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Sample records for free-solution label-free detection

  1. Label-free solution-based kinetic study of aptamer-small molecule interactions by kinetic capillary electrophoresis with UV detection revealing how kinetics control equilibrium.

    Science.gov (United States)

    Bao, Jiayin; Krylova, Svetlana M; Reinstein, Oren; Johnson, Philip E; Krylov, Sergey N

    2011-11-15

    Here we demonstrate a label-free solution-based approach for studying the kinetics of biopolymer-small molecule interactions. The approach utilizes kinetic capillary electrophoresis (KCE) separation and UV light absorption detection of the unlabeled small molecule. In this proof-of-concept work, we applied KCE-UV to study kinetics of interaction between a small molecule and a DNA aptamer. From the kinetic analysis of a series of aptamers, we found that dissociation rather than binding controls the stability of the complex. Because of its label-free features and generic nature, KCE-UV promises to become a practical tool for challenging kinetic studies of biopolymer-small molecule interactions.

  2. Optical tweezers for free-solution label-free single bio-molecule studies

    Science.gov (United States)

    Kotnala, Abhay; Al-Balushi, Ahmed A.; Gordon, Reuven

    2014-09-01

    Nanoaperture based trapping has developed as a significant tool among the various optical tweezer systems for trapping of very small particles down to the single nanometer range. The double nanohole aperture based trap provides a method for efficient, highly-sensitive, label-free, low-cost, free-solution single molecule trapping and detection. We use the double nanohole tweezer to understand biomolecular phenomena like protein unfolding, binding, structural conformation of DNA, protein-DNA interactions, and protein small molecule interactions.

  3. Free-solution, label-free molecular interactions studied by back-scattering interferometry

    DEFF Research Database (Denmark)

    Bornhop, D.J.; Latham, J.C.; Kussrow, A.

    2007-01-01

    Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules...

  4. Label-free free-solution nanoaperture optical tweezers for single molecule protein studies.

    Science.gov (United States)

    Al Balushi, Ahmed A; Kotnala, Abhay; Wheaton, Skyler; Gelfand, Ryan M; Rajashekara, Yashaswini; Gordon, Reuven

    2015-07-21

    Nanoaperture optical tweezers are emerging as useful label-free, free-solution tools for the detection and identification of biological molecules and their interactions at the single molecule level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward, high-speed and highly sensitive (SNR ∼ 33) platform to observe real-time dynamics and to quantify binding kinetics of protein-small molecule interactions without the need to use tethers or labeling. Such nanoaperture-based optical tweezers, which are 1000 times more efficient than conventional optical tweezers, have been used to trap and isolate single DNA molecules and to study proteins like p53, which has been claimed to be in mutant form for 75% of human cancers. More recently, nanoaperture optical tweezers have been used to probe the low-frequency (in the single digit wavenumber range) Raman active modes of single nanoparticles and proteins. Here we review recent developments in the field of nanoaperture optical tweezers and how they have been applied to protein-antibody interactions, protein-small molecule interactions including single molecule binding kinetics, and protein-DNA interactions. In addition, recent works on the integration of nanoaperture optical tweezers at the tip of optical fiber and in microfluidic environments are presented.

  5. Optimizing end-labeled free-solution electrophoresis by increasing the hydrodynamic friction of the drag-tag

    OpenAIRE

    Grass, Kai; Holm, Christian; Slater, Gary W.

    2009-01-01

    We study the electrophoretic separation of polyelectrolytes of varying lengths by means of end-labeled free-solution electrophoresis (ELFSE). A coarse-grained molecular dynamics simulation model, using full electrostatic interactions and a mesoscopic Lattice Boltzmann fluid to account for hydrodynamic interactions, is used to characterize the drag coefficients of different label types: linear and branched polymeric labels, as well as transiently bound micelles. It is specifically shown that t...

  6. Biopatterning for label-free detection.

    Science.gov (United States)

    Goddard, Julie M; Mandal, Sudeep; Nugen, Sam R; Baeumner, Antje J; Erickson, David

    2010-03-01

    We present a biopatterning technique suitable for applications which demand a high degree of surface cleanliness, such as immobilization of biological recognition elements onto label-free biosensors. In the case of label-free biosensing, the mechanism of signal transduction is based on surface bound matter, making them highly sensitive to surface contamination including residues left during the biopatterning process. In this communication we introduce a simple, rapid processing step that removes 98% of the residues that often remain after standard parylene lift-off patterning. Residue-free parylene biopatterning is combined with microfluidics to localize biomolecule immobilization onto the sensing region and to enable multiplexed biopatterning. We demonstrate the applicability of this method to multiplexed label-free detection platforms by patterning nucleic acid capture probes corresponding to the four different serotypes of Dengue virus onto parallel 1D photonic crystal resonator sensors. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to quantify surface cleanliness and uniformity. In addition to label-free biosensors, this technique is well suited to other nanobiotechnology patterning applications which demand a pristine, residue-free surface, such as immobilization of enzymes, antibodies, growth factors, or cell cultures.

  7. Theory of end-labeled free-solution electrophoresis: is the end effect important?

    Science.gov (United States)

    Chubynsky, Mykyta V; Slater, Gary W

    2014-03-01

    In the theory of free-solution electrophoresis of a polyelectrolyte (such as the DNA) conjugated with a "drag-tag," the conjugate is divided into segments of equal hydrodynamic friction and its electrophoretic mobility is calculated as a weighted average of the mobilities of individual segments. If all the weights are assumed equal, then for an electrically neutral drag-tag, the elution time t is predicted to depend linearly on the inverse DNA length 1/M. While it is well-known that the equal-weights assumption is approximate and in reality the weights increase toward the ends, this "end effect" has been assumed to be small, since in experiments the t(1/M) dependence seems to be nearly perfectly linear. We challenge this assumption pointing out that some experimental linear fits do not extrapolate to the free (i.e. untagged) DNA elution time in the limit 1/M→0, indicating nonlinearity outside the fitting range. We show that a theory for a flexible polymer taking the end effect into account produces a nonlinear curve that, however, can be fitted with a straight line over a limited range of 1/M typical of experiments, but with a "wrong" intercept, which explains the experimental results without additional assumptions. We also study the influence of the flexibilities of the charged and neutral parts.

  8. Photonic crystal microcapsules for label-free multiplex detection.

    Science.gov (United States)

    Ye, Baofen; Ding, Haibo; Cheng, Yao; Gu, Hongcheng; Zhao, Yuanjin; Xie, Zhuoying; Gu, Zhongze

    2014-05-28

    A novel suspension array, which possesses the joint advantages of photonic crystal encoded technology, bioresponsive hydrogels, and photonic crystal sensors with capability of full multiplexing label-free detection is developed.

  9. Label Free Chromosome Translocation Detection with Silicon nanowires

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Andersen, Karsten Brandt; Frøhling, Kasper Bayer;

    is a Fluorescent In Situ Hybridization, which is laborious and involves use of expensive reagents [1]. Here we present a label free technique for detection of chromosome translocations. As a proof of concept detection of chromosome translocation between chromosome 3 (Chr3) and chromosome 9 (Chr9) was chosen....

  10. Label-free disposable immunosensor for detection of atrazine.

    Science.gov (United States)

    Belkhamssa, Najet; Justino, Celine I L; Santos, Patrícia S M; Cardoso, Susana; Lopes, Isabel; Duarte, Armando C; Rocha-Santos, Teresa; Ksibi, Mohamed

    2016-01-01

    This work reports the construction of a fast, disposable, and label-free immunosensor for the determination of atrazine. The immunosensor is based on a field effect transistor (FET) where a network of single-walled carbon nanotubes (SWCNTs) acts as the conductor channel, constituting carbon nanotubes field effect transistors (CNTFETs). Anti-atrazine antibodies were adsorbed onto the SWCNTs and subsequently the SWCNTs were protected with Tween 20 to prevent the non-specific binding of bacteria or proteins. The principle of the immunoreaction consists in the direct adsorption of atrazine specific antibodies (anti-atrazine) to SWCNTs networks. After exposed to increasing concentrations of atrazine, the CNTFETs could be used as useful label-free platforms to detect atrazine. Under the optimal conditions, a limit of detection as low as 0.001 ng mL(-1) was obtained, which is lower than that of other methods for the atrazine detection, and in a working range between 0.001 and 10 ng mL(-1). The average recoveries obtained for real water samples spiked with atrazine varied from 87.3% to 108.0%. The results show that the constructed sensors display a high sensitivity and could be useful tools for detecting pesticides like atrazine at low concentrations. They could be also applied to the determination of atrazine in environmental aqueous samples, such as seawater and riverine water. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Label-Free Aptasensors for the Detection of Mycotoxins

    Directory of Open Access Journals (Sweden)

    Amina Rhouati

    2016-12-01

    Full Text Available Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins.

  12. Label-Free Aptasensors for the Detection of Mycotoxins.

    Science.gov (United States)

    Rhouati, Amina; Catanante, Gaelle; Nunes, Gilvanda; Hayat, Akhtar; Marty, Jean-Louis

    2016-12-18

    Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins.

  13. Label-Free Aptasensors for the Detection of Mycotoxins

    Science.gov (United States)

    Rhouati, Amina; Catanante, Gaelle; Nunes, Gilvanda; Hayat, Akhtar; Marty, Jean-Louis

    2016-01-01

    Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins. PMID:27999353

  14. Label-free DNA sequencing using Millikan detection.

    Science.gov (United States)

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-10-15

    A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.

  15. Label-Free DNA Sequencing Using Millikan Detection

    OpenAIRE

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-01-01

    A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucl...

  16. Label-free detection of breast masses using multiphoton microscopy.

    Directory of Open Access Journals (Sweden)

    Xiufeng Wu

    Full Text Available Histopathology forms the gold standard for the diagnosis of breast cancer. Multiphoton microscopy (MPM has been proposed to be a potentially powerful adjunct to current histopathological techniques. A label-free imaging based on two- photon excited fluorescence and second-harmonic generation is developed for differentiating normal breast tissues, benign, as well as breast cancer tissues. Human breast biopsies (including human normal breast tissues, benign as well as breast cancer tissues that are first imaged (fresh, unfixed, and unstained with MPM and are then processed for routine H-E histopathology. Our results suggest that the MPM images, obtained from these unprocessed biopsies, can readily distinguish between benign lesions and breast cancers. In the tissues of breast cancers, MPM showed that the tumor cells displayed marked cellular and nuclear pleomorphism. The tumor cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, infiltrated into disrupted connective tissue, leading to the loss of second-harmonic generation signals. For breast cancer, MPM diagnosis was 100% correct because the tissues of breast cancers did not have second-harmonic generation signals in MPM imaging. On the contrary, in benign breast masses, second-harmonic generation signals could be seen easily in MPM imaging. These observations indicate that MPM could be an important potential tool to provide label-free noninvasive diagnostic impressions that can guide surgeon in biopsy and patient management.

  17. Photonic Crystal Biosensor Chip for Label-Free Detection of Bacteria

    DEFF Research Database (Denmark)

    Kristensen, Martin; Krüger, Asger Christian; Groothoff, Nathaniel

    Narrow polarization-mixing resonances in planar photonic crystals are studied as candidate components for label-free refractive index sensors for detecting bacteria causing sepsis through the identification of DNA strands....

  18. Photonic Crystal Biosensor Chip for Label-Free Detection of Bacteria

    DEFF Research Database (Denmark)

    Kristensen, Martin; Krüger, Asger Christian; Groothoff, Nathaniel

    Narrow polarization-mixing resonances in planar photonic crystals are studied as candidate components for label-free refractive index sensors for detecting bacteria causing sepsis through the identification of DNA strands.......Narrow polarization-mixing resonances in planar photonic crystals are studied as candidate components for label-free refractive index sensors for detecting bacteria causing sepsis through the identification of DNA strands....

  19. Label-free detection of hybridization of oligonucleotides by oblique-incidence reflectivity difference method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.

  20. A detection instrument for enhanced-fluorescence and label-free imaging on photonic crystal surfaces.

    Science.gov (United States)

    Block, Ian D; Mathias, Patrick C; Ganesh, Nikhil; Jones, Sarah I; Dorvel, Brian R; Chaudhery, Vikram; Vodkin, Lila O; Bashir, Rashid; Cunningham, Brian T

    2009-07-20

    We report on the design and demonstration of an optical imaging system capable of exciting surface-bound fluorophores within the resonant evanescent electric field of a photonic crystal surface and gathering fluorescence emission that is directed toward the imaging objective by the photonic crystal. The system also has the ability to quantify shifts in the local resonance angle induced by the adsorption of biomolecules on the photonic crystal surface for label-free biomolecular imaging. With these two capabilities combined within a single detection system, we demonstrate label-free images self-registered to enhanced fluorescence images with 328x more sensitive fluorescence detection relative to a glass surface. This technique is applied to a DNA microarray where label-free quantification of immobilized capture DNA enables improved quality control and subsequent enhanced fluorescence detection of dye-tagged hybridized DNA yields 3x more genes to be detected versus commercially available microarray substrates.

  1. Combined enhanced fluorescence and label-free biomolecular detection with a photonic crystal surface.

    Science.gov (United States)

    Mathias, Patrick C; Ganesh, Nikhil; Chan, Leo L; Cunningham, Brian T

    2007-04-20

    A 2D photonic crystal surface with a different period in each lateral direction is demonstrated to detect biomolecules using two distinct sensing modalities. The sensing mechanisms both rely on the generation of a resonant reflection peak at one of two specific wavelengths, depending on the polarization of light that is incident on the photonic crystal. One polarization results in a resonant reflection peak in the visible spectrum to coincide with the excitation wavelength of a fluorophore, while the orthogonal polarization results in a resonant reflection peak at an infrared wavelength which is used for label-free detection of adsorbed biomolecules. The photonic crystal resonance for fluorescence excitation causes enhanced near fields at the structure surface, resulting in increased signal from fluorophores within 100 nm of the device surface. Label-free detection is performed by illuminating the photonic crystal with white light and monitoring shifts in the peak reflected wavelength of the infrared resonance with a high-resolution imaging detection instrument. Rigorous coupled-wave analysis was used to determine optimal dimensions for the photonic crystal structure, and devices were fabricated using a polymer-based nanoreplica molding approach. Fluorescence-based and label-free detection were demonstrated using arrays of spots of dye-conjugated streptavidin. Quantification of the fluorescent signal showed that the fluorescence output from protein spots on the photonic crystal was increased by up to a factor of 35, and deposited spots were also imaged in the label-free detection mode.

  2. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2015-10-29

    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  3. Label-free virus detection using silicon photonic microring resonators

    OpenAIRE

    McClellan, Melinda S.; Domier, Leslie L; Bailey, Ryan C.

    2011-01-01

    Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and featur...

  4. Detection of ochratoxin A in beer samples with a label-free monolithically integrated optoelectronic biosensor

    NARCIS (Netherlands)

    Pagkali, Varvara; Petrou, Panagiota S.; Salapatas, Alexandros; Makarona, Eleni; Peters, Jeroen; Haasnoot, Willem; Jobst, Gerhard; Economou, Anastasios; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios E.

    2016-01-01

    An optical biosensor for label-free detection of ochratoxin A (OTA) in beer samples is presented. The biosensor consists of an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources on the same Si chip (37mm2

  5. Label-free protein detection using a microfluidic Coulter-counter device

    DEFF Research Database (Denmark)

    Rodriguez-Trujíllo, Romén; Ajine, Mohammad Akram; Orzan, A.;

    2014-01-01

    A new method for measuring specific protein concentrations in solutions has been developed. The technique makes use of the Coulter effect for detecting and sizing of micro-scaled objects suspended in a buffer fluid. The method is completely label-free as it is only based on the electrical readout...

  6. Detection of ochratoxin A in beer samples with a label-free monolithically integrated optoelectronic biosensor

    NARCIS (Netherlands)

    Pagkali, Varvara; Petrou, Panagiota S.; Salapatas, Alexandros; Makarona, Eleni; Peters, Jeroen; Haasnoot, Willem; Jobst, Gerhard; Economou, Anastasios; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios E.

    2017-01-01

    An optical biosensor for label-free detection of ochratoxin A (OTA) in beer samples is presented. The biosensor consists of an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources on the same Si chip (37mm2

  7. Multiplex surface plasmon resonance imaging platform for label-free detection of foodborne pathogens

    Science.gov (United States)

    Salmonellae are among the leading causes of foodborne outbreaks in the United States, and more rapid and efficient detection methods are needed. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multiple targets simultaneous...

  8. Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

    Science.gov (United States)

    Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

    2016-10-01

    Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

  9. Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2008-02-01

    Full Text Available Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA.

  10. Label-free amino acid detection based on nanocomposites of graphene oxide hybridized with gold nanoparticles.

    Science.gov (United States)

    Zhang, Qian; Zhang, Diming; Lu, Yanli; Xu, Gang; Yao, Yao; Li, Shuang; Liu, Qingjun

    2016-03-15

    Nanocomposites of graphene oxide and gold nanoparticles (GO/GNPs) were synthesized for label-free detections of amino acids. Interactions between the composites and amino acids were investigated by both naked-eye observation and optical absorption spectroscopy. The GO/GNPs composites displayed apparent color changes and absorption spectra changes in presences of amino acids including glutamate, aspartate, and cysteine. The interaction mechanisms of the composites and amino acids were discussed and explored with sulfhydryl groups and non-α-carboxylic groups on the amino acids. Sensing properties of the composites were tested, while pure gold particles were used as the control. The results suggested that the GO/GNPs composites had better linearity and stability in dose-dependent responses to the amino acids than those of the particles, especially in detections for acidic amino acids. Therefore, the nanocomposites platform can provide a convenient and efficient approach for label-free optical detections of important molecules such as amino acids.

  11. Label-Free Electrical Detection Using Carbon Nanotube-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Kenzo Maehashi

    2009-07-01

    Full Text Available Label-free detections of biomolecules have attracted great attention in a lot of life science fields such as genomics, clinical diagnosis and practical pharmacy. In this article, we reviewed amperometric and potentiometric biosensors based on carbon nanotubes (CNTs. In amperometric detections, CNT-modified electrodes were used as working electrodes to significantly enhance electroactive surface area. In contrast, the potentiometric biosensors were based on aptamer-modified CNT field-effect transistors (CNTFETs. Since aptamers are artificial oligonucleotides and thus are smaller than the Debye length, proteins can be detected with high sensitivity. In this review, we discussed on the technology, characteristics and developments for commercialization in label-free CNT-based biosensors.

  12. Reproducible E. coli detection based on label-free SERS and mapping.

    Science.gov (United States)

    Yang, Danting; Zhou, Haibo; Haisch, Christoph; Niessner, Reinhard; Ying, Yibin

    2016-01-01

    The biosensing for rapid detection of bacteria based on surface-enhanced Raman scattering (SERS) has been widely explored for recent years. It is still a challenge to achieve a high sensitive, reproducible label free detection method for bacteria. In this work, a label-free SERS detection method of Escherichia coli based on incubation with silver colloid was reported. Optimized incubation conditions including shaking speed, time and temperature were used to help construct a rapid SERS method for E. coli analysis. It was found that the enhancement of the Raman signal of E. coli could be achieved to 1.8×10(4) cps (counts per second) with high reproducibility. Three strains of E. coli DSM 1116/498/5695 could be successfully discriminated using such SERS method combining discriminant analysis. Finally, the lowest concentration of E. coli at 1×10(5) cell/mL can be detected by SERS mapping. Thus, our detection method offers higher sensitivity and reproducibility compared to previously reported label free simple-mixing methods, opening an avenue for developing various SERS-based biosensor.

  13. Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

    Science.gov (United States)

    Lee, Kuang-Li; You, Meng-Lin; Tsai, Chia-Hsin; Lin, En-Hung; Hsieh, Shu-Yi; Ho, Ming-Hsun; Hsu, Ju-Chun; Wei, Pei-Kuen

    2016-01-15

    The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques.

  14. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Yunxia Luan

    2015-12-01

    Full Text Available A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA was developed using a cationic polymer and gold nanoparticles (AuNPs. The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection.

  15. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles.

    Science.gov (United States)

    Luan, Yunxia; Chen, Jiayi; Li, Cheng; Xie, Gang; Fu, Hailong; Ma, Zhihong; Lu, Anxiang

    2015-12-10

    A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA) was developed using a cationic polymer and gold nanoparticles (AuNPs). The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection.

  16. Label-free detection of small-molecule binding to a GPCR in the membrane environment.

    Science.gov (United States)

    Heym, Roland G; Hornberger, Wilfried B; Lakics, Viktor; Terstappen, Georg C

    2015-08-01

    Evaluation of drug-target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained. In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (k(on)=1.83×10(6)M(-1)s(-1), k(off)=2.79×10(-4) s(-1)) and the small molecule AMD3100 (k(on)=5.46×10(5)M(-1)s(-1), k(off)=1.01×10(-2)s(-1)) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.

  17. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Claudia Meindl

    2013-01-01

    Full Text Available Cytotoxicity testing of nanoparticles (NPs by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA and automated microscopy (Cell-IQ compared to formazan bioreduction (MTS assay. For validation of the label-free systems different concentrations of ethanol and of amine (AMI polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (20 nm CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

  18. Label-free fluorescence detection of melamine with a truncated aptamer.

    Science.gov (United States)

    Gu, Chunmei; Xiang, Yu; Guo, Hongli; Shi, Hanchang

    2016-07-21

    The 2008 Chinese milk scandal caused by the adulteration of melamine encouraged the public to pay attention to melamine detection in milk products and other food stuffs. To allow simple and rapid detection of melamine, we previously isolated an 88 nt melamine aptamer (called Rd29C33) using the structure-switching SELEX. However, this 88 nt oligonucleotide is costly to synthesize, and may also complicate the rational design of biosensors for melamine detection. To overcome this obstacle, we truncated Rd29C33 at several sites, and a 34 nt Rd29C33-T7 melamine aptamer was finally found to show comparable binding affinity and better selectivity to melamine compared to the original 88 nt Rd29C33. Furthermore, a label-free bioassay method for melamine detection was designed by using Rd29C33-T7 and thiazole orange (TO). The addition of melamine to a mixture of Rd29C33-T7 and TO caused the release of TO from Rd29C33-T7, resulting in a decrease of the fluorescence intensity of the solution. A detection limit of 0.12 μM for melamine was achieved using this label-free method. Good recovery ranging from 82.6% to 97.2% for melamine detection in whole milk samples suggested the promise of this bioassay method for application in monitoring melamine in real food stuffs.

  19. CSI-FID: high throughput label-free detection of DNA binding molecules.

    Science.gov (United States)

    Hauschild, Karl E; Stover, James S; Boger, Dale L; Ansari, Aseem Z

    2009-07-15

    Determining the sequence specifity of DNA binding molecules is a non-trivial task. Here we describe the development of a platform for assaying the sequence specificity of DNA ligands using label free detection on high density DNA microarrays. This is achieved by combining Cognate Site Identification (CSI) with Fluorescence Intercalation Displacement (FID) to create CSI-FID. We use the well-studied small molecule DNA ligand netropsin to develop this high throughput platform. Analysis of the DNA binding properties of protein- and small molecule-based libraries with CSI-FID will advance the development of genome-anchored molecules for therapeutic purposes.

  20. High-contrast grating resonators for label-free detection of disease biomarkers

    Science.gov (United States)

    Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

    2016-06-01

    A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

  1. Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance.

    Science.gov (United States)

    Martínez-Rivas, Adrián; Chinestra, Patrick; Favre, Gilles; Pinaud, Sébastien; Séverac, Childérick; Faye, Jean-Charles; Vieu, Christophe

    2010-09-07

    We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (<2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules.

  2. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  3. Nanogap biosensors for electrical and label-free detection of biomolecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Kyu Kim, Sang; Cho, Hyunmin; Park, Hye-Jung; Kwon, Dohyoung; Min Lee, Jeong; Hyun Chung, Bong, E-mail: chungbh@kribb.re.k [BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yuseong, Daejeon 305-600 (Korea, Republic of)

    2009-11-11

    We demonstrate nanogap biosensors for electrical and label-free detection of biomolecular interactions. Parallel fabrication of nanometer distance gaps has been achieved using a silicon anisotropic wet etching technique on a silicon-on-insulator (SOI) wafer with a finely controllable silicon device layer. Since silicon anisotropic wet etching resulted in a trapezoid-shaped structure whose end became narrower during the etching, the nanogap structure was simply fabricated on the device layer of a SOI wafer. The nanogap devices were individually addressable and a gap size of less than 60 nm was obtained. We demonstrate that the nanogap biosensors can electrically detect biomolecular interactions such as biotin/streptavidin and antigen/antibody pairs. The nanogap devices show a current increase when the proteins are bound to the surface. The current increases proportionally depending upon the concentrations of the molecules in the range of 100 fg ml{sup -1}-100 ng ml{sup -1} at 1 V bias. It is expected that the nanogap developed here could be a highly sensitive biosensor platform for label-free detection of biomolecular interactions.

  4. Label-free detection of cardiac troponin I with a photonic crystal biosensor.

    Science.gov (United States)

    Zhang, Bailin; Morales, Andres W; Peterson, Ralph; Tang, Liang; Ye, Jing Yong

    2014-08-15

    A biosensor has been developed with a photonic crystal structure used in a total-internal-reflection (PC-TIR) configuration for label-free detection of a cardiac biomarker: Troponin I (cTnI). In contrast to a conventional optical microcavity that has a closed structure with its cavity layer sandwiched between two high-reflection surfaces, the PC-TIR configuration creates a unique open microcavity, which allows its cavity layer (sensing layer) to be easily functionalized and directly exposed to analyte molecules for bioassays. In this study, a PC-TIR sensor has been used for the label-free measurements of cardiac biomarkers by monitoring the changes in the resonant condition of the cavity due to biomolecular binding processes. Antibodies against cTnI are immobilized on the sensor surface for specific detection of cTnI with a wide range of concentrations. Detection limit of cTnI with a concentration as low as 0.1ngmL(-1) has been achieved.

  5. Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction.

    Science.gov (United States)

    Hiep, Ha Minh; Kerman, Kagan; Endo, Tatsuro; Saito, Masato; Tamiya, Eiichi

    2010-02-19

    In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 microm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5'-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng microL(-1) was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3-4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23-25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.

  6. Label-free detection of DNA on silicon surfaces using Brewster angle straddle interferometry (BASI)

    Science.gov (United States)

    Wang, Xiao; Rothberg, Lewis

    2012-02-01

    Label-free sensing of biomolecular interactions is of great importance for drug screening and a variety of clinical assays. Ultrasensitive detection of dsDNA on silicon substrates can be achieved using our new label-free sensing method - Brewster angle straddle interferometry (BASI) which exploits the removal of destructive interference to detect binding of target molecules on a silicon surface functionalized by probe molecules. By exploiting the fact that reflections of p-polarization undergo 180 degree phase shifts above the Brewster angle and none below it, we are able to use unprocessed silicon substrates with native oxide serving as the interference layer. Destructive interference in the geometry we use results in reflectivities ˜ 0.01%. Reflectivity from the chip is a quantitative measure of the amount of bound target molecules and can be imaged in real time in microarray format. We demonstrate detection of DNA intercalation on pyrene modified surfaces. The substrates are shown to exhibit excellent binding toward dsDNAs. This work provides an avenue for understanding the binding specificity of small molecule-DNA interactions that can be potentially helpful in developing anticancer agents.

  7. A compact and portable optofluidic device for detection of liquid properties and label-free sensing

    Science.gov (United States)

    Lahoz, F.; Martín, I. R.; Walo, D.; Gil-Rostra, J.; Yubero, F.; Gonzalez-Elipe, A. R.

    2017-06-01

    Optofluidic lasers have been widely investigated over the last few years mainly because they can be easily integrated in sensor devices. However, high power pulse lasers are required as excitation sources, which, in practice, limit the portability of the system. Trying to overcome some of these limitations, in this paper we propose the combined use of a small CW laser with a Fabry-Perot optofluidic planar microcavity showing high sensitivity and versatility for detection of liquid properties and label-free sensing. Firstly, a fluorescein solution in ethanol is used to demonstrate the high performances of the FP microcavity as a temperature sensor both in the laser (high pump power above laser threshold) and in the fluorescence (low pump power) regimes. A shift in the wavelength of the resonant cavity modes is used to detect changes in the temperature and our results show that high sensitivities could be already obtained using cheap and portable CW diode lasers. In the second part of the paper, the demonstration of this portable device for label-free sensing is illustrated under low CW pumping. The wavelength positions of the optofluidic resonant modes are used to detect glucose concentrations in water solutions using a protein labelled with a fluorescent dye as the active medium.

  8. Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

    Science.gov (United States)

    Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2013-01-01

    Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

  9. Label Free Detection of CD4+ and CD8+ T Cells Using the Optofluidic Ring Resonator

    Directory of Open Access Journals (Sweden)

    John T. Gohring

    2010-06-01

    Full Text Available We have demonstrated label free detection of CD4+ and CD8+ T-Lymphocyte whole cells and CD4+ T-Lymphocyte cell lysis using the optofluidic ring resonator (OFRR sensor. The OFRR sensing platform incorporates microfluidics and photonics in a setup that utilizes small sample volume and achieves a fast detection time. In this work, white blood cells were isolated from healthy blood and the concentrations were adjusted to match T-Lymphocyte levels of individuals infected with HIV. Detection was accomplished by immobilizing CD4 and CD8 antibodies on the inner surface of the OFRR. Sensing results show excellent detection of CD4+ and CD8+ T-Lymphocyte cells at medically significant concentrations with a detection time of approximately 30 minutes. This work will lead to a rapid and low-cost sensing device that can provide a CD4 and CD8 count as a measure of HIV progression.

  10. Label-free optical detection of cells grown in 3D silicon microstructures.

    Science.gov (United States)

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  11. Label-free detection of C-reactive protein using an electrochemical DNA immunoassay

    Directory of Open Access Journals (Sweden)

    Temsiri Songjaroen

    2016-05-01

    Full Text Available A label-free electrochemical immunoassay that combines DNA-directed immobilization (DDI with electrochemical impedance spectroscopy (EIS on microwire sensors is reported for the detection of C-reactive protein (CRP. CRP is an acute-phase protein that is strongly correlated with systemic inflammation. Since inflammation plays a role in pathogenesis of cardiovascular diseases, CRP can be used to predict the likelihood of coronary events. To demonstrate the new chemistry, 25-μm Au electrodes were modified with single strand DNA (ssDNA and exposed to a solution containing complementary ssDNA conjugated to monoclonal anti-CRP. The charge-transfer resistance of the [Fe(CN6]3−/4− redox couple was used to determine the CRP concentration after binding. A stepwise increase in the charge transfer resistance was observed using EIS for each modification step, ssDNA, ssDNA-anti-CRP hybridization and the final CRP capture. Cyclic voltammetry (CV was used to verify the EIS results, and showed an increase in peak potential splitting in a similar stepwise manner for each modification step. Finally, fluorescence microscopy was used to confirm the DNA hybridization and CRP binding. Standard addition of CRP revealed that EIS could be used to detect CRP at clinically relevant levels in serum samples. This new form of electrochemical DNA immunoassay (eDI has significant potential as a simple, label-free sensor for proteins in microfluidic devices.

  12. Original Research: Label-free detection for radiation-induced apoptosis in glioblastoma cells.

    Science.gov (United States)

    Qi, Dandan; Feng, Jingwen; Yang, Chengwen; Jin, Changrong; Sa, Yu; Feng, Yuanming

    2016-10-01

    Current flow cytometry (FCM) requires fluorescent dyes labeling cells which make the procedure costly and time consuming. This manuscript reports a feasibility study of detecting the cell apoptosis with a label-free method in glioblastoma cells. A human glioma cell line M059K was exposed to 8 Gy dose of radiation, which enables the cells to undergo radiation-induced apoptosis. The rates of apoptosis were studied at different time points post-irradiation with two different methods: FCM in combination with Annexin V-FITC/PI staining and a newly developed technique named polarization diffraction imaging flow cytometry. Totally 1000 diffraction images were acquired for each sample and the gray level co-occurrence matrix (GLCM) algorithm was used in morphological characterization of the apoptotic cells. Among the feature parameters extracted from each image pair, we found that the two GLCM parameters of angular second moment (ASM) and sum entropy (SumEnt) exhibit high sensitivities and consistencies as the apoptotic rates (Pa) measured with FCM method. In addition, no significant difference exists between Pa and ASM_S, Pa and SumEnt_S, respectively (P > 0.05). These results demonstrated that the new label-free method can detect cell apoptosis effectively. Cells can be directly used in the subsequent biochemical experiments as the structure and function of cells and biomolecules are well-preserved with this new method.

  13. Molecularly imprinted photonic hydrogels as colorimetric sensors for rapid and label-free detection of vanillin.

    Science.gov (United States)

    Peng, Hailong; Wang, Shenqi; Zhang, Zhong; Xiong, Hua; Li, Jinhua; Chen, Lingxin; Li, Yanbin

    2012-02-29

    A novel colorimetric sensor for the rapid and label-free detection of vanillin, based on the combination of photonic crystal and molecular imprinting technique, was developed. The sensing platform of molecularly imprinted photonic hydrogel (MIPH) was prepared by a noncovalent and self-assembly approach using vanillin as a template molecule. Morphology characterization by scanning electron microscope (SEM) showed that the MIPH possessed a highly ordered three-dimensional (3D) macroporous structure with nanocavities. The vanillin recognition events of the created nonocavities could be directly transferred into readable optical signals through a change in Bragg diffraction of the ordered macropores array of MIPH. The Bragg diffraction peak shifted from 451 to 486 nm when the concentration of the vanillin was increased from 10⁻¹² to 10⁻³ mol L⁻¹ within 60 s, whereas there were no obvious peak shifts for methyl and ethyl vanillin, indicating that the MIPH had high selectivity and rapid response for vanillin. The adsorption results showed that the hierarchical porous structure and homogeneous layers were formed in the MIPH with higher adsorption capacity. The application of such a label-free sensor with high selectivity, high sensitivity, high stability, and easy operation might offer a potential method for rapid real-time detection of trace vanillin.

  14. Asymmetric split-ring resonator-based biosensor for detection of label-free stress biomarkers

    Science.gov (United States)

    Lee, Hee-Jo; Lee, Jung-Hyun; Choi, Suji; Jang, Ik-Soon; Choi, Jong-Soon; Jung, Hyo-Il

    2013-07-01

    In this paper, an asymmetric split-ring resonator, metamaterial element, is presented as a biosensing transducer for detection of highly sensitive and label-free stress biomarkers. In particular, the two biomarkers, cortisol and α-amylase, are used for evaluating the sensitivity of the proposed biosensor. In case of cortisol detection, the competitive reaction between cortisol-bovine serum albumin and free cortisol is employed, while alpha-amylase is directly detected by its antigen-antibody reaction. From the experimental results, we find that the limit of detection and sensitivity of the proposed sensing device are about 1 ng/ml and 1.155 MHz/ng ml-1, respectively.

  15. Label-free nanopore proximity bioassay for platelet-derived growth factor detection.

    Science.gov (United States)

    Zhang, Ling; Zhang, Kaixiang; Liu, Guangchao; Liu, Mengjia; Liu, Yang; Li, Jinghong

    2015-06-02

    Rapid and sensitive detection of biomarkers with ultralow concentrations remains a great challenge in disease diagnostics. Herein, we present a label-free α-hemolysin (α-HL) nanopore proximity bioassay for protein biomarker detection by a binding-induced DNA strand displacement strategy. In this bioassay, an individual target protein, platelet-derived growth factor B-chain (PDGF-BB), was selectively recognized by two oligonucleotide affinity ligands in which an output DNA was released and translocated through α-HL nanopore with a spikelike short current block. The frequency of the current block events had a linear relationship with the concentration of PDGF-BB with a wide linear dynamic range of 5 orders of magnitude and a detection limit at 500 fM. The selectivity and anti-interference capability of this bioassay show great potential for biomarker detection in bioanalytical chemistry.

  16. Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays

    Science.gov (United States)

    Li, Yifen; Syed, Lateef; Liu, Jianwei; Hua, Duy H.; Li, Jun

    2012-01-01

    We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis-Menten heterogeneous enzymatic model with a specific constant, kcat/Km, of (2.1 ± 0.1) × 107 M−1 s−1. Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4 nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1 s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions. PMID:22935373

  17. Molecular imprinted photonic crystal hydrogels for the rapid and label-free detection of imidacloprid.

    Science.gov (United States)

    Wang, Xuan; Mu, Zhongde; Liu, Ran; Pu, Yuepu; Yin, Lihong

    2013-12-15

    A novel sensor for the rapid and label-free detection of imidacloprid was developed based on the combination of a colloidal crystal templating method and a molecular imprinting technique. The molecular imprinted photonic hydrogel film was prepared with methacrylic acid as monomers, ethylene glycol dimethylacrylate as cross-linkers and imidacloprid as imprinting template molecules. When the colloidal crystal template and the molecularly imprinted template was removed, the resulted MIPH film possessed a highly ordered three-dimensional macroporous structure with nanocavities. The response of the MIPH film to imidacloprid in aqueous solution can be detected through a readable Bragg diffraction red shift. When the concentration of imidacloprid increased from 10(-13) to 10(-7) g/mL, the Bragg diffraction peak shifted from 551 to 589 nm, while there were no obvious peak shifts for thiamethoxam and acetamiprid. This sensor which comprises of no label techniques and expensive instruments has potential application for the detection of trace imidacloprid.

  18. Portable microfluidic raman system for rapid, label-free early disease signature detection

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Meiye [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Davis, Ryan Wesley [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Hatch, Anson [Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

    2015-09-01

    In the early stages of infection, patients develop non-specific or no symptoms at all. While waiting for identification of the infectious agent, precious window of opportunity for early intervention is lost. The standard diagnostics require affinity reagents and sufficient pathogen titers to reach the limit of detection. In the event of a disease outbreak, triaging the at-risk population rapidly and reliably for quarantine and countermeasure is more important than the identification of the pathogen by name. To expand Sandia's portfolio of Biological threat management capabilities, we will utilize Raman spectrometry to analyze immune subsets in whole blood to rapidly distinguish infected from non-infected, and bacterial from viral infection, for the purpose of triage during an emergency outbreak. The goal of this one year LDRD is to determine whether Raman spectroscopy can provide label-free detection of early disease signatures, and define a miniaturized Raman detection system meeting requirements for low- resource settings.

  19. Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor.

    Science.gov (United States)

    Chen, Xuejuan; Pan, Yangang; Liu, Huiqing; Bai, Xiaojing; Wang, Nan; Zhang, Bailin

    2016-05-15

    Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300 cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability.

  20. Piezoelectric Cantilever Biosensors for Label-free, Real-time Detection of DNA and RNA.

    Science.gov (United States)

    Haring, Alexander P; Cesewski, Ellen; Johnson, Blake N

    2017-01-01

    This chapter reviews the design, fabrication, characterization, and application of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. The sensor transduction mechanism, sensing principle, and mode of operation are discussed. Bio-recognition strategies and surface functionalization methods for detection of DNA and RNA are discussed with a focus on self-assembly-based approaches. Methods for the verification of biosensor response via secondary binding assays, reversible binding assays, and the integration of complementary transduction mechanisms are presented. Sensing applications for medical diagnostics, food safety, and environmental monitoring are provided. PEMC sensor technology provides a robust platform for the real-time, label-free detection of DNA and RNA in complex matrices over nanomolar (nM) to attomolar (aM) concentration ranges.

  1. Label free detection of DNA on Au/ZnO/Ag hybrid structure based SERS substrate

    Science.gov (United States)

    Pal, Anil Kumar; Mohan, D. Bharathi

    2016-04-01

    Au/ZnO/Ag based SERS substrate was fabricated for the label free detection of DNA of Escherichia Coli bacteria. The SERS substrate was fabricated by growing ZnO nanorod arrays on thermally evaporated ultrathin Ag film of 5 nm thickness using hydrothermal process. Non-spherical like Au nanoparticles were decorated on ZnO nanorod arrays by sputtering technique with sputtering time of 45 sec. The surface of Au/ZnO/Ag was observed to be nearly superhydrophobic exhibiting the contact angle of 144 °. A low volume (5 µl) of aqueous solution of DNA of laboratory strain Escherichia Coli with very low concentration was adsorbed on fabricated SERS substrate by drop casting. The SERS detection of DNA molecules was achieved up to lower concentration of 10-8 M due to strong local electric field enhancement at the nanometer gap among Au nanoparticles and superhydrophobic nature of Au/ZnO/Ag surface.

  2. Label-free electrochemical immunosensor based on cerium oxide nanowires for Vibrio cholerae O1 detection

    Energy Technology Data Exchange (ETDEWEB)

    Tam, Phuong Dinh, E-mail: phuongdinhtam@gmail.com; Thang, Cao Xuan, E-mail: thang.caoxuan@hust.edu.vn

    2016-01-01

    This paper developed a label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application. The CeO{sub 2} nanowires were synthesized by hydrothermal reaction. The immobilization of Anti-V. cholerae O1 onto CeO{sub 2} nanowire-deposited sensor was performed via an amino ester, which was created by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and sulfo-N-hydroxysuccinimide. The electrochemical responses of the immunosensor were studied by electrochemical impedance spectroscopy with [Fe (CN) {sub 6}] {sup 3−/4−} as redox probe. A linear response in electron transfer resistance for cell of V. cholerae O1 concentration was found in the range of 1.0 × 10{sup 2} CFU/mL to 1.0 × 10{sup 4} CFU/mL. The detection limit of the immunosensor was 1.0 × 10{sup 2} CFU/mL. The immunosensor sensitivity was 56.82 Ω/CFU·mL{sup −1}. Furthermore, the parameters affecting immunosensor response were also investigated, as follows: pH value, immunoreaction time, incubation temperature, and anti-V. cholerae O1 concentration. - Highlights: • A label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application was developed. • A linear response was found in the range of 1.0 × 10{sup 2} CFU/mL to 1.0 × 10{sup 4} CFU/mL. • The detection limit of the immunosensor was 1.0 × 10{sup 2} CFU/mL. • The immunosensor sensitivity was 56.82 Ω/CFU.mL{sup −1}.

  3. Label-free SnO2 nanowire FET biosensor for protein detection

    Science.gov (United States)

    Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

    2017-06-01

    Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

  4. Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

    Science.gov (United States)

    Gao, Shuyan; Jia, Xiaoxia; Chen, Yanli

    2013-01-01

    Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag+/Ag (0.7996 V) and Hg2+/Hg2 2+ (0.920 V) that makes colorless Hg2+ ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg2+ ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg2+ ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg2+ ions sensing motif shows great promise for sensing Hg2+ ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

  5. A microfluidic laser scattering sensor for label-free detection of waterborne pathogens

    Science.gov (United States)

    Wei, Huang; Yang, Limei; Li, Feng

    2016-10-01

    A microfluidic-based multi-angle laser scattering (MALS) sensor capable of acquiring scattering pattern of single particle is demonstrated. The size and relative refractive index (RI) of polystyrene (PS) microspheres were deduced with accuracies of 60 nm and 0.001 by analyzing the scattering patterns. We measured scattering patterns of waterborne parasites i.e., cryptosporidium parvum (c.parvum) and giardia lamblia (g.lamblia), and some other representative species in 1 L water within 1 hour, and the waterborne parasites were identified with accuracy better than 96% by classification of distinctive scattering patterns with a support-vector-machine (SVM) algorithm. The system provides a promising tool for label-free and rapid detection of waterborne parasites.

  6. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Science.gov (United States)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  7. A simple and sensitive approach for ochratoxin A detection using a label-free fluorescent aptasensor.

    Directory of Open Access Journals (Sweden)

    Zhenzhen Lv

    Full Text Available Ochratoxin A(OTA is found to be one of the predominant contaminating mycotoxins in a wide variety of food commodities. To avoid the risk of OTA consumption, the detection and quantitation of OTA level are of great significance. Based on the fact that ssDNA aptamer has the ability to form a double-strand structure with its complementary sequence, a simple and rapid aptamer-based label-free approach for highly sensitive and selective fluorescence detection of OTA was developed by using ultra-sensitive double-strand DNA specific dyes PicoGreen. The results showed that as low as 1 ng/mL of OTA could be detected with a dynamic range of more than 5 orders of magnitude which satisfies the requirements for OTA maximum residue limit in various food regulated by European Commission. With the specificity of aptamer, the assay exhibited high selectivity for OTA against two other analogues (N-acetyl-l-phenylalanine and zearalenone. We also tested the aptasensor practicability using real sample of 1% beer spiked with a series of concentration of OTA and the results show good tolerance to matrix effect. All detections could be achieved in less than 30 min, which provides a simple, quick and sensitive detection method for OTA screening in food safety and could be easily extend to other small molecular chemical compounds detection which aptamer has been selected.

  8. A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

    Science.gov (United States)

    Sykora, Sabine; Correro, M Rita; Moridi, Negar; Belliot, Gaël; Pothier, Pierre; Dudal, Yves; Corvini, Philippe F-X; Shahgaldian, Patrick

    2017-06-01

    The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Sensitive and label-free detection of miRNA-145 by triplex formation.

    Science.gov (United States)

    Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

  10. A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries.

    Science.gov (United States)

    Mishra, Rupesh K; Hayat, Akhtar; Catanante, Gaëlle; Ocaña, Cristina; Marty, Jean-Louis

    2015-08-19

    Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15-2.5 ng/mL, with an acceptable recovery percentage (91-95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection

    Science.gov (United States)

    Patel, Manoj Kumar; Ali, Md. Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, AbdulAziz A.; Fouad, H.; Ansari, Z.A.; Ansari, S. G.; Malhotra, Bansi D.

    2015-01-01

    Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R2) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera. PMID:26611737

  12. Porous silicon membrane-modified electrodes for label-free voltammetric detection of MS2 bacteriophage.

    Science.gov (United States)

    Reta, Nekane; Michelmore, Andrew; Saint, Christopher; Prieto-Simón, Beatriz; Voelcker, Nicolas H

    2016-06-15

    A proof of concept for the label-free detection of bacteriophage MS2, a model indicator of microbiological contamination, is validated in this work as a porous silicon (pSi) membrane-based electrochemical biosensor. PSi membranes were used to afford nanochannel architectures. The sensing mechanism was based on the nanochannel blockage caused by MS2 binding to immobilized capture antibodies. This blockage was quantified by measuring the oxidation current of the electroactive species reaching the electrode surface, by means of differential pulse voltammetry (DPV). The immunosensor showed a limit of detection of 6 pfu/mL in buffer, allowing the detection of MS2 to levels commonly found in real-world applications, and proved to be unaffected by matrix effects when analyzing MS2 in reservoir water. This platform enables the straightforward, direct and sensitive detection of a broad range of target analytes and constitutes a promising approach towards the development of portable electronic point of sample analysis devices.

  13. Label-free electronic detection of bio-toxins using aligned carbon nanotubes.

    Science.gov (United States)

    Palaniappan, Al; Goh, W H; Fam, D W H; Rajaseger, G; Chan, C E Z; Hanson, B J; Moochhala, S M; Mhaisalkar, S G; Liedberg, B

    2013-05-15

    A facile route for sensitive label-free detection of bio-toxins using aligned single walled carbon nanotubes is described. This approach involves patterning of a catalyst on the surface of a quartz substrate using a sub-100 μm stripe-patterned polydimethylsiloxane stamp for aligned carbon nanotube generation followed by fabrication of field effect transistor (FET). Atomic force microscopy, field emission scanning electron microscopy and Raman spectroscopy are employed to characterize the synthesized nanotubes. Unlike previous reports, the adopted approach enables direct electronic detection of bio-toxins with sensitivities comparable to ELISA. As a proof of concept, the fabricated FET responds to nM concentration levels (with a LOD of ∼2 nM) of epsilon toxin produced by Clostridium perfringens and a prominent food toxin. This facile approach could be customized to detect other classes of toxins and biomarkers upon appropriate functionalization of the aligned carbon nanotubes. Finally, we demonstrate the use of the FET-platform for detection of toxin in more complex matrices such as orange juice.

  14. Glycosylation of quinone-fused polythiophene for reagentless and label-free detection of E. coli.

    Science.gov (United States)

    Ma, Fen; Rehman, Abdul; Liu, Haiying; Zhang, Jingtuo; Zhu, Shilei; Zeng, Xiangqun

    2015-02-03

    In this report, a new polythiophene interface is fabricated containing fused quinone moieties which are then glycosylated to form a carbohydrate platform for bacterial detection. Very importantly, this interface can be used for label-free and reagentless detection, both by electrochemical and Quartz Crystal Microbalance (QCM) transducers and by using the direct pili-mannose binding as well as Concanavalin A (Con A) mediated lipopolysaccharides (LPS)-mannose binding. The conductive polymer's unique collective properties are very sensitive to very minor perturbations, which result in significant changes of electrical conductivity and providing amplified sensitivity and improved limits of detection (i.e., 25 cell/mL for electrochemical sensor and 50 cells/mL for QCM sensor), a widened logarithmic range of detection (i.e., 3-7 for pili-mannose binding and 2-8 for Con A mediated binding), high specificity and selectivity, and an extraordinary reliability by a mechanism of internal validation. With these analytical performances, the described biosensor is envisaged for being capable of differentiating Gram-negative bacterial strain and species, for many important applications.

  15. A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection

    Science.gov (United States)

    Patel, Manoj Kumar; Ali, Md. Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, Abdulaziz A.; Fouad, H.; Ansari, Z. A.; Ansari, S. G.; Malhotra, Bansi D.

