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Sample records for dot-based fluorescence assay

  1. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    Science.gov (United States)

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  2. Quantum Dot-Based Luminescent Oxygen Channeling Assay for Potential Application in Homogeneous Bioassays.

    Science.gov (United States)

    Zhuang, Si-Hui; Guo, Xin-Xin; Wu, Ying-Song; Chen, Zhen-Hua; Chen, Yao; Ren, Zhi-Qi; Liu, Tian-Cai

    2016-01-01

    The unique photoproperties of quantum dots are promising for potential application in bioassays. In the present study, quantum dots were applied to a luminescent oxygen channeling assay. The reaction system developed in this study was based on interaction of biotin with streptavidin. Carboxyl-modified polystyrene microspheres doped with quantum dots were biotinylated and used as acceptors. Photosensitizer-doped carboxyl-modified polystyrene microspheres were conjugated with streptavidin and used as donors. The results indicated that the singlet oxygen that was released from the donor beads diffused into the acceptor beads. The acceptor beads were then exited via thioxene, and were subsequently fluoresced. To avoid generating false positives, a high concentration (0.01 mg/mL) of quantum dots is required for application in homogeneous immunoassays. Compared to a conventional luminescent oxygen channeling assay, this quantum dots-based technique requires less time, and would be easier to automate and miniaturize because it requires no washing to remove excess labels.

  3. Carbon dots based immunosorbent assay for the determination of GFAP in human serum

    Science.gov (United States)

    Ma, Yunsu; Xu, Guanhong; Wei, Fangdi; Cen, Yao; Song, Yueyue; Ma, Yujie; Xu, Xiaoman; Shi, Menglan; Sohail, Muhammad; Hu, Qin

    2018-04-01

    Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors. So, serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors. In this study, a new sensor based on carbon dots (CDs) linked with antibodies to specifically detect GFAP in human serum was developed. Anti-GFAP (Ab1) linked with protein A/G agarose resin (PA/G) as a capture antibody (PA/G-Ab1) and anti-GFAP (Ab2) labeled with CDs as a detection antibody (CDs-Ab2) were prepared firstly. Then the CD-linked antibody immunosorbent assay (CLAISA) method was constructed based on the sandwich conjunction reaction among PA/G-Ab1, GFAP, and CDs-Ab2. CLAISA, using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal, enabled the proposed immunosensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng ml-1 and a detection limit of 25 pg ml-1. This method was applied to the determination of GFAP in human serum by the standard addition method, and the results showed high accuracy and precision. Considering the easy synthetic process and excellent performance of CLAISA, this method has great potential to be used to monitor GFAP in the clinic.

  4. Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, Mukeshchand, E-mail: mukeshchandthakur@yahoo.com [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Mewada, Ashmi [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India); Pandey, Sunil, E-mail: gurus.spandey@gmail.com [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Bhori, Mustansir, E-mail: mustansyrr@gmail.com [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); Singh, Kanchanlata [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); Sharon, Maheshwar [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India); Sharon, Madhuri, E-mail: sharonmadhuri@gmail.com [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India)

    2016-10-01

    An economical green-chemistry approach was used for the synthesis of aqueous soluble graphene quantum dots (GQDs) from cow milk for simultaneous imaging and drug delivery in cancer. The GQDs synthesized using one-pot microwave-assisted heating were multi-fluorescent, spherical in shape having a lateral size of ca. 5 nm. The role of processing parameters such as heating time and ionic strength showed a profound effect on photoluminescence properties of GQDs. The GQDs were N-doped and oxygen-rich as confirmed by X-ray photoelectron spectroscopy (XPS) analysis. Cysteamine hydrochloride (Cys) was used to attach an anti-cancer drug berberine hydrochloride (BHC) on GQDs forming GQDs@Cys-BHC complex with c.a. 88% drug loading efficiency. In vitro drug release was studied at the acidic-basic environment and drug kinetics was studied using pharmacokinetic statistical models. The GQDs were biocompatible on L929 cells whereas theranostic GQDs@Cys-BHC complex showed a potent cytotoxic effect on different cancerous cell line models: cervical cancer cell lines such as HeLa cells and breast cancer cells such as MDA-MB-231 confirmed by Trypan blue and MTT-based cytotoxic assays. Furthermore, multi-excitation based cellular bioimaging was demonstrated using confocal laser scanning microscopy (CLSM) and fluorescence microscopy using GQDs as well as GQDs@Cys-BHC complex. Thus, drug delivery (therapeutic) and bioimaging (diagnostic) properties of GQDs@Cys-BHC complex are thought to have a potential in vitro theranostic application in cancer therapy. - Highlights: • Facile green synthesis of bright dual-florescent GQDs using cow milk as a precursor • Microwave irradiation time and pH have profound effects on fluorescent properties of GQDs. • Decoration of anti-cancer drug BHC onto GQDs via Cys-linker as theranostic platform • A pH responsive in vitro anti-cancer drug release and drug release kinetic study • Multi-photon bioimaging, cell cycle analysis, and apoptosis study

  5. Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system

    International Nuclear Information System (INIS)

    Thakur, Mukeshchand; Mewada, Ashmi; Pandey, Sunil; Bhori, Mustansir; Singh, Kanchanlata; Sharon, Maheshwar; Sharon, Madhuri

    2016-01-01

    An economical green-chemistry approach was used for the synthesis of aqueous soluble graphene quantum dots (GQDs) from cow milk for simultaneous imaging and drug delivery in cancer. The GQDs synthesized using one-pot microwave-assisted heating were multi-fluorescent, spherical in shape having a lateral size of ca. 5 nm. The role of processing parameters such as heating time and ionic strength showed a profound effect on photoluminescence properties of GQDs. The GQDs were N-doped and oxygen-rich as confirmed by X-ray photoelectron spectroscopy (XPS) analysis. Cysteamine hydrochloride (Cys) was used to attach an anti-cancer drug berberine hydrochloride (BHC) on GQDs forming GQDs@Cys-BHC complex with c.a. 88% drug loading efficiency. In vitro drug release was studied at the acidic-basic environment and drug kinetics was studied using pharmacokinetic statistical models. The GQDs were biocompatible on L929 cells whereas theranostic GQDs@Cys-BHC complex showed a potent cytotoxic effect on different cancerous cell line models: cervical cancer cell lines such as HeLa cells and breast cancer cells such as MDA-MB-231 confirmed by Trypan blue and MTT-based cytotoxic assays. Furthermore, multi-excitation based cellular bioimaging was demonstrated using confocal laser scanning microscopy (CLSM) and fluorescence microscopy using GQDs as well as GQDs@Cys-BHC complex. Thus, drug delivery (therapeutic) and bioimaging (diagnostic) properties of GQDs@Cys-BHC complex are thought to have a potential in vitro theranostic application in cancer therapy. - Highlights: • Facile green synthesis of bright dual-florescent GQDs using cow milk as a precursor • Microwave irradiation time and pH have profound effects on fluorescent properties of GQDs. • Decoration of anti-cancer drug BHC onto GQDs via Cys-linker as theranostic platform • A pH responsive in vitro anti-cancer drug release and drug release kinetic study • Multi-photon bioimaging, cell cycle analysis, and apoptosis study

  6. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    Science.gov (United States)

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-02

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.

  7. A ratiometric fluorescent quantum dots based biosensor for organophosphorus pesticides detection by inner-filter effect.

    Science.gov (United States)

    Yan, Xu; Li, Hongxia; Han, Xiaosong; Su, Xingguang

    2015-12-15

    In this work, we develop a novel and sensitive sensor for the detection of organophosphorus pesticides based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs). The RF-QDs has been designed by hybridizing two differently colored CdTe QDs, in which the red emissive QDs entrapped in the silica sphere acting as the reference signal, and the green emissive QDs covalently attached on the silica surface serving as the response signal.The fluorescence of RF-QDs could be quenched by AuNPs based on IFE. Protamine could effectively turn on the fluorescence due to the electrostatic attraction between protamine and AuNPs. Trypsin can easily hydrolyze protamine, leading to the quench of the fluorescence. Then, the fluorescence could be recovered again by the addition of parathion-methyl (PM) which could inhibit the activity of trypsin. By measuring the fluorescence of RF-QDs, the inhibition efficiency of PM to trypsin activity was evaluated. Under the optimized conditions, the inhibition efficiency was proportional to the logarithm of PM concentration in the range of 0.04-400 ng mL(-1), with a detection limit of 0.018 ng mL(-1). Furthermore, the simple and convenient method had been used for PM detection in environmental and agricultural samples with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Fluorescent blood glucose monitor by hemin-functionalized graphene quantum dots based sensing system

    Energy Technology Data Exchange (ETDEWEB)

    He, Yuezhen; Wang, Xiaoxun; Sun, Jian; Jiao, Shoufeng; Chen, Hongqi; Gao, Feng; Wang, Lun, E-mail: wanglun@mail.ahnu.edu.cn

    2014-01-31

    Graphical abstract: -- Highlights: •Hemin is assembled onto the surfaces of graphene quantum dots (GQDs). •With the aid of hemin, H{sub 2}O{sub 2} could quench the FL signal of GQDs obviously. •Based on this effect, a fluorescent platform is proposed for the sensing of glucose. •The proposed method provides a new pathway to explore practical application of GQDs. -- Abstract: In the present work, a highly sensitive and specific fluorescent biosensor for blood glucose monitoring is developed based on hemin-functionalized graphene quantum dots (GQDs) and glucose oxidase (GOx) system. The GQDs which are simply prepared by pyrolyzing citric acid exhibit strong fluorescence and good water-solubility. Due to the noncovalent assembly between hemin and GQDs, the addition of hemin can make hydrogen peroxide (H{sub 2}O{sub 2}) to destroy the passivated surface of GQDs, leading to significant fluorescence quenching of GQDs. Based on this effect, a novel fluorescent platform is proposed for the sensing of glucose. Under the optimized conditions, the linear range of glucose is from 9 to 300 μM, and the limit of detection is 0.1 μM. As unique properties of GQDs, the proposed biosensor is green, simple, cost-efficient, and it is successfully applied to the determination of glucose in human serum. In addition, the proposed method provides a new pathway to further design the biosensors based on the assembly of GQDs with hemin for detection of biomolecules.

  9. ZnSe quantum dots based fluorescence quenching method for determination of paeoniflorin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi [Center of Analysis, Guangdong Medical College, Dongguan 523808 (China); School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006 (China); Chen, Jiayi; Liang, Qiaowen [School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006 (China); Wu, Dudu [Center of Analysis, Guangdong Medical College, Dongguan 523808 (China); Zeng, Yuaner, E-mail: zengyuaner@126.com [School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006 (China); Jiang, Bin, E-mail: gzjiangbin@hotmail.com [School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006 (China)

    2014-01-15

    Water soluble ZnSe quantum dots (QDs) modified by mercaptoacetic acid (MAA) were used to determinate paeoniflorin in aqueous solutions by the fluorescence spectroscopic technique. The results showed that the fluorescence of the modified ZnSe QDs could be quenched by paeoniflorin effectively in physiological buffer solution. The optimum fluorescence intensity was found to be at incubation time 10 min, pH 7.0 and temperature 25 °C. Under the optimal conditions, the detection limit of paeoniflorin was 7.30×10{sup −7} mol L{sup −1}. Moreover, the quenching mechanism was discussed to be a static quenching procedure, which was proved by quenching rate constant K{sub q} (1.02×10{sup 13} L mol{sup −1} s{sup −1}). -- Highlights: • The fluorescence intensity of ZnSe QDs could be quenched by paeoniflorin. • Foreign substance showed insignificant effect for determination of paeoniflorin. • The quenching mechanism was discussed to be a static quenching procedure.

  10. ZnSe quantum dots based fluorescence quenching method for determination of paeoniflorin

    International Nuclear Information System (INIS)

    Chen, Zhi; Chen, Jiayi; Liang, Qiaowen; Wu, Dudu; Zeng, Yuaner; Jiang, Bin

    2014-01-01

    Water soluble ZnSe quantum dots (QDs) modified by mercaptoacetic acid (MAA) were used to determinate paeoniflorin in aqueous solutions by the fluorescence spectroscopic technique. The results showed that the fluorescence of the modified ZnSe QDs could be quenched by paeoniflorin effectively in physiological buffer solution. The optimum fluorescence intensity was found to be at incubation time 10 min, pH 7.0 and temperature 25 °C. Under the optimal conditions, the detection limit of paeoniflorin was 7.30×10 −7 mol L −1 . Moreover, the quenching mechanism was discussed to be a static quenching procedure, which was proved by quenching rate constant K q (1.02×10 13 L mol −1 s −1 ). -- Highlights: • The fluorescence intensity of ZnSe QDs could be quenched by paeoniflorin. • Foreign substance showed insignificant effect for determination of paeoniflorin. • The quenching mechanism was discussed to be a static quenching procedure

  11. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    Science.gov (United States)

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-10-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  12. Nitrogen-doped graphene quantum dots-based fluorescence molecularly imprinted sensor for thiacloprid detection.

    Science.gov (United States)

    Liu, Yang; Cao, Nan; Gui, Wenying; Ma, Qiang

    2018-06-01

    In this paper, a test strip-based sensor was developed for thiacloprid quantitative detection based on PDA molecularly imprinted polymer (MIP) and nitrogen-doped graphene quantum dots (N-GQDs). Thiacloprid is a new type of nicotine insecticide, which can block the normal neurotransmitter delivery process in insects. In the sensing system, N-GQDs were immersed into filter paper at first. Then, dopamine (DA) with thiacloprid can be self-polymerized on test strip surface to form the uniform PDA film. After removed thiacloprid template, the established poly dopamine (PDA) MIP can selectively recognize thiacloprid. As a result, captured thiacloprid can enhance the fluorescence intensity of N-GQDs into the test strip. As a result, the fluorescence intensity of N-GQDs can be linearly related within a certain range of thiacloprid concentration. Under the optimum conditions, the proposed sensor for thiacloprid detection exhibited a linear ranging from 0.1 mg/L to 10 mg/L with a low detection limit of 0.03 mg/L. The N-GQDs based test strip-based sensor for thiaclopridis reported for the first time. The sensing system has high selectivity to thiacloprid and provides new opportunities in the pesticide detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Carbon-dot-based fluorescent turn-on sensor for selectively detecting sulfide anions in totally aqueous media and imaging inside live cells.

    Science.gov (United States)

    Hou, Xianfeng; Zeng, Fang; Du, Fangkai; Wu, Shuizhu

    2013-08-23

    Sulfide anions are generated not only as a byproduct from industrial processes but also in biosystems. Hence, robust fluorescent sensors for detecting sulfide anions which are fast-responding, water soluble and biocompatible are highly desirable. Herein, we report a carbon-dot-based fluorescent sensor, which features excellent water solubility, low cytotoxicity and a short response time. This sensor is based on the ligand/Cu(II) approach so as to achieve fast sensing of sulfide anions. The carbon dot (CD) serves as the fluorophore as well as the anchoring site for the ligands which bind with copper ions. For this CD-based system, as copper ions bind with the ligands which reside on the surface of the CD, the paramagnetic copper ions efficiently quench the fluorescence of the CD, affording the system a turn-off sensor for copper ions. More importantly, the subsequently added sulfide anions can extract Cu(2+) from the system and form very stable CuS with Cu(2+), resulting in fluorescence enhancement and affording the system a turn-on sensor for sulfide anions. This fast-responding and selective sensor can operate in totally aqueous solution or in physiological milieu with a low detection limit of 0.78 μM. It displays good biocompatibility, and excellent cell membrane permeability, and can be used to monitor S(2-) levels in running water and living cells.

  14. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  15. Carbon dots based dual-emission silica nanoparticles as ratiometric fluorescent probe for nitrite determination in food samples.

    Science.gov (United States)

    Xiang, Guoqiang; Wang, Yule; Zhang, Heng; Fan, Huanhuan; Fan, Lu; He, Lijun; Jiang, Xiuming; Zhao, Wenjie

    2018-09-15

    In this work, a simple and effective strategy for designing a ratiometric fluorescent nanosensor was described. A carbon dots (CDs) based dual-emission nanosensor for nitrite was prepared by coating the CDs on to dye-doped silica nanoparticles. Dual-emission silica nanoparticles fluorescence was quenched in sulfuric acid using potassium bromate (KBrO 3 ). The nitrite present catalyzed the KBrO 3 oxidation, resulting in ratiometric fluorescence response of the dual-emission silica nanoparticles. Several important parameters affecting the performance of the nanosensor were investigated. Under optimized conditions, the limit of detection was 1.0 ng mL -1 and the linear range 10-160 ng mL -1 . Furthermore, the sensor was suitable for nitrite determination in different food samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. A novel ultrasensitive carboxymethyl chitosan-quantum dot-based fluorescence "turn on-off" nanosensor for lysozyme detection.

    Science.gov (United States)

    Song, Yu; Li, Yang; Liu, Ziping; Liu, Linlin; Wang, Xinyan; Su, Xingguang; Ma, Qiang

    2014-11-15

    In this work, we developed an ultrasensitive "turn on-off" fluorescence nanosensor for lysozyme (Lyz) detection. The novel nanosensor was constructed with the carboxymethyl chitosan modified CdTe quantum dots (CMCS-QDs). Firstly, the CMCS-QDs were fabricated via the electrostatic interaction between amino groups in CMCS polymeric chains and carboxyl groups on the surface of QDs. In the fluorescence "turn-on" step, the strong binding ability between Zn(2+) and CMCS on the surface of QDs can enhance the photoluminescence intensity (PL) of QDs. In the following fluorescence "turn-off" step, the N-acetyl-glucosamine (NAG) section along the CMCS chains was hydrolyzed by Lyz. As a result, Zn(2+) was released from the surface of QDs, and the Lyz-QDs complexes were formed to quench the QDs PL. Under the optimal conditions, there was a good linear relationship between the PL of QDs and the Lyz concentration (0.1-1.2 ng/mL) with the detection limit of 0.031 ng/mL. The developed method was ultrasensitive, highly selective and fast. It has been successfully employed in the detection of Lyz in the serum with satisfactory results. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A “Turn-On” thiol functionalized fluorescent carbon quantum dot based chemosensory system for arsenite detection

    Energy Technology Data Exchange (ETDEWEB)

    Pooja, D., E-mail: poojaiitr@csio.res.in [Academy of Scientific and Innovative Research (AcSIR), Council of Scientific and Industrial Research, New Delhi (India); Central Scientific Instruments Organisation, Sectro-30 C, Chandigarh 160030 (India); Saini, Sonia; Thakur, Anupma; Kumar, Baban; Tyagi, Sachin [Central Scientific Instruments Organisation, Sectro-30 C, Chandigarh 160030 (India); Nayak, Manoj K. [Academy of Scientific and Innovative Research (AcSIR), Council of Scientific and Industrial Research, New Delhi (India); Central Scientific Instruments Organisation, Sectro-30 C, Chandigarh 160030 (India)

    2017-04-15

    Highlights: • Environmental friendly carbon quantum dots grafted with thiol moieties. • The functionalized CQDs demonstrated for optical detection of arsenite in water. • High analytical performance in terms of sensitivity, selectivity and detection limit (0.086 ppb). - Abstract: Carbon quantum dots (CQDs) have emerged out as promising fluorescent probes for hazardous heavy metals detection in recent past. In this study, water soluble CQDs were synthesized by facile microwave pyrolysis of citric acid & cysteamine, and functionalized with ditheritheritol to impart thiol functionalities at surface for selective detection of toxic arsenite in water. Microscopic analysis reveals that the synthesized CQDs are of uniform size (diameter ∼5 nm) and confirmed to have surface −SH groups by FT-IR. The functionalized probe is then demonstrated for arsenite detection in water by “Turn-On” read out mechanism, which reduces the possibility of false positive signals associated with “turn off’ probes reported earlier. The blue luminescent functionalized CQDs exhibit increase in fluorescence intensity on arsenite addition in 5–100 ppb wide detection range. The probe can be used for sensitive detection of arsenite in environmental water to a theoretical detection limit (3s) of 0.086 ppb (R{sup 2} = 0.9547) with good reproducibility at 2.6% relative standard deviation. The presented reliable, sensitive, rapid fCQDs probe demonstrated to exhibit high selectivity towards arsenite and exemplified for real water samples as well. The analytical performance of the presented probe is comparable to existing organic & semiconductor based optical probes.

  18. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  19. Fabrication of L-cysteine-capped CdTe quantum dots based ratiometric fluorescence nanosensor for onsite visual determination of trace TNT explosive

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Jing; Hua, Mengjuan [School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [Changzhou College of Information Technology, Changzhou 213164 (China); Wang, Kan; Liu, Qian; Hao, Nan [School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    2016-11-23

    New strategies for onsite determination of trace 2,4,6-trinitrotoluene (TNT) explosives have become a research hotspot for homeland security needs against terrorism and environmental concerns. Herein, we designed a ratiometric fluorescence nanohybrid comprising 3-mercaptopropionic acid-capped green-emitting CdTe quantum dots (gQDs) encapsulated into SiO{sub 2} sphere and L-cysteine (Lcys)-capped red-emitting CdTe QDs (rQDs) conjugated onto SiO{sub 2} surface. The surface Lcys can be used as not only the stabilizer of the rQDs but also the primary amine provider which can react with TNT to form Meisenheimer complexes. Without any additional surface modification procedure, the fluorescence of rQDs equipped with Lcys was selectively quenched by TNT because electrons of the rQDs transferred to TNT molecules due to the formation of Meisenheimer complexes. Meanwhile, the embedded gQDs always remained constant. Upon exposure to increasing amounts of TNT, the fluorescence of rQDs could be gradually quenched and consequently the logarithm of the dual emission intensity ratios exhibited a good linear negative correlation with TNT concentration over a range of 10 nM–8 μM with a low detection limit of 3.3 nM. One can perform onsite visual determination of TNT with high resolution because the ratiometric fluorescence nanosensing system exhibited obvious fluorescence color changes. This sensing strategy has been successfully applied in real samples and already integrated in a filter paper-based assay, which enables potential fields use application featuring easy handling and cost-effectiveness. - Highlights: • A facile strategy for preparing Lcys-capped rQDs based hybrid spheres was reported. • Lcys serves as the stabilizer of rQDs and primary amine provider to react with TNT. • One can perform onsite visual determination of TNT by using such probe. • The nanosensor exhibited a wide linear range and a low detection limit. • This sensing strategy can be fully

  20. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  1. A fluorescence sedimentation assay for dsDNA antibodies

    DEFF Research Database (Denmark)

    Duus, K; Draborg, A H; Güven, E

    2017-01-01

    The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other...... on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

  2. Development of fluorescent methods for DNA methyltransferase assay

    Science.gov (United States)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  3. Carbon-dot-based dual-emission silica nanoparticles as a ratiometric fluorescent probe for vanadium(V) detection in mineral water samples

    Science.gov (United States)

    He, Lijun; Zhang, Heng; Fan, Huanhuan; Jiang, Xiuming; Zhao, Wenjie; Xiang, Guo Qiang

    2018-01-01

    Herein, we propose a simple and effective strategy for designing a ratiometric fluorescent nanosensor. We designed and developed a carbon dots (CDs) based dual-emission nanosensor for vanadium(V) by coating the surface of dye-doped silica nanoparticles with CDs. The fluorescence of dual-emission silica nanoparticles was quenched in acetic acid through potassium bromate (KBrO3) oxidation. V(V) could catalyze KBrO3 oxidation reaction process, resulting in the ratiometric fluorescence quenching of dual-emission silica nanoparticles. We investigated several important parameters affecting the performance of the nanosensor. Under the optimized conditions, the detection limit of this nanosensor reached 1.1 ng mL- 1 and the linear range from 10 to 800 ng mL- 1. Furthermore, we found that the sensor was suitable for determination of V(V) in different mineral water samples with satisfactory results.

  4. CsPbBr3 Perovskite Quantum Dots-Based Monolithic Electrospun Fiber Membrane as an Ultrastable and Ultrasensitive Fluorescent Sensor in Aqueous Medium.

    Science.gov (United States)

    Wang, Yuanwei; Zhu, Yihua; Huang, Jianfei; Cai, Jin; Zhu, Jingrun; Yang, Xiaoling; Shen, Jianhua; Jiang, Hao; Li, Chunzhong

    2016-11-03

    Perovskite quantum dots with excellent optical properties and robust durability stand as an appealing and desirable candidate for fluorescence resonance energy transfer (FRET) based fluorescence detection, a powerful technique featuring excellent accuracy and convenience. In this work, a monolithic superhydrophobic polystyrene fiber membrane with CsPbBr 3 perovskite quantum dots encapsulated within (CPBQDs/PS FM) was prepared via one-step electrospinning. Coupling CPBQDs with PS matrix, this CPBQDs/PS FM composite exhibits high quantum yields (∼91%), narrow half-peak width (∼16 nm), nearly 100% fluorescence retention after being exposed to water for 10 days and 79.80% fluorescence retention after 365 nm UV-light (1 mW/cm 2 ) illumination for 60 h. Thanks to the outstanding optical property of CPBQDs, an ultralow detection limit of 0.01 ppm was obtained for Rhodamine 6G (R6G) detection, with the FRET efficiency calculated to be 18.80% in 1 ppm R6G aqueous solution. Electrospun as well-designed fiber membranes, CPBQDs/PS FM composite also possesses good tailorability and recyclability, showing exciting potential for future implementation into practical applications.

  5. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    International Nuclear Information System (INIS)

    Hua, Mengjuan; Wang, Chengquan; Qian, Jing; Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-01-01

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg 2+ . The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg 2+ , the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg 2+ in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg 2+ contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg 2+ quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg 2+ detection. • The Hg 2+ content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg 2+ detection. • The ratiometric probe is of good simplicity, low toxicity, and excellent stability

  6. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Mengjuan [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013 (China); Qian, Jing, E-mail: qianj@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg{sup 2+}. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg{sup 2+}, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg{sup 2+} in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg{sup 2+} contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg{sup 2+} quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg{sup 2+} detection. • The Hg{sup 2+} content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg{sup 2+} detection. • The ratiometric probe is of good simplicity, low toxicity, and

  7. Enhancing molecular logic through modulation of temporal and spatial constraints with quantum dot-based systems that use fluorescent (Förster) resonance energy transfer

    Science.gov (United States)

    Claussen, Jonathan C.; Algar, W. Russ; Hildebrandt, Niko; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2013-10-01

    Luminescent semiconductor nanocrystals or quantum dots (QDs) contain favorable photonic properties (e.g., resistance to photobleaching, size-tunable PL, and large effective Stokes shifts) that make them well-suited for fluorescence (Förster) resonance energy transfer (FRET) based applications including monitoring proteolytic activity, elucidating the effects of nanoparticles-mediated drug delivery, and analyzing the spatial and temporal dynamics of cellular biochemical processes. Herein, we demonstrate how unique considerations of temporal and spatial constraints can be used in conjunction with QD-FRET systems to open up new avenues of scientific discovery in information processing and molecular logic circuitry. For example, by conjugating both long lifetime luminescent terbium(III) complexes (Tb) and fluorescent dyes (A647) to a single QD, we can create multiple FRET lanes that change temporally as the QD acts as both an acceptor and donor at distinct time intervals. Such temporal FRET modulation creates multi-step FRET cascades that produce a wealth of unique photoluminescence (PL) spectra that are well-suited for the construction of a photonic alphabet and photonic logic circuits. These research advances in bio-based molecular logic open the door to future applications including multiplexed biosensing and drug delivery for disease diagnostics and treatment.

  8. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

    Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

    2011-09-01

    A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. A fluorescence-based rapid screening assay for cytotoxic compounds

    International Nuclear Information System (INIS)

    Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

    2004-01-01

    A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

  10. 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

  11. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Science.gov (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  12. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Directory of Open Access Journals (Sweden)

    D Ransom Hardison

    Full Text Available Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs. One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R in certain labs. A fluorescence based receptor binding assay (RBA(F was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2 for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1. Fish (N = 61 of six different species were screened using the RBA(F. Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a correlated well (R2 = 0.71 with those of the RBA(F, given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F, which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F advantages include the long-term (> 5 years stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R. The RBA(F is cost-effective, allows high sample

  13. Evaluation of Robust Estimators Applied to Fluorescence Assays

    Directory of Open Access Journals (Sweden)

    U. Ruotsalainen

    2007-12-01

    Full Text Available We evaluated standard robust methods in the estimation of fluorescence signal in novel assays used for determining the biomolecule concentrations. The objective was to obtain an accurate and reliable estimate using as few observations as possible by decreasing the influence of outliers. We assumed the true signals to have Gaussian distribution, while no assumptions about the outliers were made. The experimental results showed that arithmetic mean performs poorly even with the modest deviations. Further, the robust methods, especially the M-estimators, performed extremely well. The results proved that the use of robust methods is advantageous in the estimation problems where noise and deviations are significant, such as in biological and medical applications.

