat anti-rabbit IgG, with fluorescent detection as the interrogating approach. This microcarrier-based method also exhibited improved analytical performance compared with a microarray technique. This approach will provide new opportunities for multiplexed target assay development. PMID:22745929

  4. Chip-Based Measurements of Brownian Relaxation of Magnetic Beads Using a Planar Hall Effect Magnetic Field Sensor

    DEFF Research Database (Denmark)

    Østerberg, Frederik Westergaard; Dalslet, Bjarke Thomas; Snakenborg, Detlef;

    2010-01-01

    We present a simple 'click-on' fluidic system with integrated electrical contacts, which is suited for electrical measurements on chips in microfluidic systems. We show that microscopic magnetic field sensors based on the planar Hall effect can be used for detecting the complex magnetic response...... using only the self-field arising from the bias current applied to the sensors as excitation field. We present measurements on a suspension of magnetic beads with a nominal diameter of 250 nm vs. temperature and find that the observations are consistent with the Cole-Cole model for Brownian relaxation...... biosensor based on the detection of the dynamic response of magnetic beads....

  5. Chip-Based Measurements of Brownian Relaxation of Magnetic Beads Using a Planar Hall Effect Magnetic Field Sensor

    Science.gov (United States)

    Østerberg, Frederik W.; Dalslet, Bjarke T.; Snakenborg, Detlef; Johansson, Christer; Hansen, Mikkel F.

    2010-12-01

    We present a simple `click-on' fluidic system with integrated electrical contacts, which is suited for electrical measurements on chips in microfluidic systems. We show that microscopic magnetic field sensors based on the planar Hall effect can be used for detecting the complex magnetic response using only the self-field arising from the bias current applied to the sensors as excitation field. We present measurements on a suspension of magnetic beads with a nominal diameter of 250 nm vs. temperature and find that the observations are consistent with the Cole-Cole model for Brownian relaxation with a constant hydrodynamic bead diameter when the temperature dependence of the viscosity of water is taken into account. These measurements demonstrate the feasibility of performing measurements of the Brownian relaxation response in a lab-on-a-chip system and constitute the first step towards an integrated biosensor based on the detection of the dynamic response of magnetic beads.

  6. Magnetic hydrogel beads based on PVA/sodium alginate/laponite RD and studying their BSA adsorption.

    Science.gov (United States)

    Mahdavinia, Gholam Reza; Mousanezhad, Sedigheh; Hosseinzadeh, Hamed; Darvishi, Farshad; Sabzi, Mohammad

    2016-08-20

    In this study double physically crosslinked magnetic hydrogel beads were developed by a simple method including solution mixing of sodium alginate and poly(vinyl alcohol) (PVA) containing magnetic laponite RD (Rapid Dispersion). Sodium alginate and PVA were physically crosslinked by Ca(2+) and freezing-thawing cycles, respectively. Magnetic laponite RD nanoparticles were incorporated into the system to create magnetic response and strengthen the hydrogels. All hybrids double physically crosslinked hydrogel beads were stable under different pH values without any disintegration. Furthermore, adsorption of bovine serum albumin (BSA) on the hydrogel beads was investigated on the subject of pH, ion strength, initial BSA concentration, and temperature. Nanocomposite beads exhibited maximum adsorption capacity for BSA at pH=4.5. The experimental adsorption isotherm data were well followed Langmuir model and based on this model the maximum adsorption capacity was obtained 127.3mgg(-1) at 308K. Thermodynamic parameters revealed spontaneous and monolayer adsorption of BSA on magnetic nanocomposites beads.

  7. Magnetic hydrogel beads based on PVA/sodium alginate/laponite RD and studying their BSA adsorption.

    Science.gov (United States)

    Mahdavinia, Gholam Reza; Mousanezhad, Sedigheh; Hosseinzadeh, Hamed; Darvishi, Farshad; Sabzi, Mohammad

    2016-08-20

    In this study double physically crosslinked magnetic hydrogel beads were developed by a simple method including solution mixing of sodium alginate and poly(vinyl alcohol) (PVA) containing magnetic laponite RD (Rapid Dispersion). Sodium alginate and PVA were physically crosslinked by Ca(2+) and freezing-thawing cycles, respectively. Magnetic laponite RD nanoparticles were incorporated into the system to create magnetic response and strengthen the hydrogels. All hybrids double physically crosslinked hydrogel beads were stable under different pH values without any disintegration. Furthermore, adsorption of bovine serum albumin (BSA) on the hydrogel beads was investigated on the subject of pH, ion strength, initial BSA concentration, and temperature. Nanocomposite beads exhibited maximum adsorption capacity for BSA at pH=4.5. The experimental adsorption isotherm data were well followed Langmuir model and based on this model the maximum adsorption capacity was obtained 127.3mgg(-1) at 308K. Thermodynamic parameters revealed spontaneous and monolayer adsorption of BSA on magnetic nanocomposites beads. PMID:27178944

  8. Microsphere-based immunoassay integrated with a microfluidic network to perform logic operations

    International Nuclear Information System (INIS)

    Lab on a chip (LOC) intelligent diagnostics can be described by molecular logic-based circuits. We report on the development of an LOC approach with logic capability for screening combinations of antigen and antibody in the same sample. A microsphere-based immunoassay was integrated with a microfluidic network device to perform the logic operations AND and INHIBIT. Using the clinically relevant biomarkers TNF-α cytokine and anti-TNF-α antibody, we obtained a fluorescent output in the presence of both inputs. This results in an AND operation, while the presence of only one specific input results in a different fluorescent signal, thereby indicating the INHIBIT operation. This approach demonstrates the effective use of molecular logic computation for developing portable, point-of-care technologies for diagnostic purposes due to fast detection times, minimal reagent consumption and low costs. This model system may be further expanded to screening of multiple disease markers, combinatorial logic applications, and developing “smart” sensors and therapeutic technologies. (author)

  9. Sensitive QD@SiO2-based immunoassay for triplex determination of cereal-borne mycotoxins.

    Science.gov (United States)

    Beloglazova, Natalia V; Foubert, Astrid; Gordienko, Anna; Tessier, Mickael D; Aubert, Tangi; Drijvers, Emile; Goryacheva, Irina; Hens, Zeger; De Saeger, Sarah

    2016-11-01

    A sensitive tool for simultaneous quantitative determination of three analytes in one single well of a microtiter plate is shown for the first time. The developed technique is based on use of colloidal quantum dot enrobed into a silica shell (QD@SiO2) derivatives as a highly responsive label. Silica-coated quantum dots were prepared and subsequently modified via the co-hydrolysis with tetraethylorthosilicate (TEOS) and various organosilane reagents. Different surface modification schemes were compared in terms of applicability of the obtained particles for the multiplex immunoassay, e.g. stability and simplicity of their conjugation with biomolecules. As model system a multiplex immunosorbent assay for screening of three mycotoxins (deoxynivalenol, zearalenone and aflatoxin B1) in cereal-based products was realized via a co-immobilization of three different specific antibodies (anti- deoxynivalenol, anti-zearalenone and anti-aflatoxin B1) in one single well of a microtiter plate. Mycotoxins were simultaneously determined by labelling their conjugates with QD@SiO2 emitting in different parts of the visible spectrum. The limits of detection for the simultaneous determination were 6.1 and 5.3, 5.4 and 4.1, and 2.6 and 1.9µgkg(-1) for deoxynivalenol, zearalenone and aflatoxin B1 in maize and wheat, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used. PMID:27591588

  10. Dual-layered and double-targeted nanogold based lateral flow immunoassay for influenza virus

    International Nuclear Information System (INIS)

    We report on a highly sensitive lateral flow immunoassay (LFIA) for influenza A which serves as a model antigen. Gold nanoparticles conjugated to monoclonal antibodies specific to the two most abundant influenza A proteins, nucleoprotein and matrix protein, were used as detector probes. Using this approach, the nucleoprotein and matrix protein in the virion were detected simultaneously. The signal was further amplified via a signal amplification strategy that is making use of two-layered nanogold in combination with a double-targeted detection format. Under optimized conditions, the system is capable of detecting influenza A antigens in infected cells at levels as low as 47 TCID50 · mL−1 (50 % tissue culture infectious dose) within 15 min. Compared to the conventional LFIA based on single-targeted detection, the detection capability of this system is better by a factor of 8 without requiring additional steps or instruments. In addition to its simplicity and rapidity, this LFIA also can detect the target analyte in even complex biological matrix. This proof-of-principle of a dual-layered and double-targeted nanogold-based LFIA is deemed to be useful for developing single-step, rapid, and sensitive tests for screening and diagnosis. (author)

  11. Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots.

    Science.gov (United States)

    Malashikhina, Natalia; Garai-Ibabe, Gaizka; Pavlov, Valeri

    2013-07-16

    In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) with S(2-) ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL(-1) of analyte antibody with a linear range up to 10 ng mL(-1). The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric p-nitrophenyl phosphate assay.

  12. Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay

    OpenAIRE

    Kohlgraf, Karl G.; Ackermann, Abbey R.; Burnell, Kindra K.; Srikantha, Rupasree N.; Joly, Sophie A.; Bartlett, Jennifer; Gakhar, Lokesh; Johnson, Georgia K.; McCray, Paul B.; Guthmiller, Janet M.; Brogden, Kim A

    2011-01-01

    The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1...

  13. Toward development of a surface-enhanced Raman scattering (SERS)-based cancer diagnostic immunoassay panel.

    Science.gov (United States)

    Granger, Jennifer H; Granger, Michael C; Firpo, Matthew A; Mulvihill, Sean J; Porter, Marc D

    2013-01-21

    Proteomic analyses of readily obtained human fluids (e.g., serum, urine, and saliva) indicate that the diagnosis of complex diseases will be enhanced by the simultaneous measurement of multiple biomarkers from such samples. This paper describes the development of a nanoparticle-based multiplexed platform that has the potential for simultaneous read-out of large numbers of biomolecules. For this purpose, we have chosen pancreatic adenocarcinoma (PA) as a test bed for diagnosis and prognosis. PA is a devastating form of cancer in which an estimated 86% of diagnoses resulted in death in the United States in 2010. The high mortality rate is due, in part, to the asymptomatic development of the disease and the dearth of sensitive diagnostics available for early detection. One promising route lies in the development of a serum biomarker panel that can generate a signature unique to early stage PA. We describe the design and development of a proof-of-concept PA biomarker immunoassay array coupled with surface-enhanced Raman scattering (SERS) as a sensitive readout method. PMID:23150876

  14. Spectrum-Based Electrochemiluminescent Immunoassay with Ternary CdZnSe Nanocrystals as Labels.

    Science.gov (United States)

    Zhang, Xin; Tan, Xiao; Zhang, Bin; Miao, Wujian; Zou, Guizheng

    2016-07-01

    Conventional electrochemiluminescence (ECL) research has been performed by detecting the total photons (i.e., the ECL intensity). Herein, systematic spectral exploration on the ECL of dual-stabilizers-capped ternary CdZnSe nanocrystals (NCs) and its sensing application were carried out on a homemade ECL spectral acquiring system. The ternary CdZnSe NCs could be repeatedly injected with electrons via some electrochemical ways and then result in strong cathodic ECL with the coupling of ammonium persulfate. ECL spectrum of the CdZnSe NCs was almost identical to corresponding photoluminescence spectrum, indicating that the excited states of CdZnSe NCs in ECL were essentially the same as those in photoluminescence. Importantly, after being labeled to the probe antibody (Ab2) of α-fetal protein (AFP) antigen, the ternary NCs in the Ab2|NCs conjugates could preserve their ECL spectrum very well. A spectrum-based ECL immunoassay was consequently proposed with the CdZnSe NCs as ECL tags and AFP as target molecules. The limit of detection is 0.010 pg/mL, with a signal-to-noise (S/N) ratio of 3, indicating a sensitive ECL sensing strategy that was different from the conventional ones. This work might open a pathway to the spectrally resolved ECL analysis with even-higher S/N ratios than the fluorescent analysis. PMID:27266486

  15. Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

    Science.gov (United States)

    Melero, Mar; Giménez-Lirola, Luis G; Rubio-Guerri, Consuelo; Crespo-Picazo, José Luis; Sierra, Eva; García-Párraga, Daniel; García-Peña, Francisco Javier; Arbelo, Manuel; Álvaro, Teresa; Valls, Mónica; Sánchez-Vizcaíno, José Manuel

    2016-01-13

    A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed. PMID:26758657

  16. Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

    Science.gov (United States)

    Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

    2014-07-01

    In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity. PMID:24756606

  17. Plastic-Chip-Based Magnetophoretic Immunoassay for Point-of-Care Diagnosis of Tuberculosis.

    Science.gov (United States)

    Kim, Jeonghyo; Jang, Minji; Lee, Kyoung G; Lee, Kil-Soo; Lee, Seok Jae; Ro, Kyung-Won; Kang, In Sung; Jeong, Byung Do; Park, Tae Jung; Kim, Hwa-Jung; Lee, Jaebeom

    2016-09-14

    Tuberculosis (TB) remains a relevant infectious disease in the 21st century, and its extermination is still far from being attained. Due to the extreme infectivity of incipient TB patients, a rapid sensing system for proficient point-of-care (POC) diagnostics is required. In our study, a plastic-chip-based magnetophoretic immunoassay (pcMPI) is introduced using magnetic and gold nanoparticles (NPs) modified with Mycobacterium tuberculosis (MTB) antibodies. This pcMPI offers an ultrasensitive limit of detection (LOD) of 1.8 pg·ml(-1) for the detection of CFP-10, an MTB-secreted antigen, as a potential TB biomarker with high specificity. In addition, by combining the plastic chip with an automated spectrophotometer setup, advantages include ease of operation, rapid time to results (1 h), and cost-effectiveness. Furthermore, the pcMPI results using clinical sputum culture filtrate samples are competitively compared with and integrated with clinical data collected from conventional tools such as the acid-fast bacilli (AFB) test, mycobacteria growth indicator tube (MGIT), polymerase chain reaction (PCR), and physiological results. CFP-10 concentrations were consistently higher in patients diagnosed with MTB infection than those seen in patients infected with nontuberculosis mycobacteria (NTM) (P < 0.05), and this novel test can distinguish MTB and NTM while MGIT cannot. All these results indicate that this pcMPI has the potential to become a new commercial TB diagnostic POC platform in view of its sensitivity, portability, and affordability.

  18. A New Surface Plasmon Resonance-Based Immunoassay for Rapid, Reproducible and Sensitive Quantification of Pentraxin-3 in Human Plasma

    Directory of Open Access Journals (Sweden)

    Mara Canovi

    2014-06-01

    Full Text Available A new immunoassay based on surface plasmon resonance (SPR for the rapid, reproducible and sensitive determination of pentraxin-3 (PTX3 levels in human plasma has been developed and characterized. The method involves a 3-min flow of plasma over a sensor chip pre-coated with a monoclonal anti-PTX3 antibody (MNB4, followed by a 3-min flow of a polyclonal anti-PTX3 antibody (pAb, required for specific recognition of captured PTX3. The SPR signal generated with this secondary antibody linearly correlates with the plasma PTX3 concentration, in the range of 5–1500 ng/mL, with a lowest limit of detection of 5 ng/mL. The PTX3 concentrations determined with the SPR-based immunoassay in the plasma of 21 patients with sepsis, ranging 15–1600 ng/mL, were superimposable to those found in a classic ELISA immunoassay. Since the PTX3 concentration in the plasma of healthy subjects is <2 ng/mL, but markedly rises in certain medical conditions, the method is useful to quantify pathological levels of this important biomarker, with important diagnostic applications. In comparison with the classic ELISA, the SPR-based approach is much faster (30 min versus 4–5 h and could be exploited for the development of new cost-effective SPR devices for point-of-care diagnosis.

  19. Investigation of voltammetric enzyme-linked immunoassay based on new system of ODA-H2O2-HRP

    Institute of Scientific and Technical Information of China (English)

    焦奎; 张书圣; 韦璐

    1996-01-01

    A voltammetric enzyme-linked immunoassay based on a new system of ODA-H2O2-HRP has first been developed and used in the detection of HRP and labelled HRP. By this method, the enzyme-catalyzing reaction of H2O2 oxidizing odianisidine (ODA) couples the electrode-reduction reaction of the oxidizing product of odianisidine, which produces a sensitive polarographic wave at potential of -0.56V (SCE) in Britton-Robinson buffer solution. In using this polarographic wave, a detection limit to HRP is 3.7×10-12g/mL and a linear range 1.0×10-11-2.0×10-9g/mL. And the mechanisms of the coupling reaction and the process of electro-reduction in the ODA-H2O2-HRP voltammetric enzyme-linked immunoassay system have also been carefully studied.

  20. Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR

    OpenAIRE

    Sirdah, Mahmoud Mohammed

    2014-01-01

    Introduction: Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS).

  1. Fabrication and characterization of tosyl-activated magnetic and nonmagnetic monodisperse microspheres for use in microfluic-based ferritin immunoassay.

    Science.gov (United States)

    Reymond, Frédéric; Vollet, Christine; Plichta, Zdeněk; Horák, Daniel

    2013-01-01

    This article describes the preparation of tosyl-activated nonmagnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [P(HEMA-GMA)] microspheres by dispersion polymerization and tosyl-activated magnetic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) [P(HEMA-EDMA)] microspheres by multistep swelling polymerization method and precipitation of iron oxide inside the pores. These new approaches show that monodisperse microspheres, 2.3 µm, respectively 4.1 µm, in diameter can be produced in high yields avoiding aggregation and with the advantage of being free of aromatic moieties. To demonstrate their potential for diagnostic applications, both types of microparticles have been coated with capture and detection antibodies (DAs), respectively. Immunoassay protocols have then been developed for the dosage of ferritin using an automated affinity platform combining microchannel chips and electrochemical detection. The assay performance using the above magnetic microspheres has been compared with that obtained with commercial tosyl-activated beads. Finally, the possibility to combine functionalized magnetic and nonmagnetic microspheres has been evaluated in view of amplifying the number of enzymatic labels in the immuno-complex. At a ferritin concentration of 119.6 ng/mL, a signal-to-noise ratio of 150.5 is obtained using 0.2 mg/mL of anti-ferritin-coated P(HEMA-GMA)-DA microspheres against a value of 158.8 using free DA in solution.

  2. Layer-by-layer multienzyme assembly for highly sensitive electrochemical immunoassay based on tyramine signal amplification strategy.

    Science.gov (United States)

    Zhou, Jun; Tang, Juan; Chen, Guonan; Tang, Dianping

    2014-04-15

    A new sandwich-type electrochemical immunosensor based on nanosilver-doped bovine serum albumin microspheres (Ag@BSA) with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for quantitative monitoring of biomarkers (carcinoembryonic antigen, CEA, used in this case) by coupling enzymatic biocatalytic precipitation with tyramine signal amplification strategy on capture antibody-modified glassy carbon electrode. Two immunosensing protocols (with and without tyramine signal amplification) were also investigated for the detection of CEA and improved analytical features were acquired with tyramine signal amplification strategy. With the labeling method, the performance and factors influencing the electrochemical immunoassay were studied and evaluated in detail. Under the optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-80 ng mL(-1) toward CEA standards with a low detection limit of 5.0 pg mL(-1). Intra- and inter-assay coefficients of variation were below 11%. No significant differences at the 0.05 significance level were encountered in the analysis of 6 clinical serum specimens and 6 spiked new-born cattle serum samples between the electrochemical immunoassay and the commercialized electrochemiluminescent immunoassay method for the detection of CEA.

  3. Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

    Science.gov (United States)

    Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

    2016-06-01

    A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as

  4. Good performance of an immunoassay based method for nevirapine measurements in human breast milk

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Persson Theilgaard, Zahra; Chiduo, Mercy;

    2011-01-01

    Understanding the distribution of antiretro-virals in breastfeeding HIV-positive mothers is essential, both for prevention of mother-to-child HIV transmission and for research on the development of drug resistance. The ARK nevirapine (NVP)-test is an immunoassay method for nevirapine measurements...

  5. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

    Science.gov (United States)

    The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

  6. On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

    Science.gov (United States)

    Kongsuphol, Patthara; Liu, Yunxiao; Ramadan, Qasem

    2016-10-01

    Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

  7. Taguchi design-based optimization of sandwich immunoassay microarrays for detecting breast cancer biomarkers.

    Science.gov (United States)

    Luo, Wen; Pla-Roca, Mateu; Juncker, David

    2011-07-15

    Taguchi design, a statistics-based design of experiment method, is widely used for optimization of products and complex production processes in many different industries. However, its use for antibody microarray optimization has remained underappreciated. Here, we provide a brief explanation of Taguchi design and present its use for the optimization of antibody sandwich immunoassay microarray with five breast cancer biomarkers: CA15-3, CEA, HER2, MMP9, and uPA. Two successive optimization rounds with each 16 experimental trials were performed. We tested three factors (capture antibody, detection antibody, and analyte) at four different levels (concentrations) in the first round and seven factors (including buffer solution, streptavidin-Cy5 dye conjugate concentration, and incubation times for five assay steps) with two levels each in the second round; five two-factor interactions between selected pairs of factors were also tested. The optimal levels for each factor as measured by net assay signal increase were determined graphically, and the significance of each factor was analyzed statistically. The concentration of capture antibody, streptavidin-Cy5, and buffer composition were identified as the most significant factors for all assays; analyte incubation time and detection antibody concentration were significant only for MMP9 and CA15-3, respectively. Interactions between pairs of factors were identified, but were less influential compared with single factor effects. After Taguchi optimization, the assay sensitivity was improved between 7 and 68 times, depending on the analyte, reaching 640 fg/mL for uPA, and the maximal signal intensity increased between 1.8 and 3 times. These results suggest that Taguchi design is an efficient and useful approach for the rapid optimization of antibody microarrays.

  8. Ultrasensitive electrochemiluminescence immunoassay for tumor marker based on quantum dots coated carbon nanospheres

    Energy Technology Data Exchange (ETDEWEB)

    Li, Long; Zhang, Yan; Li, Shuai; Wang, Xiu; Li, Chen [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Ge, Shenguang [Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, University of Jinan, Jinan 250022 (China); Yu, Jinghua, E-mail: ujn.yujh@gmail.com [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Yan, Mei [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Song, Xianrang [Cancer Research Center, Shandong Tumor Hospital, Jinan 250012 (China)

    2013-12-15

    In this work, a novel electrochemiluminescence (ECL) immunosensor based on CdTe quantum dots (QDs) coated carbon nanosphere (CN/QDs) for the detection of carcinoembryonic antigen (CEA) was developed. The carbon nanospheres (CNs) with good monodispersity and uniform structure were synthetized by a hydrothermal method using glucose as raw material. Then QDs functionized CNs were prepared and employed for signal amplification to improve the sensitivity and the detection limit of immunosensor. For this proposed immunosensor, chitosan was firstly deposited on the pretreated indium tin oxide (ITO) electrode surface, which promoted the electron transfer. Subsequently, gold nanoparticles (AuNPs) were assembled onto chitosan film modified electrode to improve the absorption capacity of antibodies. Then, primary antibodies were immobilized onto the electrode through the reaction between AuNPs and amino. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was obtained on the prepared CN/QDs. The CEA was determined in the range of 0.005–200 ng mL{sup −1}, with a low detection limit of 1.2 pg mL{sup −1} (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which could be applied in more bioanalytical systems. -- Highlights: • A sandwich-type electrochemluminence immunosensor was fabricated. • CdTe quantum dots coated carbon nanospheres were used to amplify signals. • Au–chitosan biocompatible membrane modified on ITO electrode to capture antibodies.

  9. Ultrasensitive electrochemiluminescence immunoassay for tumor marker based on quantum dots coated carbon nanospheres

    International Nuclear Information System (INIS)

    In this work, a novel electrochemiluminescence (ECL) immunosensor based on CdTe quantum dots (QDs) coated carbon nanosphere (CN/QDs) for the detection of carcinoembryonic antigen (CEA) was developed. The carbon nanospheres (CNs) with good monodispersity and uniform structure were synthetized by a hydrothermal method using glucose as raw material. Then QDs functionized CNs were prepared and employed for signal amplification to improve the sensitivity and the detection limit of immunosensor. For this proposed immunosensor, chitosan was firstly deposited on the pretreated indium tin oxide (ITO) electrode surface, which promoted the electron transfer. Subsequently, gold nanoparticles (AuNPs) were assembled onto chitosan film modified electrode to improve the absorption capacity of antibodies. Then, primary antibodies were immobilized onto the electrode through the reaction between AuNPs and amino. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was obtained on the prepared CN/QDs. The CEA was determined in the range of 0.005–200 ng mL−1, with a low detection limit of 1.2 pg mL−1 (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which could be applied in more bioanalytical systems. -- Highlights: • A sandwich-type electrochemluminence immunosensor was fabricated. • CdTe quantum dots coated carbon nanospheres were used to amplify signals. • Au–chitosan biocompatible membrane modified on ITO electrode to capture antibodies

  10. SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles.

    Science.gov (United States)

    Lopez, Arielle; Lovato, Francis; Oh, Soon Hwan; Lai, Yen H; Filbrun, Seth; Driskell, Elizabeth A; Driskell, Jeremy D

    2016-01-01

    A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200 nm and AuNP diameter of 80 nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9 ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody-antigen system. Furthermore, this assay required only 60 min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection. PMID:26695280

  11. Hot embossed polyethylene through-hole chips for bead-based microfluidic devices

    Science.gov (United States)

    Chou, Jie; Du, Nan; Ou, Tina; Floriano, Pierre N.; Christodoulides, Nicolaos; McDevitt, John T.

    2013-01-01

    Over the past decade, there has been a growth of interest in the translation of microfluidic systems into real-world clinical practice, especially for use in point-of-care or near patient settings. While initial fabrication advances in microfluidics involved mainly the etching of silicon and glass, the economics of scaling of these materials is not amendable for point-of-care usage where single-test applications forces cost considerations to be kept low and throughput high. As such, a materials base more consistent with point-of-care needs is required. In this manuscript, the fabrication of a hot embossed, through-hole low-density polyethylene ensembles derived from an anisotropically etched silicon wafer is discussed. This semi-opaque polymer that can be easily sterilized and recycled provides low background noise for fluorescence measurements and yields more affordable cost than other thermoplastics commonly used for microfluidic applications such as cyclic olefin copolymer (COC). To fabrication through-hole microchips from this alternative material for microfluidics, a fabrication technique that uses a high-temperature, high-pressure resistant mold is described. This aluminum-based epoxy mold, serving as the positive master mold for embossing, is casted over etched arrays of pyramidal pits in a silicon wafer. Methods of surface treatment of the wafer prior to casting and PDMS casting of the epoxy are discussed to preserve the silicon wafer for future use. Changes in the thickness of polyethylene are observed for varying embossing temperatures. The methodology described herein can quickly fabricate 20 disposable, single use chips in less than 30 minutes with the ability to scale up 4x by using multiple molds simultaneously. When coupled as a platform supporting porous bead sensors, as in the recently developed Programmable Bio-Nano-Chip, this bead chip system can achieve limits of detection, for the cardiac biomarker C-reactive protein, of 0.3 ng/mL, thereby

  12. Vaccine potential of plasma bead-based dual antigen delivery system against experimental murine candidiasis.

    Science.gov (United States)

    Ahmad, Ejaj; Zia, Qamar; Fatima, Munazza Tamkeen; Owais, Mohammad; Saleemuddin, Mohammed

    2015-11-01

    The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

  13. IDENTIFICATION OF ENCODED BEADS

    DEFF Research Database (Denmark)

    2005-01-01

    The present invention is relates to methods for the identification of spatially encoded beaded or granulated matrices comprising a plurality of immobilised particles. The identification is based on a distance matrix determination or based on a set of geometrical figures, such a triangles, on the ......The present invention is relates to methods for the identification of spatially encoded beaded or granulated matrices comprising a plurality of immobilised particles. The identification is based on a distance matrix determination or based on a set of geometrical figures, such a triangles...

  14. IFSA: a microfluidic chip-platform for frit-based immunoassay protocols

    Science.gov (United States)

    Hlawatsch, Nadine; Bangert, Michael; Miethe, Peter; Becker, Holger; Gärtner, Claudia

    2013-03-01

    Point-of-care diagnostics (POC) is one of the key application fields for lab-on-a-chip devices. While in recent years much of the work has concentrated on integrating complex molecular diagnostic assays onto a microfluidic device, there is a need to also put comparatively simple immunoassay-type protocols on a microfluidic platform. In this paper, we present the development of a microfluidic cartridge using an immunofiltration approach. In this method, the sandwich immunoassay takes place in a porous frit on which the antibodies have immobilized. The device is designed to be able to handle three samples in parallel and up to four analytical targets per sample. In order to meet the critical cost targets for the diagnostic market, the microfluidic chip has been designed and manufactured using high-volume manufacturing technologies in mind. Validation experiments show comparable sensitivities in comparison with conventional immunofiltration kits.

  15. Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

    DEFF Research Database (Denmark)

    Barken, K.B.; Cabig-Ciminska, M.; Holmgren, A.;

    2004-01-01

    Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding sit....... Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.......Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site...

  16. Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform.

    Science.gov (United States)

    Zhou, Liping; Zhu, Anna; Lou, Xuening; Song, Dan; Yang, Rong; Shi, Hanchang; Long, Feng

    2016-01-28

    A universal sandwich-like immunoassay strategy based on quantum-dots immunoprobe (QD-labeled anti-mouse IgG antibody) was developed for rapid and ultrasensitive detection of small molecules. A portable and reusable optofluidic nano-biosensing platform was applied to investigate the sandwich-like immunoassay mechanism and format of small molecules, as well as the binding kinetics between QD immunoprobe and anti-small molecule antibody. A two-step immunoassay method that involves pre-incubation mixture of different concentration of small molecule and anti-small molecule antibody, and subsequent introduction of QD immunoprobe into the optofluidic cell was conducted for small molecule determination. Compared with the one-step immunoassay method, the two-step immunoassay method can obtain higher fluorescence signal and higher sensitivity index, thus improving the nano-biosensing performance. Based on the proposed strategy, two mode targets, namely, microcystin-LR (MC-LR) and Bisphenol A (BPA) were tested with high sensitivity, rapidity, and ease of use. A higher concentration of small molecules in the sample led to less anti-small molecule antibody bound with antigen-carrier protein conjugate immobilized onto the sensor surface, and less QD immunoprobes bound with anti-small molecule antibody. This phenomenon lowered the fluorescence signal detected by nano-biosensing platform. Under optimal operating conditions, MC-LR and BPA exhibited a limit of detection of 0.003 and 0.04 μg/L, respectively. The LODs were better than those of the indirect competitive immunoassay method for small molecules via Cy5.5-labeled anti-small molecule antibody. The proposed QD-based sandwich-like immunoassay strategy was evaluated in spiked water samples, and showed good recovery, precision and accuracy without complicated sample pretreatments. All these results demonstrate that the new detection strategy could be readily applied to the other trace small molecules in real water samples.

  17. Tyramine-based enzymatic conjugate repeats for ultrasensitive immunoassay accompanying tyramine signal amplification with enzymatic biocatalytic precipitation.

    Science.gov (United States)

    Hou, Li; Tang, Yun; Xu, Mingdi; Gao, Zhuangqiang; Tang, Dianping

    2014-08-19

    A new impedimetric immunoassay protocol based on enzyme-triggered formation of tyramine-enzyme repeats on gold nanoparticle (AuNP) was designed for highly sensitive detection of carcinoembryonic antigen (CEA, as a model) by virtue of utilizing enzymatic biocatalytic precipitation toward 4-chloro-1-naphthol (4-CN) on anti-CEA antibody (Ab1)-modified immunosensor. Initially, AuNP was functionalized with horseradish peroxidase and detection antibody (HRP-AuNP-Ab2), and then HRP-tyramine conjugate was utilized for the formation of tyramine-HRP repeats through the triggering of the immobilized HRP on the AuNP with the aid of H2O2. In the presence of target CEA, the carried HRP-tyramine repeats accompanying the sandwiched immunocomplex catalyzed the 4-CN oxidation to produce an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Three signal-transduction tags including HRP-Ab2, HRP-AuNP-Ab2, and HRP-AuNP-Ab2 with HRP-tyramine repeats were employed for target CEA evaluation, and improved analytical properties were achieved by HRP-AuNP-Ab2 with HRP-tyramine repeats. Using the unique signal-transduction tag, the analytical performance of the impedimetric immunoassay was studied in detail. Under the optimal conditions, the impedimetric immunosensor displayed a wide dynamic working range of between 0.5 pg mL(-1) and 40 ng mL(-1) with a detection limit (LOD) of 0.38 pg mL(-1) relative to target CEA. The coefficients of variation (CVs) were ≤9.3% and 13.3% for the intra-assay and interassay, respectively. The levels of CEA in eight clinical serum specimens were measured by using the developed impedimetric immunosensor. The obtained results correlated well with those from the electrochemiluminescent (ECL)-based immunoassay with a correlation coefficient of 0.998.

  18. A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

    Directory of Open Access Journals (Sweden)

    Bokken Gertie CAM

    2012-03-01

    Full Text Available Abstract Background A novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against Toxoplasma gondii and Trichinella spiralis in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one T. spiralis and three T. gondii ELISAs. For this purpose, sera from T. gondii and T. spiralis experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively. Results Results indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for T. gondii and T. spiralis, respectively, while that of the T. gondii ELISAs ranged between 0.837 and 0.930 and the T. spiralis ELISA was 0.879. Bead-based T. gondii assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based T. spiralis assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the T. gondii bead-based test and one of the T. gondii ELISAs. Moreover, in this test combination and between T. spiralis bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of

  19. A comparison of horseradish peroxidase, gold nanoparticles and quantum dots as labels in non-instrumental gel-based immunoassay

    International Nuclear Information System (INIS)

    We have developed a column gel-based immunoassays for the model analyte benzo[a]pyrene (B[a]P), and have evaluated three different kinds of labels, i. e., horseradish peroxidase, (HRP), colloidal gold (AuNPs), and quantum dots (QDs) with respect to rapid visual on-site testing. In case of HRP, a derivative of B[a]P-BA (benzo[a]pyrenebutyric acid) was directly coupled to HRP. In case of QDs and AuNPs, protein conjugates of B[a]P-BA were synthesized and used for surface functionalization. With the latter, previous coverage of the gold surface with 11-mercaptoundecanoic acid turned out to be advantageous in terms of antibody recognition and of preventing nonspecific binding to the gel layer. The assays based on the use of particle labels requires 4 consecutive working steps only, while those based on HRP require 5 additions of reagent. The lower limit of detection for B[a]P is 5 ng L-1 in case of using HPR or QDs as a label, but 25 ng L-1 when using AuNPs. We believe that the use of QDs constitutes the most promising label for qualitative and semi-quantitative gel-based immunoassays. (author)

  20. Hydrogel-based protein and oligonucleotide microchips on metal-coated surfaces: enhancement of fluorescence and optimization of immunoassay.

    Science.gov (United States)

    Zubtsova, Zh I; Zubtsov, D A; Savvateeva, E N; Stomakhin, A A; Chechetkin, V R; Zasedatelev, A S; Rubina, A Yu

    2009-10-26

    Manufacturing of hydrogel-based microchips on metal-coated substrates significantly enhances fluorescent signals upon binding of labeled target molecules. This observation holds true for both oligonucleotide and protein microchips. When Cy5 is used as fluorophore, this enhancement is 8-10-fold in hemispherical gel elements and 4-5-fold in flattened gel pads, as compared with similar microchips manufactured on uncoated glass slides. The effect also depends on the hydrophobicity of metal-coated substrate and on the presence of a layer of liquid over the gel pads. The extent of enhancement is insensitive to the nature of formed complexes and immobilized probes and remains linear within a wide range of fluorescence intensities. Manufacturing of gel-based protein microarrays on metal-coated substrates improves their sensitivity using the same incubation time for immunoassay. Sandwich immunoassay using these microchips allows shortening the incubation time without loss of sensitivity. Unlike microchips with probes immobilized directly on a surface, for which the plasmon mechanism is considered responsible for metal-enhanced fluorescence, the enhancement effect observed using hydrogel-based microchips on metal-coated substrates might be explained within the framework of geometric optics.

  1. Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatoceUular carcinoma marker glypican-3

    Institute of Scientific and Technical Information of China (English)

    Qian-Yun Zhang; Hui Chen; Zhen Lin; Jin-Ming Lin

    2011-01-01

    Glypican-3 (GPC3) is reported as a great promising tumor marker for hepatocellular carcinoma (HCC) diagnosis. Highly sensitive and accurate analysis of serum GPC3 (sGPC3), in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP), is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles (MnPs) and magnetic microparticles (MmPs) with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay (CLEIA). After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

  2. On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis

    Science.gov (United States)

    Florescu, Octavian; Wang, Kevan; Au, Patrick; Tang, Jimmy; Harris, Eva; Beatty, P. Robert; Boser, Bernhard E.

    2010-03-01

    We have demonstrated a postprocessed complementary metal oxide semiconductor (CMOS) integrated circuit (IC) capable of on-chip magnetic separation, i.e., removing via magnetic forces the nonspecifically bound magnetic beads from the detection area on the surface of the chip. Initially, 4.5 μm wide superparamagnetic beads sedimenting out of solution due to gravity were attracted to the detection area by a magnetic concentration force generated by flowing current through a conductor embedded in the IC. After sedimentation, the magnetic beads that did not bind strongly to the functionalized surface of the IC through a specific biochemical complex were removed by a magnetic separation force generated by flowing current through another conductor placed laterally to the detection area. As the spherical bead pivoted on the surface of the chip, the lateral magnetic force was further amplified by mechanical leveraging, and 50 mA of current flowing through the separation conductor placed 18 μm away from the bead resulted in 7.5 pN of tensile force on the biomolecular tether immobilizing the bead. This force proved high enough to break nonspecific interactions while leaving specific antibody-antigen bonds intact. A sandwich capture immunoassay on purified human immunoglobulin G showed strong correlation with a control enzyme linked immunosorbent assay and a detection limit of 10 ng/ml or 70 pM. The beads bound to the detection area after on-chip magnetic separation were detected optically. To implement a fully integrated molecular diagnostics platform, the on-chip magnetic separation functionality presented in this work can be readily combine with state-of-the art CMOS-based magnetic bead detection technology.

  3. Laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay for cotinine detection in mouse serum.

    Science.gov (United States)

    Liu, Wei; Cassano, Christopher L; Xu, Xin; Fan, Z Hugh

    2013-11-01

    Laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay have been developed for the detection of cotinine, a secondhand smoke (SHS) biomarker. The devices were fabricated by a craft-cutter to define flow channels, followed by lamination. This approach of cutting/lamination to fabricate LPAD is very similar to making an identification card, offering advantages in simplicity and rugged backing when compared to the common method of patterning paper using SU-8 or wax. We also developed a protocol of localized incision and paper-folding to isolate the detection zone from flow channels; the simple origami step eliminated possible reagent diffusion and flow during antibody immobilization steps and numerous washings. By incorporating luminol-based chemiluminescence for detecting horseradish peroxidase-conjugated cotinine, we employed origami-enabled LPAD to detect cotinine in mouse serum using competitive immunoassay. The detection limit was determined to be 5 ng/mL, a clinically relevant concentration. We believe that LPAD with chemiluminescence detection provides a new platform of low cost and sensitive assays for cotinine detection.

  4. Good performance of an immunoassay based method for nevirapine measurements in human breast milk

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy;

    2011-01-01

    from HIV-uninfected women. Clinical samples from HIV-infected women receiving a single-dose of nevirapine were analyzed. Results: Precision and accuracy were evaluated with two concentrations of quality control materials analyzed in three replicates on four different days and was......, requires complicated extraction techniques. The ARK method employs an immunoassay technology and requires a small sample volume (40 μL) and no pre-treatment of the samples. Methods: Commercial enzyme and antibody were used and calibration standards and quality controls were prepared from pooled breast milk...

  5. Facile synthesis of magnetic-/pH-responsive hydrogel beads based on Fe3O4 nanoparticles and chitosan hydrogel as MTX carriers for controlled drug release.