    2015-11-01

    Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R2) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

  16. A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection.

    Science.gov (United States)

    Patel, Manoj Kumar; Ali, Md Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, AbdulAziz A; Fouad, H; Ansari, Z A; Ansari, S G; Malhotra, Bansi D

    2015-11-27

    Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R(2)) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

  17. A Label-Free Immunosensor for Ultrasensitive Detection of Ketamine Based on Quartz Crystal Microbalance

    Directory of Open Access Journals (Sweden)

    Ya Yang

    2015-04-01

    Full Text Available In this study, we have developed a label-free immunosensor with the variation of resonance frequency (Δf of a quartz crystal microbalance (QCM as readout signal for ultrasensitive detection of Ketamine (KT. An optimized strategy for immobilization of KT antibody on the surface of the QCM chip was implemented via the self-assembly modification of 3-mercaptopropionic acid, and then activated with 1-ethyl-3- (3-dimethylaminoprophl carbodiimide and n-hydroxysuccinimide. The specific affinity between the antibody and the antigen ensured a selective response toward KT. The Δf linearly related to the concentration of KT in the range of 1 to 40 pg/mL, with a detection limit of 0.86 pg/mL (S/N = 3. The obtained immunosensor was applied to detect the KT in spiked human urine without any pretreatment but dilution with recoveries from 91.8% to 108%. The developed sensor is promising to perform the portable or on-spot KT detection in clinic or forensic cases.

  18. Label-free signal-on aptasensor for sensitive electrochemical detection of arsenite.

    Science.gov (United States)

    Cui, Lin; Wu, Jie; Ju, Huangxian

    2016-05-15

    A signal-on aptasensor was fabricated for highly sensitive and selective electrochemical detection of arsenite with a label-free Ars-3 aptamer self-assembled on a screen-printed carbon electrode (SPCE) via Au-S bond. The Ars-3 aptamer could adsorb cationic polydiallyldimethylammonium (PDDA) via electrostatic interaction to repel other cationic species. In the presence of arsenite, the change of Ars-3 conformation due to the formation of Ars-3/arsenite complex led to less adsorption of PDDA, and the complex could adsorb more positively charged [Ru(NH3)6](3+) as an electrochemically active indicator on the aptasensor surface, which produced a sensitive "turn-on" response. The target-induced structure switching could be used for sensitive detection of arsenite with a linear range from 0.2 nM to 100 nM and a detection limit down to 0.15 nM. Benefiting from Ars-3 aptamer, the proposed system exhibited excellent specificity against other heavy metal ions. The SPCE-based aptasensor exhibited the advantages of low cost and simple fabrication, providing potential application of arsenite detection in environment.

  19. Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance

    Directory of Open Access Journals (Sweden)

    Adrián Martínez-Rivas

    2010-09-01

    Full Text Available Adrián Martínez-Rivas1,2, Patrick Chinestra3,4, Gilles Favre3,4, Sébastien Pinaud1, Childérick Séverac1,2, Jean-Charles Faye3,4, Christophe Vieu1,21LAAS-CNRS; Université de Toulouse, Toulouse, France; 2Université de Toulouse, UPS, INSA, INP, ISAE; LAAS; Toulouse, France; 3INSERM U563, Université de Toulouse, CPTP, “Signalisation Cellulaire, GTPase Rho et cancers”, Toulouse, France; 4Institut Claudius Regaud, Biology Department, Toulouse, FranceAbstract: We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane (<2 nm thick and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules.Keywords: nickel nanoislands, cancer biomarkers, quartz crystal microbalance, PEG-silane, RhoA protein, nanobiosensor

  20. Label free selective detection of estriol using graphene oxide-based fluorescence sensor

    Science.gov (United States)

    Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

    2014-07-01

    Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

  1. A sensitive, label-free electrochemical detection of telomerase activity without modification or immobilization.

    Science.gov (United States)

    Liu, Xu; Wei, Min; Xu, Ensheng; Yang, Haitang; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2017-05-15

    Telomerase has become one of the most typical tumor marker because it is closely related to cancers. In this paper, a simple label-free electrochemical detection of telomerase activity by using methylene blue (MB) as a G-quadruplex binding probe was proposed, avoiding commonly used complex label procedures, nano-probe synthesis, complicated electrode modification, probe immobilization or signal amplification. In the presence of telomerase substrate (TS) primer, the binding of MB on primer was weak. When repeats of (TTAGGG) were extended on the TS primer under the action of telomerase, they formed multiple G-quadruplexes with the help of K(+). As a result, a large amount of MB bounded to multiple G-quadruplexes because they have more strong interaction with G-quadruplexes than TS primer. As a result, the diffusion current of MB decreased sharply, which was strongly dependent on the telomerase activity. The DPV current change has a linear correlation with the logarithm of HeLa cell number in the range of 10-10,000 cells, with the detection limit of 3 cells. The high sensitivity was due to the formed multiple G-quadruplexes. Using indium tin oxide (ITO) as working electrode without modification ensured the good reproducibility of the method. The method was also simple, rapid, and has been successfully applied in the telomerase activity detection in urine with good selectivity and reproducibility, which is significant for cancer diagnosis, anticancer drugs screening, and cancer therapy evaluation.

  2. Handheld imaging photonic crystal biosensor for multiplexed, label-free protein detection.

    Science.gov (United States)

    Jahns, Sabrina; Bräu, Marion; Meyer, Björn-Ole; Karrock, Torben; Gutekunst, Sören B; Blohm, Lars; Selhuber-Unkel, Christine; Buhmann, Raymund; Nazirizadeh, Yousef; Gerken, Martina

    2015-10-01

    We present a handheld biosensor system for the label-free and specific multiplexed detection of several biomarkers employing a spectrometer-free imaging measurement system. A photonic crystal surface functionalized with multiple specific ligands forms the optical transducer. The photonic crystal slab is fabricated on a glass substrate by replicating a periodic grating master stamp with a period of 370 nm into a photoresist via nanoimprint lithography and deposition of a 70-nm titanium dioxide layer. Capture molecules are coupled covalently and drop-wise to the photonic crystal surface. With a simple camera and imaging optics the surface-normal transmission is detected. In the transmission spectrum guided-mode resonances are observed that shift due to protein binding. This shift is observed as an intensity change in the green color channel of the camera. Non-functionalized image sections are used for continuous elimination of background drift. In a first experiment we demonstrate the specific and time-resolved detection of 90.0 nm CD40 ligand antibody, 90.0 nM EGF antibody, and 500 nM streptavidin in parallel on one sensor chip. In a second experiment, aptamers with two different spacer lengths are used as receptor. The binding kinetics with association and dissociation of 250 nM thrombin and regeneration of the sensor surface with acidic tris-HCl-buffer (pH 5.0) is presented for two measurement cycles.

  3. Label-free detection of sex determining region Y (SRY) via capacitive biosensor

    KAUST Repository

    Sivashankar, Shilpa

    2016-10-20

    In this work, we present for the first time, the use of a simple fractal capacitive biosensor for the quantification and detection of sex-determining region Y (SRY) genes. This section of genetic code, which is found on the Y chromosome, finds importance for study as it causes fetuses to develop characteristics of male sex-like gonads when a mutation occurs. It is also an important genetic code in men, and disorders involving the SRY gene can cause infertility and sexual malfunction that lead to a variety of gene mutational disorders. We have therefore designed silicon-based, label-free fractal capacitive biosensors to quantify various proteins and genes. We take advantage of a good dielectric material, Parylene C for enhancing the performance of the sensors. We have integrated these sensors with a simple microchannel for easy handling of fluids on the detection area. The read-out value of an Agilent LCR meter used to measure capacitance of the sensor at a frequency of 1 MHz determined gene specificity and gene quantification. These data revealed that the capacitance measurement of the capacitive biosensor for the SRY gene depended on both the target and the concentration of DNA. The experimental outcomes in the present study can be used to detect DNA and its variations in crucial fields that have a great impact on our daily lives, such as clinical and veterinary diagnostics, industrial and environmental testing and forensic sciences.

  4. Label-free bacteria detection using evanescent mode of a suspended core terahertz fiber

    CERN Document Server

    Mazhorova, Anna; Ng, Andy; Chinnappan, Raja; Zourob, Mohammed; Skorobogatiy, Maksim

    2011-01-01

    We propose for the first time an E. coli bacteria sensor based on the evanescent field of the fundamental mode of a suspended-core terahertz fiber. The sensor is capable of E. coli detection at concentrations in the range of 104-109 cfu/ml. The polyethylene fiber features a 150 {\\mu}m core suspended by three deeply sub-wavelength bridges in the center of a 5.1 mm-diameter cladding tube. The fiber core is biofunctionalized with T4 bacteriophages which bind and eventually destroy (lyse) their bacterial target. Using environmental SEM we demonstrate that E. coli is first captured by the phages on the fiber surface. After 25 minutes, most of the bacteria is infected by phages and then destroyed with ~1{\\mu}m-size fragments remaining bound to the fiber surface. The bacteria-binding and subsequent lysis unambiguously correlate with a strong increase of the fiber absorption. This signal allows the detection and quantification of bacteria concentration. Presented bacteria detection method is label-free and it does no...

  5. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

    2007-01-01

    We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format. PMID:17951434

  6. A label-free fluorescence turn-on sensor for rapid detection of cysteine.

    Science.gov (United States)

    Chen, Xia; Liu, Hongli; Wang, Chen; Hu, Hui; Wang, Yuhui; Zhou, Xiaodong; Hu, Jiming

    2015-06-01

    A Hg(2+)-mediated fluorescence turn-on sensor for cysteine (Cys) detection was developed using the nucleic acid minor groove binding dye DAPI. In this work, two fully complementary DNA sequences, a T-rich single-stranded molecule (ssDNA) and an A-rich single-stranded molecule, were employed to constitute consecutive "AT/TA" base pairs, which could strongly enhance the fluorescence of DAPI. In the absence of cysteine, Hg(2+) reacted with T-rich single-stranded DNA and "T-Hg(2+)-T" base pairs formed, this seriously disrupted consecutive AT base pairs. As a result, the fluorescence of DAPI was not increased efficiently. However, considering that cysteine binds strongly to Hg(2+), the structure of the "T-Hg(2+)-T" complexes was destroyed in the presence of cysteine, resulting in the re-formation of consecutive AT base pairs and increased DAPI fluorescence. Obviously, the amount of cysteine could be easily measured based on the enhancement of DAPI fluorescence, and it took only 20 min to complete the whole cysteine-sensing process. Therefore, a label-free fluorescent "turn-on" sensor for the rapid detection of cysteine was designed, and the detection limit of this sensor was as low as 2.4 nM, which was much lower than those of the most of the previously reported cysteine sensors.

  7. A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

    Science.gov (United States)

    Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

    2016-03-01

    Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

  8. Label free detection of white spot syndrome virus using lead magnesium niobate-lead titanate piezoelectric microcantilever sensors.

    Science.gov (United States)

    Capobianco, Joseph A; Shih, Wei-Heng; Leu, Jiann-Horng; Lo, Grace Chu-Fang; Shih, Wan Y

    2010-11-15

    We have investigated rapid, label free detection of white spot syndrome virus (WSSV) using the first longitudinal extension resonance peak of five lead-magnesium niobate-lead titanate (PMN-PT) piezoelectric microcantilever sensors (PEMS) 1050-700 μm long and 850-485 μm wide constructed from 8 μm thick PMN-PT freestanding films. The PMN-PT PEMS were encapsulated with a 3-mercaptopropyltrimethoxysilane (MPS) insulation layer and further coated with anti-VP28 and anti-VP664 antibodies to target the WSSV virions and nucleocapsids, respectively. By inserting the antibody coated PEMS in a flowing virion or nucleocapsid suspension, label free detection of the virions and nucleocapsids were respectively achieved by monitoring the PEMS resonance frequency shift. We showed that positive label free detection of both the virion and the nucleocapsid could be achieved at a concentration of 100virions(nucleocapsids)/ml or 10 virions(nucleocapsids)/100 μl, comparable to the detection sensitivity of polymerase chain reaction (PCR). However, in contrast to PCR, PEMS detection was label free, in situ and rapid (less than 30 min), potentially requiring minimal or no sample preparation.

  9. Towards a high-throughput label-free detection system combining localized-surface plasmon resonance and microfluidics.

    Science.gov (United States)

    Zhang, Yi; Tang, Yunfang; Hsieh, Yi-Heui; Hsu, Chuen-Yuan; Xi, Jianzhong; Lin, Kuan-Jiuh; Jiang, Xingyu

    2012-09-07

    This work reports an integrated platform combining localized-surface plasmon resonance (LSPR) and microfluidic chips to carry out multiplexed and label-free protein analysis. We fabricated an optical bench to enable detection using only a laboratory UV-Vis spectrophotometer. This assay not only improves throughput, but also allows quantitative analysis.

  10. Label-free detection in a lab-on-a-chip with a three-dimensional Mach-Zehnder interferometer

    NARCIS (Netherlands)

    Crespi, A.; Gu, Y.; Ngamson, B.; Dongre, C.; Hoekstra, H.J.W.M.; Vlekkert, van den H.H.; Watts, P.; Pollnau, M.; Cerullo, G.; Osellame, R.

    2010-01-01

    A Mach-Zehnder refractive index sensor is inscribed in a microfluidic lab-on-a-chip by exploiting the unique three-dimensional capabilities of femtosecond laser fabrication. This enables high sensitivity and spatially resolved label-free detection of biomolecules.

  11. Detection of Myoglobin with an Open-Cavity-Based Label-Free Photonic Crystal Biosensor.

    Science.gov (United States)

    Zhang, Bailin; Tamez-Vela, Juan Manuel; Solis, Steven; Bustamante, Gilbert; Peterson, Ralph; Rahman, Shafiqur; Morales, Andres; Tang, Liang; Ye, Jing Yong

    2013-01-01

    The label-free detection of one of the cardiac biomarkers, myoglobin, using a photonic-crystal-based biosensor in a total-internal-reflection configuration (PC-TIR) is presented in this paper. The PC-TIR sensor possesses a unique open optical microcavity that allows for several key advantages in biomolecular assays. In contrast to a conventional closed microcavity, the open configuration allows easy functionalization of the sensing surface for rapid biomolecular binding assays. Moreover, the properties of PC structures make it easy to be designed and engineered for operating at any optical wavelength. Through fine design of the photonic crystal structure, biochemical modification of the sensor surface, and integration with a microfluidic system, we have demonstrated that the detection sensitivity of the sensor for myoglobin has reached the clinically significant concentration range, enabling potential usage of this biosensor for diagnosis of acute myocardial infarction. The real-time response of the sensor to the myoglobin binding may potentially provide point-of-care monitoring of patients and treatment effects.

  12. Detection of Myoglobin with an Open-Cavity-Based Label-Free Photonic Crystal Biosensor

    Directory of Open Access Journals (Sweden)

    Bailin Zhang

    2013-01-01

    Full Text Available The label-free detection of one of the cardiac biomarkers, myoglobin, using a photonic-crystal-based biosensor in a total-internal-reflection configuration (PC-TIR is presented in this paper. The PC-TIR sensor possesses a unique open optical microcavity that allows for several key advantages in biomolecular assays. In contrast to a conventional closed microcavity, the open configuration allows easy functionalization of the sensing surface for rapid biomolecular binding assays. Moreover, the properties of PC structures make it easy to be designed and engineered for operating at any optical wavelength. Through fine design of the photonic crystal structure, biochemical modification of the sensor surface, and integration with a microfluidic system, we have demonstrated that the detection sensitivity of the sensor for myoglobin has reached the clinically significant concentration range, enabling potential usage of this biosensor for diagnosis of acute myocardial infarction. The real-time response of the sensor to the myoglobin binding may potentially provide point-of-care monitoring of patients and treatment effects.

  13. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  14. Label-free detection of DNA hybridization using InAs μ-Hall sensors

    Science.gov (United States)

    Aledealat, Khaled; Hira, S.; Chen, K.; Strouse, G. F.; Chase, P. B.; Xiong, P.; von Molnar, S.; Mihajlovic, G.; Field, M.; Sullivan, G.

    2010-03-01

    We present results on label-free detection of DNA hybridization using InAs μ-Hall sensors. The μ-Hall sensor consisted of six 1-μm Hall crosses defined on an InAs quantum well substrate. The sensor was then covered with sputter-deposited SiO2 and Au pads were patterned on top of some of the Hall crosses. Thiolated ssDNA strands that are complementary to one end of the target ssDNA were assembled on the Au pads and the rest of the device platform was passivated with PEG-silane. Biotinylated and fluorescently-tagged complementary ssDNA to the other end of the target ssDNA were labeled with commercial streptavidin-coated 350 nm superparamagnetic beads. Labeled ssDNA were found to assemble selectively onto the Au pads after mixing with the target ssDNA, indicating successful hybridization of the three ssDNA sequences. The presence of the assembled beads was successfully detected via the Hall sensor and confirmed using laser scanning confocal microscopy. This work was supported by NIH NIGMS GM079592.

  15. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  16. Label-Free Detection of Ag+ Based on Gold Nanoparticles and Ag+-Specific DNA.

    Science.gov (United States)

    Pu, Wendan; Zhao, Zhao; Wu, Liping; Liu, Yue; Zhao, Huawen

    2015-08-01

    A sensitive label-free method was presented for the determination of silver ion (Ag+) in this paper. Cytosine-rich DNA (C-DNA) was used as Ag+ specific DNA. Without Ag+ in the solution, fluorescence of fluorescein (FAM) is quenched by C-DNA stabilized gold nanoparticles (AuNPs) in high salt environment. When Ag+ is present in the solution, however, Ag+-mediated cytosine-Ag+-cytosine (C-Ag+-C) base pairs induced the C-DNA folding into a hairpin structure, which can not stabilize AuNPs in high salt environment, thus causing AuNPs aggregation. After centrifugation to remove the aggregated AuNPs, the quenching ability of the supernatant for FAM is decreased and the fluorescence intensity of solution increases with increasing the Ag+ concentration. Due to the highly specific interaction of the C-DNA towards Ag+ and the strong fluorescent quenching ability of AuNPs for FAM, the method has high selectivity and sensitivity for Ag+. Under the optimal conditions, the fluorescence intensity at 515 nm increased linearly with the concentration of Ag+ ranging from 15 nM to 700 nM, and the detection limit was determined as 6 nM based on 3 σ/slope. This method is simple, sensitive, and may be applied to other detection systems by selecting the appropriate DNA sequences.

  17. Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice.

    Science.gov (United States)

    Miao, Hong; Wang, Lan; Zhuo, Yan; Zhou, Zinan; Yang, Xiaoming

    2016-12-15

    A facile-green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 13.9% has been built up, while tomato juice served as the carbon source. Interestingly, not only the precursor of CDs and the whole synthesis procedure were environmental-friendly, but this type of CDs also exhibited multiple advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Significantly, a label-free sensitive assay for detecting carcinoembryonic antigen (CEA) in a continuous and recyclable way has been proposed on the basis of adsorption and desorption of aptamers by the surface of CDs through a competitive mechanism. To be specific, the richness of carboxyl groups of the CDs enabled strong adsorption of ssDNA to the surface of CDs through π-π stacking interactions, resulting in the effective fluorescence quenching by forming CDs-aptamer complexes. The stronger binding affinity between CEA and CEA-aptamer than the π-π stacking interactions has been taken advantage to achieve immediate recovery of the fluorescence of CDs once CEA was introduced. Thereby, quantitative evaluation of CEA concentration in a broad range from 1ngmL(-1) to 0.5ngmL(-1) with the detection limit of 0.3ngmL(-1) was realized in this way. This strategy can be applied in a recyclable way, broadening the sensing application of CDs with biocompatibility. Besides, the CDs were used for cell imaging, potentiating them towards diverse purposes.

  18. Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy.

    Science.gov (United States)

    Liang, Gang; Man, Yan; Jin, Xinxin; Pan, Ligang; Liu, Xinhui

    2016-09-14

    A label-free sensing assay for ethanolamine (EA) detection based on G-quadruplex-EA binding interaction is presented by using G-rich aptamer DNA (Ap-DNA) and electrochemical impedance spectroscopy (EIS). The presence of K(+) induces the Ap-DNA to form a K(+)-stabilized G-quadruplex structure which provides binding sites for EA. The sensing mechanism was further confirmed by circular dichroism (CD) spectroscopy and EIS measurement. As a result, the charge transfer resistance (RCT) is strongly increased as demonstrated by using the ferro/ferricyanide ([Fe(CN)6](3-/4-)) as a redox probe. Under the optimized conditions, a linear relationship between ΔRCT and EA concentration was obtained over the range of 0.16 nM and 16 nM EA, with a detection limit of 0.08 nM. Interference by other selected chemicals with similar structure was negligible. Analytical results of EA spiked into tap water and serum by the sensor suggested the assay could be successfully applied to real sample analysis. With the advantages of high sensitivity, selectivity and simple sensor construction, this method is potentially suitable for the on-site monitoring of EA contamination.

  19. A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Rupesh K. [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Hayat, Akhtar [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS Institute of Information Technology (CIIT), Lahore 54000 (Pakistan); Catanante, Gaëlle; Ocaña, Cristina [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Marty, Jean-Louis, E-mail: jlmarty@univ-perp.fr [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France)

    2015-08-19

    Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up. - Highlights: • Simple and facile method to detect OTA. • The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL. • The first report on OTA detection in cocoa beans using impedimetric aptasensor.

  20. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    Science.gov (United States)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  1. New function of exonuclease and highly sensitive label-free colorimetric DNA detection.

    Science.gov (United States)

    Li, Hongbo; Wang, Suqin; Wu, Zaisheng; Xu, Jianguo; Shen, Guoli; Yu, Ruqin

    2016-03-15

    Enzymatic manipulation and modulation of nucleic acids are a central part of cellular function, protection, and reproduction, while rapid and accurate detection of ultralow amount of nucleic acids remains a major challenge in molecular biology research and clinic diagnosis of genetic diseases. Herein, we reported that exonuclease III can degrade the G-quadruplex structure, indicating the new exonuclease's function. Basing on the function of exonuclease III, a novel G-quadruplex-hemin DNAzyme-based colorimetric detection of tumor suppressor gene p53 was successfully developed. Although only one oligonucleotide probe was involved, the sensing strategy could suppress the optical background and achieve an efficient G-quadruplex-hemin DNAzyme-based signal amplification. Specifically, a label-free functional nucleic acid probe (called THzyme probe) was designed via introducing target DNA probe-contained hairpin structure into G-quadruplex DNAzyme. Even if this probe can fold into G-quadruplex structure in the presence of hemin very different from the double-stranded DNA, it is easily degraded by exonuclease III. Thus, no change in UV-vis absorption intensity is detected in the absence of target DNA. However, the hybridization of target DNA can protect the integrity and catalytic activity of THzyme probe, producing the DNAzyme-amplified colorimetric signal. As a result, the p53 gene was able to be detected down to 1.0 pM (final concentration in the signal-generating solution: 50.0 fM) and mismatched target DNAs were easily distinguished. It is expected that this simple sensing methodology for DNA detection can find its utility in point-of-care applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy

    Science.gov (United States)

    Ji, Minbiao; Orringer, Daniel A.; Freudiger, Christian W.; Ramkissoon, Shakti; Liu, Xiaohui; Lau, Darryl; Golby, Alexandra J.; Norton, Isaiah; Hayashi, Marika; Agar, Nathalie Y.R.; Young, Geoffrey S.; Spino, Cathie; Santagata, Sandro; Camelo-Piragua, Sandra; Ligon, Keith L.; Sagher, Oren; Xie, X. Sunney

    2013-01-01

    Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. Here we describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from non-neoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and H&E microscopy for detection of glioma infiltration (κ=0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct. PMID:24005159

  3. Ultrasensitive, label-free detection of cardiac biomarkers with optical SIS sensor.

    Science.gov (United States)

    Diware, Mangesh S; Cho, Hyun Mo; Chegal, Won; Cho, Yong Jai; Kim, Dong Soo; O, Sang Won; Kim, Kyeong-Suk; Paek, Se-Hwan

    2017-01-15

    Acute myocardial infarction (MI) is the leading cause of high mortality and morbidity rate worldwide, early and accurate diagnosis can increase the chances of survival. In this work, we report a simple, ultrasensitive, label-free, and high-throughput solution immersed silicon (SIS) immunosensor based on non-reflection condition (NRC) for p-polarized wave for early diagnosis of MI. SIS sensor chips are just a thin dielectric polymer layer on the silicon surface, which can be functionalized for specific application. At NRC, SIS sensors are extremely sensitive to the growing thickness of a bio-layer on the sensor surface while independent of refractive index change of the surrounding medium. Therefore, SIS signal is free from thermal noise, unlike surface plasmon resonance based sensor. Also, there is no need of reference signal which facilitates fast and accurate interaction measurement. Here, SIS technology is applied to tackle two issues in MI diagnosis: high sensitivity with the direct assay and the ability to measure in human serum. Myoglobin, creatine kinase-MB, and cardiac troponin I (cTnI) proteins were used as the MI biomarkers. We were able to measure over a broad concentration range with the detection limit of 5 and 10pg/ml for cTnI in PBS and blood serum, respectively. The response time is about 5min. This novel technique is a suitable candidate for cost effective point-of-care application.

  4. Tapered Optical Fiber Sensor for Label-Free Detection of Biomolecules

    Directory of Open Access Journals (Sweden)

    Xingwei Wang

    2011-03-01

    Full Text Available This paper presents a fast, highly sensitive and low-cost tapered optical fiber biosensor that enables the label-free detection of biomolecules. The sensor takes advantage of the interference effect between the fiber’s first two propagation modes along the taper waist region. The biomolecules bonded on the taper surface were determined by demodulating the transmission spectrum phase shift. Because of the sharp spectrum fringe signals, as well as a relatively long biomolecule testing region, the sensor displayed a fast response and was highly sensitive. To better understand the influence of various biomolecules on the sensor, a numerical simulation that varied biolayer parameters such as thickness and refractive index was performed. The results showed that the spectrum fringe shift was obvious to be measured even when the biolayer was only nanometers thick. A microchannel chip was designed and fabricated for the protection of the sensor and biotesting. Microelectromechanical systems (MEMS fabrication techniques were used to precisely control the profile and depth of the microchannel on the silicon chip with an accuracy of 2 μm. A tapered optical fiber biosensor was fabricated and evaluated with an Immune globulin G (IgG antibody-antigen pair.

  5. Singular phase nano-optics in plasmonic metamaterials for label-free single-molecule detection.

    Science.gov (United States)

    Kravets, V G; Schedin, F; Jalil, R; Britnell, L; Gorbachev, R V; Ansell, D; Thackray, B; Novoselov, K S; Geim, A K; Kabashin, A V; Grigorenko, A N

    2013-04-01

    The non-trivial behaviour of phase is crucial for many important physical phenomena, such as, for example, the Aharonov-Bohm effect and the Berry phase. By manipulating the phase of light one can create 'twisted' photons, vortex knots and dislocations which has led to the emergence of the field of singular optics relying on abrupt phase changes. Here we demonstrate the feasibility of singular visible-light nano-optics which exploits the benefits of both plasmonic field enhancement and the peculiarities of the phase of light. We show that properly designed plasmonic metamaterials exhibit topologically protected zero reflection yielding to sharp phase changes nearby, which can be employed to radically improve the sensitivity of detectors based on plasmon resonances. By using reversible hydrogenation of graphene and binding of streptavidin-biotin, we demonstrate an areal mass sensitivity at a level of fg mm(-2) and detection of individual biomolecules, respectively. Our proof-of-concept results offer a route towards simple and scalable single-molecule label-free biosensing technologies.

  6. Ultrasensitive and label-free molecular level detection enabled by light phase control in magnetoplasmonic nanoantennas

    Science.gov (United States)

    Maccaferri, Nicolò; Gregorczyk, Keith; de Oliveira, Thales V. A. G.; Kataja, Mikko; van Dijken, Sebastiaan; Pirzadeh, Zhaleh; Dmitriev, Alexandre; Åkerman, Johan; Knez, Mato; Vavassori, Paolo

    2015-01-01

    Systems allowing label-free molecular detection are expected to have enormous impact on biochemical sciences. Research focuses on materials and technologies based on exploiting localized surface plasmon resonances in metallic nanostructures. The reason for this focused attention is their suitability for single molecule sensing, arising from intrinsically nanoscopic sensing volume, and the high sensitivity to the local environment. Here we propose an alternative route, which enables radically improved sensitivity compared torecently reported plasmon-based sensors. Such high sensitivity is achieved by exploiting the control of the phase of light in magnetoplasmonic nanoantennas. We demonstrate a manifold improvement of refractometric sensing figure-of-merit. Most remarkably, we show a raw surface sensitivity (i.e., without applying fitting procedures) of two orders of magnitude higher than the current values reported for nanoplasmonic sensors. Such sensitivity corresponds to a mass of ~0.8 ag per nanoantenna of polyamide-6.6 (n=1.51), which is representative for a large variety of polymers, peptides and proteins. PMID:25639190

  7. Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke.

    Science.gov (United States)

    Liu, Huanling; Jablonska, Anna; Li, Yuguo; Cao, Suyi; Liu, Dexiang; Chen, Hanwei; Van Zijl, Peter Cm; Bulte, Jeff W M; Janowski, Miroslaw; Walczak, Piotr; Liu, Guanshu

    2016-01-01

    ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion.

  8. Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke

    Science.gov (United States)

    Liu, Huanling; Jablonska, Anna; Li, Yuguo; Cao, Suyi; Liu, Dexiang; Chen, Hanwei; Van Zijl, Peter CM; Bulte, Jeff W.M.; Janowski, Miroslaw; Walczak, Piotr; Liu, Guanshu

    2016-01-01

    ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion. PMID:27446492

  9. A dual-channel detection of mercuric ions using a label free G-quadruplex-based DNAzyme molecule.

    Science.gov (United States)

    Ma, Long; Liu, Haiyan; Wu, Guanrong; Sun, Nana; Meng, Lingpei; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2016-06-20

    We have constructed a 'turn-off' and label free bio-sensor using a DNAzyme molecule. This facile bio-sensor is capable of selective detection of mercuric ions with a high sensitivity and satisfactory dynamic range. More importantly, it is able to generate both fluorescent and colourimetric signals for detection. This dual-channel bio-sensor is expected to afford high detection confidence and overcome false-positive readout especially when assaying complex biological samples.

  10. Whole-cell based label-free capacitive biosensor for rapid nanosize-dependent toxicity detection.

    Science.gov (United States)

    Qureshi, Anjum; Pandey, Ashish; Chouhan, Raghuraj S; Gurbuz, Yasar; Niazi, Javed H

    2015-05-15

    Despite intensive studies on examining the toxicity of nanomaterials (NMs), our current understanding on potential toxicity in relation to size and cellular responses has remained limited. In this work, we have developed a whole-cell based capacitive biosensor (WCB) to determine the biological toxicity of nanoparticles (NPs) using iron oxide (Fe3O4) NPs as models. This WCB chip comprised of an array of capacitor sensors made of gold interdigitated microelectrodes on which living Escherichia coli cells were immobilized. Cells-on-chip was then allowed to interact with different sizes of Fe3O4 NPs (5, 20 and 100 nm) and concentration-depended cellular-responses were measured in terms of change in dielectric properties (capacitance) as a function of applied AC frequency. The WCB response showed smaller-sized Fe3O4 NPs (5 nm) induced maximum change in surface capacitance because of their effective cellular interaction with E. coli cells-on-chip indicating that the cells suffered from severe cellular deformation, which was confirmed by scanning electron microscopic (SEM) examination. Further our results were validated through their cell viability and E. coli responses at the interface of cell-membrane and NPs as a proof-of-concept. WCB response showed a size-dependent shift in maximum response level from 2 µg/ml of 5 nm sized NPs to 4 µg/ml with NP-sizes greater than 20 nm. The developed WCB offered real-time, label-free and noninvasive detection of cellular responses against Fe3O4 NPs' toxicity with speed, simplicity and sensitivity that can be extended to toxicity screening of various other NPs. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Selection of an aptamer against Muscovy duck parvovirus for highly sensitive rapid visual detection by label-free aptasensor.

    Science.gov (United States)

    Lu, Taofeng; Ma, Qin; Yan, Wenzhuo; Wang, Yuanzhi; Zhang, Yuanyuan; Zhao, Lili; Chen, Hongyan

    2018-01-01

    Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in ducks. This study investigated a novel aptamer-based, label-free aptasensor detection of MDPV. In this study, we developed an ssDNA aptamer using the filtration partition and lambda exonuclease method with an affinity-based monitor and counter-screening process. After 15 rounds of SELEX (systematic evolution of ligands by exponential enrichment), the ssDNA aptamer Apt-10, which specifically bound to MDPV with high affinity (Kd = 467nM) was successfully screened, and the aptamer was also found to be good specific to MDPV. The selected Apt-10 aptamer can be used to distinguish MDPV and goose parvovirus (GPV). Three-dimensional structural analysis of the Apt-10 aptamer indicated that it folded into a compact stem-loop motif, which was related to its high affinity. Finally, a label-free detection method based on unmodified gold nanoparticles and Apt-10 aptamer was developed for MDPV determination. The concentration of Apt-10 aptamer at 5μM was optimal for MDPV determination in the label-free aptasensor. Excellent linearity was acquired and the lowest detection limit was 1.5 or 3 EID50 (50% egg infection dose) of MDPV, respectively, depending upon spectrophotometry or the naked eye were used. These results show the potential of the aptamer for the rapid detection of MDPV and antiviral research. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Single walled carbon nanotube-based electrical biosensor for the label-free detection of pathogenic bacteria

    DEFF Research Database (Denmark)

    Yoo, S. M.; Baek, Y. K.; Shin, S.

    2016-01-01

    We herein describe the development of a single-walled carbon nanotube (SWNT)-based electrical biosensor consisting of a two-terminal resistor, and report its use for the specific, label-free detection of pathogenic bacteria via changes in conductance. The ability of this biosensor to recognize....... This SWNT-based electrical biosensor will prove useful for the development of highly sensitive and specific handheld pathogen detectors....

  13. Direct and label-free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor.

    Science.gov (United States)

    González-Guerrero, Ana Belén; Maldonado, Jesús; Dante, Stefania; Grajales, Daniel; Lechuga, Laura M

    2017-01-01

    A label-free interferometric transducer showing a theoretical detection limit for homogeneous sensing of 5 × 10(-8) RIU, being equivalent to a protein mass coverage resolution of 2.8 fg mm(-2) , is used to develop a high sensitive biosensor for protein detection. The extreme sensitivity of this transducer combined with a selective bioreceptor layer enables the direct evaluation of the human growth hormone (hGH) in undiluted urine matrix in the 10 pg mL(-1) range.

  14. Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

    Science.gov (United States)

    Qi, Yingying; Li, Li; Li, Baoxin

    2009-09-01

    A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV-vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10 -13 to 4.5 × 10 -6 mol/L. The detection limit (3 σ) of this method has decreased to pico-molar level.

  15. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Chong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Han, Qiaorong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Wang, Daoying; Xu, Weimin [Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Wang, Weijuan [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhao, Wenbo, E-mail: zhaowenbo@njnu.edu.cn [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhou, Min, E-mail: zhouminnju@126.com [Department of Vascular Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)

    2014-11-19

    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN){sub 6}]{sup 3−/4−}. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

  16. Label-free image-based detection of drug resistance with optofluidic time-stretch microscopy (Conference Presentation)

    Science.gov (United States)

    Kobayashi, Hirofumi; Lei, Cheng; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

    2017-02-01

    Acquired drug resistance is a fundamental predicament in cancer therapy. Early detection of drug-resistant cancer cells during or after treatment is expected to benefit patients from unnecessary drug administration and thus play a significant role in the development of a therapeutic strategy. However, the development of an effective method of detecting drug-resistant cancer cells is still in its infancy due to their complex mechanism in drug resistance. To address this problem, we propose and experimentally demonstrate label-free image-based drug resistance detection with optofluidic time-stretch microscopy using leukemia cells (K562 and K562/ADM). By adding adriamycin (ADM) to both K562 and K562/ADM (ADM-resistant K562 cells) cells, both types of cells express unique morphological changes, which are subsequently captured by an optofluidic time-stretch microscope. These unique morphological changes are extracted as image features and are subjected to supervised machine learning for cell classification. We hereby have successfully differentiated K562 and K562/ADM solely with label-free images, which suggests that our technique is capable of detecting drug-resistant cancer cells. Our optofluidic time-stretch microscope consists of a time-stretch microscope with a high spatial resolution of 780 nm at a 1D frame rate of 75 MHz and a microfluidic device that focuses and orders cells. We compare various machine learning algorithms as well as various concentrations of ADM for cell classification. Owing to its unprecedented versatility of using label-free image and its independency from specific molecules, our technique holds great promise for detecting drug resistance of cancer cells for which its underlying mechanism is still unknown or chemical probes are still unavailable.

  17. Label-free glucose detection using cantilever sensor technology based on gravimetric detection principles.

    Science.gov (United States)

    Hsieh, Shuchen; Hsieh, Shu-Ling; Hsieh, Chiung-Wen; Lin, Po-Chiao; Wu, Chun-Hsin

    2013-01-01

    Efficient maintenance of glucose homeostasis is a major challenge in diabetes therapy, where accurate and reliable glucose level detection is required. Though several methods are currently used, these suffer from impaired response and often unpredictable drift, making them unsuitable for long-term therapeutic practice. In this study, we demonstrate a method that uses a functionalized atomic force microscope (AFM) cantilever as the sensor for reliable glucose detection with sufficient sensitivity and selectivity for clinical use. We first modified the AFM tip with aminopropylsilatrane (APS) and then adsorbed glucose-specific lectin concanavalin A (Con A) onto the surface. The Con A/APS-modified probes were then used to detect glucose by monitoring shifts in the cantilever resonance frequency. To confirm the molecule-specific interaction, AFM topographical images were acquired of identically treated silicon substrates which indicated a specific attachment for glucose-Con A and not for galactose-Con A. These results demonstrate that by monitoring the frequency shift of the AFM cantilever, this sensing system can detect the interaction between Con A and glucose, one of the biomolecule recognition processes, and may assist in the detection and mass quantification of glucose for clinical applications with very high sensitivity.

  18. Label-Free Glucose Detection Using Cantilever Sensor Technology Based on Gravimetric Detection Principles

    Directory of Open Access Journals (Sweden)

    Shuchen Hsieh

    2013-01-01

    Full Text Available Efficient maintenance of glucose homeostasis is a major challenge in diabetes therapy, where accurate and reliable glucose level detection is required. Though several methods are currently used, these suffer from impaired response and often unpredictable drift, making them unsuitable for long-term therapeutic practice. In this study, we demonstrate a method that uses a functionalized atomic force microscope (AFM cantilever as the sensor for reliable glucose detection with sufficient sensitivity and selectivity for clinical use. We first modified the AFM tip with aminopropylsilatrane (APS and then adsorbed glucose-specific lectin concanavalin A (Con A onto the surface. The Con A/APS-modified probes were then used to detect glucose by monitoring shifts in the cantilever resonance frequency. To confirm the molecule-specific interaction, AFM topographical images were acquired of identically treated silicon substrates which indicated a specific attachment for glucose-Con A and not for galactose-Con A. These results demonstrate that by monitoring the frequency shift of the AFM cantilever, this sensing system can detect the interaction between Con A and glucose, one of the biomolecule recognition processes, and may assist in the detection and mass quantification of glucose for clinical applications with very high sensitivity.

  19. Instrument-Free Synthesizable Fabrication of Label-Free Optical Biosensing Paper Strips for the Early Detection of Infectious Keratoconjunctivitides.

    Science.gov (United States)

    Kim, Wansun; Lee, Jae-Chul; Shin, Jae-Ho; Jin, Kyung-Hyun; Park, Hun-Kuk; Choi, Samjin

    2016-05-17

    We introduce a surface-enhanced Raman scattering (SERS)-functionalized, gold nanoparticle (GNP)-deposited paper strip capable of label-free biofluid sensing for the early detection of infectious eye diseases. The GNP biosensing paper strip was fabricated by the direct synthesis and deposition of GNPs on wax-divided hydrophilic areas of a permeable porous substrate through a facile, power-free synthesizable, and highly reproducible successive ionic layer absorption and reaction (SILAR) technique. To maximize localized surface plasmon resonance-generated SERS activity, the concentration of the reactive solution and number of SILAR cycles were optimized by controlling the size and gap distance of GNPs and verified by computational modeling with geometrical hypotheses of Gaussian-estimated metallic nanoparticles. The responses of our SERS-functionalized GNP paper strip to Raman intensities exhibited an enhancement factor of 7.8 × 10(8), high reproducibility (relative standard deviation of 7.5%), and 1 pM 2-naphthalenethiol highly sensitive detection limit with a correlation coefficient of 0.99, achieved by optimized SILAR conditions including a 10/10 mM/mM HAuCl4/NaBH4 concentration and six SILAR cycles. The SERS-functionalized GNP paper is supported by a multivariate statistics-preprocessed machine learning-judged bioclassification system to provide excellent label-free chemical structure sensitivity for identifying infectious keratoconjunctivitis. The power-free synthesizable fabrication, label-free, rapid analysis, and high sensitivity feature of the SILAR-fabricated SERS-functionalized GNP biosensing paper strip makes it an excellent alternative in point-of-care applications for the early detection of various infectious diseases.

  20. Aptamer biosensors for label-free colorimetric detection of human IgE based on polydiacetylene (PDA) supramolecules.

    Science.gov (United States)

    Kim, Jun Pyo; Park, Cheol Hee; Sim, Sang Jun

    2011-05-01

    In this study, we demonstrate an aptamer-based biosensor (apta-biosensor) using PDA liposomes for label-free detection of allergy diagnosis by hIgE detection. In order to detect the target hIgE, the surface of PDA liposome were functionalized with hIgE antibody and anti-hIgE aptamer as a receptor, and the target hIgE onto the receptors was detected by the change of fluorescence signal. The hIgE antibody-modified PDA liposome biosensor had a serious problem that the immune reaction between receptor and target could not powerfully affect the change of florescence signal on PDA liposome. In order to solve this problem, the anti-hIgE aptamer which was far smaller than whole antibody was introduced as the receptor for the PDA liposome system. An aptamer-based PDA liposome biosensor was able to measure a quantity of target protein with various concentrations and at this time the detection limit was 141 ng/mL of the hIgE concentration. These results enabled diagnosis of allergy disease by an aptamer-based PDA liposome biosensor because real allergic patients showed high concentration of hIgE in serum (greater than 290 ng/mL). Therefore, we suggest that aptamer-modified PDA supramolecules as promising candidates for development of label-free colorimetric biosensors.

  1. Trifunctional fluorescent unnatural nucleoside: Label free detection of T-T/C-C base mismatches, abasic site and bulge DNA.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Pradhan, Manoj Kumar; Talukdar, Sangita

    2017-08-01

    The detection and targeting of both the mismatched and abasic DNA is highly important which would ultimately help in designing new diagnostics and chemotherapeutics. Furthermore, sensing and targeting the bulge sequence with a fluorescent probe would be useful to study the role of bulges in nucleic acid function or could have significant therapeutic potential. Thus, detection of specific bulges by small fluorescent molecules is an attractive research area since the past several years. Many attempts have been made to prepare such compounds. We report herein a label free strategy for the detection of pyrimidine base mismatches (T/T and C/C), sensing of abasic site, and pyrimidine base bulge DNA using an unnatural tetrazolylpyrene nucleoside ((TPy)B(Do)) as a bare fluorescent probe. The H-bonding/hydrophobic force mediated interactions allow the sensing of all three deformed DNA via an enhancement of fluorescence signal using our simple "Just-Mix and Read" strategy. The binding of the probe to all the three deformed DNA duplexes is accompanied by an increase in the thermal melting stability of the deformed DNAs. That the probe binds efficiently to the minor groove near the deformed site was evident from spectroscopic studies. All the spectral evidences open up a multitude of possibilities for using our probe, tetrazolylpyrene nucleoside, as an efficient fluorescent light-up bio-probe for label free DNA detection. Copyright © 2017. Published by Elsevier B.V.

  2. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection.

    Science.gov (United States)

    Sun, Chong; Han, Qiaorong; Wang, Daoying; Xu, Weimin; Wang, Weijuan; Zhao, Wenbo; Zhou, Min

    2014-11-19

    In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)6](3-/4-). Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10fM-100nM) and a detection limit on the order of 0.90fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

  3. A novel label-free electrochemical immunosensor based on functionalized nitrogen-doped graphene quantum dots for carcinoembryonic antigen detection.

    Science.gov (United States)

    Yang, Yuying; Liu, Qing; Liu, Yan; Cui, Jianjian; Liu, Hui; Wang, Ping; Li, Yueyun; Chen, Lei; Zhao, Zengdian; Dong, Yunhui

    2017-04-15

    A novel and ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of carcino-embryonic antigen (CEA). The nitrogen-doped graphene quantum dots (N-GQDs) supported PtPd bimetallic nanoparticles (PtPd/N-GQDs) were synthesized by a simple and green hydrothermal procedure. Subsequently, PtPd/N-GQDs functionalized Au nanoparticles (PtPd/N-GQDs@Au) were prepared successfully via a self-assembly approach. Because of the synergetic effect present in PtPd/N-GQDs@Au, this novel nanocomposites has shown excellent electrocatalytic activity towards hydrogen peroxide (H2O2) reduction. Featuring good biocompatibility, excellent conductivity and large surface area, PtPd/N-GQDs@Au was applied as transducing materials to efficiently conjugate capture antibodies and amplify electrochemical signal. Under the optimal conditions, the proposed immunosensor was used for the detection of CEA with wide dynamic range in the range from 5 fg/mL to 50ng/mL with a low detection limit of 2fg/mL (S/N=3). Furthermore, this label-free immunosensor possesses high sensitivity, special selectivity and long-term stability, which shows promising application in bioassay analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Label-free detection of 3-nitro-l-tyrosine with nickel-doped graphene localized surface plasmon resonance biosensor.

    Science.gov (United States)

    Ng, Siu Pang; Qiu, Guangyu; Ding, Ning; Lu, Xiaoqing; Wu, Chi-Man Lawrence

    2017-03-15

    3-nitro-l-tyrosine (3-NT) is believed to be a biomarker of neurodegenerative diseases and metal doped graphene possess exceptionally high binding energy of 3-NT with metal-nitro chemisorption. Here we report a novel label-free detection scheme of 3-NT via nickel-doped graphene (NDG) as the functionalized receptor on our phase detecting localized surface plasmon resonance (LSPR) biosensor. When compared with reported 3-NT immunoassay with enzyme-linked immunosorbent assay (ELISA), our NDG-LSPR platform offers two advantages i.e. 1) label-free and 2) capture of 3-NT by direct chemisorption. Our limit of detection for 3-NT in PBS was found to be 0.13pg/ml and the linear dynamic range of response was from 0.5pg/ml to 1ng/ml, i.e. four orders of magnitude. The specificity of our NDG receptor to 3-NT was also verified with l-tyrosine of equivalent concentrations in PBS and diluted human serum, for which the NDG receptor shows negligible responses. In addition, the adsorption of 3-NT and l-tyrosine to the NDG receptor were also investigated by atomic force microscopy and further verified by surface enhanced Raman spectroscopy. Therefore, our NDG-LSPR biosensor competes favorably against ELISA and we believe it should be an attractive and economical solution to early diagnostic of 3-NT related disorders for clinical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Label-free triple-helix aptamer as sensing platform for "signal-on" fluorescent detection of thrombin.

    Science.gov (United States)

    Xu, Nan; Wang, Quanbo; Lei, Jianping; Liu, Lin; Ju, Huangxian

    2015-01-01

    The design of a label-free aptamer for separation of recognition sequence from signal reporter is significant to ensure the high-efficiency affinity between aptamer and target. This work develops a label-free triple-helix aptamer (THA) as sensing platform for "signal-on" fluorescent detection of thrombin. THA was composed of aptamer sequence and help DNA 1 (H1), which contained the complementary sequence of hexachloro-fluorescein (HEX) labeled help DNA 2 (H2). The specific recognition event between aptamer and thrombin triggered the dismission of THA to release H1. The released H1 then reacted with the signal probe of H2/graphene oxide (GO) nanocomposite to form H1-H2 duplex, leading to the fluorescence recovery of H2 due to the detachment of H1-H2 duplex from the surface of GO. With employment of THA as a signal transducer and GO as a "superquencher", this method shows a sensitive response to thrombin with a wide concentration range from 5 to 1200 nM. The limit of detection is 1.8 nM (S/N=3) with excellent selectivity. Considering the universality of THA, the proposed aptasensor would provide a platform for homogeneous fluorescent detection of a wide range of analytes.

  6. An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.

    Science.gov (United States)

    Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

    2015-01-15

    A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

  7. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  8. Label-free genetic and proteomic marker detection within a single flowcell assay.

    Science.gov (United States)

    Kastl, Katja F; Lowe, Christopher R; Norman, Carl E

    2010-12-15

    No one technique for multiplexing 100+ label-free measurements in a single well or flowcell has yet gained wide acceptance, probably because the added complexity introduced by the multiplexing element is seen to outweigh any possible cost advantage, or the multiplexing scheme itself is not flexible enough to accommodate the desired combinations of immobilization conditions and target/analyte molecules. Here, we demonstrate a simple yet highly versatile technology which uses microparticles, each bearing both an identifier code and an optical grating, to permit the inclusion of diverse molecules such as protein A, IgG and DNA, on chemically diverse -OH, -NH(2) and -COOH-terminated surfaces, within a single flowcell assay. Binding of avidin is used to reveal the presence of the immobilized biotinylated species, and to compare directly the binding of similar molecules on dissimilar surfaces, and vice-versa. Though we report the results of a 26-plex assay here, the technique itself has scope for increased throughput up to the level of hundreds of molecular species. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Label-free optical detection of bacteria on a 1-D photonic crystal of porous silicon

    Science.gov (United States)

    Wu, Chia-Chen; Alvarez, Sara D.; Rang, Camilla U.; Chao, Lin; Sailor, Michael J.