  14. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, s...... flippase activities in the plasma membrane of cells, using yeast as an example.......P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases......, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  15. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Jennifer R. McCall

    2014-09-01

    Full Text Available Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/ veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

  16. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  17. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Science.gov (United States)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  18. Evaluation of furocoumarins as photosynthetic inhibitor by chlorophyll a fluorescence assay

    OpenAIRE

    Sampaio, Olívia Moreira; Silva, Maria Fátima das Graças Fernandes da; Veiga, Thiago Andre Moura [UNIFESP; King-Díaz, Beatriz; Lotina-Hennsen, Blas

    2012-01-01

    The evaluations of Chorophyll a fluorescence emitted by superior plants carry structural information and photosynthetic apparatus function. Quantitative analysis apparatus of fluorescence kinetic were measured by energy flows (ABS), (TR), (ET) and (DI), known as phenomenological phenomena of OJIP test. Four furocoumarins were isolated from Ruta graveolens (Rutaceae), and chorophyll a (Chl a) fluorescence assays were performed with these compounds to evaluate the photosynthesis inhibition pote...

  19. Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

    International Nuclear Information System (INIS)

    Kokko, Tiina; Kokko, Leena; Soukka, Tero; Loevgren, Timo

    2007-01-01

    A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2-2 nmol L -1 . In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay

  20. Assay of flippase activity in proteoliposomes using fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Marek, Magdalena; Günther-Pomorski, Thomas

    2016-01-01

    Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use...... of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level....

  1. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    International Nuclear Information System (INIS)

    Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

    2015-01-01

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs

  2. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yi; Keegan, Gemma L., E-mail: gemmakeegan@gmail.com [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland); Stranik, Ondrej [Leibniz Institute of Photonic Technology, Department of NanoBiophotonics (Germany); Brennan-Fournet, Margaret E. [CMP-EMSE, MOC, Department of Bioelectronics, Ecole Nationale Superieure des Mines (France); McDonagh, Colette [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland)

    2015-07-15

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs.

  3. Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Huimin; Gao, Sheng; Liu, Meng; Chang, Yangyang; Fan, Xinfei; Quan, Xie [Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, Dalian University of Technology, Dalian, 116024 (China)

    2013-07-15

    We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers. (author)

  4. Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

    International Nuclear Information System (INIS)

    Zhao, Huimin; Gao, Sheng; Liu, Meng; Chang, Yangyang; Fan, Xinfei; Quan, Xie

    2013-01-01

    We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers. (author)

  5. Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

    Science.gov (United States)

    Ding, Baoqing; Yuan, Yao-Wu

    2016-04-01

    The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

  6. Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

    Science.gov (United States)

    Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

    2017-09-01

    Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

  7. Development of laser-induced fluorescence detection to assay DNA damage

    International Nuclear Information System (INIS)

    Sharma, M.; Freund, H.G.

    1991-01-01

    A precolumn derivation method has been developed for high performance liquid chromatographic (HPLC) analysis of DNA damage using fluorescence detection. The modified nucleotide, having excised enzymatically from the exposed DNA, is enriched from the normal nucleotides and labeled with a fluorescent reagent. The labeling procedure involves phosphoramidation of the nucleotide with ethylenediamine (EDA) followed by conjugation of the free amino end of the phosphoramidate with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride. The dansylated nucleotide can be analyzed with a sub-picomole limit of detection (LOD) by conventional HPLC using a conventional fluorescence detector. By combining microbore HPLC with laser-induced fluorescence (LIF) detection, the authors present the development of an analytical system that has sub-femtomole LOD for real-time analysis of the dansylated nucleotide. In this paper the application of the developed system in fluorescence postlabeling assay of a small alkyl-modified nucleotide (5-methyl CMP) in calf-thymus DNA is discussed

  8. Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

    Science.gov (United States)

    Machida, Kazuya; Liu, Bernard

    2017-01-01

    Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

  9. Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays

    Directory of Open Access Journals (Sweden)

    Crabb Brendan S

    2010-06-01

    Full Text Available Abstract Background Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. Methods The D10 P. falciparum line was transfected to express green fluorescent protein (GFP. In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Results Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Conclusions Flow cytometry based assays using GFP-fluorescent

  10. Assay of cyclic nucleotide phosphodiesterase using radiolabeled and fluorescent substrates

    International Nuclear Information System (INIS)

    Kincaid, R.L.; Manganiello, V.C.

    1988-01-01

    There are four major classes of phosphodiesterase with different specificities for cAMP and cGMP and different allosteric regulators. Type I phosphodiesterase is activated by calmodulin plus Ca/sup 2+/ and has a higher affinity for cGMP than cAMP. Type II phosphodiesterase likewise has a higher affinity for cGMP than cAMP, but the activity toward one substrate is markedly stimulated by low (micromolar) concentrations of the other nucleotide. Type III phosphodiesterase has a higher affinity for cAMP than cGMP; its activity is increased in responsive cells by certain hormones, e.g., insulin, isoproterenol. Type IV phosphodiesterase is the cGMP-specific enzyme, which also has an allosteric binding site for cGMP. An example of this class of enzyme is the one from retinal rod outer segments, which is activated by light via rhodopsin and the guanine nucleotide-binding protein transducin. There appears to be little structural relatedness among these enzymes based on immunologic analysis, consistent with the possibility that divergent forms evolved from an ancestral enzyme. Determination of the amount of a specific form of phosphodiesterase in crude material is often difficult. Modification of assay conditions by judicious choice of substrate and/or inhibitor concentrations may selectively favor (or reduce) the activity of a particular form; in many instances, however, some fractionation of enzymes may be necessary. This is discussed more fully in the final section of this chapter

  11. Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

    Science.gov (United States)

    Wargocki, Piotr; Deng, Wei; Anwer, Ayad G; Goldys, Ewa M

    2015-05-20

    Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  12. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki

    2015-05-01

    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  13. Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication

    NARCIS (Netherlands)

    Wieten, Rosanne W.; Jonker, Emile F. F.; Pieren, Daan K. J.; Hodiamont, Caspar J.; van Thiel, Pieter P. A. M.; van Gorp, Eric C. M.; de Visser, Adriëtte W.; Grobusch, Martin P.; Visser, Leo G.; Goorhuis, Abraham

    2016-01-01

    The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and

  14. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Probe transfer with and without membrane fusion in a fluorescence fusion assay

    NARCIS (Netherlands)

    Ohki, S; Flanagan, TD; Hoekstra, D

    1998-01-01

    An analysis of the R(18) fusion assay was made during the fusion of the Sendai virus with erythrocyte ghosts. The increase in R(18) fluorescence, reflecting the interaction process, was evaluated in terms of the different processes that in principle may contribute to this increase, that is,

  16. Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence

    Directory of Open Access Journals (Sweden)

    Stefan Seeger

    2013-02-01

    Full Text Available We present an immunoassay for Interferon-γ (IFN-γ with a limit of detection of 1.9 pM (30 pg/mL and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA. The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

  17. Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

    International Nuclear Information System (INIS)

    Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Gryczynski, Ignacy; Gryczynski, Zygmunt; Luchowski, Rafal; Laursen, Bo W

    2013-01-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes. (paper)

  18. Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

    Science.gov (United States)

    Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

    2013-06-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

  19. Azadioxatriangulenium (ADOTA+): A long fluorescence lifetime fluorophore for large biomolecule binding assay

    Science.gov (United States)

    Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

    2013-01-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes. PMID:24058730

  20. Fluorescence-based assay as a new screening tool for toxic chemicals

    Science.gov (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

    2016-09-01

    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

  1. Quantum-Dot-Based Lateral Flow Immunoassay for Detection of Neonicotinoid Residues in Tea Leaves.

    Science.gov (United States)

    Wang, Shuangjie; Liu, Ying; Jiao, Shasha; Zhao, Ying; Guo, Yirong; Wang, Mengcen; Zhu, Guonian

    2017-11-22

    Neonicotinoid insecticides are commonly used for pest control on tea plantations as a result of their broad-spectrum activity. However, neonicotinoid residues released from tea leaves into tea infusions pose a dietary risk to consumers. Therefore, a rapid, sensitive, and reliable on-site detection method for neonicotinoids is needed. We developed a quantum-dot-based fluorescent lateral flow immunochromatographic strip (LFICS) combined with a broad-specific antibody for detection of typical neonicotinoids (imidacloprid, imidaclothiz, and clothianidin), with sensitivities [50% inhibitory concentration (IC 50 )] of 0.104-0.33 ng/mL and visual detection limits of 0.5-1 ng/mL. The strip assay could be completed in less than 30 min. Using the LFICS to analyze spiked tea samples (green tea, black tea, and oolong tea), the average recovery of the three neonicotinoids ranged between 71 and 111%, with the coefficient of variation below 12%. The results from the LFICS tests for field samples were consistent with results from ultraperformance liquid chromatography-tandem mass spectrometry. The newly developed strip is a useful tool for the on-site detection of neonicotinoid residues in tea.

  2. A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery

    Directory of Open Access Journals (Sweden)

    Yi Huan

    2013-04-01

    Full Text Available The sodium/glucose cotransporter 2 (SGLT2 is responsible for the majority of glucose reabsorption in the kidney, and currently, SGLT2 inhibitors are considered as promising hypoglycemic agents for the treatment of type 2 diabetes mellitus. By constructing CHO cell lines that stably express the human SGLT2 transmembrane protein, along with a fluorescent glucose transporter assay that uses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino]2-deoxyglucose (2-NBDG as a glucose analog, we have developed a nonradioactive, cell-based assay for the discovery and characterization of SGLT2 inhibitors.

  3. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    OpenAIRE

    Wargocki, Piotr; Deng, Wei; Anwer, Ayad G.; Goldys, Ewa M.

    2015-01-01

    Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate s...

  4. The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

    Science.gov (United States)

    Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

    2018-03-13

    Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

  5. A simple fluorescence based assay for quantification of human immunodeficiency virus particle release

    Directory of Open Access Journals (Sweden)

    Heuser Anke-Mareil

    2010-04-01

    Full Text Available Abstract Background The assembly and release of human immunodeficiency virus (HIV particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1 particle assembly and/or release. Results Using a fluorescently labelled HIV-derivative, which carries an eYFP domain within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release. Conclusions The fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here

  6. Cucurbiturils: molecular nanocapsules for time-resolved fluorescence-based assays.

    Science.gov (United States)

    Marquez, Cesar; Huang, Fang; Nau, Werner M

    2004-03-01

    A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4 +/- 1) x 10(5) M(-1) along with the resistance of the CB7.DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7.DBO to different environmental conditions and possible quenchers are described.

  7. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    Directory of Open Access Journals (Sweden)

    Novielli Nicole M

    2011-10-01

    Full Text Available Abstract A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells, images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P

  8. Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication.

    Science.gov (United States)

    Wieten, Rosanne W; Jonker, Emile F F; Pieren, Daan K J; Hodiamont, Caspar J; van Thiel, Pieter P A M; van Gorp, Eric C M; de Visser, Adriëtte W; Grobusch, Martin P; Visser, Leo G; Goorhuis, Abraham

    2016-03-04

    The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and the Plaque Reduction Neutralization Test) in a group of Dutch immune-compromised travellers with a median of 33 days (IQR [28-49]) after primary YF vaccination. We collected samples of 15 immune-compromised vaccinees vaccinated with the 17D yellow fever vaccine between 2004 and 2012. All samples measured in the plaque reduction neutralization test yielded positive results (>80% virus neutralization with a 1:10 serum dilution). Immune Fluorescence Assay sensitivity was 28% (95% CI [0.12-0.49]). No adverse events were reported. All immune-compromised patients mounted an adequate response with protective levels of virus neutralizing antibodies to the 17-D YF vaccine. No adverse effects were reported. Compared to the plaque reduction neutralization test, the sensitivity of the Immune Fluorescence Assay test was low. Further research is needed to ascertain that 17D vaccination in immune-compromised patients is safe. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

    Science.gov (United States)

    Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

    2012-06-01

    N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    Science.gov (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

    2016-04-01

    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  11. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  12. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

    Science.gov (United States)

    Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

    2005-05-31

    High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

  13. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

    Directory of Open Access Journals (Sweden)

    Zachary J Farino

    Full Text Available Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.

  14. Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer.

    Science.gov (United States)

    Liu, Yingxiong; Zhao, Qiang

    2017-06-01

    Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

  15. Rapid fluorescence assay for Sudan dyes using polyethyleneimine-coated copper nanoclusters

    International Nuclear Information System (INIS)

    Ling, Yu; Li, Jia Xing; Li, Nian Bing; Luo, Hong Qun; Qu, Fei

    2014-01-01

    We report that the intensity of the blue fluorescence of copper nanoclusters coated with polyethyleneimine (PEI) is strongly reduced in the presence of the food dyestuffs Sudan I-IV. This finding was exploited in a label-free fluorescence assay for these Sudan dyes both in ethanol and aqueous solutions. The PEI-capped nanoclusters have an average diameter of 1.8 nm and are displaying, under 355 nm excitation, a blue emission at 480 nm that matches the absorption bands of the Sudan dyes. The clusters are stable in solution for at least 1 month. Under optimum conditions, this assay can be applied to the quantification of the dyes Sudan I, II, III, and IV, respectively, in the 0.1−30, 0.1–30, 0.1–25, and 0.1–25 μM concentration ranges, and the detection limits (3σ/slope) are 65, 70, 45, and 50 nM, respectively. The capability of reducing the fluorescence of the PEI-capped copper nanoclusters is directly related to the number of the functional groups in that Sudan III and IV give lower detection limits. This analytical scheme exhibits a remarkably high selectivity for the Sudan dyes over potentially interfering substances. The method was successfully applied to determine Sudan I, II, III, and IV in hot chilli powder. (author)

  16. Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates

    DEFF Research Database (Denmark)

    Robardet, E.; Andrieu, S.; Rasmussen, Thomas Bruun

    2013-01-01

    Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive...

  17. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

    Science.gov (United States)

    Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

    2018-06-05

    The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

  18. Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

    Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

  19. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    Directory of Open Access Journals (Sweden)

    Hovhannisyan Galina G

    2010-09-01

    Full Text Available Abstract Comet assay and micronucleus (MN test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.

  20. Precious metal assay analysis of fresh reforming catalyst by x-ray fluorescence spectrometry

    International Nuclear Information System (INIS)

    McElroy, F.C.; Mulhall, J.M.

    1991-01-01

    This paper reports that precious metal analysis of fresh reforming catalysts are typically performed by both the catalyst manufacturer and buyer to arrive at a financial settlement on the quantity of metal in each lot of commercial catalyst. Traditional assay methods involve a variety of fire assay or wet chemical acid digestion schemes coupled with gravimetric, colorimetic, or titrimetric measurement for precious metals. Methods must have sufficient precision and accuracy to afford interlaboratory agreement of within one half of one percent relative between the catalyst supplier and purchaser. To meet this requirement many laboratories rely on classical methods. Unfortunately these proceeders are labor intensive and time consuming. X-ray fluorescence has the inherent instrument precision to achieve typical intralaboratory precision of 0.5% RSD on a wide variety of elements and numerous sample types. We have developed an X-ray fluorescence method for the assay quality analysis of fresh reforming catalyst containing platinum, rhenium, and iridium. This method was applied to numerous samples over the past five years

  1. A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

    International Nuclear Information System (INIS)

    Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

    2011-01-01

    Highlights: ► A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. ► We have established an activity assay for ubiquitin C-terminal hydrolases. ► This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub F45W -Xaa) and di-ubiquitin chains (Ub F45W -diUb). After removal of the intact substrate by Ni 2+ -NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub F45W product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

  2. Detection of NT-pro BNP using fluorescent protein modified by streptavidin as a label in immunochromatographic assay

    Directory of Open Access Journals (Sweden)

    Haixia Li

    2016-12-01

    Full Text Available A novel fluorescent immunochromatographic assay for the detection of NT-proBNP in human serum has been developed. Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody labeled with fluorescent protein and “sandwiched” by another monoclonal antibody immobilized on the nitrocellulose membrane, the fluorescence and concentration of analytes were measured and then calculated by fluoroanalyzer. The fluorescent protein is a fusion protein and was prepared through the application of Streptavidin gene SA, β subunit cpcB of Phycocyanin, lyase alr0617, and phycoerythrobilin synthetase gene ho1, pebA, pebB for covalent binding. It is characterized with higher stability, good solubility in water and it is not easy to quench fluorescence. Take the advantages of fluorescent protein, the immunochromatographic assay exhibited a wide linear range for NT-proBNP from 200 pg ml−1 to 26,000 pg ml−1, with a detection limit of 47 pg ml−1 under optimal conditions. Compared with chemiluminescence immunoassay (CLIA, 131 human serum samples were analyzed and the correlation coefficient of the developed immunoassay was 0.978. These results demonstrated that fluorescent immunochromatographic assay is a more rapid, sensitive, specific method and could be developed into a platform for more biomarkers determination in clinical practice. Keywords: NT-pro BNP, Fluorescent protein, Immunochromatographic assay

  3. A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

    Science.gov (United States)

    Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

    2014-05-15

    In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

    Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

  5. Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

    Science.gov (United States)

    Schnabel, Christiane L; Babasyan, Susanna; Freer, Heather; Wagner, Bettina

    2017-06-01

    Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the

  6. Development of fluorescence imaging-based assay for screening cardioprotective compounds from medicinal plants.

    Science.gov (United States)

    Wang, Yi; Zhao, Xiaoping; Gao, Xiumei; Nie, Xiaojing; Yang, Yingxin; Fan, Xiaohui

    2011-09-19

    Medicinal plants have been widely recognized as a renewable resource for the discovery of novel leads and drug. In this study, an approach for screening and identification compounds with cardioprotective activity from medicinal plant extracts by cellular-fluorescence imaging technique was developed. It is a cell-based assay for measuring mitochondrial membrane potential changes in H9c2 cardiac muscle cells exposed to H(2)O(2) by using a fluorescence automatic microscopy screening platform. Rhodamine 123 was used as the fluorescent dye to indicate the change of mitochondrial membrane potential. The sensitivity and linear range of the proposed approach were evaluated and validated using vitamin C, an antioxidative compound. The method was applied to screen active components with potent cardioprotective effects from a traditional Chinese formula. The potential cardioprotective components were identified by liquid chromatography coupled with mass spectrometry (LC/MS). Moreover, the utility of the proposed approach was further validated by three compounds (salvianolic acid B, protocatechuic aldehyde, and tanshinone II A) identified from the formula which showed cardioprotective effects in a dose-dependent manner. These applications suggested that the proposed rapid and sensitive screening approach offers an efficient way to discover active components or compounds from medicinal plants. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Science.gov (United States)

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  8. A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels

    Directory of Open Access Journals (Sweden)

    Cerrone Cabanos

    2017-08-01

    Full Text Available Lipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP2 and phosphatidic acid (PA has emerged as a central cellular mechanism for controlling ion channels and the excitability of nerves. A lack of robust assays suitable for facile detection of a lipid bound to a channel has hampered the probing of the lipid binding sites and measuring the pharmacology of putative lipid agonists for ion channels. Here, we show a fluorescent PIP2 competition assay for detergent-purified potassium channels, including TWIK-1-related K+-channel (TREK-1. Anionic lipids PA and phosphatidylglycerol (PG bind dose dependently (9.1 and 96 μM, respectively and agonize the channel. Our assay shows PIP2 binds with high affinity (0.87 μM but surprisingly can directly antagonize TREK-1 in liposomes. We propose a model for TREK-1 lipid regulation where PIP2 can compete with PA and PG agonism based on the affinity of the lipid for a site within the channel.

  9. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2018-06-01

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  10. A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

    Science.gov (United States)

    Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

    2014-11-11

    Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

  11. Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

    International Nuclear Information System (INIS)

    Gang, Jong Back

    2015-01-01

    Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO

  12. A note on the assay of special nuclear materials in solution by x-ray fluorescence

    International Nuclear Information System (INIS)

    Canada, T.R.; Hsue, S.T.

    1982-01-01

    Presents a formulation that allows empirical results of the ''internal standard'' approach to be understood in a quantifiable manner, and suggests an alternative measurement procedure that removes many of the technique's undesirable features while maintaining those that add to instrumental accuracy. Assumes that the reader is familiar with x-ray fluorescence (XRF) technology. Promises a more detailed presentation, including proof-of-principle experimental results, in the future. Points out that practical applications of this approach may be achieved with both K- and L-x-ray fluorescence. Concludes that the formulation and alternative measurement procedure suggested indicates that the ''internal standard'' approach may be improved by making measurements at one or more additional x-ray energies of the element to be assayed. Effects of solution acidity variations and the relative concentrations of plutonium and uranium may be avoided. Because of the inherent stability of ratio techniques, little or no modification to this formulation is anticipated for cylindrical near-field geometries

  13. BMVC test, an improved fluorescence assay for detection of malignant pleural effusions

    International Nuclear Information System (INIS)

    Lin, I-Ting; Tsai, Yu-Lin; Kang, Chi-Chih; Huang, Wei-Chun; Wang, Chiung-Lin; Lin, Mei-Ying; Lou, Pei-Jen; Shih, Jin-Yuan; Wang, Hao-Chien; Wu, Huey-Dong; Tsai, Tzu-Hsiu; Jan, I-Shiow; Chang, Ta-Chau

    2014-01-01

    The diagnosis of malignant pleural effusions is an important issue in the management of malignancy patients. Generally, cytologic examination is a routine diagnostic technique. However, morphological interpretation of cytology is sometimes inconclusive. Here an ancillary method named BMVC test is developed for rapid detection of malignant pleural effusion to improve the diagnostic accuracy at low cost. A simple assay kit is designed to collect living cells from clinical pleural effusion and a fluorescence probe, 3,6-Bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), is used to illuminate malignant cells. The fluorescence intensity is quantitatively analyzed by ImageJ program. This method yields digital numbers for the test results without any grey zone or ambiguities in the current cytology tests due to intra-observer and inter-observer variability. Comparing with results from double-blind cytologic examination, this simple test gives a good discrimination between malignant and benign specimens with sensitivity of 89.4% (42/47) and specificity of 93.3% (56/60) for diagnosis of malignant pleural effusion. BMVC test provides accurate results in a short time period, and the digital output could assist cytologic examination to become more objective and clear-cut. This is a convenient ancillary tool for detection of malignant pleural effusions

  14. Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

    Science.gov (United States)

    Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

    2007-08-15

    In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

  15. Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

    Science.gov (United States)

    Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-12-26

    The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

  16. Exploration of the Fluorescent Properties and the Modulated Activities against Sirtuin Fluorogenic Assays of Chromenone-Derived Natural Products

    Directory of Open Access Journals (Sweden)

    Hui Wen

    2018-05-01

    Full Text Available Chromenone-derived natural products include chromones (flavone, isoflavone and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC. Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 μM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.

  17. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts

    Directory of Open Access Journals (Sweden)

    Luc Séro

    2013-11-01

    Full Text Available Advanced glycation end-products (AGEs are involved in the pathogenesis of numerous diseases. Among them, cellular accumulation of AGEs contributes to vascular complications in diabetes. Besides using drugs to lower blood sugar, a balanced diet and the intake of herbal products potentially limiting AGE formation could be considered beneficial for patients’ health. The current paper presents a simple and cheap high-throughput screening (HTS assay based on AGE fluorescence and suitable for plant extract screening. We have already implemented an HTS assay based on vesperlysines-like fluorescing AGEs quickly (24 h formed from BSA and ribose under physiological conditions. However, interference was noted when fluorescent compounds and/or complex mixtures were tested. To overcome these problems and apply this HTS assay to plant extracts, we developed a technique for systematic quantification of both vesperlysines (λexc 370 nm; λem 440 nm and pentosidine-like (λexc 335 nm; λem 385 nm AGEs. In a batch of medicinal and food plant extracts, hits were selected as soon as fluorescence decreased under a fixed threshold for at least one wavelength. Hits revealed during this study appeared to contain well-known and powerful anti-AGE substances, thus demonstrating the suitability of this assay for screening crude extracts (0.1 mg/mL. Finally, quercetin was found to be a more powerful reference compound than aminoguanidine in such assay.

  18. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Directory of Open Access Journals (Sweden)

    Prerna Grover

    Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

  19. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

    OpenAIRE

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-thr...

  20. A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

    Science.gov (United States)

    Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

    2015-12-15

    Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Kristiansen, Uffe

    2004-01-01

    In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput scr...... not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.......In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput...... ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited alpha1 GlyR function, the potentiation of alpha1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may...

  2. A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Dyhring, Tino

    2011-01-01

    PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ...

  3. Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

    Science.gov (United States)

    Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

    2015-06-01

    Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Statistical uncertainties of nondestructive assay for spent nuclear fuel by using nuclear resonance fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Shizuma, Toshiyuki, E-mail: shizuma.toshiyuki@jaea.go.jp [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Hayakawa, Takehito; Angell, Christopher T.; Hajima, Ryoichi [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Minato, Futoshi; Suyama, Kenya [Nuclear Science and Engineering Directorate, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Seya, Michio [Integrated Support Center for Nuclear Nonproliferation and Nuclear Security, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1198 (Japan); Johnson, Micah S. [Lawrence Livermore National Laboratory, 7000 East Ave. Livermore, CA 94550 (United States); Department of Physics and Astronomy, San Jose State University, One Washington Square, San Jose, CA 9519 (United States); McNabb, Dennis P. [Lawrence Livermore National Laboratory, 7000 East Ave. Livermore, CA 94550 (United States)

    2014-02-11

    We estimated statistical uncertainties of a nondestructive assay system using nuclear resonance fluorescence (NRF) for spent nuclear fuel including low-concentrations of actinide nuclei with an intense, mono-energetic photon beam. Background counts from radioactive materials inside the spent fuel were calculated with the ORIGEN2.2-UPJ burn-up computer code. Coherent scattering contribution associated with Rayleigh, nuclear Thomson, and Delbrück scattering was also considered. The energy of the coherent scattering overlaps with that of NRF transitions to the ground state. Here, we propose to measure NRF transitions to the first excited state to avoid the coherent scattering contribution. Assuming that the total NRF cross-sections are in the range of 3–100 eV b at excitation energies of 2.25, 3.5, and 5 MeV, statistical uncertainties of the NRF measurement were estimated. We concluded that it is possible to assay 1% actinide content in the spent fuel with 2.2–3.2% statistical precision during 4000 s measurement time for the total integrated cross-section of 30 eV b at excitation energies of 3.5–5 MeV by using a photon beam with an intensity of 10{sup 6} photons/s/eV. We also examined both the experimental and theoretical NRF cross-sections for actinide nuclei. The calculation based on the quasi-particle random phase approximation suggests the existence of strong magnetic dipole resonances at excitation energies ranging from 2 to 6 MeV with the scattering cross-sections of tens eV b around 5 MeV in {sup 238}U.

  5. Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

    International Nuclear Information System (INIS)

    Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

    2010-01-01

    A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

  6. Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

    Science.gov (United States)

    Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

    2004-12-01

    The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

  7. 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.

  8. A Novel Quantum Dots-Based Point of Care Test for Syphilis

    Science.gov (United States)

    Yang, Hao; Li, Ding; He, Rong; Guo, Qin; Wang, Kan; Zhang, Xueqing; Huang, Peng; Cui, Daxiang

    2010-05-01

    One-step lateral flow test is recommended as the first line screening of syphilis for primary healthcare settings in developing countries. However, it generally shows low sensitivity. We describe here the development of a novel fluorescent POC (Point Of Care) test method to be used for screening for syphilis. The method was designed to combine the rapidness of lateral flow test and sensitiveness of fluorescent method. 50 syphilis-positive specimens and 50 healthy specimens conformed by Treponema pallidum particle agglutination (TPPA) were tested with Quantum Dot-labeled and colloidal gold-labeled lateral flow test strips, respectively. The results showed that both sensitivity and specificity of the quantum dots-based method reached up to 100% (95% confidence interval [CI], 91-100%), while those of the colloidal gold-based method were 82% (95% CI, 68-91%) and 100% (95% CI, 91-100%), respectively. In addition, the naked-eye detection limit of quantum dot-based method could achieve 2 ng/ml of anti-TP47 polyclonal antibodies purified by affinity chromatography with TP47 antigen, which was tenfold higher than that of colloidal gold-based method. In conclusion, the quantum dots were found to be suitable for labels of lateral flow test strip. Its ease of use, sensitiveness and low cost make it well-suited for population-based on-the-site syphilis screening.