    Science.gov (United States)

    Wu, Juan; Jiang, Wei; Tian, Renbing; Shen, Yewen; Jiang, Wei

    2016-10-01

    In the present study, methotrexate (MTX)-encapsulated magnetic-/pH-responsive hydrogel beads based on Fe3O4 nanoparticles and chitosan were successfully prepared through a one-step gelation process, which is a very facile, economic and environmentally friendly route. The developed hydrogel beads exhibited homogeneous porous structure and super-paramagnetic responsibility. MTX can be successfully encapsulated into magnetic chitosan hydrogel beads, and the drug encapsulation efficiency (%) and encapsulation content (%) were 93.8 and 6.28%, respectively. In addition, the drug release studies in vitro indicated that the MTX-encapsulated magnetic chitosan hydrogel beads had excellent pH-sensitivity, 90.6% MTX was released from the magnetic chitosan hydrogel beads within 48 h at pH 4.0. WST-1 assays in human liver hepatocellular carcinoma cells (HepG2) demonstrated that the MTX-encapsulated magnetic chitosan hydrogel beads had good cytocompatibility and high anti-tumor activity. Therefore, our results revealed that the MTX-encapsulated magnetic chitosan hydrogel beads would be a competitive candidate for controlled drug release in the area of targeted cancer therapy in the near future. PMID:27464586

  6. Upconversion Nanoparticles and Monodispersed Magnetic Polystyrene Microsphere Based Fluorescence Immunoassay for the Detection of Sulfaquinoxaline in Animal-Derived Foods.

    Science.gov (United States)

    Hu, Gaoshuang; Sheng, Wei; Zhang, Yan; Wang, Junping; Wu, Xuening; Wang, Shuo

    2016-05-18

    A novel fluorescence immunoassay for detecting sulfaquinoxaline (SQX) in animal-derived foods was developed using NaYF4:Yb/Tm upconversion nanoparticles (UCNPs) conjugated with antibodies as fluorescence signal probes, and monodisperse magnetic polystyrene microspheres (MMPMs) modified with coating antigen as immune-sensing capture probes for trapping and separating the signal probes. Based on a competitive immunoassay format, the detection limit of the proposed method for detecting SQX was 0.1 μg L(-1) in buffer and 0.5 μg kg(-1) in food samples. The recoveries of SQX in spiked samples ranged from 69.80 to 133.00%, with coefficients of variation of 0.24-25.06%. The extraction procedure was fast, simple, and environmentally friendly, requiring no organic solvents. In particular, milk samples can be analyzed directly after simple dilution. This method has appealing properties, such as sensitive fluorescence response, a simple and fast extraction procedure, and environmental friendliness, and could be applied to detecting SQX in animal-derived foods.

  7. New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B(1) based on using peptide-MBP fusion protein as substitute for coating antigen.

    Science.gov (United States)

    Xu, Yang; Chen, Bo; He, Qing-hua; Qiu, Yu-Lou; Liu, Xing; He, Zhen-yun; Xiong, Zheng-ping

    2014-08-19

    Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are "clonable" homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 ± 0.13 ng/mL and 1.26 ± 0.08 ng/mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1-BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

  8. [Multisite-based approach to assure inter-assay system compatibility among different exclusive-typed immunoassay systems through determining exchanged calibrators].

    Science.gov (United States)

    Yamauchi, Megumi S; Yamane, Nobuhisa; Toshimitsu, Shoji; Sato, Hisatsune; Fujino, Tatsuya

    2010-01-01

    It is well known that most exclusive-typed immunoassay systems are highly precise but are poor in compatibility of their determinations. Thus, it is difficult to compare the determinations among different systems, posing problems when a patient is transferred to different hospitals or when a laboratory intends to change the system currently used. In the study, we tried to approach how to assure inter-immunoassay compatibility among four different systems through determination of the exchanged calibrators. First, determinations of total protein and albumin, and electrophoretic fractionation demonstrated marked differences among calibrators in their protein constituent. Some calibrators were prepared with human sera, but others were with inorganic or non-human albumin-based solution. Regression analysis of calibrators between the indicated concentrations by manufacturers and those actually determined by the different immunoassay systems revealed that; most slopes were closed to 1.0 for alpha-fetoprotein and prostate-specific antigen, but widely dissociated from 0.28 to 4.71 for CA19-9. In evaluation of clinical serum samples, determinations by one immunoassay system were compared with those converted based on a linear regression equation that was obtained by determination of the exchanged calibrators. However, this procedure could not improve compatibility, and positive effects of conversion varied by immunoassay systems combined, and also by test parameters. With these, we concluded that simple conversion of determinations by using the exchanged calibrators and a statistical linear regression could not provide us with the expected compatibility. Thus, standardization of target molecules or probes, and of calibrator constituent were urgent issue to assure inter-immunoassay compatibility. PMID:20169939

  9. Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals.

    Science.gov (United States)

    Oswald, S; Karsunke, X Y Z; Dietrich, R; Märtlbauer, E; Niessner, R; Knopp, D

    2013-08-01

    An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55-80 and 58-79%, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127-132 and 82-120% higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography-mass spectrometry and found to be in good agreement. PMID:23620369

  10. Ultrasensitive Detection of Angiogenin Using Surface-Enhanced Raman Scattering Immunoassay Platform

    Energy Technology Data Exchange (ETDEWEB)

    Chon, Hyangah; Lim, Dong Woo; Choo, Jaebum [Hanyang Univ., Ansan (Korea, Republic of); Chang, Sooik [Chungbuk National Univ., Cheongju (Korea, Republic of)

    2013-11-15

    Our proposed SERS-based immunoassay technique, using HGNs and magnetic beads, shows a strong potential for the early diagnosis of angiogenic disease because the ultrasensitive detection to attomolar concentration level is possible. Angiogenesis, the process of new blood-vessel growth, plays an important role in normal physiological process. To date, angiogenin (ANG) is known to be a key factor in induction of angiogenesis by activation of endothelial and smooth muscle cells as well as by triggering a number of biological processes. It is also well known that the expression of ANG is up-regulated in various types of human cancers.

  11. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    Science.gov (United States)

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  12. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species.

    Directory of Open Access Journals (Sweden)

    Naveen Kumar

    Full Text Available We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR as the gold standard, we found that the diagnostic sensitivity (DSn and specificity (DSp of the new assay was high [bovine (DSn = 94.2% & DSp = 100%; porcine (DSn = 94.6% & DSp = 93.3%; poultry (DSn = 74.2% & DSp = 97.7% and human (DSn = 82.1% & DSp = 98.7%]. The concordance with RT-PCR was also high [weighted kappa (k = 0.831-0.956 at 95% CI = 0.711-1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE. The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings.

  13. Encapsulation of lactase (β-galactosidase) into κ-carrageenan-based hydrogel beads: Impact of environmental conditions on enzyme activity.

    Science.gov (United States)

    Zhang, Zipei; Zhang, Ruojie; Chen, Long; McClements, David Julian

    2016-06-01

    Encapsulation of enzymes in hydrogel beads may improve their utilization and activity in foods. In this study, the potential of carrageenan hydrogel beads for encapsulating β-galactosidase was investigated. Hydrogel beads were fabricated by injecting an aqueous solution, containing β-galactosidase (26 U) and carrageenan (1 wt%), into a hardening solution (5% potassium chloride). Around 63% of the β-galactosidase was initially encapsulated in the hydrogel beads. Encapsulated β-galactosidase had a higher activity than that of the free enzyme over a range of pH and thermal conditions, which was attributed to the stabilization of the enzyme structure by K(+) ions within the carrageenan beads. Release of the enzyme from the beads was observed during storage in aqueous solutions, which was attributed to the relatively large pore size of the hydrogel matrix. Our results suggest that carrageenan hydrogel beads may be useful encapsulation systems, but further work is needed to inhibit enzyme leakage. PMID:26830562

  14. Encapsulation of lactase (β-galactosidase) into κ-carrageenan-based hydrogel beads: Impact of environmental conditions on enzyme activity.

    Science.gov (United States)

    Zhang, Zipei; Zhang, Ruojie; Chen, Long; McClements, David Julian

    2016-06-01

    Encapsulation of enzymes in hydrogel beads may improve their utilization and activity in foods. In this study, the potential of carrageenan hydrogel beads for encapsulating β-galactosidase was investigated. Hydrogel beads were fabricated by injecting an aqueous solution, containing β-galactosidase (26 U) and carrageenan (1 wt%), into a hardening solution (5% potassium chloride). Around 63% of the β-galactosidase was initially encapsulated in the hydrogel beads. Encapsulated β-galactosidase had a higher activity than that of the free enzyme over a range of pH and thermal conditions, which was attributed to the stabilization of the enzyme structure by K(+) ions within the carrageenan beads. Release of the enzyme from the beads was observed during storage in aqueous solutions, which was attributed to the relatively large pore size of the hydrogel matrix. Our results suggest that carrageenan hydrogel beads may be useful encapsulation systems, but further work is needed to inhibit enzyme leakage.

  15. A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

    Science.gov (United States)

    Fu, Wei; Wang, Huiyu; Wang, Chenguang; Mei, Lin; Lin, Xiangmei; Han, Xueqing; Zhu, Shuifang

    2016-03-15

    The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants.

  16. A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

    Science.gov (United States)

    Fu, Wei; Wang, Huiyu; Wang, Chenguang; Mei, Lin; Lin, Xiangmei; Han, Xueqing; Zhu, Shuifang

    2016-03-15

    The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants. PMID:26499065

  17. A highly sensitive quartz crystal microbalance immunosensor based on magnetic bead-supported bienzymes catalyzed mass enhancement strategy.

    Science.gov (United States)

    Akter, Rashida; Rhee, Choong Kyun; Rahman, Md Aminur

    2015-04-15

    A highly sensitive quartz crystal microbalance (QCM) immunosensor based on magnetic bead-supported bienzyme catalyzed mass enhanced strategy was developed for the detection of human immunoglobulin G (hIgG) protein. The high sensitive detection was achieved by increasing the deposited mass on the QCM crystal through the enhanced precipitation of 4-chloro-1-naphthol (CN) using higher amounts of horseradish peroxidase (HRP) and glucose oxidase (GOx) bienzymes attached on the magnetic beads (MB). The protein A (PA) and capture antibody (monoclonal anti-human IgG antibody produced in mouse, Ab1)-based QCM probe and the detection antibody (anti-human IgG antibody produced in goat, Ab2)-based MB/HRP/GOx bienzymatic bioconjugates were characterized using scanning electron microscope, transmission electron microscope, cyclic voltammetry, and electrochemical impedance spectroscopy techniques. Under the optimized experimental condition, the linear range and the detection limit of hIgG immunosensor were determined to be 5.0pg/mL-20.0ng/mL and 5.0±0.18pg/mL, respectively. The applicability of the present hIgG immunosensor was examined in hIgG spiked human serum samples and excellent recoveries of hIgG were obtained. PMID:25506902

  18. Liquid carry-over in an injection moulded all-polymer chip system for immiscible phase magnetic bead-based solid-phase extraction

    Energy Technology Data Exchange (ETDEWEB)

    Kistrup, Kasper, E-mail: kkis@nanotech.dtu.dk [Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, DK-2800 Kongens Lyngby (Denmark); Skotte Sørensen, Karen, E-mail: karen@nanotech.dtu.dk [Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, DK-2800 Kongens Lyngby (Denmark); Center for Integrated Point of Care Technologies (CiPoC), DELTA, Venlighedsvej 4, DK-2870 Hørsholm (Denmark); Wolff, Anders, E-mail: anders.wolff@nanotech.dtu.dk [Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, DK-2800 Kongens Lyngby (Denmark); Fougt Hansen, Mikkel, E-mail: mikkel.hansen@nanotech.dtu.dk [Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, DK-2800 Kongens Lyngby (Denmark)

    2015-04-15

    We present an all-polymer, single-use microfluidic chip system produced by injection moulding and bonded by ultrasonic welding. Both techniques are compatible with low-cost industrial mass-production. The chip is produced for magnetic bead-based solid-phase extraction facilitated by immiscible phase filtration and features passive liquid filling and magnetic bead manipulation using an external magnet. In this work, we determine the system compatibility with various surfactants. Moreover, we quantify the volume of liquid co-transported with magnetic bead clusters from Milli-Q water or a lysis-binding buffer for nucleic acid extraction (0.1 (v/v)% Triton X-100 in 5 M guanidine hydrochloride). A linear relationship was found between the liquid carry-over and mass of magnetic beads used. Interestingly, similar average carry-overs of 1.74(8) nL/µg and 1.72(14) nL/µg were found for Milli-Q water and lysis-binding buffer, respectively. - Highlights: • We present an all-polymer mass producible passive filled microfluidic chip system. • Rapid system fabrication is obtained by injection moulding and ultrasonic welding. • The system is made for single-use nucleic acid extraction using magnetic beads. • We systematically map compatibility of the chip system with various surfactants. • We quantify the volume carry-over of magnetic beads in water and 0.1% triton-X solution.

  19. Sensing characteristics of plasmonic structure based on transferring process of polystyrene nano-beads

    Science.gov (United States)

    Kim, Doo Gun; Hwang, Jeongwoo; Kim, Seon Hoon; Ki, Hyun Chul; Kim, Tae Un; Shin, Jae Cheol; Jeong, Dae-Cheol; Jeon, Seungwon; Kim, Hong-Seung; Choi, Young-Wan

    2016-04-01

    We analyzed and demonstrated the double layered metallic nano-structures using polystyrene lift-off process on the conventional surface plasmon resonance (SPR) sensor to enhance the sensitivity of an SPR surface. The double layered plasmonic structures are optimized using the three-dimensional finite-difference time-domain method for the width, thickness, and period of the polystyrene beads. The thickness of the metal film and the metallic nano-hole is 20 and 20 nm in the 305 nm wide nano-hole size, respectively. The double layered metallic nano-structures are fabricated with monolayer polystyrene beads of chloromethyl latex 4% w/v 0.4 μm. The sensitivities of the conventional SPR sensor and the double layered plasmonic sensor are obtained to 42.2 and 60 degree/RIU, respectively. The SPR devices are also applied to the lead ion sensor. The resonance shifts of SPR sensors with and without a poly(vinyl chloride) membrane are 1328 RU and 788 RU from 10-5 M to 10-2 M concentration, respectively.

  20. Enzyme-catalyzed reaction of voltammetric enzyme-linked immunoassay system based on OAP as substrate

    Institute of Scientific and Technical Information of China (English)

    张书圣; 陈洪渊; 焦奎

    1999-01-01

    The o-aminophenol (OAP)-H2O2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay new system has extremely high sensitivity. HRP can be measured with a detection limit of 6.0×10-(10) g/L and a linear range of 1.0×10-9—4.0×10-6 g/L. The pure product of H2O2 oxidizing OAP catalyzed by HRP was prepared with chemical method. The enzyme-catalyzed reaction has been investigated with electroanalytical chemistry, UV/Vis spectrum, IR spectrum, 13C NMR, 1H NMR, mass spectrum, elemental analysis, etc. Under the selected enzyme-catalyzed reaction conditions, the oxidation product of OAP with H2O2 catalyzed by HRP is 2-aminophe-noxazine-3-one. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzymecatalyzed reaction have been described.

  1. Silver-coated silica bead-based dual tagging sensors operating via surface-enhanced Raman scattering and metal-enhanced fluorescence

    International Nuclear Information System (INIS)

    Complete text of publication follows. We have developed a new type of dual-tag sensors, operating via both surface-enhanced Raman scattering (SERS) and metal enhanced fluorescence (MEF), for immunoassays. Initially, a very simple electroless-plating method was applied to prepare Ag-coated silica beads. After adsorbing Raman labels (benzenethiol or 4-aminobenzenethiol), poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) were deposited onto them by the layer-by-layer (LBL) method: The beads became fluorescent due to the incorporation of rhodamine B isothiocyanate (RhBITC)-grafted PAH in the latter process. In the final stage, the outermost PAH was reacted with glutaraldehyde (GA), and then either goat anti-human-IgG (anti-h-IgG) or mouse anti-rabbit-IgG (anti-r-IgG) was immobilized thereon. Thereafter, we confirmed, by monitoring the SERS peaks of benzenethiol (BT) and the confocal laser scanning microscopy (CLSM) of RhBITC, that anti-h-IgG-grafted SiO2aAg particles interact exclusively with h-IgG. Similarly, we confirmed from the SERS peaks of 4-aminobenzenethiol (4-ABT), as well as the CLSM of RhBITC, that anti-r-IgG-grafted SiO2aAg beads interact solely with r-IgG. These clearly illustrate that firstly, the binding event of the target molecule and then the specific type of antigen can be recognized consecutively by MEF and SERS, respectively. The application prospects of these materials are thus expected to be very high especially in the areas of biological sensing and recognition that rely heavily on optical and spectroscopic properties. This work was supported by the Korea Science and Engineering Foundation (Grant No. R11-2007-012-02002-0) and the Korea Research Foundation (Grant KRF-2008-313-C00390).

  2. An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate

    International Nuclear Information System (INIS)

    Graphical abstract: A new fluorescent polarization immunoassay (FPIA) method for ATP was structured. The fluorescence polarization method has ultrahigh sensitivity and good selectivity. The method could be applied for the ATP detection in bioanalysis. -- Highlights: •A new fluorescent polarization immunoassay (FPIA) method for ATP was structured. •The fluorescence polarization method has ultrahigh sensitivity and good selectivity. •The method could be applied for the ATP detection in bioanalysis. -- Abstract: In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8 × 10−12 M to 2.40 × 10−4 M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems

  3. An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate

    Energy Technology Data Exchange (ETDEWEB)

    He, Yanlong; Tian, Jianniao, E-mail: birdtjn@sina.com; Hu, Kun; Zhang, Juanni; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun, E-mail: yanchunzh@sina.com; Zhao, Shulin

    2013-11-13

    Graphical abstract: A new fluorescent polarization immunoassay (FPIA) method for ATP was structured. The fluorescence polarization method has ultrahigh sensitivity and good selectivity. The method could be applied for the ATP detection in bioanalysis. -- Highlights: •A new fluorescent polarization immunoassay (FPIA) method for ATP was structured. •The fluorescence polarization method has ultrahigh sensitivity and good selectivity. •The method could be applied for the ATP detection in bioanalysis. -- Abstract: In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8 × 10{sup −12} M to 2.40 × 10{sup −4} M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems.

  4. A giant magnetoresistance ring-sensor based microsystem for magnetic bead manipulation and detection

    KAUST Repository

    Gooneratne, Chinthaka P.

    2011-03-28

    In this paper a novel spin valvegiant magnetoresistance(GMR) ring-sensor integrated with a microstructure is proposed for concentrating, trapping, and detecting superparamagnetic beads (SPBs). Taking advantage of the fact that SPBs can be manipulated by an external magnetic field, a unique arrangement of conducting microrings is utilized to manipulate the SPBs toward the GMR sensing area in order to increase the reliability of detection. The microrings are arranged and activated in such a manner so as to enable the detection of minute concentrations of SPBs in a sample. Precise manipulation is achieved by applying current sequentially to the microrings. The fabricated ring-shaped GMR element is located underneath the innermost ring and has a magnetoresistance of approximately 5.9%. By the performed experiments it was shown that SPBs could be successfully manipulated toward the GMR sensing zone.

  5. Weld pool boundary and weld bead shape reconstruction based on passive vision in P-GMAW

    Institute of Scientific and Technical Information of China (English)

    Yan Zhihong; Zhang Guangjun; Gao Hongming; Wu Lin

    2006-01-01

    A passive visual sensing system is established in this research, and clear weld pool images in pulsed gas metal arc welding ( P-GMA W) can be captured with this system. The three-dimensional weld pool geometry, especially the weld height,is not only a crucial factor in determining workpiece mechanical properties, but also an important parameter for reflecting the penetration. A new three-dimensional (3D) model is established to describe the weld pool geometry in P-GMAW. Then, a series of algorithms are developed to extract the model geometrical parameters from the weld pool images. Furthermore, the method to reconstruct the 3D shape of weld pool boundary and weld bead from the two-dimensional images is investigated.

  6. Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

    Science.gov (United States)

    Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

    2016-04-01

    The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice.

  7. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

    Science.gov (United States)

    Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

    2015-12-01

    Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

  8. Microfluidic bead-based assay for microRNAs using quantum dots as labels and enzymatic amplification

    International Nuclear Information System (INIS)

    We report on a microfluidic assay for microRNA using quantum dots as labels and capture probes immobilized in a bead array. Target microRNA flows along the microfluidic channel to hit the beads array where it hybridizes with the immobilized capture probes. Next, the hybrid is labeled by using the bound microRNAs as a primer for enzymatic elongation with biotin-labeled nucleotides. Due to the specificity of (a) the hybridization assay and (b) the enzymatic elongation step, this assay is quite selective and only the completely matched duplex can be labeled, in a final step, with streptavidin-labeled quantum dots. The method was applied to the specific detection of microRNAs that occur in the miRNA-29 family and display minute differences only in their nucleotide sequence. It does not require (a) a labeling step before hybridization and (b) no amplification. This on-chip assay for microRNA can detect concentrations as low as 0.1 pmol·L−1 (at an SNR of >3) when using synthetic microRNA. The 200-fold better sensitivity than that of an off-chip test is ascribed to the microfluidic-based signal enhancement. Other features include rapid binding kinetics, the advantages of a homogeneous assay in a suspended microbead array, the detection sensitivity resulting from the use of quantum dots, small reagent consumption, short assay time, and parallel detection. (author)

  9. 3D origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device.

    Science.gov (United States)

    Ge, Lei; Wang, Shoumei; Song, Xianrang; Ge, Shenguang; Yu, Jinghua

    2012-09-01

    A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics. PMID:22763468

  10. Coated Aerogel Beads

    Science.gov (United States)

    Littman, Howard (Inventor); Plawsky, Joel L. (Inventor); Paccione, John D. (Inventor)

    2014-01-01

    Methods and apparatus for coating particulate material are provided. The apparatus includes a vessel having a top and a bottom, a vertically extending conduit having an inlet in the vessel and an outlet outside of the vessel, a first fluid inlet in the bottom of the vessel for introducing a transfer fluid, a second fluid inlet in the bottom of the vessel for introducing a coating fluid, and a fluid outlet from the vessel. The method includes steps of agitating a material, contacting the material with a coating material, and drying the coating material to produce a coated material. The invention may be adapted to coat aerogel beads, among other materials. A coated aerogel bead and an aerogel-based insulation material are also disclosed.

  11. Diagnostic performance of line-immunoassay based algorithms for incident HIV-1 infection

    Directory of Open Access Journals (Sweden)

    Schüpbach Jörg

    2012-04-01

    Full Text Available Abstract Background Serologic testing algorithms for recent HIV seroconversion (STARHS provide important information for HIV surveillance. We have previously demonstrated that a patient's antibody reaction pattern in a confirmatory line immunoassay (INNO-LIA™ HIV I/II Score provides information on the duration of infection, which is unaffected by clinical, immunological and viral variables. In this report we have set out to determine the diagnostic performance of Inno-Lia algorithms for identifying incident infections in patients with known duration of infection and evaluated the algorithms in annual cohorts of HIV notifications. Methods Diagnostic sensitivity was determined in 527 treatment-naive patients infected for up to 12 months. Specificity was determined in 740 patients infected for longer than 12 months. Plasma was tested by Inno-Lia and classified as either incident ( Results The 10 best algorithms had a mean raw sensitivity of 59.4% and a mean specificity of 95.1%. Adjustment for overrepresentation of patients in the first quarter year of infection further reduced the sensitivity. In the preferred model, the mean adjusted sensitivity was 37.4%. Application of the 10 best algorithms to four annual cohorts of HIV-1 notifications totalling 2'595 patients yielded a mean IIR of 0.35 in 2005/6 (baseline and of 0.45, 0.42 and 0.35 in 2008, 2009 and 2010, respectively. The increase between baseline and 2008 and the ensuing decreases were highly significant. Other adjustment models yielded different absolute IIR, although the relative changes between the cohorts were identical for all models. Conclusions The method can be used for comparing IIR in annual cohorts of HIV notifications. The use of several different algorithms in combination, each with its own sensitivity and specificity to detect incident infection, is advisable as this reduces the impact of individual imperfections stemming primarily from relatively low sensitivities and

  12. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    Science.gov (United States)

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format. PMID:26291091

  13. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    Science.gov (United States)

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

  14. Development of a Multiplexed, Bead-Based Assessment Tool for Rapid Identification and Quantitation of Microorganisms in Field Samples. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Lowe, M.; Halden, R.

    2002-10-09

    This was the final report for DOE NABIR grant DE-FG02-01ER63264 (PI Mary Lowe). The grant was entitled ''Development of a Multiplexed Bead-Based Assessment Tool for Rapid Identification and Quantitation of Microorganisms in Field Samples.'' The grant duration was one year. The purpose was to develop a bead-based assay for measuring analyte DNAs in environmental PCR products and to apply the method to a field experiment. The primary experiment was located at the UMTRA Old Rifle site.

  15. Renewable Surface Fluorescence Sandwich Immunoassay Biosensor for Rapid Sensitive Botulinum Toxin Detection in an Automated Fluidic Format

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Warner, Marvin G.; Ozanich, Richard M.; Miller, Keith D.; Colburn, Heather A.; Dockendorff, Brian P.; Antolick, Kathryn C.; Anheier, Norman C.; Lind, Michael A.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-03-05

    A renewable surface biosensor for rapid detection of botulinum toxin is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant fragment of the toxin heavy chain as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate epitopes of both this fragment and the holotoxin. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by the sequential injection flow system, creating a 3.6 microliter column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degree angle to one another delivered excitation light from a HeNe laser and collected fluorescent emission light for detection. After each measurement, the used sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes.

  16. Spherical porphyrin sensor array based on encoded colloidal crystal beads for VOC vapor detection.

    Science.gov (United States)

    Xu, Hua; Cao, Kai-Di; Ding, Hai-Bo; Zhong, Qi-Feng; Gu, Hong-Cheng; Xie, Zhuo-Ying; Zhao, Yuan-Jin; Gu, Zhong-Ze

    2012-12-01

    A spherical porphyrin sensor array using colloidal crystal beads (CCBs) as the encoding microcarriers has been developed for VOC vapor detection. Six different porphyrins were coated onto the CCBs with distinctive encoded reflection peaks via physical adsorption and the sensor array was fabricated by placing the prepared porphyrin-modified CCBs together. The change in fluorescence color of the porphyrin-modified CCBs array serves as the detection signal for discriminating between different VOC vapors and the reflection peak of the CCBs serves as the encoding signal to distinguish between different sensors. It was demonstrated that the VOC vapors detection using the prepared sensor array showed excellent discrimination: not only could the compounds from the different chemical classes be easily differentiated (e.g., alcohol vs acids vs ketones) but similar compounds from the same chemical family (e.g., methanol vs ethanol) and the same compound with different concentration ((e.g., Sat. ethanol vs 60 ppm ethanol vs 10 ppm ethanol) could also be distinguished. The detection reproducibility and the humidity effect were also investigated. The present spherical sensor array, with its simple preparation, rapid response, high sensitivity, reproducibility, and humidity insensitivity, and especially with stable and high-throughput encoding, is promising for real applications in artificial olfactory systems. PMID:23163575

  17. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    Directory of Open Access Journals (Sweden)

    Daniela Höfler

    Full Text Available The Federation of European Laboratory Animal Science Association (FELASA recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  18. Pre-packing of cost effective antibiotic cement beads for the treatment of traumatic osteomyelitis in the developing world - an in-vitro study based in Cambodia.

    Science.gov (United States)

    Noor, S; Gilson, A; Kennedy, K; Swanson, A; Vanny, V; Mony, K; Chaudhry, T; Gollogly, J

    2016-04-01

    The developing world often lacks the resources to effectively treat the most serious injuries including osteomyelitis following open fractures or surgical fracture treatment. Antibiotic cement beads are a widely accepted method of delivering antibiotics locally to the infected area following trauma. This study is based in Cambodia, a low income country struggling to recover from a recent genocide. The study aims to test the effectiveness of locally made antibiotic beads and analyse their effectiveness after being gas sterilised, packaged and kept in storage Different antibiotic beads were manufactured locally using bone cement and tested against MRSA bacteria grown from a case of osteomyelitis. Each antibiotic was tested before and after a process of gas sterilisation as well as later being tested after storage in packaging up to 42 days. The gentamicin, vancomycin, amikacin and ceftriaxone beads all inhibited growth of the MRSA on the TSB and agar plates, both before and after gas sterilisation. All four antibiotics continued to show similar zones of inhibition after 42 days of storage. The results show significant promise to produce beads with locally obtainable ingredients in an austere environment and improve cost effectiveness by storing them in a sterilised condition. PMID:26899719

  19. Graphene oxide-labeled sandwich-type impedimetric immunoassay with sensitive enhancement based on enzymatic 4-chloro-1-naphthol oxidation.

    Science.gov (United States)

    Hou, Li; Cui, Yuling; Xu, Mingdi; Gao, Zhuangqiang; Huang, Jianxin; Tang, Dianping

    2013-09-15

    A new sandwich-type impedimetric immunosensor based on functionalized graphene oxide nanosheets with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for the detection of carcinoembryonic antigen (CEA) by coupling with enzymatic biocatalytic precipitation of 4-chloro-1-naphthol (4-CN) on the captured antibody-modified glassy carbon electrode. Two molecular tags (with and without the graphene oxide nanosheets) were investigated for the detection of CEA and improved analytical features were acquired with the graphene-based labeling. With the labeling method, the performance and factors influencing the properties of the impedimetric immunosensors were also studied and evaluated. Under the optimal conditions, the dynamic concentration range of the impedimetric immunosensors spanned from 1.0pgmL(-1) to 80ngmL(-1) CEA with a detection limit (LOD) of 0.64pgmL(-1). Intra- and inter-assay coefficients of variation were less than 7.5% and 11%, respectively. Additionally, the methodology was evaluated for CEA analysis of 10 clinical serum samples and 5 diluted serum samples, receiving in a good accordance with the results obtained by the impedimetric immunoassay and the commercialized electrochemiluminescent method.

  20. Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting

    NARCIS (Netherlands)

    Molema, Ingrid; Mesander, G; Kroesen, Bart-Jan; Helfrich, Wijnand; Meijer, D.K F; de Leij, Lou

    1998-01-01

    In this report, we describe a new and simple method for flow cytometric quantitation of lymphocyte numbers in lymphocute-endothelial adhesion/transendothelial migration assays. The method exploits fluorescent flow cytometer alignment beads as a counting reference. Known amounts of beads are added to

  1. Proteomic profiling of renal allograft rejection in serum using magnetic bead-based sample fractionation and MALDI-TOF MS.

    Science.gov (United States)

    Sui, Weiguo; Huang, Liling; Dai, Yong; Chen, Jiejing; Yan, Qiang; Huang, He

    2010-12-01

    Proteomics is one of the emerging techniques for biomarker discovery. Biomarkers can be used for early noninvasive diagnosis and prognosis of diseases and treatment efficacy evaluation. In the present study, the well-established research systems of ClinProt Micro solution incorporated unique magnetic bead sample preparation technology, which, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), have become very successful in bioinformatics due to its outstanding performance and reproducibility for discovery disease-related biomarker. We collected fasting blood samples from patients with biopsy-confirmed acute renal allograft rejection (n = 12), chronic rejection (n = 12), stable graft function (n = 12) and also from healthy volunteers (n = 13) to study serum peptidome patterns. Specimens were purified with magnetic bead-based weak cation exchange chromatography and analyzed with a MALDI-TOF mass spectrometer. The results indicated that 18 differential peptide peaks were selected as potential biomarkers of acute renal allograft rejection, and 6 differential peptide peaks were selected as potential biomarkers of chronic rejection. A Quick Classifier Algorithm was used to set up the classification models for acute and chronic renal allograft rejection. The algorithm models recognize 82.64% of acute rejection and 98.96% of chronic rejection episodes, respectively. We were able to identify serum protein fingerprints in small sample sizes of recipients with renal allograft rejection and establish the models for diagnosis of renal allograft rejection. This preliminary study demonstrated that proteomics is an emerging tool for early diagnosis of renal allograft rejection and helps us to better understand the pathogenesis of disease process.

  2. Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips.

    Science.gov (United States)

    Liang, Rong-Liang; Xu, Xu-Ping; Liu, Tian-Cai; Zhou, Jian-Wei; Wang, Xian-Guo; Ren, Zhi-Qi; Hao, Fen; Wu, Ying-Song

    2015-09-01

    Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.

  3. Sirtuin 1 evaluation with a novel immunoassay approach based on TiO2-Au label and hyperbranched polymer hybrid.

    Science.gov (United States)

    An, Yarui

    2016-08-15

    Accurate and highly sensitive evaluation of the sirtuin 1 (SirT1) level is becoming increasingly important for understanding the contribution of SirT1 in metabolism pathways. Here, a novel electrochemical immunoassay of SirT1 based on crosslinked hyperbranched azo-polymer decorated with gold colloids (Au-HAP) as sensing platform and titanium dioxide (TiO2)-Au nanocomposites to immobilize secondary antibody-horseradish peroxidase (Ab2-HRP) as electrochemical labels has been designed. Greatly enhanced sensitivity was achieved by exploiting the excellent conductivity of Au nanoparticle, the amplification effect of Au-HAP and TiO2-Au, and the favorable catalytic ability of HRP. The nanocomposites of Au-HAP and TiO2-Au could attach numerous capture antibodies on the surface for significant immune recognition efficiency. Meanwhile, the TiO2-Au-labeled Ab2-HRP using an HRP-thionine-H2O2 (hydrogen peroxide) detection system could further induce signal readout. Under optimal conditions, the signal intensity was linearly related to the concentration of SirT1 in the range of 1-500 ng ml(-1), and the limit of detection was 0.28 ng ml(-1). The developed biosensor exhibits attractive performance for the analysis of SirT1, with rapid response, high sensitivity, and high accuracy, and could become a promising technique for protein detection. PMID:27264194

  4. A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles.

    Science.gov (United States)

    Qian, Jing; Wang, Chengquan; Pan, Xiaohu; Liu, Songqin

    2013-02-01

    A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1-110 ng mL(-1) for CEA, with a detection limit of 0.3 ng mL(-1). The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers. PMID:23340285

  5. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination.

  6. A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions.

    Science.gov (United States)

    Li, Taihua; Choi, Yo Han; Shin, Yong-Beom; Kim, Hwa-Jung; Kim, Min-Gon

    2016-05-01

    A fluorescence enhancement-based immunoassay has been developed for the detection of the polycyclic aromatic hydrocarbons (PAH), benzo[a]pyrene (BaP), in aqueous solutions. The results of this study show that BaP, which inefficiently fluoresces in aqueous solution, displays enhanced fluorescence when bound to the anti-BaP antibody (anti-BaP), as part of a label-free immunoassay system. Binding to anti-BaP results in a 3.12-fold increase in the fluorescence intensity of BaP, which emits at 435 nm when excited at 280 nm, due to the hydrophobic interaction and fluorescence resonance energy transfer (FRET) between antibody and antigen. As result of this phenomenon, the antibody-based fluorescence immunoassay system can be used to detect BaP specifically with a limit of detection (LOD) of 0.06 ng mL(-1). Finally, extraction recoveries of BaP from spiked wheat and barley samples were found to be in the range of 80.5-87.0% and 92.9-92.1%, respectively.

  7. Immunoassays in Biotechnology

    Science.gov (United States)

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  8. Direct friction measurement in draw bead testing

    DEFF Research Database (Denmark)

    Olsson, David Dam; Bay, Niels; Andreasen, Jan Lasson

    2005-01-01

    The application of draw beads in sheet metal stamping ensures controlled drawing-in of flange parts. Lubrication conditions in draw beads are severe due to sliding under simultaneous bending. Based on the original draw bead test design by Nine [1] comprehensive studies of friction in draw beads...... have been reported in literature. A major drawback in all these studies is that friction is not directly measured, but requires repeated measurements of the drawing force with and without relative sliding between the draw beads and the sheet material. This implies two tests with a fixed draw bead tool......-in piezoelectric torque transducer. This technique results in a very sensitive measurement of friction, which furthermore enables recording of lubricant film breakdown as function of drawing distance. The proposed test is validated in an experimental investigation of the influence of lubricant viscosity...

  9. Simple and robust antibody microarray-based immunoassay platform for sensitive and selective detection of PSA and hK2 toward accurate diagnosis of prostate cancer

    Directory of Open Access Journals (Sweden)

    S.W. Lee

    2015-03-01

    Full Text Available This paper reports the development of an easy to use antibody microarray-based immunoassay platform for sensitive and selective duplex detection of PSA (prostate specific antigen and hK2 (human kallikrein 2. Using PDMS wells in a 3 × 9 array on epoxy-coated glass slides 27 duplex immunoassays can be performed in parallel. Automated microarraying provided fast and reproducible antibody arraying in each assay well. To achieve highly sensitive and selective detection of each biomarker, we evaluated and optimized the density of each of the immobilized capture antibodies. The assay platform showed a limit of detection (LOD of each biomarker (PSA and hK2 of less than 10 pg/mL and a dynamic range of 104–105 orders of magnitude. Neither the PSA nor the hK2 antibody array showed any cross-reaction against each others target proteins or other plasma proteins. These results emphasize the importance of density optimization of capture antibody on the surface in order to achieve a sensitive and selective multiplex immunoassay.

  10. A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

    International Nuclear Information System (INIS)

    Graphical abstract: A Förster resonance energy transfer system by using polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor for sensitive detection of tumor marker was proposed. Highlights: ► A homogeneous immunosensing strategy based on FRET for detection of tumor marker was proposed. ► Close of QDs and AuNPs allow the occurrence of quenching the photoluminescence of nano-bio-probes. ► Signal quenching was monitored by a self-developed image analyzer. ► The fluorometric assay format is attractive for widespread carcinoma screening and even field use. -- Abstract: A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL−1 for CEA, with a detection limit of 0.3 ng mL−1. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers

  11. A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Jing [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); School of Chemistry and Chemical Engineering, Jiangsu University, Zhengjiang 212013 (China); Wang, Chengquan [Changzhou College of Information Technology, Changzhou 213164 (China); Pan, Xiaohu [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Liu, Songqin, E-mail: liusq@seu.edu.cn [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China)

    2013-02-06

    Graphical abstract: A Förster resonance energy transfer system by using polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor for sensitive detection of tumor marker was proposed. Highlights: ► A homogeneous immunosensing strategy based on FRET for detection of tumor marker was proposed. ► Close of QDs and AuNPs allow the occurrence of quenching the photoluminescence of nano-bio-probes. ► Signal quenching was monitored by a self-developed image analyzer. ► The fluorometric assay format is attractive for widespread carcinoma screening and even field use. -- Abstract: A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL{sup −1} for CEA, with a detection limit of 0.3 ng mL{sup −1}. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.

  12. Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

    International Nuclear Information System (INIS)

    We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL-1 and a 0.07 ng∙mL-1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin. (author)

  13. A bare-eye-based lateral flow immunoassay based on the use of gold nanoparticles for simultaneous detection of three pesticides

    International Nuclear Information System (INIS)

    We present a novel lateral flow immunoassay (LFIA) for the simultaneous detection of the pesticides imidacloprid, chlorpyrifos-methyl and isocarbophos based on three competitive immunoreactions. In contrast to previously reported LFIAs, the method is based on the use of four strips. Each has three red channels (three test lines dispensed with different capture reagent) to detect imidacloprid, chlorpyrifos-methyl and isocarbophos respectively. Different channels on each strip are the key to multi-detection, and four strips of LFIA are needed for visual and semi-quantitative read-outs. Under optimized conditions, the LFIA was applied to the determination of three pesticides. The detection time is within 7 min and the detection limits are 50, 100, and 100 μg L−1, respectively. Furthermore, the LFIA was applied to the analysis of spiked Chinese cabbage and soil samples and results were validated by HPLC. (author)

  14. Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Wei, Bo; Li, Fang; Yang, Huicui; Yu, Lei; Zhao, Kaihong; Zhou, Rui; Hu, Yonggang

    2012-05-15

    In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p'-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160-1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N=3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.

  15. Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges

    International Nuclear Information System (INIS)

    A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 μg kg-1 were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities

  16. Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges

    Energy Technology Data Exchange (ETDEWEB)

    Kolosova, Anna Yu. [Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent (Belgium)], E-mail: anna_kolosova@hotmail.com; Sibanda, Liberty [TOXI-TEST NV, Industrielaan 9a, 9990 Maldegem (Belgium); Dumoulin, Frederic [Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent (Belgium); Lewis, Janet; Duveiller, Etienne [International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F. (Mexico); Van Peteghem, Carlos; Saeger, Sarah de [Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent (Belgium)

    2008-06-02

    A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 {mu}g kg{sup -1} were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.