    2009-02-01

    The construction of a specific, label-free, bacteria biosensor using porous silicon 1-D photonic crystals will be described. Bacteria resident on the surface of porous silicon act as scattering centers for light resonant with the photonic crystal; the diffusely scattered light possesses the optical spectrum of the underlying photonic crystal. Using a spectrometer fitted to a light microscope, the bacteria are imaged without using exogenous dyes or labels and are quantified by measuring the intensity of scattered light. In order to selectively bind and identify bacteria using porous Si, we use surface modifications to reduce nonspecific binding to the surface and to engineer bacteria specificity onto the surface. Bovine serum albumin (BSA) was adsorbed to the porous Si surface to reduce nonspecific binding of bacteria. The coatings were then chemically activated to immobilize polyclonal antibodies specific to Escherichia coli. Two E. coli strains were used in our study, E. coli DH5α and non-pathogenic enterohemorrhagic Escherichia coli (EHEC) strain. The nonpathogenic Vibrio cholerae O1 strain was used to test for antibody specificity. Successful attachment of antibodies was measured using fluorescence microscopy and the scattering method was used to test for bacteria binding specificity.

  10. Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANInt) and poly-L-lysine (pLys).The composite of pLys and PANInt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane.The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH,but also possesses fine biocompatibility for bio-analytes.DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity.The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition.The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method.This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)6]3-/4as an indicator.

  11. Label-free detection of native proteins by surface-enhanced Raman spectroscopy using iodide-modified nanoparticles.

    Science.gov (United States)

    Xu, Li-Jia; Zong, Cheng; Zheng, Xiao-Shan; Hu, Pei; Feng, Jia-Min; Ren, Bin

    2014-02-18

    Proteins perform vital functional and structural duties in living systems, and the in-depth investigation of protein in its native state is one of the most important challenges in the postgenomic era. Surface-enhanced Raman spectroscopy (SERS) can provide the intrinsic fingerprint information of samples with ultrahigh sensitivity but suffers from the reproducibility and reliability issues. In this paper, we proposed an iodide-modified Ag nanoparticles method (Ag IMNPs) for label-free detection of proteins. The silver nanoparticles provide the huge enhancement to boost the Raman signal of proteins, and the coated iodide layer offers a barrier to prevent the direct interaction between the proteins and the metal surface, helping to keep the native structures of proteins. With this method, highly reproducible and high-quality SERS signals of five typical proteins (lysozyme, avidin, bovine serum albumin, cytochrome c, and hemoglobin) have been obtained, and the SERS features of the proteins without chromophore were almost identical to the respective normal Raman spectra. This unique feature allows the qualitative identification of them by simply taking the intensity ratio of the Raman peaks of tryptophan to phenylalanine residues. We further demonstrated that the method can also be used for label-free multiplex analysis of protein mixture as well as to study the dynamic process of protein damage stimulated by hydrogen peroxide. This method proves to be very promising for further applications in proteomics and biomedical research.

  12. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    Science.gov (United States)

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-07-29

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

  13. Multimodal CARS and SHG microscopy for label-free detection of collagen produced by hDFs in fibrin gel

    CERN Document Server

    Mortati, Leonardo; Sassi, Maria Paola

    2011-01-01

    Label-free combined CARS and SHG microscopy techniques are used as powerful tool to follow the cells behavior in cell-scaffold construct for regeneration of tissues. Imaging of histological section of hDFs seeded in fibrin gel scaffold and imaging of collagen produced by hDFs in a time course experiment at different culture days (0, 7, 21, 42) is performed. A study on the limit of collagen detection of the imaging system is reported using sample prepared with different collagen concentrations. The results show that also the small amount of collagen produced by hDFs after few hours of incubation in fibrin gel is detected. Co-localization of hDFs and collagen is also reported in function of the culture days.

  14. Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique.

    Science.gov (United States)

    Nava, G; Ceccarello, E; Giavazzi, F; Salina, M; Damin, F; Chiari, M; Buscaglia, M; Bellini, T; Zanchetta, G

    2016-05-21

    Rapid and quantitative detection of the binding of nucleic acids to surface-immobilized probes remains a challenge in many biomedical applications. We investigated the hybridization of a set of fully complementary and defected 12-base long DNA oligomers by using the Reflective Phantom Interface (RPI), a recently developed multiplexed label-free detection technique. Based on the simple measurement of reflected light intensity, this technology enables to quantify the hybridization directly as it occurs on the surface with a sensitivity of 10 pg mm(-2). We found a strong effect of single-base mismatches and of their location on hybridization kinetics and equilibrium binding. In line with previous studies, we found that DNA-DNA binding is weaker on a surface than in the bulk. Our data indicate that this effect is a consequence of weak nonspecific binding of the probes to the surface.

  15. "Molecular beacon"-hosted thioflavin T: Applications for label-free fluorescent detection of iodide and logic operations.

    Science.gov (United States)

    Li, Yan-Yun; Jiang, Xiao-Qin; Lu, Ling-Fei; Zhang, Min; Shi, Guoyue

    2016-04-01

    In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I(-)) detection based on "molecular beacon (MB)"-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I(-) ions over other anions interference due to the strong binding force between I(-) ions with Hg(2+). Upon the addition of I(-) ions, it would capture Hg(2+) from a T-Hg(2+)-T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I(-) in real samples such as human urine.

  16. "Peak tracking chip" for label-free optical detection of bio-molecular interaction and bulk sensing.

    Science.gov (United States)

    Bougot-Robin, Kristelle; Li, Shunbo; Zhang, Yinghua; Hsing, I-Ming; Benisty, Henri; Wen, Weijia

    2012-10-21

    A novel imaging method for bulk refractive index sensing or label-free bio-molecular interaction sensing is presented. This method is based on specially designed "Peak tracking chip" (PTC) involving "tracks" of adjacent resonant waveguide gratings (RWG) "micropads" with slowly evolving resonance position. Using a simple camera the spatial information robustly retrieves the diffraction efficiency, which in turn transduces either the refractive index of the liquids on the tracks or the effective thickness of an immobilized biological layer. Our intrinsically multiplex chip combines tunability and versatility advantages of dielectric guided wave biochips without the need of costly hyperspectral instrumentation. The current success of surface plasmon imaging techniques suggests that our chip proposal could leverage an untapped potential to routinely extend such techniques in a convenient and sturdy optical configuration toward, for instance for large analytes detection. PTC design and fabrication are discussed with challenging process to control micropads properties by varying their period (step of 2 nm) or their duty cycle through the groove width (steps of 4 nm). Through monochromatic imaging of our PTC, we present experimental demonstration of bulk index sensing on the range [1.33-1.47] and of surface biomolecule detection of molecular weight 30 kDa in aqueous solution using different surface densities. A sensitivity of the order of 10(-5) RIU for bulk detection and a sensitivity of the order of ∼10 pg mm(-2) for label-free surface detection are expected, therefore opening a large range of application of our chip based imaging technique. Exploiting and chip design, we expect as well our chip to open new direction for multispectral studies through imaging.

  17. Ultrasensitive electrochemical aptasensor based on sandwich architecture for selective label-free detection of colorectal cancer (CT26) cells.

    Science.gov (United States)

    Hashkavayi, Ayemeh Bagheri; Raoof, Jahan Bakhsh; Ojani, Reza; Kavoosian, Saeid

    2017-06-15

    Colorectal cancer is one of the most common cancers in the world and has no effective treatment. Therefore, development of new methods for early diagnosis is instantly required. Biological recognition probes such as synthetic receptor and aptamer is one of the candidate recognition layers to detect important biomolecules. In this work, an electrochemical aptasensor was developed by fabricating an aptamer-cell-aptamer sandwich architecture on an SBA-15-3-aminopropyltriethoxysilane (SBA-15-pr-NH2) and Au nanoparticles (AuNPs) modified graphite screen printed electrode (GSPE) surface for the selective, label-free detection of CT26 cancer cells. Based on the incubation of the thiolated aptamer with CT26 cells, the electron-transfer resistance of Fe (CN)6(3-/4-) redox couple increased considerably on the aptasensor surface. The results obtained from cyclic voltammetry and electrochemical impedance spectroscopy studies showed that the fabricated aptasensor can specifically identify CT26 cells in the concentration ranges of 10-1.0×10(5)cells/mL and 1.0×10(5)-6.0×10(6) cells/mL, respectively, with a detection limit of 2cells/mL. Applying the thiol terminated aptamer (5TR1) as a recognition layer led to a sensor with high affinity for CT26 cancer cells, compared to control cancer cells of AGS cells, VERO Cells, PC3 cells and SKOV-3 cells. Therefore a simple, rapid, label free, inexpensive, excellent, sensitive and selective electrochemical aptasensor based on sandwich architecture was developed for detection of CT26 Cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Detecting and identifying DNA via the THz backbone frequency using a metamaterial-based label-free biosensor

    Science.gov (United States)

    Mirzaei, Sahar; Green, Nicolas G.; Rotaru, Mihai; Pu, Suan Hui

    2017-02-01

    In genetic diagnostics, laboratory-based equipment generally uses analytical techniques requiring complicated and expensive fluorescent labelling of target DNA molecules. Intense research effort into, and commercial development of, Point-of-Care diagnostics and Personalized Healthcare are driving the development of simple, fast and cost-effective detection methods. One potential label-free DNA detection method uses Terahertz (THz) spectroscopy of the natural responses of DNA in metamaterial structures, which are engineered to have properties that are impossible to obtain in natural materials. This paper presents a study of the development of metamaterials based on asymmetric X-shaped resonator inclusions as a functional sensor for DNA. Gold X-shaped resonator structures with dimensions of 90/85 μm were demonstrated to produce trapped mode resonant frequency in the correct range for DNA detection. Realistic substrate materials in the form of 375 μm thick quartz were investigated, demonstrating that the non-transparent nature of the material resulted in the production of standing waves, affecting the system response, as well as requiring a reduction in scale of the resonator of 85%. As a result, the effect of introducing etched windows in the substrate material were investigated, demonstrating that increased window size significantly reduces the effect of the substrate on the system response. The device design showed a good selectivity when RNA samples were introduced to the model, demonstrating the potential for this design of device in the development of sensors capable of performing cheap and simple genetic analysis of DNA, giving label-free detection at high sensitivity.

  19. Label-free Detection of Protein Released during Platelet Activation by CNT-Enhanced Love Mode SAW Sensors.

    Science.gov (United States)

    Wu, Huiyan; Zu, Hongfei; Wang, Qing-Ming; Zhao, Gangyi; Wang, Jamesu H-C

    2014-09-01

    Platelet-rich plasma (PRP) has been applied in a series of clinical treatments. PRP contains high-concentrated platelets, which, when activated, could secret a variety of growth factors and cytokines, to promote and/or enhance healing of injured tissues. Non-activated platelets suspension could be prepared by an isolation method of centrifugation and washing currently. However, it is not clear whether platelets, if any, are already activated during this process and there is no simple method to monitor their activation accordingly. Shear-Horizontal Surface Acoustic Wave sensors (SH-SAW, Love Mode) are promising in fundamental biology as well as biomedical engineering, detecting cell behaviors in liquid in a non-invasive, simple and quantitative manner. In this study, Love mode sensors are adopted for the label-free detection of protein secreted by platelets. Carbon nanotube (CNT) is reported as an advisable platform of both non-specific protein adsorption and specific protein binding. For further improvement of Love mode sensor performance, novel CNT -coated parylene-C film is prepared on its surface as both the acoustic-wave-guiding layer and bio-interface layer. The S21 loss curves of Love mode sensors were recorded and the corresponding resonance frequencies were extracted. The results showed that the CNT-enhanced sensor possessed an increased resonance frequency shift when compared to normal sensor with single parylene-C film under identical collagen concentrations. Then, the modified sensor is used for label-free detection of protein released by various concentrations of platelets. The results revealed high sensitivity and consistency, indicating the potential of CNT-enhanced Love mode sensors in cell-based applications.

  20. Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.

    Science.gov (United States)

    Li, Kan; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-05-15

    Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.

  1. Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

    Science.gov (United States)

    Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

    2016-03-01

    Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

  2. A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide.

    Science.gov (United States)

    Zhao, Jingjin; Zhang, Liangliang; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2012-05-11

    A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.

  3. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    Science.gov (United States)

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  4. Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor.

    Science.gov (United States)

    Zhang, Decai; Yan, Yurong; Li, Qing; Yu, Tianxiao; Cheng, Wei; Wang, Long; Ju, Huangxian; Ding, Shijia

    2012-08-31

    A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.

  5. A novel label-free microfluidic paper-based immunosensor for highly sensitive electrochemical detection of carcinoembryonic antigen.

    Science.gov (United States)

    Wang, Yang; Xu, Huiren; Luo, Jinping; Liu, Juntao; Wang, Li; Fan, Yan; Yan, Shi; Yang, Yue; Cai, Xinxia

    2016-09-15

    In this work, a highly sensitive label-free paper-based electrochemical immunosensor employing screen-printed working electrode (SPWE) for detection of carcinoembryonic antigen (CEA) was fabricated. In order to raise the detection sensitivity and immobilize anti-CEA, amino functional graphene (NH2-G)/thionine (Thi)/gold nanoparticles (AuNPs) nanocomposites were synthesized and coated on SPWE. The principle of the immunosensor determination was based on the fact that the decreased response currents of Thi were proportional to the concentrations of corresponding antigens due to the formation of antibody-antigen immunocomplex. Experimental results revealed that the immunoassay enabled the determination of standard CEA solutions with linear working ranges of 50pgmL(-1) to 500ngmL(-1), the limit of detections for CEA is 10pgmL(-1) (S/N=3) and its corresponding correlation coefficients were 0.996. Furthermore, the proposed immunosensor could be used for the determination of clinical serum samples. A large number of clinical serum samples were detected and the relative errors between measured values and reference concentrations were calculated. Results showed that this novel paper-based electrochemical immunosensor could provide a new platform for low cost, sensitive, specific, and point-of-care diagnosis in cancer detection.

  6. Label-free fluorescent sensor for lead ion detection based on lead(II)-stabilized G-quadruplex formation.

    Science.gov (United States)

    Zhan, Shenshan; Wu, Yuangen; Luo, Yanfang; Liu, Le; He, Lan; Xing, Haibo; Zhou, Pei

    2014-10-01

    A label-free fluorescent DNA sensor for the detection of lead ions (Pb(2+)) based on lead(II)-stabilized G-quadruplex formation is proposed in this article. A guanine (G)-rich oligonucleotide, T30695, was used as a recognition probe, and a DNA intercalator, SYBR Green I (SG), was used as a signal reporter. In the absence of Pb(2+), the SG intercalated with the single-stranded random-coil T30695 and emitted strong fluorescence. While in the presence of Pb(2+), the random-coil T30695 would fold into a G-quadruplex structure and the SG could barely show weak fluorescence, and the fluorescence intensity was inversely proportional to the involving amount of Pb(2+). Based on this, a selective lead ion sensor with a limit of detection of 3.79 ppb (parts per billion) and a detection range from 0 to 600 ppb was constructed. Because detection for real samples was also demonstrated to be reliable, this simple, low-cost, sensitive, and selective sensor holds good potential for Pb(2+) detection in real environmental samples.

  7. Detection of ESAT-6 by a label free miniature immuno-electrochemical biosensor as a diagnostic tool for tuberculosis.

    Science.gov (United States)

    Diouani, Mohamed Fethi; Ouerghi, Oussama; Refai, Amira; Belgacem, Kamel; Tlili, Chaker; Laouini, Dhafer; Essafi, Makram

    2017-05-01

    Tuberculosis is a worldwide disease considered as a major health problem with high morbidity and mortality rates. Poor detection of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis remains a major obstacle to the global control of this disease. Here we report the development of a new test based on the detection of the major virulent factor of Mtb, namely the early secreted antigenic target 6-kDa protein or ESAT-6. A label free electrochemical immunosensor using an anti-ESAT-6 monoclonal antibody as a bio-receptor is described herein. Anti-ESAT-6 antibodies were first covalently immobilized on the surface of a gold screen-printed electrode functionalized via a self-assembled thiol monolayer. Interaction between the bio-receptor and ESAT-6 antigen was evaluated by square wave voltammetry method using [Fe(CN)6](3-/4-) as redox probe. The detection limit of ESAT-6 antigen was 7ng/ml. The immunosensor has also been able to detect native ESAT-6 antigen secreted in cell culture filtrates of three pathogenic strains of Mtb (CDC1551, H37RV and H8N8). Overall, this work describes an immune-electrochemical biosensor, based on ESAT-6 antigen detection, as a useful diagnostic tool for tuberculosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Label-Free Direct Detection of miRNAs with Poly-Silicon Nanowire Biosensors

    Science.gov (United States)

    Gong, Changguo; Qi, Jiming; Xiao, Han; Jiang, Bin; Zhao, Yulan

    2015-01-01

    Background The diagnostic and prognostic value of microRNAs (miRNAs) in a variety of diseases is promising. The novel silicon nanowire (SiNW) biosensors have advantages in molecular detection because of their high sensitivity and fast response. In this study, poly-crystalline silicon nanowire field-effect transistor (poly-SiNW FET) device was developed to achieve specific and ultrasensitive detection of miRNAs without labeling and amplification. Methods The poly-SiNW FET was fabricated by a top–down Complementary Metal Oxide Semiconductor (CMOS) wafer fabrication based technique. Single strand DNA (ssDNA) probe was bind to the surface of the poly-SiNW device which was silanated and aldehyde-modified. By comparing the difference of resistance value before and after ssDNA and miRNA hybridization, poly-SiNW device can be used to detect standard and real miRNA samples. Results Poly-SiNW device with different structures (different line width and different pitch) was applied to detect standard Let-7b sample with a detection limitation of 1 fM. One-base mismatched sequence could be distinguished meanwhile. Furthermore, these poly-SiNW arrays can detect snRNA U6 in total RNA samples extracted from HepG2 cells with a detection limitation of 0.2 μg/mL. In general, structures with pitch showed better results than those without pitch in detection of both Let-7b and snRNA U6. Moreover, structures with smaller pitch showed better detection efficacy. Conclusion Our findings suggest that poly-SiNW arrays could detect standard and real miRNA sample without labeling or amplification. Poly-SiNW biosensor device is promising for miRNA detection. PMID:26709827

  9. Label-free assay for the detection of glucose mediated by the effects of narrowband absorption on quantum dot photoluminescence

    Science.gov (United States)

    Khan, Saara A.; Smith, Gennifer T.; Ellerbee, Audrey K.

    2014-03-01

    We present a novel strategy for label-free detection of glucose based on CdSe/ZnS core/shell quantum dots (QDs). We exploit the concentration-dependent, narrowband absorption of the hexokinase-glucose 6-phosphate dehydrogenase enzymatic assay to selectively filter a 365-nm excitation source, leading to a proportional decrease in the photoluminescence intensity of the QDs. The visible wavelength emission of the QDs enables quantitative readout using standard visible detectors (e.g., CCD). Experimental results show highly linear QD photoluminescence over the clinically relevant glucose concentration range of 1-25mM, in excellent agreement with detection methods demonstrated by others. The method has a demonstrated limit of detection of 3.5μM, also on par with the best proposed methods. A significant advantage of our strategy is the complete elimination of QDs as a consumable. In contrast with other methods of QD-based measurement of glucose, our system does not require the glucose solution to be mixed with the QDs, thereby decreasing its overall cost and making it an ideal strategy for point-of-care detection of glucose in low-resource areas. Furthermore, readout can be accomplished with low-cost, portable detectors such as cellular phones, eliminating the need for expensive and bulky spectrophotometers to output quantitative information. The general strategy we present is useful for other biosensing applications involving chemistries with unique absorption peaks falling within the excitation band of available QDs.

  10. Label-Free MicroRNA Detection Based on Fluorescence Quenching of Gold Nanoparticles with a Competitive Hybridization.

    Science.gov (United States)

    Wang, Wei; Kong, Tao; Zhang, Dong; Zhang, Jinan; Cheng, Guosheng

    2015-11-01

    MicroRNAs (miRNAs), critical biomarkers of acute and chronic diseases, play key regulatory roles in many biological processes. As a result, there is great demand for robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biological samples. In this work, a label-free and Au nanoparticles (NPs) quenching-based competition assay system was developed. In the designed system, Au NPs with diameter sizes of 10 and 20 nm displayed fluorescence quenching efficiencies of 84% and 82% for Cy3 dye on slide surface, whereas the quenching efficiency of commercial BHQ2 quencher was roughly 50%. Assay conditions were optimized for miRNA-205 detection. A limit of detection of 3.8 pM and a detection range covering from 3.8 pM to 10 nM were achieved. Furthermore, the proposed system was capable of specifically discriminating miRNAs with slight variations in their nucleotide sequence and was also qualified for assessing miRNA levels in human serum. Our strategy has the potential to provide new perspectives in profiling the pattern of miRNA expression and biomedical utilizations.

  11. Labeling-free fluorescent detection of DNA hybridization through FRET from pyrene excimer to DNA intercalator SYBR green I.

    Science.gov (United States)

    Zhou, Ruyi; Xu, Chen; Dong, Jie; Wang, Guojie

    2015-03-15

    A novel labeling-free fluorescence complex probe has been developed for DNA hybridization detection based on fluorescence resonance energy transfer (FRET) mechanism from pyrene excimer of pyrene-functionalized poly [2-(N, N-dimethylamino) ethyl methacrylate] (PFP) to SYBR Green I (SG, a specific intercalator of double-stranded DNA) in a cost-effective, rapid and simple manner. The complex probe consists of the positively charged PFP, SG and negatively charged single-stranded DNA (ssDNA). Upon adding a complementary strand to the complex probe solution, double-stranded DNA (dsDNA) was formed, followed by the intercalation of SG into dsDNA. The pyrene excimer emission was overlapped with the absorption of SG very well and the electrostatic interactions between PFP and dsDNA kept them in close proximity, enabling efficient FRET from pyrene excimer to SG. The fluorescence of SG in the duplex DNA resulting from FRET can be successfully applied to detect DNA hybridization with high sensitivity for a very low detection limit of 10nM and excellent selectivity for detection of single base pair mismatch. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Label-Free Toxin Detection by Means of Time-Resolved Electrochemical Impedance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Paul Takhistov

    2010-01-01

    Full Text Available The real-time detection of trace concentrations of biological toxins requires significant improvement of the detection methods from those reported in the literature. To develop a highly sensitive and selective detection device it is necessary to determine the optimal measuring conditions for the electrochemical sensor in three domains: time, frequency and polarization potential. In this work we utilized a time-resolved electrochemical impedance spectroscopy for the detection of trace concentrations of Staphylococcus enterotoxin B (SEB. An anti-SEB antibody has been attached to the nano-porous aluminum surface using 3-aminopropyltriethoxysilane/glutaraldehyde coupling system. This immobilization method allows fabrication of a highly reproducible and stable sensing device. Using developed immobilization procedure and optimized detection regime, it is possible to determine the presence of SEB at the levels as low as 10 pg/mL in 15 minutes.

  13. Angular spectrum detection instrument for label-free photonic crystal sensors.

    Science.gov (United States)

    Liu, Longju; Xu, Zhen; Dong, Liang; Lu, Meng

    2014-05-01

    An angular spectrum analysis system was demonstrated to monitor the optical resonant mode of a photonic crystal (PC) sensor comprised of a one-dimensional grating structure. Exposed to solutions with different refractive indices or adsorbed with biomaterials, the PC sensor exhibited changes of the optical resonant modes. The developed detection system utilized a focused laser beam to detect shifts of the resonant angle, and thereby allowed a kinetic analysis of chemical absorption. Such a detection apparatus offers an adjustable angular resolution and a tunable detection range for a wide variety of refractometric sensing applications. A limit of detection of 6.57×10(-5) refractive index unit has been observed. The instrument also offers an imaging capability of rapidly characterizing low-contrast samples deposited on the PC surface with a spatial resolution of 10 μm.

  14. Label-Free Toxin Detection by Means of Time-Resolved Electrochemical Impedance Spectroscopy

    Science.gov (United States)

    Chai, Changhoon; Takhistov, Paul

    2010-01-01

    The real-time detection of trace concentrations of biological toxins requires significant improvement of the detection methods from those reported in the literature. To develop a highly sensitive and selective detection device it is necessary to determine the optimal measuring conditions for the electrochemical sensor in three domains: time, frequency and polarization potential. In this work we utilized a time-resolved electrochemical impedance spectroscopy for the detection of trace concentrations of Staphylococcus enterotoxin B (SEB). An anti-SEB antibody has been attached to the nano-porous aluminum surface using 3-aminopropyltriethoxysilane/glutaraldehyde coupling system. This immobilization method allows fabrication of a highly reproducible and stable sensing device. Using developed immobilization procedure and optimized detection regime, it is possible to determine the presence of SEB at the levels as low as 10 pg/mL in 15 minutes. PMID:22315560

  15. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection

    KAUST Repository

    Loan, Phan Thi Kim

    2017-07-19

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a residue-free graphene film using a thin gold supporting layer. A Hall effect device made of this gold-transferred graphene was demonstrated to significantly enhance the sensitivity (≈ 5 times) for hybridization detection, with a linear detection range of 1 pM – 100nM for DNA target. Our findings provide an efficient method to boost the sensitivity of graphene-based biosensors for DNA recognition.

  16. A label-free G-quadruplex-based luminescent switch-on assay for the selective detection of histidine.

    Science.gov (United States)

    He, Hong-Zhang; Wang, Modi; Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Qiu, Jian-Wen; Ma, Dik-Lung

    2013-12-15

    A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5'-TG4AG3TG4AG3TG4A2G2-3')/cupric ion (Cu(2+)) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu(2+) ions due to the Cu(2+)-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu(2+) ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 μM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated.

  17. Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Moreno, Maria de Lourdes; Cebolla, Angel; Lechuga, Laura M

    2016-05-15

    Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients.

  18. A facile label-free G-quadruplex based fluorescent aptasensor method for rapid detection of ATP

    Science.gov (United States)

    Liu, Haisheng; Ma, Changbei; Ning, Feng; Chen, Hanchun; He, Hailun; Wang, Kemin; Wang, Jun

    2017-03-01

    The present work demonstrates a simple, rapid and label-free ATP detection method using a fluorescent aptasensor that is based on G-quadruplex formation. In the absence of ATP, the Thioflavin T (ThT) dye binds to the G-rich ATP aptamer and forms an ATP aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of ATP, the ATP aptamer/ThT complex will be replaced by the formation of an ATP aptamer/ATP complex. During this process, separation of the ThT dye from the ATP aptamer/ThT complex decreases the fluorescence intensity of the reaction mixture dramatically. This fluorescence aptasensor is highly sensitive and rapid, with a detection limit of 18 nM and a total reaction time of only 10 min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

  19. 4-Fluoro-3-nitrophenyl grafted gold electrode based platform for label free electrochemical detection of interleukin-2 protein.

    Science.gov (United States)

    Arya, Sunil K; Park, Mi Kyoung

    2014-11-15

    A new platform based on 4-Fluoro-3-nitrophenyl (FNP) grafted gold disk electrode prepared via electrochemical reduction of 4-fluoro-3-nitrobenzene diazonium ion has been developed and utilized for biosensor fabrication. Anti-interleukin-2 (anti-IL2) antibody has been covalently immobilized onto FNP/Au surface and utilized for label free electrochemical impedance based detection of cytokine IL2. FNP acts as a bridge (cross-linker) between gold surface and anti-IL2, where fluoro group of FNP undergoes nucleophilic substitution by amino group of biomolecule and results in its covalent immobilization. The immobilization process and fabricated electrode have been characterized using contact angle (CA) measurements, cyclic voltammetry (CV) and electrochemical impedance (EIS) technique. CV studies show that FNP grafted surface provides conductive surface for anti-IL2 immobilization. The EIS response of studies as a function of IL2 concentrations exhibits a detection in linear range from 1 pg ml(-1) to 10 ng ml(-1) with minimum detectable concentration of 1 pg ml(-1). The electrode has been found to be selective against other cytokine molecules.

  20. A label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots (GQDs).

    Science.gov (United States)

    Tuteja, Satish K; Chen, Rui; Kukkar, Manil; Song, Chung Kil; Mutreja, Ruchi; Singh, Suman; Paul, Ashok K; Lee, Haiwon; Kim, Ki-Hyun; Deep, Akash; Suri, C Raman

    2016-12-15

    A label-free immunosensor based on electrochemical impedance spectroscopy has been developed for the sensitive detection of a cardiac biomarker myoglobin (cMyo). Hydrothermally synthesized graphene quantum dots (GQDs) have been used as an immobilized template on screen printed electrodes for the construction of an impedimetric sensor platform. The GQDs-modified electrode was conjugated with highly specific anti-myoglobin antibodies to develop the desired immunosensor. The values of charge transfer resistance (Rct) were monitored as a function of varying antigen concentration. The Rct value of the immunosensor showed a linear increase (from 0.20 to 0.31kΩ) in the range of 0.01-100ng/mL cMyo. The specific detection of cMyo was also made in the presence of other competing proteins. The limit of detection for the proposed immunosensor was estimated as 0.01ng/mL which is comparable to the standard ELISA techniques.

  1. Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

    Science.gov (United States)

    Wang, Yaoguang; Zhang, Yong; Wu, Dan; Ma, Hongmin; Pang, Xuehui; Fan, Dawei; Wei, Qin; Du, Bin

    2017-01-01

    In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe3O4-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe3O4-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe3O4 NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe3O4 NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe3O4-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10−5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers. PMID:28186128

  2. Label-Free Dengue Detection Utilizing PNA/DNA Hybridization Based on the Aggregation Process of Unmodified Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Samsulida Abdul Rahman

    2014-01-01

    Full Text Available A label-free optical detection method based on PNA/DNA hybridization using unmodified gold nanoparticles (AuNPs for dengue virus detection has been successfully developed. In this study, no immobilization method is involved and the hybridization of PNA/DNA occurs directly in solution. Unmodified AuNPs undergo immediate aggregation in the presence of neutral charge peptide nucleic acid (PNA due to the coating of PNA on AuNPs surface. However, in the presence of complementary targets DNA, the hybridization of PNA probe with target DNA forms negatively charged complexes due to the negatively charged phosphate backbone of the target DNA. The negatively charged complexes adsorbed onto the AuNPs surface ensure sufficient charge repulsion, need for AuNPs dispersion, and stability in solution. The detection procedure is a naked eye method based on immediate color changes and also through UV-vis adsorption spectra. The selectivity of the proposed method was studied successfully by single base mismatch and noncomplementary target DNA.

  3. Label-free electrochemical immunosensor based on gold-silicon carbide nanocomposites for sensitive detection of human chorionic gonadotrophin.

    Science.gov (United States)

    Yang, Long; Zhao, Hui; Fan, Shuangmei; Deng, Shuangsheng; Lv, Qi; Lin, Jie; Li, Can-Peng

    2014-07-15

    Uniform and highly dispersed gold-silicon carbide (Au@SiC) nanocomposites were prepared via simple way and used for fabrication of label-free electrochemical immunosensor for sensitive detection of human chorionic gonadotrophin (hCG). Using Au@SiC as electrode material and using ferricyanide as mediator, the proposed immunosensor provides a simple and economic method with higher sensitivity and a wider concentration range for detection of hCG. Under the optimal condition, the approach provided a good linear response range from 0.1 to 5 IU/L and 5 to 1000 IU/L with a low detection limit of 0.042 IU/L. The immunosensor showed good selectivity, acceptable stability and reproducibility. Satisfactory results were obtained for determination of hCG in human serum samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules. In addition, the bio-functionalization of SiC combined with other nanomaterials will provide promising approach for electrochemical sensing and biosensing platform.

  4. Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.

    Science.gov (United States)

    Kusić, Dragana; Rösch, Petra; Popp, Jürgen

    2016-03-01

    Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species.

  5. Label-free silver nanoparticles for the naked eye detection of entecavir

    Science.gov (United States)

    Gao, Mengmeng; Lin, Rui; Li, Lili; Jiang, Li; Ye, Baofen; He, Hua; Qiu, Lanlan

    A simple, rapid, field-portable colorimetric method for the detection of entecavir was proposed based on the color change caused by the aggregation of silver nanoparticles. Neutralization of the electrostatic repulsion from each silver nanoparticle resulted in the aggregation of AgNPs and a consequent color change of AgNPs from yellow to wine-red, which provided a platform for rapid and field-portable colorimetric detection of entecavir. The concentration of entecavir could be determined with naked eye or UV-vis spectrometer. The proposed method can be used to detect entecavir in human urine with a detection limit of 1.51 μg mL-1, within 25 min by naked eye observation without the aid of any advanced instrument or complex pretreatment. Results from UV-vis spectra showed that the absorption ratio was linear with the concentration of entecavir in the range of 5.04-25.2 μg mL-1 and 1.01-5.04 μg mL-1 with linear coefficients of 0.9907 and 0.9955, respectively. The selectivity of AgNPs detection system for entecavir is excellent comparing with other ions and analytes. Due to its rapid, visible color changes, and excellent selectivity, the AgNPs synthesized in this study are suitable to be applied to on-site screening of entecavir in human urine.

  6. Label-free mitosis detection in tumor spheroids using tissue dynamics imaging

    Science.gov (United States)

    An, Ran; Jeong, Kwan; Turek, John; Nolte, David

    2012-03-01

    The detection of cellular mitosis inside three-dimensional living tissue at depths up to 1 mm has been beyond the detection limits of conventional microscopies. In this paper, we demonstrate the use of motility contrast imaging and fluctuation spectroscopy to detect motional signatures that we attribute to mitotic events within groups of 100 cells in multicellular tumor spheroids. Motility contrast imaging is a coherence-domain speckle-imaging technique that uses low-coherence off-axis holography as a coherence gate to localize dynamic light scattering from selected depths inside tissue. Fluctuation spectroscopy is performed on a pervoxel basis to generate micro-spectrograms that display frequency content vs. time. Mitosis, especially in Telophase and Cytokinesis, is a relatively fast and high-amplitude phenomenon that should display energetic features within the micro-spectrograms. By choosing an appropriate frequency range and threshold, we detect energetic events with a density and rate that are comparable to the expected mitotic fraction in the UMR cell line. By studying these mitotic events in tumors of two different sizes, we show that micro-spectrograms contain characteristically different information content than macro-spectrograms (averaged over many voxels) in which the mitotic signatures (which are overall a low-probability event) are averaged out. The detection of mitotic fraction in thick living tissue has important consequences for the use of tissue-based assays for drug discovery.

  7. Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria.

    Science.gov (United States)

    El Ichi, Sarra; Leon, Fanny; Vossier, Ludivine; Marchandin, Helene; Errachid, Abdelhamid; Coste, Joliette; Jaffrezic-Renault, Nicole; Fournier-Wirth, Chantal

    2014-04-15

    Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3) CFU mL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3) CFU mL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum.

  8. Label-free detection of single nanoparticles and biological molecules using microtoroid optical resonators

    Institute of Scientific and Technical Information of China (English)

    Judith Su; Alexander FG Goldberg; Brian M Stoltz

    2016-01-01

    Single-molecule detection is one of the fundamental challenges of modern biology.Such experiments often use labels that can be expensive,difficult to produce,and for small analytes,might perturb the molecular events being studied.Analyte size plays an important role in determining detectability.Here we use laser-frequency locking in the context of sensing to improve the signal-to-noise ratio of microtoroid optical resonators to the extent that single nanoparticles 2.5 nm in radius,and 15.5 kDa molecules are detected in aqueous solution,thereby bringing these detectors to the size limits needed for detecting the key macromolecules of the cell.Our results,covering several orders of magnitude of particle radius (100 nm to 2 nm),agree with the 'reactive' model prediction for the frequency shift of the resonator upon particle binding.This confirms that the main contribution of the frequency shift for the resonator upon particle binding is an increase in the effective path length due to part of the evanescent field coupling into the adsorbed particle.We anticipate that our results will enable many applications,including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time.

  9. Label-Free Detection of Soybean Rust Spores using Photonic Crystal Biosensors

    Science.gov (United States)

    Soybean rust, caused by the fungus Phakopsora pachyrhizi, is one of the most devastating foliar diseases affecting soybeans grown worldwide. The disease was reported for the first time in the United States in 2004. Early spore detection, prior to the appearance of visible symptoms, is critical to ef...

  10. Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, M; Heiskanen, Arto

    2012-01-01

    optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both...

  11. Label-Free Detection and Serotyping of Salmonellae by Surface Enhanced Raman Spectroscopy with Immunomagnetic Separation

    Science.gov (United States)

    Salmonella spp. are one of the leading causes of foodborne outbreaks in the United States and globally. Current detection and characterization techniques for Salmonella are time consuming and rapid methods could greatly benefit outbreak investigation, new case prevention and disease treatment. In th...

  12. Label-free biochips for rapid detection of soybean allergen GlymBd ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research April 2017; 16 (4): 755-760 ... Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals. (DOAJ), African ..... allergy: a clinical and therapeutic dilemma. Allergy 2000; ... XY, Wan LJ, Jin G. Phage M13KO7 detection with biosensor ...

  13. Label-free high-throughput detection and content sensing of individual droplets in microfluidic systems.

    Science.gov (United States)

    Yesiloz, Gurkan; Boybay, Muhammed Said; Ren, Carolyn L

    2015-10-21

    This study reports a microwave-microfluidics integrated approach capable of performing droplet detection at high-throughput as well as content sensing of individual droplets without chemical or physical intrusion. The sensing system consists of a custom microwave circuitry and a spiral-shaped microwave resonator that is integrated with microfluidic chips where droplets are generated. The microwave circuitry is very cost effective by using off-the-shelf components only. It eliminates the need for bulky benchtop equipment, and provides a compact, rapid and sensitive tool compatible for Lab-on-a-Chip (LOC) platforms. To evaluate the resonator's sensing capability, it was first applied to differentiate between single-phase fluids which are aqueous solutions with different concentrations of glucose and potassium chloride respectively by measuring its reflection coefficient as a function of frequency. The minimum concentration assessed was 0.001 g ml(-1) for potassium chloride and 0.01 g ml(-1) for glucose. In the droplet detection experiments, it is demonstrated that the microwave sensor is able to detect droplets generated at as high throughput as 3.33 kHz. Around two million droplets were counted over a period of ten minutes without any missing. For droplet sensing experiments, pairs of droplets that were encapsulated with biological materials were generated alternatively in a double T-junction configuration and clearly identified by the microwave sensor. The sensed biological materials include fetal bovine serum, penicillin antibiotic mixture, milk (2% mf) and d-(+)-glucose. This system has significant advantages over optical detection methods in terms of its cost, size and compatibility with LOC settings and also presents significant improvements over other electrical-based detection techniques in terms of its sensitivity and throughput.

  14. Label-free SERS detection of relevant bioanalytes on silver-coated carbon nanotubes: The case of cocaine

    Science.gov (United States)

    Sanles-Sobrido, Marcos; Rodríguez-Lorenzo, Laura; Lorenzo-Abalde, Silvia; González-Fernández, África; Correa-Duarte, Miguel A.; Alvarez-Puebla, Ramón A.; Liz-Marzán, Luis M.

    2009-09-01

    Surface-enhanced Raman scattering (SERS) spectroscopy can be used for the label-free determination and quantification of relevant small biometabolites that are hard to identify by conventional immunological methods, in the absence of labelling. In this work, detection is based on monitoring the vibrational changes occurring at a specific biointerface (a monoclonal antibody, mAb) supported on silver-coated carbon nanotubes (CNT@Ag). Engineered CNT@Ag play a key role, as they offer a stable substrate to support the biointerface, with a high density of hot spots. Proof of concept is demonstrated through the analysis and quantification of the main cocaine metabolite benzoylecgonine. These results open a new avenue toward the generation of portable sensors for fast ultradetection and quantification of relevant metabolites. The use of discrete particles (CNT@Ag@mAb) rather than rough films, or other conventional SERS supports, will also enable a safe remote interrogation of highly toxic sources in environmental problems or in biological fluids.Surface-enhanced Raman scattering (SERS) spectroscopy can be used for the label-free determination and quantification of relevant small biometabolites that are hard to identify by conventional immunological methods, in the absence of labelling. In this work, detection is based on monitoring the vibrational changes occurring at a specific biointerface (a monoclonal antibody, mAb) supported on silver-coated carbon nanotubes (CNT@Ag). Engineered CNT@Ag play a key role, as they offer a stable substrate to support the biointerface, with a high density of hot spots. Proof of concept is demonstrated through the analysis and quantification of the main cocaine metabolite benzoylecgonine. These results open a new avenue toward the generation of portable sensors for fast ultradetection and quantification of relevant metabolites. The use of discrete particles (CNT@Ag@mAb) rather than rough films, or other conventional SERS supports, will also

  15. Label-Free, Single Molecule Resonant Cavity Detection: A Double-Blind Experimental Study

    Directory of Open Access Journals (Sweden)

    Maria V. Chistiakova

    2015-03-01

    Full Text Available Optical resonant cavity sensors are gaining increasing interest as a potential diagnostic method for a range of applications, including medical prognostics and environmental monitoring. However, the majority of detection demonstrations to date have involved identifying a “known” analyte, and the more rigorous double-blind experiment, in which the experimenter must identify unknown solutions, has yet to be performed. This scenario is more representative of a real-world situation. Therefore, before these devices can truly transition, it is necessary to demonstrate this level of robustness. By combining a recently developed surface chemistry with integrated silica optical sensors, we have performed a double-blind experiment to identify four unknown solutions. The four unknown solutions represented a subset or complete set of four known solutions; as such, there were 256 possible combinations. Based on the single molecule detection signal, we correctly identified all solutions. In addition, as part of this work, we developed noise reduction algorithms.

  16. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe.

    Science.gov (United States)

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Patterned Array of Poly(ethylene glycol) Silane Monolayer for Label-Free Detection of Dengue

    OpenAIRE

    Nor Zida Rosly; Shahrul Ainliah Alang Ahmad; Jaafar Abdullah; Nor Azah Yusof

    2016-01-01

    In the present study, the construction of arrays on silicon for naked-eye detection of DNA dengue was demonstrated. The array was created by exposing a polyethylene glycol (PEG) silane monolayer to 254 nm ultraviolet (UV) light through a photomask. Formation of the PEG silane monolayer and photomodifed surface properties was thoroughly characterized by using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. The results of XPS confirmed that...

  18. Noninvasive Label-Free Detection of Micrometastases in the Lymphatics with Ultrasound-Guided Photoacoustic Imaging

    Science.gov (United States)

    2015-10-01

    Ultrasound and Photoacoustic Imaging of Anatomical and Functional Indicators of Lymph Node Metastasis,” Biomedical Engineering Society Annual Meeting...imaging system that will detect functional changes associated with lymph node metastasis in breast cancer patients. Our efforts in the first year have...imaging can be used to guide dissection . We have also successfully integrated a programmable ultrasound machine (Verasonics Vantage) and tunable pulsed

  19. Disulfide-induced self-assembled targets : A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    NARCIS (Netherlands)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-01-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol-modified probes, each of which specifically

  20. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    NARCIS (Netherlands)

    Shokri, E. (Ehsan); M. Hosseini (Morteza); Davari, M.D. (Mehdi D.); Ganjali, M.R. (Mohammad R.); M.P. Peppelenbosch (Maikel); F. Rezaee (Farhad)

    2017-01-01

    textabstractA modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which

  1. Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection.

    Science.gov (United States)

    Li, Bing; Pan, Genhua; Avent, Neil D; Lowry, Roy B; Madgett, Tracey E; Waines, Paul L

    2015-10-15

    A novel printed graphene electrode modified with electrochemically reduced graphene oxide was developed for the detection of a specific oligonucleotide sequence. The graphene oxide was immobilized onto the surface of a graphene electrode via π-π bonds and electrochemical reduction of graphene oxide was achieved by cyclic voltammetry. A much higher redox current was observed from the reduced graphene oxide-graphene double-layer electrode, a 42% and 36.7% increase, respectively, in comparison with that of a bare printed graphene or reduced graphene oxide electrode. The good electron transfer activity is attributed to a combination of the large number of electroactive sites in reduced graphene oxide and the high conductivity nature of graphene. The probe ssDNA was further immobilized onto the surface of the reduced graphene oxide-graphene double-layer electrode via π-π bonds and then hybridized with its target cDNA. The change of peak current due to the hybridized dsDNA could be used for quantitative sensing of DNA concentration. It has been demonstrated that a linear range from 10(-7)M to 10(-12)M is achievable for the detection of human immunodeficiency virus 1 gene with a detection limit of 1.58 × 10(-13)M as determined by three times standard deviation of zero DNA concentration.

  2. Patterned Array of Poly(ethylene glycol Silane Monolayer for Label-Free Detection of Dengue

    Directory of Open Access Journals (Sweden)

    Nor Zida Rosly

    2016-08-01

    Full Text Available In the present study, the construction of arrays on silicon for naked-eye detection of DNA dengue was demonstrated. The array was created by exposing a polyethylene glycol (PEG silane monolayer to 254 nm ultraviolet (UV light through a photomask. Formation of the PEG silane monolayer and photomodifed surface properties was thoroughly characterized by using atomic force microscopy (AFM, X-ray photoelectron spectroscopy (XPS, and contact angle measurements. The results of XPS confirmed that irradiation of ultraviolet (UV light generates an aldehyde functional group that offers conjugation sites of amino DNA probe for detection of a specific dengue virus target DNA. Employing a gold enhancement process after inducing the electrostatic interaction between positively charged gold nanoparticles and the negatively charged target DNA hybridized to the DNA capture probe allowed to visualize the array with naked eye. The developed arrays demonstrated excellent performance in diagnosis of dengue with a detection limit as low as 10 pM. The selectivity of DNA arrays was also examined using a single base mismatch and noncomplementary target DNA.

  3. Tunable and label-free virus enrichment for ultrasensitive virus detection using carbon nanotube arrays

    Science.gov (United States)

    Yeh, Yin-Ting; Tang, Yi; Sebastian, Aswathy; Dasgupta, Archi; Perea-Lopez, Nestor; Albert, Istvan; Lu, Huaguang; Terrones, Mauricio; Zheng, Si-Yang

    2016-01-01

    Viral infectious diseases can erupt unpredictably, spread rapidly, and ravage mass populations. Although established methods, such as polymerase chain reaction, virus isolation, and next-generation sequencing have been used to detect viruses, field samples with low virus count pose major challenges in virus surveillance and discovery. We report a unique carbon nanotube size-tunable enrichment microdevice (CNT-STEM) that efficiently enriches and concentrates viruses collected from field samples. The channel sidewall in the microdevice was made by growing arrays of vertically aligned nitrogen-doped multiwalled CNTs, where the intertubular distance between CNTs could be engineered in the range of 17 to 325 nm to accurately match the size of different viruses. The CNT-STEM significantly improves detection limits and virus isolation rates by at least 100 times. Using this device, we successfully identified an emerging avian influenza virus strain [A/duck/PA/02099/2012(H11N9)] and a novel virus strain (IBDV/turkey/PA/00924/14). Our unique method demonstrates the early detection of emerging viruses and the discovery of new viruses directly from field samples, thus creating a universal platform for effectively remediating viral infectious diseases. PMID:27730213

  4. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin

    2016-08-01

    Full Text Available This study introduces the use of an IgA isotype aflatoxin (AF specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide/N-hydroxy succinimide (EDC/NHS method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

  5. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Science.gov (United States)

    Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

    2014-11-01

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml-1 with a limit of detection of 0.16 ng ml-1.

  6. Label-Free Detection of Chondroitin Sulphate Proteoglycan 4 by a Polyaniline/Graphene Nanocomposite Functionalized Impedimetric Immunosensor

    Directory of Open Access Journals (Sweden)

    JingJing Fu

    2016-01-01

    Full Text Available The chondroitin sulphate proteoglycan 4 (CSPG4, also known as high molecular weight-melanoma associated antigen (HMW-MAA, is a tumor-associated antigen that is expressed in more than 85% of surgically removed melanoma lesions but has restricted distribution in normal tissues. The diagnostic and therapeutic value of CSPG4 drives a need for sensitive and low-cost detection approaches. To this end, we developed a polyaniline/graphene oxide nanocomposite (PANI@GO that was electrochemically codeposited on indium tin oxide (ITO electrode. Glutaraldehyde mediated the covalent immobilization of CSPG4 specific antibody mAbD2.8.5 to construct a CSPG4 immunosensor using cell culture media and cell lysate as samples. The fully assembled impedimetric immunosensor was used to detect CSPG4 in CSPG4-positive cell lines M14/CSPG4 and MV3. No impedance signal changes could be observed from CSPG4-negative cell lines M14 and mAbMk2-23 showing the specificity of the CSPG4-impedimetric immunosensor. This low-cost, simple, and label-free analytical method is an alternative to enzyme-linked immunosorbent assay and flow cytometry in screening of CSPG4 in complex biological samples.

  7. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Swati; Kumar, Ashok, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007 (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India); Khare, Shashi [National Centre for Disease Control, Sham Nath Marg, Delhi 110054 (India); Mulchandani, Ashok [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States); Rajesh, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-National Physical Laboratory, Dr. K. S. Krishnan Road, New Delhi 110012 (India)

    2014-11-24

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml{sup −1} with a limit of detection of 0.16 ng ml{sup −1}.

  8. Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay.

    Science.gov (United States)

    Wang, Xiu-Li; Zhu, Ying; Fang, Qun

    2014-01-07

    In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensin I and angiotensin II as model samples, and the repeatabilities of peak area for angiotensin I and angiotensin II were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.

  9. Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay.

    Science.gov (United States)

    Braissant, Olivier; Keiser, Jennifer; Meister, Isabel; Bachmann, Alexander; Wirz, Dieter; Göpfert, Beat; Bonkat, Gernot; Wadsö, Ingemar

    2015-03-01

    Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications. Our results show that the sensitivity of the instrument allows the detection of 3 × 10(4) bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 μW. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h(-1) , respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 μW was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 μm. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs. For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.