  9. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Science.gov (United States)

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  10. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

    Full Text Available A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10% were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  11. Smart Drug Delivery System-Inspired Enzyme-Linked Immunosorbent Assay Based on Fluorescence Resonance Energy Transfer and Allochroic Effect Induced Dual-Modal Colorimetric and Fluorescent Detection.

    Science.gov (United States)

    Miao, Luyang; Zhu, Chengzhou; Jiao, Lei; Li, He; Du, Dan; Lin, Yuehe; Wei, Qin

    2018-02-06

    Numerous analytical techniques have been undertaken for the detection of protein biomarkers because of their extensive and significant applications in clinical diagnosis, whereas there are few strategies to develop dual-readout immunosensors to achieve more accurate results. To the best of our knowledge, inspired by smart drug delivery system (DDS), a novel pH-responsive modified enzyme-linked immunosorbent assay (ELISA) was innovatively developed for the first time, realizing dual-modal colorimetric and fluorescent detection of cardiac troponin I (cTnI). Curcumin (CUR) was elaborately selected as a reporter molecule, which played the same role of drugs in DDS based on the following considerations: (1) CUR can be used as a kind of pH indicator by the inherited allochroic effect induced by basic pH value; (2) the fluorescence of CUR can be quenched by certain nanocarriers as the acceptor because of the occurrence of fluorescence resonance energy transfer (FRET), while recovered by the stimuli of basic pH value, which can produce "signal-on" fluorescence detection. Three-dimensional MoS 2 nanoflowers (3D-MoS 2 NFs) were employed in immobilizing CUR to constitute a nanoprobe for the determination of cTnI by virtue of good biocompatibility, high absorption capacity, and fluorescence quench efficiency toward CUR. The proposed DDS-inspired ELISA offered dual-modal colorimetric and fluorescent detection of cTnI, thereby meeting the reliable and precise analysis requirements. We believe that the developed dual-readout ELISA will create a new avenue and bring innovative inspirations for biological detections.

  12. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    International Nuclear Information System (INIS)

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-01-01

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

  13. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    Energy Technology Data Exchange (ETDEWEB)

    Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  14. Real-time fluorescence assay of alkaline phosphatase in living cells using boron-doped graphene quantum dots as fluorophores.

    Science.gov (United States)

    Chen, Li; Yang, Guancao; Wu, Ping; Cai, Chenxin

    2017-10-15

    This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce 3+ ions because of the coordination of Ce 3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce 3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL -1 ). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce 3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    Directory of Open Access Journals (Sweden)

    Atul Asati

    Full Text Available Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA

  16. Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.

    Science.gov (United States)

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M

    2011-08-01

    The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.

  17. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-01-01

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  18. Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

    Science.gov (United States)

    Jacobs, K R; Guillemin, G J; Lovejoy, D B

    2018-02-01

    Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

  19. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    Science.gov (United States)

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

  20. Highly sensitive and selective fluorescent assay for guanine based on the Cu(2+)/eosin Y system.

    Science.gov (United States)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-15

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu(2+)/eosin Y. Cu(2+) interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu(2+)/eosin Y system, guanine reacted with Cu(2+) to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L(-1) and a linear range of 3.3-116 nmol L(-1). The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    Science.gov (United States)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  2. Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay.

    Science.gov (United States)

    Li, Qunyi; Rottländer, Mario; Xu, Mingkai; Christoffersen, Claus Tornby; Frederiksen, Kristen; Wang, Ming-Wei; Jensen, Henrik Sindal

    2011-11-01

    To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC(50) values of the positive control compound (retigabine) determined by the thallium and (86)rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC(50) values of less than 20 μmol/L and 2 demonstrated EC(50) values of less than 1 μmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75(NTR)

    DEFF Research Database (Denmark)

    Skeldal, Sune; Kjaergaard, Maj M; Alwasel, Saleh

    2015-01-01

    the vps10p domain receptor sortilin and the neurotrophin receptor p75(NTR). However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay...

  4. A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays

    Directory of Open Access Journals (Sweden)

    Linda G. Lee

    2013-10-01

    Full Text Available We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting.

  5. A Dansyl Fluorescence-Based Assay for Monitoring Kinetics of Lipid Extraction and Transfer

    Science.gov (United States)

    Ran, Yong

    2008-01-01

    Lipid transfer proteins (LTPs) play important roles in cellular biology, and fluorescence spectroscopy has found wide range use as a facile means for time-resolved monitoring of protein-lipid interactions[1]. Here, we show how the fluorescence emission properties of dansyl-DHPE can be exploited to characterize lipid extraction and lipid transfer kinetics. The GM2 activator protein serves as an example LTP where the ability to independently characterize lipid extraction from donor vesicles, formation of a protein:lipid complex in solution, and release of lipid from the complex to acceptor liposomes is crucial for full kinetic characterization of lipid transfer. PMID:18694718

  6. Rapid algal toxicity assay using variable chlorophyll fluorescence for Chlorella kessleri (Chlorophyta)

    Czech Academy of Sciences Publication Activity Database

    Kvíderová, Jana

    2010-01-01

    Roč. 25, č. 6 (2010), s. 554-562 ISSN 1520-4081 R&D Projects: GA MŠk 1M0571 Institutional research plan: CEZ:AV0Z60050516 Keywords : bioassay * variable chlorophyll fluorescence * Chlorella kessleri Subject RIV: EF - Botanics Impact factor: 1.932, year: 2010

  7. Macromolecule biosynthesis assay and fluorescence spectroscopy methods to explore antimicrobial peptide mode(s) of action

    DEFF Research Database (Denmark)

    Jana, Bimal; Baker, Kristin Renee; Guardabassi, Luca

    2017-01-01

    the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods...

  8. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    Science.gov (United States)

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

  9. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    Energy Technology Data Exchange (ETDEWEB)

    Otero, Paz; Alfonso, Amparo [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain); Alfonso, Carmen [CIFGA Laboratorio, Plaza de Santo Domingo, 1, 27001 Lugo (Spain); Araoz, Romulo; Molgo, Jordi [CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie et Developpement UPR3294, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex (France); Vieytes, Mercedes R. [Departamento de Fisiologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo (Spain); Botana, Luis M., E-mail: luis.botana@usc.es [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain)

    2011-09-09

    Highlights: {yields} A direct assay based in the binding of nAChR to spirolide toxins by FP is described. {yields} A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. {yields} FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. {yields} FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples. - Abstract: In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 {mu}g kg{sup -1} meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

  10. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    International Nuclear Information System (INIS)

    Otero, Paz; Alfonso, Amparo; Alfonso, Carmen; Araoz, Romulo; Molgo, Jordi; Vieytes, Mercedes R.; Botana, Luis M.

    2011-01-01

    Highlights: → A direct assay based in the binding of nAChR to spirolide toxins by FP is described. → A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. → FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. → FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples. - Abstract: In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg -1 meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

  11. Homogeneous competitive assay of ligand affinities based on quenching fluorescence of tyrosine/tryptophan residues in a protein via Főrster-resonance-energy-transfer

    Science.gov (United States)

    Xie, Yanling; Yang, Xiaolan; Pu, Jun; Zhao, Yunsheng; Zhang, Ying; Xie, Guoming; Zheng, Jun; Yuan, Huidong; Liao, Fei

    2010-11-01

    A new homogeneous competitive assay of ligand affinities was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Főrster-resonance-energy-transfer using a fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a protein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate ligand. Chemometrics for deriving the binding ratio of the acceptor with either fluorescence signal was discussed; the dissociation constant ( Kd) of a nonfluorescent candidate ligand was calculated from its concentration to displace 50% binding of the acceptor. N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) and N-biotinyl-N'-dansyl-ethylenediamine (BDEDA) were used as the reference ligands and acceptors to streptavidin to test this new homogeneous competitive assay. Upon binding of an acceptor to streptavidin, there were the quench of streptavidin fluorescence at 340 nm and the characteristic fluorescence at 430 nm for BNEDA or at 525 nm for BDEDA. Kd of BNEDA and BDEDA was obtained via competitive binding against biotin. By quantifying BNEDA fluorescence, Kd of each tested nonfluorescent biotin derivative was consistent with that by quantifying streptavidin fluorescence using BNEDA or BDEDA as the acceptor. The overall coefficients of variation were about 10%. Therefore, this homogeneous competitive assay was effective and promising to high-throughput-screening.

  12. Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

    International Nuclear Information System (INIS)

    Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

    2016-01-01

    A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed a RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive 14 C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of

  13. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  14. A novel fluorescent assay for edaravone with aqueous functional CdSe quantum dots

    Science.gov (United States)

    Liao, Ping; Yan, Zheng-Yu; Xu, Zhi-Ji; Sun, Xiao

    2009-06-01

    Aqueous thiol-capped CdSe QDs with a narrow, symmetric emission were prepared under a low temperature. Based on the fluorescence enhancement of thiol-stabilized CdSe quantum dots (QDs) caused by edaravone, a simple, rapid and specific quantitative method was proposed to the edaravone determination. The concentration dependence of fluorescence intensity followed the binding of edaravone to surface of the thiol-capped CdSe QDs was effectively described by a modified Langmuir-type binding isotherm. Factors affecting the fluorescence detection for edaravone with thiol-stabilized CdSe QDs were studied, such as the effect of pH, reaction time, the concentration of CdSe QDs and so on. Under the optimal conditions, the calibration plot of C/( I - I0) with concentration of edaravone was linear in the range of (1.45-17.42) μg/mL (0.008-0.1 μmol/L) with correlation coefficient of 0.998. The limit of detection (LOD) (3 σ/ κ) was 0.15 μg/mL (0.0009 μmol/mL). Possible interaction mechanism was discussed.

  15. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

    Science.gov (United States)

    One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

  16. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two rec...

  17. A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

    Science.gov (United States)

    Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

    2012-01-01

    Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

  18. Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

    Science.gov (United States)

    Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-01-15

    Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

  19. Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

    Science.gov (United States)

    Dato, Florian M; Maaßen, Andreas; Goldfuß, Bernd; Pietsch, Markus

    2018-04-01

    Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V max /K m of 1.09 and 0.134 mL min -1 mg -1 , respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL -1 ) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively). Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites.

    Science.gov (United States)

    Deng, Haiyan; Liu, Huayin; Krogstad, Frances M; Krogstad, Donald J

    2006-04-03

    A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid-solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients > or = 0.997 (r2 > or = 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) < or = 3.8% for AQ-13 and its metabolites, and < or =2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 microl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 microl.

  1. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucas, Rodrigo Ippolito [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Trindade, Edvaldo S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, Parana (Brazil); Tersariol, Ivarne L.S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP (Brazil); Dietrich, Carl P. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Nader, Helena B. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil)], E-mail: hbnader.bioq@epm.br

    2008-06-23

    Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

  2. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

    International Nuclear Information System (INIS)

    Boucas, Rodrigo Ippolito; Trindade, Edvaldo S.; Tersariol, Ivarne L.S.; Dietrich, Carl P.; Nader, Helena B.

    2008-01-01

    Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture

  3. Combining reflectometry and fluorescence microscopy: an assay for the investigation of leakage processes across lipid membranes.

    Science.gov (United States)

    Stephan, Milena; Mey, Ingo; Steinem, Claudia; Janshoff, Andreas

    2014-02-04

    The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.

  4. Fluorescence assay for mitochondrial permeability transition in cardiomyocytes cultured in a microtiter plate

    DEFF Research Database (Denmark)

    Christensen, Marie Louise Muff; Braunstein, Thomas Hartig; Treiman, Marek

    2008-01-01

    Mitochondrial permeability transition pore (MPTP) is a voltage-dependent, large-conductance channel of the inner mitochondrial membrane with an important role in a range of pathophysiological conditions. To facilitate studies of pharmacological pore modulation, we describe an assay in a model using......, dequenching occurred on the distribution of rhodamine-123 into the extracellular volume. The addition of a small buffer volume containing digitonin (final concentration 10 microg/ml) and Ca(2+) (final concentrations up to 100 microM free Ca(2+)) caused dequenching (Delta F) due to Delta Psi m dissipation...

  5. Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays.

    Science.gov (United States)

    Hau, Jean Christophe; Fontana, Patrizia; Zimmermann, Catherine; De Pover, Alain; Erdmann, Dirk; Chène, Patrick

    2011-06-01

    The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

  6. Establishment of 60Co dose calibration curve using fluorescent in situ hybridization assay technique: Result of preliminary study

    International Nuclear Information System (INIS)

    Rahimah Abdul Rahim; Noriah Jamal; Noraisyah Mohd Yusof; Juliana Mahamad Napiah; Nelly Bo Nai Lee

    2010-01-01

    This study aims at establishing an in-vitro 60 Co dose calibration curve using Fluorescent In-Situ Hybridization assay technique for the Malaysian National Bio dosimetry Laboratory. Blood samples collected from a female healthy donor were irradiated with several doses of 60 Co radiation. Following culturing of lymphocytes, microscopic slides are prepared, denatured and hybridized. The frequencies of translocation are estimated in the metaphases. A calibration curve was then generated using a regression technique. It shows a good fit to a linear-quadratic model. The results of this study might be useful in estimating absorbed dose for the individual exposed to ionizing radiation retrospectively. This information may be useful as a guide for medical treatment for the assessment of possible health consequences. (author)

  7. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  8. Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays.

    Science.gov (United States)

    Serrot, Patricia H; Sabater, Bartolomé; Martín, Mercedes

    2012-09-01

    Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3' end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5' end shortened by 18 codons with respect to that of angiosperms. Copyright © Physiologia Plantarum 2012.

  9. Assessment of the Inhibitory Effect of Rifampicin on Amyloid Formation of Hen Egg White Lysozyme: Thioflavin T Fluorescence Assay versus FTIR Difference Spectroscopy

    Directory of Open Access Journals (Sweden)

    Gang Ma

    2014-01-01

    Full Text Available The inhibitory effect of rifampicin on the amyloid formation of hen egg white lysozyme was assessed with both Thioflavin T (ThT fluorescence assay and Fourier transform infrared (FTIR difference spectroscopy. We reveal that ThT fluorescence assay gives a false positive result due to rifampicin interference, while FTIR difference spectroscopy provides a reliable assessment. With FTIR, we show that rifampicin only has marginally inhibitory effect. We then propose that FTIR difference spectroscopy can potentially be a convenient method for inhibitor screening in amyloid study.

  10. Ultrasensitive Quantum Dot Fluorescence quenching Assay for Selective Detection of Mercury Ions in Drinking Water

    Science.gov (United States)

    Ke, Jun; Li, Xinyong; Zhao, Qidong; Hou, Yang; Chen, Junhong

    2014-07-01

    Mercury is one of the most acutely toxic substances at trace level to human health and living thing. Developing a rapid, cheap and water soluble metal sensor for detecting mercury ions at ppb level remains a challenge. Herein, a metal sensor consisting of MPA coated Mn doped ZnSe/ZnS colloidal nanoparticles was utilized to ultrasensitively and selectively detect Hg2+ ions with a low detection limit (0.1 nM) over a dynamic range from 0 to 20 nM. According to strong interaction between thiol(s) and mercury ions, mercaptopropionic acid (MPA) was used as a highly unique acceptor for mercury ions in the as-obtained ultrasensitive sensor. In the presence of mercury ions, colloidal nanoparticles rapidly agglomerated due to changes of surface chemical properties, which results in severe quenching of fluorescent intensity. Meanwhile, we find that the original ligands are separated from the surface of colloidal nanoparticles involving strongly chelation between mercury ion and thiol(s) proved by controlled IR analysis. The result shows that the QD-based metal ions sensor possesses satisfactory precision, high sensitivity and selectivity, and could be applied for the quantification analysis of real samples.

  11. An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

    Science.gov (United States)

    He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

    2016-04-18

    Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    Science.gov (United States)

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-01-01

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

  13. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

    Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

  14. Quantum dot-based molecular beacon to monitor intracellular microRNAs.

    Science.gov (United States)

    Lee, Jonghwan; Moon, Sung Ung; Lee, Yong Seung; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Ali, Daoud; Ahmed, Javed; Al Salem, Abdullah M; Kim, Soonhag

    2015-06-02

    Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

  15. Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs

    Directory of Open Access Journals (Sweden)

    Jonghwan Lee

    2015-06-01

    Full Text Available Fluorescence monitoring of endogenous microRNA (miRNA or miR activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon. The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide. The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1. In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

  16. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    Science.gov (United States)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  17. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Directory of Open Access Journals (Sweden)

    Xingmei Xie

    Full Text Available Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR. Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY, five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377, one X/Y-common STR (X22, and two autosomal STRs (D13S305 and D21S11. Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

  18. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  19. Chromosome analysis of nuclear power plant workers using fluorescence in situ hybridization and Giemsa assay

    International Nuclear Information System (INIS)

    Hristova, Rositsa; Hadjidekova, Valeria; Grigorova, Mira; Nikolova, Teodora; Bulanova, Minka; Popova, Ljubomira; Staynova, Albena; Benova, Donka

    2013-01-01

    The aim of this study was to evaluate the genotoxic effects of ionizing radiation in vivo in exposed Bulgarian nuclear power plant workers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes. Chromosome analysis using fluorescence in situ hybrydization (FISH) and Giemsa techniques was undertaken on 63 workers and 45 administrative staff controls from the Bulgarian Nuclear Power Plant. Using the Giemsa method, the frequencies of cells studied with chromosome aberrations, dicentrics plus rings and chromosome fragments in the radiation workers were significantly higher compared with the control group (P=0.044, P=0.014, and P=0.033, respectively). A significant association between frequencies of dicentrics plus rings and accumulated doses was registered (P<0.01). In the present study, a FISH cocktail of whole chromosome paints for chromosomes 1, 4 and 11 was used. A significant association between frequency of translocations and accumulated doses was also observed (P<0.001). Within the control group, a correlation was found between age and the spontaneous frequency of translocations. No correlation was found between smoking status and frequency of translocations. When compared with the control group, workers with accumulated doses up to 100 mSv showed no increase in genome translocation frequency, whereas workers with accumulated doses from 101 to 200 mSv showed a statistically significant doubling of genome translocation frequency (P=0.009). Thus, in cases of chronic exposure and for purposes of retrospective dosimetry, the genome frequency of translocations is a more useful marker for evaluation of genotoxic effects than dicentric frequency. (author)

  20. Multiple exciton generation in quantum dot-based solar cells

    Science.gov (United States)

    Goodwin, Heather; Jellicoe, Tom C.; Davis, Nathaniel J. L. K.; Böhm, Marcus L.

    2018-01-01

    Multiple exciton generation (MEG) in quantum-confined semiconductors is the process by which multiple bound charge-carrier pairs are generated after absorption of a single high-energy photon. Such charge-carrier multiplication effects have been highlighted as particularly beneficial for solar cells where they have the potential to increase the photocurrent significantly. Indeed, recent research efforts have proved that more than one charge-carrier pair per incident solar photon can be extracted in photovoltaic devices incorporating quantum-confined semiconductors. While these proof-of-concept applications underline the potential of MEG in solar cells, the impact of the carrier multiplication effect on the device performance remains rather low. This review covers recent advancements in the understanding and application of MEG as a photocurrent-enhancing mechanism in quantum dot-based photovoltaics.

  1. STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

    Science.gov (United States)

    Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

    2012-01-01

    To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

  2. A new assay format for NF-kappaB based on a DNA triple helix and a fluorescence resonance energy transfer.

    Science.gov (United States)

    Altevogt, Dominik; Hrenn, Andrea; Kern, Claudia; Clima, Lilia; Bannwarth, Willi; Merfort, Irmgard

    2009-10-07

    Herein we report a feasibility study for a new concept to detect DNA binding protein NF-kappaB based on a DNA triple helix formation in combination with a fluorescence resonance energy transfer (FRET). The new principle avoids expensive antibodies and radioactivity and might have implications for assays of other DNA binding proteins.

  3. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  4. SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

    Science.gov (United States)

    A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

  5. Aptamer based fluorescent cocaine assay based on the use of graphene oxide and exonuclease III-assisted signal amplification

    International Nuclear Information System (INIS)

    Zhang, Yulin; Zhang, Guo-Jun; Sun, Zhongyue; Tang, Lina; Zhang, Hong

    2016-01-01

    The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12

  6. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  7. Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Bräuner-Osborne, Hans

    2004-01-01

    We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential...... (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics...

  8. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

    Science.gov (United States)

    Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

    2016-01-01

    In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

  9. Quantum Dot-based Immunohistochemistry for Pathological Applications

    Directory of Open Access Journals (Sweden)

    Li Zhou

    2016-01-01

    Full Text Available Quantum dots (QDs are novel light emitting semiconductor nanocrystals with diameter ranging from 2 to 20 nm. In comparison with traditional organic dyes and fluorescent proteins, QDs possess unique optical properties including extremely high fluorescence efficiency and minimal photobleaching which make them emerge as a new class of fluorescent labels for molecular imaging and biomedical analysis. Herein, recent advances in fundamental mechanisms and pathological applications of QD were reviewed.

  10. Exploring the dynamics of fluorescence staining of bacteria with cyanine dyes for the development of kinetic assays

    Science.gov (United States)

    Thomas, Marlon Sheldon

    Bacterial infections continue to be one of the major health risks in the United States. The common occurrence of such infection is one of the major contributors to the high cost of health care and significant patient mortality. The work presented in this thesis describes spectroscopic studies that will contribute to the development of a fluorescent assay that may allow the rapid identification of bacterial species. Herein, the optical interactions between six bacterial species and a series of thiacyanine dyes are investigated. The interactions between the dyes and the bacterial species are hypothesized to be species-specific. For this thesis, two Gram-negative strains, Escherichia coli (E. coli) TOP10 and Enterobacter aerogenes; two Gram-positive bacterial strains, Bacillus sphaericus and Bacillus subtilis; and two Bacillus endospores, B. globigii and B. thuringiensis, were used to test the proposed hypothesis. A series of three thiacyanine dyes---3,3'-diethylthiacyanine iodide (THIA), 3,3'-diethylthiacarbocyanine iodide (THC) and thiazole orange (THO)---were used as fluorescent probes. The basis of our spectroscopic study was to explore the bacterium-induced interactions of the bacterial cells with the individual thiacyanine dyes or with a mixture of the three dyes. Steady-state absorption spectroscopy revealed that the different bacterial species altered the absorption properties of the dyes. Mixed-dye solutions gave unique absorption patterns for each bacteria tested, with competitive binding observed between the bacteria and spectrophotometric probes (thiacyanine dyes). Emission spectroscopy recorded changes in the emission spectra of THIA following the introduction of bacterial cells. Experimental results revealed that the emission enhancement of the dyes resulted from increases in the emission quantum yield of the thiacyanine dyes upon binding to the bacteria cellular components. The recorded emission enhancement data were fitted to an exponential (mono

  11. Sensitive diagnosis of bovine tuberculosis in a farmed cervid herd with use of an MPB70 protein fluorescence polarization assay.

    Science.gov (United States)

    Surujballi, Om; Lutze-Wallace, Cyril; Turcotte, Claude; Savic, Mirjana; Stevenson, Dan; Romanowska, Anna; Monagle, Wendy; Berlie-Surujballi, Gloria; Tangorra, Erin

    2009-07-01

    After histopathological examination of a lesion found in a herd member returned a diagnosis of mycobacteriosis, a farmed herd (n = 47) of elk (Cervus elaphus nelsoni) and red deer (C. elaphus elaphus) was investigated for bovine tuberculosis with a battery of antemortem and postmortem diagnostic tests. Every animal was tested with the mid-cervical tuberculin skin test; all 47 had negative results. All of the 16 adult animals and 15 of the 31 calves (approximately 2-years-old) were blood-tested with a lymphocyte stimulation test (LST) and a fluorescence polarization assay (FPA), which detects antibody to the MPB70 protein antigen. At necropsy of the 31 blood-tested animals, tissues were harvested for histopathological examination and culture of mycobacteria. Mycobacterium bovis was isolated from 16 of the 31 animals, and a scotochromogen was also isolated from 1 of the 16 whose tissues yielded M. bovis. Each of these 16 animals, 15 of which were calves, also received a histopathological diagnosis of mycobacteriosis. Other species of mycobacteria, including those belonging to the M. avium and M. terrae complexes, were isolated from an additional 7 animals. The FPA was scored "positive" or "suspect" for 16 animals, 13 (81%) of which were culture-positive for M. bovis. The other 3 animals that were culture-positive for M. bovis had negative FPA results. Of the 3 FPA-positive or FPA-suspect animals that were culture-negative, 2 were suspected to have mycobacteriosis on the basis of the histopathological examination. The 7 animals from which Mycobacterium species other than M. bovis were cultured were all FPA-negative. The only animal with positive LST results was also FPA-positive and culture-positive for M. bovis. The M. bovis isolates had an identical spoligotype pattern, with an octal code of 664073777777600. This is the first report of the isolation and identification of this strain type in Canada.

  12. Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Strange, Mette; Bräuner-Osborne, Hans; Jensen, Anders A.

    2006-01-01

    We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay...... assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors....

  13. Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Mullasseril, Praseeda; Dawit, Sara

    2010-01-01

    N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe...... a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal...

  14. Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay.

    Science.gov (United States)

    Mazáň, Marián; Ragni, Enrico; Popolo, Laura; Farkaš, Vladimír

    2011-09-01

    BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

  15. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  16. Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

    Science.gov (United States)

    Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

    2014-01-15

    Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Quantifying sublethal effects of glyphosate and Roundup® to Daphnia magna using a fluorescence based enzyme activity assay and video tracking

    DEFF Research Database (Denmark)

    Roslev, Peter; R. Hansen, Lone; Ørsted, Michael

    Glyphosate (N-(phosphonomethyl)glycine) is the active ingredient in a range of popular broad-spectrum, non-selective herbicide formulations. The toxicity of this herbicide to non-target aquatic organisms such as Daphnia magna is often evaluated using conventional toxicity assays that focus...... on endpoints such as immobility and mortality. In this study, we investigated sublethal effects of glyphosate and Roundup® to D. magna using video tracking for quantifying behavioral changes, and a novel fluorescence based assay for measuring in vivo hydrolytic enzyme activity (FLEA assay). Roundup® exposure...... resulted in concentration-dependent inhibition of alkaline phosphatase activity in D. magna. The inhibition of alkaline phosphatase by Roundup® was temperature-dependent with lowest inhibition at 14 °C and greater inhibition at 20 and 26 °C. Exposure of D. magna to sublethal concentrations of glyphosate...

  18. A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y).

    Science.gov (United States)

    Santillo, Michael F; Liu, Yitong

    2015-01-01

    Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells. Published by Elsevier Inc.

  19. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2010-01-01

    Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

  20. A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

    Science.gov (United States)

    Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

    2011-06-01

    The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.

  1. A sensitive and selective fluorescence assay for metallothioneins by exploiting the surface energy transfer between rhodamine 6G and gold nanoparticles

    International Nuclear Information System (INIS)

    Yan, Yu-Qian; Tang, Xian; Wang, Yong-Sheng; Li, Ming-Hui; Cao, Jin-Xiu; Chen, Si-Han; Zhu, Yu-Feng; Wang, Xiao-Feng; Huang, Yan-Qin

    2015-01-01

    We report on a sensitive and selective strategy for the determination of metallothioneins (MTs). The assay is based on the suppression of the surface energy transfer that occurs between rhodamine 6G (Rh6G) and gold nanoparticles (AuNPs). If Rh6G is adsorbed onto the surface of AuNPs in water solution of pH 3.0, its fluorescence is quenched due to surface energy transfer. However, on addition of MTs to the Rh6G-AuNPs system, fluorescence is recovered owing to the formation of the MTs-AuNPs complex and the release of Rh6G into the solution. Under optimized conditions, the increase in fluorescence intensity is directly proportional to the concentration of the MTs in the range from 9.68 to 500 ng mL −1 , with a detection limit as low as 2.9 ng mL −1 . The possible mechanism of this assay is discussed. The method was successfully applied to the determination of MTs in (spiked) human urine. (author)

  2. Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

    Science.gov (United States)

    Meng, Juncai; Lai, Ming-Tain; Munshi, Vandna; Grobler, Jay; McCauley, John; Zuck, Paul; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Adam, Gregory C

    2015-06-01

    HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. © 2015 Society for Laboratory Automation and Screening.

  3. A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

    Science.gov (United States)

    Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

    2012-03-01

    A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

  4. Dual Functional Core-Shell Fluorescent Ag2S@Carbon Nanostructure for Selective Assay of E. coli O157:H7 and Bactericidal Treatment.