  17. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Science.gov (United States)

    Behnert, Astrid; Fritzler, Marvin J; Teng, Beina; Zhang, Meifeng; Bollig, Frank; Haller, Hermann; Skoberne, Andrej; Mahler, Michael; Schiffer, Mario

    2013-01-01

    The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  18. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Directory of Open Access Journals (Sweden)

    Astrid Behnert

    Full Text Available The phospholipase A2 receptor (PLA2R was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN. Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA utilizing indirect immunofluorescence (IIF on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA platform. Since reactive domains of PLA2R (i.e. epitopes could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  19. Performance Improvement of the One-Dot Lateral Flow Immunoassay for Aflatoxin B1 by Using a Smartphone-Based Reading System

    OpenAIRE

    Jihea Moon; Giyoung Kim; Sangdae Lee

    2013-01-01

    This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisi...

  20. A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples.

    Science.gov (United States)

    Yuan, Meng; Sheng, Wei; Zhang, Yan; Wang, Junping; Yang, Yijin; Zhang, Shuguang; Goryacheva, Irina Yu; Wang, Shuo

    2012-11-01

    A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1 μg L(-1). The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement.

  1. Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

    International Nuclear Information System (INIS)

    A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styren sulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays. (author)

  2. Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

    OpenAIRE

    Moreno Tamarit, Mª José; PLANA ANDANI, EMMA; Manclus Ciscar, Juan José; Montoya Baides, Ángel

    2014-01-01

    [EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(be...

  3. Nanowell-based immunoassays for measuring single-cell secretion: characterization of transport and surface binding.

    Science.gov (United States)

    Torres, Alexis J; Hill, Abby S; Love, J Christopher

    2014-12-01

    Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.

  4. A quantum dot-based immunoassay for screening of tylosin and tilmicosin in edible animal tissues.

    Science.gov (United States)

    Le, Tao; Zhu, Liqian; Yang, Xian

    2015-01-01

    A rapid, indirect competitive fluorescence-linked immunosorbent assay (ic-FLISA) based on quantum dots (QDs) as the fluorescent marker was developed for the detection of tylosin and tilmicosin in edible animal tissues. The end point fluorescent detection system was carried out using QDs conjugated with goat anti-mouse secondary antibody. The limits of detection (LODs) for the determination of tylosin and tilmicosin were 0.02 and 0.04 μg kg(-1), respectively. This detection method was used to analyse spiked samples and the recoveries ranged from 83.5% to 98.7% for tylosin and from 81.8% to 98.2% for tilmicosin. In real porcine tissue sample analysis, the results of ic-FLISA were similar to those obtained from an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to an HPLC method indicating its potential for tylosin and tilmicosin screening in edible animal tissues. PMID:25822697

  5. Electrochemical immunoassay of cotinine in serum based on nanoparticle probe and immunochromatographic strip

    Energy Technology Data Exchange (ETDEWEB)

    Nian Hungchi [Pacific Norwest National Laboratory, Richland, WA 99352 (United States); Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013, Taiwan (China); Wang Jun; Wu Hong [Pacific Norwest National Laboratory, Richland, WA 99352 (United States); Lo, Jiunn-Guang [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013, Taiwan (China); Chiu, Kong-Hwa [Department of Applied Science, National DongHwa University, Hualien, 970, 30013, Taiwan (China); Pounds, Joel G. [Pacific Norwest National Laboratory, Richland, WA 99352 (United States); Lin Yuehe, E-mail: yuehe.lin@pnnl.gov [Pacific Norwest National Laboratory, Richland, WA 99352 (United States)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Using nanoparticle probe in immunochromatographic strip. Black-Right-Pointing-Pointer Enhanced sensitivity by using nanoparticle label. Black-Right-Pointing-Pointer Rapid and sensitive detection of cotinine in serum. - Abstract: A disposable sensor for the determination of cotinine in human serum was developed based on immunochromatographic test strip and quantum dot label. In this assay, cotinine linked with quantum dot competes with cotinine in sample to bind to anti-cotinine antibody in the test strip and the quantum dots serve as signal vehicles for electrochemical readout. Some parameters governing the performance of the sensor were optimized. The sensor shows a wide linear range from 1 ng mL{sup -1} to 100 ng mL{sup -1} cotinine with a detection limit of 1.0 ng mL{sup -1}. The sensor was validated with spiked human serum samples and it was found that this method was reliable in measuring cotinine in human serum. The results demonstrate that this sensor is rapid, accurate, and less expensive and has the potential for point of care (POC) detection of cotinine and fast screening of tobacco smoke exposure.

  6. Electrochemical Immunoassay of Cotinine in Serum Based on Nanoparticle Probe and Immunochromatographic Strip

    Energy Technology Data Exchange (ETDEWEB)

    Nian, Hung-Chi; Wang, Jun; Wu, Hong J.; Lo, Jiunn-Guang; Chiu, Kong-Hwa; Pounds, Joel G.; Lin, Yuehe

    2012-02-03

    A disposable sensor for the determination of cotinine in human serum was developed based on immunochromatographic test strip and quantum dot label. In this assay, cotinine modified on quantum dot competes with cotinine in sample to bind to anti-cotinine antibody in the test strip and the quantum dots serve as signal vehicles for electrochemical readout. Some parameters governing the performance of the sensor were optimized. The sensor shows a wide linear range from 1 ng mL-1 to 100 ng mL-1 cotinine with a detection limit of 1.0 ng mL-1. The relative standard deviation (R.S.D.) of the sensor was less than 2% for cotinine. The sensor was validated with spiked human serum samples and it was found that this method was reliable in measuring cotinine in human serum with average recovery of 100.99%. The results demonstrate that this sensor is a rapid, clinically accurate, and less expensive and has the potential for point of care (POC) detection of cotinine and fast screening of tobacco smoke exposure.

  7. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    Directory of Open Access Journals (Sweden)

    Seiji Iwasa

    2012-11-01

    Full Text Available Enzyme-linked immunosorbent assay (ELISA based on a monoclonal antibody (MoAb was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl-1,3-thiazol-2-ylthio] propionic acid was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA. The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64% to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  8. Simple and Sensitive Detection of HBsAg by Using a Quantum Dots Nanobeads Based Dot-Blot Immunoassay

    OpenAIRE

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; HAN, HUANXING; MA, Wei

    2014-01-01

    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection meth...

  9. Evaluation of LIAISON Treponema Screen, a Novel Recombinant Antigen-Based Chemiluminescence Immunoassay for Laboratory Diagnosis of Syphilis

    OpenAIRE

    Marangoni, Antonella; Sambri, Vittorio; Accardo, Silvia; Cavrini, Francesca; D'Antuono, Antonietta; Moroni, Alessandra; Storni, Elisa; Cevenini, Roberto

    2005-01-01

    The purpose of this study was to evaluate the diagnostic performance of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with that of rapid plasma reagin (RPR) and the following currently used treponemal tests: hemagglutination test (TPHA), immunoenzymatic assay (EIA), and Western blot (WB). First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically charac...

  10. Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus.

    OpenAIRE

    Pacini, D L; Brady, M T; Budde, C T; Connell, M J; Hamparian, V V; Hughes, J. H.

    1988-01-01

    Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE we...

  11. Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay

    OpenAIRE

    Szkola, A.; Linares, E. M.; Worbs, Sylvia; Dorner, Brigitte; R. Dietrich; Märtlbauer, E.; Niessner, R.; Seidel, Michael

    2014-01-01

    Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an a...

  12. Antibody-based magnetic nanoparticle immunoassay for quantification of Alzheimer's disease pathogenic factor

    Science.gov (United States)

    Kim, Chang-Beom; Choi, Yu Yong; Song, Woo Keun; Song, Ki-Bong

    2014-05-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that leads to a decline in cognitive and intellectual abilities and an irreversible mental deterioration. Based on multidisciplinary AD research, the most universally accepted hypotheses on AD pathogenesis are the intracerebral aggregate formation of beta-amyloid (Aβ) peptides. According to medical paradigmatic transition from medical treatment to early diagnostic prevention, scientists have considered physiological body fluid as a biomarker medium, in which the promising AD biomarkers could be verified. Recently, use of saliva has been considered as one of the diagnostic fluids over the past decade with meaningful diagnostic potential. We utilized saliva as a biomarker medium to correlate the salivary Aβ levels to AD pathological aspects, especially to the mild cognitive impairment group among AD patients, and to verify our detecting system to be sensitive enough for an early diagnostic tool. The identification of the salivary AD biomarkers using a facile microarraying method would motivate this study with the assistance of magnetically assembled antibody-conjugated nanoparticles and a photomultiplier tube as an optical detector. This simple magnetoimmunoassay system measures the photointensity generated by fluorescence, enables the quantification of the Aβ peptides from AD salivary samples, and consequently classifies the salivary Aβ levels into AD pathological aspects. This method demonstrates a facile approach enabling it to simply detect salivary Aβ peptides at a concentration as low as ˜20 pg/ml. It is expected that our simple magnetoimmunoassay system may have a potential as a detector for low-level Aβ peptides with weak-fluorescence emission.

  13. Surfactant Mediated Cationic and Anionic Suspension Polymerization of PEG-Based Resins in Silicon Oil: Beaded SPOCC 1500 and POEPOP 1500.

    Science.gov (United States)

    Grøtli; Rademan; Groth; Lubell; Miranda; Meldal

    2001-01-01

    A novel surfactant has been synthesized for use in cationic and anionic ring-opening suspension polymerization of PEG-based macromonomers in silicon oil. A polymer of acrylate esters containing pentamethyldisiloxane and PEG was prepared by radical polymerization. The surfactant can stabilize an emulsion of PEG-based macromonomers, initiator, and solvent in silicon oil such that polymer beads are obtained by ring-opening polymerization, initiated either by a Lewis acid (cationic ring opening) or potassium tert-butoxide (anionic ring opening). The average bead size could be controlled by varying the stirring rate and the amount of surfactant and solvent. The surfactant does not interfere with the polymerization and can be removed together with residual silicon oil by a simple washing procedure. PMID:11148061

  14. Theoretical analysis of a new, efficient microfluidic magnetic bead separator based on magnetic structures on multiple length scales

    DEFF Research Database (Denmark)

    Smistrup, Kristian; Bu, Minqiang; Wolff, Anders;

    2008-01-01

    We present a theoretical analysis of a new design for microfluidic magnetic bead separation. It combines an external array of mm-sized permanent magnets with magnetization directions alternating between up and down with mu m-sized soft magnetic structures integrated in the bottom of the separation...... channel. The concept is studied analytically for simple representative geometries and by numerical simulation of an experimentally realistic system geometry. The array of permanent magnets provides long-range magnetic forces that attract the beads to the channel bottom, while the soft magnetic elements...

  15. Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

    Science.gov (United States)

    Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

    2016-05-15

    In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk.

  16. Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes.

    Science.gov (United States)

    Towbin, H; Motz, J; Oroszlan, P; Zingel, O

    1995-04-26

    Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1. PMID:7745246

  17. Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay.

    Science.gov (United States)

    Gobi, K Vengatajalabathy; Matsumoto, Kiyoshi; Toko, Kiyoshi; Ikezaki, Hidekazu; Miura, Norio

    2007-04-01

    This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde-ovalbumin conjugate (BZ-OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ-OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ-OVA bound on the mixed SAM. The BZ-OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ-OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL(-1)) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin-bovine serum albumin (Bio-BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response

  18. A Magnetic Bead Actuator

    NARCIS (Netherlands)

    Derks, R.; Prins, M.W.J.; Wimberger-Friedl, R.

    2006-01-01

    Actuation principles of superparamagnetic beads applicable on biosensing (at single beads and chain orderning) are studied in this report. This research can be used to develop new techniques that are able to accelerate bio-assays. An experimental setup containing a sub-microliter fluid volume surrou

  19. Immunoassay as a screening tool for industrial toxicants

    Energy Technology Data Exchange (ETDEWEB)

    Pierce, T.

    1986-08-01

    Immunoassay techniques may represent useful screening tools to assist analysts interested in the presence and amounts of organic toxicants in biological fluids. The widespread application of immunoassay methods in medicinal and forensic (drugs of abuse) chemistry has resulted in such screening methodologies. Four methodologies of potential benefit are considered: the free radical assay technique, the enzyme-mediated immunoassay technique, radioimmunoassay, and hemagglutination. Each of these immunoassays is based on the competitive displacement of the labeled drug (or toxicant) from the antibody complex by the unlabeled drug-toxicant in the sample.

  20. Graphene-based rapid and highly-sensitive immunoassay for C-reactive protein using a smartphone-based colorimetric reader.

    Science.gov (United States)

    Vashist, Sandeep Kumar; Marion Schneider, E; Zengerle, Roland; von Stetten, Felix; Luong, John H T

    2015-04-15

    A novel immunoassay (IA) has been developed for human C-reactive protein (CRP), an important biomarker and tissue preserving factor for infection and inflammation. Graphene nanoplatelets (GNP) and 3-aminopropyltriethoxysilane (APTES) were admixed and covalently attached to a polystyrene based-microtiter plate (MTP), pretreated with KOH. The resulting surface served as a stable layer for the covalent attachment of the anti-human CRP antibody. The IA procedure was based on the one-step kinetics-based sandwich IA employing a minimum number of process steps, whereas the enzymatic reaction solution was monitored by a smartphone-based colorimetric reader. With a limit of detection and a limit of quantification of 0.07ngmL(-1) and 0.9ngmL(-1), it precisely detected CRP spiked in diluted human whole blood and plasma as well as the CRP levels in clinical plasma samples. The results obtained for "real-world" patient samples agreed well with those of the conventional immunosorbent assay and the clinically-accredited analyzer-based IA. The antibody-bound GNP-functionalized MTPs retained its original activity after 6 weeks of storage in 0.1M PBS, pH 7.4 at 4°C.

  1. Liquid carry-over in an injection moulded all-polymer chip system for immiscible phase magnetic bead-based solid-phase extraction

    DEFF Research Database (Denmark)

    Kistrup, Kasper; Sørensen, Karen Skotte; Wolff, Anders;

    2014-01-01

    -binding buffer for nucleic acid extraction (0.1 (v/v)% Triton X-100 in 5 M guanidine hydrochloride). A linear relationship was found between the liquid carry-over and mass of magnetic beads used. Interestingly, similar average carry-overs of 1.74(8) nL/µg and 1.72(14) nL/µg were found for Milli-Q water and lysis......We present an all-polymer, single-use microfluidic chip system produced by injection moulding and bonded by ultrasonic welding. Both techniques are compatible with low-cost industrial mass-production. The chip is produced for magnetic bead-based solid-phase extraction facilitated by immiscible......-binding buffer, respectively....

  2. Evaluation of an Immunoassay-Based Algorithm for Screening and Identification of Giardia and Cryptosporidium Antigens in Human Faecal Specimens from Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Yousry Hawash

    2014-01-01

    Full Text Available An immunoassay-based algorithm, involving three commercial kits, was introduced and evaluated for screening and identification of Giardia/Cryptosporidium antigens in human stool specimens. Initially, Giardia/Cryptosporidium Chek kit (TechLab, an enzyme-linked immunosorbent assay (ELISA, was adopted for screening. The ELISA-positive reactions were subsequently characterised by RIDA Quick Giardia and RIDA Quick Cryptosporidium immunochromatographic kits (R-Biopharm. A gold standard test comprising PCR and microscopy was used for preparing control samples. Performance of individual kits was tested against these samples which included 50 Giardia-positive, 40 Cryptosporidium-positive, and 70 Cryptosporidium/Giardia-negative. For Cryptosporidium, specificities of the ELISA and RIDA Quick Cryptosporidium kits were 95.71% and 100%, respectively. Both kits demonstrated sensitivity of 95%. For Giardia, the ELISA and RIDA Quick Giardia kits showed sensitivities of 100% and 97.5%, respectively. Specificities obtained by the ELISA and RIDA Quick Giardia were 95.7% and 100%, respectively. Based on the results of two reference PCRs, on 250 random samples, the algorithm exhibited sensitivity, specificity, positive predictive value, and negative predictive value of 97.06%, 100.00%, 100.00%, and 98.91%, respectively. In conclusion, this immunoassay-based algorithm can be used as routine test in diagnostic laboratories for screening and identification of a large number of samples.

  3. Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yiwen; Xiao, Guangyang; Hu, Bin, E-mail: binhu@whu.edu.cn

    2015-04-01

    The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L{sup −1} and 0.054 μg L{sup −1} with the relative standard deviations (RSDs, n = 7, c = 5 μg L{sup −1}) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2–50 μg L{sup −1}. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications. - Highlights: • 4-Mercaptophenylboronic acid functionalized magnetic beads were prepared and characterized. • ICP-MS based magnetic immunoassay approach was developed for quantification of glycoproteins. • AFP and CEA were quantified simultaneously with Au and Ag NPs as element tags. • The developed method exhibited good selectivity and sensitivity for target glycoproteins.

  4. Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

    International Nuclear Information System (INIS)

    The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L−1 and 0.054 μg L−1 with the relative standard deviations (RSDs, n = 7, c = 5 μg L−1) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2–50 μg L−1. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications. - Highlights: • 4-Mercaptophenylboronic acid functionalized magnetic beads were prepared and characterized. • ICP-MS based magnetic immunoassay approach was developed for quantification of glycoproteins. • AFP and CEA were quantified simultaneously with Au and Ag NPs as element tags. • The developed method exhibited good selectivity and sensitivity for target glycoproteins

  5. Magnetic Particle-Based Immunoassay of Phosphorylated p53 Using Protein-Cage Templated Lead Phosphate and Carbon Nanospheres for Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Aiqiong; Bao, Yuanwu; Ge, Xiaoxiao; Shin, Yongsoon; Du, Dan; Lin, Yuehe

    2012-11-20

    Phosphorylated p53 at serin 15 (phospho-p53-15) is a potential biomarker of Gamma-radiation exposure. In this paper, we described a new magnetic particles (MPs)-based electrochemical immunoassay of human phospho-p53-15 using carbon nanospheres (CNS) and protein-cage templated lead phosphate nanoparticles for signal amplification. Greatly enhanced sensitivity was achieved by three aspects: 1) The protein-cage nanoparticle (PCN) and p53-15 signal antibody (p53-15 Ab2) are linked to CNS (PCNof each apoferritin; 3) MPs capture a large amount of primary antibodies. Using apoferritin templated metallic phosphate instead of enzyme as label has the advantage of eliminating the addition of mediator or immunoreagents and thus makes the immunoassay system simpler. The subsequent stripping voltammetric analysis of the released lead ions were detected on a disposable screen printed electrode. The response current was proportional to the phospho-p53-15 concentration in the range of 0.02 to 20 ng mL-1 with detection limit of 0.01 ng mL-1. This method shows a good stability, reproducibility and recovery.

  6. An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate.

    Science.gov (United States)

    He, Yanlong; Tian, Jianniao; Hu, Kun; Zhang, Juanni; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

    2013-11-13

    In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8×10(-12) M to 2.40×10(-4) M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems. PMID:24176506

  7. Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics

    Science.gov (United States)

    Brazhnik, Kristina; Grinevich, Regina; Efimov, Anton E.; Nabiev, Igor; Sukhanova, Alyona

    2014-05-01

    Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of

  8. Aptamer-based microfluidic beads array sensor for simultaneous detection of multiple analytes employing multienzyme-linked nanoparticle amplification and quantum dots labels.

    Science.gov (United States)

    Zhang, He; Hu, Xinjiang; Fu, Xin

    2014-07-15

    This study reports the development of an aptamer-mediated microfluidic beads-based sensor for multiple analytes detection and quantification using multienzyme-linked nanoparticle amplification and quantum dots labels. Adenosine and cocaine were selected as the model analytes to validate the assay design based on strand displacement induced by target-aptamer complex. Microbeads functionalized with the aptamers and modified electron rich proteins were arrayed within a microfluidic channel and were connected with the horseradish peroxidases (HRP) and capture DNA probe derivative gold nanoparticles (AuNPs) via hybridization. The conformational transition of aptamer induced by target-aptamer complex contributes to the displacement of functionalized AuNPs and decreases the fluorescence signal of microbeads. In this approach, increased binding events of HRP on each nanosphere and enhanced mass transport capability inherent from microfluidics are integrated for enhancing the detection sensitivity of analytes. Based on the dual signal amplification strategy, the developed aptamer-based microfluidic bead array sensor could discriminate as low as 0.1 pM of adenosine and 0.5 pM cocaine, and showed a 500-fold increase in detection limit of adenosine compared to the off-chip test. The results proved the microfluidic-based method was a rapid and efficient system for aptamer-based targets assays (adenosine (0.1 pM) and cocaine (0.5 pM)), requiring only minimal (microliter) reagent use. This work demonstrated the successful application of aptamer-based microfluidic beads array sensor for detection of important molecules in biomedical fields. PMID:24534576

  9. Simple and sensitive detection of HBsAg by using a quantum dots nanobeads based dot-blot immunoassay.

    Science.gov (United States)

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; Han, Huanxing; Ma, Wei

    2014-01-01

    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection method for the detection of infectious disease markers that couples the dot-blot immunoassay with quantum dots nanobeads (QDNBs) as a reporter. First, the QDNBs were prepared by an oil-in-water emulsion-evaporation technique. Because of the encapsulation of several QDs in one particle, the fluorescent signal of reporter can be amplified with QDNBs in a one-step test and be read using a UV lamp obviating the need for complicated instruments. Detection of disease-associated markers in complex mixture is possible, which demonstrates the potential of developing QDNBs into a sensitive diagnostic kit. PMID:24505238

  10. Improved Peak Capacity for Capillary Electrophoretic Separations of Enzyme Inhibitors with Activity-Based Detection Using Magnetic Bead Microreactors

    OpenAIRE

    Yan, Xiaoyan; Gilman, S. Douglass

    2010-01-01

    A technique for separating and detecting enzyme inhibitors was developed using capillary electrophoresis with an enzyme microreactor. The on-column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase-coated superparamagnetic beads (2.8 μm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme ...

  11. Development of a sensitive enzyme immunoassay for human epidermal growth factor (urogastrone).

    Science.gov (United States)

    Kurobe, M; Tokida, N; Furukawa, S; Ishikawa, E; Hayashi, K

    1986-04-15

    A sensitive two-site enzyme immunoassay (EIA) for human epidermal growth factor (hEGF) was developed, based on the sandwiching of an antigen between anti-hEGF IgG-coated polystyrene beads and anti-hEGF Fab'-linked peroxidase complex (horseradish peroxidase, EC. 1.11.1.7). This method has four advantages: the anti-hEGF Fab'-linked peroxidase complex is more stable than 125I-labelled antibody; the procedure is simple and rapid compared to bioassay; its discriminatory sensitivity is as low as 0.1 pg/assay tube; and serial dilution curves of unextracted human serum and urine samples all paralleled that of standard hEGF. The validity of the measurement of hEGF-like immunoreactivity in human serum and plasma is discussed.

  12. Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

    International Nuclear Information System (INIS)

    Graphical abstract: A microfluidic beads-based nucleic acid sensor for sensitive detection of circulating tumor cells (CTCs) in the blood using multienzyme-nanoparticle amplification and quantum dots labels was developed. The chip-based CTCs analysis could detect reverse transcription-polymerase chain reaction (RT-PCR) products of tumor cell as low as 1 tumor cell (e.g. CEA expressing cell) in 1 mL blood sample. This microfluidic beads-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence. -- Highlights: •Combination of microfluidic bead-based platform and enzyme–probe–AuNPs is proposed. •The developed nucleic acid sensor could respond to 5 fM of tumor associated DNA. •Microfluidic platform and multienzyme-labeled AuNPs greatly enhanced sensitivity. •The developed nucleic acid sensor could respond to RT-PCR products of tumor cell as low as 1 tumor cell in 1 mL blood sample. •We report a sensitive nucleic acid sensor for detection of circulating tumor cells. -- Abstract: This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro

  13. Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, He, E-mail: mzhang_he@126.com; Fu, Xin; Hu, Jiayi; Zhu, Zhenjun

    2013-05-24

    Graphical abstract: A microfluidic beads-based nucleic acid sensor for sensitive detection of circulating tumor cells (CTCs) in the blood using multienzyme-nanoparticle amplification and quantum dots labels was developed. The chip-based CTCs analysis could detect reverse transcription-polymerase chain reaction (RT-PCR) products of tumor cell as low as 1 tumor cell (e.g. CEA expressing cell) in 1 mL blood sample. This microfluidic beads-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence. -- Highlights: •Combination of microfluidic bead-based platform and enzyme–probe–AuNPs is proposed. •The developed nucleic acid sensor could respond to 5 fM of tumor associated DNA. •Microfluidic platform and multienzyme-labeled AuNPs greatly enhanced sensitivity. •The developed nucleic acid sensor could respond to RT-PCR products of tumor cell as low as 1 tumor cell in 1 mL blood sample. •We report a sensitive nucleic acid sensor for detection of circulating tumor cells. -- Abstract: This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro

  14. Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels.

    Science.gov (United States)

    Zhang, He; Fu, Xin; Hu, Jiayi; Zhu, Zhenjun

    2013-05-24

    This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N)3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence. PMID:23663673

  15. Mass spectrometric immunoassay

    Science.gov (United States)

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2007-12-04

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  16. Superparamagnetic bead interactions with functionalized surfaces characterized by an immunomicroarray

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Hansen, Mikkel Fougt; Moresco, Jacob Lange;

    2010-01-01

    Magneto-resistive sensors capable of detecting superparamagnetic micro-/nano-sized beads are promising alternatives to standard diagnostic assays based on absorbance or fluorescence and streptavidin-functionalized beads are widely used as an integral part of these sensors. Here we have developed an...... SiO2 performed better than polyethylene glycol-modified surfaces Two beads, Masterbeads and M-280 beads, were found to give superior results compared with other bead types. Antibody/ antigen interactions, Illustrated by C-reactive protein, were best performed with Masterbeads The results provide...... important information concerning the surface binding properties of streptavidin-functionalized beads and the immunomicroarray can be used when optimizing the performance of bead-based biosensors....

  17. In-bead screening

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to screening of one-bead-one-compound (OBOC) combinatorial libraries which is useful for the discovery of compounds displaying molecular interactions with a biological or a physicochemical system, such as substrates and inhibitors of enzymes and the like. The invention...... provides a method for screening a library of compounds for their interaction with a physico- chemical or biological system and a corresponding kit for performing the method of screening a one-bead-one-compound library of compounds....

  18. Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres.

    Science.gov (United States)

    Meimaridou, Anastasia; Haasnoot, Willem; Shelver, Weilin L; Franek, Milan; Nielen, Michel W F

    2013-01-01

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs - instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples.

  19. Comparison of two extractable nuclear antigen testing algorithms: ALBIA versus ELISA/line immunoassay.

    Science.gov (United States)

    Chandratilleke, Dinusha; Silvestrini, Roger; Culican, Sue; Campbell, David; Byth-Wilson, Karen; Swaminathan, Sanjay; Lin, Ming-Wei

    2016-08-01

    Extractable nuclear antigen (ENA) antibody testing is often requested in patients with suspected connective tissue diseases. Most laboratories in Australia use a two step process involving a high sensitivity screening assay followed by a high specificity confirmation test. Multiplexing technology with Addressable Laser Bead Immunoassay (e.g., FIDIS) offers simultaneous detection of multiple antibody specificities, allowing a single step screening and confirmation. We compared our current diagnostic laboratory testing algorithm [Organtec ELISA screen / Euroimmun line immunoassay (LIA) confirmation] and the FIDIS Connective Profile. A total of 529 samples (443 consecutive+86 known autoantibody positivity) were run through both algorithms, and 479 samples (90.5%) were concordant. The same autoantibody profile was detected in 100 samples (18.9%) and 379 were concordant negative samples (71.6%). The 50 discordant samples (9.5%) were subdivided into 'likely FIDIS or current method correct' or 'unresolved' based on ancillary data. 'Unresolved' samples (n = 25) were subclassified into 'potentially' versus 'potentially not' clinically significant based on the change to clinical interpretation. Only nine samples (1.7%) were deemed to be 'potentially clinically significant'. Overall, we found that the FIDIS Connective Profile ENA kit is non-inferior to the current ELISA screen/LIA characterisation. Reagent and capital costs may be limiting factors in using the FIDIS, but potential benefits include a single step analysis and simultaneous detection of dsDNA antibodies.

  20. Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

    Directory of Open Access Journals (Sweden)

    Ludmila A. Frank

    2010-12-01

    Full Text Available Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

  1. X-Aptamers: A bead-based selection method for random incorporation of drug-like moieties onto next-generation aptamers for enhanced binding

    OpenAIRE

    He, WeiGuo; Elizondo-Riojas, Miguel-Angel; LI, XIN; Lokesh, Ganesh Lakshmana Rao; Somasunderam, Anoma; Thiviyanathan, Varatharasa; Volk, David E.; Durland, Ross H.; Englehardt, Johnnie; Cavasotto, Claudio N.; Gorenstein, David G.

    2012-01-01

    By combining pseudo-random bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities to the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) demonstrated to bind to CD44-HABD, to a complete monothioate backbone substituted aptamer to increase its binding affini...

  2. New synthesis and characterization of (+)-lysergic acid diethylamide (LSD) derivatives and the development of a microparticle-based immunoassay for the detection of LSD and its metabolites.

    Science.gov (United States)

    Li, Z; Goc-Szkutnicka, K; McNally, A J; Pilcher, I; Polakowski, S; Vitone, S; Wu, R S; Salamone, S J

    1997-01-01

    In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis of several analytical characterization studies, the most stable derivative has been selected and a procedure to covalently link the derivative to polystyrene microparticles through a carrier protein has been developed. In addition, two new LSD immunogens have been synthesized and characterized, and from these immunogens antibodies that recognize not only LSD but also several major LSD metabolites have been generated. Using the selected derivative and antibody, a homogeneous microparticle-based immunoassay has been developed for the detection of LSD in human urine with the required sensitivity and specificity for an effective screening assay. The performance of this LSD OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.

  3. Improved electrochemiluminescence labels for heterogeneous microbead immunoassay.

    Science.gov (United States)

    Yu, Linpo; Liu, Yang; Zhou, Ming

    2016-10-01

    Ruthenium(II) complexes with carboxylic acid as a bioconjugatable group, i.e., [Ru(bathophenanthroline disulfonate)(2,2'-bipyridine)(4-methyl-4'-(3-carboxypropyl)-2,2'-bipyridine)](0), (C49H38N6O8S2Ru), and [Ru(bathophenanthroline disulfonate)2(4-methyl-4'-(3-carboxypropyl)-2,2'-bipyridine)](2-) · 2Na(+), (C63H44N6O14S4RuNa2) were characterized spectroscopically and electrochemically. As potential labels for electrochemiluminescence (ECL) immunoassays, the ECL intensities of the free labels in homogenous aqueous buffer solutions were compared under a condition that is similar to the one employed by a commercial clinical immunoassay system. The two labels were found to be more emissive and, thus, can be detected at 10(- 12) pM compared with 5× 10(-12) pM of the label currently used in the commercial ECL system. Furthermore, the improved ECL emission of the free labels in homogenous solutions was proven to be translated into more intense ECL signal in heterogeneous sandwich immunoassay and, thus, leading to a lower limit of detection in immunoassay. The data obtained from these ECL labels shed light on the further development of ECL-based clinical immunoassay technology. Graphical abstract Electrochemiluminescence immunoassays were carried out with three different ruthenium(II) complex labels. It was proved that the higher signal intensities found with the novel labels in homogeneous solutions were maintained in heterogeneous sandwich format. PMID:27178555

  4. A compact and integrated immunoassay with on-chip dispensing and magnetic particle handling.

    Science.gov (United States)

    Zirath, Helene; Peham, Johannes R; Schnetz, Guntram; Coll, Albert; Brandhoff, Lukas; Spittler, Andreas; Vellekoop, Michael J; Redl, Heinz

    2016-02-01

    We present a compact diagnostic platform for a rapid and sensitive detection of plasma biomarkers. The platform consists of a disposable microfluidic polymer chip, a processing device including a lens-free and cost efficient sensor system and a setup for dispersion of magnetic particles. The biomarkers of interest are quantified by magnetic bead based immunoassays with chemiluminescent readout technology. With a novel system for dispersion and manipulation of the magnetic particles in combination with chemiluminescence detection, the sensitivity of the immunoassay is improved and enables a rapid assay in a microfluidic format. In the disposable chip, extra chambers for storage and dispensing of biomarker specific reagents are integrated, which reduce the need of external dosing devices and thereby the cost of the platform is decreased. Plasma biomarkers for monitoring of sepsis could be quantified at 10 pg/mL concentrations within a total time of 30 min by the present system. This contribution is a fundamental step towards the development of an automatic and compact Point-of-Care testing device for monitoring of patients at the intensive care unit.

  5. Chemiluminescence immunoassay for chloramphenicol

    International Nuclear Information System (INIS)

    A simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of Chloramphenicol(CAP) in foodstuffs is described. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled conjugant is horseradish peroxidase(HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/mL), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8 and <20%, respectively, and the analytical recovery of the method was 87% 100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The assay range for the method was 0.1-10 ng/mL, and it displayed good linearity. (authors)

  6. Electrochemical Sandwich Immunoassay for the Ultrasensitive Detection of Human MUC1 Cancer Biomarker

    Directory of Open Access Journals (Sweden)

    Zahra Taleat

    2013-01-01

    Full Text Available A new electrochemical sandwich immunoassay for the ultrasensitive detection of human MUC1 cancer biomarker using protein G-functionalized magnetic beads (MBs and graphite-based screen-printed electrodes (SPEs was developed. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process. A pair of primary and secondary antibodies was used to capture the MUC1 protein. After labeling with a third antibody conjugated with horseradish peroxidase (HRP, the resulting conjugate was trapped at the surface of the graphite-based SPEs and MUC1 determination was carried out by differential pulse voltammetry (DPV at 0.4 V upon H2O2 addition using acetaminophen (APAP as the redox mediator. A linear relationship was obtained for the detection of human MUC1 over a range of 0–25 ppb with the lowest detection limit of 1.34 ppb when HRP was applied as a label. Preliminary experiments were performed using disposable electrochemical sensors in order to optimize some parameters (i.e., incubation times, concentrations, and blocking agent.

  7. Method of immunoassay

    International Nuclear Information System (INIS)

    The invention relates to a method of immunoassay for prolactin using monoclonal lgG antibodies. The method preferably comprises the use of two different monoclonal antibodies which bind respectively at different antigenic sites on the prolactin molecule. One antibody is labelled and the other is immobilised on a water-insoluble carrier material, whereby an immunochemical complex comprising labelled antibody, prolactin and immobilised antibody is formed. (author)

  8. Simultaneous detection of forbidden chemical residues in milk using dual-label time-resolved reverse competitive chemiluminescent immunoassay based on amine group functionalized surface.

    Directory of Open Access Journals (Sweden)

    Dongdong Zhang

    Full Text Available In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP and clenbuterol (CLE in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP and alkaline phosphatase (ALP in chemiluminesecence (CL systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP and CLE (ALP-CLE. The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 10(5 of CLE as background for HRP CL signal value (about 10(7 of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC50 values of CAP and CLE in milk samples were 0.00501 µg L(-1 and 0.0128 µg L(-1, with the ranges from 0.0003 µg L(-1 to 0.0912 µg L(-1 and from 0.00385 µg L(-1 to 0.125 µg L(-1, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE.

  9. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

    Science.gov (United States)

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

    2016-08-01

    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%. PMID:27403652

  10. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

    Science.gov (United States)

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

    2016-08-01

    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%.

  11. An enzyme-free and resettable platform for the construction of advanced molecular logic devices based on magnetic beads and DNA.

    Science.gov (United States)

    Zhang, Siqi; Wang, Kun; Huang, Congcong; Li, Zhenyu; Sun, Ting; Han, De-Man

    2016-08-25

    A series of multiple logic circuits based on magnetic beads and DNA are constructed to perform resettable nonarithmetic functions, including a digital comparator, 4-to-2 encoder and 2-to-3 decoder, 2-to-1 encoder and 1-to-2 decoder. The signal reporter is composed of a G-quadruplex/NMM complex and a AuNP-surface immobilized molecular beacon. It is the first time that the designed DNA-based nonarithmetic nanodevices can share the same DNA platform with a reset function, which has great potential application in information processing at the molecular level. Another novel feature of the designed system is that the developed nanodevices are operated on a simple DNA/magnetic bead platform and share a constant threshold setpoint without the assistance of any negative logic conversion. The reset function is realized by heating the output system and the magnetic separation of the computing modules. Due to the biocompatibility and design flexibility of DNA, these investigations may provide a new route towards the development of resettable advanced logic circuits in biological and biomedical fields. PMID:27524500

  12. A new strategy for label-free electrochemical immunoassay based on "gate-effect" of β-cyclodextrin modified electrode.

    Science.gov (United States)

    Deng, Huan; Li, Jianping; Zhang, Yun; Pan, Hongcheng; Xu, Guobao

    2016-07-01

    A novel label-free electrochemical immunoassay was developed for prostate-specific antigen (PSA) detection via using β-cyclodextrin (β-CD) assembled layer created gates for the electron transfer of probe. To construct the sensor, a gold electrode was self-assembled with monoclonal anti-PSA antibody labeled 6-mercapto-β-cyclodextrin. Interspaces among β-CD molecules in the layer were automatically formed on gold electrode, which act as the channel of the electron transfer of [Fe(CN)6](3-/4-) probe. When PSA bind with anti-PSA, it can block these channels on the electrode surface due to their steric hindrance effect, resulting in the decrease in redox current of the probe. Through such a gate-controlled effect, ultra trace amount of PSA may make the currents change greatly after the immunoreaction, which enhanced the signal-to-noise ratio to achieve the amplification effect. By evaluating the logarithm of PSA concentrations, the immunosensor had a good linear response to the current changes with a detection limit of 0.3 pg/mL (S/N = 3) when PSA concentration ranged from 1.0 pg/mL to 1.0 ng/mL. The label-free immunosensor exhibited satisfactory performances in sensitivity, repeatability as well as specificity. PMID:27216392

  13. A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I

    Directory of Open Access Journals (Sweden)

    Seung-Wan Kim

    2016-05-01

    Full Text Available The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA was coupled with the fluorescent BODIPY dye (Red BSA, and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL for 30 min, the fluorescent signal was significantly increased (3.5-fold compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

  14. A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I.

    Science.gov (United States)

    Kim, Seung-Wan; Cho, Il-Hoon; Park, Ji-Na; Seo, Sung-Min; Paek, Se-Hwan

    2016-01-01

    The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA) was coupled with the fluorescent BODIPY dye (Red BSA), and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL) for 30 min, the fluorescent signal was significantly increased (3.5-fold) compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold) by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm) for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI) in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

  15. Strip-based immunoassay for the simultaneous detection of the neonicotinoid insecticides imidacloprid and thiamethoxam in agricultural products.