  10. A Label-Free Electrochemical Immunosensor for Carbofuran Detection Based on a Sol-Gel Entrapped Antibody

    Directory of Open Access Journals (Sweden)

    Qingqing Li

    2011-10-01

    Full Text Available In this study, an anti-carbofuran monoclonal antibody (Ab was immobilized on the surface of a glassy carbon electrode (GCE using silica sol-gel (SiSG technology. Thus, a sensitive, label-free electrochemical immunosensor for the direct determination of carbofuran was developed. The electrochemical performance of immunoreaction of antigen with the anti-carbofuran monoclonal antibody was investigated by cyclic voltammetry (CV and electrochemical impedance spectroscopy (EIS, in which phosphate buffer solution containing [Fe(CN6]3−/4− was used as the base solution for test. Because the complex formed by the immunoreaction hindered the diffusion of [Fe(CN6]3−/4− on the electrode surface, the redox peak current of the immunosensor in the CV obviously decreased with the increase of the carbofuran concentration. The pH of working solution, the concentration of Ab and the incubation time of carbofuran were studied to ensure the sensitivity and conductivity of the immunosensor. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of carbofuran was from 1 ng/mL to 100 μg/mL and from 50 μg/mL to 200 μg/mL with a detection limit of 0.33 ng/mL (S/N = 3. The proposed immunosensor exhibited good high sensitivity and stability, and it was thus suitable for trace detection of carbofuran pesticide residues.

  11. A label-free electrochemical immunosensor for carbofuran detection based on a sol-gel entrapped antibody.

    Science.gov (United States)

    Sun, Xia; Du, Shuyuan; Wang, Xiangyou; Zhao, Wenping; Li, Qingqing

    2011-01-01

    In this study, an anti-carbofuran monoclonal antibody (Ab) was immobilized on the surface of a glassy carbon electrode (GCE) using silica sol-gel (SiSG) technology. Thus, a sensitive, label-free electrochemical immunosensor for the direct determination of carbofuran was developed. The electrochemical performance of immunoreaction of antigen with the anti-carbofuran monoclonal antibody was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), in which phosphate buffer solution containing [Fe(CN)(6)](3-/4-) was used as the base solution for test. Because the complex formed by the immunoreaction hindered the diffusion of [Fe(CN)(6)](3-/4-) on the electrode surface, the redox peak current of the immunosensor in the CV obviously decreased with the increase of the carbofuran concentration. The pH of working solution, the concentration of Ab and the incubation time of carbofuran were studied to ensure the sensitivity and conductivity of the immunosensor. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of carbofuran was from 1 ng/mL to 100 μg/mL and from 50 μg/mL to 200 μg/mL with a detection limit of 0.33 ng/mL (S/N = 3). The proposed immunosensor exhibited good high sensitivity and stability, and it was thus suitable for trace detection of carbofuran pesticide residues.

  12. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  13. Organic Liquids-Responsive β-Cyclodextrin-Functionalized Graphene-Based Fluorescence Probe: Label-Free Selective Detection of Tetrahydrofuran

    Directory of Open Access Journals (Sweden)

    Huawen Hu

    2014-06-01

    Full Text Available In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  14. Label-free silicon quantum dots as fluorescent probe for selective and sensitive detection of copper ions.

    Science.gov (United States)

    Zhao, Jiangna; Deng, Jianhui; Yi, Yinhui; Li, Haitai; Zhang, Youyu; Yao, Shouzhuo

    2014-07-01

    In this work, label-free silicon quantum dots (SiQDs) were used as a novel fluorescence probe for the sensitive and selective detection of Cu(2+). The fluorescence of the SiQDs was effectively quenched by H2O2 from the reaction of ascorbic acid with O2, and hydroxyl radicals from Fenton reaction between H2O2 and Cu(+). The fluorescence intensity of SiQDs was quenched about 25% in 15 min after the addition of H2O2 (1mM). While the SiQDs was incubated with AA (1mM) and Cu(2+) (1 µM) under the same conditions, the fluorescence intensity of SiQDs decreased about 55%. Obviously, the recycling of Cu(2+) in the test system may lead to a dramatical decrease in the fluorescence of SiQDs. Under the optimized experimental conditions, the rate of fluorescence quenching of SiQDs was linearly dependent on the Cu(2+) concentration ranging from 25 to 600 nM with the limit of detection as low as 8 nM, which was much lower than that of existing methods. Moreover, the probe was successfully applied to the determination of Cu(2+) in different environmental water samples and human hair.

  15. Graphene-Plasmonic Hybrid Platform for Label-Free SERS Biomedical Detection

    Science.gov (United States)

    Wang, Pu

    Surface Enhanced Raman Scattering (SERS) has attracted explosive interest for the wealth of vibrational information it provides with minimal invasive effects to target analyte. Nanotechnology, especially in the form of noble metal nanoparticles exhibit unique electromagnetic and chemical characteristics that are explored to realize ultra-sensitive SERS detection in chemical and biological analysis. Graphene, atom-thick carbon monolayer, exhibits superior chemical stability and bio-compatibility. A combination of SERS-active metal nanostructures and graphene will create various synergies in SERS. The main objective of this research was to exploit the applications of the graphene-Au tip hybrid platform in SERS. The hybrid platform consists of a periodic Au nano-pyramid substrate to provide reproducible plasmonic enhancement, and the superimposed monolayer graphene sheet, serving as "built-in" Raman marker. Extensive theoretical and experimental studies were conducted to determine the potentials of the hybrid platform as SERS substrate. Results from both Finite-Domain Time-Domain (FDTD) numerical simulation and Raman scattering of graphene suggested that the hybrid platform boosted a high density of hotspots yielding 1000 times SERS enhancement of graphene bands. Ultra-high sensitivity of the hybrid platform was demonstrated by bio-molecules including dye, protein and neurotransmitters. Dopamine and serotonin can be detected and distinguished at 10-9 M concentration in the presence of human body fluid. Single molecule detection was obtained using a bi-analyte technique. Graphene supported a vibration mode dependent SERS chemical enhancement of ˜10 to the analyte. Quantitative evaluation of hotspots was presented using spatially resolved Raman mapping of graphene SERS enhancement. Graphene plays a crucial role in quantifying SERS hotspots and paves the path for defining SERS EF that could be universally applied to various SERS systems. A reproducible and statistically

  16. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

    2011-05-13

    We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  17. Colorimetric Sensor for Label Free Detection of Porcine PCR Product (ID: 18)

    Science.gov (United States)

    Ali, M. E.; Hashim, U.; Bari, M. F.; Dhahi, Th. S.

    2011-05-01

    This report described the use of 40±5 nm in diameter citrate-coated gold nanoparticles (GNPs) as colorimetric sensor to visually detect the presence of a 17-base swine specific conserved sequence and nucleotide mismatch in the mixed PCR products of pig, deer and shad cytochrome b genes. The size of these PCR amplicons was 109 base-pair and was amplified with a pair of common primers. Colloidal GNPs changed color from pinkish- red to purple-gray in 2 mM PBS buffer by losing its characteristic surface plasmon resonance peak at 530 nm and gaining new features between 620 and 800 nm in the absorption spectrum indicating strong aggregation. The particles were stabilized against salt induced aggregation, retained spectral features and characteristic color upon adsorption of single-stranded DNA. The PCR products without any additional processing were hybridized with a 17-nucleotide swine probe prior to exposure to GNPs. At a critical annealing temperature (55° C) that differentiated between the match and mismatch pairing, the probe was hybridized with the pig PCR product and dehybridized from the deer's and shad's. The interaction of dehybridized probe to GNPs prevented them from salt-induced aggregation, retaining their characteristic red color. The assay did not need any surface modification chemistry or labeling steps. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The assay obviated the need of complex RFLP, sequencing or blotting to differentiate the same size PCR products. We find the application of the assay for species assignment in food analysis, mismatch detection in genetic screening and homology study among closely related species.

  18. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Science.gov (United States)

    Ali, M. E.; Hashim, U.; Mustafa, S.; Che Man, Y. B.; Yusop, M. H. M.; Bari, M. F.; Islam, Kh N.; Hasan, M. F.

    2011-05-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml - 1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  19. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples.

    Science.gov (United States)

    Ali, M E; Hashim, U; Mustafa, S; Man, Y B Che; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-05-13

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  20. Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry.

    Science.gov (United States)

    Wu, Nai-ying; Gao, Wei; He, Xu-lun; Chang, Zhu; Xu, Mao-tian

    2013-01-15

    A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.

  1. The Label-Free Unambiguous Detection and Symbolic Display of Single Nucleotide Polymorphisms on DNA Origami

    Science.gov (United States)

    Subramanian, Hari K. K.; Chakraborty, Banani; Sha, Ruojie; Seeman, Nadrian C.

    2011-01-01

    Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair inhibits the progress of branch migration in the direction of the mispair. We have combined the effectiveness of kinetic methods with AFM of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four nucleotide alphabetic characters, A, T, G and C, and the symbol containing the test nucleotide identity vanishes in the presence of the probe. The system also works with pairs of probes, corresponding to heterozygous diploid genomes. PMID:21235216

  2. Label-free detection of gliadin food allergen mediated by real-time apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Henry, Olivier; Redondo, M Carmen Bermudo; Svobodova, Marketa; O'Sullivan, Ciara K

    2014-01-01

    Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL(-1)). The specificity of the selected aptamer was

  3. Label-Free Detection of Cardiac Troponin-I Using Carbon Nanofiber Based Nanoelectrode Arrays

    Science.gov (United States)

    Periyakaruppan, Adaikkappan; Koehne, Jessica Erin; Gandhiraman, Ram P.; Meyyappan, M.

    2013-01-01

    A sensor platform based on vertically aligned carbon nanofibers (CNFs) has been developed. Their inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. A carbon nanofiber (CNF) multiplexed array has been fabricated with 9 sensing pads, each containing 40,000 carbon nanofibers as nanoelectrodes. Here, we report the use of vertically aligned CNF nanoelectrodes for the detection of cardiac Troponin-I for the early diagnosis of myocardial infarction. Antibody, antitroponin, probe immobilization and subsequent binding to human cardiac troponin-I were characterized using electrochemical impedance spectroscopy and cyclic voltammetry techniques. Each step of the modification process resulted in changes in electrical capacitance or resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific antigen. This sensor demonstrates high sensitivity, down to 0.2 ng/mL, and good selectivity making this platform a good candidate for early stage diagnosis of myocardial infarction.

  4. Highly sensitive label-free dual sensor array for rapid detection of wound bacteria.

    Science.gov (United States)

    Sheybani, Roya; Shukla, Anita

    2017-06-15

    Wound infections are a critical healthcare concern worldwide. Rapid and effective antibiotic treatments that can mitigate infection severity and prevent the spread of antibiotic resistance are contingent upon timely infection detection. In this work, dual electrochemical pH and cell-attachment sensor arrays were developed for the real-time spatial and temporal monitoring of potential wound infections. Biocompatible polymeric device coatings were integrated to stabilize the sensors and promote bacteria attachment while preventing non-specific cell and protein fouling. High sensitivity (bacteria concentration of 10(2) colony forming units (CFU)/mL and -88.1±6.3mV/pH over a pH range of 1-13) and stability over 14 days were achieved without the addition of biological recognition elements. The dual sensor array was demonstrated to successfully monitor the growth of both gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) over time through lag and log growth phases and following antibiotic administration and in simulated shallow wounds conditions. The versatile fabrication methods utilized in sensor development, superior sensitivity, prolonged stability, and lack of non-specific sensor fouling may enable long-term in situ sensor array operation in low resource settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Epi-detected quadruple-modal nonlinear optical microscopy for label-free imaging of the tooth

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zi; Zheng, Wei; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore); Stephen Hsu, Chin-Ying [Department of Dentistry, Faculty of Dentistry, National University of Singapore and National University Health System, Singapore 119083 (Singapore)

    2015-01-19

    We present an epi-detected quadruple-modal nonlinear optical microscopic imaging technique (i.e., coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon excited fluorescence (TPEF)) based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of the tooth. We demonstrate that high contrast ps-CARS images covering both the fingerprint (500–1800 cm{sup −1}) and high-wavenumber (2500–3800 cm{sup −1}) regions can be acquired to uncover the distributions of mineral and organic biomaterials in the tooth, while high quality TPEF, SHG, and THG images of the tooth can also be acquired under ps laser excitation without damaging the samples. The quadruple-modal nonlinear microscopic images (CARS/SHG/THG/TPEF) acquired provide better understanding of morphological structures and biochemical/biomolecular distributions in the dentin, enamel, and the dentin-enamel junction of the tooth without labeling, facilitating optical diagnosis and characterization of the tooth in dentistry.

  6. Label-free optical characterization methods for detecting amine silanization-driven gold nanoparticle self-assembly.

    Science.gov (United States)

    Roy, Shibsekhar; Dixit, Chandra K; Woolley, Robert; O'Kennedy, Richard; McDonagh, Colette

    2011-09-06

    Fluorescence lifetime correlation spectroscopy (FLCS) is presented as a single-step label-free detection method for probing the amine silanization-driven spontaneous 3D self-assembly of freestanding gold nanoparticles (GNPs) in solution. Unlike the conventional methods of studying self-assembly, for example, UV-vis spectroscopy and electron microscopy, FLCS utilizes the intrinsic gold fluorescence. The significance of this approach is to amalgamate the measurement of optical and hydrodynamic size properties simultaneously to achieve a more coherent description of the self-assembly pathway. GNP self-assembly has two-stage kinetics. Electrostatic interaction drives the initial amine silanization, and this is followed by siloxane bond formation between hydrolyzed ethoxy groups of GNP-attached APTES, resulting in the formation of micrometer-sized superstructures. The self-assembly has resulted in a 5-fold increase in the fluorescence lifetime (FL), and the FLCS study has shown an 8- to 10-fold increase in the diffusion coefficient using the pure diffusion model. This result is consistent with the transmission electron microscopy (TEM) observation, which shows a few hundred fold increase in the diameter due to assembly formation by the GNPs.

  7. DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.

    Science.gov (United States)

    Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli

    2014-02-15

    A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies.

  8. Label-free detection and identification of waterborne parasites using a microfluidic multi-angle laser scattering system

    Science.gov (United States)

    Huang, Wei; Yang, Limei; Lei, Lei; Li, Feng

    2017-10-01

    A microfluidic-based multi-angle laser scattering (MALS) system capable of acquiring scattering patterns of a single particle is designed and demonstrated. The system includes a sheathless nozzle microfluidic glass chip, and an on-chip MALS unit being in alignment with the nozzle exit in the chip. The size and relative refractive indices (RI) of polystyrene (PS) microspheres were deduced with accuracies of 60 nm and 0.002 by comparing the experimental scattering patterns with theoretical ones. We measured scattering patterns of waterborne parasites i.e., Cryptosporidium parvum (C.parvum) and Giardia lamblia (G. lamblia), and some other representative species suspended in deionized water at a maximum flow rate of 12 μL/min, and a maximum of 3000 waterborne parasites can be identified within one minute with a mean accuracy higher than 96% by classification of distinctive scattering patterns using a support-vector-machine (SVM) algorithm. The system provides a promising tool for label-free detection of waterborne parasites and other biological contaminants.

  9. Aptamer-based label-free impedimetric biosensor for detection of progesterone.

    Science.gov (United States)

    Contreras Jiménez, Gastón; Eissa, Shimaa; Ng, Andy; Alhadrami, Hani; Zourob, Mohammed; Siaj, Mohamed

    2015-01-20

    Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products, or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect public health. In this work, we present the selection, identification, and characterization of ssDNA aptamers with high binding affinity to P4. The aptamers were selected in vitro from a single-stranded DNA library of 1.8 × 10(15) oligonucleotides showing dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17β-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4, which results in an increase in electron transfer resistance. Aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. Significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4, as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for concentrations of P4 from 10 to 60 ng/mL with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples and showed good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications

  10. Cost-effective and label-free holographic biosensor for detection of herpes simplex virus (Conference Presentation)

    Science.gov (United States)

    Ray, Aniruddha; Ho, Ha; Daloglu, Mustafa; Torres, Avee; McLeod, Euan; Ozcan, Aydogan

    2017-03-01

    Herpes is one of the most widespread sexually transmitted viral diseases. Timely detection of Herpes Simplex Virus (HSV) can help prevent the rampant spreading of the virus. Current detection techniques such as viral culture, immuno-assays or Polymerase-Chain-Reaction, are time extensive and require expert handling. Here we present a field-portable, easy-to-use, and cost-effective biosensor for the detection of HSV based on holographic imaging. The virus is first captured from a target solution onto specifically developed substrates, prepared by coating glass coverslips with HSV-specific antibodies, and imaged using a lensfree holographic microscope. Several light-emitting-diodes (LEDs), coupled to multi-mode optical-fibers, are used to illuminate the sample containing the viruses. A micro-controller is used to activate the LEDs one at a time and in-line holograms are recorded using a CMOS imager placed immediately above the substrate. These sub-pixel shifted holograms are used to generate a super-resolved hologram, which is reconstructed to obtain the phase and amplitude images of the viruses. The signal of the viruses is enhanced using self-assembled PEG-based nanolenses, formed around the viral particles. Based on the phase information of the reconstructed images we can estimate the size of the viral particles, with an accuracy of +/- 11 nm, as well as quantify the viral load. The limit-of-detection of this system is estimated to be <500 viral copies per 100 μL sample volume that is imaged over 30 mm^2 field-of-view. This holographic microscopy based biosensor is label-free, cost-effective and field-portable, providing results in 2 hours, including sample preparation and imaging time.

  11. Label-free detection of rheumatoid factor using YbYxOy electrolyte-insulator-semiconductor devices.

    Science.gov (United States)

    Pan, Tung-Ming; Lin, Ting-Wei; Chen, Ching-Yi

    2015-09-03

    In this study, we investigated the effect of yttrium content on the structural properties and sensing characteristics of YbYxOy sensing membranes for electrolyte-insulator-semiconductor (EIS) sensors to detect the rheumatoid factor (RF). The YbYxOy EIS device prepared at the 60 W plasma condition exhibited a higher sensitivity of 65.77 mV/pH, a lower hysteresis voltage of ∼1 mV, and a smaller drift rate of 0.14 mV/h than did those prepared at the other conditions. We attribute this behavior to the optimal yttrium content in the YbYxOy film forming a smooth surface. Furthermore, we used a novel YbTixOy EIS biosensor to measure the RF antigen in human serum because of its rapid and label-free detection. Two different techniques were used for the immobilization of RF antibody onto the surface of an YbTixOy EIS sensor. The RF antibody was directly immobilized on the EIS surface modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA). In contrast, a mixture of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution was used to functionalize the carboxyl groups at the tail of RF antibodies. RF antibodies functionalized with the active NHS esters were covalently immobilized on the APTES-modified YbTixOy surface. The immobilized RF antibodies on the EIS that are functionalized with the EDC and NHS exhibit higher (41.11mV/pCRF) for detection of serum RF antigen in the range 10(-7) to 10(-3) M, compared to traditional antibody immobilization technique via APTES and GA linkage. The YbTixOy EIS biosensor is a promising analytical tool for RF antigen monitoring due to its good sensitivity, stability and repeatability.

  12. Label-free vascular imaging in a spontaneous hamster cheek pouch carcinogen model for pre-cancer detection (Conference Presentation)

    Science.gov (United States)

    Hu, Fangyao; Morhard, Robert; Liu, Heather; Murphy, Helen; Farsiu, Sina; Ramanujam, Nimmi

    2016-03-01

    Inducing angiogenesis is one hallmark of cancer. Tumor induced neovasculature is often characterized as leaky, tortuous and chaotic, unlike a highly organized normal vasculature. Additionally, in the course of carcinogenesis, angiogenesis precedes a visible lesion. Tumor cannot grow beyond 1-2 mm in diameter without inducing angiogenesis. Therefore, capturing the event of angiogenesis may aid early detection of pre-cancer -important for better treatment prognoses in regions that lack the resources to manage invasive cancer. In this study, we imaged the neovascularization in vivo in a spontaneous hamster cheek pouch carcinogen model using a, non-invasive, label-free, high resolution, reflected-light spectral darkfield microscope. Hamsters' cheek pouches were painted with 7,12-Dimethylbenz[a]anthracene (DMBA) to induce pre-cancerous to cancerous changes, or mineral oil as control. High resolution spectral darkfield images were obtained over the course of pre-cancer development and in control cheek pouches. The vasculature was segmented with a multi-scale Gabor filter with an 85% accuracy compared with manually traced masks. Highly tortuous vasculature was observed only in the DMBA treated cheek pouches as early as 6 weeks of treatment. In addition, the highly tortuous vessels could be identified before a visible lesion occurred later during the treatment. The vessel patterns as determined by the tortuosity index were significantly different from that of the control cheek pouch. This preliminary study suggests that high-resolution darkfield microscopy is promising tool for pre-cancer and early cancer detection in low resource settings.

  13. Development of a Microfluidic-Based Optical Sensing Device for Label-Free Detection of Circulating Tumor Cells (CTCs Through Their Lactic Acid Metabolism

    Directory of Open Access Journals (Sweden)

    Tzu-Keng Chiu

    2015-03-01

    Full Text Available This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs, a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner.

  14. Label-free antibody detection using band edge fringes in SOI planar photonic crystal waveguides in the slow-light regime.

    Science.gov (United States)

    García-Rupérez, Jaime; Toccafondo, Veronica; Bañuls, María José; Castelló, Javier García; Griol, Amadeu; Peransi-Llopis, Sergio; Maquieira, Ángel

    2010-11-08

    We report experimental results of label-free anti-bovine serum albumin (anti-BSA) antibody detection using a SOI planar photonic crystal waveguide previously bio-functionalized with complementary BSA antigen probes. Sharp fringes appearing in the slow-light regime near the edge of the guided band are used to perform the sensing. We have modeled the presence of these band edge fringes and demonstrated the possibility of using them for sensing purposes by performing refractive index variations detection, achieving a sensitivity of 174.8 nm/RIU. Then, label-free anti-BSA biosensing experiments have been carried out, estimating a surface mass density detection limit below 2.1 pg/mm2 and a total mass detection limit below 0.2 fg.

  15. Label-free RNA aptamer-based capacitive biosensor for the detection of C-reactive protein.

    Science.gov (United States)

    Qureshi, Anjum; Gurbuz, Yasar; Kallempudi, Saravan; Niazi, Javed H

    2010-08-28

    In this study, we report a novel aptamer-based capacitive label-free biosensor for monitoring transducing aptamer-protein recognition events, based on charge distribution under the applied frequency by non-Faradaic impedance spectroscopy (NFIS). This approach to capacitive biosensors is reported for the first time in this study, is reagent-less in processing and is developed using gold interdigitated (GID) capacitor arrays functionalized with synthetic RNA aptamers. The RNA atpamers served as biorecognition elements for C-reactive protein (CRP), a biomarker for cardiovascular disease risk (CVR). The signal is generated as a result of the change in relative capacitance occurring as a result of the formation of an RNA-CRP complex on GID capacitors with the applied AC electrical frequency (50-350 MHz). The dispersion peak of the capacitance curve was dependent on the CRP concentration and tends to shift toward lower frequencies, accompanied by the increase in relaxation time due to the increased size of the aptamer-CRP complex. The dissociation constant (K(d)) calculated from the non-linear regression analysis of the relative capacitance change with the applied frequency showed that strong binding of CRP occurred at 208 MHz (K(d) = 1.6 microM) followed by 150 MHz (K(d) = 4.2 microM) and 306 MHz (K(d) = 3.4 microM) frequencies. The dynamic detection range for CRP is determined to be within 100-500 pg ml(-1). Our results demonstrates the behavior of an RNA-protein complex on GID capacitors under an applied electric field, which can be extended to other pairs of affinity biomolecules as well as for the development of electrical biosensor systems for different applications, including the early diagnosis of diseases.

  16. Real-Time, Label-Free Detection of Biomolecular Interactions in Sandwich Assays by the Oblique-Incidence Reflectivity Difference Technique

    Directory of Open Access Journals (Sweden)

    Yung-Shin Sun

    2014-12-01

    Full Text Available One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR. The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

  17. Fundamental research on the label-free detection of protein adsorption using near-infrared light-responsive plasmonic metal nanoshell arrays with controlled nanogap

    OpenAIRE

    Uchida, Shuhei; Zettsu, Nobuyuki; Endo, Katsuyoshi; Yamamura, Kazuya

    2013-01-01

    In this work, we focused on the label-free detection of simple protein binding using near-infrared light-responsive plasmonic nanoshell arrays with a controlled interparticle distance. The nanoshell arrays were fabricated by a combination of colloidal self-assembly and subsequent isotropic helium plasma etching under atmospheric pressure. The diameter, interparticle distance, and shape of nanoshells can be tuned with nanometric accuracy by changing the experimental conditions. The Au, Ag, and...

  18. A highly sensitive and facile graphene oxide-based nucleic acid probe: Label-free detection of telomerase activity in cancer patient's urine using AIEgens.

    Science.gov (United States)

    Ou, Xiaowen; Hong, Fan; Zhang, Zhenyu; Cheng, Yong; Zhao, Zujin; Gao, Pengcheng; Lou, Xiaoding; Xia, Fan; Wang, Shutao

    2017-03-15

    Molecular beacon (MB)-based sensing platforms that consist of a fluorogen-quencher pair play an important role in medical and biological researches. However, the synthesis of both fluorogen and quencher in the nucleic acid probes will increase the burden of organic synthesis works and induce the difficulties for precisely controlling the relative distance between fluorogen and quencher, which may lead to false-positive and false-negative results. In this work, initially we report a single labeled MB (FAM-MB, with carboxyfluorescein as fluorogen and without quencher) thus simplifies MBs with the aid of graphene oxide (GO) to detect telomerase activity. To further simplify this structure, namely label-free strategy, we design a facile, sensitive and selective platform using a label-free beacon (AIE-MB, without fluorogen and quencher), based on aggregation-induced emission fluorogen (silole-R). Upon the addition of telomerase, AIE-MB induced comb-like DNA structure leads to high aggregation of silole-R and thus exhibits strong fluorescence emission. By exploitation of this, we can detect telomerase with superior sensitivity and demonstrate their applications in bladder cancer diagnosis. Compared to single-labeled FAM-MB based telomerase activity assay, the label-free AIE-MB induced method could perform the sensitive detection with high signal-to-background ratio.

  19. Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

    Science.gov (United States)

    Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

    2017-03-15

    This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb(2+)) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb(2+) ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb(2+) with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb(2+) monitoring is enabled with promising application potential.

  20. Label-free porous silicon immunosensor for broad detection of opiates in a blind clinical study and results comparison to commercial analytical chemistry techniques.

    Science.gov (United States)

    Bonanno, Lisa M; Kwong, Tai C; DeLouise, Lisa A

    2010-12-01

    In this work, we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n = 50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was <9% both within-run and between-day at and above the cutoff limit of 300 ng mL(-1). This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semiquantitative demonstration of the technology's successful clinical use. These results motivate future development of label-free PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting.

  1. A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch.

    Science.gov (United States)

    Xie, Qingyun; Zhao, Fulin; Liu, Hongrui; Shan, Yanke; Liu, Fei

    2015-07-02

    Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM.

  2. Whispering gallery mode bio-sensor for label-free detection of single molecules: thermo-optic vs. reactive mechanism.

    Science.gov (United States)

    Arnold, S; Shopova, S I; Holler, S

    2010-01-04

    Thermo-optic and reactive mechanisms for label-free sensing of bio-particles are compared theoretically for Whispering Gallery Mode (WGM) resonators (sphere, toroid) formed from silica and stimulated into a first order equatorial mode. Although it has been expected that a thermo-optic mechanism should "greatly enhance" wavelength shift signals [A.M. Armani et al, Science 317, 783-787 (2007)] accompanying protein binding on a silica WGM cavity having high Q (10(8)), for a combination of wavelength (680 nm), drive power (1 mW), and cavity size (43 microm radius), our calculations find no such enhancement. The possible reasons for this disparity are discussed.

  3. Quinone-Based Polymers for Label-Free and Reagentless Electrochemical Immunosensors: Application to Proteins, Antibodies and Pesticides Detection

    Directory of Open Access Journals (Sweden)

    Minh-Chau Pham

    2013-01-01

    Full Text Available Polyquinone derivatives are widely recognized in the literature for their remarkable properties, their biocompatibility, simple synthesis, and easy bio-functionalization. We have shown that polyquinones present very stable electroactivity in neutral aqueous medium within the cathodic potential domain avoiding side oxidation of interfering species. Besides, they can act as immobilized redox transducers for probing biomolecular interactions in sensors. Our group has been working on devices based on such modified electrodes with a view to applications for proteins, antibodies and organic pollutants using a reagentless label-free electrochemical immunosensor format. Herein, these developments are briefly reviewed and put into perspective.

  4. Label-free detection of aflatoxin M1 with electrochemical Fe{sub 3}O{sub 4}/polyaniline-based aptasensor

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Binh Hai [Institute of Material Science, Vietnam Academy of Science and Technology, 18, Hoang Quoc Viet Road, Cau Giay, Hanoi (Viet Nam); Tran, Lam Dai, E-mail: lamtd@ims.vast.ac.vn [Institute of Material Science, Vietnam Academy of Science and Technology, 18, Hoang Quoc Viet Road, Cau Giay, Hanoi (Viet Nam); Do, Quan Phuc [Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science, Hanoi (Viet Nam); Nguyen, Huy Le [School of Chemical Engineering, Hanoi University of Science and Technology, 1, Dai Co Viet Road, Hanoi (Viet Nam); Tran, Ngoc Huan [Department of Chemistry, Hanyang University, Seoul 133-791 (Korea, Republic of); Nguyen, Phuc Xuan [Institute of Material Science, Vietnam Academy of Science and Technology, 18, Hoang Quoc Viet Road, Cau Giay, Hanoi (Viet Nam)

    2013-05-01

    The selective detection of ultratrace amounts of aflatoxin M1 (AFM1) is extremely important for food safety since it is the most toxic mycotoxin class that is allowed to be present on cow milk with strictly low regulatory levels. In this work, Fe{sub 3}O{sub 4} incorporated polyaniline (Fe{sub 3}O{sub 4}/PANi) film has been polymerized on interdigitated electrode (IDE) as sensitive film for AFM1 electrochemical biosensor. The immobilized aptamers as an affinity capture reagent and magnetic nanoparticles for signal amplification element have been employed in the sensing platform. Label-free and direct detection of the aptamer-AFM1 on Fe{sub 3}O{sub 4}/PANi interface were performed via electrochemical signal change, acquired by cyclic and square wave voltammetries. With a simplified strategy, this electrochemical aptasensor shows a good sensitivity to AFM1 in the range of 6–60 ng·L{sup −1}, with the detection limit of 1.98 ng·L{sup −1}. The results open up the path for designing cost effective aptasensors for other biomedical applications. Highlights: ► Label-free detection of the aptamer-AFM1 on Fe{sub 3}O{sub 4}/PANi interface ► Electrochemical aptasensor with a good sensitivity to AFM1 in the range of 6–60 ng·L{sup −1}, with detection limit of 1.98 ng·L{sup −1} ► Advantages over other analytical techniques in terms of label free format, sensitivity, stability and analysis time.

  5. An ultra-facile and label-free immunoassay strategy for detection of copper (II) utilizing chemiluminescence self-enhancement of Cu (II)-ethylenediaminetetraacetate chelate.

    Science.gov (United States)

    Ouyang, Hui; Shu, Qi; Wang, Wenwen; Wang, Zhenxing; Yang, Shijia; Wang, Lin; Fu, Zhifeng

    2016-11-15

    The establishment of facile, rapid, sensitive and cost-effective protocols for the detection of heavy metals is of great significance for human health and environmental monitoring. Hereby, an ultra-facile and label-free immunoassay strategy was designed for detecting heavy metal ion by using Cu (II) as the model analyte. Cu (II) reacted previously with ethylenediaminetetraacetate (EDTA) was captured by immobilized monoclonal antibody for Cu (II)-EDTA chelate. Then Cu (II) was detected based on the self-enhancing effect of Cu (II)-EDTA chelate to luminol-H2O2 chemiluminescence reaction. The CL intensity is linear relative with Cu (II) concentration in a very wide range of 1.0-1000ng/mL, with a detection limit of 0.33ng/mL (S/N=3). Since the specificity of this proposed strategy relied on both the specificity of monoclonal antibody and the specificity of luminol-H2O2 system, it could avoid interference from most common ions. The proposed method was used successfully to detect Cu (II) in traditional Chinese medicine and environmental water samples with acceptable recovery values of 82-113%. This proof-of-principle work demonstrated the feasibility of the label-free immunoassay for heavy metal ions, and opened a new avenue for rapid screening and field assay for drug safety, environmental monitoring and clinical diagnosis.

  6. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    Science.gov (United States)

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  7. Multiplexed specific label-free detection of NCI-H358 lung cancer cell line lysates with silicon based photonic crystal microcavity biosensors.

    Science.gov (United States)

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Drabkin, Harry A; Gemmill, Robert M; Simon, George R; Chin, Steve H; Chen, Ray T

    2013-05-15

    We experimentally demonstrate label-free photonic crystal (PC) microcavity biosensors in silicon-on-insulator (SOI) to detect the epithelial-mesenchymal transition (EMT) transcription factor, ZEB1, in minute volumes of sample. Multiplexed specific detection of ZEB1 in lysates from NCI-H358 lung cancer cells down to an estimated concentration of 2 cells per micro-liter is demonstrated. L13 photonic crystal microcavities, coupled to W1 photonic crystal waveguides, are employed in which resonances show high Q in the bio-ambient phosphate buffered saline (PBS). When the sensor surface is derivatized with a specific antibody, the binding of the corresponding antigen from a complex whole-cell lysate generates a change in refractive index in the vicinity of the photonic crystal microcavity, leading to a change in the resonance wavelength of the resonance modes of the photonic crystal microcavity. The shift in the resonance wavelength reveals the presence of the antigen. The sensor cavity has a surface area of ∼11μm(2). Multiplexed sensors permit simultaneous detection of many binding interactions with specific immobilized antibodies from the same bio-sample at the same instant of time. Specificity was demonstrated using a sandwich assay which further amplifies the detection sensitivity at low concentrations. The device represents a proof-of-concept demonstration of label-free, high throughput, multiplexed detection of cancer cells with specificity and sensitivity on a silicon chip platform.

  8. Direct-writing colloidal photonic crystal microfluidic chips by inkjet printing for label-free protein detection.

    Science.gov (United States)

    Shen, Weizhi; Li, Mingzhu; Ye, Changqing; Jiang, Lei; Song, Yanlin

    2012-09-07

    Integrating photonic crystals (PC) into microfluidic systems has attracted immense interest for its novel functions. However, it is still a great challenge to fabricate PC microfluidic chips rapidly with complex functions. In this work, a direct-writing colloidal PC microchannel was firstly achieved by inkjet printing and was used for the surface-tension-confined microfluidic immune assay. PC channels with different structure colors have been successfully integrated on one chip. The fabricated chip has the advantages of rapid fabrication, quick fluidic transport and can monitor the fluidic fluxion using the naked eye. Utilizing this PC microfluidic chip, a colorimetric label-free immune assay was realized without nonspecific adsorption interference of the target.

  9. Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/fluorescence platform based on Bloch surface waves.

    Science.gov (United States)

    Sinibaldi, Alberto; Sampaoli, Camilla; Danz, Norbert; Munzert, Peter; Sibilio, Leonardo; Sonntag, Frank; Occhicone, Agostino; Falvo, Elisabetta; Tremante, Elisa; Giacomini, Patrizio; Michelotti, Francesco

    2017-06-15

    We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of ERBB2/neu/Her2 in cell lysates. ERBB2 is a pivotal breast cancer biomarker and targetable oncogenic driver associated with aggressive breast cancer subtypes. To quantitate soluble ERBB2, we developed an optical platform that combines label-free and fluorescence detection modes. Such platform makes use of a sandwich assay in which the one-dimensional photonic crystals sustaining Bloch surface waves are tailored with a monoclonal antibody for highly specific biological recognition (BSW biochip). In a second step, a second antibody to ERBB2 quantitatively detects the bound analyte. The strategy of the present approach takes advantage of the combination of label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3ng/mL (1.5pM) for ERBB2 in cell lysates. Such resolution meets the international guidelines and recommendations (15ng/mL) for diagnostic ERBB2 assays that in the future may help to more precisely assign therapies counteracting cancer cell proliferation and metastatic spread.

  10. A cytokine immunosensor for Multiple Sclerosis detection based upon label-free electrochemical impedance spectroscopy using electroplated printed circuit board electrodes.

    Science.gov (United States)

    Bhavsar, Kinjal; Fairchild, Aaron; Alonas, Eric; Bishop, Daniel K; La Belle, Jeffrey T; Sweeney, James; Alford, T L; Joshi, Lokesh

    2009-10-15

    A biosensor for the serum cytokine, Interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy (EIS) monitoring approach is described. Overexpression of IL-12 has been correlated to the diagnosis of Multiple Sclerosis (MS). An immunosensor has been fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode and immobilizing anti-IL-12 monoclonal antibodies (MAb) onto the surface of the electrode. This approach yields a robust sensor that facilitates reproducible mass fabrication and easy alteration of the electrode shape. Results indicate that this novel PCB sensor can detect IL-12 at physiological levels, <100 fM with f-values of 0.05 (typically <0.0001) in a label-free and rapid manner. A linear (with respect to log concentration) detectable range was achieved. Detection in a complex biological solution is also explored; however, significant loss of dynamic range is noted in the 100% complex solution. The cost effective approach described here can be used potentially for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.

  11. Label-free detection of Cu(2+ and Hg(2+ ions using reconstructed Cu(2+-specific DNAzyme and G-quadruplex DNAzyme.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu(2+-specific DNA-cleaving DNAzyme (Cu(2+-specific DNAzyme with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s, linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu(2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS-H2O2 reaction. Based on these, two label-free, cost-effective and simple Cu(2+ sensors were designed. These two sensors followed different detection modes: 'turn-on' and 'turn-off'. As for the 'turn-on' sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu(2+-specific DNAzyme and G-quadruplex DNAzyme provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu(2+ with a detection limit of 3.9 nM. Visual detection was possible. Although the 'turn-off' sensor gave lower detection sensitivity than the 'turn-on' one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg(2+ ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg(2+ sensor design. The proposed sensor allowed the specific detection of Hg(2+ ion with a detection of 4.8 nM. Visual detection was also possible.

  12. Low fouling label-free DNA sensor based on polyethylene glycols decorated with gold nanoparticles for the detection of breast cancer biomarkers.

    Science.gov (United States)

    Wang, Wenting; Fan, Xiaojian; Xu, Shenghao; Davis, Jason J; Luo, Xiliang

    2015-09-15

    A label-free and low fouling biosensor based on functional polyethylene glycols selective for breast cancer susceptibility gene (BRCA1) is reported. Sensory interfaces were prepared through the modification of a glassy carbon electrode with highly cross-linked polyethylene glycol (PEG) film containing amine groups, followed by the self-assembly of gold nanoparticles and the immobilization of BRCA1 complementary single-strand 19-mer oligonucleotides. In the presence of a specific BRCA1 sequence capture and hybridization results in interfacial change sensitively monitored using electrochemical impedance spectroscopy. The combined utilization of a PEG polymer film and gold nanoparticle mixed interface enables very high levels of sensitivity and a highly effective assaying in patient samples. Assay linear range was from 50.0 fM to 1.0 nM, with a limit of detection of 1.72 fM. Furthermore, this label-free DNA sensor has been used for assaying BRCA1 in serum samples, showing its feasible potential for diagnostic applications in clinical analysis of breast cancer gene BRCA1. Foreseeable, this sensor made on this basis undoubtedly provide the most effective and sensitive detection for BRCA1.

  13. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhong Feng; Chen, Dong Mei [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2015-10-15

    Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. - Highlights: • Conformational switch of i-motif is used for the detection of glucose and urea. • The sensor can be regenerated. • The proposed method is successfully applied in real sample assay. • Our method is label-free and inexpensive.

  14. A multi-parameter decoupling method with a Lamb wave sensor for improving the selectivity of label-free liquid detection.

    Science.gov (United States)

    Zhou, Lianqun; Wu, Yihui; Xuan, Ming; Manceau, Jean-François; Bastien, François

    2012-01-01

    In this paper, a liquid multi-parameter decoupling method with only one Lamb wave sensor is presented. In a Lamb wave sensor, antisymmetric modes (A(01) mode for low frequency, A(03) mode for high frequency) and symmetric modes (S(0) mode) are used to detect multiple parameters of a liquid, such as its density, sound velocity, and viscosity. We found they can play very different roles in the detections. For example, the A(01) mode is very sensitive to the liquid's density but the A(03) mode is sensitive to the sound velocity. Here, the A(0) mode is used to identify the density of the detected liquid and with this density value we obtained the viscosity by the amplitude shifts of the S(0) mode. This could be a way to distinguish an unknown liquid with high sensitivity or to solve the problem of selectivity of label-free detection on biosensors.

  15. Cratylia mollis lectin nanoelectrode for differential diagnostic of prostate cancer and benign prostatic hyperplasia based on label-free detection.

    Science.gov (United States)

    Silva, Priscila M S; Lima, Amanda L R; Silva, Bárbara V M; Coelho, Luana C B B; Dutra, Rosa F; Correia, Maria T S

    2016-11-15

    The research for new biomarkers of cancer has studied the role of fetuin glycoprotein on the metastatic disease diagnosis. Cratylia mollis is a lectin with high finity to fetuin, and used here to differentiate prostate cancer and benign prostatic hyperplasia. A label-free electrochemical nanosensor based on assembled carboxylated carbon nanotubes (COOH-CNTs) and poly-L-lysine (PLL) film was developed and applied to serum samples of prostate cancer positive for Gleason score. The electrode analytical response to fetuin in PBS samples, obtained by square wave voltammetry, exhibited a linear range from 0.5 to 25µgmL(-1), with a high correlation coefficient (r=0.994, pprostate cancer patients with known the Gleason score were tested showing a significant statistically correlation. Thus, the lectin nanoelectrode was able to distinguish the degree of staging prostate cancer, providing the diagnostic differentiation of benign and malign hyperplasia. To the best of our knowledge, it is the first biosensor for this application using a lectin.

  16. Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

    Science.gov (United States)

    Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

    2014-12-01

    The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification

  17. A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2016-08-17

    In this work, we report a novel label-free fluorescence “turn off-on” biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS{sub 2} quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS{sub 2} QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS{sub 2} QDs surface were interacted with the amino groups (−NH{sub 2}), carboxyl groups (−COOH) and hydroxyl groups (−OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively “turned on”. Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I{sub 0} (I and I{sub 0} were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2–192.5 nmol L{sup −1}, And the detection limit could be down to 0.08 nmol L{sup −1}. Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. - Graphical abstract: A label-free fluorescence biosensor for highly sensitive detection of lectin based on the integration of carboxymethyl chitosan, CuInS{sub 2} quantum dots and gold nanoparticles. - Highlights: • A label-free near-infrared fluorescence “turn off-on” biosensor for detection of lectin was established. • The highly sensitive biosensor was based on the

  18. Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

    Science.gov (United States)

    Donmez, Soner; Arslan, Fatma; Arslan, Halit

    2016-05-01

    In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

  19. Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

    Science.gov (United States)

    Liang, Boying; Ju, Yue; Joubert, James R; Kaleta, Erin J; Lopez, Rodrigo; Jones, Ian W; Hall, Henry K; Ratnayaka, Saliya N; Wysocki, Vicki H; Saavedra, S Scott

    2015-04-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

  20. Electrochemical Label-Free Nucleotide Sensors.

    Science.gov (United States)

    Aoki, Hiroshi

    2015-12-01

    Numerous researchers have devoted a great deal of effort over the last few decades to the development of electrochemical oligonucleotide detection techniques, owing to their advantages of simple design, inherently small dimensions, and low power requirements. Their simplicity and rapidity of detection makes label-free oligonucleotide sensors of great potential use as first-aid screening tools in the analytical field of environmental measurements and healthcare management. This review article covers label-free oligonucleotide sensors, focusing specifically on topical electrochemical techniques, including intrinsic redox reaction of bases, conductive polymers, the use of electrochemical indicators, and highly ordered probe structures.

  1. A Label-Free Microelectrode Array Based on One-Step Synthesis of Chitosan–Multi-Walled Carbon Nanotube–Thionine for Ultrasensitive Detection of Carcinoembryonic Antigen

    Directory of Open Access Journals (Sweden)

    Huiren Xu

    2016-07-01

    Full Text Available Carcinoembryonic antigen (CEA has been an extensively used tumor marker responsible for clinical early diagnosis of cervical carcinomas, and pancreatic, colorectal, gastric and lung cancer. Combined with micro-electro mechanical system (MEMS technology, it is important to develop a novel immune microelectrode array (MEA not only for rapid analysis of serum samples, but also for cell detection in vitro and in vivo. In this work, we depict a simple approach to modify chitosan–multi-walled carbon nanotubes–thionine (CS–MWCNTs–THI hybrid film through one-step electrochemical deposition and the CS-MWCNTs-THI hybrid films are successfully employed to immobilize anti-CEA for fabricating simple, label-free, and highly sensitive electro-chemical immune MEAs. The detection principle of immune MEA was based on the fact that the increasing formation of the antigen-antibody immunocomplex resulted in the decreased response currents and the relationship between the current reductions with the corresponding CEA concentrations was directly proportional. Experimental results indicated that the label-free MEA had good selectivity and the limit of detection for CEA is 0.5 pg/mL signal to noise ratio (SNR = 3. A linear calibration plot for the detection of CEA was obtained in a wide concentration range from 1 pg/mL to 100 ng/mL (r = 0.996. This novel MEA has potential applications for detecting CEA for the research on cancer cells and cancer tissue slices as well as for effective early diagnosis.

  2. New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.

    Science.gov (United States)

    Wu, Haiyun; Ohnuki, Hitoshi; Ota, Shirei; Murata, Masataka; Yoshiura, Yasutoshi; Endo, Hideaki

    2017-07-15

    Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml(-1) (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Low-cost label-free electrical detection of artificial DNA nanostructures using solution-processed oxide thin-film transistors.

    Science.gov (United States)

    Kim, Si Joon; Jung, Joohye; Lee, Keun Woo; Yoon, Doo Hyun; Jung, Tae Soo; Dugasani, Sreekantha Reddy; Park, Sung Ha; Kim, Hyun Jae

    2013-11-13

    A high-sensitivity, label-free method for detecting deoxyribonucleic acid (DNA) using solution-processed oxide thin-film transistors (TFTs) was developed. Double-crossover (DX) DNA nanostructures with different concentrations of divalent Cu ion (Cu(2+)) were immobilized on an In-Ga-Zn-O (IGZO) back-channel surface, which changed the electrical performance of the IGZO TFTs. The detection mechanism of the IGZO TFT-based DNA biosensor is attributed to electron trapping and electrostatic interactions caused by negatively charged phosphate groups on the DNA backbone. Furthermore, Cu(2+) in DX DNA nanostructures generates a current path when a gate bias is applied. The direct effect on the electrical response implies that solution-processed IGZO TFTs could be used to realize low-cost and high-sensitivity DNA biosensors.

  4. Label-free C-reactive protein electronic detection with an electrolyte-gated organic field-effect transistor-based immunosensor.

    Science.gov (United States)

    Magliulo, Maria; De Tullio, Donato; Vikholm-Lundin, Inger; Albers, Willem M; Munter, Tony; Manoli, Kyriaki; Palazzo, Gerardo; Torsi, Luisa

    2016-06-01

    In this contribution, we propose a label-free immunosensor, based on a novel type of electrolyte-gated field-effect transistor (EGOFET), for ultrasensitive detection of the C-reactive protein (CRP). The recognition layer of the biosensor is fabricated by physical adsorption of the anti-CRP monoclonal antibody onto a poly-3-hexyl thiophene (P3HT) organic semiconductor surface. A supplementary nonionic hydrophilic polymer is used as a blocking agent preventing nonspecific interactions and allowing a better orientation of the antibodies immobilized onto the P3HT surface. The whole biomolecule immobilization procedure does not require any pretreatment of the organic semiconductor surface, and the whole functionalization process is completed in less than 30 min. Surface plasmon resonance (SPR) measurements were performed to assess the amount of biomolecules physisorbed onto the P3HT and to evaluate the CRP binding proprieties of the deposited anti-CRP layer. A partial surface coverage of about 23 % of adsorbed antibody molecules was found to most efficiently sense the CRP. The electrical performance of the EGOFET immunosensor was comparable to that of a bare P3HT EGOFET device, and the obtained CRP calibration curve was linear over six orders of magnitude (from 4 pM to 2 μM). The relative standard deviation of the individual calibration points, measured on immunosensors fabricated on different chips, ranged between 1 and 14 %, and a detection limit of 2 pM (220 ng/L) was established. The novel electronic immunosensor is compatible with low-cost fabrication procedures and was successfully employed for the detection of the CRP biomarker in the clinically relevant matrix serum. Graphical abstract Schematic of the EGOFET immunosensor for CRP detection. The anti-CRP monoclonal antibody layer is physisorbed on the P3HT organic semiconductor and the CRP is directly measured by a label-free electronic EGOFET transducer.

  5. Label-free optical detection of C-reactive protein by nanoimprint lithography-based 2D-photonic crystal film.

    Science.gov (United States)

    Endo, Tatsuro; Kajita, Hiroshi; Kawaguchi, Yukio; Kosaka, Terumasa; Himi, Toshiyuki

    2016-06-01

    The development of high-sensitive, and cost-effective novel biosensors have been strongly desired for future medical diagnostics. To develop novel biosensor, the authors focused on the specific optical characteristics of photonic crystal. In this study, a label-free optical biosensor, polymer-based two-dimensional photonic crystal (2D-PhC) film fabricated using nanoimprint lithography (NIL), was developed for detection of C-reactive protein (CRP) in human serum. The nano-hole array constructed NIL-based 2D-PhC (hole diameter: 230 nm, distance: 230, depth: 200 nm) was fabricated on a cyclo-olefin polymer (COP) film (100 µm) using thermal NIL and required surface modifications to reduce nonspecific adsorption of target proteins. Antigen-antibody reactions on the NIL-based 2D-PhC caused changes to the surrounding refractive index, which was monitored as reflection spectrum changes in the visible region. By using surface modified 2D-PhC, the calculated detection limit for CRP was 12.24 pg/mL at an extremely short reaction time (5 min) without the need for additional labeling procedures and secondary antibody. Furthermore, using the dual-functional random copolymer, CRP could be detected in a pooled blood serum diluted 100× with dramatic reduction of nonspecific adsorption. From these results, the NIL-based 2D-PhC film has great potential for development of an on-site, high-sensitivity, cost-effective, label-free biosensor for medical diagnostics applications.