    Science.gov (United States)

    Wang, Ning; Wei, Xing; Zheng, An-Qi; Yang, Ting; Chen, Ming-Li; Wang, Jian-Hua

    2017-03-24

    A dual functional fluorescent core-shell Ag 2 S@Carbon nanostructure is prepared by a hydrothermally assisted multi-amino synthesis approach with folic acid (FA), polyethylenimine (PEI), and mannoses (Mans) as carbon and nitrogen sources (FA-PEI-Mans-Ag 2 S nanocomposite shortly as Ag 2 S@C). The nanostructure exhibits strong fluorescent emission at λ ex /λ em = 340/450 nm with a quantum yield of 12.57 ± 0.52%. Ag 2 S@C is bound to E. coli O157:H7 via strong interaction with the Mans moiety in Ag 2 S@C with FimH proteins on the fimbriae tip in E. coli O157:H7. Fluorescence emission from Ag 2 S@C/E. coli conjugate is closely related to the content of E. coli O157:H7. Thus, a novel procedure for fluorescence assay of E. coli O157:H7 is developed, offering a detection limit of 330 cfu mL -1 . Meanwhile, the Ag 2 S@C nanostructure exhibits excellent antibacterial performance against E. coli O157:H7. A 99.9% sterilization rate can be readily achieved for E. coli O157:H7 at a concentration of 10 6 -10 7 cfu mL -1 with 3.3 or 10 μg mL -1 of Ag 2 S@C with an interaction time of 5 or 0.5 min, respectively.

  5. High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity : application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

    NARCIS (Netherlands)

    Stijntjes, G.J.; te Koppele, J.M.; Vermeulen, N P

    1992-01-01

    An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid,

  6. FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

    Science.gov (United States)

    Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

    2017-09-01

    The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

  7. A sensitive fluorescent sensor for quantification of alpha-fetoprotein based on immunosorbent assay and click chemistry.

    Science.gov (United States)

    Xie, Qunfang; Weng, Xiuhua; Lu, Lijun; Lin, Zhenyu; Xu, Xiongwei; Fu, Caili

    2016-03-15

    A novel fluoresencent immunosensor for determination of cancer biomarkers such as alpha-fetoprotein (AFP) was designed by utilizing both the high specificity of antigen-antibody sandwich structure and the high sensitivity of the click chemistry based fluorescence detection. Instead of an enzyme or fluorophore, the CuO nanoparticles are labeled on the detection antibody, which was not susceptible to the change of the external environments. The CuO nanoparticles which were modified on the sandwich structure can be dissolved to produce Cu(2+) ions with the help of HCl and then the Cu(2+) ions were reduced by sodium ascorbate to produce Cu(+) ions which triggered the Cu(+) catalyzed alkyne-azide cycloaddition (CuAAC) reaction between the weak fluorescent compound (3-azido-7-hydroxycoumarin) and propargyl alcohol to form a strong fluorescent compound. A good linear relationship was observed between the fluorescence increase factor of the system and the concentration of AFP in the range of 0.025-5.0 ng/mL with a detection limit of 12 pg/mL (S/N=3). The proposed fluorescent sensor had been applied to detect AFP in the human serum samples and gave satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

    2013-02-05

    A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

  9. Preliminary Study of the Efficacy of Using Nuclear Resonance Fluorescence with Quasi-Monoenergetic Gamma-Ray Sources for Nuclear Safeguards Assay

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, M S; McNabb, D P; Hall, J M; Gonzalez, J J

    2011-02-17

    We have studied the efficacy of using nuclear resonance fluorescence (NRF)-based techniques to assay spent nuclear fuel for Pu content using quasi-monoenergetic sources. We have developed two techniques to precisely determine the Pu content in a fuel rod/pin. One of our approaches is virtually free of systematic uncertainties. Using analytical models, we have determined the amount of time required to measure the Pu content in spent nuclear fuel rods and spent fuel assemblies to within 1% precision. We note that Pu content can be determined in a fuel assembly about as fast as in a single fuel pin. The performance of NRF-based assay techniques with improved photon sources, which are currently under development, will also estimated. For follow-on research we propose to: (1) Construct research prototype detection systems for both of the NRF-based assay systems proposed in this paper and measure their calibration curves; (2) Determine the systematic errors associated with both assay methods, explore ways to reduce the errors and fold the results into future performance calculations; (3) Develop an algorithm to assay a fuel assembly; (4) Perform validation measurements using a single pin and scaled assemblies; (5) Research and develop current-mode detection and/or threshold detection techniques to improve assay times; (6) Characterize the flux of newly constructed sources and fold the results into the calculations presented here to determine the feasibility of a variety of proposed sources; and (7) Collaborate with others in the safeguards community to build a prototype system and perform an NRF-based assay demonstration on spent fuel.

  10. Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing

    OpenAIRE

    Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

    1999-01-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTI...

  11. Quantum-dot based nanothermometry in optical plasmonic recording media

    International Nuclear Information System (INIS)

    Maestro, Laura Martinez; Zhang, Qiming; Li, Xiangping; Gu, Min; Jaque, Daniel

    2014-01-01

    We report on the direct experimental determination of the temperature increment caused by laser irradiation in a optical recording media constituted by a polymeric film in which gold nanorods have been incorporated. The incorporation of CdSe quantum dots in the recording media allowed for single beam thermal reading of the on-focus temperature from a simple analysis of the two-photon excited fluorescence of quantum dots. Experimental results have been compared with numerical simulations revealing an excellent agreement and opening a promising avenue for further understanding and optimization of optical writing processes and media

  12. On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

    2013-07-25

    A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2013-08-06

    A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

  14. A homogeneous and “off–on” fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Yang-Bao [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Ren, Hong-Xia [Key Laboratory of Asymmetric Synthesis and Chirotechnology of Sichuan Province, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 10049 (China); Gan, Ning, E-mail: ganning@nbu.edu.cn [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Zhou, You [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Cao, Yuting, E-mail: caoyuting@nbu.edu.cn [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Li, Tianhua [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Chen, Yinji [Faculty of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210000 (China)

    2016-07-27

    In this work, a novel homogeneous and signal “off–on” aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in “off” state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the “off” signal of SSB/L-QD tracer into “on” state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability. - Highlights: • Homogeneous and “off–on” fluorescence aptamer-based assay was developed to detect chloramphenicol (CAP) residues in food. • This probe was fabricated based on a vesicle QDs signal tracer (SSB/L-QD) combining with Au-Aptamer. • The detection mechanism was based on FRET with high specificity. • The results for CAP detection in the milk samples agreed well with those from ELISA, while

  15. Significantly improving nuclear resonance fluorescence non-destructive assay by using the integral resonance transmission method and photofission

    International Nuclear Information System (INIS)

    Angell, Christopher T.; Hayakawa, Takehito; Shizuma, Toshiyuki; Hajima, Ryoichi

    2013-01-01

    Non-destructive assay (NDA) of 239 Pu in spent nuclear fuel or melted fuel using a γ-ray beam is possible using self absorption and the integral resonance transmission method. The method uses nuclear resonance absorption where resonances in 239 Pu remove photons from the beam, and the selective absorption is detected by measuring the decrease in scattering in a witness target placed in the beam after the fuel, consisting of the isotope of interest, namely 239 Pu. The method is isotope specific, and can use photofission or scattered γ-rays to assay the 239 Pu. It overcomes several problems related to NDA of melted fuel, including the radioactivity of the fuel, and the unknown composition and geometry. This talk will explain the general method, and how photofission can be used to assay specific isotopes, and present example calculations. (author)

  16. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications.

    Science.gov (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo

    2017-03-15

    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C 3 N 4 ) and transition metal dichalcogenides (e.g. MoS 2 , MnO 2, and WS 2 ). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

    DEFF Research Database (Denmark)

    Ørsted, Michael; Roslev, Peter

    2015-01-01

    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl...... or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2Cr2O7, or the herbicide formulation Roundup®. Toxicant induced changes in hydrolytic enzyme activity were compared to changes in mobility (ISO 6341). The results...... showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna, and fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup® resulted in loss of whole body enzyme activity, and release of cell...

  18. Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Alquicer, Glenda; Sedlák, David; Byun, Y.; Pavlíček, Jiří; Stathis, M.; Rojas, C.; Slusher, B.; Pomper, M.G.; Bartůněk, Petr; Bařinka, Cyril

    2012-01-01

    Roč. 17, č. 8 (2012), s. 1030-1040 ISSN 1087-0571 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378050 Keywords : fluorescence polarization * glutamate carboxypeptidase II * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.207, year: 2012

  19. Highly selective detection of p-nitrophenol using fluorescence assay based on boron, nitrogen co-doped carbon dots.

    Science.gov (United States)

    Xiao, Na; Liu, Shi Gang; Mo, Shi; Li, Na; Ju, Yan Jun; Ling, Yu; Li, Nian Bing; Luo, Hong Qun

    2018-07-01

    p-Nitrophenol (p-NP) contaminants seriously endanger environmental and living beings health, hence to establish a sensitive and selective method is of great importance for the determination of p-NP. In this work, boron and nitrogen co-doped carbon dots (B,N-CDs) were synthesized by one-step hydrothermal method using 3-aminophenylboronic acid as the sole precursor. The product was characterized through high-resolution transmission electron microscopy, fluorescence spectroscopy, UV-visible absorption spectroscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. Without any functionalized modification, B,N-CDs can be directly applied as a 'turn-off' fluorescent probe for rapid, highly selective, and sensitive detection of p-NP. The fluorescent sensor based on the B,N-CDs exhibited a broad linear response to the concentration of p-NP in the range of 0.5 - 60 μM and 60 - 200 μM, respectively, and provided a detection limit of 0.2 μM. It was found that only the absorption spectrum of p-NP has a wide overlap with the fluorescence excitation and emission spectra of B,N-CDs compared to those of other representative analogues. The response mechanism was due to the inner filter effect and the formation of dynamic covalent B-O bonds between B,N-CDs and p-NP, which endowed the sensing platform with the rapid response and high selectivity to p-NP. Finally, the sensor showed the practicability of p-NP determination in environmental water samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Unique Nanoparticle Optical Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Screening and Ranking

    Science.gov (United States)

    Nanoparticles (NPs) are novel materials having at least one dimension less than 100 nm and display unique physicochemical properties due to their nanoscale size. An emphasis has been placed on developing high throughput screening (HTS) assays to characterize and rank the toxiciti...

  1. Unique Nanoparticle Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Testing and Ranking

    Science.gov (United States)

    Nanomaterials are a diverse collection of novel materials that exhibit at least one dimension less than 100 nm and display unique chemical and physical properties due to their nanoscale size. An emphasis has been put on developing high throughput screening (HTS) assays to charac...

  2. Interfacial chemistry and the design of solid-phase nucleic acid hybridization assays using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Algar, W Russ; Krull, Ulrich J

    2011-01-01

    The use of quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) offer several advantages for the development of multiplexed solid-phase QD-FRET nucleic acid hybridization assays. Designs for multiplexing have been demonstrated, but important challenges remain in the optimization of these systems. In this work, we identify several strategies based on the design of interfacial chemistry for improving sensitivity, obtaining lower limits of detection (LOD) and enabling the regeneration and reuse of solid-phase QD-FRET hybridization assays. FRET-sensitized emission from acceptor dyes associated with hybridization events at immobilized QD donors provides the analytical signal in these assays. The minimization of active sensing area reduces background from QD donor PL and allows the resolution of smaller amounts of acceptor emission, thus lowering the LOD. The association of multiple acceptor dyes with each hybridization event can enhance FRET efficiency, thereby improving sensitivity. Many previous studies have used interfacial protein layers to generate selectivity; however, transient destabilization of these layers is shown to prevent efficient regeneration. To this end, we report a protein-free interfacial chemistry and demonstrate the specific detection of as little as 2 pmol of target, as well as an improved capacity for regeneration.

  3. Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

    Science.gov (United States)

    Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

    2017-06-15

    Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R 2 =0.9984) between the fluorescent intensity and the target DNA concentration in the samples. Copyright © 2016. Published by Elsevier B.V.

  4. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides’ activity on various yeast species

    Czech Academy of Sciences Publication Activity Database

    Kodedová, Marie; Sychrová, Hana

    2016-01-01

    Roč. 233, Sep 10 (2016), s. 26-33 ISSN 0168-1656 R&D Projects: GA TA ČR(CZ) TA04010638; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : diS-C3(3) assay * antimicrobial peptide * Candida * membrane potential * microplate reader Subject RIV: EE - Microbiology, Virology Impact factor: 2.599, year: 2016

  5. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  6. Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

    Science.gov (United States)

    Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2018-05-15

    Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

    Science.gov (United States)

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  8. Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

    International Nuclear Information System (INIS)

    Chang, Kyeong-Ok; Takahashi, Daisuke; Prakash, Om; Kim, Yunjeong

    2012-01-01

    Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI–GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

  9. Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

    Science.gov (United States)

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

  10. Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

    Science.gov (United States)

    Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

    2014-10-01

    Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

  11. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    Science.gov (United States)

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies. © 2016 Elsevier Inc. All rights reserved.

  12. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

    Science.gov (United States)

    Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

    2016-10-21

    Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  13. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Directory of Open Access Journals (Sweden)

    Xia Li

    2016-10-01

    Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  14. Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells

    International Nuclear Information System (INIS)

    Moore, L.E.; Warner, M.L.; Smith, A.H.

    1996-01-01

    The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN-) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. The assay was used on 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 μg As/L) and 18 matched controls (average level, 16 μg As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN-) and those containing whole chromosomes (MN+) both increased, suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN-frequency increased 2.06-fold (P =0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN and MN+ were positively associated with urinary arsenic and its metabolites. The association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can not be determined in epithelial tissues. 35 refs., 4 tabs

  15. Dependence of the modulation response of quantum dot based nanocavity devices on the number of emitters

    DEFF Research Database (Denmark)

    Lorke, Michael; Nielsen, Torben Roland; Mørk, Jesper

    2011-01-01

    A microscopic theory is used to study the dynamical properties of semiconductor quantum dot based nanocavity laser systems. The carrier kinetics and photon populations are determined using a fully quantum mechanical treatment of the light‐matter coupling. In this work, we investigate the dependency...... of the modulation response in such devices on the number of emitters coupled to the cavity mode. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)...

  16. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-02-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  17. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-05-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  18. Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

    Science.gov (United States)

    Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Reddy, P Vijay Anand

    2012-03-01

    Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value

  19. Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Chen Meijun; Wu Yingsong; Lin Guanfeng; Hou Jingyuan; Li Ming [Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, 510515 (China); Liu Tiancai, E-mail: liutc@smu.edu.cn [Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, 510515 (China)

    2012-09-05

    Highlights: Black-Right-Pointing-Pointer QDs-based homogeneous time-resolved fluoroimmunoassay was developed to detect AFP. Black-Right-Pointing-Pointer The conjugates were prepared with QDs-doped microspheres and anti-AFP McAb. Black-Right-Pointing-Pointer The conjugates were prepared with LTCs and another anti-AFP McAb. Black-Right-Pointing-Pointer Excess amounts of conjugates were used for detecting AFP without rinsing. Black-Right-Pointing-Pointer The wedding of QPs and LTCs was suitable for HTRFIA to detect AFP. - Abstract: Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Foerster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1 Angstrom-Sign . Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was log Y = 3.65786 + 0.43863{center_dot}log X (R = 0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0

  20. Application of the Fluorescence Polarization Assay for Detection of Caprine Antibodies to Brucella melitensis in Areas of High Prevalence and Widespread Vaccination▿

    Science.gov (United States)

    Ramírez-Pfeiffer, C.; Nielsen, K.; Smith, P.; Marín-Ricalde, F.; Rodríguez-Padilla, C.; Gomez-Flores, R.

    2007-01-01

    The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats. PMID:17267588

  1. Development and evaluation of a real-time fluorescent polymerase chain reaction assay for the detection of bovine contaminates in cattle feed.

    Science.gov (United States)

    Rensen, Gabriel; Smith, Wayne; Ruzante, Juliana; Sawyer, Mary; Osburn, Bennie; Cullor, James

    2005-01-01

    A real-time fluorescent polymerase chain reaction assay for detecting prohibited ruminant materials such as bovine meat and bone meal (BMBM) in cattle feed using primers and FRET probes targeting the ruminant specific mitochondrial cytochrome b gene was developed and evaluated on two different types of cattle feed. Common problems involved with PCR based testing of cattle feed include the presence of high levels of PCR inhibitors and the need for certain pre-sample processing techniques in order to perform DNA extractions. We have developed a pre-sample processing technique for extracting DNA from cattle feed which does not require the feed sample to be ground to a fine powder and utilizes materials that are disposed of between samples, thus, reducing the potential of cross contamination. The DNA extraction method utilizes Whatman FTA card technology, is adaptable to high sample throughput analysis and allows for room temperature storage with established archiving of samples of up to 14 years. The Whatman FTA cards are subsequently treated with RNAse and undergo a Chelex-100 extraction (BioRad, Hercules, CA), thus removing potential PCR inhibitors and eluting the DNA from the FTA card for downstream PCR analysis. The detection limit was evaluated over a period of 30 trials on calf starter mix and heifer starter ration feed samples spiked with known concentrations of BMBM. The PCR detection assay detected 0.05% wt/wt BMBM contamination with 100% sensitivity, 100% specificity, and 100% confidence. Concentrations of 0.005% and 0.001% wt/wt BMBM contamination were also detected in both feed types but with varying levels of confidence.

  2. Gene protein detection platform--a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

    Science.gov (United States)

    Stålhammar, Gustav; Farrajota, Pedro; Olsson, Ann; Silva, Cristina; Hartman, Johan; Elmberger, Göran

    2015-08-01

    Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing.

    Directory of Open Access Journals (Sweden)

    Dawn M Roellig

    Full Text Available Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC, the Association of Public Health Laboratories (APHL, and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent "head to head" re-testing using both RCA and direct fluorescence assay (DFA. Community level results from three sites indicated that 54.4% (166/305 of Meridian ImmunoCard STAT! positives and 87.0% (67/77 of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186 of Meridian ImmunoCard STAT! positives and 95.2% (60/63 of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US.

  4. Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples.

    Science.gov (United States)

    Stefan, A; Scaramagli, S; Bergami, R; Mazzini, C; Barbanera, M; Perelle, S; Fach, P

    2007-03-01

    This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157 for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

  5. Effective delivery of hydrophobic drugs to breast (MCF-7) and Liver (HepG2) cancer cells: A detailed investigation using Cytotoxicity assays, fluorescence imaging and flow cytometry.

    Science.gov (United States)

    Manatunga, Danushika C; de Silva, Rohini M; Nalin de Silva, K M; Neelika Malavige, Gathsaurie; Wijeratne, Dulharie T; Williams, Gareth R; Jayasinghe, Chanika D; Udagama, Preethi V

    2018-04-03

    This study aimed to develop a drug carrier system consisting of a polymer containing hydroxyapatite (HAp) shell and a magnetic core of iron oxide nanoparticles. Doxorubicin and/or curcumin were loaded into the carrier via a simple diffusion deposition approach, with encapsulation efficiencies (EE) for curcumin and doxorubicin of 93.03 ± 0.3% and 97.37 ± 0.12% respectively. The co-loading of curcumin and doxorubicin led to a total EE of 76.02 ± 0.48%. Release studies were carried out at pH 7.4 and 5.3, and revealed higher release was at pH 5.3 expressing the potential application in tumor microenvironments. Cytotoxicity assays, fluorescence imaging and flow cytometry showed the formulations could effectively inhibit the growth of MCF-7 and HEpG2 cancer cells, being more potent than the free drug molecules both in dose and time dependent manner. Additionally, hemolysis tests and cytotoxicity evaluations determined the drug-loaded carriers to be non-toxic towards non-cancerous cells. These formulations thus have great potential in the development of new cancer therapeutics. Copyright © 2018. Published by Elsevier B.V.

  6. A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  7. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    Science.gov (United States)

    Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  8. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    Science.gov (United States)

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests

  9. Pump dependence of the dynamics of quantum dot based waveguide absorbers

    Science.gov (United States)

    Viktorov, Evgeny A.; Erneux, Thomas; Piwonski, Tomasz; Pulka, Jaroslaw; Huyet, Guillaume; Houlihan, John

    2012-06-01

    The nonlinear two stage recovery of quantum dot based reverse-biased waveguide absorbers is investigated experimentally and analytically as a function of the initial ground state occupation probability of the dot. The latter is controlled experimentally by the pump pulse power. The slow stage of the recovery is exponential and its basic timescale is independent of pump power. The fast stage of the recovery is a logistic function which we analyze in detail. The relative strength of slow to fast components is highlighted and the importance of higher order absorption processes at the highest pump level is demonstrated.

  10. An inter-comparison of HO2 measured by Fluorescence Assay by Gas Expansion and Cavity Ringdown Spectroscopy in the Highly Instrumented Reactor for Atmospheric Chemistry.

    Science.gov (United States)

    Brennan, A.; Onel, L. C.; Gianella, M.; Ronnie, G.; Aguila, A. L.; Hancock, G.; Whalley, L.; Seakins, P. W.; Ritchie, G.; Heard, D. E.

    2017-12-01

    HO2 is an important species in the atmosphere, as it is involved in the HOx radical reaction cycle that is critical to the oxidation of atmospheric pollutants and the ultimate cleaning of the troposphere. One of the most widely utilised methods to measure HO2 is Fluorescence Assay by Gas Expansion (FAGE), which indirectly measures HO2 by sampling into a low pressure cell and titrating HO2 with NO to produce OH that is then detected by Laser Induced Fluorescence. This is an indirect and non-absolute detection technique that requires careful calibration to convert the measured signal into [HO2], which involves the photolysis of H2O at 185 nm to produce OH and HO2, and is subject to 30 % errors at 2σ level. The work presented here shows the validation of the FAGE technique and its calibration procedure through inter-comparison experiments between the non-absolute FAGE technique and Cavity Ringdown Spectroscopy (CRDS), an absolute absorption based method. The CRDS system was used to excite the first O-H overtone of the HO2 absorption band at 1506.43 nm, and features a cavity length of 1.2 m and a total path of 60 km. The experiments were performed inside the 2.25 m3 stainless steel Highly Instrumented Reactor for Atmospheric Chemistry (HIRAC), using a synthetic air mixture at 150 and 1000 mbar of pressure and 298 K. HO2 was generated by photolysis of Cl2 at 365 nm in the presence of CH3OH and O2, and the [HO2] was monitored using both instruments. Additionally, monitoring the temporal decay of HO2 during its self-reaction provided an alternative calibration method for the FAGE instrument, and allowed the absorption cross section of HO2 at 1506.43 nm, σHO2, to be measured. FAGE calibration factors determined through the second order decays of HO2 at 1000 mbar agreed within 8 % of the H2O photolysis method, and determinations of σHO2 at 150 and 1000 mbar agree with previously reported data within 20 % and 12 % respectively. [HO2] correlation plots between the two

  11. Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.

    Directory of Open Access Journals (Sweden)

    Zhi-Ke Zhang

    Full Text Available Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9, suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in

  12. Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Chen, Lu; Algar, W Russ; Tavares, Anthony J; Krull, Ulrich J

    2011-01-01

    The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD-oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel

  13. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    Science.gov (United States)

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  14. 2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

    Science.gov (United States)

    Ma, Jun; Ding, Changqin; Zhou, Jie; Tian, Yang

    2015-08-15

    Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis.

    Science.gov (United States)

    Kongklieng, Amornmas; Intapan, Pewpan M; Boonmars, Thidarut; Thanchomnang, Tongjit; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

    2015-03-01

    A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate. © 2015 The Author(s).

  16. Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

    International Nuclear Information System (INIS)

    Zheng Xiannuo; Weng Lixing; Tian Jing; Wang Lianhui; Wu Lei; Jin Qinghui; Zhao Jianlong

    2012-01-01

    There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core–shell QDs, and CdTe/CdS/ZnS core–shell–shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events. (paper)

  17. Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

    Science.gov (United States)

    Zheng, Xiannuo; Tian, Jing; Weng, Lixing; Wu, Lei; Jin, Qinghui; Zhao, Jianlong; Wang, Lianhui

    2012-02-01

    There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core-shell QDs, and CdTe/CdS/ZnS core-shell-shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events.

  18. Electron-longitudinal-acoustic-phonon scattering in double-quantum-dot based quantum gates

    International Nuclear Information System (INIS)

    Zhao Peiji; Woolard, Dwight L.

    2008-01-01

    We propose a nanostructure design which can significantly suppress longitudinal-acoustic-phonon-electron scattering in double-quantum-dot based quantum gates for quantum computing. The calculated relaxation rates vs. bias voltage exhibit a double-peak feature with a minimum approaching 10 5 s -1 . In this matter, the energy conservation law prohibits scattering contributions from phonons with large momenta; furthermore, increasing the barrier height between the double quantum dots reduces coupling strength between the dots. Hence, the joint action of the energy conservation law and the decoupling greatly reduces the scattering rates. The degrading effects of temperatures can be reduced simply by increasing the height of the barrier between the dots

  19. Harnessing Sun’s Energy with Quantum Dots Based Next Generation Solar Cell

    Science.gov (United States)

    Halim, Mohammad A.

    2012-01-01

    Our energy consumption relies heavily on the three components of fossil fuels (oil, natural gas and coal) and nearly 83% of our current energy is consumed from those sources. The use of fossil fuels, however, has been viewed as a major environmental threat because of their substantial contribution to greenhouse gases which are responsible for increasing the global average temperature. Last four decades, scientists have been searching for alternative sources of energy which need to be environmentally clean, efficient, cost-effective, renewable, and sustainable. One of the promising sustainable sources of energy can be achieved by harnessing sun energy through silicon wafer, organic polymer, inorganic dye, and quantum dots based solar cells. Among them, quantum dots have an exceptional property in that they can excite multiple electrons using only one photon. These dots can easily be synthesized, processed in solution, and incorporated into solar cell application. Interestingly, the quantum dots solar cells can exceed the Shockley-Queisser limit; however, it is a great challenge for other solar cell materials to exceed the limit. Theoretically, the quantum dots solar cell can boost the power conversion efficiency up to 66% and even higher to 80%. Moreover, in changing the size of the quantum dots one can utilize the Sun’s broad spectrum of visible and infrared ranges. This review briefly overviews the present performance of different materials-based solar cells including silicon wafer, dye-sensitized, and organic solar cells. In addition, recent advances of the quantum dots based solar cells which utilize cadmium sulfide/selenide, lead sulfide/selenide, and new carbon dots as light harvesting materials has been reviewed. A future outlook is sketched as to how one could improve the efficiency up to 10% from the current highest efficiency of 6.6%. PMID:28348320

  20. Harnessing Sun's Energy with Quantum Dots Based Next Generation Solar Cell.

    Science.gov (United States)

    Halim, Mohammad A

    2012-12-27

    Our energy consumption relies heavily on the three components of fossil fuels (oil, natural gas and coal) and nearly 83% of our current energy is consumed from those sources. The use of fossil fuels, however, has been viewed as a major environmental threat because of their substantial contribution to greenhouse gases which are responsible for increasing the global average temperature. Last four decades, scientists have been searching for alternative sources of energy which need to be environmentally clean, efficient, cost-effective, renewable, and sustainable. One of the promising sustainable sources of energy can be achieved by harnessing sun energy through silicon wafer, organic polymer, inorganic dye, and quantum dots based solar cells. Among them, quantum dots have an exceptional property in that they can excite multiple electrons using only one photon. These dots can easily be synthesized, processed in solution, and incorporated into solar cell application. Interestingly, the quantum dots solar cells can exceed the Shockley - Queisser limit; however, it is a great challenge for other solar cell materials to exceed the limit. Theoretically, the quantum dots solar cell can boost the power conversion efficiency up to 66% and even higher to 80%. Moreover, in changing the size of the quantum dots one can utilize the Sun's broad spectrum of visible and infrared ranges. This review briefly overviews the present performance of different materials-based solar cells including silicon wafer, dye-sensitized, and organic solar cells. In addition, recent advances of the quantum dots based solar cells which utilize cadmium sulfide/selenide, lead sulfide/selenide, and new carbon dots as light harvesting materials has been reviewed. A future outlook is sketched as to how one could improve the efficiency up to 10% from the current highest efficiency of 6.6%.