    Science.gov (United States)

    Xu, Ting; Xu, Qi Gong; Li, Hao; Wang, Jia; Li, Qing X; Shelver, Weilin L; Li, Ji

    2012-11-15

    A semiquantitative strip immunoassay was developed for the rapid detection of imidacloprid and thiamethoxam in agricultural products using specific nanocolloidal gold-labeled monoclonal antibodies. The conjugates of imidacloprid-BSA, thiamethoxam-BSA and goat anti-mouse IgG were coated on the nitro-cellulose membrane of the strip, serving as test lines and control line, respectively. The flow of the complexes of gold labeled antibodies and insecticides along the strip resulted in intensive color formed on the test lines inversely proportional to the concentrations of imidacloprid and thiamethoxam. The visual detection limits of imidacloprid and thiamethoxam in assay buffer were 0.5 and 2 ng mL(-1), respectively. Matrix interference of cucumber, tomato, lettuce, apple, and orange on the strip assay could be eliminated by diluting sample extracts with assay buffer. The strip analysis of imidacloprid and thiamethoxam in these samples was compared to liquid chromatography-mass spectrometry and the results were in good agreement. The strip was stable for storage more than 5 months at 4 °C. The strip assay is a rapid and simple method for the simultaneous screening of imidacloprid and thiamethoxam in agricultural products. PMID:23158295

  16. 利用悬液芯片系统建立一种高通量检测牛奶中头孢氨苄和莱克多巴胺残留的方法%On the Development of a Microsphere-based Multiplexed Immunoassay for Cephalexin and Ractopamine in Raw Milk

    Institute of Scientific and Technical Information of China (English)

    刘天龙; 王燕飞; 邹明强; 刘小雷; 云彩麟

    2011-01-01

    A rapid and convenient multiplexed, microsphere-based immunoassay was simultaneously developed for Cephalexin (Cex) and Ractopamine ( Rac) in raw milk. Synthetic antigen of Cephalexin and Ractopamine were covalently coupled to fluorescent-coded polystyrene microspheres as capture antigen, which were used to build a competitive detection system with standard sample of antibiotics and monoclonal antibody against Cephalexin and Ractopamine. Phycoerythrin-labeled goat anti mice IgG as fluorescent probe labeled monoclonal antibody specifically react with capture antigen. Fluorescent intensity associating microsphere and probe were detected respectively by laser on suspension array system to complete final detection. No crossreactivity of the antibody was observed in reaction system, indicating that the antibody is highly specific for Cephalexin and Ractopamine. Optimum concentration of antigen covered microsphere was set up (8. 4μg and 16. 64μg per lOOμl microspheres). Finally, the performance of beads-based immunoassay for simultaneous determination of Cephalexin and Ractopamine were evaluated demonstrating that the beads-based immunoassay is more adjective method for Cephalexin and Ractopamine determination in raw milk ( LOD Cex = 20. 59 ng/ml;LOD Rac = 23.51 ng/ml).%目的:旨在应用基于荧光编码微球技术的悬液芯片系统建立一种方便、稳定性好及高通量的检测牛奶中头孢氨苄和莱克多巴胺残留的免疫检测方法.方法:利用碳二亚胺法将抗生素合成抗原与表面具有羧基的聚苯乙烯微球通过酰胺键偶联成捕获抗原.利用捕获抗原、抗生素的单克隆抗体及抗生素标准品构建竞争性免疫检测体系.荧光标记的羊抗小鼠的IgG作为荧光探针标记与捕获抗原结合的单克隆抗体得到悬液芯片系统的检测物.悬液芯片系统的检测器由两柬特殊的激光构威,能够检测荧光探针荧光强度的同时分辨不同型号的微球以实现高通量

  17. One-step detection of pathogens and cancer biomarkers by the naked eye based on aggregation of immunomagnetic beads

    Science.gov (United States)

    Chen, Yiping; Xianyu, Yunlei; Sun, Jiashu; Niu, Yajing; Wang, Yu; Jiang, Xingyu

    2015-12-01

    This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay.This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07044a

  18. A half-ring GMR sensor for detection of magnetic beads immobilized on a circular micro-trap

    KAUST Repository

    Gooneratne, Chinthaka Pasan

    2011-11-01

    Utilizing magnetic principles in biological immunoassays is an attractive option given its ability to remotely and non-invasively manipulate and detect cells tagged with micro/nano size superparamagnetic type beads and due to the fact that even the most complex biological immunoassays will have very little magnetic effect. The presence of magnetic beads can be detected by a magnetic sensor which quantifies the amount of target cells present in the immunoassay. In order to increase the detection rate a circular conducting micro-trap is employed to attract, trap and transport the magnetic beads to the sensing area. In this research we propose a half-ring spin valve type giant magnetoresistance (GMR) sensor for the measurement of stray fields produced by 2 μm magnetic beads which are around the circular micro-trap. A couple of half-ring GMR sensors can be used to cover the entire circular border width, in order to detect the majority of the immobilized magnetic beads. Analytical and numerical analysis leading towards the fabrication of the half-ring GMR sensor are presented. DC characterization of the fabricated sensor showed a magnetoresistance of 5.9 %. Experimental results showed that the half-ring GMR sensor detected the presence of 2 μm magnetic beads. Hence, half-ring GMR sensors integrated with a circular micro-trap have great potential to be used as an effective disease diagnostic device. © 2011 IEEE.

  19. A photoacoustic immunoassay for biomarker detection.

    Science.gov (United States)

    Zhao, Yunfei; Cao, Mingfeng; McClelland, John F; Shao, Zengyi; Lu, Meng

    2016-11-15

    Challenges in protein biomarker analysis include insufficient sensitivity for detecting low-abundance biomarkers, poor measurement reproducibility, and the high costs and large footprints of detection systems. To address these issues, a new detection modality was developed for analyzing protein biomarkers based on the plasmon-enhanced photoacoustic (PA) effect. The detection modality employed a heterogeneous immunoassay scheme and used gold nanoparticles (AuNPs) as the signal reporter. Due to their localized plasmon resonance, AuNPs can strongly interact with intensity-modulated laser excitation and generate strong PA signals, which are subsequently sensed and quantified using a microphone. As an example, the performance of the PA immunoassay was evaluated by detecting the human interleukin 8 chemokine. The PA immunoassay provided approximately 143× lower limit of detection (LOD) than observed with the gold standard enzyme-linked immunosorbent assay - a decrease from 23pg/mL to 0.16pg/mL. In addition to the significant performance improvement in terms of the LOD, the PA immunoassay also offers advantages in terms of compatibility with low-cost instruments and the long-term stability of assay results. PMID:27183276

  20. Magnetic beads-based DNAzyme recognition and AuNPs-based enzymatic catalysis amplification for visual detection of trace uranyl ion in aqueous environment.

    Science.gov (United States)

    Zhang, Hongyan; Lin, Ling; Zeng, Xiaoxue; Ruan, Yajuan; Wu, Yongning; Lin, Minggui; He, Ye; Fu, FengFu

    2016-04-15

    We herein developed a novel biosensor for the visual detection of trace uranyl ion (UO2(2+)) in aqueous environment with high sensitivity and specificity by using DNAzyme-functionalized magnetic beads (MBs) for UO2(2+) recognition and gold nano-particles (AuNPs)-based enzymatic catalysis oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) for signal generation. The utilization of MBs facilitates the magnetic separation and collection of sensing system from complex sample solution, which leads to more convenient experimental operation and more strong resistibility of the biosensor to the matrix of sample, and the utilization of AuNPs-based enzymatic catalysis amplification greatly improved the sensitivity of the biosensor. Compared with the previous DNAzyme-based UO2(2+) sensors, the proposed biosensor has outstanding advantages such as relative high sensitivity and specificity, operation convenience, low cost and more strong resistibility to the matrix of sample. It can be used to detect as low as 0.02 ppb (74 pM) of UO2(2+) in aqueous environment by only naked-eye observation and 1.89 ppt (7.0 pM) of UO2(2+) by UV-visible spectrophotometer with a recovery of 93-99% and a RSD ≤ 5.0% (n=6) within 3h. Especially, the visual detection limit of 0.02 ppb (74 pM) is much lower than the maximum allowable level of UO2(2+) (130 nM) in the drinking water defined by the U.S. Environmental Protection Agency (EPA), indicating that our method meets the requirement of rapid and on-site detection of UO2(2+) in the aqueous environment by only naked-eye observation.

  1. Interference in immunoassay

    International Nuclear Information System (INIS)

    Interfering factors are evident in both limited reagent (radioimmunoassay) and excess reagent (immunometric assay) technologies and should be suspected whenever there is a discrepancy between analytical results and clinical findings in the investigation of particular diseases. The overall effect of interference in immunoassay is analytical bias in result, either positive or negative of variable magnitude. The interference maybe caused by a wide spectrum of factors from poor sample collection and handling to physiological factors e.g. lipaemia, heparin treatment, binding protein abnormalities, autoimmunity and drug treatments. The range of interfering factors is extensive and difficult to discuss effectively in a short review

  2. Enzyme immunoassay for human ferritin

    International Nuclear Information System (INIS)

    We described an enzyme immunoassay with use of β-D-galactosidase for quantitation of ferritin in human serum. The minimum detectable ferritin concentration is 0.25 μg/L of serum, which is comparable to results obtained by radioimmunoassay. The correlation coefficient between values determined by enzyme immunoassay and radioimmunoassay was 0.95

  3. Automated separation for heterogeneous immunoassays

    OpenAIRE

    Truchaud, A.; Barclay, J; Yvert, J. P.; Capolaghi, B.

    1991-01-01

    Beside general requirements for modern automated systems, immunoassay automation involves specific requirements as a separation step for heterogeneous immunoassays. Systems are designed according to the solid phase selected: dedicated or open robots for coated tubes and wells, systems nearly similar to chemistry analysers in the case of magnetic particles, and a completely original design for those using porous and film materials.

  4. Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins.

    Science.gov (United States)

    Adhikari, Rajan P; Haudenschild, Christian; Sterba, Patricia M; Sahandi, Sara; Enterlein, Sven; Holtsberg, Frederick W; Aman, M Javad

    2016-03-01

    An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton-Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens - Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.

  5. Performance improvement of the one-dot lateral flow immunoassay for aflatoxin B1 by using a smartphone-based reading system.

    Science.gov (United States)

    Lee, Sangdae; Kim, Giyoung; Moon, Jihea

    2013-01-01

    This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis. PMID:23598499

  6. Performance Improvement of the One-Dot Lateral Flow Immunoassay for Aflatoxin B1 by Using a Smartphone-Based Reading System

    Directory of Open Access Journals (Sweden)

    Jihea Moon

    2013-04-01

    Full Text Available This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1 was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis.

  7. A two-channel detection method for autofluorescence correction and efficient on-bead screening of one-bead one-compound combinatorial libraries using the COPAS fluorescence activated bead sorting system

    International Nuclear Information System (INIS)

    One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads. (technical note)

  8. An immunoassay for dibutyl phthalate based on direct hapten linkage to the polystyrene surface of microtiter plates.

    Directory of Open Access Journals (Sweden)

    Chenxi Wei

    Full Text Available BACKGROUND: Dibutyl phthalate (DBP is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. METHODOLOGY/PRINCIPAL FINDINGS: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC. Compared with conjugate coated format (IC(50=106 ng/mL, the direct hapten coated format (IC(50=14.6 ng/mL improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. CONCLUSIONS/SIGNIFICANCE: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed

  9. Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

    Institute of Scientific and Technical Information of China (English)

    BO Chun-Miao; GONG Bo-Lin; HU Wen-Zhi

    2008-01-01

    Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 μm monodisperse non-porous poly(glycidyl methacrylate-co-ethylenedimethacrylate)(PGMMEDMA)beads.The retention behavior of proteins on the metal ion chelated columns loaded with copper(Ⅱ),nickel(Ⅱ)and zin(Ⅱ)ion was studied.The effect of pH on the protein retention Was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0.Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni2+ -IDA(iminodiacetic acid)packings.The separation time was shorter than other immobilized metal affinity chromatography reported in the literature.Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked-IDA and Cu2+ -IDA columns,respectively.The purities of the purified trypsin and lysozyme were more than 92%and 95%,respectively.

  10. Polyelectrolyte-based electrochemiluminescence enhancement for Ru(bpy){sub 3}{sup 2+} loaded by SiO{sub 2} nanoparticle carrier and its high sensitive immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Zhi-Li; Song, Tian-Mei; Chen, Zhe [College of Pharmaceutical Science, Soochow University, Suzhou 215123 (China); Guo, Wu-Run [College of Pharmaceutical Science, Soochow University, Suzhou 215123 (China); College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637002 (China); Xie, Hong-Ping, E-mail: hpxie@suda.edu.cn [College of Pharmaceutical Science, Soochow University, Suzhou 215123 (China); Xie, Lian, E-mail: xielian@suda.edu.cn [College of Pharmaceutical Science, Soochow University, Suzhou 215123 (China)

    2015-03-03

    Highlights: • Preparation of strong ECL nanoparticles PAA–Ru@SiO{sub 2}/[PAA⋯Ru&Nafion⋯Ru]. • Ion-pair macromolecule PAA–Ru formed to greatly increase the doping amount. • PAA&Nafion membrane increased the amount of ion-exchanged Ru(bpy){sub 3}{sup 2+}. • PAA&Nafion membrane enhanced the ability of electron transfer. • Realized antibody labeling and established a high-sensitive immunoassay. - Abstract: In this paper the strong electrochemiluminescence (ECL) nanoparticles have been prepared based on the anionic polyelectrolyte sodium polyacrylate (PAA)-ECL enhancement for Ru(bpy){sub 3}{sup 2+}, which were loaded by the carrier of SiO{sub 2} nanoparticle. There were two kinds of Ru(bpy){sub 3}{sup 2+} for the as-prepared nanoparticles, the doped one and the exchanged one. The former was loaded inside the ECL nanoparticles by doping, in a form of ion-pair macromolecules PAA–Ru(bpy){sub 3}{sup 2+}. The corresponding ECL was enhanced about 2 times owing to the doping increase of Ru(bpy){sub 3}{sup 2+}. The latter was loaded on the PAA-doped Nafion membrane by ion exchange. The corresponding ECL was enhanced about 3 times owing to the ion-exchanging increase of Ru(bpy){sub 3}{sup 2+}. At the same time, ECL intensity of the doped-inside Ru(bpy){sub 3}{sup 2+} was further enhanced 13 times because polyelectrolyte PAA in the doped membrane could obviously enhance electron transfer between the doped Ru(bpy){sub 3}{sup 2+} and the working electrode. Furthermore, based on hydrophobic regions of the doped membrane antibody labeling could be easily realized by the as-prepared nanoparticles and then a high sensitive ECL immunoassay for HBsAg was developed. The linear range was between 1.0 and 100 pg mL{sup −1} (R{sup 2} = 0.9912). The detection limit could be as low as 0.11 pg mL{sup −1} (signal-to-noise ratio = 3)

  11. Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

    Directory of Open Access Journals (Sweden)

    Jinu Manoj

    2015-08-01

    Full Text Available Aim: To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC based enzyme-linked immunoassay (ELISA for the diagnosis of salmonellosis in poultry. Materials and Methods: Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results: The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62% samples with rOmpC antigen, while 24 (9.41% samples gave a positive reaction with both Omp and whole cell antigens. Conclusion: We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

  12. Novel circulating peptide biomarkers for esophageal squamous cell carcinoma revealed by a magnetic bead-based MALDI-TOFMS assay.

    Science.gov (United States)

    Jia, Kun; Li, Wei; Wang, Feng; Qu, Haixia; Qiao, Yuanyuan; Zhou, Lanping; Sun, Yulin; Ma, Qingwei; Zhao, Xiaohang

    2016-04-26

    Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application. PMID:26993605

  13. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

    Science.gov (United States)

    Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

  14. A capillary flow immunoassay microchip utilizing inkjet printing-based antibody immobilization onto island surfaces—toward sensitive and reproducible determination of carboxyterminal propeptide of type I procollagen

    Science.gov (United States)

    Fuchiwaki, Yusuke; Tanaka, Masato; Takaoka, Hiroki; Goya, Kenji

    2016-04-01

    A capillary-flow-driven microchip system requires no external power and has no moving off-chip components, in contrast with most microfluidic-based immunoassay systems which are complicated to operate and require external components. To accelerate the sensitive and reproducible determination of analytes required for practical point-of-care applications, we formed island microchannel surfaces on a microcapillary channel to allow stable antibody immobilization. The island surface was surrounded by a circular groove 10 μm deep and 150 μm wide and allowed uniform inkjet printing of antibody spots, complete bio-reagent replacement, and sensitive detection of luminescence intensity. Quantitative analysis of carboxyterminal propeptide of type I procollagen (PICP) concentrations using this microchannel was demonstrated between 0-600 ng·ml-1, which is adequate for the clinical estimation of PICP concentrations in the blood. This microchip system holds promise as a model diagnostic platform that is readily adaptable to hands-free operation.

  15. Lateral flow test strip based on colloidal selenium immunoassay for rapid detection of melamine in milk, milk powder, and animal feed.

    Science.gov (United States)

    Wang, Zhizeng; Zhi, Dejuan; Zhao, Yang; Zhang, Hailong; Wang, Xin; Ru, Yi; Li, Hongyu

    2014-01-01

    Although high melamine (MEL) intake has been proven to cause serious health problems, MEL is sometimes illegally added to milk products and animal feed, arousing serious food safety concerns. A satisfactory method of detecting MEL in onsite or in-home testing is in urgent need of development. This work aimed to explore a rapid, convenient, and cost-effective method of identifying MEL in milk products or other food by colloidal selenium-based lateral flow immunoassay. Colloidal selenium was synthesized by L-ascorbic acid to reduce seleninic acid at room temperature. After conjugation with a monoclonal antibody anti-MEL, a test strip was successfully prepared. The detection limit of the test strip reached 150 μg/kg, 1,000 μg/kg, and 800 μg/kg in liquid milk, milk powder, and animal feed, respectively. No cross-reactions with homologues cyanuric acid, cyanurodiamide, or ammelide were found. Moreover, the MEL test strip can remain stable after storage for 1 year at room temperature. Our results demonstrate that the colloidal selenium MEL test strip can detect MEL in adulterated milk products or animal feed conveniently, rapidly, and sensitively. In contrast with a colloidal gold MEL test strip, the colloidal selenium MEL test strip was easy to prepare and more cost-efficient.

  16. One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min.

    Science.gov (United States)

    Vashist, Sandeep Kumar; Czilwik, Gregor; van Oordt, Thomas; von Stetten, Felix; Zengerle, Roland; Marion Schneider, E; Luong, John H T

    2014-07-01

    This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml(-1) with a limit of detection of 0.4 ng ml(-1) and an analytical sensitivity of 0.7 ng ml(-1). It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

  17. A High-Throughput SU-8Microfluidic Magnetic Bead Separator

    DEFF Research Database (Denmark)

    Bu, Minqiang; Christensen, T. B.; Smistrup, Kristian;

    2007-01-01

    We present a novel microfluidic magnetic bead separator based on SU-8 fabrication technique for high through-put applications. The experimental results show that magnetic beads can be captured at an efficiency of 91 % and 54 % at flow rates of 1 mL/min and 4 mL/min, respectively. Integration...

  18. Highly sensitive electroluminescence immunoassay for Hg(II) ions based on the use of CdSe quantum dots, the methylmercury-6-mercaptonicotinic acid-ovalbumin conjugate, and a specific monoclonal antibody

    International Nuclear Information System (INIS)

    We have designed a rapid and ultrasensitive electrochemiluminescent (ECL) competitive immunoassay for the determination of mercury(II) ion. It is based on the use of CdSe quantum dots (QDs), methylmercury-6-mercaptonicotinic acid-ovalbumin as coating antigen and specific monoclonal antibodies (mAbs) against Hg(II). The latter is quite selective for Hg(II). The coating antigen was immobilized on the surface of a gold electrode via reaction between the functional groups of cysteamine and glutaraldehyde. The mercury(II) ions in a sample and the coating antigen compete for binding sites of QD-labeled monoclonal antibody which binds specifically to Hg(II) ions. The ECL of the system decreases with increasing concentration of Hg(II) because less QD-labeled mAbs are present on the surface of the electrode. Under optimal conditions, the decrease of ECL intensity is linearly related to the logarithm of the Hg(II) concentration in the range from 0.02 to 100 ng mL−1, with a detection limit of 6.2 pg mL−1. As far as we know, this is the first report on an ECL immunoassay for Hg(II) based on a specific monoclonal antibody. The favorable results obtained when this method was applied to real samples indicate that this detection scheme can widely enlarge the applicability of detecting heavy metal ions by exploiting the ECL of QDs for immunoassays. (author)

  19. Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

    2016-06-22

    In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

  20. Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

    2016-06-22

    In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources. PMID:27177195

  1. 125I-labeled protein A as a general tracer in immunoassay: suitability of goat and sheep antibodies

    International Nuclear Information System (INIS)

    The immunoassay method in which 125I-labeled staphylococcal Protein A ([125I]PA) serves as a general tracer has been extended to include goat and sheep IgG antibodies. Goat and sheep IgG normally do not react significantly with PA. However, once IgG antibody is bound to immobilized antigen or hapten, binding of [125I]PA is enhanced markedly. Binding efficiencies of [125I]PA to immune complexed goat anti-human IgM, human IgE, methotrexate and sheep anti-IgE were determined and compared quantitatively to rabbit IgG with the corresponding specificity. Immunoassays were developed based on the inhibition of [125I]PA binding as a measure of antibody inhibition by fluid-phase homologous ligand. Goat antibody to the monovalent hapten methotrexate behaved anomalously: for each concentration of IgG tested, there was an optimal amount of methotrexate beads that gave maximum binding of [125I]PA. In the other immune systems, for each antibody concentration maximum binding of tracer was a function only of the amount of immobilized anitgen added. In contrast to the results obtained with solid-phase antigen, solutions containing antibody and amounts of antigen ranging from large antigen excess to antibody excess failed to react significantly with PA or [125I]PA. (Auth.)

  2. Polyelectrolyte-based electrochemiluminescence enhancement for Ru(bpy)₃²⁺ loaded by SiO₂ nanoparticle carrier and its high sensitive immunoassay.

    Science.gov (United States)

    Ge, Zhi-Li; Song, Tian-Mei; Chen, Zhe; Guo, Wu-Run; Xie, Hong-Ping; Xie, Lian

    2015-03-01

    In this paper the strong electrochemiluminescence (ECL) nanoparticles have been prepared based on the anionic polyelectrolyte sodium polyacrylate (PAA)-ECL enhancement for Ru(bpy)3(2+), which were loaded by the carrier of SiO2 nanoparticle. There were two kinds of Ru(bpy)3(2+) for the as-prepared nanoparticles, the doped one and the exchanged one. The former was loaded inside the ECL nanoparticles by doping, in a form of ion-pair macromolecules PAA-Ru(bpy)3(2+). The corresponding ECL was enhanced about 2 times owing to the doping increase of Ru(bpy)3(2+). The latter was loaded on the PAA-doped Nafion membrane by ion exchange. The corresponding ECL was enhanced about 3 times owing to the ion-exchanging increase of Ru(bpy)3(2+). At the same time, ECL intensity of the doped-inside Ru(bpy)3(2+) was further enhanced 13 times because polyelectrolyte PAA in the doped membrane could obviously enhance electron transfer between the doped Ru(bpy)3(2+) and the working electrode. Furthermore, based on hydrophobic regions of the doped membrane antibody labeling could be easily realized by the as-prepared nanoparticles and then a high sensitive ECL immunoassay for HBsAg was developed. The linear range was between 1.0 and 100 pg mL(-1) (R(2)=0.9912). The detection limit could be as low as 0.11 pg mL(-1) (signal-to-noise ratio=3). PMID:25682425

  3. Rapid Wuchereria bancrofti-Specific Antigen Wb123-Based IgG4 Immunoassays as Tools for Surveillance following Mass Drug Administration Programs on Lymphatic Filariasis

    Science.gov (United States)

    Steel, Cathy; Golden, Allison; Kubofcik, Joseph; LaRue, Nicole; de los Santos, Tala; Domingo, Gonzalo J.

    2013-01-01

    The Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapid IgG4 enzyme-linked immunosorbent assay (ELISA) and a lateral-flow strip immunoassay, were developed based on the highly sensitive and specific Wuchereria bancrofti antigen Wb123. A comparison of W. bancrofti-infected and -uninfected patients (with or without other helminth infections) demonstrated that both tests had high sensitivities and specificities (93 and 97% [ELISA] and 92 and 96% [strips], respectively). When the W. bancrofti-uninfected group was separated into those with other filarial/helminth infections (i.e., onchocerciasis, loiasis, and strongyloidiasis) and those who were parasite uninfected, the specificities of the assays varied between 91 and 100%. In addition, the geometric mean response by ELISA of W. bancrofti-infected patients was significantly higher than the response of those without W. bancrofti infection (P < 0.0001). Furthermore, the Wb123 ELISA and the lateral-flow strips had high positive and negative predictive values, giving valuable information on the size of survey population needed to be reasonably certain whether or not transmission is ongoing. These highly sensitive and specific IgG4 tests to the W. bancrofti Wb123 protein give every indication that they will serve as useful tools for post-MDA monitoring. PMID:23740923

  4. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

    Directory of Open Access Journals (Sweden)

    Zhang Haiyu

    2007-12-01

    Full Text Available Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD, testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4 was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old" samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of

  5. Integration of Multiplex Bead Assays for Parasitic Diseases into a National, Population-Based Serosurvey of Women 15-39 Years of Age in Cambodia.

    Science.gov (United States)

    Priest, Jeffrey W; Jenks, M Harley; Moss, Delynn M; Mao, Bunsoth; Buth, Sokhal; Wannemuehler, Kathleen; Soeung, Sann Chan; Lucchi, Naomi W; Udhayakumar, Venkatachalam; Gregory, Christopher J; Huy, Rekol; Muth, Sinuon; Lammie, Patrick J

    2016-05-01

    Collection of surveillance data is essential for monitoring and evaluation of public health programs. Integrated collection of household-based health data, now routinely carried out in many countries through demographic health surveys and multiple indicator surveys, provides critical measures of progress in health delivery. In contrast, biomarker surveys typically focus on single or related measures of malaria infection, HIV status, vaccination coverage, or immunity status for vaccine-preventable diseases (VPD). Here we describe an integrated biomarker survey based on use of a multiplex bead assay (MBA) to simultaneously measure antibody responses to multiple parasitic diseases of public health importance as part of a VPD serological survey in Cambodia. A nationally-representative cluster-based survey was used to collect serum samples from women of child-bearing age. Samples were tested by MBA for immunoglobulin G antibodies recognizing recombinant antigens from Plasmodium falciparum and P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium, and Strongyloides stercoralis. Serologic IgG antibody results were useful both for generating national prevalence estimates for the parasitic diseases of interest and for confirming the highly focal distributions of some of these infections. Integrated surveys offer an opportunity to systematically assess the status of multiple public health programs and measure progress toward Millennium Development Goals. PMID:27136913

  6. Integration of Multiplex Bead Assays for Parasitic Diseases into a National, Population-Based Serosurvey of Women 15-39 Years of Age in Cambodia

    Science.gov (United States)

    Priest, Jeffrey W.; Jenks, M. Harley; Moss, Delynn M.; Mao, Bunsoth; Buth, Sokhal; Wannemuehler, Kathleen; Soeung, Sann Chan; Lucchi, Naomi W.; Udhayakumar, Venkatachalam; Gregory, Christopher J.; Huy, Rekol; Muth, Sinuon; Lammie, Patrick J.

    2016-01-01

    Collection of surveillance data is essential for monitoring and evaluation of public health programs. Integrated collection of household-based health data, now routinely carried out in many countries through demographic health surveys and multiple indicator surveys, provides critical measures of progress in health delivery. In contrast, biomarker surveys typically focus on single or related measures of malaria infection, HIV status, vaccination coverage, or immunity status for vaccine-preventable diseases (VPD). Here we describe an integrated biomarker survey based on use of a multiplex bead assay (MBA) to simultaneously measure antibody responses to multiple parasitic diseases of public health importance as part of a VPD serological survey in Cambodia. A nationally-representative cluster-based survey was used to collect serum samples from women of child-bearing age. Samples were tested by MBA for immunoglobulin G antibodies recognizing recombinant antigens from Plasmodium falciparum and P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium, and Strongyloides stercoralis. Serologic IgG antibody results were useful both for generating national prevalence estimates for the parasitic diseases of interest and for confirming the highly focal distributions of some of these infections. Integrated surveys offer an opportunity to systematically assess the status of multiple public health programs and measure progress toward Millennium Development Goals. PMID:27136913

  7. Fabrication of inorganic-organic hybrid based on polyoxometalate SiW10Fe2 and folate as peroxidases for colorimetric immunoassay of cancer cells

    Institute of Scientific and Technical Information of China (English)

    Zhong Sun; Hai-Zhou Bie; Mei-Jie Wei; Jing-Jing wang; Xu-Guang Mi; Xiao-Hong Wang; Yin Wu

    2013-01-01

    Fabrication of folate and iron-substituted polyoxometalate [(FeOH2)2SiW10O36] to form nanoparticles(FA-SiWFe2) has been achieved.This inorganic-organic hybrid possesses intrinsic peroxidase-like activity,which could be used in detection of cancer cells in colorimetric multiplexed immunoassay.

  8. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

    Directory of Open Access Journals (Sweden)

    Xiaohua He

    Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

  9. Lateral flow test strip based on colloidal selenium immunoassay for rapid detection of melamine in milk, milk powder, and animal feed

    Directory of Open Access Journals (Sweden)

    Wang ZZ

    2014-04-01

    Full Text Available Zhizeng Wang,1 Dejuan Zhi,2 Yang Zhao,1 Hailong Zhang,2 Xin Wang,2 Yi Ru,1 Hongyu Li1,2 1MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, People's Republic of China; 2Institute of Microbiology and Biochemical Pharmacy, School of Pharmaceutics, Lanzhou University, Lanzhou, People's Republic of China Abstract: Although high melamine (MEL intake has been proven to cause serious health problems, MEL is sometimes illegally added to milk products and animal feed, arousing serious food safety concerns. A satisfactory method of detecting MEL in onsite or in-home testing is in urgent need of development. This work aimed to explore a rapid, convenient, and cost-effective method of identifying MEL in milk products or other food by colloidal selenium-based lateral flow immunoassay. Colloidal selenium was synthesized by L-ascorbic acid to reduce seleninic acid at room temperature. After conjugation with a monoclonal antibody anti-MEL, a test strip was successfully prepared. The detection limit of the test strip reached 150 µg/kg, 1,000 µg/kg, and 800 µg/kg in liquid milk, milk powder, and animal feed, respectively. No cross-reactions with homologues cyanuric acid, cyanurodiamide, or ammelide were found. Moreover, the MEL test strip can remain stable after storage for 1 year at room temperature. Our results demonstrate that the colloidal selenium MEL test strip can detect MEL in adulterated milk products or animal feed conveniently, rapidly, and sensitively. In contrast with a colloidal gold MEL test strip, the colloidal selenium MEL test strip was easy to prepare and more cost-efficient. Keywords: melamine, selenium nanoparticles, test strip, milk, animal feed, dairy food

  10. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    Science.gov (United States)

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples. PMID:27376196

  11. Determination of human IgG by solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing dibromofluorescein luminescent molecules

    International Nuclear Information System (INIS)

    Luminescent silicon dioxide nano-particles with size of 20 nm, which containing dibromofluorescein (D) were synthesized by sol-gel method (symbolized by D-SiO2).The particles can emit intense and stable room temperature phosphorescence signal on polyamide membrane when Pb(Ac)2 was used as a heavy atom perturber. The λexmax/λemmax was 457/622 nm. Our research indicated that the specific immune reaction between goat-anti-human IgG antibody labeled with D-SiO2 and human IgG could be carried out on polyamide membrane quantitatively, and the phosphorescence intensity of the particle was enhanced after the immunoreactions. Thus a new method of solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human IgG was established basing on antibody labeled with the D-SiO2 nanoparticles. The linear range of this method was 0.0624-20.0 pg human IgG spot-1 (corresponding concentration: 0.156-50.0 ng ml-1, the sample volume: 0.40 μl spot-1) with a limit of detection (LD) as 0.018 pg spot-1, and the regression equation of working curve was ΔIp = 7.201 mIgG (pg spot-1) + 82.57. Samples containing 0.156 and 50.0 ng ml-1 of IgG were measured repeatedly for 11 times and R.S.D.s were 4.1 and 3.4%, respectively. Results showed that this method had the merits as sensitive, accurate and precise

  12. Determination of human IgG by solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing Rhodamine 6G luminescent molecules

    Science.gov (United States)

    Jia-Ming, Liu; hui, Zhu Guo; Aihong, Wu; Pingping, Li; Huanhuan, Xu; Li, Long-Di; Liu, Zhen-bo

    2005-03-01

    Luminescent silicon dioxide nanoparticles (R-SiO 2) with size of 50 nm containing Rhodamine 6G (R) were synthesized by sol-gel method. In the presence of Pb(Ac) 2 as a heavy atom perturber, the particle can emit intense and stable room temperature phosphorescence signal of R, respectively, on polyamide membrane, with the λexmax/λemmax=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO 2 and human IgG can be carried on polyamide membrane quantitatively, and the phosphorescence intensity was enhanced after the immunoreactions. Thus, a new method of solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human IgG was established basing on antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624-20.0 pg spot -1 of human IgG (corresponding concentration, 0.156-50.0 ng mL -1; sample volume, 0.40 μL spot -1). The regression equations of working curves are Δ Ip = 88.16. + 16.79m IgG (pg spot -1) (485/646 nm, r = 0.9997). Detection limits calculated by 3Sb/k are 0.017 pg spot -1. For samples containing 0.156 and 50.0 ng mL -1 of IgG, we measured repeatedly for 11 times, RSDs are 3.9 and 2.8%, respectively. This method is sensitive, accurate and of high precision.

  13. Rheological Modeling with Hookean Bead-Spring Cubes (SC, BBC and FCC)

    NARCIS (Netherlands)

    Denneman, A.I.M.; Jongschaap, R.J.J.; Mellema, J.

    1998-01-01

    In this study a general bead-spring model is used for predicting some rheological properties of a cubic bead-spring structure of arbitrary size immersed in a Newtonian solvent. The topology of this bead-spring structure is based upon the well-known cubic crystals (SC, BCC or FCC) and it consists of

  14. A NOVEL APPROACH TO SYNTHESIZE CHITOSAN BEADS CROSSLINKED BY EPICHLOROHYDRIN

    Institute of Scientific and Technical Information of China (English)

    WANG Yongjian; BAI Shu; SUN Yan

    2001-01-01

    The present investigation describes a novel method for preparing spherical chitosan particles based on crosslinking with epichlorohydrin. Certain amount of pre-crosslinking agent was added to form chitosan gels by traditional inverse phase suspension polymerization. Then the gels were crosslinked by epichlorohydrin at basic condition to obtain chitosan beads. The effects of reaction conditions, such as crosslinking time, the amount of crosslinking agent and the NaOtt concentration,on the physical properties of the chitosan beads were investigated. The beads were found to have more amino groups in the polymer chains than the beads crosslinked by glutaraldehyde. The capacity for copper ions is as high as 40mg/g. The beads have good mechanical strength and can be reused.

  15. A NOVEL APPROACH TO SYNTHESIZE CHITOSAN BEADS CROSSLINKED BY EPICHLOROHYDRIN

    Institute of Scientific and Technical Information of China (English)

    WANGYongjina; BAIShu; 等

    2001-01-01

    The present investigation describes a novel method for preparing spherical chitosan particles based on crosslinking with epichlorohydrin.Certain amount of pre-crosslinking agent was added to form chitosan gels by traditional inverse phase suspension polymerization.Then the gels were crosslinked by epichlorohydrin at basic condition to obtain chitosan beads.The effects of reaction conditions,such as crosslinking time,the amount of crosslinking agent and the NaOH concentration,on the physical properties of the chitosan beads were investigated.The beads were found to have more amino groups in the polymer chains than the beads crosslinked by glutaraldehyde.The capacity for copper ions in as high as 40mg/g,The beads have good mechanical strength and can be reused.

  16. Magnetic manipulation and sensing of beads for bioapplications

    DEFF Research Database (Denmark)

    Henriksen, Anders Dahl

    a minimum feature size of one micron, which limits the movable bead size to approximately one micron. Parts II and III investigate magnetic bead detection using planar Hall effect bridge (PHEB) sensors. The PHEB sensor also uses a permalloy based magnetic tacks with anisotropic magnetoresistance...... research have gone into making a customizable and multipurpose sensor platform, which could accelerate the number of practices. This thesis investigates the role of magnetic beads as candidates for use in these new biosensing devices. Magnetic beads have stable properties and can be easily coated....... By combining four resistors in a Wheatstone bridge, the sensor output is shown to be proportional to low magnetic fields. In this thesis, the PHEB sensors are used for either detection of an external homogeneous magnetic field or for detection of magnetic beads that are magnetized by the sensor self...

  17. Nanofibrous polymeric beads from aramid fibers for efficient bilirubin removal.

    Science.gov (United States)

    Peng, Zihang; Yang, Ye; Luo, Jiyue; Nie, Chuanxiong; Ma, Lang; Cheng, Chong; Zhao, Changsheng

    2016-08-16

    Polymer based hemoperfusion has been developed as an effective therapy to remove the extra bilirubin from patients. However, the currently applied materials suffer from either low removal efficiency or poor blood compatibility. In this study, we report the development of a new class of nanofibrous absorbent that exhibited high bilirubin removal efficiency and good blood compatibility. The Kevlar nanofiber was prepared by dissolving micron-sized Kevlar fiber in proper solvent, and the beads were prepared by dropping Kevlar nanofiber solutions into ethanol. Owing to the nanofiborous structure of the Kevlar nanofiber, the beads displayed porous structures and large specific areas, which would facilitate the adsorption of toxins. In the adsorption test, it was noticed that the beads possessed an adsorption capacity higher than 40 mg g(-1) towards bilirubin. In plasma mimetic solutions, the beads still showed high bilirubin removal efficiency. Furthermore, after incorporating with carbon nanotubes, the beads were found to have increased adsorption capacity for human degradation waste. Moreover, the beads showed excellent blood compatibility in terms of a low hemolysis ratio, prolonged clotting times, suppressed coagulant activation, limited platelet activation, and inhibited blood related inflammatory activation. Additionally, the beads showed good compatibility with endothelial cells. In general, the Kevlar nanofiber beads, which integrated with high adsorption capacity, good blood compatibility and low cytotoxicity, may have great potential for hemoperfusion and some other applications in biomedical fields. PMID:27481656

  18. Nanofibrous polymeric beads from aramid fibers for efficient bilirubin removal.

    Science.gov (United States)

    Peng, Zihang; Yang, Ye; Luo, Jiyue; Nie, Chuanxiong; Ma, Lang; Cheng, Chong; Zhao, Changsheng

    2016-08-16

    Polymer based hemoperfusion has been developed as an effective therapy to remove the extra bilirubin from patients. However, the currently applied materials suffer from either low removal efficiency or poor blood compatibility. In this study, we report the development of a new class of nanofibrous absorbent that exhibited high bilirubin removal efficiency and good blood compatibility. The Kevlar nanofiber was prepared by dissolving micron-sized Kevlar fiber in proper solvent, and the beads were prepared by dropping Kevlar nanofiber solutions into ethanol. Owing to the nanofiborous structure of the Kevlar nanofiber, the beads displayed porous structures and large specific areas, which would facilitate the adsorption of toxins. In the adsorption test, it was noticed that the beads possessed an adsorption capacity higher than 40 mg g(-1) towards bilirubin. In plasma mimetic solutions, the beads still showed high bilirubin removal efficiency. Furthermore, after incorporating with carbon nanotubes, the beads were found to have increased adsorption capacity for human degradation waste. Moreover, the beads showed excellent blood compatibility in terms of a low hemolysis ratio, prolonged clotting times, suppressed coagulant activation, limited platelet activation, and inhibited blood related inflammatory activation. Additionally, the beads showed good compatibility with endothelial cells. In general, the Kevlar nanofiber beads, which integrated with high adsorption capacity, good blood compatibility and low cytotoxicity, may have great potential for hemoperfusion and some other applications in biomedical fields.

  19. Microfabricated Passive Magnetic Bead separators

    DEFF Research Database (Denmark)

    Hansen, Mikkel Fougt; Lund-Olesen, Torsten; Smistrup, Kristian;

    2006-01-01

    The use and manipulation of functionalized magnetic beads for bioanalysis in lab-on-a-chip systems is receiving growing interest. We have developed microfluidic systems with integrated magnetic structures for the capture and release of magnetic beads. The systems are fabricated in silicon by deep...

  20. Protective T Cell and Antibody Immune Responses against Hepatitis C Virus Achieved Using a Biopolyester-Bead-Based Vaccine Delivery System.