  6. Fluorescent trimethyl-substituted naphthyridine as a label-free signal reporter for one-step and highly sensitive fluorescent detection of DNA in serum samples.

    Science.gov (United States)

    Wang, Jiamian; Wang, Xiuyun; Wu, Shuo; Che, Ruping; Luo, Pinchen; Meng, Changgong

    2017-01-15

    A facile label-free sensing method is developed for the one-step and highly sensitive fluorescent detection of DNA, which couples the specific C-C mismatch bonding and fluorescent quenching property of a trimethyl-substituted naphthyridine dye (ATMND) with the exonuclease III (Exo III) assisted cascade target recycling amplification strategy. In the absence of target DNA, the DNA hairpin probe with a C-C mismatch in the stem and more than 4 bases overhung at the 3' terminus could entrap and quench the fluorescence of ATMND and resist the digestion of Exo III, thus showing a low fluorescence background. In the presence of the target, however, the hybridization event between the two protruding segments and the target triggers the digestion reaction of Exo III, recycles the initial target, and simultaneously releases both the secondary target analogue and the ATMND caged in the stem. The released initial and secondary targets take part in another cycle of digestion, thus leading to the release of a huge amount of free ATMND for signal transducing. Based on the fluorescence recovery, the as-proposed label-free fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 10pM to 1μM, a low limit of detection of 6pM, good selectivity, and a facile one-step operation at room temperature. Practical sample analysis in serum samples indicates the method has good precision and accuracy, which may thus have application potentials for point-of-care screening of DNA in complex clinical and environmental samples.

  7. Label-Free Biosensors for Cell Biology

    Directory of Open Access Journals (Sweden)

    Ye Fang

    2011-01-01

    Full Text Available Label-free biosensors for studying cell biology have finally come of age. Recent developments have advanced the biosensors from low throughput and high maintenance research tools to high throughput and low maintenance screening platforms. In parallel, the biosensors have evolved from an analytical tool solely for molecular interaction analysis to powerful platforms for studying cell biology at the whole cell level. This paper presents historical development, detection principles, and applications in cell biology of label-free biosensors. Future perspectives are also discussed.

  8. Label-free, real-time detection of the dynamic processes of protein degradation using oblique-incidence reflectivity difference method

    Science.gov (United States)

    Liu, S.; Zhu, J. H.; He, L. P.; Dai, J.; Lu, H. B.; Wu, L.; Jin, K. J.; Yang, G. Z.; Zhu, H.

    2014-04-01

    Based on the requirements for studying the dynamic process of proteinase action substrates in life science, we selected six random proteins including 1L-10, SCGB2A2, CENPQ, GST, HK1, KLHL7, as well as five different concentrations of 1L-10 proteins of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml, and fabricated two types of substrate protein microarrays, respectively. We detected the dynamic processes of proteins degraded by proteinase K using oblique-incidence reflectivity difference (OIRD) method in a label-free and real-time manner. We obtained the relevant degradation velocities and the degradation time. The experimental results demonstrate that OIRD has the ability to study proteinase action substrates which is out of reach of label methods and is expected to offer opportunities to determine protease-substrate relationships on the systems biology level.

  9. Label-free detection of cardiac troponin-I using gold nanoparticles functionalized single-walled carbon nanotubes based chemiresistive biosensor

    Science.gov (United States)

    Rajesh, Sharma, Vikash; Puri, Nitin K.; Singh, Rajiv K.; Biradar, Ashok M.; Mulchanadani, Ashok

    2013-11-01

    We report a specific and ultrasensitive, label-free chemiresistive biosensor based on mercaptopropionic acid capped gold nanoparticles (GNP) functionalized single walled carbon nanotube (SWNT) hybrid for the detection of cardiac specific biomarker troponin-I (cTnI). GNPs were attached to SWNTs through a molecular linker 1-pyrenemethylamine. The highly specific cTnI antibody was covalently immobilized on GNPs through capping agent using carbodiimide coupling reaction. The cTnI interaction to its corresponding antibody was studied with respect to changes in conductance in SWNTs channel, and a detailed field-effect transistor characteristic was delineated. The device exhibited a linear response to cTnI from 0.01 to 10 ng ml-1.

  10. Sci—Fri AM: Mountain — 04: Label-free Raman spectroscopy of single tumour cells detects early radiation-induced glycogen synthesis associated with increased radiation resistance

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, Q; Lum, JJ [BC Cancer Agency — Vancouver Island Centre (Canada); Isabelle, M; Harder, S; Jirasek, A [Physics and Astronomy, University of Victoria (Australia); Brolo, AG [Chemistry, University of Victoria (Australia)

    2014-08-15

    Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, and experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.

  11. Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.

    Science.gov (United States)

    Sinville, Rondedrick; Coyne, Jennifer; Meagher, Robert J; Cheng, Yu-Wei; Barany, Francis; Barron, Annelise; Soper, Steven A

    2008-12-01

    We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele-specific ligase detection reaction (LDR) with free-solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide "drag-tags" onto the LDR primers. The additional drag of the charge-neutral drag-tag breaks the linear scaling of the charge-to-friction ratio of DNA and enables size-based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag-tags onto a set of four LDR primers designed to probe the K-ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag-tag conjugate (LDR-dt) products in a multiplexed, single-tube format with mutant:WT ratios as low as 1:100, respectively, and the single-base, high-resolution separation of all four LDR-dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the EOF. Typical analysis times for LDR-dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel-based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.

  12. Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

    Science.gov (United States)

    Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

    2017-02-01

    Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

  13. A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR.

    Science.gov (United States)

    Diakité, Mohamed Lemine Youba; Champ, Jerôme; Descroix, Stephanie; Malaquin, Laurent; Amblard, François; Viovy, Jean-Louis

    2012-11-21

    In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 μl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.

  14. Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

    Science.gov (United States)

    Gao, Ran; Lu, Dan-Feng; Cheng, Jin; Jiang, Yi; Jiang, Lan; Xu, Jian-Dong; Qi, Zhi-Mei

    2016-12-15

    An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity.

  15. A Multi-Parameter Decoupling Method with a Lamb Wave Sensor for Improving the Selectivity of Label-Free Liquid Detection

    Directory of Open Access Journals (Sweden)

    Jean-François Manceau

    2012-07-01

    Full Text Available In this paper, a liquid multi-parameter decoupling method with only one Lamb wave sensor is presented. In a Lamb wave sensor, antisymmetric modes (A01 mode for low frequency, A03 mode for high frequency and symmetric modes (S0 mode are used to detect multiple parameters of a liquid, such as its density, sound velocity, and viscosity. We found they can play very different roles in the detections. For example, the A01 mode is very sensitive to the liquid’s density but the A03 mode is sensitive to the sound velocity. Here, the A0 mode is used to identify the density of the detected liquid and with this density value we obtained the viscosity by the amplitude shifts of the S0 mode. This could be a way to distinguish an unknown liquid with high sensitivity or to solve the problem of selectivity of label-free detection on biosensors.

  16. Nanocomposites of gold nanoparticles and graphene oxide towards an stable label-free electrochemical immunosensor for detection of cardiac marker troponin-I.

    Science.gov (United States)

    Liu, Guozhen; Qi, Meng; Zhang, Yin; Cao, Chaomin; Goldys, Ewa M

    2016-02-25

    A stable label-free amperometric immunosensor is presented based on gold nanoparticles and graphene oxide nanocomposites for detection of cardiac troponin-I in the early diagnosis of myocardial infarction. For designing of the sensing platform, firstly the nanocomposites based on GO and AuNPs were prepared and anchored on electrode surfaces. The formed nanocomposites provided a platform with big surface area for loading anti-cTnI capture antibody, and worked as a bridge for fast electron transfer subsequently increased the sensitivity. Moreover, the linkages between AuNP, GO, and electrodes were based on covalent bonding by aryldiazonium salt coupling chemistry, which favors the stability of the sensing interface. Finally, the anti-cTnI detection antibody was immobilized on GO tailored with ferrocene molecules, functioning as the signal reporter for the detection of cTnI. The modification process was monitored using electrochemistry, SEM, XPS. The herein immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI, and is capable of detecting cTnI at concentrations as low as 0.05 ng mL(-1), which is 100 times lower than that possible by conventional methods. It is potential to design the portable sensing platform based on AuNPs and GO nanocomposites for future point-of-care diagnostics.

  17. Binding-induced and label-free colorimetric method for protein detection based on autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy.

    Science.gov (United States)

    Wu, Hao; Zhang, Kai; Liu, Yaling; Wang, Hongyong; Wu, Jun; Zhu, Feifan; Zou, Pei

    2015-02-15

    In this work, a new binding-induced and label-free colorimetric method for protein detection has been developed on the basis of an autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy. The system consists of two proximity probes carrying two aptamer sequences as recognition elements for target, and two hairpin structures include three-fourths and one-fourth of the G-quadruplex sequences in inactive configuration as functional elements. In the presence of target protein, two proximity probes bind to the protein simultaneously, forming a stable DNA-protein complex. Then the complex triggers an autonomous cross-opening of the two functional hairpin structures, leading to the formation of numerous hemin/G-quadruplex DNAzymes. The resulting DNAzymes catalyze the oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the green-colored ABTS(•-) with the presence of H2O2, thus providing the amplified colorimetric detection of target. Using human α-thrombin as the protein target, this binding-induced DNAzyme amplification colorimetric method affords high sensitivity with a detection limit of 1.9 pM. Furthermore, this method might be further extended to sensitive detection of other proteins by simply replacing recognition elements of proximity probes.

  18. Integrated biochip for label-free and real-time detection of DNA amplification by contactless impedance measurements based on interdigitated electrodes.

    Science.gov (United States)

    Fang, Xinxin; Jin, Qinghui; Jing, Fengxiang; Zhang, Huanqian; Zhang, Feng; Mao, Hongju; Xu, Baojian; Zhao, Jianlong

    2013-06-15

    Here, we introduce an integrated biochip which offers accurate thermal control and sensitive electrochemical detection of DNA amplification in real-time. The biochip includes a 10-μl microchamber, a temperature sensor, a heater, and a contactless impedance biosensor. A pair of interdigitated electrodes is employed as the impedance biosensor and the products of the amplification are determined directly through tracing the impedance change, without using any labels, redox indicators, or probes. Real-time monitoring of strand-displacement amplification and rolling circle amplification was successfully performed on the biochip and a detection limit of 1 nM was achieved. Amplification starting at an initial concentration of 10 nM could be discriminated from that starting at 1 nM started concentration as well as from the negative control. Since an insulation layer covers the electrodes, the electrodes are spared from erosion, hydrolysis and bubble formation on the surface, thus, ensuring a long lifetime and a high reusability of the sensor. In comparison to bench-top apparatus, our chip shows good efficiency, sensitivity, accuracy, and versatility. Our system requires only simple equipments and simple skills, and can easily be miniaturized into a micro-scale system. The system will then be suitable for a handheld portable device, which can be applied in remote areas. It covers merits such as low cost, low-power consumption, rapid response, real-time monitoring, label-free detection, and high-throughput detection.

  19. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline

    Science.gov (United States)

    Park, Ki Soo; Oh, Seung Soo; Soh, H. Tom; Park, Hyun Gyu

    2014-08-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to

  20. Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Di; Zhuo, Ming [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Zhang, Xiaoai [State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing (China); Xu, Cheng; Jiang, Jie [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Gao, Fu [State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing (China); Wan, Qing, E-mail: wanqing7686@hotmail.com [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Li, Qiuhong, E-mail: liqiuhong2004@hotmail.com [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Wang, Taihong, E-mail: thwang@hnu.cn [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China)

    2013-04-22

    Highlights: ► A highly selective label-free biosensor is established based on indium-tin-oxide thin-film transistors (ITO TFTs). ► AI H5N1 virus was successfully detected through shift in threshold voltage and field-effect mobility of ITO TFT. ► The ITO TFT is applied in biosensor for the first time and shows good reusability and stability. ► Fabrication of the platform is simple with low cost, which is suitable for mass commercial production. -- Abstract: As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (V{sub T}) and field-effect mobility. The changes of I{sub D}–V{sub G} curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5 × 10{sup −9} g mL{sup −1} to 5 × 10{sup −6} g mL{sup −1} with a detection limit of 0.8 × 10{sup −10} g mL{sup −1}. Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs

  1. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    Science.gov (United States)

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

  2. Rapid and label-free detection of ochratoxin A and aflatoxin B1 using an optical portable instrument.

    Science.gov (United States)

    Arduini, Fabiana; Neagu, Daniela; Pagliarini, Valeria; Scognamiglio, Viviana; Leonardis, Maria Antonietta; Gatto, Emanuela; Amine, Aziz; Palleschi, Giuseppe; Moscone, Danila

    2016-04-01

    In this study, we report a novel assay for the combined on site detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA), through a colorimetric biosensing system for AFB1 and a fluorimetric detection for OTA, exploiting the capability of the portable fibre optic spectrometer to perform both analyses. AFB1 was detected using the acetylcholinesterase (AChE) enzyme that is inhibited by this toxin, and the degree of inhibition was quantified by the Ellman's spectrophotometric method, obtaining a detection limit of 10 µg L(-1). OTA quantification was performed by monitoring its intrinsic fluorescence in methanol, reaching a detection limit of 0.1 µg L(-1). In order to successfully apply the analytical tool in the food analysis, immunoaffinity columns were used. Clean-up and quantification of both AFB1 and OTA in millet samples was obtained by HPLC-dedicated AflaOchra-Test HPLC™ (Vicam™) and Afla-OtaCLEAN™ (LC-Tech) immunoaffinity columns, followed by absorption/fluorescence detection. Millet samples which were fortified with both OTA (50 µg kg(-1)) and AFB1 (20 µg kg(-1)), gave recovery values of 100 ± 6% for OTA, and 110 ± 10% for AFB1, using AflaOchra-Test HPLC™. Single OTA clean-up and quantification in wine samples was obtained, using an OchraTest immunoaffinity column (Vicam™), reaching a detection limit of 0.3 µg L(-1) and recovery values between 80% and 120%. These results demonstrated the possibility of employing a single clean-up and a cost-effective, and easy to use analytical system for both AFB1 and OTA detection at µg kg(-1) (ppb) level. Furthermore, in the case of positive samples, they could be analysed further, using standard chromatographic procedures, without any additional clean-up step, since the same extraction procedure of standard method is proposed in our method.

  3. Impedance spectral fingerprint of E. coli cells on interdigitated electrodes: A new approach for label free and selective detection

    Directory of Open Access Journals (Sweden)

    Maria Mallén-Alberdi

    2016-03-01

    Full Text Available Impedance-based biosensors for bacterial detection offer a rapid and cost-effective alternative to conventional techniques that are time-consuming and require specialized equipment and trained users. In this work, a new bacteria detection scheme is presented based on impedance measurements with antibody-modified polysilicon interdigitated electrodes (3 μm pitch, IDEs. The detection approach was carried out taking advantage of the E. coli structure which, in electrical terms, is constituted by two insulating cell membranes that separate a conductive cytoplasmatic medium and a more conductive periplasm. Impedance detection of bacteria is usually analyzed using electrical equivalent circuit models that show limitations for the interpretation of such complex cell structure. Here, a differential impedance spectrum representation is used to study the unique fingerprint that arises when bacteria attach to the surface of IDEs. That fingerprint shows the dual electrical behavior, insulating and conductive, at different frequency ranges. In parallel, finite-element simulations of this system using a three-shell bacteria model are performed to explain such phenomena. Overall, a new approach to detect bacteria is proposed that also enables to differentiate viable bacteria from other components non-specifically attached to the IDE surface by just detecting their spectral fingerprints.

  4. Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification

    Science.gov (United States)

    Zhu, Xiao; Xing, Da

    2012-12-01

    A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

  5. Field-effect transistor with a chemically synthesized MoS2 sensing channel for label-free and highly sensitive electrical detection of DNA hybridization

    Institute of Scientific and Technical Information of China (English)

    Doo-Won Lee[1,6; Jinhwan Lee[2,6; II Yung Sohn[1; Bo-Yeong Kim[3; Young Min Son[4; Hunyoung Bark[3; JaehyuckJung[3; Minseok Choi[5; Tae Hyeong Kim[5; Changgu Lee[2,3; Nae-Eung Lee[1,3,4

    2015-01-01

    A field-effect transistor (FET) with two-dimensional (2D) few-layer MoS2 as a sensing-channel material was investigated for label-free electrical detection of the hybridization of deoxyribonucleic acid (DNA) molecules. The high-quality MoS2-channel pattern was selectively formedthrough the chemical reaction of the Mo layer with H2S gas. The MoS2 FET was very stable in an electrolyte and inert to pH changes due to the lack of oxygen-containing functionalities on the MoS2 surface. Hybridization of single-stranded target DNA molecules with single-stranded probe DNA molecules physically adsorbed on the MoS2 channel resulted in a shift of the threshold voltage (Vt,) in the negative direction and an increase in the drain current. The negative shift in Vth is attributed to electrostatic gating effects induced by the detachment of negatively charged probe DNA molecules from the channel surface after hybridization. A detection limit of 10 fM, high sensitivity of 17 mWdec, and high dynamic range of 106 were achieved. The results showed that a bio-FET with an ultrathin 2D MoS2 channel can be used to detect very small concentrations of target DNA molecules specifically hybridized with the probe DNA molecules.

  6. Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

    Science.gov (United States)

    Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

    2017-02-01

    Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL-1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL-1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.

  7. Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

    Science.gov (United States)

    Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

    2017-01-01

    Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL−1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL−1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples. PMID:28198459

  8. Carbon dots prepared by hydrothermal treatment of dopamine as an effective fluorescent sensing platform for the label-free detection of iron(III) ions and dopamine.

    Science.gov (United States)

    Qu, Konggang; Wang, Jiasi; Ren, Jinsong; Qu, Xiaogang

    2013-05-27

    A facile, economic and green one-step hydrothermal synthesis route using dopamine as source towards photoluminescent carbon nanoparticles (CNPs) is proposed. The as-prepared CNPs have an average size about 3.8 nm. The emission spectra of the CNPs are broad, ranging from approximately 380 (purple) to approximately 525 nm (green), depending on the excitation wavelengths. Due to the favorable optical properties, the CNPs can readily enter into A549 cells and has been used for multicolor biolabeling and bioimaging. Most importantly, the as-prepared CNPs contain distinctive catechol groups on their surfaces. Due to the special response of catechol groups to Fe(3+) ions, we further demonstrate that such wholly new CNPs can serve as a very effective fluorescent sensing platform for label-free sensitive and selective detection of Fe(3+) ions and dopamine with a detection limit as low as 0.32 μM and 68 nM, respectively. The new "mix-and-detect" strategy is simple, green, and exhibits high sensitivity and selectivity. The present method was also applied to the determination of Fe(3+) ions in real water samples and dopamine in human urine and serum samples successfully. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Science.gov (United States)

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation.

  10. Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun, E-mail: yunatswu@swu.edu.cn

    2016-04-15

    The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. - Highlights: • Amplified and sensitive detection of microRNA from tumor cells is achieved. • Signal amplification is realized by two cascaded strand displacement reactions. • The developed sensor is selective and label-free without involving any enzymes.

  11. An interference-free and label-free sandwich-type magnetic silicon microsphere -rGO-based probe for fluorescence detection of microRNA.

    Science.gov (United States)

    Li, Shiyu; He, Kui; Liao, Rong; Chen, Chunyan; Chen, Xiaoming; Cai, Changqun

    2017-11-01

    An interference-free and label-free sensing platform was developed for the highly sensitive detection of microRNA-21 (miRNA-21) in vitro by magnetic silicon microsphere (MNP)-reduced graphene oxide (rGO)-based sandwich probe. In this method, DNA capture probes (P1) were connected with MNPs at the 5' end and hybridized with completely complementary target miRNA. Subsequently, rGO was retained and induced the fluorescence quenching in the supernatant. Through the magnetic separation, the supernatant environment was simplified and the interference to analytical signal was eliminated. When DNA capture probe-modified magnetic silicon microspheres (MNP-P1) were adsorbed through rGO in the absence of a target and formed a sandwich structure, the formed nanostructure was easily removed from the solution by a magnetic field and the fluorescence intensity was maximally recovered. This proposed strategy, which both overcame the expensive and cumbersome fluorescent labeling, and eliminated interference to analytical signal for guaranteeing high signal-to-background ratio, exhibited high sensitivity with a detection limit as low as 0.098nM and special selectivity toward miRNA-21. The method was potentially applicable for not only detection of miRNA-21 but also various biomarker analyses just by changing capture probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Label-free impedimetric aptasensor for detection of femtomole level acetamiprid using gold nanoparticles decorated multiwalled carbon nanotube-reduced graphene oxide nanoribbon composites.

    Science.gov (United States)

    Fei, Airong; Liu, Qian; Huan, Juan; Qian, Jing; Dong, Xiaoya; Qiu, Baijing; Mao, Hanping; Wang, Kun

    2015-08-15

    Gold nanoparticles (Au NPs) decorated multiwalled carbon nanotube-reduced graphene oxide nanoribbon (Au/MWCNT-rGONR) composites were synthesized by a one-pot reaction. By employing the resulting Au/MWCNT-rGONR composites as the support for aptamer immobilization, we developed an ultrasensitive label-free electrochemical impedimetric aptasensor for acetamiprid detection, which was based on that the variation of electron transfer resistance was relevant to the formation of acetamiprid-aptamer complex at the modified electrode surface. Compared with pure Au NPs and MWCNT-rGONR, the Au/MWCNT-rGONR composites modified electrode was the most sensitive aptasensing platform for the determination of acetamiprid. The proposed aptasensor displayed a linear response for acetamiprid in the range from 5×10(-14) M to 1×10(-5) M with an extremely low detection limit of 1.7×10(-14) M (S/N=3). In addition, this impedimetric aptasensor possessed great advantages including the simple operation process, low-cost, selectivity and sensitivity, which provided a promising model for the aptamer-based detection with a direct impedimetric method.

  13. Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tianshu [College of Physics, Jilin University, Changchun, Jilin 130012 (China); State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China); Liu, Jiyang; Gu, Xiaoxiao; Li, Dan [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China); Wang, Jin, E-mail: jin.wang.1@stonybrook.edu [College of Physics, Jilin University, Changchun, Jilin 130012 (China); State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China); Department of Chemistry, Physics and Applied Mathematics, State University of New York at Stony Brook, Stony Brook, NY 11794-3400 (United States); Wang, Erkang, E-mail: ekwang@ciac.jl.cn [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China)

    2015-07-02

    Highlights: • Fc-PAH was modified on the surface of graphene to prepare hybid nanocomposite (Fc-PAH-G). • A cytosensor was constructed with Fc-PAH-G, PSS and aptamer AS1411 by LBL technology. • The sensing interface introduced more redox probe and enhanced current signal on electrode. • The sensor showed a detection range of 10–10{sup 6} cells/mL with a detection limit of 10 cells/mL. - Abstract: Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10{sup 6} cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells.

  14. A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold

    Directory of Open Access Journals (Sweden)

    Christopher Gwenin

    2013-08-01

    Full Text Available Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring the activity of the toxin is the mouse bioassay, which is lengthy and has ethical issues due to the use of live animals. This report demonstrates a novel assay that utilises the endopeptidase activity of the toxin to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of 373 fg/mL and has been further developed into a high throughput method using a microplate reader detecting down to 600 fg/mL of active toxin. The results show clear differences between the toxin product and the placebo, which contains the pharmaceutical excipients human serum albumin and lactose, showing that the assay detects the active form of the toxin.

  15. Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells.

    Science.gov (United States)

    Wang, Tianshu; Liu, Jiyang; Gu, Xiaoxiao; Li, Dan; Wang, Jin; Wang, Erkang

    2015-07-02

    Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10(6) cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells.

  16. Epi-detecting label-free multimodal imaging platform using a compact diode-pumped femtosecond solid-state laser

    DEFF Research Database (Denmark)

    Andreana, Marco; Le, Tuan; Hansen, Anders Kragh

    2017-01-01

    We have developed an epi-detected multimodal nonlinear optical microscopy platform based on a compact and cost-effective laser source featuring simultaneous acquisition of signals arising from hyperspectral coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence, and second harmonic...... for these three modalities, achieving sufficient spectral resolution for CARS in the lipid region. The experimental results on a biological tissue reveal that the combination of the epi-detection scheme and the use of a compact diode-pumped femtosecond solid-state laser in the nonlinear optical microscope...

  17. Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

    KAUST Repository

    Wang, Zhijuan

    2011-05-01

    Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

  18. Simultaneous detection and serotyping of Salmonellae by immunomagnetic separation and label-free surface enhanced Raman spectroscopy

    Science.gov (United States)

    Salmonella spp. are one of the leading causes of foodborne outbreaks in the United States and globally. Current detection and characterization techniques for Salmonellae are time consuming and costly, and rapid methods could greatly benefit outbreak investigation, new case prevention and disease tre...

  19. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences

    Directory of Open Access Journals (Sweden)

    Natália Oliveira

    2015-07-01

    Full Text Available Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS. Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3. DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM. Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

  20. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences.

    Science.gov (United States)

    Oliveira, Natália; Souza, Elaine; Ferreira, Danielly; Zanforlin, Deborah; Bezerra, Wessulla; Borba, Maria Amélia; Arruda, Mariana; Lopes, Kennya; Nascimento, Gustavo; Martins, Danyelly; Cordeiro, Marli; Lima-Filho, José

    2015-07-01

    Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3). DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV) was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM). Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

  1. Label-Free and Multiplex Detection of Antibiotic Residues in Milk Using Imaging Surface Plasmon Resonance-Based Immunosensor

    NARCIS (Netherlands)

    Raz, Sabina Rebe; Bremer, Maria G. E. G.; Haasnoot, Willem; Norde, Willem

    2009-01-01

    Monitoring of antimicrobial drug residues in foods relies greatly on the availability of adequate analytical techniques. Currently, there is a need for a high-throughput screening method with a broad-spectrum detection range. This paper describes the development of a microarray biosensor, based on a

  2. Label-Free and Multiplex Detection of Antibiotic Residues in Milk Using Imaging surface Plasmon Resonance-Based immunosensor

    NARCIS (Netherlands)

    Rebe, S.; Bremer, M.G.E.G.; Haasnoot, W.; Norde, W.

    2009-01-01

    Monitoring of antimicrobial drug residues in foods relies greatly on the availability of adequate analytical techniques. Currently, there is a need for a high-throughput screening method with a broad-spectrum detection range. This paper describes the development of a microarray biosensor, based on a

  3. A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

    Directory of Open Access Journals (Sweden)

    Junsheng Wang

    2013-11-01

    Full Text Available Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis.

  4. Label-free detection and characterization of the binding of hemagglutinin protein and broadly neutralizing monoclonal antibodies using terahertz spectroscopy

    Science.gov (United States)

    Sun, Yiwen; Zhong, Junlan; Zhang, Cunlin; Zuo, Jian; Pickwell-MacPherson, Emma

    2015-03-01

    Hemagglutinin (HA) is the main surface glycoprotein of the influenza A virus. The H9N2 subtype influenza A virus is recognized as the most possible pandemic strain as it has crossed the species barrier, infecting swine and humans. We use terahertz spectroscopy to study the hydration shell formation around H9 subtype influenza A virus's HA protein (H9 HA) as well as the detection of antigen binding of H9 HA with the broadly neutralizing monoclonal antibody. We observe a remarkable concentration dependent nonlinear response of the H9 HA, which reveals the formation process of the hydration shell around H9 HA molecules. Furthermore, we show that terahertz dielectric properties of the H9 HA are strongly affected by the presence of the monoclonal antibody F10 and that the terahertz dielectric loss tangent can be used to detect the antibody binding at lower concentrations than the standard ELISA test.

  5. Label-Free Detection of Chondroitin Sulphate Proteoglycan 4 by a Polyaniline/Graphene Nanocomposite Functionalized Impedimetric Immunosensor

    OpenAIRE

    JingJing Fu; ZhuanZhuan Shi; Man Li; Yangyang Wang; Ling Yu

    2016-01-01

    The chondroitin sulphate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a tumor-associated antigen that is expressed in more than 85% of surgically removed melanoma lesions but has restricted distribution in normal tissues. The diagnostic and therapeutic value of CSPG4 drives a need for sensitive and low-cost detection approaches. To this end, we developed a polyaniline/graphene oxide nanocomposite (PANI@GO) that was electrochemically cod...

  6. Label-free voltammetric aptasensor for the sensitive detection of microcystin-LR using graphene-modified electrodes.

    Science.gov (United States)

    Eissa, Shimaa; Ng, Andy; Siaj, Mohamed; Zourob, Mohammed

    2014-08-05

    The development of successful biosensing platforms is highly dependent upon the biorecognition properties of the recognition receptor and the sensitivity of the transducer of the binding signal. The integration of the high affinity and specificity of DNA aptamers with the unique properties of the carbon nanomaterial graphene offers an excellent avenue for sensitive and selective biosensing architectures. In this work, a highly sensitive and selective aptasensor which utilizes an unlabeled DNA aptamer assembled on a graphene electrode for microcystin-LR detection was developed. A facile strategy was used for the aptasensor fabrication on the basis of the noncovalent assembly of DNA aptamer on graphene-modified screen printed carbon electrodes. Assembly of the DNA aptamer on the graphene-modified electrodes caused a marked drop in the square wave voltammetric reduction signal of the [Fe(CN)6](4-/3-) redox couple. The presence of microcystin-LR, on the other hand, caused a dose-responsive increase in peak current, allowing the quantification of microcystin-LR through the measurement of peak current change. Under optimal conditions, the detection limit of the developed aptasensor was 1.9 pM in buffer, a concentration much lower than those offered by previously reported biosensors for microcystin-LR. The developed aptasensor also exhibited excellent selectivity for microcystin-LR with no detectable cross-reactivity to okadaic acid, microcystin-LA, and microcystin-YR. Moreover, the proposed aptasensor has been applied for the analysis of spiked tap water and fish samples showing good recovery percentages. This novel, simple, high-performance, and low-cost detection platform would facilitate the routine monitoring of microcystin-LR in real samples.

  7. High sensitive and selective electrochemical biosensor: Label-free detection of human norovirus using affinity peptide as molecular binder.

    Science.gov (United States)

    Hwang, Hye Jin; Ryu, Myung Yi; Park, Chan Young; Ahn, Junki; Park, Hyun Gyu; Choi, Changsun; Ha, Sang-Do; Park, Tae Jung; Park, Jong Pil

    2017-01-15

    Norovirus is known as the major cause of highly infection for gastrointestinal tracts. In this study, robust and highly sensitive biosensors for detecting human norovirus by employing a recognition affinity peptide-based electrochemical platform were described. A series of amino acid-substituted and cysteine-incorporated recognition peptides isolated from evolutionary phage display technique was chemically synthesized and immobilized to a gold sensor layer, the detection performance of the gold-immobilized synthetic peptide-based sensor system was assessed using QCM, CV and EIS. Using EIS, the limit of detection with Noro-1 as a molecular binder was found to be 99.8nM for recombinant noroviral capsid proteins (rP2) and 7.8copies/mL for human norovirus, thereby demonstrating a high degree of sensitivity for their corresponding targets. These results suggest that a biosensor which consists of affinity peptides as a molecular binder and miniaturized microdevices as diagnostic tool could be served as a new type of biosensing platform for point-of-care testing.

  8. Label-free attomolar detection of lactate based on radio frequency sputtered of nickel oxide thin film field effect transistor.

    Science.gov (United States)

    Mansouri Majd, Samira; Salimi, Abdollah; Astinchap, Bandar

    2017-06-15

    The radio frequency sputtered nickel oxide thin film nanostrtablucture deposited on glass substrate was used as a potential matrix for the realization of highly sensitive and selective field effect transistor-type lactate biosensor. Firstly, NiO-FET was tested for NADH detection showing a linear concentration range 1aM to 1nM and a low detection limit of 0.2aM. Then, NiO surface modified with chitosan and functionalized with glutaraldehyde and lactate dehydrogenase enzyme was immobilized on the aldehyde terminal. The biosensor is found to exhibit highly efficient sensing response characteristics with good linearity of 1aM to 1pM and low limit of detection of 0.5aM. The biosensor shows high stability without interferences from commonly interfering compounds in biological fluids, including uric acid, ascorbic acid, glucose and acetaminophen. Furthermore, the application of the proposed biosensor for analysis of lactate in artificial serum samples was evaluated with good satisfactory results. This protocol can be used to develop of disposable, low cost, and portable various types of dehydrogenase based biosensor devices using metal oxide nanomaterials.

  9. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity.

    Science.gov (United States)

    Sharma, R; Deacon, S E; Nowak, D; George, S E; Szymonik, M P; Tang, A A S; Tomlinson, D C; Davies, A G; McPherson, M J; Wälti, C

    2016-06-15

    Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35 ± 10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5-10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential.

  10. Core-Shell Structure of Gold Nanoparticles with Inositol Hexaphosphate Nanohybrids for Label-Free and Rapid Detection by SERS Nanotechnology

    Directory of Open Access Journals (Sweden)

    Andreas H. H. Mevold

    2015-01-01

    Full Text Available Gold nanoparticles bound with inositol hexaphosphate (IP6 (AuNPs/IP6 were prepared by in situ reduction of various concentrations of IP6 (0~320 µM through modified Frens method for surface-enhanced Raman scattering (SERS detection. The resultant AuNPs/IP6 were subject to characterization including UV/Vis spectroscopy, transmission electron microscopy (TEM, dynamic light scattering (DLS, zeta potential, and X-ray photoelectron spectroscopy (XPS. The results showed that AuNPs with 65 µM of IP6 would result in a core AuNPs-shell (IP6 layer structure, which exhibited the strongest SERS signal, due to the “hot spot effect” generated from the 1-2 nm interparticle gaps of AuNPs/IP6 nanohybrids (ionic interaction of IP6 and Au+. Furthermore, the reaction kinetics of Au and IP6 were also investigated in this work. Higher concentration of IP6 (190 and 260 µM will make AuNPs become irregularly shaped, because IP6 is a basic salt and served as a pH mediator. The morphology and distribution of AuNPs were greatly improved by addition of 65 µM of IP6. This novel AuNPs/IP6 nanohybrid showed great stability and Raman enhancement. It is promising in the application of rapid and label-free biological detection of bacteria or tumor cells.

  11. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    Science.gov (United States)

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Label-free Electrochemiluminescent Immunosensor for Detection of Prostate Specific Antigen based on Aminated Graphene Quantum Dots and Carboxyl Graphene Quantum Dots.

    Science.gov (United States)

    Wu, Dan; Liu, Yixin; Wang, Yaoguang; Hu, Lihua; Ma, Hongmin; Wang, Guoqin; Wei, Qin

    2016-02-04

    Prostate-specific antigen (PSA) was used as the model, an ultrasensitive label-free electrochemiluminescent immunosensor was developed based on graphene quantum dots. Au/Ag-rGO was sythsized and used as electrode material to load a great deal of graphene quantum dots due to the large surface area and excellent electron conductivity. After aminated graphene quantum dots and acarboxyl graphene quantum dots were modified onto the electrode, the ECL intensity was much high using K2S2O8 as coreactant. Then, antibody of PSA was immobilized on the surface of modified electrode surface through the adsorption of Au/Ag toward proteins, leading to the decrease of the ECL intensity. As proven by ECL spectra test and electrochemical impedance spectroscopy (EIS) analysis, the fabrication process of the immunosensor is successful. Under the optimal conditions, the ECL intensity decreased linearly with the logarithm of PSA concentration in the range of 1 pg/mL ~ 10 ng/mL. The detection limit achieved is 0.29 pg/mL. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results. Due to excellent stability, high sensitivity, acceptable repeatability and selectivity, the immunosensor has promising applications in disease and drug analysis.

  13. First improvements in the detection and quantification of label-free nucleic acids by laser-induced breakdown spectroscopy: Application to the deoxyribonucleic acid micro-array technology

    Energy Technology Data Exchange (ETDEWEB)

    Le Meur, Julien [Laboratoire de Cancerologie Experimentale, Commissariat a l' Energie Atomique de Fontenay-aux-Roses, Direction des Sciences du Vivant, Departement de Radiobiologie et Radiopathologie, Fontenay-aux-Roses (France); Menut, Denis [Laboratoire de Reactivite des Surfaces et des Interfaces, Commissariat a l' Energie Atomique de Saclay, Direction de l' Energie Nucleaire, Departement de Physico-Chimie, Gif sur Yvette (France); Wodling, Pascal [Laboratoire d' Interaction Laser-Matiere, Commissariat a l' Energie Atomique de Saclay, Direction de l' Energie Nucleaire, Departement de Physico-Chimie, Gif sur Yvette (France); Salmon, Laurent [Laboratoire de Reactivite des Surfaces et des Interfaces, Commissariat a l' Energie Atomique de Saclay, Direction de l' Energie Nucleaire, Departement de Physico-Chimie, Gif sur Yvette (France); Thro, Pierre-Yves [Laboratoire d' Interaction Laser-Matiere, Commissariat a l' Energie Atomique de Saclay, Direction de l' Energie Nucleaire, Departement de Physico-Chimie, Gif sur Yvette (France); Chevillard, Sylvie [Laboratoire de Cancerologie Experimentale, Commissariat a l' Energie Atomique de Fontenay-aux-Roses, Direction des Sciences du Vivant, Departement de Radiobiologie et Radiopathologie, Fontenay-aux-Roses (France); Ugolin, Nicolas [Laboratoire de Cancerologie Experimentale, Commissariat a l' Energie Atomique de Fontenay-aux-Roses, Direction des Sciences du Vivant, Departement de Radiobiologie et Radiopathologie, Fontenay-aux-Roses (France)], E-mail: nugolin@cea.fr

    2008-04-15

    The accurate quantification of nucleic acids is essential in many fields of modern biology and industry, and in some cases requires the use of fluorescence labeling. Yet, in addition to standardization problems and quantification reproducibility, labeling can modify the physicochemical properties of molecules or affect their stability. To address these limitations, we have developed a novel method to detect and quantify label-free nucleic acids. This method is based on stoichiometric proportioning of phosphorus in the nucleic acid skeleton, using laser-induced breakdown spectroscopy, and a specific statistical analysis, which indicates the error probability for each measurement. The results obtained appear to be quantitative, with a limit of detection of 10{sup 5} nucleotides/{mu}m{sup 2} (i.e. 2 x 10{sup 13} phosphorus atoms/cm{sup 2}). Initial micro-array analysis has given very encouraging results, which point to new ways of quantifying hybridized nucleic acids. This is essential when comparing molecules of different sequences, which is presently very difficult with fluorescence labeling.

  14. Enzyme-free and label-free ultra-sensitive colorimetric detection of Pb(2+) using molecular beacon and DNAzyme based amplification strategy.

    Science.gov (United States)

    Yun, Wen; Cai, Dingzhou; Jiang, JiaoLai; Zhao, Pengxiang; Huang, Yu; Sang, Ge

    2016-06-15

    An enzyme-free and label-free colorimetric Pb(2+) sensor based on DNAzyme and molecular beacon (MB) has been developed and demonstrated by recycle using enzyme strand for signal amplification. The substrate strand DNA (S-DNA) of DNAzyme could be converted into MB structure with base pairs of stem part at the both ends. The MB could hybridize with enzyme strand DNA (E-DNA) to form DNAzyme, and be activated and cleaved in the presence of Pb(2+). The cleaved MB is much less stable, releasing from the DNAzyme as two product pieces. The product pieces of MB are flexible and could bind to unmodified AuNPs to effectively stabilize them against salt-induced aggregation. Then, the E-DNA is liberated to catalyze the next reaction and amplify the response signal. By taking advantage of repeated using of E-DNA, our proposed method exhibited high sensitive for Pb(2+) detection in a linear range from 0.05 to 5 nM with detection limit of 20 pM by UV-vis spectrometer. Moreover, this method was also used for determination of Pb(2+) in river water samples with satisfying results. Importantly, this strategy could reach high sensitivity without any modification and complex enzymatic or hairpins based amplification procedures.

  15. A label-free colorimetric aptasensor for simple, sensitive and selective detection of Pt (II) based on platinum (II)-oligonucleotide coordination induced gold nanoparticles aggregation.

    Science.gov (United States)

    Fan, Daoqing; Zhai, Qingfeng; Zhou, Weijun; Zhu, Xiaoqing; Wang, Erkang; Dong, Shaojun

    2016-11-15

    Herein, a gold nanoparticles (AuNPs) based label-free colorimetric aptasensor for simple, sensitive and selective detection of Pt (II) was constructed for the first time. Four bases (G-G mismatch) mismatched streptavidin aptamer (MSAA) was used to protect AuNPs from salt-induced aggregation and recognize Pt (II) specifically. Only in the presence of Pt (II), coordination occurs between G-G bases and Pt (II), leading to the activation of streptavidin aptamer. Streptavidin coated magnetic beads (MBs) were used as separation agent to separate Pt (II)-coordinated MSAA. The residual less amount of MSAA could not efficiently protect AuNPs anymore and aggregation of AuNPs will produce a colorimetric product. With the addition of Pt (II), a pale purple-to-blue color variation could be observed by the naked eye. A detection limit of 150nM and a linear range from 0.6μM to 12.5μM for Pt (II) could be achieved without any amplification.

  16. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    Science.gov (United States)

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.

  17. Label-free and multiplex detection of antibiotic residues in milk using imaging surface plasmon resonance-based immunosensor.

    Science.gov (United States)

    Rebe Raz, Sabina; Bremer, Maria G E G; Haasnoot, Willem; Norde, Willem

    2009-09-15

    Monitoring of antimicrobial drug residues in foods relies greatly on the availability of adequate analytical techniques. Currently, there is a need for a high-throughput screening method with a broad-spectrum detection range. This paper describes the development of a microarray biosensor, based on an imaging surface plasmon resonance (iSPR) platform, for quantitative and simultaneous immunodetection of different antibiotic residues in milk. Model compounds from four major antibiotic families: aminoglycosides (Neomycin, Gentamicin, Kanamycin, and Streptomycin), sulfonamides (Sulfamethazine), fenicols (Chloramphenicol), and fluoroquinolones (Enrofloxacin) were detected using a single sensor chip. By multiplexing seven immunoassays in a competitive format, we were able to measure all the target compounds at parts per billion (ppb) levels in buffer and in 10x-diluted milk. The assays for Neomycin, Kanamycin, Streptomycin, Enrofloxacin, and Sulfamethazine were sensitive enough for milk control at maximum residue levels as established in the European Union. The overall performance of the biosensor was determined to be comparable to that of conventional four-channel surface plasmon resonance (SPR)-based biosensors, in terms of assay sensitivity and robustness. Combining the advantages of a SPR sensor and a microarray, utilization of the biosensor described here offers a promising alternative to the existing methods and is highly relevant for multianalyte food profiling.

  18. Highly sensitive and stable Ag@SiO2 nanocubes for label-free SERS-photoluminescence detection of biomolecules

    Science.gov (United States)

    Nguyen, Minh-Kha; Su, Wei-Nien; Chen, Ching-Hsiang; Rick, John; Hwang, Bing-Joe

    2017-03-01

    Surface-enhanced Raman scattering (SERS) and fluorescence microscopy are a widely used biological and chemical characterization techniques. However, the peak overlapping in multiplexed experiments and rapid photobleaching of fluorescent organic dyes is still the limitations. When compared to Ag nanocubes (NCs), higher SERS sensitivities can be obtained with thin shelled silica Ag@SiO2 NCs, in contrast metal-enhanced photoluminescence (MEPL) is only found with NCs that have thicker silica shells. A 'dual functionality' represented by the simultaneous strengthening of SERS and MEPL signals can be achieved by mixing Ag@SiO2 NCs, with a silica shell thickness of 1.5 nm and 4.4 nm. This approach allows both the Ag@SiO2 NCs SERS and MEPL sensitivities to be maintained at 90% after 12 weeks of storage. Based on the distinguished detection of creatinine and flavin adenine dinucleotide in the mixture, the integration of SERS and MEPL together on a stable single plasmonic nanoparticle platform offers an opportunity to enhance both biomarker detection sensitivity and specificity.

  19. Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

    Science.gov (United States)

    Souza, Elaine; Nascimento, Gustavo; Santana, Nataly; Ferreira, Danielly; Lima, Manoel; Natividade, Edna; Martins, Danyelly; Lima-Filho, José

    2011-01-01

    A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. PMID:22163916

  20. Label-free detection of ApoE4-mediated β-amyloid aggregation on single nanoparticle uncovering Alzheimer's disease.

    Science.gov (United States)

    Kang, Min Kyung; Lee, Jeewon; Nguyen, Anh H; Sim, Sang Jun

    2015-10-15

    Beta amyloid (Aβ) deposition is a pathological milestone of Alzheimer's disease (AD). This is facilitated by an isoform of Apolipoprotein E4 (ApoE4), which is a dominant risk factor for AD. However, current in vitro Aβ aggregation assays were performed in extreme conditions not linked to physiological conditions, to understand the mechanism of Aβ induced neurotoxicity. Here, we present a simple method for the ApoE4-mediated Aβ aggregation at physiological conditions using single gold nanoparticle based on localized surface plasmon resonance (LSPR). It can be directly observed by dark-field microscope or even by the naked eye. Following LSPR principles, we used ApoE4 inducing Aβ42 self-assemblies on gold nanoparticles (AuNPs) surface via their surface charge interaction. Using physiologically mimic cerebrospinal fluid, we determined a detection limit of 1.5 pM for Aβ42 corresponding to the ~2.9 nm LSPR-peak shift under ApoE4. Interestingly, the result also shows that ApoE4 induces the aggregation of Aβ42 more specifically and rapidly than that of Aβ40. This is the first biomimetic platform for real-time detection of Aβ aggregation, mimicking biological conditions, which can be used to investigate AD directly.

  1. Linear-array-based photoacoustic tomography for label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters

    Science.gov (United States)

    Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Wang, Lihong V.

    2017-03-01

    Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

  2. Label-free detection of glycated haemoglobin in human blood using silicon-based photonic crystal nanocavity biosensor

    Science.gov (United States)

    Olyaee, Saeed; Seifouri, Mahmood; Mohsenirad, Hamideh

    2016-07-01

    In this paper, we describe a two-dimensional photonic crystal-based biosensor that consists of a waveguide and a nanocavity with high sensitivity. A new method is employed for increasing sensitivity of the biosensor. The simulation results show that biosensor is highly sensitive to the refractive index (RI) variations due to injected biomaterials, like glycated haemoglobin, into the sensing surface. The proposed biosensor is designed for the wavelength range of 1514.4-1896.3 nm. The sensitivity and the quality factor are calculated to be 3000 and 272.43 nm/RIU, respectively. The designed structure can detect a 0.002 change in the RI via resonant wavelength shift of 0.9 nm. The band diagram and transmission spectra are computed using plane wave expansion and finite difference time domain methods.

  3. Label-free detection of proteins in ternary mixtures using surface-enhanced Raman scattering and protein melting profiles

    Science.gov (United States)

    Keskin, Sercan; Efeoğlu, Esen; Keçeci, Kaan; Çulha, Mustafa

    2013-03-01

    The multiplex detection of biologically important molecules such as proteins in complex mixtures has critical importance not only in disease diagnosis but also in other fields such as proteomics and biotechnology. Surface-enhanced Raman scattering (SERS) is a powerful technique for multiplex identification of molecular components in a mixture. We combined the multiplexing power of SERS and heat denaturation of proteins to identify proteins in ternary protein mixtures. The heat denaturation profiles of four model blood proteins, transferrin, human serum albumin, fibrinogen, and hemoglobin, were studied with SERS. Then, two ternary mixtures of these four proteins were used to test the feasibility of the approach. It was demonstrated that unique denaturation profiles of each protein could be used for their identification in the mixture.

  4. Label-free detection of protein-ligand interactions in real time using micromachined bulk acoustic resonators

    Science.gov (United States)

    Zhang, Hao; Pang, Wei; Marma, Mong S.; Lee, Chuang-Yuan; Kamal-Bahl, Sanat; Kim, Eun Sok; McKenna, Charles E.

    2010-03-01

    In this paper, we present a micromachined film bulk acoustic resonator (FBAR) to detect protein-ligand interactions in real-time. The surface of the FBAR device has a thin layer of gold deposited on it to immobilize thiol-modified biotin. The resonant frequency of the biotin modified FBAR was measured to decrease by 170 ppm when exposed to streptavidin solution with a concentration of 5×10-7 M, corresponding to an added mass of 120 pg on the FBAR surface due to the biotin-streptavidin interaction. Consequently, the biotin modified FBAR can be used to observe in real time the biotin-streptavidin interaction without the use of labeling or molecular tags. The FBAR can be used in a variety of protein-ligand systems, and be designed for testing in array formats to give high throughput screening for drug discovery.

  5. Label-Free Optical Detection of Acute Myocardial Infarction Based on Blood Plasma Surface-Enhanced Raman Spectroscopy

    Science.gov (United States)

    Chen, Y. X.; Chen, M. W.; Lin, J. Y.; Lai, W. Q.; Huang, W.; Chen, H. Y.; Weng, G. X.