  1. An intercomparison of HO2 measurements by fluorescence assay by gas expansion and cavity ring-down spectroscopy within HIRAC (Highly Instrumented Reactor for Atmospheric Chemistry

    Directory of Open Access Journals (Sweden)

    L. Onel

    2017-12-01

    Full Text Available The HO2 radical was monitored simultaneously using two independent techniques in the Leeds HIRAC (Highly Instrumented Reactor for Atmospheric Chemistry atmospheric simulation chamber at room temperature and total pressures of 150 and 1000 mbar of synthetic air. In the first method, HO2 was measured indirectly following sampling through a pinhole expansion to 3 mbar when sampling from 1000 mbar and to 1 mbar when sampling from 150 mbar. Subsequent addition of NO converted it to OH, which was detected via laser-induced fluorescence spectroscopy using the FAGE (fluorescence assay by gas expansion technique. The FAGE method is used widely to measure HO2 concentrations in the field and was calibrated using the 185 nm photolysis of water vapour in synthetic air with a limit of detection at 1000 mbar of 1.6 × 106 molecule cm−3 for an averaging time of 30 s. In the second method, HO2 was measured directly and absolutely without the need for calibration using cavity ring-down spectroscopy (CRDS, with the optical path across the entire ∼ 1.4 m width of the chamber, with excitation of the first O-H overtone at 1506.43 nm using a diode laser and with a sensitivity determined from Allan deviation plots of 3.0 × 108 and 1.5 × 109 molecule cm−3 at 150 and 1000 mbar respectively, for an averaging period of 30 s. HO2 was generated in HIRAC by the photolysis of Cl2 using black lamps in the presence of methanol in synthetic air and was monitored by FAGE and CRDS for ∼ 5–10 min periods with the lamps on and also during the HO2 decay after the lamps were switched off. At 1000 mbar total pressure the correlation plot of [HO2]FAGE versus [HO2]CRDS gave an average gradient of 0.84 ± 0.08 for HO2 concentrations in the range ∼ 4–100 × 109 molecule cm−3, while at 150 mbar total pressure the corresponding gradient was 0.90 ± 0.12 on average for HO2 concentrations in the range

  2. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    Science.gov (United States)

    Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  3. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    Directory of Open Access Journals (Sweden)

    Xin Zhang

    Full Text Available Fusarium oxysporum f. sp. cubense (Foc, the causal agent of Fusarium wilt (Panama disease, is one of the most devastating diseases of banana (Musa spp.. The Foc tropical race 4 (TR4 is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05. Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  4. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

    Directory of Open Access Journals (Sweden)

    Puisieux Alain

    2003-10-01

    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  5. Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics.

    Science.gov (United States)

    Miao, Peng; Tang, Yuguo; Wang, Bidou; Meng, Fanyu

    2016-08-02

    Dysregulation of miRNA expression is correlated with the development and progression of many diseases. These miRNAs are regarded as promising biomarkers. However, it is challenging to measure these low abundant molecules without employing time-consuming radioactive labeling or complex amplification strategies. Here, we present a DNA logic gate platform for miRNA diagnostics with fluorescence outputs from near-infrared (NIR) Ag2S quantum dots (QDs). Carefully designed toehold exchange-mediated strand displacements with different miRNA inputs occur on a solid-state interface, which control QDs release from solid-state interface to solution, responding to multiplex information on initial miRNAs. Excellent fluorescence emission properties of NIR Ag2S QDs certify the great prospect for amplification-free and sensitive miRNA assay. We demonstrate the potential of this platform by achieving femtomolar level miRNA analysis and the versatility of a series of logic circuits computation.

  6. Colorimetric assay for on-the-spot alcoholic strength sensing in spirit samples based on dual-responsive lanthanide coordination polymer particles with ratiometric fluorescence

    International Nuclear Information System (INIS)

    Deng, Jingjing; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    This study demonstrates a new strategy for colorimetric detection of alcoholic strength (AS) in spirit samples based on dual-responsive lanthanide infinite coordination polymer (Ln-ICP) particles with ratiometric fluorescence. The ICP used in this study are composed of two components: one is the supramolecular Ln-ICP network formed by the coordination between the ligand 2,2’-thiodiacetic acid (TDA) and central metal ion Eu"3"+; and the other is a fluorescent dye, i.e., coumarin 343 (C343), both as the cofactor ligand and as the sensitizer, doped into the Ln-ICP network through self-adaptive chemistry. Upon being excited at 300 nm, the red fluorescence of Ln-ICP network itself at 617 nm is highly enhanced due to the concomitant energy transfer from C343 to Eu"3"+, while the fluorescence of C343 at 495 nm is supressed. In pure ethanol solvent, the as-formed C343@Eu-TDA is well dispersed and quite stable. However, the addition of water into ethanolic dispersion of C343@Eu-TDA destructs Eu-TDA network structure, resulting in the release of C343 from ICP network into the solvent. Consequently, the fluorescence of Eu-TDA turns off and the fluorescence of C343 turns on, leading to the fluorescent color change of the dispersion from red to blue, which constitutes a new mechanism for colorimetric sensing of AS in commercial spirit samples. With the method developed here, we could clearly distinguish the AS of different spirit samples within a wide linear range from 10% vol to 100% vol directly by “naked eye” with the help of UV-lamp (365 nm). This study not only offers a new method for on-the-spot visible detection of AS, but also provides a strategy for dual-responsive sensing mode by rational designing the optical properties of the Ln-ICP network and the guest, respectively. - Highlights: • Dual responsive lanthanide coordination polymer particles C343@Eu-TDA were synthesized. • The guest molecular coumarin 343 sensitized the luminescence of Eu-TDA network

  7. Colorimetric assay for on-the-spot alcoholic strength sensing in spirit samples based on dual-responsive lanthanide coordination polymer particles with ratiometric fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Jingjing, E-mail: jjdeng@des.ecnu.edu.cn [School of Ecological and Environmental Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Shi, Guoyue [Department of Chemistry, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Zhou, Tianshu, E-mail: tszhou@des.ecnu.edu.cn [School of Ecological and Environmental Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China)

    2016-10-26

    This study demonstrates a new strategy for colorimetric detection of alcoholic strength (AS) in spirit samples based on dual-responsive lanthanide infinite coordination polymer (Ln-ICP) particles with ratiometric fluorescence. The ICP used in this study are composed of two components: one is the supramolecular Ln-ICP network formed by the coordination between the ligand 2,2’-thiodiacetic acid (TDA) and central metal ion Eu{sup 3+}; and the other is a fluorescent dye, i.e., coumarin 343 (C343), both as the cofactor ligand and as the sensitizer, doped into the Ln-ICP network through self-adaptive chemistry. Upon being excited at 300 nm, the red fluorescence of Ln-ICP network itself at 617 nm is highly enhanced due to the concomitant energy transfer from C343 to Eu{sup 3+}, while the fluorescence of C343 at 495 nm is supressed. In pure ethanol solvent, the as-formed C343@Eu-TDA is well dispersed and quite stable. However, the addition of water into ethanolic dispersion of C343@Eu-TDA destructs Eu-TDA network structure, resulting in the release of C343 from ICP network into the solvent. Consequently, the fluorescence of Eu-TDA turns off and the fluorescence of C343 turns on, leading to the fluorescent color change of the dispersion from red to blue, which constitutes a new mechanism for colorimetric sensing of AS in commercial spirit samples. With the method developed here, we could clearly distinguish the AS of different spirit samples within a wide linear range from 10% vol to 100% vol directly by “naked eye” with the help of UV-lamp (365 nm). This study not only offers a new method for on-the-spot visible detection of AS, but also provides a strategy for dual-responsive sensing mode by rational designing the optical properties of the Ln-ICP network and the guest, respectively. - Highlights: • Dual responsive lanthanide coordination polymer particles C343@Eu-TDA were synthesized. • The guest molecular coumarin 343 sensitized the luminescence of Eu

  8. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    International Nuclear Information System (INIS)

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  9. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  10. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    Science.gov (United States)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  11. Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

    Science.gov (United States)

    Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

    2013-03-15

    We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

    Science.gov (United States)

    Dean, Kimberly M; Grayhack, Elizabeth J

    2012-12-01

    We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

  13. Non-linear effects and thermoelectric efficiency of quantum dot-based single-electron transistors.

    Science.gov (United States)

    Talbo, Vincent; Saint-Martin, Jérôme; Retailleau, Sylvie; Dollfus, Philippe

    2017-11-01

    By means of advanced numerical simulation, the thermoelectric properties of a Si-quantum dot-based single-electron transistor operating in sequential tunneling regime are investigated in terms of figure of merit, efficiency and power. By taking into account the phonon-induced collisional broadening of energy levels in the quantum dot, both heat and electrical currents are computed in a voltage range beyond the linear response. Using our homemade code consisting in a 3D Poisson-Schrödinger solver and the resolution of the Master equation, the Seebeck coefficient at low bias voltage appears to be material independent and nearly independent on the level broadening, which makes this device promising for metrology applications as a nanoscale standard of Seebeck coefficient. Besides, at higher voltage bias, the non-linear characteristics of the heat current are shown to be related to the multi-level effects. Finally, when considering only the electronic contribution to the thermal conductance, the single-electron transistor operating in generator regime is shown to exhibit very good efficiency at maximum power.

  14. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  15. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  16. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    Science.gov (United States)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  17. Fluorescent dye labeled influenza virus mainly infects innate immune cells and activated lymphocytes and can be used in cell-mediated immune response assay

    OpenAIRE

    Xie, Dongxu

    2009-01-01

    Early results have recognized that influenza virus infects the innate and adaptive immune cells. The data presented in this paper demonstrated that influenza virus labeled with fluorescent dye not only retained the ability to infect and replicate in host cells, but also stimulated a similar human immune response as did unlabeled virus. Influenza virus largely infected the innate and activated adaptive immune cells. Influenza B type virus was different from that of A type virus. B type virus w...

  18. Quantitative fluorescence-polymerase chain reaction assay for the detection of the duplication of the Charcot Marie Tooth disease type 1A critical region.

    Science.gov (United States)

    De Toffol, Simona; Bellone, Emilia; Dulcetti, Francesca; Ruggeri, Anna Maria; Maggio, Pietro Paolo; Pulimeno, Maria Rosaria; Mandich, Paola; Maggi, Federico; Simoni, Giuseppe; Grati, Francesca Romana

    2010-04-01

    Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

  19. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    Science.gov (United States)

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  20. Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

    Science.gov (United States)

    Wu, Szu-Huei; Yao, Chun-Hsu; Hsieh, Chieh-Jui; Liu, Yu-Wei; Chao, Yu-Sheng; Song, Jen-Shin; Lee, Jinq-Chyi

    2015-07-10

    Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 μM by high-throughput screening of 41,000 compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Preparation of K+-Doped Core-Shell NaYF4:Yb, Er Upconversion Nanoparticles and its Application for Fluorescence Immunochromatographic Assay of Human Procalcitonin.

    Science.gov (United States)

    Tang, Jie; Lei, Lijiang; Feng, Hui; Zhang, Hongman; Han, Yuwang

    2016-11-01

    In the present study, we reported a convenient route to prepare well dispersed and functionalized K + -doped core-shell upconversion nanoparticles (UCP) by layer-by-layer (LbL) assembly of polyelectrolytes. UCP was firstly transferred to aqueous phase using cationic surfactant cetyl trimethyl ammonium bromide (CTAB) via hydrophobic interaction without removing the existing oleic acid (OA). Then the positively charged hydrophilic UCP@CTAB was further alternately deposited with negatively charged [poly (sodium 4-styrenesulfonate)] (PSS), positively charged [poly (allylamine hydrochloride)] (PAH) and negatively charged [poly (acrylic acid)] (PAA). The final carboxyl functionalized UCP@CTAB@PSS@PAH@PAA was then conjugated with monoclonal antibody1 (AB1) of procalcitonin (PCT), resulting in successful detection of PCT antigens based on the immunochromatographic assay (ICA). Linear response was achieved from 0 to 10 ng/mL, and the lowest limit of detection (LLD) was 0.18 ng/mL.

  2. Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

    Science.gov (United States)

    Shu, Chang; Ding, Li; Zhong, Wenying

    2014-10-01

    In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

  3. Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

    Directory of Open Access Journals (Sweden)

    Francis M. F. Nunes

    2013-01-01

    Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  4. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  5. Break-apart interphase fluorescence in situ hybridization assay in papillary thyroid carcinoma: on the road to optimizing the cut-off level for RET/PTC rearrangements.

    Science.gov (United States)

    Colato, Chiara; Vicentini, Caterina; Cantara, Silvia; Pedron, Serena; Brazzarola, Paolo; Marchetti, Ivo; Di Coscio, Giancarlo; Chilosi, Marco; Brunelli, Matteo; Pacini, Furio; Ferdeghini, Marco

    2015-05-01

    Chromosomal rearrangements of the RET proto-oncogene is one of the most common molecular events in papillary thyroid carcinoma (PTC). However, their pathogenic role and clinical significance are still debated. This study aimed to investigate the prevalence of RET/PTC rearrangement in a cohort of BRAF WT PTCs by fluorescence in situ hybridization (FISH) and to search a reliable cut-off level in order to distinguish clonal or non-clonal RET changes. Forty BRAF WT PTCs were analyzed by FISH for RET rearrangements. As controls, six BRAFV600E mutated PTCs, 13 follicular adenomas (FA), and ten normal thyroid parenchyma were also analyzed. We performed FISH analysis on formalin-fixed, paraffin-embedded tissue using a commercially available RET break-apart probe. A cut-off level equivalent to 10.2% of aberrant cells was accepted as significant. To validate FISH results, we analyzed the study cohort by qRT-PCR. Split RET signals above the cut-off level were observed in 25% (10/40) of PTCs, harboring a percentage of positive cells ranging from 12 to 50%, and in one spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in five cases showing a frequency of rearrangement very close to the cut-off. FISH approach represents a powerful tool to estimate the ratio between broken and non-broken RET tumor cells. Establishing a precise FISH cut-off may be useful in the interpretation of the presence of RET rearrangement, primarily when this strategy is used for cytological evaluation or for targeted therapy. © 2015 European Society of Endocrinology.

  6. Characterization of the commercially-available fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a marker for chloroquine resistance and uptake in a 96-well plate assay.

    Directory of Open Access Journals (Sweden)

    Cheryl C Y Loh

    Full Text Available Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.

  7. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  8. Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

    OpenAIRE

    Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

    2015-01-01

    We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based mi...

  9. Multifunctional quantum dots-based cancer diagnostics and stem cell therapeutics for regenerative medicine.

    Science.gov (United States)

    Onoshima, Daisuke; Yukawa, Hiroshi; Baba, Yoshinobu

    2015-12-01

    A field of recent diagnostics and therapeutics has been advanced with quantum dots (QDs). QDs have developed into new formats of biomolecular sensing to push the limits of detection in biology and medicine. QDs can be also utilized as bio-probes or labels for biological imaging of living cells and tissues. More recently, QDs has been demonstrated to construct a multifunctional nanoplatform, where the QDs serve not only as an imaging agent, but also a nanoscaffold for diagnostic and therapeutic modalities. This review highlights the promising applications of multi-functionalized QDs as advanced nanosensors for diagnosing cancer and as innovative fluorescence probes for in vitro or in vivo stem cell imaging in regenerative medicine. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

    Science.gov (United States)

    Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

    2015-10-01

    Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. Copyright © 2015. Published by Elsevier Ltd.

  11. Aptamer/quantum dot-based simultaneous electrochemical detection of multiple small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Haixia [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Jiang Bingying [School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400040 (China); Xiang Yun, E-mail: yunatswu@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Zhang Yuyong; Chai Yaqin [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Yuan Ruo, E-mail: yuanruo@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2011-03-04

    A novel strategy for 'signal on' and sensitive one-spot simultaneous detection of multiple small molecular analytes based on electrochemically encoded barcode quantum dot (QD) tags is described. The target analytes, adenosine triphosphate (ATP) and cocaine, respectively, are sandwiched between the corresponding set of surface-immobilized primary binding aptamers and the secondary binding aptamer/QD bioconjugates. The captured QDs yield distinct electrochemical signatures after acid dissolution, whose position and size reflect the identity and level, respectively, of the corresponding target analytes. Due to the inherent amplification feature of the QD labels and the 'signal on' detection scheme, as well as the sensitive monitoring of the metal ions released upon acid dissolution of the QD labels, low detection limits of 30 nM and 50 nM were obtained for ATP and cocaine, respectively, in our assays. Our multi-analyte sensing system also shows high specificity to target analytes and promising applicability to complex sample matrix, which makes the proposed assay protocol an attractive route for screening of small molecules in clinical diagnosis.

  12. Quantum dot based probing of mannitol: An implication in clinical diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Debasmita; Ghosh, Srabanti [UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8 Bidhannagar, Kolkata 700098 (India); Saha, Abhijit, E-mail: abhijit@alpha.iuc.res.in [UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8 Bidhannagar, Kolkata 700098 (India)

    2010-08-24

    We demonstrate that mannitol, an important diuretic, can be determined with cysteine-capped CdS quantum dots (QDs) using optical spectroscopy. Unique luminescence enhancement of QDs was observed on interaction with mannitol. The binding between QDs and mannitol was monitored by UV-vis, Fourier transform infra-red and Raman spectroscopy. The binding constant was determined following the Langmuir binding isotherm. A quantitative correlation between mannitol concentration and PL enhancement of CdS QDs has been established. We also observed that possible interfering agents, such as, urea, uric acid, creatinine, some metal ions, glucose, sorbitol or sucrose had no significant effect on luminescence of CdS QDs. The proposed strategy can be a very fast, simple and potential tool for the monitoring of diuretics and assaying intestinal permeability.

  13. Development of quantum dots-based biosensor towards on-farm detection of subclinical ketosis.

    Science.gov (United States)

    Weng, Xuan; Chen, Longyan; Neethirajan, Suresh; Duffield, Todd

    2015-10-15

    Early detection of dairy animal health issues allows the producer or veterinarian to intervene before the animals' production levels, or even survival, is threatened. An increased concentration of β-hydroxybutyrate (βHBA) is a key biomarker for diagnosis of subclinical ketosis (SCK), and provides information on the health stress in cows well before any external symptoms are observable. In this study, quantum dots (QDs) modified with cofactor nicotinamide adenine dinucleotide (NAD(+)) were prepared for the sensing event, by which the βHBA concentration in the cow's blood and milk samples was determined via fluorescence analysis of the functionalized QDs. The detection was performed on a custom designed microfluidic platform combining with a low cost and miniaturized optical sensor. The sensing mechanism was first validated by a microplate reader method and then applied to the microfluidic platform. Standard βHBA solution, βHBA in blood and milk samples from cows were successfully measured by this novel technology with a detection limit at a level of 35 µM. Side by side comparison of the developed microfluidic biosensor with a commercial kit presented its good performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Phototoxicity free quantum dot-based niosome formulation for controlled drug release and its monitoring

    Science.gov (United States)

    Kumar, Sunil; Kang, T. W.; Bala, Suman; Kamboj, Sunil; Jeon, H. C.

    2018-04-01

    A novel niosomes-based system composed of Hypromellose (HPMC) functionalized fluorescent, biocompatible ZnS:Mn quantum dots (QDs), and anti-HIV drug Tenofovir disoproxil fumarate (TDF) was designed. An appropriate ratio of surfactant Sorbitan Monostearate (SPAN-60) and cholesterol was used to obtain an optimal entrapment efficiency. Initially, after observing the successful interaction of HPMC with SPAN-60, the noisome formulation including (QDs + drug) and HPMC-coated QDs was synthesized by a wet chemical route and characterized by X-ray diffraction (XRD), Transmission electron microscope (TEM) and Selected Electron Diffraction (SAED). Secondly, (QDs + drug) loaded niosome formulations were studied by varying the ratio of SPAN-60 and cholesterol. Multiple studies were done to characterize the shape, size, viscosity, colloidal stability, and entrapment efficiency of (QDs + drug) loaded niosomes. Lastly, pH-dependent (QDs + drug) release profiles were studied by a spectroscopic technique considering the pH of the human gastrointestinal region to obtain the formulation stability of (QDs + drug) release from the niosome vesicles. These studies also include pH-dependent photo-stability measurements based on laser-induced multiphoton excitation technique in the Infrared region. The multiphoton time-resolved studies were completed to avoid the UV induced phototoxicity in the drug delivery modules. Current studies on the formulation of niosomes-based (QDs + drug) system laid a foundation to make a complete phototoxicity free system for tracking controlled drug release and its imaging.

  15. Data transformation methods for multiplexed assays

    Science.gov (United States)

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  16. A quantum-dot-based fluoroassay for detection of food-borne pathogens.

    Science.gov (United States)

    Mohamadi, Elaheh; Moghaddasi, Mohammadali; Farahbakhsh, Afshin; Kazemi, Abbass

    2017-09-01

    Evaluation of the distribution capability of food-borne pathogens existing in food products by taking the advantage of quantum dots (QDs) for their photoluminescence properties was carried out. Bacteria namely Escherichia coli (E. coli) labelled with CdSe-QDs were examined both on an Agar nutrient and ground fish substrates in order to observe their growth rate in different environments in the Lab. A sample with an appropriate concentration ratio 10 7 CFU/mL of bacteria/CdSe-QDs was empirically selected from the samples which were grown on the Agar containing plates. The selected sample was also tested on a ground fish substrate as a real food sample. The bacterial growth was observed under the irradiation of UV light and the growth patterns were investigated for 3 successive days. The growth patterns indicated that E. coli can stay alive and can be distributed on food products so that the growth can be easily monitored. This approach makes bacterial growth on food products detectable so that it can be used as a bacteria-QD assay for an easy detection of food borne pathogens grown on a food sample. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Comparison of the fluorescent polarization (TDx and the enzymatic competitive (EMIT 2000 immune assays for the measurement of cyclosporin A blood concentration Comparação dos imuno-ensaios de fluorescência polarizada (TDx e enzimático competitivo (EMIT 2000 na dosagem da concentração de ciclosporina A no sangue total

    Directory of Open Access Journals (Sweden)

    Elias David-Neto

    2000-12-01

    Full Text Available Evaluation of Cyclosporin A (CyA blood concentration is imperative in solid organ transplantation in order to achieve maximal immunosuppression with the least side effects. We compared the results of whole blood concentrations of CyA in 50 blood samples simultaneously evaluated by the fluorescent polarization immune assay (TDx and the enzymatic competitive immune assay (EMIT 2000. There was a strong correlation between both kits for any range of CyA blood concentration (R=0.99, pA avaliação da concentração sanguínea de ciclosporina A (CyA é necessária em transplantes de órgãos sólidos para obter-se máxima imunosupressão e mínimos efeitos colaterais. Nós comparamos os resultados da concentração de CyA em 50 amostras sanguíneas analisadas pelos métodos dos imuno-ensaios de fluorescência polarizada (TDx e enzimático competitivo (EMIT 2000. Houve uma forte correlação entre ambos métodos para qualquer faixa de concentração de CyA (R=0.99, p<0.001. Os coeficientes de variação intra e interensaio foram menores que 4% para ambos os métodos. O custo para cada análise de CyA foi 50% menor para o EMIT em comparação com o TDx. Concluímos que a metodologia EMIT é tão acurada quanto a de TDx para dosar CyA em amostras sanguíneas. Além disso, a metodologia EMIT é mais prática para ser realizada e mais barata, sendo ainda possível adotá-la para dosagens de CyA em vários horários de um mesmo dia.

  18. Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans

    Science.gov (United States)

    Corlu, Alper; Choe, Regine; Durduran, Turgut; Rosen, Mark A.; Schweiger, Martin; Arridge, Simon R.; Schnall, Mitchell D.; Yodh, Arjun G.

    2007-05-01

    We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Phantom and in vivo studies confirm the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. Fluorescence images of breast tumors were in good agreement with those of MRI, and with DOT based on endogenous contrast. Tumorto- normal tissue contrast based on ICG fluorescence was two-to-four-fold higher than contrast based on hemoglobin and scattering parameters. In total the measurements demonstrate that FDOT of breast cancer is feasible and promising.

  19. Fluorescence lifetime based bioassays

    Science.gov (United States)

    Meyer-Almes, Franz-Josef

    2017-12-01

    Fluorescence lifetime (FLT) is a robust intrinsic property and material constant of fluorescent matter. Measuring this important physical indicator has evolved from a laboratory curiosity to a powerful and established technique for a variety of applications in drug discovery, medical diagnostics and basic biological research. This distinct trend was mainly driven by improved and meanwhile affordable laser and detection instrumentation on the one hand, and the development of suitable FLT probes and biological assays on the other. In this process two essential working approaches emerged. The first one is primarily focused on high throughput applications employing biochemical in vitro assays with no requirement for high spatial resolution. The second even more dynamic trend is the significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging. The latter approach is currently pursued to enable not only the investigation of immortal tumor cell lines, but also specific tissues or even organs in living animals. This review tries to give an actual overview about the current status of FLT based bioassays and the wide range of application opportunities in biomedical and life science areas. In addition, future trends of FLT technologies will be discussed.

  20. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  1. Optical assay for biotechnology and clinical diagnosis.

    Science.gov (United States)

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  2. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  3. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  4. Carbon-Dots-Based Lab-On-a-Nanoparticle Approach for the Detection and Differentiation of Antibiotics.

    Science.gov (United States)

    Qiao, Li'na; Qian, Sihua; Wang, Yuhui; Yan, Shifeng; Lin, Hengwei

    2018-03-26

    Fluorescent carbon dots (CDs) have received considerable attention in recent years due to their superior optical properties. To take further advantages of these unique features, herein, a CDs-based "lab-on-a-nanoparticle" approach for the detection and discrimination of antibiotics is developed. The sensing platform was designed based on the different channel's fluorescence recoveries or further quenching of the full-color emissive CDs (F-CDs) and metal ion ensembles upon the addition of antibiotics. The F-CDs exhibited unusually comparable emission intensity nearly across the entire visible spectrum even as the excitation wavelength is shifted, making it very suitable for the construction of multi-channel sensing systems. The sensing platform was fabricated on the basis of the competing interaction of metal ions with the F-CDs and antibiotics. Three metal ions (i.e., Cu 2+ , Ce 3+ and Eu 3+ ) can efficiently quench the fluorescence of the F-CDs. Upon the addition of antibiotics, the fluorescent intensities either recovered at different emission wavelengths or were further quenched to various degrees. The fluorescence response patterns at different emission wavelength were characteristic for each antibiotic and can be quantitatively differentiated by standard statistical methods (e.g., hierarchical clustering analysis and principal component analysis). Moreover, as an example, the proposed method was applied for quantitative detection of oxytetracycline with a limit of detection to be 0.06 μm. Finally, the sensing system was successfully employed for residual antibiotics detection and identification in real food samples. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Fluorescent quantification of melanin.

    Science.gov (United States)

    Fernandes, Bruno; Matamá, Teresa; Guimarães, Diana; Gomes, Andreia; Cavaco-Paulo, Artur

    2016-11-01

    Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  7. Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C3(3) fluorescence assay

    Czech Academy of Sciences Publication Activity Database

    Maláč, J.; Urbánková, E.; Sigler, Karel; Gášková, D.

    2005-01-01

    Roč. 37, - (2005), s. 2536-2543 ISSN 1357-2725 R&D Projects: GA ČR GD204/03/H066; GA ČR GP202/04/P110 Institutional research plan: CEZ:AV0Z50200510 Keywords : mdr- pump s * yeast * fluriometric assay Subject RIV: EE - Microbiology, Virology Impact factor: 3.871, year: 2005

  8. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    Science.gov (United States)

    2013-07-12

    assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979, 16:710–718. 3. Noedl H, Attlmayr B...40:685–691. 32. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM, Ward SA: Relationship between antimalarial drug activity , accumulation, and...success rate when testing DHA, AS, MQ, QN, CQ, and PPQ activities . A “successful” IC50 assay result for each P. falciparum clinical isolate was defined as

  9. Detection of metronidazole in honey and metronidazole tablets using carbon dots-based sensor via the inner filter effect.

    Science.gov (United States)

    Zhao, Jianru; Pan, Xiaohua; Sun, Xiaobo; Pan, Wei; Yu, Guifeng; Wang, Jinping

    2018-06-01

    In this work, carbon dots (CDs) with a high quantum yield (22.3%) were easily prepared by hydrothermal pyrolysis of acid fuchsin 6B and hydrogen peroxide at 180°C for 10 h. The resultant CDs possess a narrow size distribution in the range of 2.6 to 3.2 nm and emit blue fluorescence. Interestingly, the absorption band of metronidazole (MTZ) centered at 318 nm can complementary overlap with the excitation band of the as-prepared CDs centered at 320 nm, resulting in an inner filter effect (IFE) in high efficiency. In fact, the fluorescence quenching of the CDs depends on the concentration of MTZ. Therefore, a simple method for the detection of MTZ can be established using the CDs-based sensor via the IFE. The linear range of the proposed method was 0-10 μg mL -1 with the limit of detection as low as 0.257 μg mL -1 . This CDs-based sensor had been applied for the detection of MTZ in honey and MTZ tablets with the recoveries in the range of 98.0% to 105.1% and 95.7% to 106.5%, respectively. Therefore, the as-prepared CDs have a potential to be developed as a MTZ sensor with high selectivity, sensitivity and accuracy. Copyright © 2018 John Wiley & Sons, Ltd.