    Science.gov (United States)

    Martínez-Donato, G; Piniella, B; Aguilar, D; Olivera, S; Pérez, A; Castañedo, Y; Alvarez-Lajonchere, L; Dueñas-Carrera, S; Lee, J W; Burr, N; Gonzalez-Miro, M; Rehm, B H A

    2016-04-01

    Hepatitis C virus (HCV) infection is a major worldwide problem. Chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocellular carcinoma, and liver failure. Although new, directly acting antiviral therapies are suggested to overcome the low efficacy and adverse effects observed for the current standard of treatment, an effective vaccine would be the only way to certainly eradicate HCV infection. Recently, polyhydroxybutyrate beads produced by engineeredEscherichia colishowed efficacy as a vaccine delivery system. Here, an endotoxin-freeE. colistrain (ClearColi) was engineered to produce polyhydroxybutyrate beads displaying the core antigen on their surface (Beads-Core) and their immunogenicity was evaluated in BALB/c mice. Immunization with Beads-Core induced gamma interferon (IFN-γ) secretion and a functional T cell immune response against the HCV Core protein. With the aim to target broad T and B cell determinants described for HCV, Beads-Core mixed with HCV E1, E2, and NS3 recombinant proteins was also evaluated in BALB/c mice. Remarkably, only three immunization with Beads-Core+CoE1E2NS3/Alum (a mixture of 0.1 μg Co.120, 16.7 μg E1.340, 16.7 μg E2.680, and 10 μg NS3 adjuvanted in aluminum hydroxide [Alum]) induced a potent antibody response against E1 and E2 and a broad IFN-γ secretion and T cell response against Core and all coadministered antigens. This immunological response mediated protective immunity to viremia as assessed in a viral surrogate challenge model. Overall, it was shown that engineered biopolyester beads displaying foreign antigens are immunogenic and might present a particulate delivery system suitable for vaccination against HCV. PMID:26888185

  1. Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Martin H.F. [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany); Krause, Hans-Joachim [Institute of Bio-and Nanosystems (IBN-2), Research Center Juelich (Germany); Hartmann, Markus [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany); Miethe, Peter [SENOVA GmbH, Jena (Germany); Oster, Juergen [chemagen GmbH, Baesweiler (Germany); Keusgen, Michael [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany)]. E-mail: Keusgen@staff.uni-marburg.de

    2007-04-15

    A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP[reg] polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10{sup 4}-10{sup 6} cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.

  2. Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

    Science.gov (United States)

    Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

    2015-01-01

    The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

  3. Immunoassays in monitoring biotechnological drugs.

    Science.gov (United States)

    Gygax, D; Botta, L; Ehrat, M; Graf, P; Lefèvre, G; Oroszlan, P; Pfister, C

    1996-08-01

    For the evaluation and interpretation of pharmacokinetic data reliable quantitative determinations are a requirement that can only be met by well-characterized and fully validated analytical methods. To cope with these requirements a method is being established that is based on an integrated and automated fiber-optic biospecific interaction analysis system (FOBIA) for immunoassays. Performance characteristics of this system used in monitoring of recombinant hirudin (CGP 39 393) are presented. Recombinant hirudin is a highly potent and selective inhibitor of human thrombin. Owing to its size and charge, recombinant hirudin is mainly eliminated by glomerular filtration. But only a fraction of the hirudin dose seems to be reabsorbed at the proximal tubule by luminal endocytosis and hydrolyzed by lysosomal enzymes, leaving approximately 50% of the dose to be extracted in the urine. Thus, renal clearance of recombinant hirudin in the absence of renal insufficiency appears to depend primarily on the glomerular filtration rate. During a 3-month i.v. tolerability study in dogs, some of the dogs developed antibodies against recombinant hirudin. The hirudin-antibody complex accumulated in plasma and apparent hirudin plasma concentrations were therefore much higher than expected from single-dose kinetics. Hirudin captured by antibodies showed an extended half-life and the hirudin-antibody complex is still pharmacologically active, as demonstrated by the observed increase in thrombin time. In conclusion, only appropriate analytical methods allow adequate monitoring and pharmacokinetic characterization of biotechnology drugs in biological materials. PMID:8857560

  4. Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

    Directory of Open Access Journals (Sweden)

    W.H.M. van der Poel

    2014-04-01

    Full Text Available Hepatitis E virus (HEV is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3 infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1 antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

  5. Development of an improved immunoassay for detection of sorLA in cells and biological samples

    DEFF Research Database (Denmark)

    Andersen, Olav Michael; Thakurta, Ishita Guha; West, Mark J.

    bead releases singlet oxygen which triggers a series of chemical reactions in the acceptor beads causing a sharp peak of light emission at 615 nm. A series of experiments were designed to optimize the assay by conjugation of the beads to various anti-sorLA antibodies, cross titrations of the antibodies......, spike and recovery experiments to check matrix interference, signal to noise ratio determined for the counts, and comparison of our novel immunoassay in terms of sensitivity with existing methods. Results: Our results show that as compared to traditional methods, AlphaLISA is a sensitive and rapid assay......, which can be automated suitably for determination of sorLA in large sample batches. It also shows high recovery and signal to noise ratio. Conclusions: The results support the development of an improved method for measuring sorLA quantitatively, which could further prove as an important tool...

  6. Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.

    Science.gov (United States)

    Fraga, María; Vilariño, Natalia; Louzao, M Carmen; Fernández, Diego A; Poli, Mark; Botana, Luis M

    2016-01-15

    Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 μg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes. PMID:26709295

  7. Dual-signal amplified electrochemiluminescence immunoassay for salbutamol based on quantum dots and gold nanoparticle-labeled horseradish peroxidase.

    Science.gov (United States)

    Cai, Fudong; Wang, Nan; Dong, Tiantian; Deng, Anping; Li, Jianguo

    2015-09-01

    This study describes a novel electrochemical immunosensor to amplify the electrochemiluminescence (ECL) signal for the ultrasensitive detection of salbutamol (SAL) using quantum dots (QDs) and gold nanoparticle (AuNP) conjugated horseradish peroxidase (HRP). The electrochemical detection was based on the HRP catalyzed consumption of self-produced H2O2, which has been extensively used as a co-reactant of QDs, by o-phenylenediamine (OPD). The enzymatic reaction rate is proportional to the amount of HRP bound to the electrode. In the presence of a SAL standard solution, the immobilized SAL coating antigens competed with the SAL solution for the Ab-AuNPs-HRP complexes. With an increase in the SAL concentration, the amount of immobilized HRP decreases, which leads to an increase in the ECL intensity. Under optimized conditions, the ECL intensity changes linearly with the logarithm of the SAL concentration in the range of 0.05-500 ng mL(-1) with a detection limit of 0.017 ng mL(-1) (S/N = 3). The ECL immunosensor possesses high sensitivity, satisfactory reproducibility and selectivity, and may provide a feasible route for practical application.

  8. Suspension column for recovery and separation of substances using ultrasound-assisted retention of bead sorbents.

    Science.gov (United States)

    Spivakov, Boris Ya; Shkinev, Valeriy M; Danilova, Tatiana V; Knyazkov, Nikolai N; Kurochkin, Vladimir E; Karandashev, Vasiliy K

    2012-12-15

    A novel approach to sorption recovery and separation of different substances is proposed which is based on the use of suspended bead sorbents instead of conventional packed beds of such sorbents. This makes it possible to employ small-sized beads which are trapped in a low-pressure column due to ultrasound-assisted retention, without any frits to hold the sorption material. A flow system including a separation mini-column, named herein a suspension column, has been developed and tested by the studies of solid phase extraction (SPE) of trace metals from bi-distilled water and sea water using a 150-μL column with a silica-based sorbent containing iminodiacetic groups (DIAPAK IDA) and having a grain size of 6 μm. The adsorption properties of DIAPAK IDA suspension (9.5mg) were evaluated through adsorption/desorption experiments, where the effect of solution pH and eluent on the SPE of trace metals were examined by ICP-MS or ICP-AES measurements. When sample solution was adjusted to pH 8.0 and 1 mol L(-1) nitric acid was used as eluent, very good recoveries of more than 90% were obtained for a number of elements in a single-step extraction. To demonstrate the versatility of the approach proposed and to show another advantage of ultrasonic field (acceleration of sorbate/sorbent interaction), a similar system was used for heterogeneous immunoassays of some antigens in ultrasonic field using agarose sorbents modified by corresponding antibodies. It has been shown that immunoglobulins, chlamidia, and brucellos bacteria can be quantitatively adsorbed on 15-μm sorbent (15 particles in 50 μL) and directly determined in a 50-μL mini-chamber using fluorescence detection. PMID:23182579

  9. Selective collection and detection of MCF-7 breast cancer cells using aptamer-functionalized magnetic beads and quantum dots based nano-bio-probes

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Xin [State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Zhou, Zhenxian [Nanjing Second Hospital, Nanjing 210083 (China); Yuan, Liang [State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Liu, Songqin, E-mail: liusq@seu.edu.cn [State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China)

    2013-07-25

    Graphical abstract: -- Highlights: •Aptamer–cell affinity interaction was employed for selective collection and detection of MCF-7. •CdTe QDs and aptamer were coated on SiO{sub 2} NPs for bio-labeling. •Good sensitivity was achieved due to the signal amplification of SiO{sub 2} NPs. -- Abstract: A novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer–cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO{sub 2} NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to nucleolin overexpressed cells through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were used to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using these two aptamers together as recognition elements simultaneously, compared to using any single aptamer. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO{sub 2}), the detection sensitivity was enhanced and a detection limit of 201 and 85 cells mL{sup −1} by PL and SWV method were achieved, respectively. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.

  10. An Ultrasensitive Electrochemiluminescent Immunoassay for Aflatoxin M1 in Milk, Based on Extraction by Magnetic Graphene and Detection by Antibody-Labeled CdTe Quantumn Dots-Carbon Nanotubes Nanocomposite

    Directory of Open Access Journals (Sweden)

    Ning Gan

    2013-04-01

    Full Text Available An ultrasensitive electrochemiluminescent immunoassay (ECLIA for aflatoxins M1 (ATM1 in milk using magnetic Fe3O4-graphene oxides (Fe-GO as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs as the signal tag is presented. Firstly, Fe3O4 nanoparticles were immobilized on GO to fabricate the magnetic nanocomposites, which were used as absorbent to ATM1. Secondly, aflatoxin M1 antibody (primary antibody, ATM1 Ab1, was attached to the surface of the CdTe QDs-carbon nanotubes nanocomposite to form the signal tag (ATM1 Ab1/CdTe-CNT. The above materials were characterized. The optimal experimental conditions were obtained. Thirdly, Fe-GO was employed for extraction of ATM1 in milk. Results indicated that it can adsorb ATM1 efficiently and selectively within a large extent of pH from 3.0 to 8.0. Adsorption processes reached 95% of the equilibrium within 10 min. Lastly, the ATM1 with a serial of concentrations absorbed on Fe-GO was conjugated with ATM1 Ab1/CdTe-CNT signal tag based on sandwich immunoassay. The immunocomplex can emit a strong ECL signal whose intensity depended linearly on the logarithm of ATM1 concentration from 1.0 to 1.0 × 105 pg/mL, with the detection limit (LOD of 0.3 pg/mL (S/N = 3. The method was more sensitive for ATM1 detection compared to the ELISA method. Finally, ten samples of milk were tested based on the immunoassay. The method is fast and requires very little sample preparation, which was suitable for high-throughput screening of mycotoxins in food.

  11. An ultrasensitive electrochemiluminescent immunoassay for aflatoxin M1 in milk, based on extraction by magnetic graphene and detection by antibody-labeled CdTe quantumn dots-carbon nanotubes nanocomposite.

    Science.gov (United States)

    Gan, Ning; Zhou, Jing; Xiong, Ping; Hu, Futao; Cao, Yuting; Li, Tianhua; Jiang, Qianli

    2013-05-01

    An ultrasensitive electrochemiluminescent immunoassay (ECLIA) for aflatoxins M1 (ATM1) in milk using magnetic Fe3O4-graphene oxides (Fe-GO) as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs) as the signal tag is presented. Firstly, Fe3O4 nanoparticles were immobilized on GO to fabricate the magnetic nanocomposites, which were used as absorbent to ATM1. Secondly, aflatoxin M1 antibody (primary antibody, ATM1 Ab1), was attached to the surface of the CdTe QDs-carbon nanotubes nanocomposite to form the signal tag (ATM1 Ab1/CdTe-CNT). The above materials were characterized. The optimal experimental conditions were obtained. Thirdly, Fe-GO was employed for extraction of ATM1 in milk. Results indicated that it can adsorb ATM1 efficiently and selectively within a large extent of pH from 3.0 to 8.0. Adsorption processes reached 95% of the equilibrium within 10 min. Lastly, the ATM1 with a serial of concentrations absorbed on Fe-GO was conjugated with ATM1 Ab1/CdTe-CNT signal tag based on sandwich immunoassay. The immunocomplex can emit a strong ECL signal whose intensity depended linearly on the logarithm of ATM1 concentration from 1.0 to 1.0 × 10(5) pg/mL, with the detection limit (LOD) of 0.3 pg/mL (S/N = 3). The method was more sensitive for ATM1 detection compared to the ELISA method. Finally, ten samples of milk were tested based on the immunoassay. The method is fast and requires very little sample preparation, which was suitable for high-throughput screening of mycotoxins in food. PMID:23628784

  12. Evaluation of an immunoassay for determination of plasma efavirenz concentrations in resource-limited settings

    DEFF Research Database (Denmark)

    Abdissa, Alemseged; Wiesner, Lubbe; McIlleron, Helen;

    2014-01-01

    to be implemented in resource-limited settings. This study evaluated a commercially available immunoassay for measurement of plasma efavirenz. METHODS: The immunoassay-based method was applied to measure efavirenz using a readily available Humastar 80 chemistry analyzer. We compared plasma efavirenz concentrations...

  13. Improvement of immunoassay detection system by using alternating current magnetic susceptibility

    Science.gov (United States)

    Kawabata, R.; Mizoguchi, T.; Kandori, A.

    2016-03-01

    A major goal with this research was to develop a low-cost and highly sensitive immunoassay detection system by using alternating current (AC) magnetic susceptibility. We fabricated an improved prototype of our previously developed immunoassay detection system and evaluated its performance. The prototype continuously moved sample containers by using a magnetically shielded brushless motor, which passes between two anisotropic magneto resistance (AMR) sensors. These sensors detected the magnetic signal in the direction where each sample container passed them. We used the differential signal obtained from each AMR sensor's output to improve the signal-to-noise ratio (SNR) of the magnetic signal measurement. Biotin-conjugated polymer beads with avidin-coated magnetic particles were prepared to examine the calibration curve, which represents the relation between AC magnetic susceptibility change and polymer-bead concentration. For the calibration curve measurement, we, respectively, measured the magnetic signal caused by the magnetic particles by using each AMR sensor installed near the upper or lower part in the lateral position of the passing sample containers. As a result, the SNR of the prototype was 4.5 times better than that of our previous system. Moreover, the data obtained from each AMR sensor installed near the upper part in the lateral position of the passing sample containers exhibited an accurate calibration curve that represented good correlation between AC magnetic susceptibility change and polymer-bead concentration. The conclusion drawn from these findings is that our improved immunoassay detection system will enable a low-cost and highly sensitive immunoassay.

  14. A High-Throughput SU-8Microfluidic Magnetic Bead Separator

    OpenAIRE

    Bu, Minqiang; Christensen, T. B.; Smistrup, Kristian; Wolff, Anders; Hansen, Mikkel Fougt

    2007-01-01

    We present a novel microfluidic magnetic bead separator based on SU-8 fabrication technique for high through-put applications. The experimental results show that magnetic beads can be captured at an efficiency of 91 % and 54 % at flow rates of 1 mL/min and 4 mL/min, respectively. Integration of soft magnetic elements in the chip leads to a slightly higher capturing efficiency and a more uniform distribution of captured beads over the separation chamber than the system without soft magnetic el...

  15. A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads on gold surface for the detection of atrazine

    International Nuclear Information System (INIS)

    A disposable immunomagnetic electrochemical sensor involving magnetic particles was developed and applied for the detection of atrazine. The sensor was based on a magnetic monolayer of magnetic particles coated with streptavidin, formed on a gold electrode after application of a magnetic field. The magnetic monolayer was characterized using faradaic impedance spectroscopy, cyclic voltammetry and atomic force microscopy (AFM) techniques. The magnetic monolayer formation leads to an important change in constant phase element due to a change in thickness. AFM images show that the magnetic monolayer is formed and is very dense. The atrazine interacts with biotinyl-Fab fragment K47 and the immunoreaction was characterized by impedance spectroscopy. A decrease in electron transfer resistance was observed which could be attributed to rearrangements in the magnetic monolayer. This approach leads to a sensitive detection of atrazine, acting as an antigen, with a good linear response in the range 10-600 ng/ml

  16. Microstructure-based calculations and experimental results for sound absorbing porous layers of randomly packed rigid spherical beads

    Science.gov (United States)

    Zieliński, Tomasz G.

    2014-07-01

    Acoustics of stiff porous media with open porosity can be very effectively modelled using the so-called Johnson-Champoux-Allard-Pride-Lafarge model for sound absorbing porous media with rigid frame. It is an advanced semi-phenomenological model with eight parameters, namely, the total porosity, the viscous permeability and its thermal analogue, the tortuosity, two characteristic lengths (one specific for viscous forces, the other for thermal effects), and finally, viscous and thermal tortuosities at the frequency limit of 0 Hz. Most of these parameters can be measured directly, however, to this end specific equipment is required different for various parameters. Moreover, some parameters are difficult to determine. This is one of several reasons for the so-called multiscale approach, where the parameters are computed from specific finite-element analyses based on some realistic geometric representations of the actual microstructure of porous material. Such approach is presented and validated for layers made up of loosely packed small identical rigid spheres. The sound absorption of such layers was measured experimentally in the impedance tube using the so-called two-microphone transfer function method. The layers are characterised by open porosity and semi-regular microstructure: the identical spheres are loosely packed by random pouring and mixing under the gravity force inside the impedance tubes of various size. Therefore, the regular sphere packings were used to generate Representative Volume Elements suitable for calculations at the micro-scale level. These packings involve only one, two, or four spheres so that the three-dimensional finite-element calculations specific for viscous, thermal, and tortuous effects are feasible. In the proposed geometric packings, the spheres were slightly shifted in order to achieve the correct value of total porosity which was precisely estimated for the layers tested experimentally. Finally, in this paper some results based on

  17. Using permalloy based planar hall effect sensors to capture and detect superparamagnetic beads for lab on a chip applications

    Energy Technology Data Exchange (ETDEWEB)

    Volmer, Marius, E-mail: volmerm@unitbv.ro [Transilvania University of Brasov, Electrical Engineering and Applied Physics Department. Eroilor 29, Brasov 500036 (Romania); Avram, Marioara [National Institute for Research and Development in Microtechnologies, Str. Erou Iancu Nicolae 32B, 72996 Bucharest (Romania)

    2015-05-01

    Experimental studies have been carried out on planar Hall effect (PHE) sensors used to detect magnetic nanoparticles employed as labels for biodetection applications. Disk shaped sensors, 1 mm diameter, were structured on Permalloy film, 20 nm thick. To control the sensor magnetisation state and thus the field sensitivity and linearity, a DC biasing field has been applied parallel to the driving current. Maghemite nanoparticles (10 nm) functionalised with Polyethylene glycol (PEG) 6000 were immobilised over the sensor surface using particular magnetisation state and applied magnetic fields. In order to obtain a higher response from the magnetic nanoparticles, it was used a detection setup which allows the application of magnetic fields larger than 100 Oe but avoiding saturation of the PHE signal. Based on this setup, two field scanning methods are presented in this paper. During our experiments, low magnetic moments, of about 1.87×10{sup −5} emu, have been easily detected. This value corresponds to a mass of 9.35 µg of maghemite nanoparticles functionalised with PEG 6000. The results suggest that this type of structure is feasible for building low cost micrometer sized PHE sensors to be used for high-resolution bio sensing applications. - Highlights: • Disk-shaped Permalloy planar Hall effect sensors have been obtained and tested. • Two field scanning methods have been proposed. • The magnetic nanoparticles can be trapped on the sensor surface. • High detection sensitivity has been obtained.

  18. Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium.

    Science.gov (United States)

    Belščak-Cvitanović, Ana; Komes, Draženka; Karlović, Sven; Djaković, Senka; Spoljarić, Igor; Mršić, Gordan; Ježek, Damir

    2015-01-15

    Alginate-based blends consisting of carrageenan, pectin, chitosan or psyllium husk powder were prepared for assessment of the best formulation aimed at encapsulation of caffeine. Alginate-pectin blend exhibited the lowest viscosity and provided the smallest beads. Alginate-psyllium husk blend was characterised with higher viscosity, yielding the largest bead size and the highest caffeine encapsulation efficiency (83.6%). The release kinetics of caffeine indicated that the porosity of alginate hydrogel was not reduced sufficiently to retard the diffusion of caffeine from the beads. Chitosan coated alginate beads provided the most retarded release of caffeine in water. Morphological characteristics of beads encapsulating caffeine were adversely affected by freeze drying. Bitterness intensity of caffeine-containing beads in water was the lowest for alginate-psyllium beads and chitosan coated alginate beads. Higher sodium alginate concentration (3%) for production of hydrogel beads in combination with psyllium or chitosan coating would present the most favourable carrier systems for immobilization of caffeine.

  19. An Ultrasensitive Electrochemiluminescence Immunoassay for Carbohydrate Antigen 19-9 in Serum Based on Antibody Labeled Fe3O4 Nanoparticles as Capture Probes and Graphene/CdTe Quantum Dot Bionanoconjugates as Signal Amplifiers

    Science.gov (United States)

    Gan, Ning; Zhou, Jing; Xiong, Ping; Li, Tianhua; Jiang, Shan; Cao, Yuting; Jiang, Qianli

    2013-01-01

    The CdTe quantum dots (QDs), graphene nanocomposite (CdTe-G) and dextran–Fe3O4 magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL) immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9) in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe3O4) for enriching CA 19-9 were synthesized by immobilizing the CA 19-9’s first antibody (CA 19-9 Ab1) on magnetic nanoparticles (dextran-Fe3O4). Secondly, the signal probes (CA 19-9 Ab2/CdTe-G), which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2) to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA). The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor. PMID:23685872

  20. An Ultrasensitive Electrochemiluminescence Immunoassay for Carbohydrate Antigen 19-9 in Serum Based on Antibody Labeled Fe3O4 Nanoparticles as Capture Probes and Graphene/CdTe Quantum Dot Bionanoconjugates as Signal Amplifiers

    Directory of Open Access Journals (Sweden)

    Ning Gan

    2013-05-01

    Full Text Available The CdTe quantum dots (QDs, graphene nanocomposite (CdTe-G and dextran–Fe3O4 magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9 in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe3O4 for enriching CA 19-9 were synthesized by immobilizing the CA 19-9’s first antibody (CA 19-9 Ab1 on magnetic nanoparticles (dextran-Fe3O4. Secondly, the signal probes (CA 19-9 Ab2/CdTe-G, which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2 to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA. The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor.

  1. Uptake of mercury by thiol-grafted chitosan gel beads.

    Science.gov (United States)

    Merrifield, John D; Davids, William G; MacRae, Jean D; Amirbahman, Aria

    2004-07-01

    This study describes the synthesis and characterization of thiol-grafted chitosan beads for use as mercury (Hg) adsorbents. Chitosan flakes were dissolved and formed into spherical beads using a phase inversion technique, then crosslinked to improve their porosity and chemical stability. Cysteine was grafted onto the beads in order to improve the adsorption affinity of Hg to the beads. The beads possessed an average diameter of 3.2 mm, porosity of 0.9, specific surface area of approximately 100 m2/g, average pore size of approximately 120 angstroms, and specific gravity of 2.0. Equilibrium and kinetic uptake experiments were conducted to study the uptake of Hg by the beads. The adsorption capacity was approximately 8.0 mmol-Hg/g-dry beads at pH 7, and decreased with decreasing pH. Hg adsorption kinetics was modeled as radial pore diffusion into a spherical bead with nonlinear adsorption. Use of the nonlinear Freundlich isotherm in the diffusion equation allowed modeling of the uptake kinetics with a single tortuosity factor of 1.5 +/- 0.3 as the fitting parameter for all initial Hg concentrations, chitosan loadings, and agitation rates. At agitation rates of 50 and 75 rpm, where uptake rate was reduced significantly due to the boundary layer effect, the mass transfer coefficient at the outside boundary was also used as a fitting parameter to model the kinetic data. At agitation rates higher than 150 rpm, pore diffusion was the rate-limiting step. The beads exhibited a high initial uptake rate followed by a slower uptake rate suggesting pore diffusion as the rate-determining step especially at high agitation rates. Higher uptake rates observed in this study compared to those in a previous study of chitosan-based crab shells indicate that dissolution and gel formation increase the porosity and pore accessibility of chitosan.

  2. Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer

    OpenAIRE

    Maine, G T; Stricker, R; Schuler, M.; Spesard, J.; Brojanac, S.; Iriarte, B.; Herwig, K.; Gramins, T.; Combs, B.; Wise, J.; Simmons, H.; Gram, T.; Lonze, J.; Ruzicki, D.; Byrne, B

    2000-01-01

    A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical condit...

  3. Archaeological study of ostrich eggshell beads collected from SDG site

    Institute of Scientific and Technical Information of China (English)

    WANG ChunXue; ZHANG Yue; GAO Xing; ZHANG XiaoLing; WANG HuiMin

    2009-01-01

    Ostrich eggshell beads and fragments collected from SDG site reflect primordial art and a kind of symbolic behavior of modern humans.Based on stratigraphic data and OSL dating,these ostrich eggshell beads are probably in Early Holocene (<10 ka BP).Two different prehistoric manufacturing pathways are usually used in the manufacture of ostrich eggshell beads in Upper Paleolithic.According to statistic analysis of the characteristics of ostrich eggshell beads,Pathway 1 is identified from these collections.In pathway 1,blanks are drilled prior to being trimmed to rough discs.They exhibit great potential for the study of the origin of primordial art and the development of ancient cultures and provide important data for studying behavioral options adopted by hominids in SDG area.In addition,they bear important implications for the origin of modern humans in East Asia.

  4. Optimization of GMAW process of AA 6063-T5 aluminum alloy butt joints based on the response surface methodology and on the bead geometry

    International Nuclear Information System (INIS)

    The geometry of the weld beads is characterized by the overhead, the width and the penetration. These values are indices of the behavior of the welded joint and therefore, they can be considered as factors that control the process. This work is performed to optimize the GMAW process of the aluminum alloy AA 6063-T5 by means of the response surface methodology (RSM). The variables herein considered are the arc voltage, the welding speed, the wire feed speed and the separation between surfaces in butt joints. The response functions that are herein studied are the overhead, the width, the penetration and the angle of the bead. The obtained results by RSM show high grade of agreement with the experimental values. The procedure is experimentally validated by welding for the theoretically obtained optimized technological conditions and a wide agreement between theoretical and experimental values is found. (Author) 16 refs.

  5. A Novel Inherently Radiopaque Bead for Transarterial Embolization to Treat Liver Cancer - A Pre-clinical Study

    Science.gov (United States)

    Duran, Rafael; Sharma, Karun; Dreher, Matthew R.; Ashrafi, Koorosh; Mirpour, Sahar; Lin, MingDe; Schernthaner, Ruediger E.; Schlachter, Todd R.; Tacher, Vania; Lewis, Andrew L.; Willis, Sean; den Hartog, Mark; Radaelli, Alessandro; Negussie, Ayele H.; Wood, Bradford J.; Geschwind, Jean-François H.

    2016-01-01

    Purpose: Embolotherapy using microshperes is currently performed with soluble contrast to aid in visualization. However, administered payload visibility dimishes soon after delivery due to soluble contrast washout, leaving the radiolucent bead's location unknown. The objective of our study was to characterize inherently radiopaque beads (RO Beads) in terms of physicomechanical properties, deliverability and imaging visibility in a rabbit VX2 liver tumor model. Materials and Methods: RO Beads, which are based on LC Bead® platform, were compared to LC Bead. Bead size (light microscopy), equilibrium water content (EWC), density, X-ray attenuation and iodine distribution (micro-CT), suspension (settling times), deliverability and in vitro penetration were investigated. Fifteen rabbits were embolized with either LC Bead or RO Beads + soluble contrast (iodixanol-320), or RO Beads+dextrose. Appearance was evaluated with fluoroscopy, X-ray single shot, cone-beam CT (CBCT). Results: Both bead types had a similar size distribution. RO Beads had lower EWC (60-72%) and higher density (1.21-1.36 g/cc) with a homogeneous iodine distribution within the bead's interior. RO Beads suspension time was shorter than LC Bead, with durable suspension (>5 min) in 100% iodixanol. RO Beads ≤300 µm were deliverable through a 2.3-Fr microcatheter. Both bead types showed similar penetration. Soluble contrast could identify target and non-target embolization on fluoroscopy during administration. However, the imaging appearance vanished quickly for LC Bead as contrast washed-out. RO Beads+contrast significantly increased visibility on X-ray single shot compared to LC Bead+contrast in target and non-target arteries (P=0.0043). Similarly, RO beads demonstrated better visibility on CBCT in target arteries (P=0.0238) with a trend in non-target arteries (P=0.0519). RO Beads+dextrose were not sufficiently visible to monitor embolization using fluoroscopy. Conclusion: RO Beads provide better

  6. Fabrication of multiwalled carbon nanotubes-magnetite nanocomposite as an effective ultra-sensing platform for the early screening of nasopharyngeal carcinoma by luminescence immunoassay.

    Science.gov (United States)

    Liu, Chia-Ching; Sadhasivam, S; Savitha, S; Lin, Feng-Huei

    2014-05-01

    The hybrid nanocomposite that consists of multiwalled carbon nanotubes (MWCNTs) and magnetite (Fe₃O4) was fabricated by chemical co-precipitation method. Briefly, CNTs were oxidized with acids to form carboxylic group and then co-precipitated with Fe₃O4 to form CNT-Fe₃O4 nanocomposites. The nanocomposites were characterized by SEM, HRTEM, XRD, FTIR X-ray photoelectron spectrometry (XPS) and SQUID. The XRD results indicated the high crystallinity of Fe₃O₄ nanoparticles with spinel structure and the transmission electron microscope images depicted the intercalated iron oxide magnetic particles on the surface of CNTs. The MWCNTs-Fe₃O₄ was applied as a sensing interface to perform luminescence enzyme immunoassays. Firstly, EBNA-1 antigen was immobilized onto the carboxyl group functionalized MWCNTs-Fe₃O₄, followed by binding with anti-EBNA-1 IgA antibodies. The diluted secondary antibodies (anti-human IgA-HRP) were then added to the CNTs/Fe₃O₄-PEG-EBNA-1-anti-EBV IgA ab complex and act as a catalyst to produce a visible light upon reaction with the substrate luminol. The formed RLU is proportional to the amount of IgA anti-EBV antiobodies on the MWCNTs. The detection limit of proposed CNTs/Fe₃O₄ based luminescence enzyme immunoassay was in the order of 0.00128 EU/mL (1:100,000 fold dilution) for the detection of anti-EBV IgA antibodies, whereas the commercial ELISA and magnetic beads' assay was accounted for up to the dilution fold of 1000 (i.e., 0.128 EU/mL). The initial findings showed that CNTs/Fe₃O₄ nanocomposites have a great potential in luminescent enzyme immunoassays and could be used as a sensing platform for the early screening of nasopharyngeal carcinoma. PMID:24720983

  7. Glass-bead peen plating

    Science.gov (United States)

    Graves, J. R.

    1974-01-01

    Peen plating of aluminum, copper, and nickel powders was investigated. Only aluminum was plated successfully within the range of peen plating conditions studied. Optimum plating conditions for aluminum were found to be: (1) bead/powder mixture containing 25 to 35% powder by weight, (2) peening intensity of 0.007A as measured by Almen strip, and (3) glass impact bead diameter of at least 297 microns (0.0117 inches) for depositing-100 mesh aluminum powder. No extensive cleaning or substrate preparation is required beyond removing loose dirt or heavy oil.

  8. Hyaluronidase treatment of synovial fluid to improve assay precision for biomarker research using multiplex immunoassay platforms.

    Science.gov (United States)

    Jayadev, Chethan; Rout, Raj; Price, Andrew; Hulley, Philippa; Mahoney, David

    2012-12-14

    Synovial fluid (SF) is a difficult biological matrix to analyse due to its complex non-Newtonian nature. This can result in poor assay repeatability and potentially inefficient use of precious samples. This study assessed the impact of SF treatment by hyaluronidase and/or dilution on intra-assay precision using the Luminex and Meso Scale Discovery (MSD) multiplex platforms. SF was obtained from patients with knee osteoarthritis at the time of joint replacement surgery. Aliquots derived from the same sample were left untreated (neat), 2-fold diluted, 4-fold diluted or treated with 2mg/ml testicular hyaluronidase (with 2-fold dilution). Preparation methods were compared in a polysterene-bead Luminex 10-plex (N=16), magnetic-bead Luminex singleplex (N=7) and MSD 4-plex (N=7). Each method was assessed for coefficient of variation (CV) of replicate measurements, number of bead events (for Luminex assays) and dilution-adjusted analyte concentration. Percentage recovery was calculated for dilutions and HAse treatment. Hyaluronidase treatment significantly increased the number of wells with satisfactory bead events/region (95%) compared to neat (48%, pmagnetic-bead Luminex assay achieved ≥50 bead events irrespective of treatment method. Hyaluronidase treatment resulted in lower intra-assay CVs for detectable ligands (group average CVmagnetic-bead Luminex assays. In addition, measured sample concentrations were higher and recovery was poor (elevated) after hyaluronidase treatment. In the MSD 4-plex, within-group comparison of the intra-assay CV or concentration was not conclusively influenced by SF preparation. However, only hyaluronidase treatment resulted in CV<25% for all samples for TNF-α. There was no effect on analyte concentrations or recovery. Hyaluronidase treatment can improve intra-assay precision and assay signal of SF analysis by multiplex immunoassays and should be recommended for SF biomarker research, particularly using the Luminex platform. PMID:22955210

  9. Fluorescence Polarization Immunoassay of Mycotoxins: A Review

    OpenAIRE

    Maragos, Chris

    2009-01-01

    Immunoassays are routinely used in the screening of commodities and foods for fungal toxins (mycotoxins). Demands to increase speed and lower costs have lead to continued improvements in such assays. Because many reported mycotoxins are low molecular weight (below 1 kDa), immunoassays for their detection have generally been constructed in competitive heterogeneous formats. An exception is fluorescence polarization immunoassay (FPIA), a homogeneous format that does not require the separation o...

  10. Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

    Science.gov (United States)

    Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

    2012-10-16

    The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

  11. A Highly Sensitive Porous Silicon (P-Si-Based Human Kallikrein 2 (hK2 Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Sang Wook Lee

    2015-05-01

    Full Text Available Levels of total human kallikrein 2 (hK2, a protein involved the pathology of prostate cancer (PCa, could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7 was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS at three different antibody densities (75, 100 and 145 µg/mL. The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL. The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL.

  12. A sensitive magnetic nanoparticle-based immunoassay of phosphorylated acetylcholinesterase using protein cage templated lead phosphate for signal amplification with graphite furnace atomic absorption spectrometry detection.

    Science.gov (United States)

    Liang, Pei; Kang, Caiyan; Yang, Enjian; Ge, Xiaoxiao; Du, Dan; Lin, Yuehe

    2016-04-01

    We developed a new magnetic nanoparticle sandwich-like immunoassay using protein cage nanoparticles (PCN) for signal amplification together with graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of an organophosphorylated acetylcholinesterase adduct (OP-AChE), the biomarker of exposure to organophosphate pesticides (OPs) and nerve agents. OP-AChE adducts were firstly captured by titanium dioxide coated magnetic nanoparticles (TiO2-MNPs) from the sample matrixes through metal chelation with phospho-moieties, and then selectively recognized by anti-AChE antibody labeled on PCN which was packed with lead phosphate in its cavity (PCN-anti-AChE). The sandwich-like immunoreaction was performed among TiO2-MNPs, OP-AChE and PCN-anti-AChE to form a TiO2-MNP/OP-AChE/PCN-anti-AChE immunocomplex. The complex could be easily isolated from the sample solution with the help of magnet, and the released lead ions from PCN were detected by GFAAS for the quantification of OP-AChE. Greatly enhanced sensitivity was achieved because PCN increased the amount of metal ions in the cavity of each apoferritin. The proposed immunoassay yielded a linear response over a broad range of OP-AChE concentrations from 0.01 nM to 2 nM, with a detection limit of 2 pM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This new method showed an acceptable stability and reproducibility and was validated with OP-AChE spiked human plasma.

  13. Sensitive magnetic nanoparticle-based immunoassay of phosphorylated acetylcholinesterase using protein cage templated lead phosphate for signal amplification with graphite furnace atomic absorption spectrometry detection

    Science.gov (United States)

    Liang, Pei; Kang, Caiyan; Yang, Enjian; Ge, Xiaoxiao; Du, Dan; Lin, Yuehe

    2016-01-01

    We developed a new magnetic nanoparticles sandwich-like immunoassay using protein cage nanoparticles (PCN) for signal amplification together with graphite furnace atomic absorption spectrometry (GFAAS) for quantification of organophosphorylated acetylcholinesterase adduct (OP-AChE), the biomarker of exposure to organophosphate pesticides (OPs) and nerve agents. OP-AChE adducts were firstly captured by titanium dioxide coated magnetic nanoparticles (TiO2-MNPs) from the sample matrixes through metal chelation with phospho-moieties, and then selectively recognized by anti-AChE antibody labeled on PCN which was packed with lead phosphate in its cavity (PCN-anti-AChE). The sandwich-like immunoreaction was performed among TiO2-MNPs, OP-AChE and PCN-anti-AChE to form TiO2-MNPs/OP-AChE/PCN-anti-AChE immunocomplex. The complex could be easily isolated from the sample solution with the help of magnet, and the released lead ions from PCN were detected by GFAAS for the quantification of OP-AChE. Greatly enhanced sensitivity was achieved because PCN increased the amount of metal ions in the cavity of each apoferritin. The proposed immunoassay yielded a linear response over a broad OP-AChE concentrations from 0.01 nM to 2 nM, with a detection limit of 2 pM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This new method showed an acceptable stability and reproducibility and was validated with OP-AChE spiked human plasma. PMID:26953358

  14. What Can a Urine Drug Screening Immunoassay Really Tell Us?

    Science.gov (United States)

    Nelson, Zachary J; Stellpflug, Samuel J; Engebretsen, Kristin M

    2016-10-01

    Urine drug screening has become standard of care in many medical practice settings to assess compliance, detect misuse, and/or to provide basis for medical or legal action. The antibody-based enzymatic immunoassays used for qualitative analysis of urine have significant drawbacks that clinicians are often not aware of. Recent literature suggests that there is a lack of understanding of the shortcomings of these assays by clinicians who are ordering and/or interpreting them. This article addresses the state of each of the individual immunoassays that are most commonly used today in order to help the reader become proficient in the interpretation and application of the results. Some literature already exists regarding sources of "false positives" and "false negatives," but none seem to present the material with the practicing clinician in mind. This review aims to avoid overwhelming the reader with structures and analytical chemistry. The reader will be presented relevant clinical knowledge that will facilitate appropriate interpretation of immunoassays regardless of practice settings. Using this review as a learning tool and a reference, clinicians will be able to interpret the results of commonly used immunoassays in an evidence-based, informed manner and minimize the negative impact that misinterpretation has on patient care.

  15. Glass bead cultivation of fungi

    DEFF Research Database (Denmark)

    Droce, Aida; Sørensen, Jens Laurids; Giese, H.;

    2013-01-01

    Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum...