    2016-11-01

    This study is intended to explore the potential of silver (Ag) nanoparticle-based plasma surface-enhanced Raman spectroscopy (SERS) for providing a rapid and simple "Yes/No" assessment to detect acute myocardial infarction (AMI). A simple, rapid, and accurate method of diagnosing AMI is critical to reduce mortality and improve prognosis. Techniques such as electrocardiography examination and use of cardiac troponins have not yet met the current clinical need. Therefore, alternative approaches need to be developed. Plasma samples from 32 patients with AMI and 32 healthy control (Clt) subjects were assessed. Multivariate statistical techniques, including principal component (PC) analysis and linear discriminant analysis (PCA-LDA), were employed to develop a diagnostic algorithm for differentiating between patients with AMI and Clt subjects. Furthermore, the receiver operating characteristic was tested to evaluate the performance of the PCA-LDA algorithm for AMI detection. Each plasma sample was mixed with an equal volume of Ag colloidal solution, and the SERS measurement of each plasma sample was performed. The plasma SERS spectrum showed much stronger and sharper peaks compared with the normal Raman spectrum. Tentative assignments of Raman spectroscopy bands showed specific biomolecular (e.g., proteins, adenosine, adenine, and uric acid) changes. PC analysis and LDA were employed to discriminate patients with AMI from Clt subjects, yielding a sensitivity of 87.5% and a specificity of 93.8%. The findings of this study suggest that plasma SERS has a great potential for improving AMI in the future, and this will certainly reduce the difficulty, time to draw blood, and patients' pain to a great extent.

  6. Nanowell surface enhanced Raman scattering arrays fabricated by soft-lithography for label-free biomolecular detections in integrated microfluidics

    Science.gov (United States)

    Liu, Gang L.; Lee, Luke P.

    2005-08-01

    We describe a low-cost, ultrasensitive surface-enhanced Raman scattering (SERS) substrate in microfluidic biochips fabricated by soft lithography. A batch nanofabrication method is developed to create nanopillars structures on a silicon wafer as a master copy of molding, then the complementary nanowells structures on polydimethylsiloxane (PDMS) are created by soft lithography. The selective deposition of Ag thin film on the nanowells is applied to create SERS active sites before the integration with a glass-based microfluidic chip which functions as a sample delivery device and a transparent optical window for SERS spectroscopic imaging. Detections of Rhodamine 6G and adenosine SERS spectra are accomplished by using a 785nm laser with 300μW excitation power. The Raman scattering signal enhancement on the nanowell-based Ag SERS substrate is more than 107 times higher than the control sample (i.e. the smooth Ag layer on PDMS). Fabrication of ultrasensitive nanowell SERS substrate by economical and repeatable soft lithography method can contribute to the future microdevices for high throughput screening of functional genomics, proteomics, and cellular activities.

  7. DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

    Science.gov (United States)

    Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

    2016-11-15

    Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification.

    Science.gov (United States)

    Guo, Yuna; Wang, Yu; Liu, Su; Yu, Jinghua; Wang, Hongzhi; Wang, Yalin; Huang, Jiadong

    2016-01-15

    In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K(+) and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8cfumL(-1) and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Graphene-interfaced electrical biosensor for label-free and sensitive detection of foodborne pathogenic E. coli O157:H7.

    Science.gov (United States)

    Pandey, Ashish; Gurbuz, Yasar; Ozguz, Volkan; Niazi, Javed H; Qureshi, Anjum

    2017-05-15

    E. coli O157:H7 is an enterohemorrhagic bacteria responsible for serious foodborne outbreaks that causes diarrhoea, fever and vomiting in humans. Recent foodborne E. coli outbreaks has left a serious concern to public health. Therefore, there is an increasing demand for a simple, rapid and sensitive method for pathogen detection in contaminated foods. In this study, we developed a label-free electrical biosensor interfaced with graphene for sensitive detection of pathogenic bacteria. This biosensor was fabricated by interfacing graphene with interdigitated microelectrodes of capacitors that were biofunctionalized with E. coli O157:H7 specific antibodies for sensitive pathogenic bacteria detection. Here, graphene nanostructures on the sensor surface provided superior chemical properties such as high carrier mobility and biocompatibility with antibodies and bacteria. The sensors transduced the signal based on changes in dielectric properties (capacitance) through (i) polarization of captured cell-surface charges, (ii) cells' internal bioactivity, (iii) cell-wall's electronegativity or dipole moment and their relaxation and (iv) charge carrier mobility of graphene that modulated the electrical properties once the pathogenic E. coli O157:H7 captured on the sensor surface. Sensitive capacitance changes thus observed with graphene based capacitors were specific to E. coli O157:H7 strain with a sensitivity as low as 10-100 cells/ml. The proposed graphene based electrical biosensor provided advantages of speed, sensitivity, specificity and in-situ bacterial detection with no chemical mediators, represents a versatile approach for detection of a wide variety of other pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. A label-free fluorescence strategy for selective detection of nicotinamide adenine dinucleotide based on a dumbbell-like probe with low background noise.

    Science.gov (United States)

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Wang, Yiru; Chen, Xi

    2016-03-15

    In this work we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide (NAD(+)) based on a dumbbell-like DNA probe designed for both ligation reaction and digestion reaction with low background noise. SYBR Green I (SG I), a double-helix dye, was chosen as the readout fluorescence signal. In the absence of NAD(+), the ligation reaction did not occur, but the probe was digested to mononucleotides after the addition of exonuclease I (Exo I) and exonuclease I (Exo III), resulting in a weak fluorescence intensity due to the weak interaction between SG I and mononucleotides. In the presence of NAD(+), the DNA probe was ligated by Escherichia coli DNA ligase, blocking the digestion by Exo I and Exo III. As a result, SG I was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved. This method was simple in design, fast to operate, with good sensitivity and selectivity which could discriminate NAD(+) from its analogs.

  11. A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing.

    Science.gov (United States)

    Ewald, Melanie; Fechner, Peter; Gauglitz, Günter

    2015-05-01

    For the first time, a multi-analyte biosensor platform has been developed using the label-free 1-lambda-reflectometry technique. This platform is the first, which does not use imaging techniques, but is able to perform multi-analyte measurements. It is designed to be portable and cost-effective and therefore allows for point-of-need testing or on-site field-testing with possible applications in diagnostics. This work highlights the application possibilities of this platform in the field of animal testing, but is also relevant and transferable to human diagnostics. The performance of the platform has been evaluated using relevant reference systems like biomarker (C-reactive protein) and serology (anti-Salmonella antibodies) as well as a panel of real samples (animal sera). The comparison of the working range and limit of detection shows no loss of performance transferring the separate assays to the multi-analyte setup. Moreover, the new multi-analyte platform allows for discrimination between sera of animals infected with different Salmonella subtypes.

  12. Enhancing sensitivity and selectivity in a label-free colorimetric sensor for detection of iron(II) ions with luminescent molybdenum disulfide nanosheet-based peroxidase mimetics.

    Science.gov (United States)

    Wang, Yong; Hu, Jie; Zhuang, Qianfen; Ni, Yongnian

    2016-06-15

    In the present study, we demonstrated that the luminescent molybdenum disulfide (MoS2) nanosheets, which were prepared hydrothermally by using sodium molybdate and thiourea as precursors, possessed peroxidase-like activity, and could catalyze the oxidation of peroxidase substrate o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2) to produce a yellow color reaction. Further addition of Fe(2+) into the nanosheets led to peroxidase mimetics with greatly enhanced catalytic activity. The observation was exploited to develop a label-free colorimetric nanozyme sensor for detection of Fe(2+). The fabricated MoS2/OPD/H2O2 sensor showed a wide linear range of 0.01-0.8 µM with a detection limit of 7 nM. Moreover, it was found that the MoS2/OPD/H2O2 sensor displayed enhanced sensitivity and selectivity toward Fe(2+) compared with the OPD/H2O2 sensor, suggesting that the MoS2 nanosheets could improve the performance of the Fe(2+) sensor. An advanced chemometrics algorithm, multivariate curve resolution by alternating least squares (MCR-ALS), was further applied to interpret the origin of enhancing sensitivity and selectivity in the Fe(2+) sensor with the MoS2 nanosheets. The time-dependent UV-vis spectral data of the studied systems were collected, and submitted to the MCR-ALS. The results showed that the increased sensitivity and selectivity of the MoS2/OPD/H2O2 sensor for Fe(2+) detection likely arose from its large reaction rate constant. Finally, the proposed MoS2/OPD/H2O2 sensor was successfully applied for detection of Fe(2+) in water samples.

  13. A novel graphene-based label-free fluorescence `turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells

    Science.gov (United States)

    Ke, Yaotang; Garg, Bhaskar; Ling, Yong-Chien

    2016-02-01

    A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti4+. The as-prepared rGO@PDA-Ti4+-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti4+. The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, β-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti4+), leading to an excellent fluorescence `turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, β-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions

  14. Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips.

    Science.gov (United States)

    Albrecht, Jennifer Coyne; Kotani, Akira; Lin, Jennifer S; Soper, Steven A; Barron, Annelise E

    2013-02-01

    We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated "sample-in/answer-out" devices with amplification, LDR, and detection all on one platform.

  15. Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1.

    Science.gov (United States)

    Guo, Di; Zhuo, Ming; Zhang, Xiaoai; Xu, Cheng; Jiang, Jie; Gao, Fu; Wan, Qing; Li, Qiuhong; Wang, Taihong

    2013-04-22

    As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (VT) and field-effect mobility. The changes of ID-VG curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5×10(-9) g mL(-1) to 5×10(-6) g mL(-1) with a detection limit of 0.8×10(-10) g mL(-1). Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs can be promising candidates for the development of label-free biosensors.

  16. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    Science.gov (United States)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-04-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3‧ and 5‧ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

  17. A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

    Science.gov (United States)

    Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

    2011-03-15

    A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Sensitive detection of microcystin-LR by using a label-free electrochemical immunosensor based on Au nanoparticles/silicon template/methylene blue nanocomposite.

    Science.gov (United States)

    Fu, Xuewen; Feng, Yajuan; Niu, Sipeng; Zhao, Chunling; Yang, Minghui; Yang, Yunhui

    2013-12-01

    A label-free electrochemical immunosensor, based on a gold nanoparticles (Au NPs)/silicon template/methylene blue (MB)/chitosan (CHIT) nanocomposite-modified electrode, was fabricated for the ultrasensitive detection of microcystin-LR (MC-LR). The nanocomposite film showed high binding affinity to the antibodies of MC-LR because gold nanoparticles have large surface area and good biocompatibility which can immobilize large amount of antibody through ionic interactions and other interactions between AuNPs and mercapto or primary amine groups of antibodies with high stability and bioactivity. MB was used as redox indicator due to its good electrochemical behavior in conductive substrate. This hybrid membrane was evaluated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) to determine its electrochemical properties in immunosensor application. A decrease in DPV responses was observed with increasing concentrations of MC-LR in standard and real samples due to the formation of immuno-complexes between MC-LR and anti-MC-LR antibodies which hindered the electron charge transfer at the electrode-electrolyte interface. At optimal conditions, this immunosensor could detect MC-LR in a linear range from 0.5 ng/mL to 25 microg/mL with a low detection limit of 0.1 ng/mL at 3 sigma. Moreover, the prepared immunosensor was applied for the analysis of MC-LR in water samples with satisfactory results. The proposed method showed high selectivity, acceptable reproducibility, stability and reliability.

  19. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    Science.gov (United States)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-01-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3′ and 5′ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10−9 mol.L−1 over a linear ranged from 20 × 10−9 to 10 × 10−7 mol.L−1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications. PMID:28387331

  20. Label-free in-situ real-time DNA hybridization kinetics detection employing microfiber-assisted Mach-Zehnder interferometer.

    Science.gov (United States)

    Song, Binbin; Zhang, Hao; Liu, Bo; Lin, Wei; Wu, Jixuan

    2016-07-15

    A label-free DNA biosensor based on microfiber-assisted Mach-Zehnder interferometer (MAMZI) for in-situ real-time DNA hybridization kinetics detection has been proposed and experimentally demonstrated. A microfiber of hundreds of microns in length is fabricated by tapering a segment of standard single-mode fiber (SMF) to construct the U-shaped microcavity between the lead-in and lead-out SMFs. Thanks to the mode field mismatching between the SMF and microfiber, the incident guided mode light would separate into two beams that respectively propagate in the air microcavity and the microfiber. Consequently, interference between different light modes would occur at the joint between the microfiber and the lead-out SMF. Experimental results indicate that owing to the participation of opening cavity modes in the modal interference process, the interferometric spectrum of our proposed microcavity sensor is highly sensitive to the variation of environmental refractive index (RI), especially for the RI range around 1.34 which is useful for most biological applications. The microfiber functionalization is achieved by stepwise modifying the microfiber with monolayer Poly-l-lysine (PLL) and single-stranded DNA (ssDNA) probes to produce the sensitive surface that could uniquely attach specific target ssDNAs. The fiber surface functionalization as well as DNA hybridization processes have been experimentally investigated for different target ssDNA solutions in real time. The interferometric transmission spectrum shows large wavelength shift for different biological phases, and a detection limit conservatively down to 0.0001pmol/μL has been acquired by employing the U-shaped microcavity of 176.88μm in length. Our proposed DNA biosensor possesses several advantages such as compact size, ease of fabrication, and strong response for DNA hybridization, which make it a promising candidate for potential applications in such rapidly expanding areas as medical diagnosis, cancer

  1. Nanocomposites of gold nanoparticles and graphene oxide towards an stable label-free electrochemical immunosensor for detection of cardiac marker troponin-I

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guozhen, E-mail: gzliu@mail.ccnu.edu.cn [Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079 (China); ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Department of Physics and Astronomy, Macquarie University, North Ryde 2109 (Australia); Qi, Meng; Zhang, Yin; Cao, Chaomin [Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079 (China); Goldys, Ewa M. [ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Department of Physics and Astronomy, Macquarie University, North Ryde 2109 (Australia)

    2016-02-25

    A stable label-free amperometric immunosensor is presented based on gold nanoparticles and graphene oxide nanocomposites for detection of cardiac troponin-I in the early diagnosis of myocardial infarction. For designing of the sensing platform, firstly the nanocomposites based on GO and AuNPs were prepared and anchored on electrode surfaces. The formed nanocomposites provided a platform with big surface area for loading anti-cTnI capture antibody, and worked as a bridge for fast electron transfer subsequently increased the sensitivity. Moreover, the linkages between AuNP, GO, and electrodes were based on covalent bonding by aryldiazonium salt coupling chemistry, which favors the stability of the sensing interface. Finally, the anti-cTnI detection antibody was immobilized on GO tailored with ferrocene molecules, functioning as the signal reporter for the detection of cTnI. The modification process was monitored using electrochemistry, SEM, XPS. The herein immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI, and is capable of detecting cTnI at concentrations as low as 0.05 ng mL{sup −1}, which is 100 times lower than that possible by conventional methods. It is potential to design the portable sensing platform based on AuNPs and GO nanocomposites for future point-of-care diagnostics. - Highlights: • Nanocomposites based on GO and AuNPs were prepared and anchored on the electrode surfaces covalently to form a stable sensing interface. • The anti-cTnI detection antibody was immobilized on GO tailored with ferrocene molecules, functioning as the signal reporter for the detection of cTnI. • The detectable concentration of cTnI is 0.05 ng mL{sup -1} in buffer with the assay time of less than 5 min. • The herein simple and novel approach for fabrication of AuNP and graphene based platform is promising for future fabrication of point-of-care devices.

  2. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu(2+) and L-cysteine.

    Science.gov (United States)

    Lin, Liping; Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru; Zhao, Li; Zhao, Tingting; Chen, Xi

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel "off-on" fluorescent probe for the label-free determination of Cu(2+) and l-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu(2+) owing to the coordination reaction between Cu(2+) and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu(2+) to L-Cys via the Cu-S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1-10 μM for Cu(2+) and 0.5-50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu(2+) and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu(2+) and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples.

  3. A label-free amperometric immunosensor for detection of zearalenone based on trimetallic Au-core/AgPt-shell nanorattles and mesoporous carbon

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Lei; Chao, Yingjun; Cao, Wei, E-mail: chm_caow@ujn.edu.cn; Wang, Yulan; Luo, Chuannan; Pang, Xuehui; Fan, Dawei; Wei, Qin, E-mail: sdjndxwq@163.com

    2014-10-17

    Highlights: • Au@AgPt nanorattles have special structure of Au-core and imperfect AgPt-shell. • Au@AgPt are proposed for the first time applied in electrochemical immunosensor. • Substrate materials MC/Au@AgPt possess excellent conductivity and high surface area. • The proposed immunosensor exhibits a low detection limit of 1.7 pg mL{sup −1}. - Abstract: A novel label-free amperometric immunosensor is proposed for the ultrasensitive detection of zearalenone (ZEN) based on mesoporous carbon (MC) and trimetallic nanorattles (core/shell particles with movable cores encapsulated in the shells). The nanorattles are composed of special Au-core and imperfect AgPt-shell structure (Au@AgPt). The Au@AgPt nanorattles are loaded onto the MC by physical adsorption. The structure of the Au@AgPt nanorattles was characterized by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Energy dispersive X-ray spectroscopy (EDS) confirmed the composition of the synthesized nanorattles. Compared with monometallic and bimetallic nanoparticles (NPs), Au@AgPt nanorattles show a higher electron transfer rate due to the synergistic effect of the Au, Ag and Pt NPs. MC further improves the sensitivity of the immunosensor because of its extraordinarily large specific surface area, suitable pore arrangement and outstanding conductivity. The large specific surface area of MC and MC@Au@AgPt were characterized by the BET method. ZEN antibodies are immobilized onto the nanorattles via Ag–NH{sub 2} bonds and Pt–NH{sub 2} bonds. Cyclic voltammetry and square wave voltammetry were used to characterize the recognizability of ZEN. Under optimum experimental conditions, the proposed immunosensor exhibited a low detection limit (1.7 pg mL{sup −1}), a wide linear range (from 0.005 to 15 ng mL{sup −1}) as well as good stability, reproducibility and selectivity. The sensor can be used in clinical analysis.

  4. Label-free detection of rheumatoid factor using YbY{sub x}O{sub y} electrolyte–insulator–semiconductor devices

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Tung-Ming, E-mail: tmpan@mail.cgu.edu.tw [Department of Electronics Engineering, Chang Gung University, Taoyuan 33302, Taiwan (China); Lin, Ting-Wei [School of Medicine, Chang Gung University, Taoyuan 33302, Taiwan (China); Chen, Ching-Yi [Department of Electronics Engineering, Chang Gung University, Taoyuan 33302, Taiwan (China)

    2015-09-03

    In this study, we investigated the effect of yttrium content on the structural properties and sensing characteristics of YbY{sub x}O{sub y} sensing membranes for electrolyte–insulator–semiconductor (EIS) sensors to detect the rheumatoid factor (RF). The YbY{sub x}O{sub y} EIS device prepared at the 60 W plasma condition exhibited a higher sensitivity of 65.77 mV/pH, a lower hysteresis voltage of ∼1 mV, and a smaller drift rate of 0.14 mV/h than did those prepared at the other conditions. We attribute this behavior to the optimal yttrium content in the YbY{sub x}O{sub y} film forming a smooth surface. Furthermore, we used a novel YbTi{sub x}O{sub y} EIS biosensor to measure the RF antigen in human serum because of its rapid and label-free detection. Two different techniques were used for the immobilization of RF antibody onto the surface of an YbTi{sub x}O{sub y} EIS sensor. The RF antibody was directly immobilized on the EIS surface modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA). In contrast, a mixture of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution was used to functionalize the carboxyl groups at the tail of RF antibodies. RF antibodies functionalized with the active NHS esters were covalently immobilized on the APTES-modified YbTi{sub x}O{sub y} surface. The immobilized RF antibodies on the EIS that are functionalized with the EDC and NHS exhibit higher (41.11 mV/pC{sub RF}) for detection of serum RF antigen in the range 10{sup −7} to 10{sup −3} M, compared to traditional antibody immobilization technique via APTES and GA linkage. The YbTi{sub x}O{sub y} EIS biosensor is a promising analytical tool for RF antigen monitoring due to its good sensitivity, stability and repeatability. - Highlights: • Effect of yttrium content on the structural and sensing properties of YbY{sub x}O{sub y} sensing membrane was explored. • YbY{sub x}O{sub y} EIS device prepared at the

  5. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu{sup 2+} and L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Liping [College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002 (China); Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); Zhao, Li; Zhao, Tingting [Xiamen Huaxia College, Xiamen, 361024 (China); Chen, Xi, E-mail: xichen@xmu.edu.cn [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361005 (China)

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel “off-on” fluorescent probe for the label-free determination of Cu{sup 2+} and L-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu{sup 2+} owing to the coordination reaction between Cu{sup 2+} and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu{sup 2+} to L-Cys via the Cu–S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1–10 μM for Cu{sup 2+} and 0.5–50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu{sup 2+} and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu{sup 2+} and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. - Highlights: • The europium-decorated graphene quantum dots (Eu-GQDs) have been successfully prepared. • Various characterizations results proved that Eu atoms were successfully introduced into graphene quantum dots. • The introduced Eu atoms changed the electron density and surface chemical activities of Eu-GQDs. • Eu-GQDs were used as an “off-on” fluorescent probe for Cu{sup 2+} and L-cysteine detection

  6. Atomic force microscopy images label-free, drug encapsulated nanoparticles in vivo and detects difference in tissue mechanical properties of treated and untreated: a tip for nanotoxicology.

    Directory of Open Access Journals (Sweden)

    Dimitrios A Lamprou

    Full Text Available Overcoming the intractable challenge of imaging of label-free, drug encapsulated nanoparticles in tissues in vivo would directly address associated regulatory concerns over 'nanotoxicology'. Here we demonstrate the utility of Atomic Force Microscopy (AFM for visualising label-free, drug encapsulated polyester particles of ∼280 nm distributed within tissues following their intravenous or peroral administration to rodents. A surprising phenomenon, in which the tissues' mechanical stiffness was directly measured (also by AFM and related to the number of embedded nanoparticles, was utilised to generate quantitative data sets for nanoparticles localisation. By coupling the normal determination of a drug's pharmacokinetics/pharmacodynamics with post-sacrifice measurement of nanoparticle localisation and number, we present for the first time an experimental design in which a single in vivo study relates the PK/PD of a nanomedicine to its toxicokinetics.

  7. Label-free Electrochemiluminescent Immunosensor for Detection of Prostate Specific Antigen based on Aminated Graphene Quantum Dots and Carboxyl Graphene Quantum Dots

    OpenAIRE

    Dan Wu; Yixin Liu; Yaoguang Wang; Lihua Hu; Hongmin Ma; Guoqin Wang; Qin Wei

    2016-01-01

    Prostate-specific antigen (PSA) was used as the model, an ultrasensitive label-free electrochemiluminescent immunosensor was developed based on graphene quantum dots. Au/Ag-rGO was sythsized and used as electrode material to load a great deal of graphene quantum dots due to the large surface area and excellent electron conductivity. After aminated graphene quantum dots and acarboxyl graphene quantum dots were modified onto the electrode, the ECL intensity was much high using K2S2O8 as coreact...

  8. Preparation of Au-Pt nanostructures by combining top-down with bottom-up strategies and application in label-free electrochemical immunosensor for detection of NMP22.

    Science.gov (United States)

    Jia, Hongying; Gao, Picheng; Ma, Hongmin; Wu, Dan; Du, Bin; Wei, Qin

    2015-02-01

    A novel label-free amperometric immunosensor for sensitive detection of nuclear matrix protein 22 (NMP22) was developed based on Au-Pt bimetallic nanostructures, which were prepared by combining top-down with bottom-up strategies. Nanoporous gold (NPG) was prepared by "top-down" dealloying of commercial Au/Ag alloy film. After deposition of NPG on an electrode, Pt nanoparticles (PtNPs) were further decorated on NPG by "bottom-up" electrodeposition. The prepared bimetallic nanostructures combine the merits of both NPG and PtNPs, and show a high electrocatalytic activity towards the reduction of H2O2. The label-free immunosensor was constructed by directly immobilizing antibody of NMP22 (anti-NMP22) on the surface of bimetallic nanostructures. The immunoreaction induced amperometric response could be detected and negatively correlated to the concentration of NMP22. Bimetallic nanostructure morphologies and detection conditions were investigated to obtain the best sensing performance. Under the optimal conditions, a linear range from 0.01ng/mL to 10ng/mL and a detection limit of 3.33pg/mL were obtained. The proposed immunosensor showed high sensitivity, good selectivity, stability, reproducibility, and regeneration for the detection of NMP22, and it was evaluated in urine samples, receiving satisfactory results.

  9. Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo.

    Science.gov (United States)

    Dislich, Bastian; Wohlrab, Felix; Bachhuber, Teresa; Müller, Stephan A; Kuhn, Peer-Hendrik; Hogl, Sebastian; Meyer-Luehmann, Melanie; Lichtenthaler, Stefan F

    2015-10-01

    Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1-/- and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1-/- and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1-/- mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.

  10. Performance limitations of label-free sensors in molecular diagnosis using complex samples

    Science.gov (United States)

    Varma, Manoj

    2016-03-01

    Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

  11. Emerging applications of label-free optical biosensors

    Science.gov (United States)

    Zanchetta, Giuliano; Lanfranco, Roberta; Giavazzi, Fabio; Bellini, Tommaso; Buscaglia, Marco

    2017-01-01

    Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

  12. Grazing incidence angle based sensing approach integrated with fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy for remote and label-free detection of medical device contaminations

    Energy Technology Data Exchange (ETDEWEB)

    Hassan, Moinuddin, E-mail: moinuddin.hassan@fda.hhs.gov; Ilev, Ilko [Optical Therapeutics and Medical Nanophotonics Laboratory, Division of Biomedical Physics, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, U.S. Food and Drug Administration, Silver Spring, Maryland 20993 (United States)

    2014-10-15

    Contamination of medical devices has become a critical and prevalent public health safety concern since medical devices are being increasingly used in clinical practices for diagnostics, therapeutics and medical implants. The development of effective sensing methods for real-time detection of pathogenic contamination is needed to prevent and reduce the spread of infections to patients and the healthcare community. In this study, a hollow-core fiber-optic Fourier transform infrared spectroscopy methodology employing a grazing incidence angle based sensing approach (FO-FTIR-GIA) was developed for detection of various biochemical contaminants on medical device surfaces. We demonstrated the sensitivity of FO-FTIR-GIA sensing approach for non-contact and label-free detection of contaminants such as lipopolysaccharide from various surface materials relevant to medical device. The proposed sensing system can detect at a minimum loading concentration of approximately 0.7 μg/cm{sup 2}. The FO-FTIR-GIA has the potential for the detection of unwanted pathogen in real time.

  13. Visual detection of lead(II) using a label-free DNA-based sensor and its immobilization within a monolithic hydrogel.

    Science.gov (United States)

    Jacobi, Zachary E; Li, Lu; Liu, Juewen

    2012-02-07

    Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors with low to sub-nM detection limits have been reported. These figures of merit were typically measured using a spectrophotometer that can detect nM DNA with a high signal-to-noise ratio. For visual detection, however, μM DNA or dye was required, making it difficult to detect low nM targets. We recently achieved a visual sensitivity of 10 nM Hg(2+) by immobilizing a DNA probe in a hydrogel. This was made possible because the gel was able to actively adsorb Hg(2+). In this work, we aim to test whether this method can be extended to the detection of Pb(2+). First, a new Pb(2+) sensor was designed based on a guanine-rich DNA and DNA binding dyes such as thiazole orange and SYBR Green I. The free DNA showed a detection limit of 8 nM Pb(2+) using 40 nM DNA. For visual detection in solution with 1 μM of the DNA probe, however, ∼300 nM Pb(2+) was required. After immobilization in a monolithic polyacrylamide hydrogel, even 20 nM Pb(2+) could be visually detected with a sample volume of 50 mL. Therefore, sensitive detection without signal amplification was achieved. Finally, we demonstrated simultaneous detection of both Hg(2+) and Pb(2+) in the same water sample with shape encoded hydrogel sensors.

  14. Lytic enzymes as selectivity means for label-free, microfluidic and impedimetric detection of whole-cell bacteria using ALD-Al2O3 passivated microelectrodes

    OpenAIRE

    Couniot, Numa; Vanzieleghem, Thomas; Rasson, Jonathan; Van Overstraeten, Nancy; Poncelet, Olivier; Mahillon, Jacques; Francis, Laurent; Flandre, Denis

    2014-01-01

    Point-of-care (PoC) diagnostics for bacterial detection offer tremendous prospects for public health care improvement. However, such tools require the complex combination of the following performances: rapidity, selectivity, sensitivity, miniaturization and affordability. To meet these specifications, this paper presents a new selectivity method involving lysostaphin together with a CMOS-compatible impedance sensor for genus-specific bacterial detection. The method enables the sample matrix t...

  15. Label-Free 3D Ag Nanoflower-Based Electrochemical Immunosensor for the Detection of Escherichia coli O157:H7 Pathogens

    Science.gov (United States)

    Huang, He; Liu, Minghuan; Wang, Xiangsheng; Zhang, Wenjie; Yang, Da-Peng; Cui, Lianhua; Wang, Xiansong

    2016-11-01

    It is highly desirable to develop a rapid and simple method to detect pathogens. Combining nanomaterials with electrochemical techniques is an efficient way for pathogen detection. Herein, a novel 3D Ag nanoflower was prepared via a biomineralization method by using bovine serum albumin (BSA) as a template. It was adopted as a sensing interface to construct an electrochemical bacteria immunosensor for the rapid detection of foodborne pathogens Escherichia coli ( E. coli) O157:H7. Bacterial antibody was immobilized onto the surface of Ag nanoflowers through covalent conjugation. Electrochemical impedance spectroscopy (EIS) was used to detect and validate the resistance changes, where [Fe(CN)6]3-/4- acted as the redox probe. A linear relation between R et and E. coli concentration was obtained in the E. coli concentration range of 3.0 × 102-3.0 × 108 cfu mL-1. The as-prepared biosensor gave rise to an obvious response to E. coli but had no distinct response to Cronobacter sakazakii, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus albus, Lactobacillus easei, and Shigella flexneri, revealing a high selectivity for the detection of the pathogens down to 100 cfu mL-1 in a short time. We believe that this BSA-conjugated 3D Ag nanoflowers could be used as a powerful interface material with good conductivity and biocompatibility for improving pathogen detection and treatment in the field of medicine, environment, and food safety.

  16. Label-free and reagentless electrochemical detection of PCR fragments using self-assembled quinone derivative monolayer: Application to Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Zhang, Q D; March, G; Noel, V;

    2012-01-01

    ), as well as with polymerase chain reaction (PCR) products related to different lineages of Mycobacterium tuberculosis strains. With pure oligonucleotides, this system achieves high sensitivity (~300pM of DNA target, i.e. 30fmol in a 100µL sample) and excellent selectivity, allowing to detect a single...

  17. A label-free aptasensor based on polyethyleneimine wrapped carbon nanotubes in situ formed gold nanoparticles as signal probe for highly sensitive detection of dopamine.

    Science.gov (United States)

    Azadbakht, Azadeh; Roushani, Mahmoud; Abbasi, Amir Reza; Menati, Saeid; Derikvand, Zohreh

    2016-11-01

    Herein, a highly sensitive and selective aptamer biosensor for quantitative detection of a model target, dopamine (DA), was developed by using a gold (Au) electrode modified with highly dispersed gold nanoparticles (AuNPs) and acid-oxidized carbon nanotubes (CNTs-COOH) functionalized with polyethyleneimine (PEI). Amine-terminated12-mercaptureprobe (ssDNA1) as a capture probe and specific DA-aptamer (ssDNA2) as a detection probe was immobilized on the surface of a modified electrode via the formation of covalent amide bond and hybridization, respectively. Methylene blue (MB) was used as the redox probe, which was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to decreased amount of adsorbed MB. The developed electrochemical DA aptasensor showed a good linear response to DA from 5 to 300nM with detection limit of 2.1nM. The biosensor also exhibited satisfactory selectivity and could be successfully used to detect DA in blood serum sample.

  18. Lytic enzymes as selectivity means for label-free, microfluidic and impedimetric detection of whole-cell bacteria using ALD-Al2O3 passivated microelectrodes.

    Science.gov (United States)

    Couniot, N; Vanzieleghem, T; Rasson, J; Van Overstraeten-Schlögel, N; Poncelet, O; Mahillon, J; Francis, L A; Flandre, D

    2015-05-15

    Point-of-care (PoC) diagnostics for bacterial detection offer tremendous prospects for public health care improvement. However, such tools require the complex combination of the following performances: rapidity, selectivity, sensitivity, miniaturization and affordability. To meet these specifications, this paper presents a new selectivity method involving lysostaphin together with a CMOS-compatible impedance sensor for genus-specific bacterial detection. The method enables the sample matrix to be directly flown on the polydopamine-covered sensor surface without any pre-treatment, and considerably reduces the background noise. Experimental proof-of-concept, explored by simulations and confirmed through a setup combining simultaneous optical and electrical real-time monitoring, illustrates the selective and capacitive detection of Staphylococcus epidermidis in synthetic urine also containing Enterococcus faecium. While providing capabilities for miniaturization and system integration thanks to CMOS compatibility, the sensors show a detection limit of ca. 10(8) (CFU/mL).min in a 1.5 μL microfluidic chamber with an additional setup time of 50 min. The potentials, advantages and limitations of the method are also discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Label-free detection of Staphylococcus aureus in skin using real-time potentiometric biosensors based on carbon nanotubes and aptamers.

    Science.gov (United States)

    Zelada-Guillén, Gustavo A; Sebastián-Avila, José Luis; Blondeau, Pascal; Riu, Jordi; Rius, F Xavier

    2012-01-15

    In this paper we report the first biosensor that is able to detect Staphylococcus aureus in real-time. A network of single-walled carbon nanotubes (SWCNTs) acts as an ion-to-electron potentiometric transducer and anti-S. aureus aptamers are the recognition element. Carbon nanotubes were functionalized with aptamers using two different approaches: (1) non-covalent adsorption of drop-casted pyrenil-modified aptamers onto the external walls of the SWCNTs; and (2) covalent bond formation between amine-modified aptamers and carboxylic groups previously introduced by oxidation at the ends of the SWCNTs. Both of these approaches yielded functional biosensors but there were large differences in the minimum detectable bacteria concentration and sensitivity values. With covalent functionalization, the minimum concentration detected was 8×10(2)colony-forming units (CFU)/mL and the sensitivity was 0.36 mV/Decade. With the non-covalent approach, the sensitivity was higher (1.52 mV/Decade) but the minimum concentration detected was greatly affected (10(7) CFU/mL). In both cases, potential as a function of Decade of bacteria concentration was linear. Functional biosensors were used to test real samples from freshly excised pig skin, contaminated with the target microorganism, as a surrogate for human skin.

  20. Optofluidic ring resonator sensor for sensitive label-free detection of breast cancer antigen CA15-3 in human serum

    Science.gov (United States)

    Zhu, Hongying; Dale, Paul S.; Fan, Xudong

    2009-05-01

    Breast cancer is the most frequently diagnosed malignancy in women worldwide. Because of its great impact on society, a lot of research funding has been used to develop novel detection tools for aiding breast cancer diagnosis and prognosis. In this work, we demonstrated a simple, fast, and sensitive detection of circulating breast cancer biomarker CA15-3 with opto-fluidic ring resonator (OFRR) sensors. The OFRR sensor employs a thin-walled capillary with wall thickness less than 4 μm. The circular cross section of the capillary forms the optical ring resonator, in which the light circulates in the form of whispering gallery modes (WGMs). The capillary wall is thin enough that the evanescent field of the WGM extends into the capillary core and responds to refractive index changes in the capillary core or close to its interior surface. The WGM spectral position will change when the biomolecules bind to the surface, yielding quantitative and kinetic information about the biomolecule interaction. Here, the direct immunoassay method was employed for the detection of CA15-3 antigen without any signal amplification steps. The sensor performance in both PBS buffer and human serum were investigated, respectively. The experimental detection limit was 5 units/mL in PBS buffer and 30 units/mL for CA15-3 spiked in serum, both of which satisfied clinical diagnosis requirements. The potential use of the OFRR as the point-of-care device for breast cancer detection was tested by measuring the CA15-3 level in blood samples collected from stage IV breast cancer patients and the results were compared with standard clinical test.

  1. Label-free turn-on fluorescent detection of melamine based on the anti-quenching ability of Hg 2+ to gold nanoclusters.

    Science.gov (United States)

    Dai, Haichao; Shi, Yan; Wang, Yilin; Sun, Yujing; Hu, Jingting; Ni, Pengjuan; Li, Zhuang

    2014-03-15

    In this work, we proposed a facile, environmentally friendly and cost-effective assay for melamine with BSA-stabilized gold nanoclusters (AuNCs) as a fluorescence reader. Melamine, which has a multi-nitrogen heterocyclic ring, is prone to coordinate with Hg(2+). This property causes the anti-quenching ability of Hg(2+) to AuNCs through decreasing the metallophilic interaction between Hg(2+) and Au(+). By this method, detection limit down to 0.15 µM is obtained, which is approximately 130 times lower than that of the US food and Drug Administration estimated melamine safety limit of 20 µM. Furthermore, several real samples spiked with melamine, including raw milk and milk powder, are analyzed using the sensing system with excellent recoveries. This gold-nanocluster-based fluorescent method could find applications in highly sensitive detection of melamine in real samples.

  2. Droplet-based label-free detection system based on guided-mode resonance and electrowetting-on-dielectric for concentration measurement

    Science.gov (United States)

    Wang, Ying-Bin; Su, Lin-Yun; Fu, Ching-Shu; Huang, Cheng-Sheng; Hsu, Wensyang

    2017-05-01

    We demonstrate a digital microfluidic system with an on-chip detection capability that is based on a guided-mode resonance filter (GMRF). Versatile droplet manipulation was achieved through the electrowetting-on-dielectric (EWOD) technique. GMRF was fabricated through replica molding and soft lithography, and the EWOD chip was fabricated through a typical photolithography process. Droplets measuring 1.125 µL were used to demonstrate merging, mixing, and cutting, and to achieve twofold and Threefold dilutions, and were then transported to the GMRF region for concentration measurement. The bulk sensitivity of GMRF is approximately 90 nm RIU-1 [17494 nm/(g/L)], resulting in a minimum detectable concentration of 4.66 × 10-5 g/L for a sucrose solution.

  3. Multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole DNA biosensor for label-free detection of genetically modified organisms by QCM and EIS.

    Science.gov (United States)

    Truong, Thi Ngoc Lien; Tran, Dai Lam; Vu, Thi Hong An; Tran, Vinh Hoang; Duong, Tuan Quang; Dinh, Quang Khieu; Tsukahara, Toshifumi; Lee, Young Hoon; Kim, Jong Seung

    2010-01-15

    In this paper, we describe DNA electrochemical detection for genetically modified organism (GMO) based on multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole (PPy). DNA hybridization is studied by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). An increase in DNA complementary target concentration results in a decrease in the faradic charge transfer resistance (R(ct)) and signifying "signal-on" behavior of MWCNTs-PPy-DNA system. QCM and EIS data indicated that the electroanalytical MWCNTs-PPy films were highly sensitive (as low as 4pM of target can be detected with QCM technique). In principle, this system can be suitable not only for DNA but also for protein biosensor construction.

  4. A simple and rapid label-free fluorimetric biosensor for protamine detection based on glutathione-capped CdTe quantum dots aggregation.

    Science.gov (United States)

    Ensafi, Ali A; Kazemifard, N; Rezaei, B

    2015-09-15

    A novel fluorescent biosensor is developed, based on glutathione-capped CdTe quantum dots aggregation, for the determination of trace amount of an important drug, protamine. In this method with increasing the protamine concentration, the fluorescence of the quantum dots was quenched due to their aggregation. Different parameters affect the sensitivity, such as pH and the amount of the quantum dots, were optimized. Using the new optical biosensor, under the optimized conditions, protamine could be measured in the range of 2.0-200 ng mL(-1) with a detection limit of 1.0 ng mL(-)(1). The relative standard deviation for five replicates determination of 30.0 ng mL(-)(1) protamine was 1.26%. The influence of common interfering species on the protamine detection was studied. The results showed that the biosensor is highly selective and sensitive for the detection of protamine. The optical biosensor was successfully used for the determination of protamine in real samples.

  5. Direct Growth Graphene on Cu Nanoparticles by Chemical Vapor Deposition as Surface-Enhanced Raman Scattering Substrate for Label-Free Detection of Adenosine

    CERN Document Server

    Xu, Shicai; Jiang, Shouzhen; Wang, Jihua; Wei, Jie; Xu, Shida; Liu, Hanping

    2015-01-01

    We present a graphene/Cu nanoparticle hybrids (G/CuNPs) system as a surface-enhanced Raman scattering (SERS) substrate for adenosine detection. The Cu nanoparticles wrapped around a monolayer graphene shell were directly synthesized on flat quartz by chemical vapor deposition in a mixture of methane and hydrogen. The G/CuNPs showed an excellent SERS enhancement activity for adenosine. The minimum detected concentration of the adenosine in serum was demonstrated as low as 5 nM, and the calibration curve showed a good linear response from 5 to 500 nM. The capability of SERS detection of adenosine in real normal human urine samples based on G/CuNPs was also investigated and the characteristic peaks of adenosine were still recognizable. The reproducible and the ultrasensitive enhanced Raman signals could be due to the presence of an ultrathin graphene layer. The graphene shell was able to enrich and fix the adenosine molecules, which could also efficiently maintain chemical and optical stability of G/CuNPs. Based...

  6. A surface enhanced Raman scattering quantitative analytical platform for detection of trace Cu coupled the catalytic reaction and gold nanoparticle aggregation with label-free Victoria blue B molecular probe.

    Science.gov (United States)

    Li, Chongning; Ouyang, Huixiang; Tang, Xueping; Wen, Guiqing; Liang, Aihui; Jiang, Zhiliang

    2017-01-15

    With development of economy and society, there is an urgent need to develop convenient and sensitive methods for detection of Cu(2+) pollution in water. In this article, a simple and sensitive SERS sensor was proposed to quantitative analysis of trace Cu(2+) in water. The SERS sensor platform was prepared a common gold nanoparticle (AuNP)-SiO2 sol substrate platform by adsorbing HSA, coupling with the catalytic reaction of Cu(2+)-ascorbic acid (H2A)-dissolved oxygen, and using label-free Victoria blue B (VBB) as SERS molecular probes. The SERS sensor platform response to the AuNP aggregations by hydroxyl radicals (•OH) oxidizing from the Cu(2+) catalytic reaction, which caused the SERS signal enhancement. Therefore, by monitoring the increase of SERS signal, Cu(2+) in water can be determined accurately. The results show that the SERS sensor platforms owns a linear response with a range from 0.025 to 25μmol/L Cu(2+), and with a detection limit of 0.008μmol/L. In addition, the SERS method demonstrated good specificity for Cu(2+), which can determined accurately trace Cu(2+) in water samples, and good recovery and accuracy are obtained for the water samples. With its high selectivity and good accuracy, the sensitive SERS quantitative analysis method is expected to be a promising candidate for determining copper ions in environmental monitoring and food safety.

  7. Label-free detection of neuronal differentiation in cell populations using high-throughput live-cell imaging of PC12 cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Weber

    Full Text Available Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over 6 days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.

  8. Label-Free Electrochemiluminescent Immunosensor for Detection of Carcinoembryonic Antigen Based on Nanocomposites of GO/MWCNTs-COOH/Au@CeO₂.

    Science.gov (United States)

    Pang, Xuehui; Li, Jianxiu; Zhao, Yongbei; Wu, Dan; Zhang, Yong; Du, Bin; Ma, Hongmin; Wei, Qin

    2015-09-01

    A high-sensitivity electrochemiluminescence (ECL) sensor was conducted to detect carcinoembryonic antigen (CEA). Nanocomposites of graphene oxide/carboxylated multiwall carbon nanotubes/gold/cerium oxide nanoparticles (GO/MWCNTs-COOH/Au@CeO2) were used as antibody carriers and sensing platforms to modify on glassy carbon electrodes (GCE). CeO2 nanoparticles were first exploited as an ECL luminescent material and the possible ECL mechanism was proposed in this work. GO/MWCNTs-COOH was used as a loading matrix for CeO2 nanoparticles because of the superior conductivity and large specific surface area. Au nanoparticles were further deposited on this matrix to attach anti-CEA and enhance the sensitivity of immunosensor. The proposed sensing platform showed excellent cathodic ECL performance and sensitive response to CEA. The effects of experimental conditions on the ECL performance were investigated. The proposed immunosensor showed the broad linear range (0.05-100 ng/mL) and the low detection limit (LOD, 0.02 ng/mL, signal-to-noise ratio = 3) according to the selected experimental conditions. The excellent analysis performance for determination of CEA in the human serum samples simplied this immunosensor displayed high sensitivity and excellent repeatability. More importantly, this conducted immunosensor broadens the use scope of CeO2 nanoparticles.

  9. Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

    Science.gov (United States)

    Geissler, David; Belder, Detlev

    2015-12-01

    One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (body polymer microfluidic devices. This was achieved by means of two-photon excitation in the visible range (λex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Label-Free LSPR Detection of Trace Lead(II) Ions in Drinking Water by Synthetic Poly(mPD-co-ASA) Nanoparticles on Gold Nanoislands.

    Science.gov (United States)

    Qiu, Guangyu; Ng, Siu Pang; Liang, Xiongyi; Ding, Ning; Chen, Xiangfeng; Wu, Chi-Man Lawrence

    2017-02-07

    Using self-assembly gold nanoislands (SAM-AuNIs) functionalized by poly(m-phenylenediamine-co-aniline-2-sulfonic acid) (poly(mPD-co-ASA)) copolymer nanoparticles as specific receptors, a highly sensitive localized surface plasmon resonance (LSPR) optochemical sensor is demonstrated for detection of trace lead cation (Pb(II)) in drinking water. The copolymer receptor is optimized in three aspects: (1) mole ratio of mPD:ASA monomers, (2) size of copolymer nanoparticles, and (3) surface density of the copolymer. It is shown that the 95:5 (mPD:ASA mole ratio) copolymer with size less than 100 nm exhibits the best Pb(II)-sensing performance, and the 200 times diluted standard copolymer solution contributes to the most effective functionalization protocol. The resulting poly(mPD-co-ASA)-functionalized LSPR sensor attains the detection limit to 0.011 ppb toward Pb(II) in drinking water, and the linear dynamic range covers 0.011 to 5000 ppb (i.e., 6 orders of magnitude). In addition, the sensing system exhibits robust selectivity to Pb(II) in the presence of other metallic cations as well as common anions. The proposed functional copolymer functionalized on AuNIs is found to provide excellent Pb(II)-sensing performance using simple LSPR instrumentation for rapid drinking-water inspection.

  11. A microfluidic device for label-free detection of Escherichia coli in drinking water using positive dielectrophoretic focusing, capturing, and impedance measurement.

    Science.gov (United States)

    Kim, Myounggon; Jung, Taekeon; Kim, Youngjin; Lee, Changgeun; Woo, Kyungchul; Seol, Jae Hun; Yang, Sung

    2015-12-15

    While sensors that allow for high-throughput enumeration of microorganisms within drinking water are useful for water quality monitoring, it is particularly challenging to accurately quantify microorganisms that are present in low numbers (water cannot be analyzed by this approach. Here, we report a positive DEP (pDEP)-based Escherichia coli detection system that is integrated with a focusing and sensing electrode. By incorporating a passivation layer, we avoided issues with adhesion of E. coli to the electrode, and achieved efficient cell focusing under high flow rate conditions (1500 μL/h). The resulting focused E. coli cells were then trapped on the sensor electrode, resulting in changes in impedance. The proposed system was evaluated using four different E. coli populations (150-1500 CFU/mL). We successfully enumerated populations as low as 300 CFU/mL within 1 min, and the signal variation was 1.13±0.37%. The device introduced in this study provides the basis for the development of portable, highly sensitive microorganism sensors that enable rapid detection of bacteria in drinking water.

  12. Ultra-sensitive label-free in-situ detection of dynamically driven self-assembly of 2D nanoplatelets on SOI chip

    CERN Document Server

    Hogan, Benjamin T; Brennan, Lorcan J; Younesy, Salma; Perova, Tatiana; Gunko, Yurii K; Craciun, Monica F; Baldycheva, Anna

    2016-01-01

    Fluid dispersed two-dimensional (2D) composite materials with dynamically tunable functional properties have recently emerged as a novel highly promising class of optoelectronic materials, opening up new routes not only for the emerging field of metamaterials but also to chip-scale multifunctional metadevices. However, in-situ monitoring and detection of the dynamic ordering of 2D nanoparticles on chip and during the device operation is still a huge challenge. Here we introduce a novel approach for on-chip, in-situ Raman characterisation of 2D-fluid composite materials incorporated into Si photonics chip. In this work the Raman signal for 2D nanoplatelets is selectively enhanced by Fabry-Perot resonator design of CMOS photonic-compatible microfluidic channels. This has then been extended to demonstrate the first in-situ Raman detection of the dynamics of individual 2D nanoplatelets, within a microfluidic channel. Our work paves the way for the first practicable realisation of 3D photonic microstructure shapin...

  13. A Label-Free Impedance Immunosensor Using Screen-Printed Interdigitated Electrodes and Magnetic Nanobeads for the Detection of E. coli O157:H7.

    Science.gov (United States)

    Wang, Ronghui; Lum, Jacob; Callaway, Zach; Lin, Jianhan; Bottje, Walter; Li, Yanbin

    2015-12-15

    Escherichia coli O157:H7 is one of the leading bacterial pathogens causing foodborne illness. In this study, an impedance immunosensor based on the use of magnetic nanobeads and screen-printed interdigitated electrodes was developed for the rapid detection of E. coli O157:H7. Magnetic nanobeads coated with anti-E. coli antibody were mixed with an E. coli sample and used to isolate and concentrate the bacterial cells. The sample was suspended in redox probe solution and placed onto a screen-printed interdigitated electrode. A magnetic field was applied to concentrate the cells on the surface of the electrode and the impedance was measured. The impedance immunosensor could detect E. coli O157:H7 at a concentration of 10(4.45) cfu·mL(-1) (~1400 bacterial cells in the applied volume of 25 μL) in less than 1 h without pre-enrichment. A linear relationship between bacteria concentration and impedance value was obtained between 10(4.45) cfu·mL(-1) and 10(7) cfu·mL(-1). Though impedance measurement was carried out in the presence of a redox probe, analysis of the equivalent circuit model showed that the impedance change was primarily due to two elements: Double layer capacitance and resistance due to electrode surface roughness. The magnetic field and impedance were simulated using COMSOL Multiphysics software.