  10. Photoluminescence Enhancement of Silole-Capped Silicon Quantum Dots Based on Förster Resonance Energy Transfer.

    Science.gov (United States)

    Kim, Seongwoong; Kim, Sungsoo; Ko, Young Chun; Sohn, Honglae

    2015-07-01

    Photoluminescent porous silicon were prepared by an electrochemical etch of n-type silicon under the illumination with a 300 W tungsten filament bulb for the duration of etch. The red photoluminescence emitting at 650 nm with an excitation wavelength of 450 nm is due to the quantum confinement of silicon quantum dots in porous silicon. HO-terminated red luminescent PS was obtained by an electrochemical treatment of fresh PS with the current of 150 mA for 60 seconds in water and sodium chloride. As-prepared PS was sonicated, fractured, and centrifuged in toluene solution to obtain photoluminescence silicon quantum dots. Dichlorotetraphenylsilole exhibiting an emission band at 520 nm was reacted with HO-terminated silicon quantum dots to give a silole-capped silicon quantum dots. The optical characterization of silole-derivatized silicon quantum dots was investigated by UV-vis and fluorescence spectrometer. The fluorescence emission efficiency of silole-capped silicon quantum dots was increased by about 2.5 times due to F6rster resonance energy transfer from silole moiety to silicon quantum dots.

  11. Fluorescent Probes and Fluorescence (Microscopy Techniques — Illuminating Biological and Biomedical Research

    Directory of Open Access Journals (Sweden)

    Gregor P. C. Drummen

    2012-11-01

    Full Text Available Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

  12. Evaporation-induced self-assembly of quantum dots-based concentric rings on polymer-based nanocomposite films.

    Science.gov (United States)

    Zhang, Shaofu; Luan, Weiling; Zhong, Qixin; Yin, Shaofeng; Yang, Fuqian

    2016-10-12

    The "ball-on-film" template is used to construct concentric rings on the surface of PMMA-QDs (polymethyl methacrylate - quantum dots) nanocomposite films via the evaporation of pure chloroform droplets, which are confined by a steel ball. The concentric rings consist of QDs, as revealed by the fluorescence images of the concentric rings. The photoluminescence intensity of the concentric rings increases with the increase of the distance to the ball center, suggesting that the amount of QDs accumulated around the contact line at individual stick state increases with the increase of the distance to the ball center. Both the wavelength and cross-sectional area (width) of the concentric rings increase approximately linearly with increasing distance to the ball center, independent of the ball size, the film thickness and the QDs concentration. For the PMMA-QDs nanocomposite films prepared from the same QDs concentration in chloroform, the thicker the PMMA-QDs nanocomposite film, the larger the wavelength for the same distance to the ball center. The effect of confinement of two steel balls on the surface patterns over the PMMA-QDs nanocomposite films is studied via a template of "two spheres on film". Symmetric surface patterns are formed. There exist two types of featureless zone between the two balls, depending on the distance between the two balls: one is the inner featureless zone and the other is the outer featureless zone. The size of both featureless zones increases with the increase of the ball distance.

  13. Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy.

    Science.gov (United States)

    Wang, Yuyuan; Wang, Yidan; Chen, Guojun; Li, Yitong; Xu, Wei; Gong, Shaoqin

    2017-09-13

    A quantum-dot (QD)-based micelle conjugated with an anti-epidermal growth factor receptor (EGFR) nanobody (Nb) and loaded with an anticancer drug, aminoflavone (AF), has been engineered for EGFR-overexpressing cancer theranostics. The near-infrared (NIR) fluorescence of the indium phosphate core/zinc sulfide shell QDs (InP/ZnS QDs) allowed for in vivo nanoparticle biodistribution studies. The anti-EGFR nanobody 7D12 conjugation improved the cellular uptake and cytotoxicity of the QD-based micelles in EGFR-overexpressing MDA-MB-468 triple-negative breast cancer (TNBC) cells. In comparison with the AF-encapsulated nontargeted (i.e., without Nb conjugation) micelles, the AF-encapsulated Nb-conjugated (i.e., targeted) micelles accumulated in tumors at higher concentrations, leading to more effective tumor regression in an orthotopic triple-negative breast cancer xenograft mouse model. Furthermore, there was no systemic toxicity observed with the treatments. Thus, this QD-based Nb-conjugated micelle may serve as an effective theranostic nanoplatform for EGFR-overexpressing cancers such as TNBCs.

  14. Nondestructive assay measurements applied to reprocessing plants

    International Nuclear Information System (INIS)

    Ruhter, Wayne D.; Lee, R. Stephen; Ottmar, Herbert; Guardini, Sergio

    1999-01-01

    Nondestructive assay for reprocessing plants relies on passive gamma-ray spectrometry for plutonium isotopic and plutonium mass values of medium-to-low-density samples and holdup deposits; on active x-ray fluorescence and densitometry techniques for uranium and plutonium concentrations in solutions; on calorimetry for plutonium mass in product; and passive neutron techniques for plutonium mass in spent fuel, product, and waste. This paper will describe the radiation-based nondestructive assay techniques used to perform materials accounting measurements. The paper will also discuss nondestructive assay measurements used in inspections of reprocessing plants [ru

  15. Simultaneous quantitative detection of multiple tumor markers with a rapid and sensitive multicolor quantum dots based immunochromatographic test strip.

    Science.gov (United States)

    Wang, Chunying; Hou, Fei; Ma, Yicai

    2015-06-15

    A novel multicolor quantum dots (QDs) based immunochromatographic test strip (ICTS) was developed for simultaneous quantitative detection of multiple tumor markers, by utilizing alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) as models. The immunosensor could realize simultaneous quantitative detection of tumor markers with only one test line and one control line on the nitrocellulose membrane (NC membrane) due to the introduction of multicolor QDs. In this method, a mixture of mouse anti-AFP McAb and mouse anti-CEA McAb was coated on NC membrane as test line and goat anti-mouse IgG antibody was coated as control line. Anti-AFP McAb-QDs546 conjugates and anti-CEA McAb-QDs620 conjugates were mixed and applied to the conjugate pad. Simultaneous quantitative detection of multiple tumor markers was achieved by detecting the fluorescence intensity of captured QDs labels on test line and control line using a test strip reader. Under the optimum conditions, AFP and CEA could be detected as low as 3 ng/mL and 2 ng/mL in 15 min with a sample volume of 80 μL, and no obvious cross-reactivity was observed. The immunosensor was validated with 130 clinical samples and in which it exhibited high sensitivity (93% for AFP and 87% for CEA) and specificity (94% for AFP and 97% for CEA). The immunosensor also demonstrated high recoveries (87.5-113% for AFP and 90-97.3% for CEA) and low relative standard deviations (RSDs) (2.8-6.2% for AFP and 4.9-9.6% for CEA) when testing spiked human serum. This novel multicolor QDs based ICTS provides an easy and rapid, simultaneous quantitative detecting strategy for point-of-care testing of tumor markers. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Investigation of some critical parameters of buffer conditions for the development of quantum dots-based optical sensors

    International Nuclear Information System (INIS)

    Yuan Jipei; Guo Weiwei; Wang Erkang

    2008-01-01

    The unique surface-sensitive properties make quantum dots (QDs) great potential in the development of sensors for various analytes. However, quantum dots are not only sensitive to a certain analyte, but also to the surrounding conditions. The controlled response to analyte may be the first step in the designing of functional quantum dots sensors. In this study, taking the quenching effect of benzoquinone (BQ) on CdTe QDs as model, several critical parameters of buffer solution conditions with potential effect on the sensors were investigated. The pH value and the concentration of sodium citrate in the buffer solution critically influenced the quenching effects of BQ. Dozens folds elevation of the quenching extents were observed with the increase of concentrations of H + and sodium citrate, and the quenching mechanisms were also fundamentally different with the changes of the surrounding buffer solutions. The quenching models were proposed and analyzed at different buffer conditions. Taking pH values for example, QDs quenching obeyed the sphere of effective quenching model with the sphere radii of 8.29 nm at pH 8.0, the linear Stern-Volmer equation with Stern-Volmer constant of 2.0 x 10 3 mol -1 L at pH 7.0, and the two binding site static quenching model at basic conditions. The elucidation of parameters for assay performance was important in the development of QDs-based optical sensors

  17. Controlling reabsorption effect of bi-color CdSe quantum dots-based white light-emitting diodes

    Science.gov (United States)

    Siao, Cyuan-Bin; Chung, Shu-Ru; Wang, Kuan-Wen

    2017-08-01

    The colloidal semiconductor quantum dots (QDs) have the potentials to be used in white light-emitting diode (WLED) as a down-converting component to replace incandescent lamps, because the traditional WLED composed of Y3Al5O12:Ce3+ (YAG:Ce) phosphor lack of red color emissions and shows low color quality. Among various QDs, CdSe has been extensively studied because it possesses attractive characteristics such as high quantum yields (QYs), narrow emission spectral bandwidth, as well as size-tunable optical characteristics. However, in order to enhance the color rendering index (CRI) of WLED, blending materials with different emission wavelengths has been used frequently. Unfortunately, these procedures are complex and time-consuming, and the emission energy of smaller QDs can be reabsorbed by larger QDs, resulting in decreasing the excitation intensity in yellowish-green region. Therefore, in this study, in order to decrease the reabsorption effect and to simplify the procedures, we have demonstrated a facile thermal pyrolyzed route to prepare bicolor CdSe QDs with dual-wavelengths. The emission wavelengths, particle sizes, and QYs of QDs can be tuned from 537/595 to 537/602 nm, 2.59/3.92 to 2.59/4.01 nm, and 27 to 40 %, for GR1 to 3 samples, respectively when the amount of Se precursor is decreased from 1.5 to 0.75 mmol. Meanwhile, the area ratio of green to red (Ag/Ar) in fluorescence spectra is gradually increased, due to the increase in growth rate, and decrease in nuclei formation in red emission. The GR1, GR2, and GR3 QDs are then encapsulated by convert types to form the LED, in which the QDs are deposited on the blue-emitting InGaN LED chip (λem = 450 nm). After encapsulation, the devices properties of Commission International d'Eclairage (CIE) chromaticity and Ag/Ar area ratio are (0.40, 0.24), 0.28/1, (0.40, 0.31), 0.52/1, and (0.40, 0.38), 1.02/1, respectively for GR1, GR2, and GR3. The results show that the green emission intensity are strongly

  18. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  19. ZnCuInS/ZnSe/ZnS Quantum Dot-Based Downconversion Light-Emitting Diodes and Their Thermal Effect

    Directory of Open Access Journals (Sweden)

    Wenyan Liu

    2015-01-01

    Full Text Available The quantum dot-based light-emitting diodes (QD-LEDs were fabricated using blue GaN chips and red-, yellow-, and green-emitting ZnCuInS/ZnSe/ZnS QDs. The power efficiencies were measured as 14.0 lm/W for red, 47.1 lm/W for yellow, and 62.4 lm/W for green LEDs at 2.6 V. The temperature effect of ZnCuInS/ZnSe/ZnS QDs on these LEDs was investigated using CIE chromaticity coordinates, spectral wavelength, full width at half maximum (FWHM, and power efficiency (PE. The thermal quenching induced by the increased surface temperature of the device was confirmed to be one of the important factors to decrease power efficiencies while the CIE chromaticity coordinates changed little due to the low emission temperature coefficients of 0.022, 0.050, and 0.068 nm/°C for red-, yellow-, and green-emitting ZnCuInS/ZnSe/ZnS QDs. These indicate that ZnCuInS/ZnSe/ZnS QDs are more suitable for downconversion LEDs compared to CdSe QDs.

  20. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  1. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen

    2017-06-01

    Systems and methods are provided for simultaneously assaying cell adhesion or cell rolling for multiple cell specimens. One embodiment provides a system for assaying adhesion or cell rolling of multiple cell specimens that includes a confocal imaging system containing a parallel plate flow chamber, a pump in fluid communication with the parallel plate flow chamber via a flow chamber inlet line and a cell suspension in fluid communication with the parallel plate flow chamber via a flow chamber outlet line. The system also includes a laser scanning system in electronic communication with the confocal imaging system, and a computer in communication with the confocal imaging system and laser scanning system. In certain embodiments, the laser scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least seven cell specimens in the parallel plate flow chamber.

  2. Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

    Science.gov (United States)

    Bogh, Sidsel A.; Bora, Ilkay; Rosenberg, Martin; Thyrhaug, Erling; Laursen, Bo W.; Just Sørensen, Thomas

    2015-12-01

    Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0  =  0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

  3. Reviews in fluorescence 2010

    CERN Document Server

    Geddes, Chris D

    2011-01-01

    ""Reviews in Fluorescence 2010"", the seventh volume of the book serial from Springer, serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence and closely related disciplines. It summarizes the year's progress in fluorescence and its applications, with authoritative analytical reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. ""Reviews in Fluorescence"" offers an essential reference material for any lab working in the fluoresc

  4. Principles of fluorescence techniques

    CERN Document Server

    2016-01-01

    Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

  5. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  6. Reviews in fluorescence 2008

    CERN Document Server

    Geddes, Chris D

    2010-01-01

    This volume serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence spectroscopy. It summarizes the year's progress in fluorescence and its applications as well as includes authoritative analytical reviews.

  7. Fluorescent optical position sensor

    Science.gov (United States)

    Weiss, Jonathan D.

    2005-11-15

    A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

  8. Safe biodegradable fluorescent particles

    Science.gov (United States)

    Martin, Sue I [Berkeley, CA; Fergenson, David P [Alamo, CA; Srivastava, Abneesh [Santa Clara, CA; Bogan, Michael J [Dublin, CA; Riot, Vincent J [Oakland, CA; Frank, Matthias [Oakland, CA

    2010-08-24

    A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

  9. Multiplexing a high-throughput liability assay to leverage efficiencies.

    Science.gov (United States)

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  10. Optimization of fluorescent proteins

    NARCIS (Netherlands)

    Bindels, D.S.; Goedhart, J.; Hink, M.A.; van Weeren, L.; Joosen, L.; Gadella (jr.), T.W.J.; Engelborghs, Y.; Visser, A.J.W.G.

    2014-01-01

    Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and microcopy

  11. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  12. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  13. Detection of nitrite based on fluorescent carbon dots by the hydrothermal method with folic acid

    Science.gov (United States)

    Lin, Haitao; Ding, Liyun; Zhang, Bingyu; Huang, Jun

    2018-05-01

    A fluorescent carbon dots probe for the detection of aqueous nitrite was fabricated by a one-pot hydrothermal method, and the transmission electron microscope, X-ray diffractometer, UV-Vis absorption spectrometer and fluorescence spectrophotometer were used to study the property of carbon dots. The fluorescent property of carbon dots influenced by the concentration of aqueous nitrite was studied. The interaction between the electron-donating functional groups and the electron-accepting nitrous acid could account for the quenching effect on carbon dots by adding aqueous nitrite. The products of the hydrolysis of aqueous nitrite performed a stronger quenching effect at lower pH. The relationship between the relative fluorescence intensity of carbon dots and the concentration of nitrite was described by the Stern-Volmer equation (I0/I - 1 = 0.046[Q]) with a fine linearity (R2 = 0.99). The carbon dots-based probe provides a convenient method for the detection of nitrite concentration.

  14. Passive nondestructive assay of nuclear materials

    International Nuclear Information System (INIS)

    Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

    1991-03-01

    The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

  15. Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming

    2015-01-01

    that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell...... and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology....

  16. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  17. Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Erin Wilson

    2018-05-01

    Full Text Available A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP. The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600 and plate counting (colony-forming units (CFUs. While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

  18. Fluorogenic dansyl-ligated gold nanoparticles for the detection of sulfur mustard by displacement assay.

    Science.gov (United States)

    Knighton, Richard C; Sambrook, Mark R; Vincent, Jack C; Smith, Simon A; Serpell, Christopher J; Cookson, James; Vickers, Matthew S; Beer, Paul D

    2013-03-21

    The dansyl fluorophore ligated to gold nanoparticles via imidazole and amine groups affords conjugates capable of detecting micromolar concentrations of the chemical warfare agent sulfur mustard by a fluorescence switching 'ON' displacement assay.

  19. Dyes assay for measuring physicochemical parameters.

    Science.gov (United States)

    Moczko, Ewa; Meglinski, Igor V; Bessant, Conrad; Piletsky, Sergey A

    2009-03-15

    A combination of selective fluorescent dyes has been developed for simultaneous quantitative measurements of several physicochemical parameters. The operating principle of the assay is similar to electronic nose and tongue systems, which combine nonspecific or semispecific elements for the determination of diverse analytes and chemometric techniques for multivariate data analysis. The analytical capability of the proposed mixture is engendered by changes in fluorescence signal in response to changes in environment such as pH, temperature, ionic strength, and presence of oxygen. The signal is detected by a three-dimensional spectrofluorimeter, and the acquired data are processed using an artificial neural network (ANN) for multivariate calibration. The fluorescence spectrum of a solution of selected dyes allows discreet reading of emission maxima of all dyes composing the mixture. The variations in peaks intensities caused by environmental changes provide distinctive fluorescence patterns which can be handled in the same way as the signals collected from nose/tongue electrochemical or piezoelectric devices. This optical system opens possibilities for rapid, inexpensive, real-time detection of a multitude of physicochemical parameters and analytes of complex samples.

  20. Highly Sensitive Ratiometric Fluorescent Sensor for Trinitrotoluene Based on the Inner Filter Effect between Gold Nanoparticles and Fluorescent Nanoparticles.

    Science.gov (United States)

    Lu, Hongzhi; Quan, Shuai; Xu, Shoufang

    2017-11-08

    In this work, we developed a simple and sensitive ratiometric fluorescent assay for sensing trinitrotoluene (TNT) based on the inner filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent nanoparticles (RFNs), which was designed by hybridizing green emissive carbon dots (CDs) and red emissive quantum dots (QDs) into a silica sphere as a fluorophore pair. AuNPs in their dispersion state can be a powerful absorber to quench CDs, while the aggregated AuNPs can quench QDs in the IFE-based fluorescent assays as a result of complementary overlap between the absorption spectrum of AuNPs and emission spectrum of RFNs. As a result of the fact that TNT can induce the aggregation of AuNPs, with the addition of TNT, the fluorescent of QDs can be quenched, while the fluorescent of CDs would be recovered. Then, ratiometric fluorescent detection of TNT is feasible. The present IFE-based ratiometric fluorescent sensor can detect TNT ranging from 0.1 to 270 nM, with a detection limit of 0.029 nM. In addition, the developed method was successfully applied to investigate TNT in water and soil samples with satisfactory recoveries ranging from 95 to 103%, with precision below 4.5%. The simple sensing approach proposed here could improve the sensitivity of colorimetric analysis by changing the ultraviolet analysis to ratiometric fluorescent analysis and promote the development of a dual-mode detection system.

  1. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  2. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  3. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  4. Characterization of antibody-chelator conjugates: Determination of chelator content by terbium fluorescence titration

    Energy Technology Data Exchange (ETDEWEB)

    Brandt, K.D.; Schnobrich, K.E.; Johnson, D.K. (Abbott Laboratories, Department 90M, Abbott Park, IL (United States))

    1991-01-01

    Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates.

  5. Characterization of antibody-chelator conjugates: Determination of chelator content by terbium fluorescence titration

    International Nuclear Information System (INIS)

    Brandt, K.D.; Schnobrich, K.E.; Johnson, D.K.

    1991-01-01

    Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates

  6. Atomic-fluorescence spectrophotometry

    International Nuclear Information System (INIS)

    Bakhturova, N.F.; Yudelevich, I.G.

    1975-01-01

    Atomic-fluorescence spectrophotometry, a comparatively new method for the analysis of trace quantities, has developed rapidly in the past ten years. Theoretical and experimental studies by many workers have shown that atomic-fluorescence spectrophotometry (AFS) is capable of achieving a better limit than atomic absorption for a large number of elements. The present review examines briefly the principles of atomic-fluorescence spectrophotometry and the types of fluorescent transition. The excitation sources, flame and nonflame atomizers, used in AFS are described. The limits of detection achieved up to the present, using flame and nonflame methods of atomization are given

  7. Fluorescence of irradiated hydrocarbons

    International Nuclear Information System (INIS)

    Gulis, I.G.; Evdokimenko, V.M.; Lapkovskij, M.P.; Petrov, P.T.; Gulis, I.M.; Markevich, S.V.

    1977-01-01

    A visible fluorescence has been found out in γ-irradiated aqueous of carbohydrates. Two bands have been distinguished in fluorescence spectra of the irradiated solution of dextran: a short-wave band lambdasub(max)=140 nm (where lambda is a wave length) at lambdasub(β)=380 nm and a long-wave band with lambdasub(max)=540 nm at lambdasub(β)=430 nm. A similar form of the spectrum has been obtained for irradiated solutions of starch, amylopectin, lowmolecular glucose. It has been concluded that a macromolecule of polysaccharides includes fluorescent centres. A relation between fluorescence and α-oxiketon groups formed under irradiation has been pointed out

  8. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  9. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  10. Factor II assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  11. Factor VII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  12. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  13. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    NARCIS (Netherlands)

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  14. Integrated bioassays in microfluidic devices: botulinum toxin assays.

    Science.gov (United States)

    Mangru, Shakuntala; Bentz, Bryan L; Davis, Timothy J; Desai, Nitin; Stabile, Paul J; Schmidt, James J; Millard, Charles B; Bavari, Sina; Kodukula, Krishna

    2005-12-01

    A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

  15. Fluorescence Enhancement on Large Area Self-Assembled Plasmonic-3D Photonic Crystals.

    Science.gov (United States)

    Chen, Guojian; Wang, Dongzhu; Hong, Wei; Sun, Lu; Zhu, Yongxiang; Chen, Xudong

    2017-03-01

    Discontinuous plasmonic-3D photonic crystal hybrid structures are fabricated in order to evaluate the coupling effect of surface plasmon resonance and the photonic stop band. The nanostructures are prepared by silver sputtering deposition on top of hydrophobic 3D photonic crystals. The localized surface plasmon resonance of the nanostructure has a symbiotic relationship with the 3D photonic stop band, leading to highly tunable characteristics. Fluorescence enhancements of conjugated polymer and quantum dot based on these hybrid structures are studied. The maximum fluorescence enhancement for the conjugated polymer of poly(5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene) potassium salt by a factor of 87 is achieved as compared with that on a glass substrate due to the enhanced near-field from the discontinuous plasmonic structures, strong scattering effects from rough metal surface with photonic stop band, and accelerated decay rates from metal-coupled excited state of the fluorophore. It is demonstrated that the enhancement induced by the hybrid structures has a larger effective distance (optimum thickness ≈130 nm) than conventional plasmonic systems. It is expected that this approach has tremendous potential in the field of sensors, fluorescence-imaging, and optoelectronic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide) interactions. Even though fluorescence...

  17. Multimodal fluorescence imaging spectroscopy

    NARCIS (Netherlands)

    Stopel, Martijn H W; Blum, Christian; Subramaniam, Vinod; Engelborghs, Yves; Visser, Anthonie J.W.G.

    2014-01-01

    Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample.

  18. Intrinsic fluorescence biomarkers in cells treated with chemopreventive drugs

    Science.gov (United States)

    Kirkpatrick, Nathaniel D.; Brands, William R.; Zou, Changping; Brewer, Molly A.; Utzinger, Urs

    2005-03-01

    Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.

  19. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-05

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  1. Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA and indirect ELISA Detecção de anticorpos anti-Toxoplasma gondii por meio das técnicas de Imunofluorescência Indireta e ELISA Indireto em primatas experimentalmente e naturalmente infectados

    Directory of Open Access Journals (Sweden)

    Andréa Bouer

    2010-03-01

    Full Text Available The Indirect Fluorescence Assay (IFA and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI with the IFA (IgG and IgM and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI. Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2% seropositive animals for this parasite and 149 (70.8% negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.Detectou-se anticorpos das classes IgG e IgM anti-Toxoplasma gondii em primatas experimentalmente e naturalmente infectados, utilizando-se como técnicas comparativas a RIFI e o ELISA-teste. No grupo dos primatas experimentalmente infectados, anticorpos de valor diagnóstico foram detectados a partir do 9º dia de infecção tanto na RIFI (IgG e IgM como no ELISA-IgG. O ELISA IgM detectou anticorpos a partir do 3º dia de infecção até o final do experimento (102 dias pós-infecção. Dos 209 soros dos primatas naturalmente infectados, de diversos zoológicos do Estado de São Paulo, 64,59 e 67,94% mostraram-se positivos na RIFI-IgG e no ELISA-IgG, respectivamente. O

  2. Quantum dots-based quantitative and in situ multiple imaging on ki67 and cytokeratin to improve ki67 assessment in breast cancer.

    Directory of Open Access Journals (Sweden)

    Jing Ping Yuan

    Full Text Available As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer (BC prognosis. Although immunohistochemical staining is the current standard method, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study was to develop a fluorescent spectrum-based quantitative analysis of Ki67 expression by quantum-dots (QDs multiple imaging technique.A QDs-based in situ multiple fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic cytokeratin (CK as green signal. Both Ki67 and CK signals were automatically separated and quantified by professional spectrum analysis software. This technique was applied to tissue microarrays from 240 BC patients. Both Ki67 and CK values, and Ki67/CK ratio were obtained for each patient, and their prognostic value on 5-year disease free survival was assessed.This method simultaneously stains nuclear Ki67 and cytoplasmic CK with clear signal contrast, making it easy for signal separation and quantification. The total fluorescent signal intensities of both Ki67 sum and CK sum were obtained, and Ki67/CK ratio calculated. Ki67 sum and Ki67/CK ratio were each attributed into two grades by X-tile software based on the best P value principle. Multivariate analysis showed Ki67 grade (P = 0.047 and Ki67/CK grade (P = 0.004 were independent prognostic factors. Furthermore, area under curve (AUC of ROC analysis for Ki67/CK grade (AUC: 0.683, 95%CI: 0.613-0.752 was higher than Ki67 grade (AUC: 0.665, 95%CI: 0.596-0.734 and HER-2 gene (AUC: 0.586, 95%CI: 0.510-0.661, but lower than N stage (AUC: 0.760, 95%CI: 0.696-0.823 and histological grade (AUC: 0.756, 95%CI: 0.692-0.820 on predicting the risk for recurrence.A QDs-based quantitative and in situ multiple imaging on Ki67 and CK was developed to improve Ki67 assessment in BC, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.

  3. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  4. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  5. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  6. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  7. A new seed-based assay for meiotic recombination in Arabidopsis thaliana.

    NARCIS (Netherlands)

    Melamed-Bessudo, C.; Yehuda, E.; Stuitje, A.R.; Levy, A.A.

    2005-01-01

    Meiotic recombination is a fundamental biological process that plays a central role in the evolution and breeding of plants. We have developed a new seed-based assay for meiotic recombination in Arabidopsis. The assay is based on the transformation of green and red fluorescent markers expressed

  8. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  9. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  10. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta

    2007-01-01

    Full Text Available Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots. This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

  11. [A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus].

    Science.gov (United States)

    Liu, Lin; Zhang, Quan-Fu; Li, Chuan; Li, Jian-Dong; Jiang, Xiao-Lin; Zhang, Fu-Shun; Wu, Wei; Liang, Mi-Fang; Li, De-Xin

    2013-06-01

    To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA. A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method. The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949). The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.

  12. Fluorescent discharge lamp

    Science.gov (United States)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-01-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  13. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  14. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  15. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  16. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  17. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  18. Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

    Science.gov (United States)

    Kidwell, David A.