  16. Preparation and characterization of chitosan/cashew gum beads loaded with Lippia sidoides essential oil

    Energy Technology Data Exchange (ETDEWEB)

    Paula, Haroldo C.B., E-mail: hpaula@ufc.br [Department of Analytical and Physical Chemistry, Federal University of Ceara, UFC, Fortaleza-CE (Brazil); Sombra, Fernanda Matoso; Cavalcante, Rafaela de Freitas; Abreu, Flavia O.M.S. [Department of Analytical and Physical Chemistry, Federal University of Ceara, UFC, Fortaleza-CE (Brazil); Paula, Regina C.M. de [Department of Organic and Inorganic Chemistry, Federal University of Ceara, UFC, Fortaleza-CE (Brazil)

    2011-03-12

    Beads based on chitosan (CH) and cashew gum (CG), were prepared and loaded with an essential oil with larvicide activity (Lippia sidoides - Ls). CH and CH-CG beads were characterized by scanning electron microscopy (SEM), infrared and UV-VIS spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), as well as, regarding their larvicide loading, swelling, in vitro and in vivo release kinetics. The oil encapsulation was evidenced by FTIR analysis and LS loading ranges from 2.4% to 4.4%. CH beads duly showed swelling degree (Q) values from 4.0 to 6.7, reaching equilibrium after 30 min, whereas crosslinked CH-CG beads showed lower swelling values, from 0.4 to 3.8, exhibiting a longer equilibrium time. Liquid transport parameters have revealed diffusion coefficient for CH-CG beads, as low as 2 x 10{sup -15} m{sup 2}/s. TGA and DSC revealed that CH:CG crosslinked beads are more thermally stable than CH beads. In vitro release follows a non-Fickian diffusion profile for both bead types, however, and a prolonged release being achieved only after beads crosslinking. In vivo release showed that both CH and CH-CG presented a prolonged larvicide effect. These aforesaid results, indicate that CH-CG beads loaded with LS are efficient for A. aegypti larval control.

  17. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL−1 with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO2 particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors

  18. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  19. Selective nanomanipulation of fluorescent polystyrene nano-beads and single quantum dots at gold nanostructures based on the AC-dielectrophoretic force

    Science.gov (United States)

    Kim, Jinsik; Hwang, Kyo Seon; Lee, Sangyoup; Park, Jung Ho; Shin, Hyun-Joon

    2015-11-01

    We introduced the selective manipulation of polystyrene (PS) nano-beads and single quantum dots (QDs) at a gold nanostructure by using the AC-dielectrophoretic (DEP) force. Manipulation in three degrees of freedom (end-facet, side, and position-selective manipulation) was accomplished in gold nanostructures between microelectrodes. A 10 μm gap between the microelectrodes, which has a 100 nm-wide nanowire and 200 nm-wide vortex nanostructures at the inside of the gap, was fabricated, and nanostructures were not connected with the electrodes. We also performed theoretical calculations to verify the selective manipulation through the floating AC-DEP force. A sufficiently high gradient of the square of the electric field (∇|E|2, ~1019 V2 m-3) was accomplished and controlled for achieving a strong attaching force of nanoparticles using the gap between microelectrodes and nanostructures as well as the rotation of structures. Fluorescent PS nano-beads and QDs were attached at the designed end facet, side, and position of nanostructures with high selectivity. A single QD attachment was also realized at gold nanostructures, and the attached QDs were verified as single using optical ``blinking'' measurements.We introduced the selective manipulation of polystyrene (PS) nano-beads and single quantum dots (QDs) at a gold nanostructure by using the AC-dielectrophoretic (DEP) force. Manipulation in three degrees of freedom (end-facet, side, and position-selective manipulation) was accomplished in gold nanostructures between microelectrodes. A 10 μm gap between the microelectrodes, which has a 100 nm-wide nanowire and 200 nm-wide vortex nanostructures at the inside of the gap, was fabricated, and nanostructures were not connected with the electrodes. We also performed theoretical calculations to verify the selective manipulation through the floating AC-DEP force. A sufficiently high gradient of the square of the electric field (∇|E|2, ~1019 V2 m-3) was accomplished and

  20. On-Chip Magnetic Bead Manipulation and Detection Using a Magnetoresistive Sensor-Based Micro-Chip: Design Considerations and Experimental Characterization

    Science.gov (United States)

    Gooneratne, Chinthaka P.; Kodzius, Rimantas; Li, Fuquan; Foulds, Ian G.; Kosel, Jürgen

    2016-01-01

    The remarkable advantages micro-chip platforms offer over cumbersome, time-consuming equipment currently in use for bio-analysis are well documented. In this research, a micro-chip that includes a unique magnetic actuator (MA) for the manipulation of superparamagnetic beads (SPBs), and a magnetoresistive sensor for the detection of SPBs is presented. A design methodology, which takes into account the magnetic volume of SPBs, diffusion and heat transfer phenomena, is presented with the aid of numerical analysis to optimize the parameters of the MA. The MA was employed as a magnetic flux generator and experimental analysis with commercially available COMPEL™ and Dynabeads® demonstrated the ability of the MA to precisely transport a small number of SPBs over long distances and concentrate SPBs to a sensing site for detection. Moreover, the velocities of COMPEL™ and Dynabead® SPBs were correlated to their magnetic volumes and were in good agreement with numerical model predictions. We found that 2.8 μm Dynabeads® travel faster, and can be attracted to a magnetic source from a longer distance, than 6.2 μm COMPEL™ beads at magnetic flux magnitudes of less than 10 mT. The micro-chip system could easily be integrated with electronic circuitry and microfluidic functions, paving the way for an on-chip biomolecule quantification device. PMID:27571084

  1. On-Chip Magnetic Bead Manipulation and Detection Using a Magnetoresistive Sensor-Based Micro-Chip: Design Considerations and Experimental Characterization.

    Science.gov (United States)

    Gooneratne, Chinthaka P; Kodzius, Rimantas; Li, Fuquan; Foulds, Ian G; Kosel, Jürgen

    2016-08-26

    The remarkable advantages micro-chip platforms offer over cumbersome, time-consuming equipment currently in use for bio-analysis are well documented. In this research, a micro-chip that includes a unique magnetic actuator (MA) for the manipulation of superparamagnetic beads (SPBs), and a magnetoresistive sensor for the detection of SPBs is presented. A design methodology, which takes into account the magnetic volume of SPBs, diffusion and heat transfer phenomena, is presented with the aid of numerical analysis to optimize the parameters of the MA. The MA was employed as a magnetic flux generator and experimental analysis with commercially available COMPEL™ and Dynabeads(®) demonstrated the ability of the MA to precisely transport a small number of SPBs over long distances and concentrate SPBs to a sensing site for detection. Moreover, the velocities of COMPEL™ and Dynabead(®) SPBs were correlated to their magnetic volumes and were in good agreement with numerical model predictions. We found that 2.8 μm Dynabeads(®) travel faster, and can be attracted to a magnetic source from a longer distance, than 6.2 μm COMPEL™ beads at magnetic flux magnitudes of less than 10 mT. The micro-chip system could easily be integrated with electronic circuitry and microfluidic functions, paving the way for an on-chip biomolecule quantification device.

  2. Survey of immunoassay techniques for biological analysis

    International Nuclear Information System (INIS)

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs

  3. Optical encoding of microbeads based on silica particle encapsulated quantum dots and its applications

    International Nuclear Information System (INIS)

    A novel method concerning the coding technology of polystyrene beads with Si encapsulated quantum dot (QD) particles (Si - QDs particles) is studied in this paper. In the reverse microemulsion system containing tetraethoxysilane (TEOS), water-soluble QDs (emission peak at 600 nm) were enveloped within the silica shell, forming Si - QDs particles. The Si - QDs particles were characterized by TEM, showing good uniform size, with an average diameter of about 167.0 nm. In comparison with the pure water-soluble QDs, the encapsulation of water-soluble QDs in the silica shell led to an enhancement in anti-photobleaching by providing inert barriers for the QDs. Images presented by SEM and confocal laser scanning microscopy demonstrated that the Si - QDs particles were equably coated on the surface of carboxyl functionalized polystyrene (PS) beads. Then, with the assistance of ethyl-3-(dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), human IgG could be successfully crosslinked to Si - QDs particle coated PS-COOH beads. Furthermore, the Si - QDs coated PS-COOH beads with human IgG were examined in immunoassay experiments, and the results indicated that these beads could be applied in the specific recognition of goat-anti-human IgG in solution. This investigation is expected to provide a new route to bead coding in the field of suspension microarrays, based on the use of QDs

  4. Fluorescent detection of C-reactive protein using polyamide beads

    Science.gov (United States)

    Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

    2016-03-01

    Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

  5. Developement of Spherical Polyurethane Beads

    Institute of Scientific and Technical Information of China (English)

    K. Maeda; H. Ohmori; H. Gyotoku

    2005-01-01

    @@ 1Results and Discussion We established a new method to produce the spherical polyurethane beads which have narrower distribution of particle size. This narrower distribution was achieved by the polyurethane prepolymer which contains ketimine as a blocked chain-extending agent. Firstly, the prepolymer is dispersed into the aqueous solution containing surfactant. Secondaly, water comes into the inside of prepolymer as oil phase. Thirdly, ketimine is hydrolyzed to amine, and amine reacts with prepolymer immediately to be polyurethane.Our spherical polyurethane beads are very suitable for automotive interior parts especially for instrument panel cover sheet producing under the slush molding method, because of good process ability, excellent durability to the sunlight and mechanical properties at low temperature. See Fig. 1 ,Fig. 2 and Fig. 3 (Page 820).

  6. On-chip measurements of Brownian relaxation of magnetic beads with diameters from 10 nm to 250 nm

    DEFF Research Database (Denmark)

    Østerberg, Frederik Westergaard; Rizzi, Giovanni; Hansen, Mikkel Fougt

    2013-01-01

    We demonstrate the use of planar Hall effect magnetoresistive sensors for AC susceptibility measurements of magnetic beads with frequencies ranging from DC to 1 MHz. This wide frequency range allows for measuring Brownian relaxation of magnetic beads with diameters ranging from 10 nm to 250 nm...... to sedimentation, magnetic trapping, and signal per bead. Among the investigated beads, we conclude that the beads with a nominal diameter of 80 nm are best suited for future on-chip volume-based biosensing experiments using planar Hall effect sensors....

  7. Controlled antiseptic release by alginate polymer films and beads.

    Science.gov (United States)

    Liakos, Ioannis; Rizzello, Loris; Bayer, Ilker S; Pompa, Pier Paolo; Cingolani, Roberto; Athanassiou, Athanassia

    2013-01-30

    Biodegradable polymeric materials based on blending aqueous dispersions of natural polymer sodium alginate (NaAlg) and povidone iodine (PVPI) complex, which allow controlled antiseptic release, are presented. The developed materials are either free standing NaAlg films or Ca(2+)-cross-linked alginate beads, which properly combined with PVPI demonstrate antibacterial and antifungal activity, suitable for therapeutic applications, such as wound dressing. Glycerol was used as the plasticizing agent. Film morphology was studied by optical and atomic force microscopy. It was found that PVPI complex forms well dispersed circular micro-domains within the NaAlg matrix. The beads were fabricated by drop-wise immersion of NaAlg/PVPI/glycerol solutions into aqueous calcium chloride solutions to form calcium alginate beads encapsulating PVPI solution (CaAlg/PVPI). Controlled release of PVPI was possible when the composite films and beads were brought into direct contact with water or with moist media. Bactericidal and fungicidal properties of the materials were tested against Escherichia coli bacteria and Candida albicans fungi. The results indicated very efficient antibacterial and antifungal activity within 48 h. Controlled release of PVPI into open wounds is highly desired in clinical applications to avoid toxic doses of iodine absorption by the wound. A wide variety of applications are envisioned such as external and internal wound dressings with controlled antiseptic release, hygienic and protective packaging films for medical devices, and polymer beads as water disinfectants.

  8. Ultra-sensitive immunoassay biosensors using hybrid plasmonic-biosilica nanostructured materials

    OpenAIRE

    Yang, Jing; Zhen, Le; Ren, Fanghui; Campbell, Jeremy; Rorrer, Gregory L.; Wang, Alan X.

    2014-01-01

    We experimentally demonstrate an ultra-sensitive immunoassay biosensor using diatom biosilica with self-assembled plasmonic nanoparticles. As the nature-created photonic crystal structures, diatoms have been adopted to enhance surface plasmon resonances of metal nanoparticles on the surfaces of diatom frustules and to increase the sensitivity of surface-enhanced Raman scattering (SERS). In this study, a sandwich SERS immunoassay is developed based on the hybrid plasmonic-biosilica nano-struct...

  9. A competitive immunoassay for ultrasensitive detection of Hg(2+) in water, human serum and urine samples using immunochromatographic test based on surface-enhanced Raman scattering.

    Science.gov (United States)

    She, Pei; Chu, Yanxin; Liu, Chunwei; Guo, Xun; Zhao, Kang; Li, Jianguo; Du, Haijing; Zhang, Xiang; Wang, Hong; Deng, Anping

    2016-02-01

    An immunochromatographic test (ICT) strip was developed for ultrasensitive competitive immunoassay of Hg(2+). This strategy was achieved by combining the easy-operation and rapidity of ICT with the high sensitivity of surface-enhanced Raman scattering (SERS). Monoclonal antibody (mAb) against Hg(2+) and Raman active substance 4-mercaptobenzoic acid (MBA) dual labelled gold nanoparticles (GNPs) were prepared as an immunoprobe. The Raman scattering intensity of MBA on the test line of the ICT strip was measured for quantitative determination of Hg(2+). The ICT was able to directly detect Hg(2+) without complexing due to the specific recognition of the mAb with Hg(2+). The IC50 and limit of detection (LOD) of the assay for Hg(2+) detection were 0.12 ng mL(-1) and 0.45 pg mL(-1), respectively. There was no cross-reactivity (CR) of the assay with other nineteen ions and the ICT strips could be kept for 5 weeks without loss of activity. The recoveries of the assay for water, human serum and urine samples spiked with Hg(2+) were in range of 88.3-107.3% with the relative standard deviations (RSD) of 1.5-9.5% (n = 3). The proposed ICT was used for the detection of Hg(2+) in urine samples collected from Occupational Disease Hospital and the results were confirmed by cold-vapor atomic fluorescence spectroscopy (CV-AFS). The assay exhibited high sensitivity, selectivity, stability, precision and accuracy, demonstrating a promising method for the detection of trace amount of Hg(2+) in environmental water samples and biological serum and urine samples.

  10. Ex vivo mucoadhesion of different zinc-pectinate hydrogel beads.

    Science.gov (United States)

    Hagesaether, Ellen; Bye, Ragnar; Sande, S Arne

    2008-01-22

    The objective of this study was to investigate the mucoadhesive properties of pre-swelled hydrogel beads made of six types of pectin from three manufacturers. The types of pectin differed mainly in the degree of methoxylation and degree of amidation. Zinc ions were used as cross-linking agent. The mucoadhesive properties were tested on an inverted fresh porcine small intestine attached to a rotating cylinder. Beads made of pectin with a high degree of methoxylation (70%) showed superior mucoadhesive results compared to the other formulations, which could be correlated to the lower amount of zinc in this formulation, subsequently leading to a lower amount of cross-linking and higher mobility of the polymer chains of these beads. This study therefore also indicated the importance of doing mucoadhesive measurements on relevant formulations, and not basing the understanding solely on investigating polymer solutions. Samples from different manufacturers produced the same results.

  11. On-chip measurement of the Brownian relaxation frequency of magnetic beads using magnetic tunneling junctions

    DEFF Research Database (Denmark)

    Donolato, M.; Sogne, E.; Dalslet, Bjarke Thomas;

    2011-01-01

    We demonstrate the detection of the Brownian relaxation frequency of 250 nm diameter magnetic beads using a lab-on-chip platform based on current lines for exciting the beads with alternating magnetic fields and highly sensitive magnetic tunnel junction (MTJ) sensors with a superparamagnetic free...

  12. Antiphospholipase A2 Receptor Autoantibodies: A Comparison of Three Different Immunoassays for the Diagnosis of Idiopathic Membranous Nephropathy

    Directory of Open Access Journals (Sweden)

    Astrid Behnert

    2014-01-01

    Full Text Available Background. The recent identification of circulating autoantibodies directed towards the M-type phospholipase A2 receptor (PLA2R has been a major advancement in the serological diagnosis of idiopathic membranous nephropathy (IMN, a common cause of nephrotic syndrome in adults. The goal of this study was to compare the performance characteristics of two commercial assays as well as the first addressable laser bead immunoassay (ALBIA developed for the detection of anti-PLA2R antibodies. Methods. Serum samples of 157 IMN patients and 142 controls were studied. Samples were tested by a cell based immunofluorescence assay (CBA-IFA, Euroimmun, Germany, by ELISA (Euroimmun, and by a novel ALBIA employing an in vivo expressed recombinant human PLA2R. Results. Overall, the three assays showed significant qualitative and quantitative correlation. As revealed by receiver operating characteristic analysis, the ALBIA correlated better with the CBA-IFA than the ELISA (P=0.0003. The clinical sensitivities/specificities for IMN were 60.0% (51.0–68.5%/98.6% (95.0–99.8% and 56.2% (47.2–64.8%/100.0% (97.4–100.0% for ALBIA and CBA-IFA, respectively. Conclusion. The ALBIA represents a promising assay for the detection of anti-PLA2R antibodies showing similar performance to the CBA-IFA and the advantage of ease of use and suitability for high throughput, rapid turnaround times, and multiplexing.

  13. A pilot-scale study on PVA gel beads based integrated fixed film activated sludge (IFAS) plant for municipal wastewater treatment.

    Science.gov (United States)

    Kumar Singh, Nitin; Singh, Jasdeep; Bhatia, Aakansha; Kazmi, A A

    2016-01-01

    In the present study, a pilot-scale reactor incorporating polyvinyl alcohol gel beads as biomass carrier and operating in biological activated sludge mode (a combination of moving bed biofilm reactor (MBBR) and activated sludge) was investigated for the treatment of actual municipal wastewater. The results, during a monitoring period of 4 months, showed effective removal of chemical oxygen demand (COD), biological oxygen demand (BOD) and NH3-N at optimum conditions with 91%, ∼92% and ∼90% removal efficiencies, respectively. Sludge volume index (SVI) values of activated sludge varied in the range of 25-72 mL/g, indicating appreciable settling characteristics. Furthermore, soluble COD and BOD in the effluent of the pilot plant were reduced to levels well below discharge limits of the Punjab Pollution Control Board, India. A culture dependent method was used to enrich and isolate abundant heterotrophic bacteria in activated sludge. In addition to this, 16S rRNA genes analysis was performed to identify diverse dominant bacterial species in suspended and attached biomass. Results revealed that Escherichia coli, Pseudomonas sp. and Nitrosomonas communis played a significant role in biomass carrier, while Acinetobactor sp. were dominant in activated sludge of the pilot plant. Identification of ciliated protozoa populations rendered six species of ciliates in the plant, among which Vorticella was the most dominant. PMID:26744941

  14. An adaptive algorithm for tracking 3D bead displacements: application in biological experiments

    International Nuclear Information System (INIS)

    This paper presents a feature-vector-based relaxation method (FVRM) to track bead displacements within a three-dimensional (3D) volume. The FVRM merges the feature vector method, a technique used in tracking bead displacements in biological gels, with the relaxation method, an algorithm employed successfully in tracking bead pairs in fluids. More specifically, the FVRM evaluates the probability of a bead pairing event based on the quasi-rigidity condition between the feature vectors of a bead and its candidate positions within a searching domain. Computational efficiency is improved via the introduction of an adaptive searching domain size and mismatches are reduced via a two-directional matching strategy. The algorithm is validated using simulated 3D bead displacements caused by a force dipole within a linear elastic gel. Results demonstrate a consistently high recovery ratio (above 98%) and low mismatch ratio (below 0.1%) for tracking parameter (mean bead distance/maximum bead displacement) greater than 0.73. (paper)

  15. Single Step Nanoplasmonic Immunoassay for the Measurement of Protein Biomarkers

    Directory of Open Access Journals (Sweden)

    Shradha Prabhulkar

    2013-02-01

    Full Text Available A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1. The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps, and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

  16. Beads, beaded-fibres and fibres: Tailoring the morphology of poly(caprolactone) using pressurised gyration.

    Science.gov (United States)

    Hong, Xianze; Edirisinghe, Mohan; Mahalingam, Suntharavathanan

    2016-12-01

    This work focuses on forming bead on string poly(caprolactone) (PCL) by using gyration under pressure. The fibre morphology of bead on string is an interesting feature that falls between bead-free fibres and droplets, and it could be effectively controlled by the rheological properties of spinning dopes and the major processing parameters of the pressurised gyration system which are working pressure and rotating speed. Bead products were not always spherical in shape and tended to be more elliptical, therefore both their width and length were measured. The average bead width and length produced spanned a range 145-660μm and 140-1060μm, respectively. The average distance between two adjacent beads (i.e. inter-bead distance) and the bead size (width and length) are shown to be a function of processing parameters and polymer concentration. An interesting morphology i.e. beads with short fibre was observed when using a high polymer concentration. Bead on string structure agglomeration was promoted by a low polymer concentration. Formation of droplets or agglomerated bead on string is promoted below 5wt% polymer concentration, and beads with short fibre were present in the microstructure beyond a polymer concentration of 20wt%. PMID:27612839

  17. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads

    Directory of Open Access Journals (Sweden)

    Harikrishnan Jayamohan

    2015-05-01

    Full Text Available In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic beads for capture and polyguanine (polyG oligonucleotide functionalized secondary (polystyrene beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead. While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\\(^{8}\\ guanine tags per secondary bead (\\(7.5\\times10^{6}\\ biotin-FITC per secondary bead, 20 guanines per oligonucleotide bound to the target (E. coli. A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridineruthenium(II (Ru(bpy\\(_{3}^{2+}\\ as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3

  18. Predicting of bead undercut defects in high-speed gas metal arc welding (GMAW)

    Institute of Scientific and Technical Information of China (English)

    Wen-jing XU; Chuan-song WU; De-gang ZOU

    2008-01-01

    In the gas metal arc welding (GMAW) process, when the welding speed reaches a certain threshold, there will be an onset of weld bead undercut defects which limit the further increase of the welding speed. Establishing a mathematical model for high-speed GMAW to predict the tendency of bead undercuts is of great significance to pre-vent such defects. Under the action of various forces, the transferred metal from filler wire to the weld pool, and the geometry and dimension of the pool itself decide if the bead undercut occurs or not. The previous model simpli-fied the pool shape too much. In this paper, based on the actual weld pool geometry and dimension calculated from a numerical model, a hydrostatic model for liquid metal surface is used to study the onset of bead undercut defects in the high-speed welding process and the effects of dif-ferent welding parameters on the bead undercut tendency.

  19. On-chip magnetic bead microarray using hydrodynamic focusing in a passive magnetic separator.

    Science.gov (United States)

    Smistrup, K; Kjeldsen, B G; Reimers, J L; Dufva, M; Petersen, J; Hansen, M F

    2005-11-01

    Implementing DNA and protein microarrays into lab-on-a-chip systems can be problematic since these are sensitive to heat and strong chemicals. Here, we describe the functionalization of a microchannel with two types of magnetic beads using hydrodynamic focusing combined with a passive magnetic separator with arrays of soft magnetic elements. The soft magnetic elements placed on both sides of the channel are magnetized by a relatively weak applied external magnetic field (21 mT) and provide magnetic field gradients attracting magnetic beads. Flows with two differently functionalized magnetic beads and a separating barrier flow are introduced simultaneously at the two channel sides and the centre of the microfluidic channel, respectively. On-chip experiments with fluorescence labeled beads demonstrate that the two types of beads are captured at each of the channel sidewalls. On-chip hybridization experiments show that the microfluidic systems can be functionalized with two sets of beads carrying different probes that selectively recognize a single base pair mismatch in target DNA. By switching the places of the two types of beads it is shown that the microsystem can be cleaned and functionalized repeatedly with different beads with no cross-talk between experiments. PMID:16234958

  20. Optical tweezers and manipulation of PMMA beads in various conditions

    Science.gov (United States)

    Kotsifaki, D. G.; Makropoulou, M.; Serafetinides, A. A.

    2009-07-01

    Laser optical trapping and micromanipulation of microparticles or cells and subcellular structures have gained remarkable interest in biomedical research and applications. Several laser sources are employed for the combination of a laser scalpel with an optical tweezers device, under microscopic control. However, although the principles and the mechanisms of pulsed laser ablation have been well described for macroscopic interventions, the microbeam operation, under microscopic guidance, necessitates further experiments and investigations. We present experimental results of controlled micro-ablation of PMMA beads of 3-8 μm diameters, trapped by laser tweezers in various media e.g. solutes of different index of refraction. An optical tweezers system, based on a continuous wave He-Ne laser emitting at 632.8 nm, was tested on beads and, despite the low power of the He-Ne laser, the optical trap was stable. Another optical system, based on a cw Nd:YAG laser emitting at 1.06 μm, was tested on microspheres too. Successful beads ablation was carried out by irradiation with multiple, or even a single nitrogen laser pulse of 7 ns pulse duration at a wavelength of 337 nm. The ablative perforation of the microspheres was estimated by controlling the laser fluence. Moreover, shape deformations of PMMA microspheres were observed. The experimentally obtained results are theoretically explained via the spatial intensity distribution based on Mie light scattering theory. Furthermore, the appearance of laser ablation holes in the back side of microspheres is explained by the ablation triggered shock waves propagation. The role of the stretching forces action is also discussed. Additionally, we report experimental results on measuring the optical trap force of PMMA beads. A powerful optical tweezers system based on a continuous wave Nd:YAG laser was used in order to estimate the trapping efficiency for several beads diameter.

  1. Current status and future developments in radiolabelled immunoassays

    International Nuclear Information System (INIS)

    Radioisotopes are used extensively in medical practice and their use in RIA or IRMA usually represent a small proportion of the total. Radiolabelled immunoassays based on 125I constitute a simple didactic, cost effective and robust technology which is still regarded as the reference method in many clinical applications. The IAEA has implemented many successful programmes using the ''bulk reagent'' approach, involving 68 countries in all the different regions. The main achievements have been in technology transfer with self sufficiency in production for some countries; training of large numbers of staff; quality control and quality assurance schemes; devolution of screening programmes for neonatal congenital hypothryoidism. Alternatives to the use of radioisotopic tracers are constrained by many factors and are often only available in restricted commercial packages. They are often not suitable for technology transfer programmes and often lack any didactic component in addition to a relative high cost. The production of radiolabels using 125I is both simple and adaptable. In addition expertise in their preparation and purification is widespread even in developing countries. Together with the ease of producing antibodies, the facts have made 125I-radiolabelled immunoassays ideal for investigative procedures for many research activities (30,31) particularly in the medical context where radioisotopes are commonly used. In conclusion, even a superficial examination of public health statistics for various countries throughout the continents indicates a need for a simple, inexpensive and robust analytical tool. In this light, there is a predicted continuing role for radiolabelled immunoassays. (author)

  2. Multiplexed electrochemical immunoassay of biomarkers using chitosan nanocomposites.

    Science.gov (United States)

    Chen, Xia; Ma, Zhanfang

    2014-05-15

    In this work, a novel and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using functionalized chitosan composites. The immunosensing platform was prepared via immobilizing capture anti-AFP and anti-CEA on chitosan-Au nanoparticles (AuNPs) through EDC/NHS linking. The signal tags were fabricated by immobilizing electroactive redox probes - Prussian blue (PB) and ferrocenecarboxylic acid (Fc) on chitosan (CHIT), following by absorbing AuNPs to immobilize labeled anti-AFP and anti-CEA, respectively. A sandwich-type immunoassay format was employed for the simultaneous detection of AFP and CEA. The assay was based on the electrochemical oxidation/reduction of the redox species in signal tags, which has a relationship with the concentration of analytes. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA with a wide range of 0.05-100 ng mL(-1) for both AFP and CEA. The detection limits (LOD) was 0.03 ng mL(-1) for AFP and 0.02 ng mL(-1) for CEA (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from standard ELISA method. And the negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis. PMID:24413402

  3. Expanded polylactide bead foaming - A new technology

    Science.gov (United States)

    Nofar, M.; Ameli, A.; Park, C. B.

    2015-05-01

    Bead foaming technology with double crystal melting peak structure has been recognized as a promising method to produce low-density foams with complex geometries. During the molding stage of the bead foams, the double peak structure generates a strong bead-to-bead sintering and maintains the overall foam structure. During recent years, polylactide (PLA) bead foaming has been of the great interest of researchers due to its origin from renewable resources and biodegradability. However, due to the PLA's low melt strength and slow crystallization kinetics, the attempts have been limited to the manufacturing methods used for expanded polystyrene. In this study, for the first time, we developed microcellular PLA bead foams with double crystal melting peak structure. Microcellular PLA bead foams were produced with expansion ratios and average cell sizes ranging from 3 to 30-times and 350 nm to 15 µm, respectively. The generated high melting temperature crystals during the saturation significantly affected the expansion ratio and cell density of the PLA bead foams by enhancing the PLA's poor melt strength and promoting heterogeneous cell nucleation around the crystals.

  4. Porous bead packings for gas chromatography

    Science.gov (United States)

    Pollock, G. E.; Woeller, F. H.

    1979-01-01

    Porous polyaromatic packing beads have low polarity, high efficiency, short retention time, and may be synthesized in size range of 50 to 150 micrometers (100 to 270 mesh). Mechanically strong beads may be produced using various materials depending on elements and compounds to be identified.

  5. Multiplex immunoassay for persistent organic pollutants in tilapia: Comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

    Science.gov (United States)

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays required a flow cytometer with sophisticated fluidics and optics. The new imaging superparamagnetic SEMs-based platform transports SEMs with considerably ...

  6. A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

    Science.gov (United States)

    Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

    2016-05-01

    The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community. PMID:27057765

  7. A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

    Science.gov (United States)

    Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

    2016-05-01

    The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

  8. Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe{sub 3}O{sub 4}/polyaniline platform and multi-enzyme-functionalized carbon sphere label

    Energy Technology Data Exchange (ETDEWEB)

    Lin Mouhong [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Liu Yingju, E-mail: liuyingju@hotmail.com [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Sun Zihong; Zhang Shenglai; Yang Zhuohong [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Ni Chunlin, E-mail: niclchem@scau.edu.cn [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China)

    2012-04-13

    Graphical abstract: Schematic representation of Fe{sub 3}O{sub 4}/PANI/Nafion-based immunosensor using multi-HRP-HCS-Ab{sub 2} bioconjugates as labels. Highlights: Black-Right-Pointing-Pointer An electrochemical immunosensor for high sensitive detection of BaP. Black-Right-Pointing-Pointer A dual amplification strategy by Fe{sub 3}O{sub 4}/PANI/Nafion film and multi-HRP-HCS-Ab{sub 2} label. Black-Right-Pointing-Pointer An accelerated electron transfer pathway by the Fe{sub 3}O{sub 4}/PANI/Nafion film. - Abstract: An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe{sub 3}O{sub 4}/polyaniline/Nafion (Fe{sub 3}O{sub 4}/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab{sub 2}) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe{sub 3}O{sub 4}/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe{sub 3}O{sub 4}/polyaniline (Fe{sub 3}O{sub 4}/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8 pM and 2 nM with the detection limit of 4 pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants.

  9. Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

    NARCIS (Netherlands)

    Meimaridou, A.; Haasnoot, W.; Shelver, W.L.; Franek, M.; Nielen, M.W.F.

    2013-01-01

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with cons

  10. Development of a MAb-based immunoassay for the simultaneous determination of O,O-diethyl and O,O-dimethyl organophosphorus pesticides in vegetable and fruit samples pretreated with QuEChERS.

    Science.gov (United States)

    Zhao, Fengchun; Hu, Chunyan; Wang, Huimin; Zhao, Longyu; Yang, Zhengyou

    2015-12-01

    To develop a broad-specificity immunoassay for organophosphorus pesticides (OPs), a broad-specificity monoclonal antibody (MAb) for OPs against a generic hapten, O,O-diethyl O-(3-carboxyphenyl) phosphorothioate with the carboxy group in the meta position of the benzene ring, was produced. Eight haptens were prepared and covalently attached to ovalbumin (OVA) for use as coating antigens, and the optimum coating antigen was selected. Then, a sensitive and broadly class selective competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on the MAb and the optimum coating antigen (hapten H-OVA, possessing an O,O-dimethyl generic structure and linked through a linear spacer arm) was developed and optimized. The MAb developed in this study showed quite different cross-reactivity and selectivity compared to previously produced anti-OPs broad-specificity MAbs. Specifically, the MAb showed high and uniform sensitivity to seven O,O-diethyl OPs and six O,O-dimethyl OPs. With the optimum ciELISA, the IC50 values of the 13 OPs were determined as 23.1∼151.2 ng mL(-1). The average IC50 and coefficient of variation (CV) for the IC50 values of the 13 OPs were 74.6 ng mL(-1) and 33.9%, respectively. For the recovery study, a QuEChERS approach based on dispersive solid-phase extraction (d-SPE) was implemented to decrease the matrix effects of vegetable and fruit samples. The recoveries of six representative OPs from the spiked samples ranged from 89.4 to 135.5%; the CV ranged from 3.5 to 15.7%. The ciELISA was also applied to real samples, followed by confirmation with gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis. The results demonstrated that the ciELISA is suitable for monitoring OP contamination in vegetable and fruit samples.

  11. BeadArray expression analysis using bioconductor.

    Directory of Open Access Journals (Sweden)

    Matthew E Ritchie

    2011-12-01

    Full Text Available Illumina whole-genome expression BeadArrays are a popular choice in gene profiling studies. Aside from the vendor-provided software tools for analyzing BeadArray expression data (GenomeStudio/BeadStudio, there exists a comprehensive set of open-source analysis tools in the Bioconductor project, many of which have been tailored to exploit the unique properties of this platform. In this article, we explore a number of these software packages and demonstrate how to perform a complete analysis of BeadArray data in various formats. The key steps of importing data, performing quality assessments, preprocessing, and annotation in the common setting of assessing differential expression in designed experiments will be covered.

  12. Acupressure Bead in the Eustachian Tube.

    Science.gov (United States)

    Igarashi, Kazunori; Matsumoto, Yu; Kakigi, Akinobu

    2015-08-01

    In this article, we aim to enlighten practitioners and patients involved with acupressure beads and to contribute to their safer use by reporting a unique case of insidious intrusion of an acupressure bead into the eustachian tube. A metallic object was found in the eustachian tube of a patient while conducting a magnetic resonance imaging (MRI) examination. The object was later confirmed to be an auricular acupressure bead, and was successfully removed by performing a tympanoplasty and a canal wall down mastoidectomy. The bead was assumed to have passed through an existing perforation of the tympanic membrane. According to previously published literature, tympanic membrane perforations exist in ∼1% of the population. Therefore, middle-ear foreign bodies are relatively common occurrences for otolaryngologists. However, metallic objects such as acupressure beads are especially important in the sense that they can cause severe burns during MRI. To avoid potential complications, acupressure-bead practitioners should be aware of the possibility that intrusions through the tympanic membrane could go unnoticed. PMID:26276456

  13. Preprogrammed, parallel on-chip immunoassay using system-level capillarity control.

    Science.gov (United States)

    Kim, Sung-Jin; Paczesny, Sophie; Takayama, Shuichi; Kurabayashi, Katsuo

    2013-07-16

    Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources.

  14. Label-free impedimetric immunoassay for trace levels of polychlorinated biphenyls in insulating oil.

    Science.gov (United States)

    Date, Yasumoto; Aota, Arata; Sasaki, Kazuhiro; Namiki, Yukie; Matsumoto, Norio; Watanabe, Yoshitomo; Ohmura, Naoya; Matsue, Tomokazu

    2014-03-18

    A rapid, ultrasensitive, and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First, we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated, and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 μg/L) and a low detection limit (LOD) of 0.001 μg/L, which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning, an LOD of 0.052 mg/kg-oil was achieved, which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally, the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs, and the results agreed well with the Japanese official method (HRGC/HRMS).

  15. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    Science.gov (United States)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  16. Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium.

    Science.gov (United States)

    Belščak-Cvitanović, Ana; Komes, Draženka; Karlović, Sven; Djaković, Senka; Spoljarić, Igor; Mršić, Gordan; Ježek, Damir

    2015-01-15

    Alginate-based blends consisting of carrageenan, pectin, chitosan or psyllium husk powder were prepared for assessment of the best formulation aimed at encapsulation of caffeine. Alginate-pectin blend exhibited the lowest viscosity and provided the smallest beads. Alginate-psyllium husk blend was characterised with higher viscosity, yielding the largest bead size and the highest caffeine encapsulation efficiency (83.6%). The release kinetics of caffeine indicated that the porosity of alginate hydrogel was not reduced sufficiently to retard the diffusion of caffeine from the beads. Chitosan coated alginate beads provided the most retarded release of caffeine in water. Morphological characteristics of beads encapsulating caffeine were adversely affected by freeze drying. Bitterness intensity of caffeine-containing beads in water was the lowest for alginate-psyllium beads and chitosan coated alginate beads. Higher sodium alginate concentration (3%) for production of hydrogel beads in combination with psyllium or chitosan coating would present the most favourable carrier systems for immobilization of caffeine. PMID:25149001

  17. A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5 '-diphosphocholine coupled dendrimers

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Pedersen, H. G.; Jensen, A. L.;

    2009-01-01

    C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP...

  18. A highly sensitive SPR biosensor based on a graphene oxide sheet modified with gold bipyramids, and its application to an immunoassay for rabbit IgG

    International Nuclear Information System (INIS)

    A new SPR sensing substrate was fabricated that is based on the use of graphene oxide (GO) and gold bipyramids (GBPs). It can substantially improve the sensitivity of wavelength modulation SPR biosensors. First, a support consisting of a sheet of single layered GO was prepared, and its morphology and thickness were determined by atomic force microscopy (AFM). GBPs were synthesized and modified with staphylococcal protein A (SPA) for the oriented immobilization of antibody. GBPs modified with SPA were assembled on GO sheets through covalent attachment. The resulting SPR biosensor exhibits a highly sensitive response to rabbit IgG in the 0.15–40 μg mL−1 concentration range. The limit of quantification is better by factors of 4 and 16 compared to those obtained with gold nanoparticle-based, and conventional gold film-based sensors, respectively. In our expectation, this GBP-based SPR biosensor has a wide scope in that it may be employed to develop detection schemes for many other biomolecules by changing the corresponding receptor on its surface. (author)

  19. Asynchronous Magnetic Bead Rotation (AMBR Microviscometer for Label-Free DNA Analysis

    Directory of Open Access Journals (Sweden)

    Yunzi Li

    2014-03-01

    Full Text Available We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

  20. Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid qualitative and semi-quantitative analysis of artesunate and dihydroartemisinin

    OpenAIRE

    He, Lishan; Nan, Tiegui; Cui, Yongliang; Guo, Suqin; Zhang, Wei; Zhang, Rui; Tan, Guiyu; Wang, Baomin; Cui, Liwang

    2014-01-01

    Background Artemisinin-based combination therapy (ACT) plays an indispensable role in malaria control and elimination. However, the circulation of counterfeit, substandard drugs has greatly threatened malaria elimination campaigns. Most methods for the analysis of artemisinin and its derivatives require expensive equipment and sophisticated instrumentation. A convenient, easy-to-use diagnostic device for rapid evaluation of the quality of artemisinin drugs at the point-of-care is still lackin...

  1. Rapid Wuchereria bancrofti-Specific Antigen Wb123-Based IgG4 Immunoassays as Tools for Surveillance following Mass Drug Administration Programs on Lymphatic Filariasis

    OpenAIRE

    Steel, Cathy; Golden, Allison; Kubofcik, Joseph; LaRue, Nicole; de los Santos, Tala; Domingo, Gonzalo J.; Nutman, Thomas B.

    2013-01-01

    The Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapi...

  2. A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Sandeep Kumar Vashist, Gregor Czilwik, Thomas van Oordt, Felix von Stetten, Roland Zengerle, E. Marion Schneider & John H.T. Luong ### Abstract A rapid one-step kinetics-based sandwich enzyme-linked immunosorbent (ELISA) procedure has been developed for highly-sensitive detection of C-reactive protein (CRP) in less than 30 min. With minimal process steps, the procedure is highly simplified and cost-effective. The analysis only involves sequentially the formation of a san...

  3. Amperometric homogeneous competitive immunoassay in a perfluorocarbon emulsion oxygen therapeutic (PEOT).

    Science.gov (United States)

    Barlag, Rebecca E; Halsall, H Brian; Heineman, William R

    2013-04-01

    The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2-20% PEOT.