  14. Emerging applications of label-free optical biosensors

    Directory of Open Access Journals (Sweden)

    Zanchetta Giuliano

    2017-01-01

    Full Text Available Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

  15. N-doped TiO2 based visible light activated label-free photoelectrochemical biosensor for detection of Hg(2+) through quenching of photogenerated electrons.

    Science.gov (United States)

    Han, Qianqian; Wang, Kewei; Xu, Lijun; Yan, Xiang; Zhang, Kunchi; Chen, Xing; Wang, Qinglin; Zhang, Lan; Pei, Renjun

    2015-06-21

    A novel photoelectrochemical (PEC) biosensor was fabricated based on N-doped TiO2 for the detection of Hg(2+) through the quenching of photogenerated electrons. The N-doped TiO2 was synthesized by a sol-gel method with urea and tetrabutyl titanate as the N and Ti sources. Compared with the undoped TiO2, the N-doped TiO2 showed an enhanced photocurrent response under visible light (λ > 420 nm). The sensing surface was functionalized with 5'-amino-modified T-rich oligonucleotides. The photoelectrochemical biosensor bound Hg(2+) on the surface by a highly specific T-Hg(2+)-T recognition. Hg(2+) on the surface of the N-doped TiO2 film withdrew the photogenerated electrons and decreased the recorded current signal. The dynamic linear range for Hg(2+) has been determined to be as low as 2-6 μM.

  16. Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV).

    Science.gov (United States)

    Molazemhosseini, Alireza; Magagnin, Luca; Vena, Pasquale; Liu, Chung-Chiun

    2016-07-01

    A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV) was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET) substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA) were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5-20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R² = 0.999) in the range of 0.1-0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated.

  17. Label-free probing of genes by time-domain terahertz sensing

    Energy Technology Data Exchange (ETDEWEB)

    Bolivar, P Haring [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Brucherseifer, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Nagel, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Kurz, H [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Bosserhoff, A [Institut fuer Pathologie, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg (Germany); Buettner, R [Institut fuer Pathologie, Universitaetsklinikum Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn (Germany)

    2002-11-07

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

  18. Label-free photoacoustic microscopy of cytochromes

    Science.gov (United States)

    Zhang, Chi; Zhang, Yu Shrike; Yao, Da-Kang; Xia, Younan; Wang, Lihong V.

    2013-02-01

    Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (˜420 nm), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.

  19. Detection of Ochratoxin A by Label-Free Liquid Crystal lmmunosensor%非标记液晶型免疫传感器检测赭曲霉素A

    Institute of Scientific and Technical Information of China (English)

    杨胜园; 谭慧; 刘艳梅; 吴朝阳; 沈国励; 俞汝勤

    2011-01-01

    A novel liquid crystal immunosensor based on the indirect competitive assay format was developed for the detection of ochratoxin A (OTA). OTA was tethered onto the self-assembled film surface on a glass slide via a cross-linker glutaradehyde. The specific binding of anti-OTA antibody and ochratoxin A induced the homeotropic-to-tilted transition of LCs, resulting in the corresponding changes of opitical images under the crossed polarized light. The LC-based imaging method had a good signal-to-noise ratio and a clear distinction between positive and negative results. When the concentration of analyte OTA exceeded a critical value(0.01 ng/mL), the opitical images showed a very strong response. The proposed sensor exhibited high sensitivityand desirable selectivity, and was label free and easy in operation.%研制了一种基于液晶取向改变的非标记液晶型免疫传感器,并用于检测赭曲霉素A(0TA).采用戊二醛交联法将OTA同定在由自组装膜修饰的玻璃肇底表面.自组装膜能诱导液晶分子垂直排列,而连接OTA抗体后则扰乱了液晶分子取向的有序排列,导致液晶分子在化学敏感膜表面的取向发生变化,使光学信号的亮度及色彩发生变化,以此实现对OTA的检测.当OTA含量超过0.01 ng/mL时,光学信号发生明显变化.本方法具有灵敏度高、特异性好、非标记和操作简单等特点.

  20. Multiplexed label-free optical biosensor for medical diagnostics.

    Science.gov (United States)

    Bottazzi, Barbara; Fornasari, Lucia; Frangolho, Ana; Giudicatti, Silvia; Mantovani, Alberto; Marabelli, Franco; Marchesini, Gerardo; Pellacani, Paola; Therisod, Rita; Valsesia, Andrea

    2014-01-01

    This paper describes a new multiplexed label-free biosensor. The detection technology is based on nanostructured gold-polymer surfaces. These surfaces support surface plasmon resonance modes that can be probed by a miniaturized optical setup. The optical characterization of the sensing chip shows the sensitivity and the limit-of-detection to refractive index changes. Moreover, by studying the progressive adhesion of molecular monolayers of polyelectrolytes, the decay of the plasmonic mode electric field above the surface has been reconstructed. A multiplexed label-free biosensing device is then described and characterized in terms of sensitivity, lateral resolution, and sensitivity to a model biological assay. The sensitivity in imaging mode of the device is of the order of 10-6 refractive index units, while the measured lateral resolution is 6.25 μm within a field of view of several tenths of mm2, making the instrument unique in terms of multiplexing capability. Finally, the proof-of-concept application of the technology as a point-of-care diagnostic tool for an inflammatory marker is demonstrated.

  1. Progress of new label-free techniques for biosensors: a review.

    Science.gov (United States)

    Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

    2016-01-01

    The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

  2. Label-free surface plasmon sensing towards cancer diagnostics

    Science.gov (United States)

    Sankaranarayanan, Goutham

    The main objective of this thesis is to develop a conventional, home-built SPR bio-sensor to demonstrate bio-sensing applications. This emphasizes the understanding of basic concepts of Surface Plasmon Resonance and various interrogation techniques. Intensity Modulation was opted to perform the label-free SPR bio-sensing experiments due to its cost-efficient and compact setup. Later, label-free surface plasmon sensing was carried out to study and understand the bio-molecular interactions between (1). BSA and Anti BSA molecules and (2). Exosome/Liposome on thin metal (Au) films. Exosomes are cell-derived vesicles present in bodily fluids like blood, saliva, urine, epididymal fluid containing miRNAs, RNA, proteins, etc., at stable quantities during normal health conditions. The exosomes comprise varied constituents based on their cell origin from where they are secreted and is specific to that particular origin. However an exacerbated release is observed during tumor or cancer conditions. This increased level of exosomes present in the sample, can be detected using the SPR bio-sensor demonstrated in this thesis and effective thickness of adsorption on Au surface can be estimated. Also, chemically synthesized liposome particles were studied to determine if they can generate an equivalent sensor response to that of exosomes to consider them as an alternate. Finally a 10ppb Mercury (Hg) sensing was performed as part of Environment Monitoring application and results have been tabulated and compared.

  3. CEST theranostics: label-free MR imaging of anticancer drugs

    Science.gov (United States)

    Xu, Jiadi; Yadav, Nirbhay N.; Chan, Kannie W. Y.; Luo, Liangping; McMahon, Michael T.; Vogelstein, Bert; van Zijl, Peter C.M.; Zhou, Shibin; Liu, Guanshu

    2016-01-01

    Image-guided drug delivery is of great clinical interest. Here, we explored a direct way, namely CEST theranostics, to detect diamagnetic anticancer drugs simply through their inherent Chemical Exchange Saturation Transfer (CEST) MRI signal, and demonstrated its application in image-guided drug delivery of nanoparticulate chemotherapeutics. We first screened 22 chemotherapeutic agents and characterized the CEST properties of representative agents and natural analogs in three major categories, i.e., pyrimidine analogs, purine analogs, and antifolates, with respect to chemical structures. Utilizing the inherent CEST MRI signal of gemcitabine, a widely used anticancer drug, the tumor uptake of the i.v.-injected, drug-loaded liposomes was successfully detected in CT26 mouse tumors. Such label-free CEST MRI theranostics provides a new imaging means, potentially with an immediate clinical impact, to monitor the drug delivery in cancer. PMID:26837220

  4. Label-free gold nanoparticles for the determination of neomycin

    Science.gov (United States)

    Apyari, Vladimir V.; Dmitrienko, Stanislava G.; Arkhipova, Viktoriya V.; Atnagulov, Aydar G.; Gorbunova, Mariya V.; Zolotov, Yury A.

    2013-11-01

    A new spectrophotometric method for the determination of neomycin has been developed. The method is based on aggregation of label-free gold nanoparticles leading to change in absorption spectra and color of the solution. Influence of different factors (the concentration of ethylenediaminetetraacetate (EDTA), pH, the concentrations of neomycin and the nanoparticles) on the aggregation and analytical performance of the method was investigated. EDTA plays an important role not only as a masking agent to eliminate interferences of metal cations but strongly affects the sensitivity of the nanoparticles relative to neomycin. The method allows to determine neomycin with detection limit of 28 ng mL-1. It was applied to analysis of eye- and ear-drops. The sample pretreatment is simply done by diluting the formulation with water.

  5. Integrated label-free silicon nanowire sensor arrays for (bio)chemical analysis

    NARCIS (Netherlands)

    De, Arpita; Nieuwkasteele, van Jan; Carlen, Edwin T.; Berg, van den Albert

    2013-01-01

    We present a label-free (bio)chemical analysis platform that uses all-electrical silicon nanowire sensor arrays integrated with a small volume microfluidic flow-cell for real-time (bio)chemical analysis and detection. The integrated sensing platform contains an automated multi-sample injection syste

  6. Chip-LC-MS for label-free profiling of human serum

    NARCIS (Netherlands)

    Horvatovich, Peter; Govorukhina, Natalia .; Reijmers, Theo H.; van der Zee, Ate G. J.; Suits, Frank; Bischoff, Rainer

    2007-01-01

    The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free

  7. Label-free bead-based metallothionein electrochemical immunosensor.

    Science.gov (United States)

    Nejdl, Lukas; Nguyen, Hoai Viet; Richtera, Lukas; Krizkova, Sona; Guran, Roman; Masarik, Michal; Hynek, David; Heger, Zbynek; Lundberg, Karin; Erikson, Kristofer; Adam, Vojtech; Kizek, Rene

    2015-08-01

    A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

  8. Scattering pulse of label free fine structure cells to determine the size scale of scattering structures

    Science.gov (United States)

    Zhang, Lu; Chen, Xingyu; Zhang, Zhenxi; Chen, Wei; Zhao, Hong; Zhao, Xin; Li, Kaixing; Yuan, Li

    2016-04-01

    Scattering pulse is sensitive to the morphology and components of each single label-free cell. The most direct detection result, label free cell's scattering pulse is studied in this paper as a novel trait to recognize large malignant cells from small normal cells. A set of intrinsic scattering pulse calculation method is figured out, which combines both hydraulic focusing theory and small particle's scattering principle. Based on the scattering detection angle ranges of widely used flow cytometry, the scattering pulses formed by cell scattering energy in forward scattering angle 2°-5° and side scattering angle 80°-110° are discussed. Combining the analysis of cell's illuminating light energy, the peak, area, and full width at half maximum (FWHM) of label free cells' scattering pulses for fine structure cells with diameter 1-20 μm are studied to extract the interrelations of scattering pulse's features and cell's morphology. The theoretical and experimental results show that cell's diameter and FWHM of its scattering pulse agree with approximate linear distribution; the peak and area of scattering pulse do not always increase with cell's diameter becoming larger, but when cell's diameter is less than about 16 μm the monotone increasing relation of scattering pulse peak or area with cell's diameter can be obtained. This relationship between the features of scattering pulse and cell's size is potentially a useful but very simple criterion to distinguishing malignant and normal cells by their sizes and morphologies in label free cells clinical examinations.

  9. Deep Learning in Label-free Cell Classification

    National Research Council Canada - National Science Library

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-01-01

    .... Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification...

  10. Diatom-based label-free optical biosensor for biomolecules.

    Science.gov (United States)

    Viji, S; Anbazhagi, M; Ponpandian, N; Mangalaraj, D; Jeyanthi, S; Santhanam, P; Devi, A Shenbaga; Viswanathan, C

    2014-10-01

    Diatoms are unicellular algae, which fabricates ornate biosilica shells called frustules that possess a surface rich in reactive silanol (Si-OH) groups. The intrinsic patterned porous structure of diatom frustules at nanoscale can be exploited in the effective detection of biomolecules. In this study, the frustules of a specific diatom Amphora sp. has been functionalized to detect bovine serum albumin (BSA). The functionalization of the diatom frustule substrate is achieved by using 3-aminopropyltriethoxysilane (APES). The field emission scanning electron microscopy (FESEM) results showed an ornately patterned surface of the frustule valve ordered at nanoscale. The Fourier transform infrared (FTIR) spectra confirmed the N-H bending and stretching of the amine group after amine functionalization. The emission peaks in the photoluminescence (PL) spectra of the amine-functionalized diatom biosilica selectively enhanced the intensity by a factor of ten when compared to that of a bare diatom biosilica. The result showed a significant quenching of PL intensity of BSA at around 445 nm due to the interaction of amine-functionalized diatom-BSA protein complex. The detection limit was found to be 3 × 10(-5) M of BSA protein. Hence, the study proves that the functionalized frustule of Amphora sp. is an effective quantitative analytical tool for optical label-free biosensing applications.

  11. Label-free monitoring of individual DNA hybridization using SERS

    Science.gov (United States)

    Qi, Ji; Zeng, Jianbo; Zhao, Fusheng; Santos, Greggy M.; Lin, Steven Hsesheng; Raja, Balakrishnan; Strych, Ulrich; Willson, Richard C.; Shih, Wei-Chuan

    2015-03-01

    Sequence-specific detection of DNA hybridization at the single-molecule level has been instrumental and gradually become a ubiquitous tool in a wide variety of biological and biomedical applications such as clinical diagnostics, biosensors, and drug development. Label-free and amplification-free schemes are of particular interest because they could potentially provide in situ monitoring of individual hybridization events, which may lead to techniques for discriminating subtle variations due to single-base modification without stringency control or repetitive thermal cycling. Surface-enhanced Raman spectroscopy (SERS) has been widely used for molecular detection and identification by exploiting the localized surface plasmon resonance effect when the target molecules are near gold or silver nanostructures. However, effective and robust SERS assays have yet become a reality for trace detection. Recently, we have developed a SERS substrate by shaping nanoporous gold thin films into monolithic submicron disks, called nanoporous gold disks (NPGD). Here we demonstrate in situ monitoring of the same immobilized ssDNA molecules and their individual hybridization events.

  12. Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

    Science.gov (United States)

    Chirvi, Sajal

    Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi

  13. Label-Free Biosensor Imaging on Photonic Crystal Surfaces

    Directory of Open Access Journals (Sweden)

    Yue Zhuo

    2015-08-01

    Full Text Available We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in “digital” diagnostics with single molecule sensing resolution. We will review PCEM’s development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity.

  14. Photonic Crystal Surfaces as a General Purpose Platform for Label-Free and Fluorescent Assays

    OpenAIRE

    Cunningham, Brian T.

    2010-01-01

    Photonic crystal surfaces can be designed to provide a wide range of functions that are used to perform biochemical and cell-based assays. Detection of the optical resonant reflections from photonic crystal surfaces enables high sensitivity label-free biosensing, while the enhanced electromagnetic fields that occur at resonant wavelengths can be used to enhance the detection sensitivity of any surface-based fluorescence assay. Fabrication of photonic crystals from inexpensive plastic material...

  15. Label-free tissue scanner for colorectal cancer screening

    Science.gov (United States)

    Kandel, Mikhail E.; Sridharan, Shamira; Liang, Jon; Luo, Zelun; Han, Kevin; Macias, Virgilia; Shah, Anish; Patel, Roshan; Tangella, Krishnarao; Kajdacsy-Balla, Andre; Guzman, Grace; Popescu, Gabriel

    2017-06-01

    The current practice of surgical pathology relies on external contrast agents to reveal tissue architecture, which is then qualitatively examined by a trained pathologist. The diagnosis is based on the comparison with standardized empirical, qualitative assessments of limited objectivity. We propose an approach to pathology based on interferometric imaging of "unstained" biopsies, which provides unique capabilities for quantitative diagnosis and automation. We developed a label-free tissue scanner based on "quantitative phase imaging," which maps out optical path length at each point in the field of view and, thus, yields images that are sensitive to the "nanoscale" tissue architecture. Unlike analysis of stained tissue, which is qualitative in nature and affected by color balance, staining strength and imaging conditions, optical path length measurements are intrinsically quantitative, i.e., images can be compared across different instruments and clinical sites. These critical features allow us to automate the diagnosis process. We paired our interferometric optical system with highly parallelized, dedicated software algorithms for data acquisition, allowing us to image at a throughput comparable to that of commercial tissue scanners while maintaining the nanoscale sensitivity to morphology. Based on the measured phase information, we implemented software tools for autofocusing during imaging, as well as image archiving and data access. To illustrate the potential of our technology for large volume pathology screening, we established an "intrinsic marker" for colorectal disease that detects tissue with dysplasia or colorectal cancer and flags specific areas for further examination, potentially improving the efficiency of existing pathology workflows.

  16. Bioplasmonic calligraphy for multiplexed label-free biodetection.

    Science.gov (United States)

    Tian, Limei; Tadepalli, Sirimuvva; Park, Sang Hyun; Liu, Keng-Ku; Morrissey, Jeremiah J; Kharasch, Evan D; Naik, Rajesh R; Singamaneni, Srikanth

    2014-09-15

    Printable multi-marker biochips that enable simultaneous quantitative detection of multiple target biomarkers in point-of-care and resource-limited settings are a holy grail in the field of biodiagnostics. However, preserving the functionality of biomolecules, which are routinely employed as recognition elements, during conventional printing approaches remains challenging. In this article, we introduce a simple yet powerful approach, namely plasmonic calligraphy, for realizing multiplexed label-free bioassays. Plasmonic calligraphy involves a regular ballpoint pen filled with biofunctionalized gold nanorods as plasmonic ink for creating isolated test domains on paper substrates. Biofriendly plasmonic calligraphy approach serves as a facile method to miniaturize the test domain size to few mm(2), which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. Furthermore, plasmonic calligraphy also serves as a simple and efficient means to isolate multiple test domains on a single test strip, which facilitates multiplexed biodetection and multi-marker biochips. Plasmonic calligraphy, which can be potentially automated by implementing with a robotic arm, serves as an alternate path forward to overcome the limitations of conventional ink-jet printing. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Advance ultra sensitive multi-layered nano plasmonic devices for label free biosensing targeting immunodiagnostics

    Science.gov (United States)

    Sharma, Divya; Dwivedi, R. P.

    2016-09-01

    The rapid advancement in technology has envisaged and drafted the use of optical bio-sensing units into label free and multiplexed bio-sensing, exploring the surface plasmon polaritons, which has turned into a gold standard on the commercial basis, but they are bulky and find difficulty in scaling up for the throughput detection. The integration of plasmonic crystals with microfluidics on the bio-sensing frontier offers a multi-level validation of results with the ease of real-time detection and imaging and holds a great promise to develop ultra-sensitive, fast, portable device for the point-of-care diagnostics. The paper describes the fast, low cost approach of designing and simulating label free biosensor using open source MEEP and other software tools targeting Immunodiagnostics.

  18. Microfluidics-integrated cascaded double-microring resonators for label-free biosensing

    Science.gov (United States)

    Chen, Yangqing; Yu, Fang; Yang, Chang; Li, Mingyu; Tang, Longhua; Song, Jinyan; He, Jian-Jun

    2014-11-01

    A highly-sensitive optical waveguide biosensor integrated with microfluidic channels based on silicon-on-insulator (SOI) was investigated in this paper. Experimental results of the label-free detection exhibits this novel biosensor with the superior reliability for quantitative and kinetic measurement of the interaction between biological molecules, dramatically improving the sensitivity due to the Vernier effect induced by cascaded double-microring resonators.

  19. Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia

    Science.gov (United States)

    2016-10-01

    regions in injured eyes 4) Measure TRPM7 and cellular/apoptosis biomarkers in retinas 5) Measure neuronal death and cell-specific biomarker in retinas...modal label-free imaging system and calibrating our system to measure blood oxygen levels in the eye . The reported problems with our coherent anti...now ready to test human hemoglobin with our CARS system. 3) Detect and map hypoxic regions in injured eyes In Quarter 1-2: We optimized our

  20. Imaging label-free biosensor with microfluidic system

    Science.gov (United States)

    Jahns, S.; Glorius, P.; Hansen, M.; Nazirizadeh, Y.; Gerken, M.

    2015-06-01

    We present a microfluidic system suitable for parallel label-free detection of several biomarkers utilizing a compact imaging measurement system. The microfluidic system contains a filter unit to separate the plasma from human blood and a functionalized, photonic crystal slab sensor chip. The nanostructure of the photonic crystal slab sensor chip is fabricated by nanoimprint lithography of a period grating surface into a photoresist and subsequent deposition of a TiO2 layer. Photonic crystal slabs are slab waveguides supporting quasi-guided modes coupling to far-field radiation, which are sensitive to refractive index changes due to biomarker binding on the functionalized surface. In our imaging read-out system the resulting resonance shift of the quasi-guided mode in the transmission spectrum is converted into an intensity change detectable with a simple camera. By continuously taking photographs of the sensor surface local intensity changes are observed revealing the binding kinetics of the biomarker to its specific target. Data from two distinct measurement fields are used for evaluation. For testing the sensor chip, 1 μM biotin as well as 1 μM recombinant human CD40 ligand were immobilized in spotsvia amin coupling to the sensor surface. Each binding experiment was performed with 250 nM streptavidin and 90 nM CD40 ligand antibody dissolved in phosphate buffered saline. In the next test series, a functionalized sensor chip was bonded onto a 15 mm x 15 mm opening of the 75 mm x 25 mm x 2 mm microfluidic system. We demonstrate the functionality of the microfluidic system for filtering human blood such that only blood plasma was transported to the sensor chip. The results of first binding experiments in buffer with this test chip will be presented.

  1. Bifunctional polydopamine thin film coated zinc oxide nanorods for label-free photoelectrochemical immunoassay.

    Science.gov (United States)

    Yang, Yan; Hu, Weihua

    2017-05-01

    Photoelectrochemical (PEC) detection is a promising method for label-free immunoassay by reporting the specific biological recognition events with electrical signals. However, it is challenging to rationally incorporate immunosensing components with a photocurrent conversion interface, which generally necessitates multistep fabrication and careful tailoring of various components such as photoactive material and biological probe. For high detection reliability and reproducibility, it is highly desirable to rationally construct an efficient PEC interface with architecture as simple as possible. In this work, a novel yet simple PEC immunosensor based on bio-inspired polydopamine (PDA) thin film-coated zinc oxide (ZnO) nanorods was reported. In this PEC immunosensor, the PDA thin film serves simultaneously as a unique sensitizer for charge separation as well as a functional layer for probe antibody attachment. The photocurrent on this electrode under illumination decreases upon the immunoreaction on the surface, possibly due to the blocking effect of formed immunocomplexes on the access of reducing reagent to the photoelectrode, thus offering a simple and reliable platform for PEC label-free immunoassay. By using an antibody-antigen pair as a model, successful label-free immunoassay was achieved with a detection limit of 10pgmL(-1) and a dynamic range from 100pgmL(-1) to 500ngmL(-1). This work demonstrates intriguing electro-optical property and bioconjugation activity of PDA film and may pave the way toward advanced PEC immunoassays.

  2. Microfluidic-optical integrated CMOS compatible devices for label-free biochemical sensing

    Science.gov (United States)

    Blanco, F. J.; Agirregabiria, M.; Berganzo, J.; Mayora, K.; Elizalde, J.; Calle, A.; Dominguez, C.; Lechuga, L. M.

    2006-05-01

    The fabrication, characterization and packaging of novel microfluidic-optical integrated biosensors for label-free biochemical detection is presented in this paper. The integrated device consists of a three-dimensional embedded microchannel network fabricated using enhanced CMOS compatible SU-8 multilevel polymer technology on top of a wafer containing Mach-Zehnder Interferometer (MZI) nanophotonic biosensor devices. PMMA housing provides connection to the macro-world and ensures robust leakage-free flow operation of the devices. This macro-microfluidic module can operate at pressure drops up to 1000 kPa. Fluid flow experiments have been performed in order to demonstrate the robustness of our microfluidic devices. The devices have been designed to operate under continuous flow. Steady-state flow rates ranging from 1 to 100 µl min-1 at pressure drops ranging from 10 to 500 kPa were measured in the laminar flow regime. Experimental results are in good agreement with laminar flow theory. The first interferometric sensing measurements are presented in order to demonstrate the functionality of these novel integrated devices for lab-on-a-chip and label-free biosensing applications. A bulk refractive index detection limit of 3.8 × 10-6 was obtained, close to the minimum detected up to now by label-free biosensor devices without microfluidic integration. As far as we know, this is the first time that a label-free biosensor device is integrated within a microfluidic network using a wafer-level CMOS compatible process technology.

  3. Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

    Science.gov (United States)

    Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

    2014-09-01

    A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

  4. Label-free characterization of biomembranes: from structure to dynamics

    NARCIS (Netherlands)

    Mashaghi, A.; Mashaghi, S.; Reviakine, I.; Heeren, R.M.A.; Sandoghdarf, V.; Bonn, M.

    2013-01-01

    We review recent progress in the study of the structure and dynamics of phospholipid membranes and associated proteins, using novel label-free analytical tools. We describe these techniques and illustrate them with examples highlighting current capabilities and limitations. Recent advances in applyi

  5. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    Science.gov (United States)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  6. Label-free profiling of skeletal muscle using high-definition mass spectrometry

    Science.gov (United States)

    Burniston, Jatin G.; Connolly, Joanne; Kainulainen, Heikki; Britton, Steven L.; Koch, Lauren G.

    2014-01-01

    We report automated and time efficient (2 h per sample) profiling of muscle using ultra-performance liquid chromatography (LC) coupled directly with high-definition mass spectrometry (HDMSE). Soluble proteins extracted from rat gastrocnemius (n=10) were digested with trypsin and analysed in duplicate using a 90 min RPLC gradient. Protein identification and label-free quantitation were performed from HDMSE spectra analysed using TransOmics Informatics for Proteomics software. In total 1,514 proteins were identified. Of these, 811 had at least 3 unique peptides and were subsequently used to assess the dynamic range and precision of LC-HDMSE label-free profiling. Proteins analysed by LC-HDMSE encompass the entire complement of glycolytic, beta-oxidation and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned 4 orders of magnitude. The correlation between technical replicates of the 10 biological samples was R2 = 0.9961 ± 0.0036 (95 % CI = 0.9940 – 0.9992) and the technical coefficient of variation averaged 7.3 ± 6.7 % (95 % CI = 6.87 – 7.79 %). This represents the most sophisticated label-free profiling of skeletal muscle to date. PMID:25065561

  7. Label-free measurements on cell apoptosis using a terahertz metamaterial-based biosensor

    Science.gov (United States)

    Zhang, Caihong; Liang, Lanju; Ding, Liang; Jin, Biaobing; Hou, Yayi; Li, Chun; Jiang, Ling; Liu, Weiwei; Hu, Wei; Lu, Yanqing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-06-01

    Label-free, real-time, and in-situ measurement on cell apoptosis is highly desirable in cell biology. We propose here a design of terahertz (THz) metamaterial-based biosensor for meeting this requirement. This metamaterial consists of a planar array of five concentric subwavelength gold ring resonators on a 10 μm-thick polyimide substrate, which can sense the change of dielectric environment above the metamaterial. We employ this sensor to an oral cancer cell (SCC4) with and without cisplatin, a chemotherapy drug for cancer treatment, and find a linear relation between cell apoptosis measured by Flow Cytometry and the relative change of resonant frequencies of the metamaterial measured by THz time-domain spectroscopy. This implies that we can determine the cell apoptosis in a label-free manner. We believe that this metamaterial-based biosensor can be developed into a cheap, label-free, real-time, and in-situ detection tool, which is of significant impact on the study of cell biology.

  8. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  9. Label-free Protein Detection Based on the Heat-Transfer Method--A Case Study with the Peanut Allergen Ara h 1 and Aptamer-Based Synthetic Receptors.

    Science.gov (United States)

    Peeters, Marloes; van Grinsven, Bart; Cleij, Thomas J; Jiménez-Monroy, Kathia Lorena; Cornelis, Peter; Pérez-Ruiz, Elena; Wackers, Gideon; Thoelen, Ronald; De Ceuninck, Ward; Lammertyn, Jeroen; Wagner, Patrick

    2015-05-20

    Aptamers are an emerging class of molecules that, because of the development of the systematic evolution of ligands by exponential enrichment (SELEX) process, can recognize virtually every target ranging from ions, to proteins, and even whole cells. Although there are many techniques capable of detecting template molecules with aptamer-based systems with high specificity and selectivity, they lack the possibility of integrating them into a compact and portable biosensor setup. Therefore, we will present the heat-transfer method (HTM) as an interesting alternative because this offers detection in a fast and low-cost manner and has the possibility of performing experiments with a fully integrated device. This concept has been demonstrated for a variety of applications including DNA mutation analysis and screening of cancer cells. To the best our knowledge, this is the first report on HTM-based detection of proteins, in this case specifically with aptamer-type receptors. For proof-of-principle purposes, measurements will be performed with the peanut allergen Ara h 1 and results indicate detection limits in the lower nanomolar regime in buffer liquid. As a first proof-of-application, spiked Ara h 1 solutions will be studied in a food matrix of dissolved peanut butter. Reference experiments with the quartz-crystal microbalance will allow for an estimate of the areal density of aptamer molecules on the sensor-chip surface.

  10. A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on label-free amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

    Science.gov (United States)

    A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogen...

  11. Au nanoparticles grafted on Fe{sub 3}O{sub 4} as effective SERS substrates for label-free detection of the 16 EPA priority polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jingjing; Xu, Jianwei; Sun, Zhenli; Jing, Chuanyong, E-mail: cyjing@rcees.ac.cn

    2016-04-07

    Several methods and materials have been explored for the sensitive and practicable detection of polycyclic aromatic hydrocarbons (PAHs). However, it is still a challenge to develop simple and cost-effective sensing techniques for PAHs. Herein we report the synthesis and construction of Fe{sub 3}O{sub 4}@Au SERS substrate. This magnetic substrate was composed by Fe{sub 3}O{sub 4} microspheres and Au NPs. The size, morphology, and surface composition of Fe{sub 3}O{sub 4}@Au were characterized by multiple complimentary techniques including scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction. The spatial distributions of electro-magnetic field enhancement around Fe{sub 3}O{sub 4}@Au was calculated using finite difference time domain (FDTD) simulations. As a result of its remarkable sensitivity, the Fe{sub 3}O{sub 4}@Au-based SERS assay has been applied to detect the 16 EPA priority PAHs. The LODs achieved by our method (100–5 nM, 16.6–1.01 μg L{sup −1}) make it promising for the rapid screening of highly contaminated cases. As a proof-of-concept study, the substrate was applied in SERS sensing of PAHs in river matrix. The 16 PAHs could be differentiated based upon their characteristic SERS peaks. Most importantly, the detection was successfully conducted using a portable Raman spectrometer, which could be used for on-site monitoring of PAHs. - Highlights: • SERS detection of the 16 EPA priority PAHs was conducted. • The satellite-core structure lead to high SERS enhancement by confined hotspots. • The approach does not require expensive instrumentation or large sample volumes. • The effective protocol is useful for the identification of hydrophobic molecules.

  12. Antibody mimetic receptor proteins for label-free biosensors.

    Science.gov (United States)

    Raina, M; Sharma, R; Deacon, S E; Tiede, C; Tomlinson, D; Davies, A G; McPherson, M J; Wälti, C

    2015-02-07

    The development of high sensitivity biosensors, for example for clinical diagnostics, requires the identification of suitable receptor molecules which offer high stability, specificity and affinity, even when embedded into solid-state biosensor transducers. Here, we present an electrochemical biosensor employing small synthetic receptor proteins (Mw pM and 6.7 nM. These findings demonstrate that these non-antibody receptor proteins are excellent candidates for recognition molecules in label-free biosensors.

  13. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis.

  14. Deep Learning in Label-free Cell Classification

    Science.gov (United States)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  15. Deep Learning in Label-free Cell Classification.

    Science.gov (United States)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-15

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  16. Issues and Applications in Label-Free Quantitative Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Xianyin Lai

    2013-01-01

    Full Text Available To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics.

  17. Comparison of serum fractionation methods by data independent label-free proteomics

    Directory of Open Access Journals (Sweden)

    D. Baiwir

    2015-12-01

    Full Text Available Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values.

  18. Label-free discrimination of normal and fibroadenomal breast tissues using second harmonic generation imaging.

    Science.gov (United States)

    Zheng, Liqin; Zhuo, Shuangmu; Chen, Gang; Zhu, Xiaoqin; Jiang, Xingshan; Yan, Jun; Chen, Jianxin; Xie, Shusen

    2011-01-01

    Early detection of fibroadenoma (FA) is critical for preventing subsequent breast cancer. In this work, we show that label-free second harmonic generation (SHG) imaging is feasible and effective in quantitatively differentiating the fibroadenomal tissue from normal breast tissue. With the advent of the clinical portability of miniature SHG microscopy, we believe that the technique has great potential in offering a noninvasive in vivo imaging tool for early detection of FA and monitoring the treatment responses of FA in clinics. Copyright © 2011 Wiley Periodicals, Inc.

  19. Immobilization and hybridization of DNA based on magnesium ion modified 2,6-pyridinedicarboxylic acid polymer and its application for label-free PAT gene fragment detection by electrochemical impedance spectroscopy

    Institute of Scientific and Technical Information of China (English)

    JIAO Kui; YANG Tao; YANG Jie; FENG YuanYuan

    2007-01-01

    A new approach for a simple electrochemical detection of PAT gene fragment is described. Poly(2,6-pyridinedicarboxylic acid) (PDC) modified glassy carbon electrode (GCE) was prepared by potential scan electropolymerization in an aqueous solution. Mg2+ ions were incorporated by immersion of the modified electrode in 0.5 mol/L aqueous solution of MgCl2 to complete the preparation of a generic "activated" electrode ready for binding the probe DNA. The ssDNA was linked to the conducting polymer by forming a bidentate complex between the carboxyl groups on the polymer and the phosphate groups of DNA via Mg2+. DNA immobilization and hybridization were characterized with differential pulse voltammetry (DPV) by using methylene blue (MB) as indicator and electrochemical impedance spectroscopy (EIS). The EIS was of higher sensitivity for DNA detection as compared with voltammetric methods in our strategy. The electron transfer resistance (Ret) of the electrode surface in EIS in [Fe(CN)6]3-/4- solution increased after the immobilization of the DNA probe on the Mg/PDC/GCE electrode. The hybridization of the DNA probe with complementary DNA (cDNA) made Ret increase further. The difference between the Ret at ssDNA/Mg/PDC/GCE and that at hybridization DNA modified electrode (dsDNA/Mg/PDC/GCE) was applied to determine the specific sequence related to the target PAT gene with the dynamic range comprised between 1.0×10-9 and 1.0×10-5 mol/L. A detection limit of 3.4×10-10 mol/L of oligonucleotides can be estimated.

  20. Immobilization and hybridization of DNA based on magnesium ion modified 2,6-pyridinedicarboxylic acid polymer and its application for label-free PAT gene fragment detection by electrochemical impedance spectroscopy

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A new approach for a simple electrochemical detection of PAT gene fragment is described. Poly(2,6-pyridinedicarboxylic acid) (PDC) modified glassy carbon electrode (GCE) was prepared by potential scan electropolymerization in an aqueous solution. Mg2+ ions were incorporated by immer-sion of the modified electrode in 0.5 mol/L aqueous solution of MgCl2 to complete the preparation of a generic "activated" electrode ready for binding the probe DNA. The ssDNA was linked to the conduct-ing polymer by forming a bidentate complex between the carboxyl groups on the polymer and the phosphate groups of DNA via Mg2+. DNA immobilization and hybridization were characterized with dif-ferential pulse voltammetry (DPV) by using methylene blue (MB) as indicator and electrochemical im-pedance spectroscopy (EIS). The EIS was of higher sensitivity for DNA detection as compared with voltammetric methods in our strategy. The electron transfer resistance (Ret) of the electrode surface in EIS in [Fe(CN)6]3-/4- solution increased after the immobilization of the DNA probe on the Mg/PDC/GCE electrode. The hybridization of the DNA probe with complementary DNA (cDNA) made Ret increase further. The difference between the Ret at ssDNA/Mg/PDC/GCE and that at hybridization DNA modified electrode (dsDNA/Mg/PDC/GCE) was applied to determine the specific sequence related to the target PAT gene with the dynamic range comprised between 1.0 × 10-9 and 1.0 × 105 mol/L. A detection limit of 3.4 × 10-10 mol/L of oligonucleotides can be estimated.

  1. Ionic liquids as precursors for highly luminescent, surface-different nitrogen-doped carbon dots used for label-free detection of Cu2+/Fe3+ and cell imaging.

    Science.gov (United States)

    Zhao, Andong; Zhao, Chuanqi; Li, Meng; Ren, Jinsong; Qu, Xiaogang

    2014-01-27

    Carbon nanodots (C-Dots) have attracted much attention in recent years due to their low cost, ready scalability, excellent chemical stability, biocompatibility and multicolor luminescence. Here, we report a facile strategy for producing highly luminescent, surface-different nitrogen-doped carbon dots (C-Dots) by using different ionic liquids (ILs). Intriguingly, the surface-different C-Dots show different selectivity for Cu(2+) and Fe(3+). To the best of our knowledge, this is the first example which shows that ILs are excellent precursors for producing luminescent nanomaterial used for detection of different metal ions. The resultant nitrogen-doped C-Dots are highly photoluminescent and can be used for multicolor bioimaging. Most notable, by taking different ILs as precursors, we obtain surface-different C-Dots, which can be directly used for selective detection of Cu(2+) and Fe(3+) without any modification. These C-Dots based sensors exhibit high sensitivity and selectivity and the sensing process can be easily accomplished with one-step rapid operation. More importantly, compared with other method using QDs, organic dyes and organic solvent, this strategy is much more eco-friendly. This work may offer a new approach for developing low cost and sensitive C-Dots-based sensors for biological and environmental applications.

  2. The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    Science.gov (United States)

    Campbell, Kate; Deery, Michael J.; Lilley, Kathryn S.; Ralser, Markus

    2014-01-01

    The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition on a TripleTOF 5600 instrument. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides. PMID:24741437

  3. Label-Free Optical Biochemical Sensors via Liquid-Cladding-Induced Modulation of Waveguide Modes.

    Science.gov (United States)

    Tran, Nhu Hoa Thi; Kim, Jisoo; Phan, Thang Bach; Khym, Sungwon; Ju, Heongkyu

    2017-09-07

    We demonstrated modulation of the waveguide mode mismatch via liquid cladding of the controllable refractive index for label-free quantitative detection of concentration of chemical or biological substances. A multimode optical fiber with its core exposed was used as the sensor head with the suitable chemical modification of its surface. Injected analyte liquid itself formed the liquid cladding for the waveguide. We found that modulation of the concentration of analyte injected enables a degree of the waveguide mode mismatch to be controlled, resulting in sensitive change in optical power transmission, which was utilized for its real-time quantitative assay. We applied the device to quantitating concentration of glycerol and bovine serum albumin (BSA) solutions. We obtained experimentally the limit of detection (LOD) of glycerol concentration, 0.001% (volume ratio), corresponding to the resolvable index resolution of ∼1.02 × 10(-6) RIU (refractive index unit). The presented sensors also exhibited reasonably good reproducibility. In BSA detection, the sensor device response was sensitive to change in the refractive indices not only of liquid bulk but also of layers just above the sensing surface with higher sensitivity, providing the LOD experimentally as ∼3.7 ng/mL (mass coverage of ∼30 pg/mm(2)). A theoretical model was also presented to invoke both mode mismatch modulation and evanescent field absorption for understanding of the transmission change, offering a theoretical background for designing the sensor head structure for a given analyte. Interestingly, the device sensing length played little role in the important sensor characteristics such as sensitivity, unlike most of the waveguide-based sensors. This unraveled the possibility of realizing a highly simple structured label-free sensor for point-of-care testing in a real-time manner via an optical waveguide with liquid cladding. This required neither metal nor dielectric coating but still produced

  4. Optimal design of label-free silicon “lab on a chip” biosensors

    Institute of Scientific and Technical Information of China (English)

    Yaping Zhang

    2013-01-01

    This paper reported the optimal design of label-free silicon on insulator (SOI)“lab on a chip”biosensors. These devices are designed on the basis of the evanescent field detection principles and interferometer technologies. The well-established silicon device process technology can be applied to fabricate and test these biosensor devices. In addition, these devices can be monolithically integrated with CMOS electronics and microfluidics. For these biosensor devices, multi-mode interferometer (MMI) was employed to combine many stand-alone biosensors to form chip-level biosensor arrays, which enable real-time and label-free monitoring and parallel detection of various analytes in multiple test samples. This sensing and detection technology features the highest detection sensitivity, which can detect analytes at extremely low concentrations instantaneously. This research can lead to innovative commercial development of the new generation of high sensitivity biosensors for a wide range of applications in many fields, such as environmental monitoring, food security control, medical and biological applications.

  5. Label-free monitoring of interaction between DNA and oxaliplatin in aqueous solution by terahertz spectroscopy

    Science.gov (United States)

    Wu, Xiaojun; E, Yiwen; Xu, Xinlong; Wang, Li

    2012-07-01

    We demonstrated the feasibility of applying terahertz time-domain spectroscopy (THz-TDS) to monitor the molecular reactions in aqueous solutions of anticancer drug oxaliplatin with λ-DNA and macrophages DNA. The reaction time dependent refractive index and absorption coefficient were extracted and analyzed. The reaction half-decaying time of about 4.0 h for λ-DNA and 12.9 h for M-DNA was established. The results suggest that the THz-TDS detection could be an effective label-free technique to sense the molecular reaction in aqueous solutions and could be very useful in biology, medicine, and pharmacy industry.

  6. Fiber optic label-free biophotonic diagnostic tool for cardiovascular disease

    Science.gov (United States)

    Rius, Cristina; Ackermann, Tobias N.; Dorado, Beatriz; Muñoz-Berbel, Xavier; Andrés, Vicente; Llobera, Andreu

    2015-06-01

    A label-free compact method for performing photonic characterization of "healthy" versus "diseased" arteries has been developed. It permits the detection of atherosclerotic lesion in living mouse arteries. Using this prototype, we observed that the spectral response (photonic fingerprint, PIN) obtained from aortas of wild-type mice differs from the response of ApoE-KO mice fed with high-fat diet (an atheroprone mouse model). Benchmark of the results against gold standard was performed by staining the aortas with Oil-Red-O to visualize atherosclerotic plaques.

  7. A Label-Free Biosensing Platform Using a PLL Circuit and Biotin-Streptavidin Binding System.

    Science.gov (United States)

    Yunseog Hong; Hee-Jo Lee; Sang-Gyu Kim; Byung-Hyun Kim; Gi-Ho Yun; Jong-Gwan Yook

    2015-06-01

    This paper proposes a novel RF biosensor that utilizes a frequency synthesizer associated with a microstrip open-loop resonator for label-free biomolecular detection. The RF biosensor consists mainly of a resonance-assisted transducer and a phase locked loop (PLL) circuit. In this work, the performance of the RF biosensor is validated using the well-known biotin-streptavidin binding system. When biotin is bound to streptavidin, the input impedance of the resonator is varied, resulting in a change in the oscillation frequency. The concentration of the streptavidin is ultimately detected by a voltage signal of the PLL's loop filter with simple measurement equipment. According to the experimental results, the RF biosensor has revealed excellent sensitivity ( ~ 61 kHz/ngml(-1)) and a low detection limit ( ~ 1 ng/ml), as well as a rapid response. These results demonstrate that the RF biosensor can be an effective sensing platform for label-free detection in a biomolecular binding system.

  8. Molybdenum carbide nanotubes: a novel multifunctional material for label-free electrochemical immunosensing.

    Science.gov (United States)

    Zhai, Qingfeng; Zhang, Xiaowei; Li, Jing; Wang, Erkang

    2016-08-18

    Herein, a multifunctional nanoarchitecture has been developed by integrating well-crystalline molybdenum carbide (Mo2C) nanotubes and an electrochemical indicator - thionin (TH). The Mo2C nanotubes were synthesized through the self-degradable template method and high-temperature calcination, and their structure and morphology were characterized through scanning electron microscopy (SEM), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Due to the high electrocatalytic properties, excellent conductivity and highly active surface area of Mo2C nanotubes, the Mo2C-based material was used as a nanocarrier to load TH molecules for the development of a label-free electrochemical immunosensor for α-fetoprotein (AFP) detection. The decorated TH probe on the Mo2C nanotubes not only acted as a bridging molecule to effectively capture and immobilize primary anti-AFP on the Mo2C nanotubes, but also acted as a signal indicator for the detection of AFP. The proposed immunosensor exhibited excellent selectivity (with a detection limit of 3 pg mL(-1)), high stability and good reproducibility by combining the unique structure and features of the Mo2C nanotubes. Furthermore, this sensing platform was finally used for the detection of AFP in human serum with satisfactory results. Therefore, the Mo2C nanotubes can be considered as a candidate carbon material for fabrication of simple, label-free and ultrasensitive electrochemical sensors, broadening the application of this material.

  9. Photonic Biosensor Chips for Label-Free Detection

    DEFF Research Database (Denmark)

    Kristensen, Martin

    Optical fibers are ideal for transmission of light due to their low loss. This is less important for optical sensors where chemical compatibility, size and price are more important. These parameters can be optimized by using planar integrated optics and fabrication methods from the semiconductor ...

  10. Photonic Biosensor Chips for Label-Free Detection

    DEFF Research Database (Denmark)

    Kristensen, Martin

    Optical fibers are ideal for transmission of light due to their low loss. This is less important for optical sensors where chemical compatibility, size and price are more important. These parameters can be optimized by using planar integrated optics and fabrication methods from the semiconductor ...... industry with adaptations to satisfy the requirements of biosensors....

  11. Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

    Directory of Open Access Journals (Sweden)

    Kamila Gawel

    2010-04-01

    Full Text Available Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications.

  12. Label-free classification of cultured cells through diffraction imaging.

    Science.gov (United States)

    Dong, Ke; Feng, Yuanming; Jacobs, Kenneth M; Lu, Jun Q; Brock, R Scott; Yang, Li V; Bertrand, Fred E; Farwell, Mary A; Hu, Xin-Hua

    2011-06-01

    Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

  13. Functionalized nanopipettes: toward label-free, single cell biosensors.

    Science.gov (United States)

    Actis, Paolo; Mak, Andy C; Pourmand, Nader

    2010-08-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.

  14. Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

    Science.gov (United States)

    Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

    2016-01-30

    Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in

  15. Label-free fluorescent aptasensor for potassium ion using structure-switching aptamers and berberine

    Science.gov (United States)

    Guo, Yanqing; Chen, Yanxia; Wei, Yanli; Li, Huanhuan; Dong, Chuan

    2015-02-01

    A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N = 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust, and stable in rough conditions.

  16. Silicon-based optoelectronic integrated circuit for label-free bio/chemical sensor.

    Science.gov (United States)

    Song, Junfeng; Luo, Xianshu; Kee, Jack Sheng; Han, Kyungsup; Li, Chao; Park, Mi Kyoung; Tu, Xiaoguang; Zhang, Huijuan; Fang, Qing; Jia, Lianxi; Yoon, Yong-Jin; Liow, Tsung-Yang; Yu, Mingbin; Lo, Guo-Qiang

    2013-07-29

    We demonstrate a silicon-based optoelectronic integrated circuit (OEIC) for label-free bio/chemical sensing application. Such on-chip OEIC sensor system consists of optical grating couplers for vertical light coupling into silicon waveguides, a thermal-tunable microring as a tunable filter, an exposed microring as an optical label-free sensor, and a Ge photodetector for a direct electrical readout. Different from the conventional wavelength-scanning method, we adopt low-cost broadband ASE light source, together with the on-chip tunable filter to generate sliced light source. The effective refractive index change of the sensing microring induced by the sensing target is traced by scanning the supplied electrical power applied onto the tracing microring, and the detected electrical signal is read out by the Ge photodetector. For bulk refractive index sensing, we demonstrate using such OEIC sensing system with a sensitivity of ~15 mW/RIU and a detection limit of 3.9 μ-RIU, while for surface sensing of biotin-streptavidin, we obtain a surface mass sensitivity of S(m) = ~192 µW/ng·mm(-2) and a surface detection limit of 0.3 pg/mm(2). The presented OEIC sensing system is suitable for point-of-care applications.

  17. Photonic Crystal Surfaces as a General Purpose Platform for Label-Free and Fluorescent Assays.

    Science.gov (United States)

    Cunningham, Brian T

    2010-04-01

    Photonic crystal surfaces can be designed to provide a wide range of functions that are used to perform biochemical and cell-based assays. Detection of the optical resonant reflections from photonic crystal surfaces enables high sensitivity label-free biosensing, while the enhanced electromagnetic fields that occur at resonant wavelengths can be used to enhance the detection sensitivity of any surface-based fluorescence assay. Fabrication of photonic crystals from inexpensive plastic materials over large surface areas enables them to be incorporated into standard formats that include microplates, microarrays, and microfluidic channels. This report reviews the design of photonic crystal biosensors, their associated detection instrumentation, and biological applications. Applications including small molecule high throughput screening, cell membrane integrin activation, gene expression analysis, and protein biomarker detection are highlighted. Recent results in which photonic crystal surfaces are used for enhancing the detection of Surface-Enhanced Raman Spectroscopy, and the development of high resolution photonic crystal-based laser biosensors are also described.

  18. Label-free selection of RNA aptamers for metabolic engineering.

    Science.gov (United States)

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment.

  19. Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma.