    1993-05-01

    A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

  19. Fluorescent cadmium sulfide nanoparticles for selective and sensitive detection of toxic pesticides in aqueous medium

    International Nuclear Information System (INIS)

    Walia, Shanka; Acharya, Amitabha

    2014-01-01

    The detection of pesticide residues in ground water, food, or soil samples is extremely important. The currently available laboratory techniques have several drawbacks and needs to be replaced. Fluorescent chemosensors for pesticide detection were reported in the literature, with few reports published on quantum dot-based pesticide sensors, but none of these were focused toward differentiating organophosphorus and organochlorine pesticides specifically. In this respect, glutathione-coated CdS nanoparticles were synthesized and characterized. The TEM studies of the nanoparticles suggested mostly monodispersed spherical particles, with size in the range of 11.5±1 nm. The prepared fluorescent nanoparticles were found to selectively recognize organochlorine pesticide dicofol among all the other pesticides studied, by increasing the fluorescence intensity of the nanoparticles ∼ 2.5 times. Similar studies carried out with organophosphorous pesticide dimethoate did not result any change in the fluorescence intensity of the nanoparticles. Further studies carried out with commercially available pesticide solutions, also confirmed similar results. The TEM, SEM, and DLS studies suggested aggregation of the nanoparticles in the presence of dicofol. Control experiments suggested possible role of both amine and carboxylic acid functional groups of glutathione in the recognition of dicofol. The limit of detection of dicofol was found to be ∼ 55±11 ppb.Graphical AbstractGlutathione-coated CdS nanoparticles selectively recognize organochlorine pesticide dicofol among all the other pesticides studied, by increasing the fluorescence intensity of the nanoparticles. The TEM, SEM, and DLS studies suggested aggregation of the nanoparticles in the presence of dicofol

  20. Fluorescent cadmium sulfide nanoparticles for selective and sensitive detection of toxic pesticides in aqueous medium

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Shanka; Acharya, Amitabha, E-mail: amitabhachem@gmail.com [CSIR-Institute of Himalayan Bioresource Technology, Biotechnology Division (India)

    2014-12-15

    The detection of pesticide residues in ground water, food, or soil samples is extremely important. The currently available laboratory techniques have several drawbacks and needs to be replaced. Fluorescent chemosensors for pesticide detection were reported in the literature, with few reports published on quantum dot-based pesticide sensors, but none of these were focused toward differentiating organophosphorus and organochlorine pesticides specifically. In this respect, glutathione-coated CdS nanoparticles were synthesized and characterized. The TEM studies of the nanoparticles suggested mostly monodispersed spherical particles, with size in the range of 11.5±1 nm. The prepared fluorescent nanoparticles were found to selectively recognize organochlorine pesticide dicofol among all the other pesticides studied, by increasing the fluorescence intensity of the nanoparticles ∼ 2.5 times. Similar studies carried out with organophosphorous pesticide dimethoate did not result any change in the fluorescence intensity of the nanoparticles. Further studies carried out with commercially available pesticide solutions, also confirmed similar results. The TEM, SEM, and DLS studies suggested aggregation of the nanoparticles in the presence of dicofol. Control experiments suggested possible role of both amine and carboxylic acid functional groups of glutathione in the recognition of dicofol. The limit of detection of dicofol was found to be ∼ 55±11 ppb.Graphical AbstractGlutathione-coated CdS nanoparticles selectively recognize organochlorine pesticide dicofol among all the other pesticides studied, by increasing the fluorescence intensity of the nanoparticles. The TEM, SEM, and DLS studies suggested aggregation of the nanoparticles in the presence of dicofol.

  1. Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

    OpenAIRE

    Feng, Sunlian; Kendall, Lon V.; Hodzic, Emir; Wong, Scott; Lorenzana, Edward; Freet, Kimberly; Ku, Karin S.; Luciw, Paul A.; Barthold, Stephen W.; Khan, Imran H.

    2004-01-01

    Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated t...

  2. Comparison of the Vidas C. difficile GDH Automated Enzyme-Linked Fluorescence Immunoassay (ELFA) with Another Commercial Enzyme Immunoassay (EIA) (Quik Chek-60), Two Selective Media, and a PCR Assay for gluD for Detection of Clostridium difficile in Fecal Samples.

    Science.gov (United States)

    Davies, K A; Berry, C E; Morris, K A; Smith, R; Young, S; Davis, T E; Fuller, D D; Buckner, R J; Wilcox, M H

    2015-06-01

    Prevention and management of Clostridium difficile infection (CDI) can be improved by rapid and reliable diagnostics. The Vidas C. difficile glutamate dehydrogenase assay had performance comparable to that of the Quik Chek-60 assay (overall agreement, 95%) and a sensitivity of >93%; thus, it is suitable as the first test in two-stage algorithms for a CDI diagnosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. [A cell-based detection of ciguatoxin using sodium fluorescence probe].

    Science.gov (United States)

    Yuan, Jian-hui; Yang, Hui; Tang, Huan-wen; Huang, Wei; Xu, Xin-yun; Liu, Jian-jun; Ke, Yue-bin; Cheng, Jin-quan; Zhuang, Zhi-xiong

    2011-04-01

    To establish a cell-based detection method of ciguatoxin using fluorescence assay. Mouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNaTM Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish. A correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 103 g/ml and 1012 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time. The cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.

  4. Reviews in fluorescence 2007

    CERN Document Server

    Lakowicz, Joseph R; Geddes, Chris D

    2009-01-01

    This fourth volume in the Springer series summarizes the year's progress in fluorescence, with authoritative analytical reviews specialized enough for professional researchers, yet also appealing to a wider audience of scientists in related fields.

  5. Introduction to fluorescence

    CERN Document Server

    Jameson, David M

    2014-01-01

    "An essential contribution to educating scientists in the principles of fluorescence. It will also be an important addition to the libraries of practitioners applying the principles of molecular fluorescence."-Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill"An exquisite compendium of fluorescence and its applications in biochemistry enriched by a very exciting historical perspective. This book will become a standard text for graduate students and other scientists."-Drs. Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center"… truly a masterwork, combining clarity, precision, and good humor. The reader, novice or expert, will be pleased with the text and will not stop reading. It is a formidable account of the fluorescence field, which has impacted the life sciences so considerably in the last 60 years."-Jerson L. Silva, M.D., Ph.D., Professor and Director, National Institute of Science and Tech...

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Liping; Zhu, Anna; Lou, Xuening; Song, Dan; Yang, Rong [School of Environment and Natural Resources, Renmin University of China, Beijing (China); Shi, Hanchang [School of Environment, Tsinghua University, Beijing (China); Long, Feng, E-mail: longf04@ruc.edu.cn [School of Environment and Natural Resources, Renmin University of China, Beijing (China)

    2016-01-28

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

  8. Fluorescence (Multiwave) Confocal Microscopy.

    Science.gov (United States)

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. The potential of a fluorescent-based approach for bioassay of antifungal agents against chili anthracnose disease in Thailand.

    Science.gov (United States)

    Chutrakul, Chanikul; Khaokhajorn, Pratoomporn; Auncharoen, Patchanee; Boonruengprapa, Tanapong; Mongkolporn, Orarat

    2013-01-01

    Severe chili anthracnose disease in Thailand is caused by Colletotrichum gloeosporioides and C. capsici. To discover anti-anthracnose substances we developed an efficient dual-fluorescent labeling bioassay based on a microdilution approach. Indicator strains used in the assay were constructed by integrating synthetic green fluorescent protein (sGFP) and Discosoma sp. red fluorescent protein (DsRedExp) genes into the genomes of C. gloeosporioides or C. capsici respectively. Survival of co-spore cultures in the presence of inhibitors was determined by the expression levels of these fluorescent proteins. This developed assay has high potential for utilization in the investigation of selective inhibition activity to either one of the pathogens as well as the broad-range inhibitory effect against both pathogens. The value of using the dual-fluorescent assay is rapid, reliable, and consistent identification of anti-anthracnose agents. Most of all, the assay enables the identification of specific inhibitors under the co-cultivation condition.

  10. Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

    Science.gov (United States)

    Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

    2016-01-01

    A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

  11. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...... PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant....

  12. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by ...

  13. Single-analyte to multianalyte fluorescence sensors

    Science.gov (United States)

    Lavigne, John J.; Metzger, Axel; Niikura, Kenichi; Cabell, Larry A.; Savoy, Steven M.; Yoo, J. S.; McDevitt, John T.; Neikirk, Dean P.; Shear, Jason B.; Anslyn, Eric V.

    1999-05-01

    The rational design of small molecules for the selective complexation of analytes has reached a level of sophistication such that there exists a high degree of prediction. An effective strategy for transforming these hosts into sensors involves covalently attaching a fluorophore to the receptor which displays some fluorescence modulation when analyte is bound. Competition methods, such as those used with antibodies, are also amenable to these synthetic receptors, yet there are few examples. In our laboratories, the use of common dyes in competition assays with small molecules has proven very effective. For example, an assay for citrate in beverages and an assay for the secondary messenger IP3 in cells have been developed. Another approach we have explored focuses on multi-analyte sensor arrays with attempt to mimic the mammalian sense of taste. Our system utilizes polymer resin beads with the desired sensors covalently attached. These functionalized microspheres are then immobilized into micromachined wells on a silicon chip thereby creating our taste buds. Exposure of the resin to analyte causes a change in the transmittance of the bead. This change can be fluorescent or colorimetric. Optical interrogation of the microspheres, by illuminating from one side of the wafer and collecting the signal on the other, results in an image. These data streams are collected using a CCD camera which creates red, green and blue (RGB) patterns that are distinct and reproducible for their environments. Analysis of this data can identify and quantify the analytes present.

  14. Fluorescence Image Segmentation by using Digitally Reconstructed Fluorescence Images

    OpenAIRE

    Blumer, Clemens; Vivien, Cyprien; Oertner, Thomas G; Vetter, Thomas

    2011-01-01

    In biological experiments fluorescence imaging is used to image living and stimulated neurons. But the analysis of fluorescence images is a difficult task. It is not possible to conclude the shape of an object from fluorescence images alone. Therefore, it is not feasible to get good manual segmented nor ground truth data from fluorescence images. Supervised learning approaches are not possible without training data. To overcome this issues we propose to synthesize fluorescence images and call...

  15. Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

    Science.gov (United States)

    Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

    2013-03-01

    Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

  16. Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis.

    Science.gov (United States)

    Smith, Stephanie A; Lawson, Corinne M; McMichael, Maureen A; Jung, Katrina; O'Brien, Mauria; Achiel, Ron

    2017-06-01

    Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma. © 2017 American Society for Veterinary Clinical Pathology.

  17. Fluorescent-labeled ligands for the benzodiazepine receptor - Part 1 : Synthesis and characterization of fluorescent-labeled benzodiazepines

    NARCIS (Netherlands)

    Janssen, M.J; Hulst, A.J R L; Kellogg, R.M; Hendriks, M.M W B; Ensing, K; de Zeeuw, R.A

    Because radioactive labeled ligands in receptor assays have several disadvantages, we synthesized a number of fluorescent-labeled benzodiazepines. Several fluorophores were attached at different positions of 1,4-benzodiazepine molecules in order to assess the impact of the fluorophores and their

  18. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  19. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  20. Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Honglei Chen

    2012-10-01

    Full Text Available Caveolin-1 (Cav-1 expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV-associated GC (EBVaGC is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029 that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032 and was positively associated with Cav-1 expression (r = 0.432, p < 0.001. EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

  1. Structural, optical and photovoltaic properties of P3HT and Mn-doped CdS quantum dots based bulk hetrojunction hybrid layers

    Science.gov (United States)

    Jabeen, Uzma; Adhikari, Tham; Pathak, Dinesh; Shah, Syed Mujtaba; Nunzi, Jean-Michel

    2018-04-01

    Cadmium sulphide (CdS) and Mn-doped CdS nanocrystals were synthesized by co-precipitation method. The nanocrystals were characterized by Fluorescence, Fourier Transformed Infra-red Spectrometer (FTIR), UV-Visible, X-ray diffraction (XRD), X-ray photoelectron spectrometer (XPS), Field Emission Scanning Electron Microscope (FESEM), and High Resolution Transmission Electron Microscope (HRTEM). A considerable blue shift of absorption band with respect to the cadmium sulphide was observed by the Mn concentration (0.5 M) in the doped sample with decreasing the size of nanocrystals. Other reason for this may be Mn doping. Subsequently the band gap was altered from 2.11 to 2.21 eV due to quantum confinement effect. Scanning electron microscope supplemented with EDAX was operated to find grain size and chemical composition of the synthesized nanomaterials. The PL spectrum of Mn-doped CdS nanocrystals displays three PL bands the first one, within the range of 500 nm and the second band at 537 nm, and the third one around 713 nm is labelled red band emission due to attributed to a 4T1→6A1 transition within the 3d shell of divalent manganese. XRD analysis showed that the material was in cubic crystalline state. A comparative study of surfaces of un-doped and metal doped CdS nanocrystals were investigated using X-ray Photoelectron Spectroscopy (XPS). The synthesized nanomaterial in combination with polymer, poly (3-hexyl thiophene) was operated in the construction of photovoltaic cells. The photovoltaic devices with CdS nanocrystals exhibited power conversion efficiency of 0.34% without annealing and 0.38% with annealing. However, the power conversion efficiency was enhanced by a factor of 0.35 without annealing and 0.42 with annealing with corporation of Mn impurity in CdS lattice. Atomic Force Microscopy was employed for morphology and packing behavior of blend of nanocrystals with organic polymer.

  2. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  3. Fluorescence-based bioassays for the detection and evaluation of food materials.

    Science.gov (United States)

    Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

    2015-10-13

    We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

  4. Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

    Directory of Open Access Journals (Sweden)

    Kentaro Nishi

    2015-10-01

    Full Text Available We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

  5. Application of fluorescent in situ hybridisation for demonstration of Coxiella burnetti in placentas from ruminant abortions

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Montgomery, Donald L.; Jaeger, Paula T.

    2007-01-01

    A fluorescent in situ hybridisation (FISH) assay targeting 16S ribosomal RNA was developed for detection of the zoonotic bacterium Coxiella burnetii in formalin-fixed, paraffin-embedded tissue, and applied on placentas from ruminant abortions. The applicability of the FISH assay was compared...

  6. Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells

    Directory of Open Access Journals (Sweden)

    Peng CW

    2017-12-01

    Full Text Available Chunwei Peng,1 Jiuyang Liu,1 Guifang Yang,2 Yan Li3 1Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center, 2Department of Pathology, Zhongnan Hospital of Wuhan University, Wuchang District, Wuhan, 3Department of Peritoneal Cancer Surgery, Cancer Center of Beijing Shijitan Hospital Affiliated to the Capital Medical University, Yangfangdian, Beijing, People’s Republic of China Purpose: Semiconductor quantum dots (QDs are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methods: A QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs including 184 gastric cancer (GC tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX. The stromal status including infiltrating of immune cells (high density of macrophages, angiogenesis (high density of microvessel density [MVD], low neovessel maturation and extracellular matrix (ECM remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9] was evaluated.Results: This study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe

  7. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    Levin, G.V.; Straat, P.A.

    1981-01-01

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  8. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  9. Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

    Directory of Open Access Journals (Sweden)

    Ji Yeon Hwang

    2016-11-01

    Full Text Available Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule or muc1 (mucin1 expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon. The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1 or BHQ2 (black hole quencher2. In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

  10. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  11. Nanosecond fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs

  12. Fluorescence uranium determination

    International Nuclear Information System (INIS)

    Fernandez Cellini, R.; Crus Castillo, F. de la; Barrera Pinero, R.

    1960-01-01

    An equipment for analysis of uranium by fluorescence was developed in order to determine it at such a low concentration that it can not be determined by the most sensible analytical methods. this new fluorimeter was adapted to measure the fluorescence emitted by the phosphorus sodium fluoride-sodium carbonate-potasium carbonate-uranyl, being excited by ultraviolet light of 3,650 A the intensity of the light emitted was measure with a photomultiplicator RCA 5819 and the adequate electronic equipment. (Author) 19 refs

  13. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  14. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  15. Monitoring by fluorescence measurements

    International Nuclear Information System (INIS)

    Malcolme-Lawes, D.J.; Gifford, L.A.

    1981-01-01

    A fluorimetric detector is described in which the fluorescence excitation source may be 3 H, 14 C, 35 S, 147 Pm or 63 Ni. Such a detector can be adapted for use with flowing liquid systems especially liquid chromatography systems. (U.K.)

  16. Fluorescent Lamp Replacement Study

    Science.gov (United States)

    2017-07-01

    not be cited for purposes of advertisement. DISPOSITION INSTRUCTIONS: Destroy this document when no longer needed. Do not return to the... recycling , and can be disposed safely in a landfill. (2) LEDs offer reduced maintenance costs and fewer bulb replacements, significantly reducing... recycling . Several fixtures, ballasts and energy efficient fluorescent bulbs that were determined to be in pristine condition were returned to ATC

  17. Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Macháň, Radek; Kapusta, Peter; Hof, Martin

    Roč. 406 , č. 20 (2014), s. 4797-4813 ISSN 1618-2642 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Filtered fluorescence correlation spectroscopy * Fluorescence lifetime correlation spectroscopy * Fluorescence spectral correlation spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.436, year: 2014

  18. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

    Full Text Available An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease. Keywords: Prions, Thickness shear mode quartz sensor

  19. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  20. Who's who in fluorescence 2008

    CERN Document Server

    Geddes, Chris D

    2008-01-01

    The Journal of Fluorescence's sixth Who's Who directory publishes the names, contact details, specialty keywords, and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. This is a unique reference.

  1. Quantitative fluorescence microscopy and image deconvolution.

    Science.gov (United States)

    Swedlow, Jason R

    2013-01-01

    Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

  2. Construction of a multiple fluorescence labeling system for use in co-invasion studies of Listeria monocytogenes

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Roldgaard, Bent; Lindner, A. B.

    2006-01-01

    strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay......, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. Conclusion The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between...... deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains...

  3. Anticholinesterase activity of the fluorescent zoanthid pigment, parazoanthoxanthin A.

    Science.gov (United States)

    Sepcić, K; Turk, T; Macek, P

    1998-06-01

    A synthetic linear tetrazacyclopent(f)azulene compound, parazoanthoxanthin A (m.w. 214.2), strongly fluorescent pigment occurring in zoanthids, was characterized and assayed for anticholinesterase activity. The pigment, emitting fluorescence at lambda(em) 420 nm, was found to be a pure competitive inhibitor of cholinesterases. At pH 8.0, a Ki value of 19 and 26 microM was determined with insect recombinant, and electric eel acetylcholinesterase. Horse serum butyrylcholinesterase was less sensitive with a Ki of 70 microM.

  4. Synthesis and Fluorescence Spectra of Triazolylcoumarin Fluorescent Dyes

    Institute of Scientific and Technical Information of China (English)

    PENG Xian-fu; LI Hong-qi

    2009-01-01

    Much attention is devoted to fluorescent dyes especially those with potential in versatile applications. Reactions under "click" conditions between nonfluorescent 3 - azidocoumarins and terminal alkynes produced 3 -(1, 2, 3- triazol- 1 - yl)cournarins, a novel type of fluorescent dyes with intense fluorescence. The structures of the new coumarins were characterized by 1H NMR, MS, and IR spectra. Fluorescence spectra measurement demonstrated excellent fluorescence performance of the triazolylcoumarins and this click reaction is a promising candidate for bioconjugation and bioimaging applications since both azide and alkynes are quite inert to biological systems.

  5. Fluorescence spectroscopy of dental calculus

    International Nuclear Information System (INIS)

    Bakhmutov, D; Gonchukov, S; Sukhinina, A

    2010-01-01

    The aim of the present study was to investigate the fluorescence properties of dental calculus in comparison with the properties of adjacent unaffected tooth structure using both lasers and LEDs in the UV-visible range for fluorescence excitation. The influence of calculus color on the informative signal is demonstrated. The optimal spectral bands of excitation and registration of the fluorescence are determined

  6. Fluorescence spectroscopy of dental calculus

    Science.gov (United States)

    Bakhmutov, D.; Gonchukov, S.; Sukhinina, A.

    2010-05-01

    The aim of the present study was to investigate the fluorescence properties of dental calculus in comparison with the properties of adjacent unaffected tooth structure using both lasers and LEDs in the UV-visible range for fluorescence excitation. The influence of calculus color on the informative signal is demonstrated. The optimal spectral bands of excitation and registration of the fluorescence are determined.

  7. Fluorescence Imaging Reveals Surface Contamination

    Science.gov (United States)

    Schirato, Richard; Polichar, Raulf

    1992-01-01

    In technique to detect surface contamination, object inspected illuminated by ultraviolet light to make contaminants fluoresce; low-light-level video camera views fluorescence. Image-processing techniques quantify distribution of contaminants. If fluorescence of material expected to contaminate surface is not intense, tagged with low concentration of dye.

  8. Who's who in fluorescence 2005

    CERN Document Server

    Geddes, Chris D

    2006-01-01

    The Journal of Fluorescence's third Who's Who directory publishes the names, contact details, specialty keywords, photographs, and a brief description of scientists employing fluorescence methodology and instrumentation in their working livesThe directory provides company contact details with a brief list of fluorescence-related products.

  9. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

    Science.gov (United States)

    Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

    2014-01-01

    The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

  10. Assay to detect lipid peroxidation upon exposure to nanoparticles.

    Science.gov (United States)

    Potter, Timothy M; Neun, Barry W; Stern, Stephan T

    2011-01-01

    This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered to be an important component of biocompatibility screens. The products of lipid peroxidation, lipid hydroperoxides, and aldehydes, such as MDA, can be measured via a thiobarbituric acid reactive substances (TBARS) assay. In this assay, which can be performed in cell culture or in cell lysate, MDA combines with thiobarbituric acid (TBA) to form a fluorescent adduct that can be detected at an excitation wavelength of 530 nm and an emission wavelength of 550 nm. The results are then expressed as MDA equivalents, normalized to total cellular protein (determined by Bradford assay).

  11. Fluorescence fluctuation spectroscopy (FFS)

    CERN Document Server

    Tetin, Sergey

    2012-01-01

    This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers fluorescence fluctuation spectroscopy Contains chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells.

  12. Application of a Homogenous Assay for the Detection of 2,4,6-Trinitrotoluene to Environmental Water Samples

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2005-01-01

    Full Text Available A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10x phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17 M significantly improved the range over which the assay functioned in several river water samples.

  13. Fluorescent nanodiamond for biomedicine

    International Nuclear Information System (INIS)

    Milos Nesladek

    2014-01-01

    NV centers in diamond have gained strong interest as a novel tool for quantum information processing, quantum computing and quantum photonics. These applications are based on fluorescent and spin properties of NV-centres. However, in some conditions NV- can lose an electron and turn to NV0. The occupation of NV0 and NV- charge states depend on the position of their ground states with respect to the Fermi level and the mechanism of the charge transfer. Interestingly, that the charge switch has important implications on applications of fluorescent nanodiamond (fND) to nano-biology and nano-medicine. fND can be used for bio-marking and bio-tracking but also for the monitoring of targeted delivery to the cells. In this presentation we review the current state-of-the art for using fND particles for fluorescent bio imaging in cells and discuss the charge transfer and its luminescence stability by using ultra high sensitive spectroscopy methods to study the NV0 and NV- state occupation. (author)

  14. Fluorescence detection of pesticides using quantum dot materials – A review

    Energy Technology Data Exchange (ETDEWEB)

    Nsibande, S.A.; Forbes, P.B.C., E-mail: patricia.forbes@up.ac.za

    2016-11-16

    High pesticide use, especially in agriculture, can lead to environmental pollution and potentially adverse health effects. As result, pesticide residues end up in different media, including water and food products, which may serve as direct routes for human exposure. There is thus a continuous drive to develop analytical methods for screening and quantification of these compounds in the different environmental media in which they may occur. Development of quantum dot (QD) based sensors for monitoring pesticides has gained momentum in recent years. QD materials have excellent and unique optical properties and have high fluorescence quantum yields compared to other fluorophores. They have thus been used in numerous studies for the development of probes for organic pollutants. In this paper we specifically review their application as fluorescence probes for pesticide detection in different media including water and in fruits and vegetables. The low detection limits reported demonstrate the potential use of these methods as alternatives to expensive and time-consuming conventional techniques. We also highlight potential limitations that these probes may present when it comes to routine application. Finally we discuss possible future improvements to enhance the selectivity and robustness of these sensors. We note that there is still a need for researchers to develop standardized QD based sensors which could lead to their commercialization and routine application. - Highlights: • Application of quantum dots as fluorescence probes in pesticide detection. • Recognition elements and modification strategies towards selective pesticide detection. • Sensitive detection below regulatory limits in various matrices. • Challenges and possible solutions towards standardization of quantum dot based analytical methods.

  15. Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH) Assay

    OpenAIRE

    Daniel Lerda; Marta Cabrera; Jorge Flores; Luis Gutierrez; Armando Chierichetti; Martin Revol; Hernan Garcia Onto

    2013-01-01

    Objetive: The overall purpose of the study was to demonstrate applicability of the Dako dual-color chromogenic in situ hybridization (CISH) assay (DAKO Denmark, Glostrup) with respect to fluorescence in situ hybridization (FISH) probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals. The dual –color CISH assay was p...

  16. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  17. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    OpenAIRE

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

  18. Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

    Directory of Open Access Journals (Sweden)

    Padalkar Vikas S

    2011-11-01

    Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

  19. Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test Estudo da reatividade cruzada em amostras de soro de cães positivos para Leishmania sp., Babesia canis e Ehrlichia canis, pelo ensaio imunoenzimático indireto e pela reação de imunofluorescência indireta

    Directory of Open Access Journals (Sweden)

    Trícia Maria F. de Sousa Oliveira

    2008-03-01

    Full Text Available To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT and Enzyme-linked immunosorbent assay (ELISA. All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA.Para verificar a existência de reação cruzada entre leishmaniose visceral, erliquiose e babesiose, nos testes sorológicos utilizados em programas de controle da leishmaniose visceral humana, amostras de soro canino provenientes de áreas endêmicas e não endêmicas para essa enfermidade, foram testadas pela Reação de Imunofluorescência (RIFI e Ensaio imunoenzimático (ELISA. Todos os soros provenientes de área endêmica foram positivos para Leishmania sp pelo ELISA e RIFI, 51% para Babesia canis e 43% para Ehrlichia canis pela RIFI. Pela RIFI, nenhum dos soros provenientes de área não endêmica foi positivo para Leishmania sp, sendo 67% positivos para B. canis e 78% para E. canis pelo mesmo teste. Quando testados pelo ELISA para Leishmania sp., quatro soros da área não endêmica foram positivos. Os cães foram localizados

  20. Fluorescent microthermographic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Barton, D.L.

    1993-09-01

    In the early days of microelectronics, design rules and feature sizes were large enough that sub-micron spatial resolution was not needed. Infrared or IR thermal techniques were available that calculated the object`s temperature from infrared emission. There is a fundamental spatial resolution limitation dependent on the wavelengths of light being used in the image formation process. As the integrated circuit feature sizes began to shrink toward the one micron level, the limitations imposed on IR thermal systems became more pronounced. Something else was needed to overcome this limitation. Liquid crystals have been used with great success, but they lack the temperature measurement capabilities of other techniques. The fluorescent microthermographic imaging technique (FMI) was developed to meet this need. This technique offers better than 0.01{degrees}C temperature resolution and is diffraction limited to 0.3 {mu}m spatial resolution. While the temperature resolution is comparable to that available on IR systems, the spatial resolution is much better. The FMI technique provides better spatial resolution by using a temperature dependent fluorescent film that emits light at 612 nm instead of the 1.5 {mu}m to 12 {mu}m range used by IR techniques. This tutorial starts with a review of blackbody radiation physics, the process by which all heated objects emit radiation to their surroundings, in order to understand the sources of information that are available to characterize an object`s surface temperature. The processes used in infrared thermal imaging are then detailed to point out the limitations of the technique but also to contrast it with the FMI process. The FMI technique is then described in detail, starting with the fluorescent film physics and ending with a series of examples of past applications of FMI.

  1. Fluorescence Imaging of Fast Retrograde Axonal Transport in Living Animals

    Directory of Open Access Journals (Sweden)

    Dawid Schellingerhout

    2009-11-01

    Full Text Available Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography. To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc. TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60 for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

  2. A fluorescence scanning electron microscope

    International Nuclear Information System (INIS)

    Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

    2009-01-01

    Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

  3. Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

    Science.gov (United States)

    Ong, Kimberly J; MacCormack, Tyson J; Clark, Rhett J; Ede, James D; Ortega, Van A; Felix, Lindsey C; Dang, Michael K M; Ma, Guibin; Fenniri, Hicham; Veinot, Jonathan G C; Goss, Greg G

    2014-01-01

    The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

  4. Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

    Directory of Open Access Journals (Sweden)

    Kimberly J Ong

    Full Text Available The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1 nanoparticle intrinsic fluorescence/absorbance, 2 interactions between nanoparticles and assay components, and 3 the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

  5. Development of a fluorescent cryocooler

    International Nuclear Information System (INIS)

    Edwards, B.C.; Buchwald, M.I.; Epstein, R.I.; Gosnell, T.R.; Mungan, C.E.