  4. Ultra-sensitive immunoassay biosensors using hybrid plasmonic-biosilica nanostructured materials.

    Science.gov (United States)

    Yang, Jing; Zhen, Le; Ren, Fanghui; Campbell, Jeremy; Rorrer, Gregory L; Wang, Alan X

    2015-08-01

    We experimentally demonstrate an ultra-sensitive immunoassay biosensor using diatom biosilica with self-assembled plasmonic nanoparticles. As the nature-created photonic crystal structures, diatoms have been adopted to enhance surface plasmon resonances of metal nanoparticles on the surfaces of diatom frustules and to increase the sensitivity of surface-enhanced Raman scattering (SERS). In this study, a sandwich SERS immunoassay is developed based on the hybrid plasmonic-biosilica nanostructured materials that are functionalized with goat anti-mouse IgG. Our experimental results show that diatom frustules improve the detection limit of mouse IgG to 10 pg/mL, which is ˜100× better than conventional colloidal SERS sensors on flat glass. Ultra-sensitive immunoassay biosensor using diatom biosilica with self-assembled plasmonic nanoparticles. PMID:25256544

  5. Ultrasensitive photoelectrochemical immunoassay of indole-3-acetic acid based on the MPA modified CdS/RGO nanocomposites decorated ITO electrode.

    Science.gov (United States)

    Sun, Bing; Chen, Lijian; Xu, Yan; Liu, Min; Yin, Huanshun; Ai, Shiyun

    2014-01-15

    A novel ultrasensitive photoelectrochemical immunosensor was fabricated based on 3-mercaptopropionic acid stabilized CdS/reduced graphene oxide (MPA-CdS/RGO) nanocomposites for indole-3-acetic acid (IAA) detection. The MPA-CdS/RGO nanocomposites were synthesized by in situ solvothermal growth of triangulated pyramidal CdS nanoparticles on the RGO sheet. 3-Mercaptopropionic acid (MPA) was employed as the modifier and bridge to immobilize the antibody. The nanocomposites were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy and UV/vis spectra. The results showed that the MPA-CdS/RGO nanocomposites revealed enhanced photocurrent response due to excellent electron transport properties of RGO and the improved assembly of CdS nanoparticles onto RGO sheet with the introduction of MPA. Based on the dependence of the photocurrent decline on the concentration of IAA, the proposed photoelectrochemical immunosensor for IAA depicted a linear range from 0.1 to 1000 ng/mL with a lower detection limit (0.05 ng/mL). The high sensitivity, reproducibility and specificity of the method permitted the method suitable to be used in real samples. PMID:23954674

  6. Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate.

    Science.gov (United States)

    Kobayashi, Masatoshi; Sato, Masahiro; Li, Yan; Soh, Nobuaki; Nakano, Koji; Toko, Kiyoshi; Miura, Norio; Matsumoto, Kiyoshi; Hemmi, Akihide; Asano, Yasukazu; Imato, Toshihiko

    2005-12-15

    A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25ppt to 25ppb, and the coefficient of variation of the SPR signals for the 25ppb TNP solution was determined to be 13% (n=4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations. PMID:18970305

  7. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    Science.gov (United States)

    Goto, Youhei; Kanda, Masato; Yamamoto, Daigo; Shioi, Akihisa

    2015-09-01

    Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms.

  8. Optimized dendrimer-encapsulated gold nanoparticles and enhanced carbon nanotube nanoprobes for amplified electrochemical immunoassay of E. coli in dairy product based on enzymatically induced deposition of polyaniline.

    Science.gov (United States)

    Zhang, Xinai; Shen, Jianzhong; Ma, Haile; Jiang, Yuxiang; Huang, Chenyong; Han, En; Yao, Boshui; He, Yunyao

    2016-06-15

    A highly sensitive immunosensor was reported for Escherichia coli assay in dairy product based on electrochemical measurement of polyaniline (PAn) that was catalytically deposited by horseradish peroxidase (HRP) labels. Herein, the immunosensor was developed by using poly(amidoamine) dendrimer-encapsulated gold nanoparticles (PAMAM(Au)) as sensing platform. Importantly, the optimal HAuCl4/PAMAM ratio was investigated to design the efficient PAMAM(Au) nanocomposites. The nanocomposites were proven to not only increase the amount of immobilized capture antibody (cAb), but also accelerate the electron transfer process. Moreover, the {dAb-CNT-HRP} nanoprobes were prepared by exploiting the amplification effect of multiwalled carbon nanotubes (CNTs) for loading detection antibody (dAb) and enormous HRP labels. After a sandwich immunoreaction, the quantitatively captured nanoprobes could catalyze oxidation aniline to produce electroactive PAn for electrochemical measurement. On the basis of signal amplification of the PAMAM(Au)-based immunosensor and the {dAb-CNT-HRP} nanoprobes, the proposed strategy exhibited a linear relationship between the peak current of PAn and the logarithmic value of E. coli concentration ranging from 1.0 × 10(2) to 1.0 × 10(6) cfu mL(-1) with a detection limit of 50 cfu mL(-1) (S/N=3), and the electrochemical detection of E. coli could be achieved in 3h. The electrochemical immunosensor was also used to determine E. coli in dairy product (pure fresh milk, infant milk powder, yogurt in shelf-life and expired yogurt), and the recoveries of standard additions were in the range of 96.8-108.7%. Overall, this method gave a useful protocol for E. coli assay with high sensitivity, acceptable accuracy and satisfying stability, and thus provided a powerful tool to estimate the quality of dairy product. PMID:26908184

  9. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    Science.gov (United States)

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  10. Identifying and reducing potentially wrong immunoassay results even when plausible and "not-unreasonable".

    Science.gov (United States)

    Ismail, Adel A A

    2014-01-01

    The primary role of the clinical laboratory is to report accurate results for diagnosis of disease and management of illnesses. This goal has, to a large extent been achieved for routine biochemical tests, but not for immunoassays which remained susceptible to interference from endogenous immunoglobulin antibodies, causing false, and clinically misleading results. Clinicians regard all abnormal results including false ones as "pathological" necessitating further investigations, or concluding iniquitous diagnosis. Even more seriously, "false-negative" results may wrongly exclude pathology, thus denying patients' necessary treatment. Analytical error rate in immunoassays is relatively high, ranging from 0.4% to 4.0%. Because analytical interference from endogenous antibodies is confined to individuals' sera, it can be inconspicuous, pernicious, sporadic, and insidious because it cannot be detected by internal or external quality assessment procedures. An approach based on Bayesian reasoning can enhance the robustness of clinical validation in highlighting potentially erroneous immunoassay results. When this rational clinical/statistical approach is followed by analytical affirmative follow-up tests, it can help identifying inaccurate and clinically misleading immunoassay data even when they appear plausible and "not-unreasonable." This chapter is largely based on peer reviewed articles associated with and related to this approach. The first section underlines (without mathematical equations) the dominance and misuse of conventional statistics and the underuse of Bayesian paradigm and shows that laboratorians are intuitively (albeit unwittingly) practicing Bayesians. Secondly, because interference from endogenous antibodies is method's dependent (with numerous formats and different reagents), it is almost impossible to accurately assess its incidence in all differently formulated immunoassays and for each analytes/biomarkers. However, reiterating the basic concepts

  11. Design criteria for developing low-resource magnetic bead assays using surface tension valves.

    Science.gov (United States)

    Adams, Nicholas M; Creecy, Amy E; Majors, Catherine E; Wariso, Bathsheba A; Short, Philip A; Wright, David W; Haselton, Frederick R

    2013-01-01

    Many assays for biological sample processing and diagnostics are not suitable for use in settings that lack laboratory resources. We have recently described a simple, self-contained format based on magnetic beads for extracting infectious disease biomarkers from complex biological samples, which significantly reduces the time, expertise, and infrastructure required. This self-contained format has the potential to facilitate the application of other laboratory-based sample processing assays in low-resource settings. The technology is enabled by immiscible fluid barriers, or surface tension valves, which stably separate adjacent processing solutions within millimeter-diameter tubing and simultaneously permit the transit of magnetic beads across the interfaces. In this report, we identify the physical parameters of the materials that maximize fluid stability and bead transport and minimize solution carryover. We found that fluid stability is maximized with ≤0.8 mm i.d. tubing, valve fluids of similar density to the adjacent solutions, and tubing with ≤20 dyn/cm surface energy. Maximizing bead transport was achieved using ≥2.4 mm i.d. tubing, mineral oil valve fluid, and a mass of 1-3 mg beads. The amount of solution carryover across a surface tension valve was minimized using ≤0.2 mg of beads, tubing with ≤20 dyn/cm surface energy, and air separators. The most favorable parameter space for valve stability and bead transport was identified by combining our experimental results into a single plot using two dimensionless numbers. A strategy is presented for developing additional self-contained assays based on magnetic beads and surface tension valves for low-resource diagnostic applications. PMID:24403996

  12. Application of ZnO quantum dots dotted carbon nanotube for sensitive electrochemiluminescence immunoassay based on simply electrochemical reduced Pt/Au alloy and a disposable device

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fang; Deng, Wenping; Zhang, Yan [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022 (China); Ge, Shenguang [Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan, 250022 (China); Yu, Jinghua, E-mail: ujn.yujh@gmail.com [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022 (China); Song, Xianrang, E-mail: sxr@vip.163.com [Cancer Research Center, Shandong Tumor Hospital, Jinan, 250012 (China)

    2014-03-01

    Highlights: • A sandwich-type electrochemluminence immunosensor was fabricated. • Simply electrochemical reduced Pt/Au alloy was selected as immunosensing probes. • ZnO@CNT composite was first employed as signal amplification label. Abstract: We report on a disposable microdevice suitable for sandwich-type electrochemiluminescence (ECL) detection of prostate specific antigen (PSA). The method is making use of ZnO quantum dots dotted carbon nanotube (ZnO@CNT) and simply electrochemical reduced Pt/Au alloy. The latter was selected as immunosensing probe to modify screen-printed carbon electrode, due to its excellent electrical property. For further ultrasensitive, low-potential and stable ECL detection, ZnO@CNT composite was first synthesized using a facile solvothermal method, and employed as signal amplification label. In this work, two working electrodes in one device were used for one determination to obtain more exact results based on screen-print technique. Taking advantage of dual-amplification effects of the Pt/Au and ZnO@CNT, this immunosensor could detect the PSA quantitatively, in the range of 0.001–500 ng mL⁻¹, with a low detection limit of 0.61 pg mL⁻¹. The resulting versatile immunosensor possesses high sensitivity, satisfactory reproducibility and regeneration. This simple and specific strategy has vast potential to be used in other biological assays.

  13. Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish.

    Science.gov (United States)

    Le Berre, Marie; Kilcoyne, Michelle; Kane, Marian

    2015-09-01

    Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes.

  14. Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

    Science.gov (United States)

    Basilicata, Pascale; Pieri, Maria; Settembre, Veronica; Galdiero, Alessandra; Della Casa, Elvira; Acampora, Antonio; Miraglia, Nadia

    2011-11-15

    According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred. PMID:21992470

  15. Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification.

    Science.gov (United States)

    Yin, Huanshun; Zhou, Yunlei; Xu, Zhenning; Wang, Mo; Ai, Shiyun

    2013-11-15

    In this work, we fabricated a novel electrochemical immunosensor for detection of DNA methylation, analysis of DNA MTase activity and screening of MTase inhibitor. The immunosensor was on the basis of methyl binding domain protein of MeCP2 as DNA CpG methylation recognization unit, anti-His tag antibody as "immuno-bridge" and horseradish peroxidase labeled immuneglobulin G functionalized gold nanoparticles (AuNPs-IgG-HRP) as signal amplification unit. In the presence of M. SssI MTase, the symmetrical sequence of 5'-CCGG-3' was methylated and then recognized by MeCP2 protein. By the immunoreactions, anti-His tag antibody and AuNPs-IgG-HRP was captured on the electrode surface successively. Under the catalysis effect of HRP towards hydroquinone oxidized by H2O2, the electrochemical reduction signal of benzoquinone was used to analyze M. SssI MTase activity. The electrochemical reduction signal demonstrated a wide linear relationship with M. SssI concentration ranging from 0.05 unit/mL to 90 unit/mL, achieving a detection limit of 0.017 unit/mL (S/N=3). The most important advantages of this method were its high sensitivity and good selectivity, which enabled the detection of even one-base mismatched sequence. In addition, we also verified that the developed method could be applied for screening the inhibitors of DNA MTase and for developing new anticancer drugs.

  16. Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish.

    Science.gov (United States)

    Le Berre, Marie; Kilcoyne, Michelle; Kane, Marian

    2015-09-01

    Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes. PMID:26169671

  17. Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

    Science.gov (United States)

    Reverté, Laia; de la Iglesia, Pablo; del Río, Vanessa; Campbell, Katrina; Elliott, Christopher T; Kawatsu, Kentaro; Katikou, Panagiota; Diogène, Jorge; Campàs, Mònica

    2015-11-01

    The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

  18. Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

    Science.gov (United States)

    Reverté, Laia; de la Iglesia, Pablo; del Río, Vanessa; Campbell, Katrina; Elliott, Christopher T; Kawatsu, Kentaro; Katikou, Panagiota; Diogène, Jorge; Campàs, Mònica

    2015-11-01

    The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities. PMID:26424329

  19. Tumour metastasis-associated gene profiling using one-dimensional microfluidic beads array

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Great efforts have been made on the early diagnosis and molecular mechanism research of tumour metastasis in recent years. In this paper, based on the one-dimensional microfluidic beads array, a novel platform for tumour metastasis-associated genes profiling has been developed by depositing nucleic acids functional beads in the microchannel. This platform is sensitive (limit of detection: 0.02 nmol/L) and can perform mRNAs analysis without PCR. Two human colon cancer cell lines (primary and metastatic) from the same patient were used as a model, and transcriptional expression profiling of multiple tumour metastasis-associated genes in these two cell lines was successfully achieved. Furthermore, the results obtained on the beads array were validated by RT-PCR. This novel beads array has advantages of high sensitivity, little sample consumption, short assay time, low cost and high throughput capability. It holds the potential in early diagnosis and mechanism research of tumour metastasis.

  20. Experimental and numerical study of restraining force development in inclined draw beads

    Science.gov (United States)

    Raghavan, K. S.; Narainen, R.; Smith, L. M.

    2013-12-01

    Inclined (angled) draw bead geometries are becoming increasingly common as body styling requirements necessitate external panel shapes with considerable curvature. The restraining force that develops as material undergoes bending and frictional contact varies with bead geometry, material strength level and ambient lubrication conditions. In this study, an FEA based parametric approach is used to model the effects of material strength, friction condition, and binder angle on draw bead restraining force (DBRF). A finite element draw bead simulation was calibrated to experimental data for a 250 MPa electro-galvanized bake-hardenable specimen. The experimental data is used to confirm that the DBRF vs. binder angle curve roughly follows a concave shaped second order function with a maximum somewhere in the positive binder angle domain.

  1. Sequential Injection/Electrochemical Immunoassay for Quantifying the Pesticide Metabolite 3, 5, 6-Trichloro-2-Pyridinol

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guodong; Riechers, Shawn L.; Timchalk, Chuck; Lin, Yuehe

    2005-12-04

    An automated and sensitive sequential injection electrochemical immunoassay was developed to monitor a potential insecticide biomarker, 3, 5, 6-trichloro-2-pyridinol. The current method involved a sequential injection analysis (SIA) system equipped with a thin-layer electrochemical flow cell and permanent magnet, which was used to fix 3,5,6-trichloro-2-pyridinol (TCP) antibody coated magnetic beads (TCP-Ab-MBs) in the reaction zone. After competitive immunoreactions among TCP-Ab-MBs, TCP analyte, and horseradish peroxidase (HRP) labeled TCP, a 3, 3?, 5, 5?-tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB-H2O2) substrate solution was injected to produce an electroactive enzymatic product. The activity of HRP tracers was monitored by a square wave voltammetric scanning electroactive enzymatic product in the thin-layer flow cell. The voltammetric characteristics of the substrate and the enzymatic product were investigated under batch conditions, and the parameters of the immunoassay were optimized in the SIA system. Under the optimal conditions, the system was used to measure as low as 6 ng L-1 (ppt) TCP, which is around 50-fold lower than the value indicated by the manufacturer of the TCP RaPID Assay? kit (0.25 ug/L, colorimetric detection). The performance of the developed immunoassay system was successfully evaluated on tap water and river water samples spiked with TCP. This technique could be readily used for detecting other environmental contaminants by developing specific antibodies against contaminants and is expected to open new opportunities for environmental and biological monitoring.

  2. Latex immunoassay for rapid detection of rotavirus.

    OpenAIRE

    Hughes, J. H.; Tuomari, A V; Mann, D R; Hamparian, V V

    1984-01-01

    A latex agglutination (LA) test was evaluated for the detection of human rotaviruses in stool specimens. Both antiserum and immunoglobulin G (IgG)-sensitized latex particles were used, with IgG-coated beads being more sensitive for human rotavirus antigen detection. Latex beads sensitized with anti-simian-SA-11 IgG were stable for at least 8 months when stored at 4 degrees C. The sensitivity of the test was compared with that of the Rotazyme (Abbott Laboratories, Diagnostics Div., North Chica...

  3. Beaded streams of Arctic permafrost landscapes

    Directory of Open Access Journals (Sweden)

    C. D. Arp

    2014-07-01

    Full Text Available Beaded streams are widespread in permafrost regions and are considered a common thermokarst landform. However, little is known about their distribution, how and under what conditions they form, and how their intriguing morphology translates to ecosystem functions and habitat. Here we report on a Circum-Arctic inventory of beaded streams and a watershed-scale analysis in northern Alaska using remote sensing and field studies. We mapped over 400 channel networks with beaded morphology throughout the continuous permafrost zone of northern Alaska, Canada, and Russia and found the highest abundance associated with medium- to high-ice content permafrost in moderately sloping terrain. In the Fish Creek watershed, beaded streams accounted for half of the drainage density, occurring primarily as low-order channels initiating from lakes and drained lake basins. Beaded streams predictably transition to alluvial channels with increasing drainage area and decreasing channel slope, although this transition is modified by local controls on water and sediment delivery. Comparison of one beaded channel using repeat photography between 1948 and 2013 indicate relatively stable form and 14C dating of basal sediments suggest channel formation may be as early as the Pleistocene–Holocene transition. Contemporary processes, such as deep snow accumulation in stream gulches effectively insulates river ice and allows for perennial liquid water below most beaded stream pools. Because of this, mean annual temperatures in pool beds are greater than 2 °C, leading to the development of perennial thaw bulbs or taliks underlying these thermokarst features. In the summer, some pools stratify thermally, which reduces permafrost thaw and maintains coldwater habitats. Snowmelt generated peak-flows decrease rapidly by two or more orders of magnitude to summer low flows with slow reach-scale velocity distributions ranging from 0.1 to 0.01 m s−1, yet channel runs still move water

  4. Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India

    Science.gov (United States)

    Ejazi, Sarfaraz Ahmad; Bhattacharya, Pradyot; Bakhteyar, Md. Asjad Karim; Mumtaz, Aquil Ahmad; Pandey, Krishna; Das, Vidya Nand Ravi; Das, Pradeep; Rahaman, Mehebubar; Goswami, Rama Prosad; Ali, Nahid

    2016-01-01

    Background Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. Methodology/principal findings We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. Conclusions/significance ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response

  5. Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(methacrylic acid

    Directory of Open Access Journals (Sweden)

    Vinokurova Ludmila G

    2001-08-01

    Full Text Available Abstract Background Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. Results New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(methacrylic acid. The beads contain up to 0.31 μmol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. Conclusions An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

  6. Chitosan and chemically modified chitosan beads for acid dyes sorption

    Institute of Scientific and Technical Information of China (English)

    AZLAN Kamari; WAN SAIME Wan Ngah; LAI KEN Liew

    2009-01-01

    The capabilities of chitosan and chitosan-EGDE (ethylene glycol diglycidyl ether) beads for removing Acid Red 37 (AR 37) and Acid Blue 25 (AB 25) from aqueous solution were examined. Chitosan beads were cross-linked with EGDE to enhance its chemical resistance and mechanical strength. Experiments were performed as a function of pH, agitation period and concentration of AR 37 and AB 25. It was shown that the adsorption capacities of chitosan were comparatively higher than chitosan-EGDE for both acid dyes. This is mainly because cross-linking using EGDE reduces the major adsorption sites -NH3+ on chitosan. Langmuir isotherm model showed best conformity compared to Freundlich and BET. The kinetic experimental data agreed very well to the pseudo second-order kinetic model. The desorption study revealed that after three cycles of adsorption and desorption by NaOH and HCl, both adsorbents retained their promising adsorption abilities. FT-IR analysis proved that the adsorption of acid dyes onto chitosan-based adsorbents was a physical adsorption. Results also showed that chitosan and chitosan-EGDE beads were favourable adsorbers and could be employed as low-cost alternatives for the removal of acid dyes in wastewater treatment.

  7. Development and validation of predictive simulation model of multi-layer repair welding process by temper bead technique

    International Nuclear Information System (INIS)

    Stress corrosion cracking (SCC) has recently been observed in the nickel base alloy weld metal of dissimilar pipe joint used in pressurized water reactor (PWR) . Temper bead technique has been developed as one of repair procedures against SCC applicable in case that post weld heat treatment (PWHT) is difficult to carry out. In this regard, however it is essential to pass the property and performance qualification test to confirm the effect of tempering on the mechanical properties at repair welds before temper bead technique is actually used in practice. Thus the appropriate welding procedure conditions in temper bead technique are determined on the basis of the property and performance qualification testing. It is necessary for certifying the structural soundness and reliability at repair welds but takes a lot of work and time in the present circumstances. Therefore it is desirable to establish the reasonable alternatives for qualifying the property and performance at repair welds. In this study, mathematical modeling and numerical simulation procedures were developed for predicting weld bead configuration and temperature distribution during multi-layer repair welding process by temper bead technique. In the developed simulation technique, characteristics of heat source in temper bead welding are calculated from weld heat input conditions through the arc plasma simulation and then weld bead configuration and temperature distribution during temper bead welding are calculated from characteristics of heat source obtained through the coupling analysis between bead surface shape and thermal conduction. The simulation results were compared with the experimental results under the same welding heat input conditions. As the results, the bead surface shape and temperature distribution, such as Acl lines, were in good agreement between simulation and experimental results. It was concluded that the developed simulation technique has the potential to become useful for accurate

  8. Comparison of polymethylmethacrylate (PMMA, native calcium sulfate, and high porous calcium sulfate beads as gentamicin carriers and osteoblast attachment

    Directory of Open Access Journals (Sweden)

    Chaiyakorn Thitiyanaporn

    2013-06-01

    Full Text Available Calcium sulfate, a bioresorbable material, has been used as a bone substitute material and antibiotic vehicle. The increasing porosity of calcium sulfate beads might improve drug delivery capacity as well as enhance the antibiotic elution property. High porous calcium sulfate (HPCS beads were fabricated using a salt leaching technique as a new bead type for antibiotic delivery system. Gentamicin-based antibiotic beads were conducted by impregnating gentamicin (3.8% w/w with polymethylmethacrylate (PMMA, or coating of PMMA, native calcium sulfate (NCS, and HPCS with gentamicin solution. Physical properties, microstructure, and gentamicin elution from gentamicin-impregnated PMMA (GI-PMMA, gentamicincoated PMMA (G-PMMA, gentamicin-coated NCS (G-NCS, and gentamicin-coated HPCS (G-HPCS were compared. The osteoblast attachment revealed that PMMA, NCS, and HPCS beads were not toxic to h-OBs after co-incubation for sevendays. Furthermore, more h-OBs attachment appeared in HPCS beads compared to PMMA and NCS beads after co-culture for 7 days. Eluted gentamicin from G-NCS and G-HPCS beads were greater than those from GI-PMMA and G-PMMA beads during the experimental period. All types of beads were able to elute gentamicin for 10 days except G-PMMA, which released gentamicin only for four days. The highest to lowest total concentrations of eluted gentamicin were from G-NCS, G-HPCS,G-PMMA, and GI-PMMA, respectively. These results suggested that the HPCS beads improved local antibiotic delivery and improved h-OBs attachment.

  9. Evaluation of an immunoassay for determination of plasma efavirenz concentrations in resource-limited settings

    Directory of Open Access Journals (Sweden)

    Alemseged Abdissa

    2014-06-01

    Full Text Available Introduction: Therapeutic drug monitoring (TDM may improve antiretroviral efficacy through adjustment of individual drug administration. This could result in reduced toxicity, prevent drug resistance, and aid management of drug–drug interactions. However, most measurement methods are too costly to be implemented in resource-limited settings. This study evaluated a commercially available immunoassay for measurement of plasma efavirenz. Methods: The immunoassay-based method was applied to measure efavirenz using a readily available Humastar 80 chemistry analyzer. We compared plasma efavirenz concentrations measured by the immunoassay with liquid chromatography tandem mass spectrometry (LC-MS/MS (reference method in 315 plasma samples collected from HIV patients on treatment. Concentrations were categorized as suboptimal4 µg/ml. Agreement between results of the methods was assessed via Bland-Altman plot and κ statistic values. Results: The median Interquartile range (IQR efavirenz concentration was 2.8 (1.9; 4.5 µg/ml measured by the LC–MS/MS method and 2.5 (1.8; 3.9 µg/ml by the immunoassay and the results were well correlated (ρ=0.94. The limits of agreement assessed by Bland–Altman plots were −2.54; 1.70 µg/ml. Although immunoassay underestimated high concentrations, it had good agreement for classification into low, normal or high concentrations (K=0.74. Conclusions: The immunoassay is a feasible alternative to determine efavirenz in areas with limited resources. The assay provides a reasonable approximation of efavirenz concentration in the majority of samples with a tendency to underestimate high concentrations. Agreement between tests evaluated in this study was clinically satisfactory for identification of low, normal and high efavirenz concentrations.

  10. A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera.

    Science.gov (United States)

    Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Czechowska, Justyna; Serdiuk, Illia E; Krzymiński, Karol; Kur, Józef

    2016-08-01

    This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

  11. ADSORPTION AND RELEASING PROPERTIES OF BEAD CELLULOSE

    Institute of Scientific and Technical Information of China (English)

    A. Morales; E. Bordallo; V. Leon; J. Rieumont

    2004-01-01

    The adsorption of some dyes on samples of bead cellulose obtained in the Unit of Research-Production "Cuba 9"was studied. Methylene blue, alizarin red and congo red fitted the adsorption isotherm of Langmuir. Adsorption kinetics at pH = 6 was linear with the square root of time indicating the diffusion is the controlling step. At pH = 12 a non-Fickian trend was observed and adsorption was higher for the first two dyes. Experiments carried out to release the methylene blue occluded in the cellulose beads gave a kinetic behavior of zero order. The study of cytochrome C adsorption was included to test a proteinic material. Crosslinking of bead cellulose was performed with epichlorohydrin decreasing its adsorption capacity in acidic or alkaline solution.

  12. Porous Ca-based bead sorbents for simultaneous removal of SO₂, fine particulate matters, and heavy metals from pilot plant sewage sludge incineration.

    Science.gov (United States)

    Han, Yosep; Hwang, Gukhwa; Kim, Donghyun; Park, Soyeon; Kim, Hyunjung

    2015-01-01

    In this study, a porous calcium-based sorbent was prepared for simultaneous removal of SO2, particulate matter (PM), and heavy metals generated during incineration of sewage sludge. The prepared sorbent was confirmed to have a 3-dimensional-network pore structure, a high specific surface area of 68.5m(2)/g, and gas permeability of 1.12 × 10(-10)m(2). Laboratory-scale tests indicated that there was an improvement in the performance of SO2 removal as the porosity and the specific surface area of the sorbent increased. Additionally, increasing reaction temperature led to greater SO2 removal. Meanwhile, the SL-4 and LS-3 sorbents prepared in this study were installed for operation during pilot tests treating the sewage sludge combustion gas generated by a fluidized incinerator in order to compare and evaluate their feasibility for use in industrial applications. The results showed that the reactivity between SO2 and the starting material of the sorbent (Ca(OH)2>CaCO3), as well as the high specific surface area of the sorbent, were confirmed to be critical factors that improved the performance of SO2 removal. Notably, the results confirmed that both fine PM (≤ 1 μm) and heavy metals were simultaneously removed with increasing efficiency over the time of operation.

  13. Optimization of an immunoassay of 2,6-dichlorobenzamide (BAM) and development of regenerative surfaces by immunosorbent modification with newly synthesised BAM hapten library

    DEFF Research Database (Denmark)

    Uthuppu, Basil; Aamand, Jens; Jørgensen, Claus;

    2012-01-01

    Dichlobenil is an extensively used herbicide worldwide which is transformed to the mobile 2,6-dichlorobenzamide (BAM) in soil. BAM has been found in many European groundwater resources that are exploited for drinking water. Currently, immunoassay based monitoring technique (plate based ELISA......) is being employed to quantitatively detect BAM in water samples. In this work, as a starting step of developing immunoassay based on-site monitoring systems for pesticide analysis, the heterogeneous BAM immunoassay is optimised in terms of surface (polymer) regeneration. We have synthesised a small library...

  14. Development of an Heterologous Immunoassay for Ciprofloxacin Residue in Milk

    Science.gov (United States)

    Jinqing, Jiang; Haitang, Zhang; Zhixing, An; Zhiyong, Xu; Xuefeng, Yang; Huaguo, Huang; Ziliang, Wang

    A heterologous immunoassay has been developed for the determination of Ciprofloxacin (CPFX) residues in milk. For this reason, carbodiimide active ester method was employed to synthesize the artificial antigen of CPFX-BSA, and mixed anhydride reaction was used to prepare the coating antigen of CPFX-OVA to pursue the heterologous sensitivity. Based on the square matrix titration, an icELISA method was developed for the quantitative detection of CPFX in cattle milk. The dynamic range was from 0.036 to 92.5 ng/mL, with LOD and IC50 value of 0.019 ng/mL and 1.8 ng/mL, respectively. Except for a high cross-reactivity (89.7%) to Enrofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 0.03 mol/L of HCl, or 10% of methanol was used in the assay buffer. 20-fold dilution in cattle milk gave an inhibition curve almost the same as that in PBS buffer. The regression equation for this assay was y = 0.9036 x + 1.4574, with a correlation coefficient (R2) of 0.9844. The results suggest the veracity of the heterologous immunoassay for detecting CPFX residue in milk.

  15. Sensitive chemiluminescent immunoassay of triclopyr by digital image analysis.

    Science.gov (United States)

    Díaz, Aurora N; Sánchez, Francisco G; Baro, Enrique N; Díaz, Ana F G; Aguilar, Alfonso; Algarra, Manuel

    2012-08-15

    An image based detection of chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the quantification of triclopyr has been developed. The immunoassay was an indirect competitive immunoassay with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence was produced by the luminol/H(2)O(2)/HRP reaction, detected by a monochrome video CCD camera and digitized with an Imagraph IC-PCI frame grabber using a custom program developed in C(++) (Microsoft Visual C(++) 6.0). Two main improvements are reported in the data processing software: the implementation of a circular mesh covering the perimeter of each well, eliminating diffuse light from the neighboring wells, and the use of volume (the integration of light intensity of all pixels that define a well) as an analytical signal instead of CL intensity or area (as usual in commercial plate readers) to improve precision for normalization of the total light output. The standard curve was produced for 0.01-10 ng/L triclopyr. The limit of detection was 0.8 ng/L and the variation coefficient was 3.07% (n=10, P=0.05). PMID:22841045

  16. Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms.

    Science.gov (United States)

    Armbrecht, L; Dincer, C; Kling, A; Horak, J; Kieninger, J; Urban, G

    2015-11-21

    In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 μm spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2. PMID:26394820

  17. “Nanofiltration” Enabled by Super-Absorbent Polymer Beads for Concentrating Microorganisms in Water Samples

    Science.gov (United States)

    Xie, Xing; Bahnemann, Janina; Wang, Siwen; Yang, Yang; Hoffmann, Michael R.

    2016-02-01

    Detection and quantification of pathogens in water is critical for the protection of human health and for drinking water safety and security. When the pathogen concentrations are low, large sample volumes (several liters) are needed to achieve reliable quantitative results. However, most microbial identification methods utilize relatively small sample volumes. As a consequence, a concentration step is often required to detect pathogens in natural waters. Herein, we introduce a novel water sample concentration method based on superabsorbent polymer (SAP) beads. When SAP beads swell with water, small molecules can be sorbed within the beads, but larger particles are excluded and, thus, concentrated in the residual non-sorbed water. To illustrate this approach, millimeter-sized poly(acrylamide-co-itaconic acid) (P(AM-co-IA)) beads are synthesized and successfully applied to concentrate water samples containing two model microorganisms: Escherichia coli and bacteriophage MS2. Experimental results indicate that the size of the water channel within water swollen P(AM-co-IA) hydrogel beads is on the order of several nanometers. The millimeter size coupled with a negative surface charge of the beads are shown to be critical in order to achieve high levels of concentration. This new concentration procedure is very fast, effective, scalable, and low-cost with no need for complex instrumentation.

  18. Artocarpus heterophyllus L. seed starch-blended gellan gum mucoadhesive beads of metformin HCl.

    Science.gov (United States)

    Nayak, Amit Kumar; Pal, Dilipkumar; Santra, Kousik

    2014-04-01

    Jackfruit (Artocarpus heterophyllus Lam., family: Moraceae) seed starch (JFSS)-gellan gum (GG) mucoadhesive beads containing metformin HCl were developed through ionotropic gelation technique. The effect of GG to JFSS ratio and CaCl2 concentration on the drug encapsulation efficiency (DEE, %) and cumulative drug release at 10h (R10h, %) was optimized and analyzed using response surface methodology based on 3(2) factorial design. The optimized JFSS-GG beads containing metformin HCl showed DEE of 92.67±4.46%, R10h of 61.30±2.37%, and mean diameter of 1.67±0.27 mm. The optimized beads showed pH-dependent swelling and mucoadhesivity with the goat intestinal mucosa. The in vitro drug release from all these JFSS-GG beads containing metformin HCl was followed zero-order pattern (R(2)=0.9907-0.9975) with super case-II transport mechanism over a period of 10 h. The beads were also characterized by SEM and FTIR. The optimized JFSS-GG beads containing metformin HCl exhibited significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged period after oral administration. PMID:24447799

  19. "Nanofiltration" Enabled by Super-Absorbent Polymer Beads for Concentrating Microorganisms in Water Samples.

    Science.gov (United States)

    Xie, Xing; Bahnemann, Janina; Wang, Siwen; Yang, Yang; Hoffmann, Michael R

    2016-01-01

    Detection and quantification of pathogens in water is critical for the protection of human health and for drinking water safety and security. When the pathogen concentrations are low, large sample volumes (several liters) are needed to achieve reliable quantitative results. However, most microbial identification methods utilize relatively small sample volumes. As a consequence, a concentration step is often required to detect pathogens in natural waters. Herein, we introduce a novel water sample concentration method based on superabsorbent polymer (SAP) beads. When SAP beads swell with water, small molecules can be sorbed within the beads, but larger particles are excluded and, thus, concentrated in the residual non-sorbed water. To illustrate this approach, millimeter-sized poly(acrylamide-co-itaconic acid) (P(AM-co-IA)) beads are synthesized and successfully applied to concentrate water samples containing two model microorganisms: Escherichia coli and bacteriophage MS2. Experimental results indicate that the size of the water channel within water swollen P(AM-co-IA) hydrogel beads is on the order of several nanometers. The millimeter size coupled with a negative surface charge of the beads are shown to be critical in order to achieve high levels of concentration. This new concentration procedure is very fast, effective, scalable, and low-cost with no need for complex instrumentation. PMID:26876979

  20. Artocarpus heterophyllus L. seed starch-blended gellan gum mucoadhesive beads of metformin HCl.

    Science.gov (United States)

    Nayak, Amit Kumar; Pal, Dilipkumar; Santra, Kousik

    2014-04-01

    Jackfruit (Artocarpus heterophyllus Lam., family: Moraceae) seed starch (JFSS)-gellan gum (GG) mucoadhesive beads containing metformin HCl were developed through ionotropic gelation technique. The effect of GG to JFSS ratio and CaCl2 concentration on the drug encapsulation efficiency (DEE, %) and cumulative drug release at 10h (R10h, %) was optimized and analyzed using response surface methodology based on 3(2) factorial design. The optimized JFSS-GG beads containing metformin HCl showed DEE of 92.67±4.46%, R10h of 61.30±2.37%, and mean diameter of 1.67±0.27 mm. The optimized beads showed pH-dependent swelling and mucoadhesivity with the goat intestinal mucosa. The in vitro drug release from all these JFSS-GG beads containing metformin HCl was followed zero-order pattern (R(2)=0.9907-0.9975) with super case-II transport mechanism over a period of 10 h. The beads were also characterized by SEM and FTIR. The optimized JFSS-GG beads containing metformin HCl exhibited significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged period after oral administration.

  1. Fast Diagnosis of Gonorrhea Witth Enhanced Luminescence Enzyme Immunoassay

    Institute of Scientific and Technical Information of China (English)

    ZHENG Heyi(郑和义); CAO Jingjiang(曹经江); SHAO Yanglin(邵燕玲)

    2002-01-01

    Objective:To evaluate the value of enhanced luminescence enzyme immunoassay in the diagnosis of Neisseria gonorrhea(NG) infection.Methods: Anti-catalase antibody for Neisseria gonorrheae combined with enhanced luminescence enzyme immunoassay were used to test for N. Gonorrhea.Results: A minimum of 1x104/CFU of GC in genital tract secretions or urine could be detected with the technique of luminescence enzyme immunoassay.Conclusion : The enhanced luninescence enzyme immunoassay has the advantage of high sensitivity and specificity for diagnosing NG from genitourinary tract secretion and urine.

  2. An enzymatic immunoassay microfluidics integrated with membrane valves for microsphere retention and reagent mixing.

    Science.gov (United States)

    Ren, Li; Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Liu, Rui; Wang, Xueqin; Xu, Juan; Wang, Yaolei; Zhang, Yanrong; Li, Li; Wang, Jinyi

    2012-05-15

    The present study presents a new microfluidic device integrated with pneumatic microvalves and a membrane mixer for enzyme-based immunoassay of acute myocardial infarction (AMI) biomarkers, namely, myoglobin, and heart-type fatty acid binding protein (H-FABP). Superparamagnetic microspheres with carboxyl groups on their surfaces were used as antibody solid carriers. A membrane mixer consisting of four ψ-type membrane valves was assembled under the reaction chamber for on-chip performing microsphere trapping and reagent mixing. The entire immunoassay process, including microsphere capture, reagent input, mixing, and subsequent reaction, was accomplished on the device either automatically or manually. The post-reaction substrate resultant was analyzed using a microplate reader. The results show that the average absorbance value is correlated with the concentration of cardiac markers, in agreement with the results obtained using a conventional microsphere-based immunoassay; this indicated that the proposed on-chip immunoassay protocol could be used to detect both myoglobin and H-FABP. The minimum detectable concentration is 5 ng/mL for myoglobin and 1 ng/mL for H-FABP.

  3. Black holes as beads on cosmic strings

    OpenAIRE

    Ashoorioon, Amjad; B. Mann, Robert

    2014-01-01

    We consider the possibility of formation of cosmic strings with black holes as beads. We focus on the simplest setup where two black holes are formed on a long cosmic string. It turns out the in absence of a background magnetic field and for observationally viable values for cosmic string tensions, $\\mu

  4. Universal approach for selective trace metal determinations via sequential injection-bead injection-lab-on-valve using renewable hydrophobic bead surfaces as reagent carriers

    DEFF Research Database (Denmark)

    Long, Xiangbao; Miró, Manuel; Hansen, Elo Harald

    2005-01-01

    A new concept is presented for selective and sensitive determination of trace metals via electrothermal atomic absorption spectrometry (ETAAS) based on the principle of bead injection (BI) with renewable reversed-phase surfaces in a sequential injection-lab-on-valve (SI-LOV) mode. The methodology...... involves the use of poly(styrene-divinylbenzene) beads containing pendant octadecyl moieties (C18-PS/DVB), which are preimpregnated with a selective organic metal chelating agent prior to the automatic manipulation of the beads in the microbore conduits of the LOV unit. By adapting this approach, the...... immobilization of the most suitable chelating agent can be effected irrespective of the kinetics involved, optimal reaction conditions can be used for implementing the chelating reaction of the target metal analyte with the immobilized reagent, and an added degree of freedom is offered in selecting the most...