    Science.gov (United States)

    Kirk, James T; Brault, Norman D; Baehr-Jones, Tom; Hochberg, Michael; Jiang, Shaoyi; Ratner, Daniel M

    2013-04-15

    A widely acknowledged goal in personalized medicine is to radically reduce the costs of highly parallelized, small fluid volume, point-of-care and home-based diagnostics. Recently, there has been a surge of interest in using complementary metal-oxide-semiconductor (CMOS)-compatible silicon photonic circuits for biosensing, with the promise of producing chip-scale integrated devices containing thousands of orthogonal sensors, at minimal cost on a per-chip basis. A central challenge in biosensor translation is to engineer devices that are both sensitive and specific to a target analyte within unprocessed biological fluids. Despite advances in the sensitivity of silicon photonic biosensors, poor biological specificity at the sensor surface remains a significant factor limiting assay performance in complex media (i.e. whole blood, plasma, serum) due to the non-specific adsorption of proteins and other biomolecules. Here, we chemically modify the surface of silicon microring resonator biosensors for the label-free detection of an analyte in undiluted human plasma. This work highlights the first application of a non-fouling zwitterionic surface coating to enable silicon photonic-based label-free detection of a protein analyte at clinically relevant sensitivities in undiluted human plasma.

  20. Capillary Microfluidics-Assembled Virus-like Particle Bionanoreceptor Interfaces for Label-Free Biosensing.

    Science.gov (United States)

    Zang, Faheng; Gerasopoulos, Konstantinos; Brown, Adam D; Culver, James N; Ghodssi, Reza

    2017-03-15

    A capillary microfluidics-integrated sensor system is developed for rapid assembly of bionanoreceptor interfaces on-chip and label-free biosensing. Genetically engineered Tobacco mosaic virus (TMV) virus-like particles (VLPs), displaying thousands copies of identical receptor peptides FLAG-tags, are utilized as nanoceptors for antibody sensing. Controlled and accelerated assembly of VLP receptor layer on impedance sensor has been achieved using capillary action and surface evaporation from an open-channel capillary microfluidic system. VLPs create a dense and localized receptor monolayer on the impedance sensor using only 5 μL of VLP sample solution (0.2 mg/mL) in only 6 min at room temperature. The VLP-functionalized impedance sensor is capable of label-free detection of target antibodies down to 55 pM concentration within 5 min. These results highlight the significant potentials of an integrated microsystem for rapid and controlled receptor-transducer interface creation and the nanoscale VLP-based sensors for fast, accurate, and decentralized pathogen detection.

  1. Spiked proteomic standard dataset for testing label-free quantitative software and statistical methods.

    Science.gov (United States)

    Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Dorssaeler, Alain Van; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

    2016-03-01

    This data article describes a controlled, spiked proteomic dataset for which the "ground truth" of variant proteins is known. It is based on the LC-MS analysis of samples composed of a fixed background of yeast lysate and different spiked amounts of the UPS1 mixture of 48 recombinant proteins. It can be used to objectively evaluate bioinformatic pipelines for label-free quantitative analysis, and their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. More specifically, it can be useful for tuning software tools parameters, but also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. The raw MS files can be downloaded from ProteomeXchange with identifier PXD001819. Starting from some raw files of this dataset, we also provide here some processed data obtained through various bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in different workflows, to exemplify the use of such data in the context of software benchmarking, as discussed in details in the accompanying manuscript [1]. The experimental design used here for data processing takes advantage of the different spike levels introduced in the samples composing the dataset, and processed data are merged in a single file to facilitate the evaluation and illustration of software tools results for the detection of variant proteins with different absolute expression levels and fold change values.

  2. Design Low Crosstalk Ring-Slot Array Structure for Label-Free Multiplexed Sensing

    Directory of Open Access Journals (Sweden)

    Lijun Huang

    2014-08-01

    Full Text Available We theoretically demonstrate a low crosstalk ring-slot array structure used for label-free multiplexed sensing. The proposed sensors array is based on an array of three ring-slot and input/output line defect coupling waveguides. Each ring-slot cavity has slightly different cavity spacing and different resonant frequency. Results obtained using two dimensional finite-difference time-domain (2D-FDTD simulation indicate that the resonant frequencies of each sensor unit in response to the refractive index variations are independent. The refractive index sensitivity is 134 ~ 145.5 nm/RIU (refractive index unit and the Q factors more than 104 can be achieved. The calculated detect limit lower than 1.13 × 10−4 RIU is obtained. In addition, an extremely small crosstalk lower than −25.8 dB is achieved among the array of three ring-slot cavities. The results demonstrate that this multiplexed sensor array is a promising platform for integrated optical devices and enables highly parallel label-free detection.

  3. Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing

    Directory of Open Access Journals (Sweden)

    Pengfei Wang

    2015-07-01

    Full Text Available Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres.

  4. Label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Villar-Vazquez, Roi; Casal, J Ignacio; Lechuga, Laura M

    2016-08-03

    Colorectal cancer is treatable and curable when detected at early stages. However there is a lack of less invasive and more specific screening and diagnosis methods which would facilitate its prompt identification. Blood circulating autoantibodies which are immediately produced by the immune system at tumor appearance have become valuable biomarkers for preclinical diagnosis of cancer. In this work, we present the rapid and label-free detection of colorectal cancer autoantibodies directly in blood serum or plasma using a recently developed nanoplasmonic biosensor. Our nanoplasmonic device offers sensitive and real-time quantification of autoantibodies with excellent selectivity and reproducibility, achieving limits of detection around 1 nM (150-160 ng mL(-1)). A preliminary evaluation of clinical samples of colorectal cancer patients has shown good correlation with ELISA. These results demonstrate the reliability of the nanobiosensor strategy and pave the way towards the achievement of a sensitive diagnostic tool for early detection of colorectal cancer.

  5. Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

    Directory of Open Access Journals (Sweden)

    Juho Pirhonen

    Full Text Available Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD. We tested whether second-harmonic generation (SHG and coherent anti-Stokes Raman scattering (CARS microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD.We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals.We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III.This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

  6. Spectroscopic Methods for Label-Free Optical Nanoscopy

    Science.gov (United States)

    Chandler, John E.

    It is becoming increasingly evident that the nanoscale organization and structure of macromolecules play a significant role in determining the function and properties of biological systems. To understand the relationships between biological structure and function at nanometer length scales, there is a need for methods which enable imaging of intact nanoscale biological structure. An ideal technique for these applications is sensitive to nanoscale structure below the resolution limit of conventional optical microscopy ( 200 nm), achieves label-free contrast, is non-perturbing to biological samples, is quantitative, is capable of molecular specificity, is high-throughput, and finally is simple, enabling widespread utilization. Existing techniques meet some of these criteria, but all have limitations. For example, super-resolution optical microscopy methods achieve molecular-specific nanoscale resolution well below the resolution limit of conventional optical microscopes, however, they rely on fluorescent labels often at high densities that can be toxic and can often require potentially damaging illumination intensities for imaging. As a result, there remains a need for label-free optical techniques to study the nanoscale structural properties of cells. To address this need, the development of instrumentation and algorithms for Partial Wave Spectroscopic (PWS) microscopy will be described. PWS is a spectroscopic, label-free, nanoscale sensitive microscope which, senses rather than resolves structure below the resolution limit of conventional microscopes ( 200nm). First, PWS has shown utility as a diagnostic screening tool for cancer due to nanoscale structural alterations that occur in cells as part of the earliest stages of carcinogenesis. Instrumentation and algorithms developed to enable high-throughput cancer screening applications will be described. Further enhancement of data acquisition and analysis speed will then be described through the development of new

  7. Integrated label-free silicon nanowire sensor arrays for (bio)chemical analysis.

    Science.gov (United States)

    De, Arpita; van Nieuwkasteele, Jan; Carlen, Edwin T; van den Berg, Albert

    2013-06-07

    We present a label-free (bio)chemical analysis platform that uses all-electrical silicon nanowire sensor arrays integrated with a small volume microfluidic flow-cell for real-time (bio)chemical analysis and detection. The integrated sensing platform contains an automated multi-sample injection system that eliminates erroneous sensor responses from sample switching due to flow rate fluctuations and provides precise sample volumes down to 10 nl. Biochemical sensing is demonstrated with real-time 15-mer DNA-PNA (peptide nucleic acid) duplex hybridization measurements from different sample concentrations in a low ionic strength, and the equilibrium dissociation constant KD ≈ 140 nM has been extracted from the experimental data using the first order Langmuir binding model. Chemical sensing is demonstrated with pH measurements from different injected samples in flow that have sensitivities consistent with the gate-oxide materials. A differential sensor measurement configuration results in a 30× reduction in sensor drift. The integrated label-free analysis platform is suitable for a wide range of small volume chemical and biochemical analyses.

  8. Molecularly imprinted polymer diffraction grating as label-free optical bio(mimetic)sensor.

    Science.gov (United States)

    Barrios, C A; Zhenhe, C; Navarro-Villoslada, F; López-Romero, D; Moreno-Bondi, M C

    2011-01-15

    Micropatterned molecularly imprinted polymer (MIP) transmissive 2D diffraction gratings (DGs) are fabricated and evaluated as label-free antibiotic bio(mimetic)sensors. Polymeric gratings are prepared by using microtransfer molding based on SiO(2)/Si molds. The morphology of the MIP gratings is studied by optical and atomic force microscopes. MIP 2D-DGs exhibit 2D optical diffraction patterns, and measurement of changes in diffraction efficiency is used as sensor response. The refractive index of the micropatterned MIP material was estimated, via solvent index matching experiments, to be 1.486. Immersion of a MIP 2D-DG in different solutions of target-antibiotic enrofloxacin leads to significant variations in diffraction efficiency, demonstrating target-molecule detection. On the other hand, no significant response is observed for both control experiments: MIP grating exposed to a non-retained analyte and an equivalent non-imprinted polymer grating exposed to the target analyte, showing highly specific antibiotic label-free optical recognition.

  9. Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex.

    Science.gov (United States)

    Wang, Sai; Liu, Jiahui; Wu, Yanxia; You, Yawen; He, Jingyi; Zhang, Jichuan; Zhang, Liqun; Dong, Yiyang

    2016-04-15

    Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy-immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.

  10. Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

    Science.gov (United States)

    Wu, Enqi; Peng, Yuan; Zhang, Xihao; Bai, Jialei; Song, Yanqiu; He, Houluo; Fan, Longxing; Qu, Xiaochen; Gao, Zhixian; Liu, Ying; Ning, Baoan

    2017-02-22

    A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10(-3) ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.

  11. Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy.

    Directory of Open Access Journals (Sweden)

    Steffen Dietzel

    Full Text Available Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200-1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label.

  12. Automated selected reaction monitoring software for accurate label-free protein quantification.

    Science.gov (United States)

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  13. Label-free aptasensor for platelet-derived growth factor (PDGF) protein

    Energy Technology Data Exchange (ETDEWEB)

    Degefa, Tesfaye Hailu [Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of); Kwak, Juhyoun [Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of)], E-mail: Juhyoun_Kwak@kaist.ac.kr

    2008-04-21

    A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN){sub 6}]{sup 3-/4-}, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R{sub ct} was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.

  14. Label-free nonlinear optical imaging of mouse retina.

    Science.gov (United States)

    He, Sicong; Ye, Cong; Sun, Qiqi; Leung, Christopher K S; Qu, Jianan Y

    2015-03-01

    A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE.

  15. Studies towards the development of label-free AC impedimetric immunosensors for healthcare and food quality control

    OpenAIRE

    Tsekenis, Georgios

    2008-01-01

    This thesis describes work focused towards the fabrication and characterisation of immunosensor platforms for the label-free detection of analytes of importance in the health and food industries. Due to their low unit cost and ease of fabrication, the immunosensor market has significantly increased recently, resulting in a constant demand for new immunosensor applications. Within this thesis, therefore, a novel fabrication protocol is reported towards the production of immunose...

  16. Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

    Directory of Open Access Journals (Sweden)

    Young Ah Goo

    2008-01-01

    Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

  17. Limitations of Label-Free Sensors in Serum Based Molecular Diagnostics

    CERN Document Server

    Varma, Manoj M

    2015-01-01

    Immunoassay formats applicable for clinical or point-of-care diagnostics fall into two broad classes. One which uses labeled secondary antibodies for signal transduction and the other which does not require the use of any labels. Comparison of the limits of detection (LoD) reported by these two sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Further, a vast majority of commercial tests and recent examples of technology translations are based on labeled assay formats. In light of this data, it is argued that extension of traditional labeled approaches and enhancing their functionality may have better clinical impact than the development of newer label-free techniques.

  18. Label-Free Vapor Selectivity in Poly(p-Phenylene Oxide) Photonic Crystal Sensors.

    Science.gov (United States)

    Lova, Paola; Bastianini, Chiara; Giusto, Paolo; Patrini, Maddalena; Rizzo, Paola; Guerra, Gaetano; Iodice, Mario; Soci, Cesare; Comoretto, Davide

    2016-11-23

    The lack of sensors for low cost, extensive, and continuous detection of vapor pollutants is a serious concern for health and safety in industrialized urban areas. Colorimetric sensors, such as distributed Bragg reflectors made of polymers, could achieve this task thanks to their low cost and easy signal transduction but are typically affected by low vapor permeability and lack of selectivity without chemical labeling. Here we demonstrate all-polymer Bragg multilayers for label-free selective detection of organic volatile compounds. The system exploits the ability of amorphous poly(p-phenylene oxide), PPO, to uptake large amount of guest molecules and to form cocrystalline phases with distinct optical properties. Bragg stacks embedding PPO active layers show selective colorimetric response to vapors of carbon tetrachloride and aromatic homologues, which can be revealed by the naked eye.

  19. Carbon Nanostructure-Based Field-Effect Transistors for Label-Free Chemical/Biological Sensors

    Directory of Open Access Journals (Sweden)

    PingAn Hu

    2010-05-01

    Full Text Available Over the past decade, electrical detection of chemical and biological species using novel nanostructure-based devices has attracted significant attention for chemical, genomics, biomedical diagnostics, and drug discovery applications. The use of nanostructured devices in chemical/biological sensors in place of conventional sensing technologies has advantages of high sensitivity, low decreased energy consumption and potentially highly miniaturized integration. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene based electrical devices have been widely developed for high performance label-free chemical/biological sensors. Here, we review the latest developments of carbon nanostructure-based transistor sensors in ultrasensitive detection of chemical/biological entities, such as poisonous gases, nucleic acids, proteins and cells.

  20. 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.

  1. A highly sensitive impedimetric label free immunosensor for Ochratoxin measurement in cocoa beans.

    Science.gov (United States)

    Malvano, Francesca; Albanese, Donatella; Pilloton, Roberto; Di Matteo, Marisa

    2016-12-01

    In this work the development and optimization of an impedimetric label free immunosensor for the detection of Ochratoxin A (OTA) is reported. Two antibody immobilization methods (oriented and not oriented) were compared highlighting a lower limit of detection (5pg/ml) for the not oriented immobilization but a closer linear range in contrast to oriented anti-OTA immunosensors which showed linearity in the range of 0.01-5ng/mL OTA. The analysis of the Atomic Force Microscopy (AFM) images showed two different nanostructures indicating that the use of oriented immobilization created a more ordered and highly dense antibody surface. Finally the oriented immunosensor was used to quantify OTA in spiked cocoa bean samples and the results were compared with those registered with competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2μg/kg that represents the lower acceptable limit of OTA established by European legislation for the common food products.

  2. High-sensitivity label-free optical fiber optrodes based on the excitation of Bloch surface waves

    Science.gov (United States)

    Scaravilli, M.; Castaldi, G.; Cusano, A.; Galdi, V.

    2016-05-01

    In this study, the possibility to excite Bloch surface waves (BSWs) on the tip of a single-mode optical fiber is explored for the first time. In particular, we first show the possibility to achieve an on-tip excitation of BSWs, with optimized characteristic of the arising resonances, via an "all-fiber" grating-coupled configuration. Furthermore, envisioning novel high-performance fiber tip nanoprobes for label-free biosensing, we introduce an ad hoc design aimed at maximizing the refractive-index sensitivity. Numerical results indicate that the estimated sensitivities are comparable with those exhibited by current plasmonic lab-on-tip bio-probes, but are accompanied by a higher spectral selectivity. Therefore, this preliminary work paves the way to the development of new classes of miniaturized surface-wave optical fiber devices for low-detection-limit label-free chemical and biological sensing.

  3. A cyclodextrin host-guest recognition approach to a label-free electrochemical DNA hybridization biosensor.

    Science.gov (United States)

    Abbaspour, Abdolkarim; Noori, Abolhassan

    2012-04-21

    A novel label-free electrochemical DNA hybridization biosensor using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified screen printed electrode (CD/PNAANI/CNT/SPE) has been developed. The proposed DNA hybridization biosensor relies on the intrinsic oxidation signals of guanine (G) and adenine (A) from single-stranded DNA entered into the cyclodextrin (CD) cavity. Due to the binding of G and A bases to complementary cytosine and thymine bases in dsDNA, the signals obtained for ssDNA were much higher than that of dsDNA. The synergistic effect of the multi-walled carbon nanotubes provides a significantly enhanced voltammetric signal, and the CD encapsulation effect makes anodic peaks of G and A shift to less positive potentials than that at the bare SPE. The peak heights of G and A signals are dependent on both the number of the respective bases in oligonucleotides and the concentration of the target DNA sequences. Hybridization of complementary strands was monitored through the measurements of oxidation signal of purine bases, which enabled the detection of target sequences from 0.01 to 1.02 nmol μl(-1) with the detection limit of target DNA as low as 5.0 pmol μl(-1) (S/N = 3). Implementation of label-free and homogeneous electrochemical hybridization detection constitutes an important step toward low-cost, simple, highly sensitive and accurate DNA assay. Discrimination between complementary, noncomplementary, and two-base mismatch targets was easily accomplished using the proposed electrode.

  4. Data from quantitative label free proteomics analysis of rat spleen

    Directory of Open Access Journals (Sweden)

    Khadar Dudekula

    2016-09-01

    Full Text Available The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides. A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

  5. Data from quantitative label free proteomics analysis of rat spleen.

    Science.gov (United States)

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

    The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

  6. Label-free structural photoacoustic tomography of intact mouse brain

    Science.gov (United States)

    Li, Lei; Xia, Jun; Li, Guo; Garcia-Uribe, Alejandro; Wang, Lihong V.

    2015-03-01

    Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

  7. Label-free screening of bio-molecular interactions.

    Science.gov (United States)

    Cooper, Matthew A

    2003-11-01

    The majority of techniques currently employed to interrogate a biomolecular interaction require some type of radio- or enzymatic- or fluorescent-labelling to report the binding event. However, there is an increasing awareness of novel techniques that do not require labelling of the ligand or the receptor, and that allow virtually any complex to be screened with minimal assay development. This review focuses on three major label-free screening platforms: surface plasmon resonance biosensors, acoustic biosensors, and calorimetric biosensors. Scientists in both academia and industry are using biosensors in areas that encompass almost all areas drug discovery, diagnostics, and the life sciences. The capabilities and advantages of each technique are compared and key applications involving small molecules, proteins, oligonucleotides, bacteriophage, viruses, bacteria, and cells are reviewed. The role of the interface between the biosensor surface (in the case of SPR and acoustic biosensors) and the chemical or biological systems to be studied is also covered with attention to the covalent and non-covalent coupling chemistries commonly employed.

  8. Label-free high-throughput imaging flow cytometry

    Science.gov (United States)

    Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

    2014-03-01

    Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

  9. Label-Free Microcavity Biosensors: Steps towards Personalized Medicine

    Directory of Open Access Journals (Sweden)

    Dragos Amarie

    2012-12-01

    Full Text Available Personalized medicine has the potential to improve our ability to maintain health and treat disease, while ameliorating continuously rising healthcare costs. Translation of basic research findings to clinical applications within regulatory compliance is required for personalized medicine to become the new foundation for practice of medicine. Deploying even a few of the thousands of potential diagnostic biomarkers identified each year as part of personalized treatment workflows requires clinically efficient biosensor technologies to monitor multiple biomarkers in patients in real time. This paper discusses a critical component of a regulatory system, a microcavity optical biosensor for label-free monitoring of biomolecular interactions at physiologically-relevant concentrations. While most current biosensor research focuses on improving sensitivity, this paper emphasizes other characteristics a biosensor technology requires to be practical in a clinical setting, presenting robust microcavity biosensors which are easy to manufacture and integrate with microfluidics into flexible and redesignable platforms making the microcavity biosensors deployable for continuous monitoring of biomarkers in body fluids in the clinic,  in dense 2D random arrays for high-throughput applications like drug-library screening in interactomics, and of the secretory behavior of single cells in the laboratory.

  10. Label-free biosensor based on long period grating

    Science.gov (United States)

    Baldini, Francesco; Chiavaioli, Francesco; Giannetti, Ambra; Brenci, Massimo; Trono, Cosimo

    2013-03-01

    Long period gratings have been recently proposed as label-free optical devices for biochemical sensing. A biochemical interaction along the grating region changes the biolayer refractive index and a change in the fiber transmission spectrum occurs. The fiber biofunctionalization was performed with a novel chemistry using Eudragit L100 copolymer as opposed to the commonly-used silanization procedure. An IgG/anti-IgG bioassay was carried out for studying the antigen/antibody interaction. The biosensor was fully characterized, monitoring the kinetics during the antibody immobilization and achieving the calibration curve of the assay. To compare the biosensor performance, two LPG-based biosensors with distinct grating periods were characterized following the same bioassay protocol. Experimental results demonstrated an enhancement of the biosensor performance when the fundamental core mode of a single-mode fiber couples with a higher order cladding mode. Considering an LPG manufactured on a bare optical fiber, in which the coupling occurs with the 7-th cladding mode, a dynamic signal range of 0.33 nm, a working range of 1.7 - 1450 mg L-1 and a LOD of 500 μg L-1 were achieved

  11. High throughput label-free platform for statistical bio-molecular sensing.

    Science.gov (United States)

    Bosco, Filippo G; Hwu, En-Te; Chen, Ching-Hsiu; Keller, Stephan; Bache, Michael; Jakobsen, Mogens H; Hwang, Ing-Shouh; Boisen, Anja

    2011-07-21

    Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing cantilever based sensors. These sensors have often been acclaimed to facilitate highly parallelized operation. Unfortunately, so far no concept has been presented which offers large datasets as well as easy liquid sample handling. We use optics and mechanics from a DVD player to handle liquid samples and to read-out cantilever deflection and resonant frequency. Also, surface roughness is measured. When combined with cantilever deflection the roughness is discovered to hold valuable additional information on specific and unspecific binding events. In a few minutes, 30 liquid samples can be analyzed in parallel, each by 24 cantilever-based sensors. The approach was used to detect the binding of streptavidin and antibodies.

  12. Low-cost label-free biosensors using photonic crystals embedded between crossed polarizers.

    Science.gov (United States)

    Nazirizadeh, Yousef; Bog, Uwe; Sekula, Sylwia; Mappes, Timo; Lemmer, Uli; Gerken, Martina

    2010-08-30

    There is a strong need for low-cost biosensors to enable rapid, on-site analysis of biological, biomedical, or chemical substances. We propose a platform for label-free optical biosensors based on applying the analyte onto a surface-functionalized photonic crystal slab and performing a transmission measurement with two crossed polarization filters. This dark-field approach allows for efficient background suppression as only the photonic crystal guided-mode resonances interacting with the functionalized surface experience significant polarization rotation. We present a compact biosensor demonstrator using a low-cost light emitting diode and a simple photodiode capable of detecting the binding kinetics of a 2.5 nM solution of the protein streptavidin on a biotin-functionalized photonic crystal surface.

  13. Analytical modeling of label free biosensor using charge plasma based gate underlap dielectric modulated MOSFET

    Science.gov (United States)

    Chanda, Manash; Das, Rahul; Kundu, Atanu; Sarkar, Chandan K.

    2017-04-01

    In this paper charge plasma based dielectric modulated four gated MOSFET (CP-GUDM-MOSFET) has been proposed for the efficacy of label free electrical detection of the biomolecules. To achieve low thermal budgeting, charge-plasma concept is employed using appropriate metal work function electrodes. Extensive simulations have been done using the Sentaurus TCAD to validate the proposed architecture. An analytical modeling has also been done on surface potential and drain current to consolidate the feasibility of the structure. Significant improvements in the on current (ION) and threshold voltage have been observed in presence of the charged biomolecules. The performance of proposed structure is found to be sensitive to gate-oxide thickness variations. High sensitivity of the proposed CP-GUDM-MOSFET based biosensor with low thermal budgeting scheme; simple structure and its compatibility with the existing CMOS processes make it an exciting alternative to the conventional FET-based biosensors.

  14. Label-free colorimetric estimation of proteins using nanoparticles of silver

    Institute of Scientific and Technical Information of China (English)

    Siddhartha Shrivastava; Debabrata Dash

    2010-01-01

    Metallic nanoparticles have received considerable attention in bioassays and diagnostics due to their unique surface plasmon resonance (SPR) properties. Gold nanoparticles have been employed for the development of SPR-based colorimetric bioassays. In the present report we have described a sensitive colorimetric approach for estimation of proteins, within a detection limit of 10~80 μg/mL, using unmodified silver nanoparticles. Besides the common advantages of colorimetric assay such as simplicity, high sensitivity, and low cost, our method has a label-free design and provides an important and attractive alternative to classical sensing probes and systems. The present work will contribute to the development of nanotechnology-based diagnostic tools.

  15. NI-78LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

    Science.gov (United States)

    Uckermann, Ortrud; Galli, Roberta; Geiger, Kathrin; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a

  16. Protein analysis of atrial fibrosis via label-free proteomics in chronic atrial fibrillation patients with mitral valve disease.

    Directory of Open Access Journals (Sweden)

    Peide Zhang

    Full Text Available BACKGROUND: Atrial fibrosis, as a hallmark of atrial structure remodeling, plays an important role in maintenance of chronic atrial fibrillation, but interrelationship of atrial fibrosis and atrial fibrillation is uncertain. Label-free proteomics can implement high throughput screening for finding and analyzing pivotal proteins related to the disease.. Therefore, we used label-free proteomics to explore and analyze differentially proteins in chronic atrial fibrillation patients with mitral valve disease. METHODS: Left and right atrial appendages obtained from patients with mitral valve disease were both in chronic atrial fibrillation (CAF, AF≥6 months, n = 6 and in sinus rhythm (SR, n = 6. One part of the sample was used for histological analysis and fibrosis quantification; other part were analyzed by label-free proteomic combining liquid chromatography with mass spectrometry (LC-MS, we utilized bioinformatics analysis to identify differential proteins. RESULTS: Degree of atrial fibrosis was higher in CAF patients than that of SR patients. 223 differential proteins were detected between two groups. These proteins mainly had vital functions such as cell proliferation, stress response, focal adhesion apoptosis. We evaluated that serine/threonine protein kinase N2 (PKN2, dermatopontin (DP, S100 calcium binding protein B (S100B, protein tyrosine kinase 2 (PTK2 and discoidin domain receptor tyrosine kinase 2 (DDR2 played important roles in fibrotic process related to atrial fibrillation. CONCLUSION: The study presented differential proteins responsible for atrial fibrosis in chronic atrial fibrillation patients through label-free proteomic analysis. We assessed some vital proteins including their characters and roles. These findings may open up new realm for mechanism research of atrial fibrillation.

  17. Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging.

    Science.gov (United States)

    Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S

    2015-11-01

    In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.

  18. Novel label-free biosensing technology for monitoring of aqueous solutions (Conference Presentation)

    Science.gov (United States)

    Kehl, Florian; Bielecki, Robert; Follonier, Stephane; Dorokhin, Denis

    2016-03-01

    Waste water, drinking water and other industrial water sources are more and more/increasingly polluted with a large variety of contaminants, such as pesticides or residuals of pharmaceuticals. These compounds can impact human and animal organisms and lead to serious health issues. Today, in order to analyze the presence and quantity of the abovementioned micropollutants, samples are typically sent to specialized centralized laboratories and their processing may take up to several days. In order to meet the demand for continuous and consistent monitoring of aqueous solutions we propose a novel label-free technology system comprising proprietary chip and reader device designs. The core of the system is constituted by a planar-grated-waveguide (PGW) chip. Label-free biosensors, based on PGWs are sensitive to effective refractive index changes caused by the adsorption of biomolecules (micropollutants) onto the sensor surface or due to refractive index changes of the bulk solution. The presented reader device operates with a novel readout concept based on a scanning MEMS mirror for the angular interrogation of input grating couplers at a high repetition rate. The reader has fully integrated optics, electronics and fluidics and at the same time consumes limited energy (portable, field use ready). In the recent experiments, the effectiveness of the technology has been demonstrated with various liquids and bioassays showing (i) an excellent refractometric sensitivity with a limit of detection towards effective refractive index changes of ▵neff < 2 x 10-7, and (ii) the capability to perform affinity measurements for large (<150 kDa) and small (<250 Da) molecules.

  19. Label free screening of enzyme inhibitors at femtomole scale using segmented flow electrospray ionization mass spectrometry.

    Science.gov (United States)

    Sun, Shuwen; Slaney, Thomas R; Kennedy, Robert T

    2012-07-03

    Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of ~0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.

  20. Label free sensing of creatinine using a 6 GHz CMOS near-field dielectric immunosensor.

    Science.gov (United States)

    Guha, S; Warsinke, A; Tientcheu, Ch M; Schmalz, K; Meliani, C; Wenger, Ch

    2015-05-07

    In this work we present a CMOS high frequency direct immunosensor operating at 6 GHz (C-band) for label free determination of creatinine. The sensor is fabricated in standard 0.13 μm SiGe:C BiCMOS process. The report also demonstrates the ability to immobilize creatinine molecules on a Si3N4 passivation layer of the standard BiCMOS/CMOS process, therefore, evading any further need of cumbersome post processing of the fabricated sensor chip. The sensor is based on capacitive detection of the amount of non-creatinine bound antibodies binding to an immobilized creatinine layer on the passivated sensor. The chip bound antibody amount in turn corresponds indirectly to the creatinine concentration used in the incubation phase. The determination of creatinine in the concentration range of 0.88-880 μM is successfully demonstrated in this work. A sensitivity of 35 MHz/10 fold increase in creatinine concentration (during incubation) at the centre frequency of 6 GHz is gained by the immunosensor. The results are compared with a standard optical measurement technique and the dynamic range and sensitivity is of the order of the established optical indication technique. The C-band immunosensor chip comprising an area of 0.3 mm(2) reduces the sensing area considerably, therefore, requiring a sample volume as low as 2 μl. The small analyte sample volume and label free approach also reduce the experimental costs in addition to the low fabrication costs offered by the batch fabrication technique of CMOS/BiCMOS process.

  1. Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction.

    Science.gov (United States)

    Zhou, Jun; Xu, Mingdi; Tang, Dianping; Gao, Zhuangqiang; Tang, Juan; Chen, Guonan

    2012-12-28

    A label-free, non-enzyme immunosensing strategy is designed for ultrasensitive electronic detection of disease-related proteins (carcinoembryonic antigen as a model) by using gold nanoparticle-based bio-bar codes and an in situ amplified DNA-based hybridization chain reaction.

  2. Rapid, label-free, electrical whole blood bioassay based on nanobiosensor systems.

    Science.gov (United States)

    Chang, Hsiao-Kang; Ishikawa, Fumiaki N; Zhang, Rui; Datar, Ram; Cote, Richard J; Thompson, Mark E; Zhou, Chongwu

    2011-12-27

    Biomarker detection based on nanowire biosensors has attracted a significant amount of research effort in recent years. However, only very limited research work has been directed toward biomarker detection directly from physiological fluids mainly because of challenges caused by the complexity of media. This limitation significantly reduces the practical impact generated by the aforementioned nanobiosensors. In this study, we demonstrate an In(2)O(3) nanowire-based biosensing system that is capable of performing rapid, label-free, electrical detection of cancer biomarkers directly from human whole blood collected by a finger prick. Passivating the nanowire surface successfully blocked the signal induced by nonspecific binding when performing active measurement in whole blood. Passivated devices showed markedly smaller signals induced by nonspecific binding of proteins and other biomaterials in serum and higher sensitivity to target biomarkers than bare devices. The detection limit of passivated sensors for biomarkers in whole blood was similar to the detection limit for the same analyte in purified buffer solutions at the same ionic strength, suggesting minimal decrease in device performance in the complex media. We then demonstrated detection of multiple cancer biomarkers with high reliability at clinically meaningful concentrations from whole blood collected by a finger prick using this sensing system.

  3. Bioaerosol analysis based on a label-free microarray readout method using surface-enhanced Raman scattering.

    Science.gov (United States)

    Schwarzmeier, Kathrin; Knauer, Maria; Ivleva, Natalia P; Niessner, Reinhard; Haisch, Christoph

    2013-06-01

    Bacterial contamination of indoor air is a serious threat to human health. Pathogenic germs can be transferred from the liquid to the aerosol phase, for instance, when water is sprayed in the air, such as in shower rooms, air conditioners, or fountains. Existing analytical methods for biological indoor air-quality assessment and contamination monitoring are mostly time consuming as they generally require a cultivation step. The need for a rapid, sensitive, and selective detection method for bioaerosols is evident. Our approach is based on the combination of a commercial wet particle sampler (Coriolis μ, Bertin Technologies, France) and a label-free microarray readout based on surface-enhanced Raman scattering (SERS) for detection, which was established in our laboratories. Heat-inactivated Escherichia coli bacteria were used as test microorganisms. An E. coli suspension was sprayed into the chamber by a jet air nebulizer. The resulting bioaerosol was dried, neutralized, and then collected by a Coriolis μ sampler. The bacteria collected were detected by a recently developed microarray readout system, based on label-free SERS detection. A special data evaluation procedure was applied in order to fully exploit the selectivity of the detection scheme, resulting in a detection limit of 144 particles per cubic centimeter.

  4. A universal label-free biosensing platform based on opto-fluidic ring resonators

    Science.gov (United States)

    Zhu, Hongying; White, Ian M.; Suter, Jonathan D.; Gohring, John; Fan, Xudong

    2009-02-01

    Rapid and accurate detection of biomolecules is important for medical diagnosis, pharmaceuticals, homeland security, food quality control, and environmental protection. A simple, low cost and highly sensitive label-free optical biosensor based on opto-fluidic ring resonator (OFRR) has been developed that naturally integrates microfluidics with ring resonators. The OFRR employs a piece of fused silica capillary with a diameter around 100 micrometers. The circular cross section of the capillary forms the ring resonator and light repeatedly travels along the resonator circumference in the form of whispering gallery modes (WGMs) through total internal reflection. When the capillary wall is as thin as a couple of micrometers (detect the target molecules with high specificity, the OFRR inner surface is functionalized with receptors, such as antibodies, peptide-displayed bacteriophage or oligonucleotide DNA probes. The WGM spectral position shifts when biomolecules bind to the OFRR inner surface and change the local refractive index, which provides quantitative and kinetic information about the biomolecule interaction near the OFRR inner surface. The OFRR has been successfully demonstrated for detection of various types of biomoelcuels. Here, we will first introduce the basic operation principle of the OFRR as a sensor and then application examples of the OFRR in the detection of proteins, disease biomarkers, virus, DNA molecules, and cells with high sensitivities will be presented.

  5. Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform

    Science.gov (United States)

    Baaske, Martin D.; Foreman, Matthew R.; Vollmer, Frank

    2014-11-01

    Biosensing relies on the detection of molecules and their specific interactions. It is therefore highly desirable to develop transducers exhibiting ultimate detection limits. Microcavities are an exemplary candidate technology for demonstrating such a capability in the optical domain and in a label-free fashion. Additional sensitivity gains, achievable by exploiting plasmon resonances, promise biosensing down to the single-molecule level. Here, we introduce a biosensing platform using optical microcavity-based sensors that exhibits single-molecule sensitivity and is selective to specific single binding events. Whispering gallery modes in glass microspheres are used to leverage plasmonic enhancements in gold nanorods for the specific detection of nucleic acid hybridization, down to single 8-mer oligonucleotides. Detection of single intercalating small molecules confirms the observation of single-molecule hybridization. Matched and mismatched strands are discriminated by their interaction kinetics. Our platform allows us to monitor specific molecular interactions transiently, hence mitigating the need for high binding affinity and avoiding permanent binding of target molecules to the receptors. Sensor lifetime is therefore increased, allowing interaction kinetics to be statistically analysed.

  6. Label-Free Biosensors Based on Bimodal Waveguide (BiMW) Interferometers.

    Science.gov (United States)

    Herranz, Sonia; Gavela, Adrián Fernández; Lechuga, Laura M

    2017-01-01

    The bimodal waveguide (BiMW) sensor is a novel common path interferometric transducer based on the evanescent field detection principle, which in combination with a bio-recognition element allows the direct detection of biomolecular interactions in a label-free scheme. Due to its inherent high sensitivity it has great potential to become a powerful analytical tool for monitoring substances of interest in areas such as environmental control, medical diagnostics and food safety, among others. The BiMW sensor is fabricated using standard silicon-based technology allowing cost-effective production, and meeting the requirements of portability and disposability necessary for implementation in a point-of-care (POC) setting.In this chapter we describe the design and fabrication of the BiMW transducer, as well as its application for bio-sensing purposes. We show as an example the biosensor capabilities two different applications: (1) the immunodetection of Irgarol 1051 biocide useful in the environmental field, and (2) the detection of human growth hormone as used in clinical diagnostics. The detection is performed in real time by monitoring changes in the intensity pattern of light exiting the BiMW transducer resulting from antigen-antibody interactions on the surface of the sensor.

  7. A distributed national network for label-free rapid identification of emerging pathogens

    Science.gov (United States)

    Robinson, J. Paul; Rajwa, Bartek P.; Dundar, M. Murat; Bae, Euiwon; Patsekin, Valery; Hirleman, E. Daniel; Roumani, Ali; Bhunia, Arun K.; Dietz, J. Eric; Davisson, V. Jo; Thomas, John G.

    2011-05-01

    Typical bioterrorism prevention scenarios assume well-known and well-characterized pathogens like anthrax or tularemia, which are serious public concerns if released into food and/or water supplies or distributed using other vectors. Common governmental contingencies include rapid response to these biological threats with predefined treatments and management operations. However, bioterrorist attacks may follow a far more sophisticated route. With the widely known and immense progress in genetics and the availability of molecular biology tools worldwide, the potential for malicious modification of pathogenic genomes is very high. Common non-pathogenic microorganisms could be transformed into dangerous, debilitating pathogens. Known pathogens could also be modified to avoid detection, because organisms are traditionally identified on the basis of their known physiological or genetic properties. In the absence of defined primers a laboratory using genetic biodetection methods such as PCR might be unable to quickly identify a modified microorganism. Our concept includes developing a nationwide database of signatures based on biophysical (such as elastic light scattering (ELS) properties and/or Raman spectra) rather than genetic properties of bacteria. When paired with a machine-learning system for emerging pathogen detection these data become an effective detection system. The approach emphasizes ease of implementation using a standardized collection of phenotypic information and extraction of biophysical features of pathogens. Owing to the label-free nature of the detection modalities ELS is significantly less costly than any genotypic or mass spectrometry approach.

  8. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    Science.gov (United States)

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  9. A novel self-powered and sensitive label-free DNA biosensor in microbial fuel cell.

    Science.gov (United States)

    Asghary, Maryam; Raoof, Jahan Bakhsh; Rahimnejad, Mostafa; Ojani, Reza

    2016-08-15

    In this work, a novel self-powered, sensitive, low-cost, and label-free DNA biosensor is reported by applying a two-chambered microbial fuel cell (MFC) as a power supply. A graphite electrode and an Au nanoparticles modified graphite electrode (AuNP/graphite electrode) were used as anode and cathode in the MFC system, respectively. The active biocatalyst in the anodic chamber was a mixed culture of microorganisms. The sensing element of the biosensor was fabricated by the well-known Au-thiol binding the ssDNA probe on the surface of an AuNP/graphite cathode. Electrons produced by microorganisms were transported from the anode to the cathode through an external circuit, which could be detected by the terminal multi-meter detector. The difference between power densities of the ssDNA probe modified cathode in the absence and presence of complementary sequence served as the detection signal of the DNA hybridization with detection limit of 3.1nM. Thereafter, this biosensor was employed for diagnosis and determination of complementary sequence in a human serum sample. The hybridization specificity studies further revealed that the developed DNA biosensor could distinguish fully complementary sequences from one-base mismatched and non-complementary sequences.

  10. Label-free electrochemical monitoring of protein addressing through electroactivated "click" chemistry on gold electrodes.

    Science.gov (United States)

    Meini, Nadir; Ripert, Micaël; Chaix, Carole; Farre, Carole; De Crozals, Gabriel; Kherrat, Rochdi; Jaffrezic-Renault, Nicole

    2014-05-01

    In this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at -300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/mL.

  11. Biconically Tapered Fiber Optic Probes for Rapid Label-Free Immunoassays

    Directory of Open Access Journals (Sweden)

    John Miller

    2015-04-01

    Full Text Available We report use of U-shaped biconically tapered optical fibers (BTOF as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G-antigen (rabbit anti-mouse IgG reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

  12. Construction of label-free electrochemical immunosensor on mesoporous carbon nanospheres for breast cancer susceptibility gene

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Haixia; Zhang, Yong; Wu, Dan; Ma, Hongmin; Li, Xiaojing; Li, Yan; Wang, Huan; Li, He; Du, Bin [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Wei, Qin, E-mail: sdjndxwq@163.com [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China)

    2013-04-03

    Highlights: ► The immunosensor is designed to determine breast cancer susceptibility gene. ► Mesoporous carbon nanospheres (MCN) have great adsorption capacity. ► MCN could enhance the electroactivity of toluidine blue. ► Room temperature ionic liquid should increase the electrochemical signal. -- Abstract: In this contribution, mesoporous carbon nanospheres (MCN) were used to fabricate a label-free electrochemical immunosensor for breast cancer susceptibility gene (BRCAl). The detection platform was constructed by conjugation of anti-BRCA1 on glassy carbon electrodes which were modified by mesoporous carbon nanospheres–toluidine blue nanocomposite (MCN–TB)/room temperature ionic-liquid (RTIL) composited film. TB was adsorbed onto MCN and acted as a redox probe. The electroactivity of TB was greatly enhanced in the presence of MCN. The good conductivity of MCN and BMIM·BF{sub 4} could promote the electron transfer and thus enhance the detection sensitivity. Moreover, the large surface area of MCN and the protein-binding properties of BMIM·BF{sub 4} could greatly increase the antibody loading. The specific antibody–antigen immunoreaction on the electrode surface resulted in a decrease of amperometric signal of the electrode. Under optimized conditions, the amperometric signal decreased linearly with BRCAl concentration in the range of 0.01–15 ng mL{sup −1} with a low detection limit of 3.97 pg mL{sup −1}. The immunosensor exhibits high sensitivity, good selectivity and stability.

  13. Rapid label-free identification of mixed bacterial infections by surface plasmon resonance

    Directory of Open Access Journals (Sweden)

    Fu Weiling

    2011-06-01

    Full Text Available Abstract Background Early detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research. Methods In this study, we developed a single-stranded DNA (ssDNA amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens. Results We constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P 2 values of >0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P Conclusions Our method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.

  14. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification.

    Science.gov (United States)

    Liu, Shufeng; Gong, Hongwei; Wang, Yanqun; Wang, Li

    2016-03-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5 fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine.

  15. Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells.

    Science.gov (United States)

    Agnarsson, Björn; Lundgren, Anders; Gunnarsson, Anders; Rabe, Michael; Kunze, Angelika; Mapar, Mokhtar; Simonsson, Lisa; Bally, Marta; Zhdanov, Vladimir P; Höök, Fredrik

    2015-12-22

    Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.

  16. Design of a New Ultracompact Resonant Plasmonic Multi-Analyte Label-Free Biosensing Platform

    Directory of Open Access Journals (Sweden)

    Francesco Dell’Olio

    2017-08-01

    Full Text Available In this paper, we report on the design of a bio-multisensing platform for the selective label-free detection of protein biomarkers, carried out through a 3D numerical algorithm. The platform includes a number of biosensors, each of them is based on a plasmonic nanocavity, consisting of a periodic metal structure to be deposited on a silicon oxide substrate. Light is strongly confined in a region with extremely small size (=1.57 μm2, to enhance the light-matter interaction. A surface sensitivity Ss = 1.8 nm/nm has been calculated together with a detection limit of 128 pg/mm2. Such performance, together with the extremely small footprint, allow the integration of several devices on a single chip to realize extremely compact lab-on-chip microsystems. In addition, each sensing element of the platform has a good chemical stability that is guaranteed by the selection of gold for its fabrication.

  17. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Directory of Open Access Journals (Sweden)

    Raf Donders

    2016-01-01

    Full Text Available In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM and second harmonic generation (SHG could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

  18. Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy.

    Science.gov (United States)

    Wu, Ching-Chou; Lin, Chia-Hung; Wang, Way-Shyan

    2009-06-30

    Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spectra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C circuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1-1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagentless and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin.

  19. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    Science.gov (United States)

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research.

  20. Label-free, turn-on fluorescent sensor for trypsin activity assay and inhibitor screening.

    Science.gov (United States)

    Zhang, Lufeng; Qin, Haiyan; Cui, Wanwan; Zhou, Yang; Du, Jianxiu

    2016-12-01

    The development of new detection methods for proteases activity assay is important in clinical diagnostics and drug development. In this work, a simple, label-free, and turn-on fluorescent sensor was fabricated for trypsin, a protease produced in the pancreas. Cytochrome c, a natural substance of trypsin, could be selectively cleaved by trypsin into heme-peptide fragment. The produced heme-peptide fragment exhibited an intensive catalytic role on the H2O2-mediated the oxidation of thiamine to form strong fluorescent thiochrome. The fluorescence intensity was closely dependent on the amount of trypsin presented. The procedure allowed the measurement of trypsin over the range of 0.5-20.0μg/mL with a detection limit of 0.125μg/mL. The sensor showed better precision with a relative standard deviation of 1.6% for the measurement of 1.0μg/mL trypsin solution (n=11). This sensing system was applied to screen the inhibitor of trypsin, the IC50 values were calculated to be 12.71ng/mL for the trypsin inhibitor from soybean and 2.0μg/mL for benzamidine hydrochloride, respectively, demonstrating its potential application in drug development and related diseases treatment.

  1. Label-Free Acetylcholine Image Sensor Based on Charge Transfer Technology for Biological Phenomenon Tracking

    Science.gov (United States)

    Takenaga, Shoko; Tamai, Yui; Okumura, Koichi; Ishida, Makoto; Sawada, Kazuaki

    2012-02-01

    A 32 ×32 charge-transfer enzyme-type acetylcholine (ACh) image sensor array was produced for label-free tracking of images of ACh distribution and its performance in repeatable measurements without enzyme deactivation was examined. The proposed sensor was based on a charge-transfer-type pH image sensor, which was modified using an enzyme membrane (acetylcholine esterase, AChE) for each pixel. The ACh image sensor detected hydrogen ions generated by the ACh-AChE reaction. A polyion complex membrane composed of poly(L-lysine) and poly(4-styrenesulfonate) was used to immobilize the enzyme on the sensor. The improved uniformity and adhesion of the polyion complex membrane were evaluated in this study. As a result, temporal and spatial fluctuations of the ACh image sensor were successfully minimized using this approach. The sensitivity of the sensor was 4.2 mV/mM, and its detection limit was 20 µM. In five repeated measurements, the repeatability was 8.8%.

  2. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Science.gov (United States)

    Paesen, Rik; Gyselaers, Wilfried; Stinissen, Piet

    2016-01-01

    In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin. PMID:27746820

  3. Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients.

    Science.gov (United States)

    Maldonado, Jesús; González-Guerrero, Ana Belén; Domínguez, Carlos; Lechuga, Laura M

    2016-11-15

    Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis.

  4. Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications.

    Science.gov (United States)

    Harrer, S; Kim, S C; Schieber, C; Kannam, S; Gunn, N; Moore, S; Scott, D; Bathgate, R; Skafidas, S; Wagner, J M

    2015-05-08

    Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular 'omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual's genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in

  5. Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications

    Science.gov (United States)

    Harrer, S.; Kim, S. C.; Schieber, C.; Kannam, S.; Gunn, N.; Moore, S.; Scott, D.; Bathgate, R.; Skafidas, S.; Wagner, J. M.

    2015-05-01

    Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular ‘omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual’s genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in

  6. iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

    Directory of Open Access Journals (Sweden)

    Hung V. Trinh

    2013-01-01

    Full Text Available Both isobaric tags for relative and absolute quantitation (iTRAQ and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC and tandem mass spectrometry (MS/MS. To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R2 correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method.

  7. Description of an Advantageous Optical Label-Free Biosensing Interferometric Read-Out Method to Measure Biological Species

    Directory of Open Access Journals (Sweden)

    Miguel Holgado

    2014-02-01

    Full Text Available In this article we report a new, simple, and reliable optical read-out detection method able to assess Rotavirus present in human sera as well as in the viral pollution sources. It is based on the interference of two interferometers used as biophotonic transducers. The method significantly improves the optical label-free biosensing response measuring both, the concentration of the AgR and its corresponding size. Two different immunoassays were carried out: Bovine Serum Albumin (BSA, and the recognition by its antibody (anti-BSA; and Rotavirus (AgR and the recognition by its antibody (anti-AgR. In the cases studied, and using as model interferometer a simple Fabry-Perot transducer, we demonstrate a biosensing enhancement of two orders of magnitude in the Limit of Detection (LoD. In fact, this read-out optical method may have significant implications to enhance other optical label-free photonic transducers reported in the scientific literature.

  8. Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing.

    Science.gov (United States)

    Hu, Yuwei; Hua, Shucheng; Li, Fenghua; Jiang, Yuanyuan; Bai, Xiaoxue; Li, Dan; Niu, Li

    2011-07-15

    Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged -COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH₂-IL) was applied to the reduction of HAuCl₄ to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH₂-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.

  9. Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates

    Directory of Open Access Journals (Sweden)

    Yannick eCharreti