  5. A Controlled Drug-Delivery Experiment Using Alginate Beads

    Science.gov (United States)

    Farrell, Stephanie; Vernengo, Jennifer

    2012-01-01

    This paper describes a simple, cost-effective experiment which introduces students to drug delivery and modeling using alginate beads. Students produce calcium alginate beads loaded with drug and measure the rate of release from the beads for systems having different stir rates, geometries, extents of cross-linking, and drug molecular weight.…

  6. Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis.

    Science.gov (United States)

    DuVall, Jacquelyn A; Cabaniss, Scott T; Angotti, Morgan L; Moore, John H; Abhyankar, Mayuresh; Shukla, Nishant; Mills, Daniel L; Kessel, Bryan G; Garner, Gavin T; Swami, Nathan S; Landers, James P

    2016-10-01

    Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

  7. Highly sensitive detection of clenbuterol using competitive surface-enhanced Raman scattering immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Zhu Guichi [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Hu Yongjun, E-mail: yjhu@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Gao Jiao; Zhong Liang [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2011-07-04

    Graphical abstract: Schemes of SERS nanoprobes preparation (a) and competitive SERS immunoassay for clenbuterol (b). Highlights: > A new method for clenbuterol detection by the use of a competitive SERS immunoassay has been developed. > 4,4'-Dipyridyl is chosen as the Raman reporter due to its fast-labeled, nontoxic and bifunctional properties. > The present method could detect clenbuterol over a wide dynamic concentration range and exhibit significant specificity in real samples. > The technique is more sensitive and simpler than the conventional method ELISA. - Abstract: In this report, we present a novel approach to detect clenbuterol based on competitive surface-enhanced Raman scattering (SERS) immunoassay. Herein, a SERS nanoprobe that relies on gold nanoparticle (GNP) is labeled by 4,4'-dipyridyl (DP) and clenbuterol antibody, respectively. The detection of clenbuterol is carried out by competitive binding between free clenbuterol and clenbuterol-BSA fastened on the substrate with their antibody labeled on SERS nanoprobes. The present method allows us to detect clenbuterol over a much wider concentration range (0.1-100 pg mL{sup -1}) with a lower limit of detection (ca. 0.1 pg mL{sup -1}) than the conventional methods. Furthermore, by the use of this new competitive SERS immunoassay, the clenbuterol-BSA (antigen) is chosen to fasten on the substrate instead of the clenbuterol antibody, which could reduce the cost of the assay. Results demonstrate that the proposed method has the wide potential applications in food safety and agonist control.

  8. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays. Applications, fundamentals, and optimization

    Energy Technology Data Exchange (ETDEWEB)

    Driskell, Jeremy Daniel [Iowa State Univ., Ames, IA (United States)

    2006-08-09

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  9. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays: applications, fundamentals, and optimization

    International Nuclear Information System (INIS)

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  10. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays: applications, fundamentals, and optimization

    Energy Technology Data Exchange (ETDEWEB)

    Jeremy Daniel Driskell

    2006-08-09

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  11. Microfluidic Sample Preparation for Immunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Visuri, S; Benett, W; Bettencourt, K; Chang, J; Fisher, K; Hamilton, J; Krulevitch, P; Park, C; Stockton, C; Tarte, L; Wang, A; Wilson, T

    2001-08-09

    Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. to support detection instruments, they are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. They are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Many of these fluidic functions are accomplished with acoustic radiation pressure or dielectrophoresis. They are integrating these technologies into packaged systems with pumps and valves to control fluid flow through the fluidic circuit.

  12. Gliadin Detection in Food by Immunoassay

    Science.gov (United States)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  13. “BACs-on-Beads:5所产前诊断实验室的前瞻性研究”点评

    Institute of Scientific and Technical Information of China (English)

    刘鹏飞; 王谢桐

    2012-01-01

    <正>1 原文摘要 Objective We previously reported on the validation of Prenatal BACs-on-Beads TM on retrospectively selected and prospective prenatal samples.This bead-based multiplex assay detects chromosome 13 , 18 , 21and X / Y aneuploidies and the nine most frequent microdeletion syndromes. We demonstrated that Prenatal BACs-on-Beads TM is a newgeneration , prenatal screening tool.Here , we describe the experience of five European prenatal diagnosis laboratories concerning the ongoing use of Prenatal BACs- on-Beads TM . Methods Some 1653samples were analyzed.

  14. Elemental analysis of bead samples using a laser-induced plasma at low pressure

    International Nuclear Information System (INIS)

    An Nd:YAG laser (1064 nm, 8 ns, 30 mJ) was focused on various types of fresh, fossilized white coral and giant shell samples, including samples of imitation shell and marble. Such samples are extremely important as material for preparing prayer beads that are extensively used in the Buddhist faith. The aim of this research was to develop a non-destructive method to distinguish original beads from their imitations by means of spectral measurements of the carbon, hydrogen, sodium and magnesium emission intensities and by measuring the hardness of the sample using the ratio between Ca (II) 396.8 nm and Ca (I) 422.6 nm. Based on these measurements, original fresh coral beads can be distinguished from any imitation made from hard wood. The same technique was also effective in distinguishing beads made of shell from its imitation. A spectral analysis of bead was also performed on a fossilized white coral sample and the result can be used to distinguish to some extent the fossilized white coral beads from any imitation made from marble. It was also found that the plasma plume should be generated at low ambient pressure to significantly improve the hydrogen and carbon emission intensity and also to avoid energy loss inside the crater during laser irradiation at atmospheric pressure. The results of this study confirm that operating the laser-induced plasma spectroscopy at reduced ambient pressure offers distinct advantage for bead analysis over the conventional laser-induced breakdown spectroscopy (LIBS) technique operated at atmospheric pressure

  15. Recovery of uranium from phosphoric acid medium by polymeric composite beads encapsulating organophosphorus extractants

    Energy Technology Data Exchange (ETDEWEB)

    Singh, D.K.; Yadav, K.K.; Varshney, L.; Singh, H. [Bhabha Atomic Research Centre, Mumbai 400 085 (India)

    2013-07-01

    The present study deals with the preparation and evaluation of the poly-ethersulfone (PES) based composite beads encapsulating synergistic mixture of D2EHPA and Cyanex 923 (at 4:1 mole ratio) for the separation of uranium from phosphoric acid medium. SEM was used for the characterization of the composite materials. Addition of 1% PVA (polyvinyl alcohol) improved the internal morphology and porosity of the beads. Additionally, microscopic examination of the composite bead confirmed central coconut type cavity surrounded by porous polymer layer of the beads through which exchange of metal ions take place. Effect of various experimental variables including aqueous acidity, metal ion concentration in aqueous feed, concentration of organic extractant inside the beads, extractant to polymer ratio, liquid to solid (L/S) ratio and temperature on the extraction of uranium was studied. Increase in acidity (1-6 M), L/S ratio (1- 10), metal ion concentration (0.2-3 g/L U{sub 3}O{sub 8}) and polymer to extractant ratio (1:4 -1:10) led to decrease in extraction of uranium. At 5.5 M (comparable to wet process phosphoric acid concentration) the extraction of uranium was about 85% at L/S ratio 5. Increase in extractant concentration inside the bead resulted in enhanced extraction of metal ion. Increase in temperature in the range of 30 to 50 Celsius degrees increased the extraction, whereas further increase to 70 C degrees led to the decrease in extraction of uranium. Amongst various reagents tested, stripping of uranium was quantitative by 12% Na{sub 2}CO{sub 3} solution. Polymeric beads were found to be stable and reusable up-to 10 cycles of extraction/stripping. (authors)

  16. Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination.

    Science.gov (United States)

    Alonso, María Cruz; Trapiella-Alfonso, Laura; Fernández, José M Costa; Pereiro, Rosario; Sanz-Medel, Alfredo

    2016-03-15

    A quantitative immunoassay for the determination of immunoglobulin E (IgE) in human serum using gold nanoclusters (AuNCs) as fluorescent label was developed. Water soluble AuNCs were synthesized using lipoic acid and then thoroughly characterized. The obtained AuNCs have a particle size of 2.7 ± 0.1 nm and maximum fluorescence emission at 710 nm. The synthesized AuNCs showed very good stability of the fluorescent signal with light exposure and at neutral and slightly basic media. A covalent bioconjugation of these AuNCs with the desired antibody was carried out by the carbodiimide reaction. After due optimization of such bioconjugation reaction, a molar ratio 1:3 (antibody:AuNCs) was selected. The bioconjugate maintained an intense luminescence emission, slightly red-shifted as compared to the free AuNCs. Two typical immunoassay configurations, competitive and sandwich, were assayed and their performance for IgE determination critically compared. After the different immunoassay steps were accomplished, the fluorescence emission of the bioconjugate was measured. While the sandwich format provided a detection limit (DL) of 10 ng/mL and a linear range between 25 and 565 ng/mL of IgE, the competitive format revealed a DL of 0.2 ng/mL with a linear range between 0.3 and 7.1 ng/mL The applicability of the more sensitive competitive fluorescent immunoassay was assessed by successful analysis of the IgE in human serum and comparison of results with those from a commercial kit. The main advantages of the proposed AuNCs-based fluorimetric method include a low DL and a simple immunoassay protocol involving few reagents.

  17. Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables.

    Science.gov (United States)

    Moreno, M J; Abad, A; Pelegrí, R; Marínez, M J; Sáez, A; Gamón, M; Montoya, A

    2001-04-01

    The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput. PMID:11308315

  18. Fungal cultivation on glass-beads

    DEFF Research Database (Denmark)

    Droce, Aida; Sørensen, Jens Laurids; Giese, Henriette;

    Transcription of various bioactive compounds and enzymes are dependent on fungal cultivation method. In this study we cultivate Fusarium graminearum and Fusarium solani on glass-beads with liquid media in petri dishes as an easy and inexpensive cultivation method, that resembles in secondary meta...... metabolite production to agar-cultivation but with an easier and more pure RNA-extraction of total fungal mycelia....

  19. RF Bead Pull Measurements of the DQW

    CERN Document Server

    Jaume, Guillaume

    2015-01-01

    This report was written within the framework of the CERN Summer Student Program. It is focused on the Radio Frequency study of the Double Quarter Wave Crab Cavity [1] considered for the crab-crossing scheme of the LHC Luminosity upgrade [2]. HFSS simulation [3] and Bead-Pull Measurements technique were used for the characterization of the higher-order terms of the main deflecting mode.

  20. Two dimensional barcode-inspired automatic analysis for arrayed microfluidic immunoassays

    OpenAIRE

    Zhang, Yi; Qiao, Lingbo; Ren, Yunke; Wang, Xuwei; Gao, Ming; Tang, Yunfang; Jeff Xi, Jianzhong; Fu, Tzung-May; Jiang, Xingyu

    2013-01-01

    The usability of many high-throughput lab-on-a-chip devices in point-of-care applications is currently limited by the manual data acquisition and analysis process, which are labor intensive and time consuming. Based on our original design in the biochemical reactions, we proposed here a universal approach to perform automatic, fast, and robust analysis for high-throughput array-based microfluidic immunoassays. Inspired by two-dimensional (2D) barcodes, we incorporated asymmetric function patt...

  1. Effect of methacrylic acid beads on the sonic hedgehog signaling pathway and macrophage polarization in a subcutaneous injection mouse model.

    Science.gov (United States)

    Lisovsky, Alexandra; Zhang, David K Y; Sefton, Michael V

    2016-08-01

    Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads promote a vascular regenerative response when used in diabetic wound healing. Previous studies reported that MAA beads modulated the expression of sonic hedgehog (Shh) and inflammation related genes in diabetic wounds. The aim of this work was to follow up on these observations in a subcutaneous injection model to study the host response in the absence of the confounding factors of diabetic wound healing. In this model, MAA beads improved vascularization in healthy mice of both sexes compared to control poly(methyl methacrylate) (MM) beads, with a stronger effect seen in males than females. MAA-induced vessels were perfusable, as evidenced from the CLARITY-processed images. In Shh-Cre-eGFP/Ptch1-LacZ non-diabetic transgenic mice, the increased vessel formation was accompanied by a higher density of cells expressing GFP (Shh) and β-Gal (patched 1, Ptch1) suggesting MAA enhanced the activation of the Shh pathway. Ptch1 is the Shh receptor and a target of the pathway. MAA beads also modulated the inflammatory cell infiltrate in CD1 mice: more neutrophils and more macrophages were noted with MAA relative to MM beads at days 1 and 7, respectively. In addition, MAA beads biased macrophages towards a MHCII-CD206+ ("M2") polarization state. This study suggests that the Shh pathway and an altered inflammatory response are two elements of the complex mechanism whereby MAA-based biomaterials effect vascular regeneration. PMID:27264502

  2. Ultrasensitive photoelectrochemical immunoassay for CA19-9 detection based on CdSe@ZnS quantum dots sensitized TiO2NWs/Au hybrid structure amplified by quenching effect of Ab2@V(2+) conjugates.

    Science.gov (United States)

    Zhu, Hua; Fan, Gao-Chao; Abdel-Halim, E S; Zhang, Jian-Rong; Zhu, Jun-Jie

    2016-03-15

    A novel, enhanced photoelectrochemical immunoassay was established for sensitive and specific detection of carbohydrate antigen 19-9 (CA19-9, Ag). In this protocol, TiO2 nanowires (TiO2NWs) were first decorated with Au nanoparticles to form TiO2NWs/Au hybrid structure, and then coated with CdSe@ZnS quantum dots (QDs) via the layer-by-layer method, producing TiO2NWs/Au/CdSe@ZnS sensitized structure, which was employed as the photoelectrochemical matrix to immobilize capture CA19-9 antibodies (Ab1); whereas, bipyridinium (V(2+)) molecules were labeled on signal CA19-9 antibodies (Ab2) to form Ab2@V(2+) conjugates, which were used as signal amplification elements. The TiO2NWs/Au/CdSe@ZnS sensitized structure could adequately absorb light energy and dramatically depress electron-hole recombination, resulting in evidently enhanced photocurrent intensity of the immunosensing electrode. While target Ag were detected, the Ab2@V(2+) conjugates could significantly decrease the photocurrent detection signal because of strong electron-withdrawing property of V(2+) coupled with evident steric hindrance of Ab2. Thanks to synergy effect of TiO2NWs/Au/CdSe@ZnS sensitized structure and quenching effect of Ab2@V(2+) conjugates, the well-established photoelectrochemical immunoassay exhibited a low detection limit of 0.0039 U/mL with a wide linear range from 0.01 U/mL to 200 U/mL for target Ag detection. This proposed photoelectrochemical protocol also showed good reproducibility, specificity and stability, and might be applied to detect other important biomarkers.

  3. Two dimensional barcode-inspired automatic analysis for arrayed microfluidic immunoassays

    Science.gov (United States)

    Zhang, Yi; Qiao, Lingbo; Ren, Yunke; Wang, Xuwei; Gao, Ming; Tang, Yunfang; Jeff Xi, Jianzhong; Fu, Tzung-May; Jiang, Xingyu

    2013-01-01

    The usability of many high-throughput lab-on-a-chip devices in point-of-care applications is currently limited by the manual data acquisition and analysis process, which are labor intensive and time consuming. Based on our original design in the biochemical reactions, we proposed here a universal approach to perform automatic, fast, and robust analysis for high-throughput array-based microfluidic immunoassays. Inspired by two-dimensional (2D) barcodes, we incorporated asymmetric function patterns into a microfluidic array. These function patterns provide quantitative information on the characteristic dimensions of the microfluidic array, as well as mark its orientation and origin of coordinates. We used a computer program to perform automatic analysis for a high-throughput antigen/antibody interaction experiment in 10 s, which was more than 500 times faster than conventional manual processing. Our method is broadly applicable to many other microchannel-based immunoassays. PMID:24404030

  4. Magnetic bead micromixer: Influence of magnetic element geometry and field amplitude

    DEFF Research Database (Denmark)

    Lund-Olesen, Torsten; Buus, Bjarke B.; Howalt, Jakob;

    2008-01-01

    A scheme for the silicon microfabrication of lab-on-a-chip systems with mixing based on dynamic plugs of magnetic beads is presented. The systems consist of a microfluidic channel integrated with a number of soft magnetic elements by the sides of the channel. The elements are magnetized by a...... homogeneous external ac magnetic field. The systems are scalable with respect to the number of magnetic bead plugs and number of parallel channels, and thus they have high potential for use in biological separation using functionalized magnetic beads. The mixing efficiency is characterized for two different...... geometries of the soft magnetic structures and found to be highly sensitive to the geometry and position of the structures....

  5. A multiplexed immunoassay for detection of antibodies to Actinobacillus pleuropneumoniae (App) in pigs

    DEFF Research Database (Denmark)

    Berger, Sanne Schou; Boas, Ulrik; Andresen, Lars Ole;

    2014-01-01

    The bacterium Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumoniae, a contagious and severe respiratory disease in pigs. Based on capsular antigens, 15 App serovars have been described, and the prevalence and morbidity of these serovars vary with geographic...... regions (1). In Denmark, the most important serovars are considered to be App 1, 2, 5, 6, 7, 10 and 12. As part of the Danish surveillance program for App, the Danish Veterinary Institute uses ELISAs and complement fixation tests (CFT) to test for porcine anti-App antibodies (2-7). In an effort to improve...... our diagnostic tools, we are currently developing a novel indirect fluorescent microsphere immunoassay that can facilitate simultaneous detection of antibodies towards multiple App serovars within a single serum sample volume. The multiplex immunoassay is based on Luminex technology (8) and has...

  6. Immunoassay of chemical contaminants in milk:A review

    Institute of Scientific and Technical Information of China (English)

    XU Fei; REN Kang; YANG Yu-ze; GUO Jiang-peng; MA Guang-peng; LIU Yi-ming; LU Yong-qiang; LI Xiu-bo

    2015-01-01

    The detection of chemical contaminants is critical to ensure dairy safety. These contaminants include veterinary medicines, antibiotics, pesticides, heavy metals, mycotoxins, and persistent organic polutants (POPs). Immunoassays have recently been used to detect contaminants in milk because of their simple operation, high speed, and low cost. This article describes the latest developments in the most important component of immunoassays—antibodies, and then reviews the four major substrates used for immunoassays (i.e., microplates, membranes, gels, and chips) as wel as their use in the detection of milk contaminants. The paper concludes with prospects for further applications of these immunoassays.

  7. Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine.

    Science.gov (United States)

    Ma, Mingfang; Wen, Kai; Beier, Ross C; Eremin, Sergei A; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

    2016-07-20

    We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.

  8. Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine.

    Science.gov (United States)

    Ma, Mingfang; Wen, Kai; Beier, Ross C; Eremin, Sergei A; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

    2016-07-20

    We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants. PMID:27362827

  9. Simulation of Enzyme Catalysis in Calcium Alginate Beads

    Directory of Open Access Journals (Sweden)

    Ameel M. R. Al-Mayah

    2012-01-01

    Full Text Available A general mathematical model for a fixed bed immobilized enzyme reactor was developed to simulate the process of diffusion and reaction inside the biocatalyst particle. The modeling and simulation of starch hydrolysis using immobilized α-amylase were used as a model for this study. Corn starch hydrolysis was carried out at a constant pH of 5.5 and temperature of . The substrate flow rate was ranging from 0.2 to 5.0 mL/min, substrate initial concentrations 1 to 100 g/L. α-amylase was immobilized on to calcium alginate hydrogel beads of 2 mm average diameter. In this work Michaelis-Menten kinetics have been considered. The effect of substrate flow rate (i.e., residence time and initial concentration on intraparticle diffusion have been taken into consideration. The performance of the system is found to be affected by the substrate flow rate and initial concentrations. The reaction is controlled by the reaction rate. The model equation was a nonlinear second order differential equation simulated based on the experimental data for steady state condition. The simulation was achieved numerically using FINITE ELEMENTS in MATLAB software package. The simulated results give satisfactory results for substrate and product concentration profiles within the biocatalyst bead.

  10. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  11. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    黄承志; 李原芳; 黄新华; 范美坤

    2000-01-01

    The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  12. HPMA and HEMA copolymer bead interactions with eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Cristina D. Vianna-Soares

    2004-09-01

    Full Text Available Two different hydrophilic acrylate beads were prepared via aqueous suspension polymerization. Beads produced of a hydroxypropyl methacrylate (HPMA and ethyleneglycol methacrylate (EDMA copolymer were obtained using a polyvinyl alcohol suspending medium. Copolymers of 2hydroxyethyl methacrylate (HEMA, methyl methacrylate (MMA and ethyleneglycol methacrylate (EDMA beads were obtained using magnesium hydroxide as the suspending agent. Following characterization by scanning electron microscopy (SEM, nitrogen sorption analysis (NSA and mercury intrusion porosimetry (MIP, the beads were cultured with monkey fibroblasts (COS7 to evaluate their ability to support cell growth, attachment and adhesion. Cell growth behavior onto small HPMA/EDMA copolymer beads and large HEMA/MMA/EDMA copolymer beads is evaluated regarding their hidrophilicity/hidrophobicity and surface roughness.

  13. A highly-sensitive multisubstrate-compatible chemiluminescent immunoassay for human fetuin A

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Sandeep Kumar Vashist ### Abstract We report a highly-sensitive chemiluminescent immunoassay (CIA) for the detection of human fetuin A (HFA), which is based on the leach-proof covalent crosslinking of anti-HFA capture antibodies on 3-aminopropyltriethoxysilane (APTES)-functionalized 96-well chemiluminescent microtiter plates (CMTP) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide. It has more than 3-fold reduced overall assay du...

  14. Evaluation of a Treponema pallidum enzyme immunoassay as a screening test for syphilis.

    OpenAIRE

    Hooper, N E; Malloy, D C; Passen, S

    1994-01-01

    The CAPTIA Syphilis-G enzyme immunoassay for the detection of antibodies to Treponema pallidum was evaluated as a screening test for syphilis in comparison with the standard rapid plasma reagin (RPR) test. One thousand samples were tested, and the standard fluorescent treponemal antibody absorption test and the standard microhemmaglutination test were used to confirm the presence of treponemal antibodies. Diagnosis of syphilis was based on traditional standard serology results. Clinical data ...

  15. Detection of ''beading faults'' in welded tubes

    International Nuclear Information System (INIS)

    In the steel tube industry the word ''beading'' refers to a highly localised leak affecting the welded zone. During the pneumatic test its flow rate is generally very low no more than a few thousandths of a mm3/second. Detection of such a fault by this test is consequently slow, and those which are choked or at the limit of leakage may escape detection. For greater safety, the tube technician is now using non-destructive testing methods such as eddy-currents and ultrasonics

  16. Controlling the magnetic field distribution on the micrometer scale and generation of magnetic bead patterns for microfluidic applications.

    Science.gov (United States)

    Yu, Xu; Feng, Xuan; Hu, Jun; Zhang, Zhi-Ling; Pang, Dai-Wen

    2011-04-19

    As is well known, controlling the local magnetic field distribution on the micrometer scale in a microfluidic chip is significant and has many applications in bioanalysis based on magnetic beads. However, it is a challenge to tailor the magnetic field introduced by external permanent magnets or electromagnets on the micrometer scale. Here, we demonstrated a simple approach to controlling the local magnetic field distribution on the micrometer scale in a microfluidic chip by nickel patterns encapsulated in a thin poly(dimethylsiloxane) (PDMS) film under the fluid channel. With the precisely controlled magnetic field, magnetic bead patterns were convenient to generate. Moreover, two kinds of fluorescent magnetic beads were patterned in the microfluidic channel, which demonstrated that it was possible to generate different functional magnetic bead patterns in situ, and could be used for the detection of multiple targets. In addition, this method was applied to generate cancer cell patterns.

  17. Development of temper bead welding by under water laser welding

    International Nuclear Information System (INIS)

    Toshiba has developed temper bead welding by under water laser welding as SCC counter measure for aged components in PWR and BWR nuclear power plants. Temper bead welding by under water laser welding technique recovers toughness of low alloy steel reactor vessel by employing proper the number of cladding layers and their welding conditions. In this report, some evaluation results of material characteristics of temper bead welded low alloy steel are presented. (author)

  18. A Novel Colorimetric Immunoassay Utilizing the Peroxidase Mimicking Activity of Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Hyun Gyu Park

    2013-05-01

    Full Text Available A simple colorimetric immunoassay system, based on the peroxidase mimicking activity of Fe3O4 magnetic nanoparticles (MNPs, has been developed to detect clinically important antigenic molecules. MNPs with ca. 10 nm in diameter were synthesized and conjugated with specific antibodies against target molecules, such as rotaviruses and breast cancer cells. Conjugation of the MNPs with antibodies (MNP-Abs enabled specific recognition of the corresponding target antigenic molecules through the generation of color signals arising from the colorimetric reaction between the selected peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB and H2O2. Based on the MNP-promoted colorimetric reaction, the target molecules were detected and quantified by measuring absorbance intensities corresponding to the oxidized form of TMB. Owing to the higher stabilities and economic feasibilities of MNPs as compared to horseradish peroxidase (HRP, the new colorimetric system employing MNP-Abs has the potential of serving as a potent immunoassay that should substitute for conventional HRP-based immunoassays. The strategy employed to develop the new methodology has the potential of being extended to the construction of simple diagnostic systems for a variety of biomolecules related to human cancers and infectious diseases, particularly in the realm of point-of-care applications.

  19. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    Science.gov (United States)

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  20. Analog modeling of Worm-Like Chain molecules using macroscopic beads-on-a-string.

    Science.gov (United States)

    Tricard, Simon; Feinstein, Efraim; Shepherd, Robert F; Reches, Meital; Snyder, Phillip W; Bandarage, Dileni C; Prentiss, Mara; Whitesides, George M

    2012-07-01

    This paper describes an empirical model of polymer dynamics, based on the agitation of millimeter-sized polymeric beads. Although the interactions between the particles in the macroscopic model and those between the monomers of molecular-scale polymers are fundamentally different, both systems follow the Worm-Like Chain theory.

  1. Ormosil Beads for Insulation of Ground Cryogenic Storage Tanks Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Organically modified silica (Ormosil) aerogel beads developed at Aspen Aerogels, Inc. offer several advantages for retrofitting perlite insulation in NASA's ground...

  2. Bead magnetorelaxometry with an on-chip magnetoresistive sensor

    DEFF Research Database (Denmark)

    Dalslet, Bjarke Thomas; Damsgaard, Christian Danvad; Donolato, Marco;

    2011-01-01

    Magnetorelaxometry measurements on suspensions of magnetic beads are demonstrated using a planar Hall effect sensor chip embedded in a microfluidic system. The alternating magnetic field used for magnetizing the beads is provided by the sensor bias current and the complex magnetic susceptibility...... spectra are recorded as the 2nd harmonic of the sensor response. The complex magnetic susceptibility signal appears when a magnetic bead suspension is injected, it scales with the bead concentration, and it follows the Cole-Cole expression for Brownian relaxation. The complex magnetic susceptibility...

  3. Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria and Dengue Fever.

    Science.gov (United States)

    Deraney, Rachel N; Mace, Charles R; Rolland, Jason P; Schonhorn, Jeremy E

    2016-06-21

    Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas coendemic for malaria and dengue fever, this assay could be used as a rapid, point-of-care diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care. PMID:27186893

  4. Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

    International Nuclear Information System (INIS)

    Highlights: ► We developed a rapid method based on a multiplex dipstick immunoassay. ► The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. ► We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg−1, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg−1, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

  5. Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

    Energy Technology Data Exchange (ETDEWEB)

    Lattanzio, Veronica M.T., E-mail: veronica.lattanzio@ispa.cnr.it [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Nivarlet, Noan [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Lippolis, Vincenzo; Gatta, Stefania Della [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Huet, Anne-Catherine; Delahaut, Philippe [Centre d' Economie Rurale (CER Groupe), Rue du Point du Jour no 8, B-6900 Marloie (Belgium); Granier, Benoit [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Visconti, Angelo [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We developed a rapid method based on a multiplex dipstick immunoassay. Black-Right-Pointing-Pointer The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. Black-Right-Pointing-Pointer We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins

  6. Characterization of some tin-contained ancient glass beads found in China by means of SEM-EDS and raman spectroscopy.

    Science.gov (United States)

    Li, Qinghui; Liu, Song; Su, Bomin; Zhao, Hongxia; Fu, Qiang; Dong, Junqing

    2013-02-01

    A total of nine tin-contained ancient glass beads were characterized by a combination of scanning electron microscopy coupled with energy-dispersive X-ray spectrometry and Raman spectroscopy. These glass beads dated from 1st century BC to 10th century AD were excavated from the Xinjiang and Guangxi provinces of China. Two kinds of tin-based opacifiers/colorants included crystalline cassiterite (SnO(2)) and lead-tin yellow types II were first found in these soda lime glass beads. The tentative chronology of the tin-based opacifiers/colorants used in ancient glasses from China and the West was compared. In addition, several transition metal ions colorants were also found in these beads. The detailed study of the glassy matrices, crystalline inclusions, and the microstructural heterogeneities for these glass beads has revealed some valuable information to trace the possible making technology and provenances.

  7. Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

    International Nuclear Information System (INIS)

    Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expression levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research. -- Highlights: •We established a 3D metastasis model mimicking the metastatic ability in vivo. •The invasion ability of cells derived from our model was increased significantly. •The model is easy to reproduce, convenient to handle, and amenable for large-scale

  8. Study on the uptake of Americium using PC88A - impregnated macroporous polymeric beads

    International Nuclear Information System (INIS)

    The prime objective of radioactive waste treatment in nuclear industry is to minimize the waste volume by efficient process without generating secondary radioactive waste for its final disposal. Among the currently available technologies, for separation and recovery of metal ions, solvent extraction, ion-exchange, membrane based technologies and solid sorbent materials are more popular means. Though, all these technologies play major role in all the bulk separation processes, their marked limitations force the separation scientists to think of advance, more efficient and technically feasible alternatives. The extractant impregnated polymeric beads (EIMPBs), impregnated with metal-specific extractants, exhibit reliable separation performances under column operation, and hence bridge the gap between solvent extraction and ion exchange techniques. In the present work, PC88A impregnated PES beads are prepared and the sorption of Am (III) from aqueous waste solutions is investigated. The synthesized EIMPBs were characterized by FTIR, TGA and SEM techniques. The physiochemical strength of the beads was found to be excellent. The sorption study of Am (III), using these beads, was carried out by batch equilibration method and the effect of various parameters, like pH, equilibration time, Am (III) concentration, etc., on the sorption process, was investigated. The synthesized polymeric beads presented fairly higher sorption capacity for Am (III) at pH 3. The kinetics of extraction is very fast. The saturation of sorption is achieved in about 60 minutes of equilibration. The sorption kinetics data fits well in the pseudo second-order model, indicating that the sorption is dominated by chemisorptions. The sorption of Am (III) is observed to follow Langmuir isotherm and the monolayer capacity was calculated as 2.498 mg/g. The quantitative stripping of the extracted Am (III) can be achieved by using 0.1M oxalic acid. The blank polymeric beads, without PC88A,have shown

  9. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    Science.gov (United States)

    Shen, Jun; Zhou, Yaofeng; Fu, Fen; Xu, Hengyi; Lv, Jiaofeng; Xiong, Yonghua; Wang, Andrew

    2015-09-01

    Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X-0.0059 (R(2)=0.9983) for low HBsAg concentrations between 75 pg mL(-1) and 4.8 ng mL(-1), and Y=0.8404 X-2.9364 (R(2)=0.9939) for high HBsAg concentrations in the range from 4.8 ng mL(-1) to 75 ng mL(-1). The detection limit of the proposed ICA sensor achieved was 75 pg mL(-1), which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL(-1), 3.75 ng mL(-1) and 18.75 ng mL(-1) ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R(2)=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers. PMID:26003704

  10. Immunoassay utilizing biochemistry reaction product via surface-enhanced Raman scattering in near field

    Institute of Scientific and Technical Information of China (English)

    ZHAO Haiying; NI Yi; JIANG Wei; LUO Peiqing; HUANG Meizheng; YIN Guangzhong; DOU Xiaoming

    2005-01-01

    We propose here a kind of applications of surface-enhanced Raman scattering (SERS) to immunology. It is a new enzyme immunoassay based on SERS. In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogenperoxide, azoaniline is generated. This azo compound is adsorbed on a silver colloid and only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product. The detection limit of this SERS enzyme immunoassay method was found to be about 10-15 mol/L.

  11. Advances in ovarian cancer diagnosis: A journey from immunoassays to immunosensors.

    Science.gov (United States)

    Sharma, Shikha; Raghav, Ragini; O'Kennedy, Richard; Srivastava, Sudha

    2016-07-01

    This review focuses on the technological advancements, challenges and trends in immunoassay technologies for ovarian cancer diagnosis. Emphasis is placed on the principles of the technologies, their merits and limitations and on the evolution from laboratory-based methods to point-of-care devices. While the current market is predominantly associated with clinical immunoassay kits, over the last decade a major thrust in development of immunosensors is evident due to their potential in point-of-care devices. Technological advancements in immunosensors, extending from labeled to label-free detection, with and without mediators, for enhancing proficiencies and reliability have been dealt with in detail. Aspects of the utilisation of nanomaterials and immobilization strategies for enhancing sensitivity and altering the detection range have also been addressed. Finally, we have discussed some distinct characteristics and limitations associated with the recently commericalised technologies used for quantitation of relevant ovarian cancer markers. PMID:27233124

  12. Evaluation of the LIAISON Chemiluminescence Immunoassay for Diagnosis of Syphilis▿

    OpenAIRE

    Knight, Carrie S.; Crum, Mary A.; Hardy, Robert W

    2007-01-01

    We report the results of an evaluation of the LIAISON Treponema pallidum-specific assay, a one-step sandwich chemiluminescence immunoassay (CLIA), as a screening test and as a confirmatory test for the diagnosis of syphilis. The assay was compared with the CAPTIA Syphilis-G enzyme immunoassay (EIA) and with a testing algorithm that also included the rapid plasma reagin (RPR) and T. pallidum particle agglutination (PA) assays. As a screening test, the CLIA showed levels of agreement with the E...

  13. A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies

    OpenAIRE

    Machard, Sophie; Theodoro, Frederic; Benech, Henri; Grognet, Jean-Marc; Ezan, Eric

    2000-01-01

    Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed o...

  14. Radio-immunoassay of somatostatin from isolated rat pancreatic islets

    International Nuclear Information System (INIS)

    Certain aspects of radio-immunoassay of somatostatin from isolated rat pancreatic islets are described. Somatostatin-14, and not somatostatin-28, is secreted from isolated rat pancreatic islets. Less somatostatin secretion is measured per islet owing to purity of tracer in the radio-immunoassay. Theophylline apparently cross-reacts with somatostatin in the assay described, and this has to be taken into consideration when studying somatostatin release induced by theophylline in isolated islets. (author)

  15. Status of immunoassay as an analytical tool in environmental investigations

    International Nuclear Information System (INIS)

    Immunoassay methods were initially applied in clinical situations where their sensitivity and selectivity were utilized for diagnostic purposes. In the 1970s, pesticide chemists realized the potential benefits of immunoassay methods for compounds difficult to analyze by gas chromatography. This transition of the technology has extended to the analysis of soil, water, food and other matrices of environmental and human exposure significance particularly for compounds difficult to analyze by chromatographic methods. The utility of radioimmunoassays and enzyme immunoassays for environmental investigations was recognized in the 1980s by the U.S. Environmental Protection Agency (U.S. EPA) with the initiation of an immunoassay development programme. The U.S. Department of Agriculture (USDA) and the U.S. Food and Drug Administration (PDA) have investigated immunoassays for the detection of residues in food both from an inspection and a contamination prevention perspective. Environmental immunoassays are providing rapid screening information as well as quantitative information to fulfill rigorous data quality objectives for monitoring programmes

  16. Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens

    Science.gov (United States)

    Saliva has an important advantage over serum as a medium for antibody detection due to non-invasive sampling, which is critical for community-based epidemiological surveys. The development of a Luminex multiplex immunoassay for measurement of salivary IgG and IgA responses to pot...

  17. In situ deposition of Prussian blue on mesoporous carbon nanosphere for sensitive electrochemical immunoassay.

    Science.gov (United States)

    Lai, Guosong; Zhang, Haili; Yu, Aimin; Ju, Huangxian

    2015-12-15

    A Prussian blue (PB) functionalized mesoporous carbon nanosphere (MCN) composite was prepared for loading signal antibody and high-content glucose oxidase (GOD) to obtain a new nanoprobe for sensitive electrochemical immunoassay. The MCN nanocarrier with an average diameter of 180 nm was synthesized by using mesoporous silica nanosphere as a hard template in combination with a hydrothermal carbonization method. This hydrophilic carbon nanomaterial provided an ideal platform for in situ deposition of high-content PB to form the MCN-PB nanocomposite. Based on the step-wise assembly of polyelectrolyte and gold nanoparticles (Au NPs) on the negative-charged nanocomposite, signal antibody and high-content GOD were loaded on this nanocarrier to obtain the nanoprobe. After a sandwich immunoreaction at an Au NPs-modified screen-printed carbon electrode based immunosensor, the nanoprobes were quantitatively captured on the electrode surface to produce sensitive electrochemical response with a PB-mediated GOD catalytic reaction for immunoassay. The high loading of PB and GOD on the nanoprobe greatly amplified the electrochemical signal, leading to the development of a new immunoassay method with high sensitivity. Using human immunoglobulin G as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a low detection limit down to 7.8 pg/mL was obtained. Additionally, the immunosensor showed high specificity, satisfactory stability and repeatability as well as acceptable reliability. The PB-mediated GOD electrochemical system well excluded the conventional interference from the dissolved oxygen. Thus this immunoassay method provides great potentials for practical applications. PMID:26201983

  18. Development of a prototype lateral flow immunoassay (LFI for the rapid diagnosis of melioidosis.

    Directory of Open Access Journals (Sweden)

    Raymond L Houghton

    2014-03-01

    Full Text Available Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb, an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI; the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml. The analytical reactivity (inclusivity of the AMD LFI was 98.7% (76/77 when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity testing determined that 97.2% of B. pseudomallei near neighbor species (35/36 were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

  19. Comparison of enzyme-multiplied immunoassay technique with fluorescence polarization immunoassay for determination of gentamicin and tobramycin levels in serum.

    OpenAIRE

    Pohlod, D J; Saravolatz, L D; Somerville, M M

    1984-01-01

    We assayed serum gentamicin and tobramycin specimens by the enzyme multiplied immunoassay technique (Syva EMIT) and the fluorescence polarization immunoassay (Abbott TDx). When interassay and intraassay control samples were evaluated, both methods gave an overall coefficient of variation of less than +/- 10%. Using patient serum samples, we obtained excellent correlation with both methods in the assay of gentamicin (correlation coefficient, 0.985) and tobramycin (correlation coefficient, 0.982).

  20. l-Arginine grafted alginate hydrogel beads: A novel pH-sensitive system for specific protein delivery

    Directory of Open Access Journals (Sweden)

    Mohamed S. Mohy Eldin

    2015-05-01

    Full Text Available Novel pH-sensitive hydrogels based on l-arginine grafted alginate (Arg-g-Alg hydrogel beads were synthesized and utilized as a new carrier for protein delivery (BSA in specific pH media. l-arginine was grafted onto the polysaccharide backbone of virgin alginate via amine functions. Evidences of grafting of alginate were extracted from FT-IR and thermal analysis, while the morphological structure of Arg-g-Alg hydrogel beads was investigated by SEM photographs. Factors affecting on the grafting process e.g. l-arginine concentration, reaction time, reaction temperature, reaction pH, and crosslinking conditions, have been studied. Whereas, grafting efficiency of each factor was evaluated. Grafting of alginate has improved both thermal and morphological properties of Arg-g-Alg hydrogel beads. The swelling behavior of Arg-g-Alg beads was determined as a function of pH and compared with virgin calcium alginate beads. The cumulative in vitro release profiles of BSA loaded beads were studied at different pHs for simulating the physiological environments of the gastrointestinal tract. The amount of BSA released from neat alginate beads at pH 2 was almost 15% after 5 h, while the Arg-g-Alg beads at the same conditions were clearly higher than 45%, then it increased to 90% at pH 7.2. Accordingly, grafting of alginate has improved its release profile behavior particularly in acidic media. The preliminary results clearly suggested that the Arg-g-Alg hydrogel may be a potential candidate for polymeric carrier for oral delivery of protein or drugs.