    1995-01-01

    Recent work at Los Alamos National Laboratory has demonstrated the physical principles for a new type of solid-state cryocooler based on anti-Stokes fluorescence. Design studies indicate that a vibration-free, low-mass ''fluorescent cryocooler'' could operate for years with efficiencies and cooling powers comparable to current commercial systems. This paper presents concepts for a fluorescent cryocooler, design considerations and expected performance

  6. Fluorescence of ceramic color standards

    International Nuclear Information System (INIS)

    Koo, Annette; Clare, John F.; Nield, Kathryn M.; Deadman, Andrew; Usadi, Eric

    2010-01-01

    Fluorescence has been found in color standards available for use in calibration and verification of color measuring instruments. The fluorescence is excited at wavelengths below about 600 nm and emitted above 700 nm, within the response range of silicon photodiodes, but at the edge of the response of most photomultipliers and outside the range commonly scanned in commercial colorimeters. The degree of fluorescence on two of a set of 12 glossy ceramic tiles is enough to introduce significant error when those tiles have been calibrated in one mode of measurement and are used in another. We report the nature of the fluorescence and the implications for color measurement.

  7. Multicolor fluorescent light-emitting diodes based on cesium lead halide perovskite quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Peng [State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012 (China); State Key Laboratory of Superhard Materials, College of Physics, Jilin University, Changchun 130012 (China); Bai, Xue, E-mail: baix@jlu.edu.cn, E-mail: yuzhang@jlu.edu.cn; Sun, Chun; Zhang, Xiaoyu; Zhang, Yu, E-mail: baix@jlu.edu.cn, E-mail: yuzhang@jlu.edu.cn [State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012 (China); Zhang, Tieqiang [State Key Laboratory of Superhard Materials, College of Physics, Jilin University, Changchun 130012 (China)

    2016-08-08

    High quantum yield, narrow full width at half-maximum and tunable emission color of perovskite quantum dots (QDs) make this kind of material good prospects for light-emitting diodes (LEDs). However, the relatively poor stability under high temperature and air condition limits the device performance. To overcome this issue, the liquid-type packaging structure in combination with blue LED chip was employed to fabricate the fluorescent perovskite quantum dot-based LEDs. A variety of monochromatic LEDs with green, yellow, reddish-orange, and red emission were fabricated by utilizing the inorganic cesium lead halide perovskite quantum dots as the color-conversion layer, which exhibited the narrow full width at half-maximum (<35 nm), the relatively high luminous efficiency (reaching 75.5 lm/W), and the relatively high external quantum efficiency (14.6%), making it the best-performing perovskite LEDs so far. Compared to the solid state LED device, the liquid-type LED devices exhibited excellent color stability against the various working currents. Furthermore, we demonstrated the potential prospects of all-inorganic perovskite QDs for the liquid-type warm white LEDs.

  8. Multicolor fluorescent light-emitting diodes based on cesium lead halide perovskite quantum dots

    International Nuclear Information System (INIS)

    Wang, Peng; Bai, Xue; Sun, Chun; Zhang, Xiaoyu; Zhang, Yu; Zhang, Tieqiang

    2016-01-01

    High quantum yield, narrow full width at half-maximum and tunable emission color of perovskite quantum dots (QDs) make this kind of material good prospects for light-emitting diodes (LEDs). However, the relatively poor stability under high temperature and air condition limits the device performance. To overcome this issue, the liquid-type packaging structure in combination with blue LED chip was employed to fabricate the fluorescent perovskite quantum dot-based LEDs. A variety of monochromatic LEDs with green, yellow, reddish-orange, and red emission were fabricated by utilizing the inorganic cesium lead halide perovskite quantum dots as the color-conversion layer, which exhibited the narrow full width at half-maximum (<35 nm), the relatively high luminous efficiency (reaching 75.5 lm/W), and the relatively high external quantum efficiency (14.6%), making it the best-performing perovskite LEDs so far. Compared to the solid state LED device, the liquid-type LED devices exhibited excellent color stability against the various working currents. Furthermore, we demonstrated the potential prospects of all-inorganic perovskite QDs for the liquid-type warm white LEDs.

  9. Immunofluorescence Plaque Assay for African Swine Fever Virus

    Science.gov (United States)

    Tessler, J.; Hess, W. R.; Pan, I. C.; Trautman, R.

    1974-01-01

    Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37°C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells. Three statistical criteria for a quantitatively reliable assay were met: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm2 coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated. PMID:4279763

  10. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  11. Sensitive detection of biothiols and histidine based on the recovered fluorescence of the carbon quantum dots–Hg(II) system

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Juan; Zhang, Fengshuang; Yan, Xu; Wang, Long; Yan, Jin [College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Ding, Hong [State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun 130012 (China); Ding, Lan, E-mail: dinglan@jlu.edu.cn [College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012 (China)

    2015-02-15

    Highlights: • Carbon quantum dots-based probe was used for detection of GSH, Cys or His. • The fluorescence of CQDs was quenched by Hg(II) and then recovered by GSH, Cys or His. • No further surface modification or purification of CQDs was required. • This sensor exhibits superior accuracy and sensitivity. • The proposed method was simple in design, fast in operation. - Abstract: In this paper, we presented a novel, rapid and highly sensitive sensor for glutathione (GSH), cysteine (Cys) and histidine (His) based on the recovered fluorescence of the carbon quantum dots (CQDs)–Hg(II) system. The CQDs were synthesized by microwave-assisted approach in one pot according to our previous report. The fluorescence of CQDs could be quenched in the presence of Hg(II) due to the coordination occurring between Hg(II) and functional groups on the surface of CQDs. Subsequently, the fluorescence of the CQDs–Hg(II) system was recovered gradually with the addition of GSH, Cys or His due to their stronger affinity with Hg(II). A good linear relationship was obtained from 0.10 to 20 μmol L{sup −1} for GSH, from 0.20 to 45 μmol L{sup −1} for Cys and from 0.50 to 60 μmol L{sup −1} for His, respectively. This method has been successfully applied to the trace detection of GSH, Cys or His in human serum samples with satisfactory results. The proposed method was simple in design and fast in operation, which demonstrated great potential in bio-sensing fields.

  12. Sensitive detection of biothiols and histidine based on the recovered fluorescence of the carbon quantum dots–Hg(II) system

    International Nuclear Information System (INIS)

    Hou, Juan; Zhang, Fengshuang; Yan, Xu; Wang, Long; Yan, Jin; Ding, Hong; Ding, Lan

    2015-01-01

    Highlights: • Carbon quantum dots-based probe was used for detection of GSH, Cys or His. • The fluorescence of CQDs was quenched by Hg(II) and then recovered by GSH, Cys or His. • No further surface modification or purification of CQDs was required. • This sensor exhibits superior accuracy and sensitivity. • The proposed method was simple in design, fast in operation. - Abstract: In this paper, we presented a novel, rapid and highly sensitive sensor for glutathione (GSH), cysteine (Cys) and histidine (His) based on the recovered fluorescence of the carbon quantum dots (CQDs)–Hg(II) system. The CQDs were synthesized by microwave-assisted approach in one pot according to our previous report. The fluorescence of CQDs could be quenched in the presence of Hg(II) due to the coordination occurring between Hg(II) and functional groups on the surface of CQDs. Subsequently, the fluorescence of the CQDs–Hg(II) system was recovered gradually with the addition of GSH, Cys or His due to their stronger affinity with Hg(II). A good linear relationship was obtained from 0.10 to 20 μmol L −1 for GSH, from 0.20 to 45 μmol L −1 for Cys and from 0.50 to 60 μmol L −1 for His, respectively. This method has been successfully applied to the trace detection of GSH, Cys or His in human serum samples with satisfactory results. The proposed method was simple in design and fast in operation, which demonstrated great potential in bio-sensing fields

  13. Comparison of three PCR-based assays for SNP genotyping in sugar beet

    Science.gov (United States)

    Background: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved t...

  14. Novel Photochrome Aptamer Switch Assay (PHASA) for adaptive binding to aptamers.

    Science.gov (United States)

    Papper, Vladislav; Pokholenko, Oleksandr; Wu, Yuanyuan; Zhou, Yubin; Jianfeng, Ping; Steele, Terry W J; Marks, Robert S

    2014-11-01

    A novel Photochrome-Aptamer Switch Assay (PHASA) for the detection and quantification of small environmentally important molecules such as toxins, explosives, drugs and pollutants, which are difficult to detect using antibodies-based assays with high sensitivity and specificity, has been developed. The assay is based on the conjugation of a particular stilbene-analyte derivative to any aptamer of interest. A unique feature of the stilbene molecule is its reporting power via trans-cis photoisomerisation (from fluorescent trans-isomer to non-fluorescent cis-isomer) upon irradiation with the excitation light. The resulting fluorescence decay rate for the trans-isomer of the stilbene-analyte depends on viscosity and spatial freedom to rotate in the surrounding medium and can be used to indicate the presence of the analyte. Quantification of the assay is achieved by calibration of the fluorescence decay rate for the amount of the tested analyte. Two different formats of PHASA have been recently developed: direct conjugation and adaptive binding. New stilbene-maleimide derivatives used in the adaptive binding format have been prepared and characterised. They demonstrate effective binding to the model thiol compound and to the thiolated Malachite Green aptamer.

  15. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

    DEFF Research Database (Denmark)

    Robey, R W; Honjo, Y; van de Laar, A

    2001-01-01

    The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compo...

  16. A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum

    DEFF Research Database (Denmark)

    Lloyd, Yukie M.; Ngati, Elise P.; Salanti, Ali

    2017-01-01

    Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation....... A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads...

  17. Enzyme activity assays within microstructured optical fibers enabled by automated alignment.

    Science.gov (United States)

    Warren-Smith, Stephen C; Nie, Guiying; Schartner, Erik P; Salamonsen, Lois A; Monro, Tanya M

    2012-12-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women's health.

  18. O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

    Science.gov (United States)

    Borodkin, Vladimir S; Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T; van Aalten, Daan M F

    2018-05-18

    The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

  19. Genetic barcoding with fluorescent proteins for multiplexed applications.

    Science.gov (United States)

    Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

    2015-04-14

    Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

  20. 3-D Image Analysis of Fluorescent Drug Binding

    Directory of Open Access Journals (Sweden)

    M. Raquel Miquel

    2005-01-01

    Full Text Available Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIPY FL-prazosin (QAPB was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or genetic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in the histogram. In the presence of 10 μM phenoxybenzamine (blocking agent, the QAPB (50 nM histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that of control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

  1. Chiral Recognition by Fluorescence: One Measurement for Two Parameters

    Directory of Open Access Journals (Sweden)

    Shanshan Yu

    2014-01-01

    Full Text Available This outlook describes two strategies to simultaneously determine the enantiomeric composition and concentration of a chiral substrate by a single fluorescent measurement. One strategy utilizes a pseudoenantiomeric sensor pair that is composed of a 1,1′-bi-2-naphthol-based amino alcohol and a partially hydrogenated 1,1′-bi-2-naphthol-based amino alcohol. These two molecules have the opposite chiral configuration with fluorescent enhancement at two different emitting wavelengths when treated with the enantiomers of mandelic acid. Using the sum and difference of the fluorescent intensity at the two wavelengths allows simultaneous determination of both concentration and enantiomeric composition of the chiral acid. The other strategy employs a 1,1′-bi-2-naphthol-based trifluoromethyl ketone that exhibits fluorescent enhancement at two emission wavelengths upon interaction with a chiral diamine. One emission responds mostly to the concentration of the chiral diamine and the ratio of the two emissions depends on the chiral configuration of the enantiomer but independent of the concentration, allowing both the concentration and enantiomeric composition of the chiral diamine to be simultaneously determined. These strategies would significantly simplify the practical application of the enantioselective fluorescent sensors in high-throughput chiral assay.

  2. Systematic random sampling of the comet assay.

    Science.gov (United States)

    McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

    2009-07-01

    The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

  3. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  4. Solution assay instrument operations manual

    International Nuclear Information System (INIS)

    Li, T.K.; Marks, T.; Parker, J.L.

    1983-09-01

    An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

  5. Radioligand assay in reproductive biology

    International Nuclear Information System (INIS)

    Korenman, S.G.; Sherman, B.M.

    1975-01-01

    Radioligand assays have been developed for the principal reproductive steroids and peptide hormones. Specific binding reagents have included antibodies, plasma binders, and intracellular receptors. In each assay, problems of specificity, sensitivity, and nonspecific inhibitors were encountered. Many features of the endocrine physiology in childhood, during puberty, and in adulthood have been characterized. Hormonal evaluations of endocrine disorders of reproduction are characterized on the basis of their characteristic pathophysiologic alterations. (U.S.)

  6. Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

    Energy Technology Data Exchange (ETDEWEB)

    Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

    2003-08-01

    Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

  7. Microcystin Detection Characteristics of Fluorescence Immunochromatography and High Performance Liquid Chromatography

    International Nuclear Information System (INIS)

    Pyo, Dong Jin; Park, Geun Young; Choi, Jong Chon; Oh, Chang Suk

    2005-01-01

    Different detection characteristics of fluorescence immunochromatography method and high performance liquid chromatography (HPLC) method for the analysis of cyanobacterial toxins were studied. In particular, low and high limits of detection, detection time and reproducibility and detectable microcystin species were compared when fluorescence immunochromatography method and high performance liquid chromatography method were applied for the detection of microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa. A Fluorescence immunochromatography assay system has the unique advantages of short detection time and low detection limit, and high performance liquid chromatography detection method has the strong advantage of individual quantifications of several species of microcystins

  8. Fluorescence detection of a protein-bound 2Fe2S cluster.

    Science.gov (United States)

    Hoff, Kevin G; Goodlitt, Rochelle; Li, Rui; Smolke, Christina D; Silberg, Jonathan J

    2009-03-02

    A fluorescent biosensor is described for 2Fe2S clusters that is composed of green fluorescent protein (GFP) fused to glutaredoxin 2 (Grx2), as illustrated here. 2Fe2S detection is based on the reduction of GFP fluorescence upon the 2Fe2S-induced dimerization of GFP-Grx2. This assay is sufficiently sensitive to detect submicromolar changes in 2Fe2S levels, thus making it suitable for high-throughput measurements of metallocluster degradation and synthesis reactions.

  9. Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

    Science.gov (United States)

    Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

    2016-04-01

    Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to

  10. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

  11. Fluorescing macerals from wood precursors

    Energy Technology Data Exchange (ETDEWEB)

    Stout, S A; Bensley, D F

    1987-01-01

    A preliminary investigation into the origin of wood-derived macerals has established the existence of autofluorescent maceral precursors in the secondary xylem of swamp-inhabiting plant species. The optical character and fluorescent properties of microtomed thin-sections of modern woods from the Florida Everglades and Okefenokee Swamp, Georgia are compared to the character and properties of their peatified equivalents from various Everglades and Okefenokee peat horizons and their lignitic equivalents from the Brandon lignite of Vermont and the Trail Ridge lignitic peat from northern Florida. The inherent fluorescence of woody cell walls is believed to be caused by lignin though other cell wall components may contribute. The fluorescence spectra for several wood and cell types had a ..gamma../sub m//sub a//sub x/ of 452 nm and Q value of 0.00. The color as observed in blue light and the spectral geometry as measured in UV light of peatified and lignitic woody cell walls (potential textinites) may change progressively during early coalification. Cell wall-derived maceral material is shown to maintain its fluorescing properties after being converted to a structureless material, perhaps a corpohuminite or humodetrinite precursor. Fluorescing xylem cell contents, such as condensed tannins or essential oils, can maintain the fluorescent character through early coalification. Xylem cell walls and xylem cell contents are shown to provide fluorescing progenitor materials which would not require subsequent infusion with 'lipid' materials to account for their fluorescence as phytoclast material or as macerals in coal. 35 references.

  12. Assessing Photosynthesis by Fluorescence Imaging

    Science.gov (United States)

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  13. Chair-side detection of Prevotella Intermedia in mature dental plaque by its fluorescence.

    Science.gov (United States)

    Nomura, Yoshiaki; Takeuchi, Hiroaki; Okamoto, Masaaki; Sogabe, Kaoru; Okada, Ayako; Hanada, Nobuhiro

    2017-06-01

    Prevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque. Thirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person's correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated. When relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia. To check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe.

    Science.gov (United States)

    Liu, Hua; Na, Weidan; Liu, Ziping; Chen, Xueqian; Su, Xingguang

    2017-06-15

    In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H 2 O 2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H 2 O 2 in the range of 3.33-500µM with a detection limit of 1.2µM. The linear detection for AA was in the range from 1.11 to 300µM with a detection limit of 0.32µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Copyright © 2017. Published by Elsevier B.V.

  15. Time-resolved fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Gustavsson, Thomas; Mialocq, Jean-Claude

    2007-01-01

    This article addresses the evolution in time of light emitted by a molecular system after a brief photo-excitation. The authors first describe fluorescence from a photo-physical point of view and discuss the characterization of the excited state. Then, they explain some basic notions related to fluorescence characterization (lifetime and decays, quantum efficiency, so on). They present the different experimental methods and techniques currently used to study time-resolved fluorescence. They discuss basic notions of time resolution and spectral reconstruction. They briefly present some conventional methods: intensified Ccd cameras, photo-multipliers and photodiodes associated with a fast oscilloscope, and phase modulation. Other methods and techniques are more precisely presented: time-correlated single photon counting (principle, examples, and fluorescence lifetime imagery), streak camera (principle, examples), and optical methods like the Kerr optical effect (principle and examples) and fluorescence up-conversion (principle and theoretical considerations, examples of application)

  16. Cysteine residue is not essential for CPM protein thermal-stability assay.

    Science.gov (United States)

    Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

    2015-05-01

    A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

  17. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    -point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method......A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...

  18. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  19. A new trend to determine biochemical parameters by quantitative FRET assays.

    Science.gov (United States)

    Liao, Jia-yu; Song, Yang; Liu, Yan

    2015-12-01

    Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

  20. Phenotypic assays for Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

    2017-10-01

    Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  1. Localized irradiations, evaluation through 'Comet Assay'

    International Nuclear Information System (INIS)

    Di Giorgio, Marina; Taja, Maria R.; Nasazzi, Nora B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

    2000-01-01

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources of Cobalt 60, Cesium 137 or Iridium 192 at work placed for industrial gammagraphy and other radiation sources. Severe skin reaction may developed at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models 'Contaminated Poisson' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. There are also different biophysical techniques that can give response in localized irradiations. Biological dosimetry is a necessary complement to physical and clinical dosimetries. Thus, there is increasing interest in the assessment of biological markers that permit the detection of radiation induced damage in the localized irradiations. The 'Comet Assay' (single cell gel electrophoresis) is a sensitive, rapid and relatively inexpensive method for measuring DNA damage in individual cells. Single cells are embedded in agarose on microscope slides, lysed to remove the majority of the proteins, electrophoresed, then stained with ethidium bromide in order to visualize the DNA. When visualized using a fluorescent microscope, DNA of undamaged cells appears as a spherical mass occupying the cavity formed by the lysed cell. Following radiation damage, the smaller the fragment size and the grater the number of fragments of DNA, the grater the percentage of DNA that it is able to migrate in an electric field, forming a comet image. The assay can be performed under alkaline conditions to examine DNA single strand breaks (SSBs), or in non denaturing (neutral) conditions to measure double strand breaks (DSBs) in individual

  2. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  3. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen; Abuelela, Ayman F.; Mohammad, Amal Jehad

    2017-01-01

    scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least

  4. Evaluation of environmental genotoxicity by comet assay in Columba livia.

    Science.gov (United States)

    González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I

    2016-01-01

    The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

  5. Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

    Directory of Open Access Journals (Sweden)

    Mann David GJ

    2012-05-01

    Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

  6. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Directory of Open Access Journals (Sweden)

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  7. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2015-02-01

    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  8. Fluorescent standards for photodynamic therapy

    Science.gov (United States)

    Belko, N.; Kavalenka, S.; Samtsov, M.

    2016-08-01

    Photodynamic therapy is an evolving technique for treatment of various oncological diseases. This method employs photosensitizers - species that lead to death of tumor cells after the photoactivation. For further development and novel applications of photodynamic therapy new photosensitizers are required. After synthesis of a new photosensitizer it is important to know its concentration in different biological tissues after its administration and distribution. The concentration is frequently measured by the extraction method, which has some disadvantages, e.g. it requires many biological test subjects that are euthanized during the measurement. We propose to measure the photosensitizer concentration in tissue by its fluorescence. For this purpose fluorescent standards were developed. The standards are robust and simple to produce; their fluorescence signal does not change with time. The fluorescence intensity of fluorescent standards seems to depend linearly on the dye concentration. A set of standards thus allow the calibration of a spectrometer. Finally, the photosensitizer concentration can be determined by the fluorescence intensity after comparing the corresponding spectrum with spectra of the set of fluorescent standards. A biological test subject is not euthanized during this kind of experiment. We hope this more humane technique can be used in future instead of the extraction method.

  9. Fluorescence molecular tomography in the presence of background fluorescence

    International Nuclear Information System (INIS)

    Soubret, Antoine; Ntziachristos, Vasilis

    2006-01-01

    Fluorescence molecular tomography is an emerging imaging technique that resolves the bio-distribution of engineered fluorescent probes developed for in vivo reporting of specific cellular and sub-cellular targets. The method can detect fluorochromes in picomole amounts or less, imaged through entire animals, but the detection sensitivity and imaging performance drop in the presence of background, non-specific fluorescence. In this study, we carried out a theoretical and an experimental investigation on the effect of background fluorescence on the measured signal and on the tomographic reconstruction. We further examined the performance of three subtraction methods based on physical models of photon propagation, using experimental data on phantoms and small animals. We show that the data pre-processing with subtraction schemes can improve image quality and quantification when non-specific background florescence is present

  10. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  11. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    Science.gov (United States)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  12. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

  13. Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yi [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China); Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Zhou, Yaofeng [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China)

    2016-09-14

    The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H{sub 2}O{sub 2}) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H{sub 2}O{sub 2} or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL{sup −1} to 625 pg mL{sup −1} with a limit of detection of 2.2 pg mL{sup −1}, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H{sub 2}O{sub 2}-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

  14. Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

    International Nuclear Information System (INIS)

    Liang, Yi; Huang, Xiaolin; Yu, Ruijin; Zhou, Yaofeng; Xiong, Yonghua

    2016-01-01

    The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H_2O_2) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H_2O_2 or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL"−"1 to 625 pg mL"−"1 with a limit of detection of 2.2 pg mL"−"1, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H_2O_2-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

  15. Fluorescent nanodiamonds and their use in biomedical research

    Science.gov (United States)

    Suarez-Kelly, Lorena P.; Rampersaud, Isaac V.; Moritz, Charles E.; Campbell, Amanda R.; Hu, Zhiwei; Alkahtani, Masfer H.; Alghannam, Fahad S.; Hemmer, Phillip; Carson, William E.; Rampersaud, Arfaan A.

    2016-03-01

    Nanodiamonds containing color-centers produce non-quenching fluorescence that is easily detected. This makes them useful for cellular, proteomic and genomic applications. However, fluorescent nanodiamonds have yet to become popular in the biomedical research community as labeling reagents. We discuss production of nanodiamonds with distinct color-centers and assess their biocompatibility and techniques for bioconjugation. Fluorescent diamonds were fabricated by electron irradiation of high-pressure, high-temperature micron-sized diamonds which generated diamonds with vacancy-related defects (V). These diamonds were annealed to create nitrogen vacancy (NV)-centers then following a milling step were fractionated into nanoparticle sizes of 30, 60, and 95 nm. Optical characterization of Vand NV-center diamonds demonstrated fluorescence in two distinct green and red channels, respectively. In vitro studies demonstrated that these nanodiamonds are biocompatible and readily taken up by murine macrophage cells. Quantification of NV-center nanodiamond uptake by flow cytometry, showed that uptake was independent of nanodiamond size. Confocal microscopy demonstrated that NV-center nanodiamonds accumulate within the cytoplasm of these cells. NV-center nanodiamonds were then conjugated with streptavidin using a short polyethylene chain as linker. Conjugation was confirmed via a catalytic assay employing biotinylated-horseradish peroxidase. We present a technique for large-scale production of biocompatible conjugated V- or NV-center nanodiamonds. Functional testing is essential for standardization of fluorescent nanodiamond bioconjugates and quality control. Large-scale production of bioconjugated fluorescent nanodiamonds is crucial to their development as novel tools for biological and medical applications.

  16. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    Science.gov (United States)

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Instructive for disposal of fluorescent

    International Nuclear Information System (INIS)

    Salazar Vargas, Gerlin

    2014-01-01

    An instructive is established for the management system of waste fluorescent lamps, ensuring the storage, collection, transportation, and final disposal. The lamp is changed by an official of the Seccion de Matenimiento Construccion of the Oficina de Servicios Generales or is produced with the support of an official of the unit. The fluorescent should be deposited in stock of materials of the building maintenance section or unit specified with the help of a staff and in appropriate conditions. The fluorescent lamp is transported according to the guidelines in the manual. A responsible company is contracted by la Vicerrectoria de Administracion of the Universidad de Costa Rica dedicated to the transport and proper handling of fluorescent lamps [es

  18. ANTAGONISTIC POTENTIAL OF FLUORESCENT Pseudomonas ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    GROWTH OF TOMATO CHALLENGED WITH PHTOPATHOGENS ... This study focused on the antagonistic potential of fluorescent Pseudomonas in vitro, and its inoculation effect on growth .... the 5 days old culture in starch agar with Lugol's.

  19. Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

    Science.gov (United States)

    Hansen, Scott D.; Zuchero, J. Bradley; Mullins, R. Dyche

    2014-01-01

    The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy. PMID:23868587

  20. Bio-Inspired Microsystem for Robust Genetic Assay Recognition

    Directory of Open Access Journals (Sweden)

    Jaw-Chyng Lue

    2008-01-01

    Full Text Available A compact integrated system-on-chip (SoC architecture solution for robust, real-time, and on-site genetic analysis has been proposed. This microsystem solution is noise-tolerable and suitable for analyzing the weak fluorescence patterns from a PCR prepared dual-labeled DNA microchip assay. In the architecture, a preceding VLSI differential logarithm microchip is designed for effectively computing the logarithm of the normalized input fluorescence signals. A posterior VLSI artificial neural network (ANN processor chip is used for analyzing the processed signals from the differential logarithm stage. A single-channel logarithmic circuit was fabricated and characterized. A prototype ANN chip with unsupervised winner-take-all (WTA function was designed, fabricated, and tested. An ANN learning algorithm using a novel sigmoid-logarithmic transfer function based on the supervised backpropagation (BP algorithm is proposed for robustly recognizing low-intensity patterns. Our results show that the trained new ANN can recognize low-fluorescence patterns better than an ANN using the conventional sigmoid function.

  1. Intermolecular G-quadruplex structure-based fluorescent DNA detection system.

    Science.gov (United States)

    Zhou, Hui; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-Qin

    2013-03-15

    Adopting multi-donors to pair with one acceptor could improve the performance of fluorogenic detection probes. However, common dyes (e.g., fluorescein) in close proximity to each other would self-quench the fluorescence, and the fluorescence is difficult to restore. In this contribution, we constructed a novel "multi-donors-to-one acceptor" fluorescent DNA detection system by means of the intermolecular G-quadruplex (IGQ) structure-based fluorescence signal enhancement combined with the hairpin oligonucleotide. The novel IGQ-hairpin system was characterized using the p53 gene as the model target DNA. The proposed system showed an improved assay performance due to the introduction of IGQ-structure into fluorescent signaling probes, which could inhibit the background fluorescence and increase fluorescence restoration amplitude of fluoresceins upon target DNA hybridization. The proof-of-concept scheme is expected to provide new insight into the potential of G-quadruplex structure and promote the application of fluorescent oligonucleotide probes in fundamental research, diagnosis, and treatment of genetic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Giemsa as a fluorescent dye for mineralizing bone-like nodules in vitro

    International Nuclear Information System (INIS)

    Querido, W; Farina, M; Balduino, A

    2012-01-01

    Giemsa was first used as a fluorescent dye for mineralized bone and cartilage in tissue sections. The aim of this study was to establish the use of Giemsa as a fluorescent dye for mineralizing bone-like nodules produced in cell cultures. Osteoblasts were grown under mineralizing conditions for 14 days, producing typical bone-like nodules. Upon staining with Giemsa stock solution for 1 min, the mineralizing nodules could be selectively visualized emitting intense green and red fluorescence when observed under blue and green illumination, respectively. The textural details of the nodules were clearly observed under fluorescence microscopy, allowing to identify regions with different degrees of mineralization. The mineralized nature of the nodules was confirmed using von Kossa's method, Alizarin Red S staining and x-ray mapping for Ca and P in a scanning electron microscope, showing a strong correlation between the mineralizing and the fluorescent nodules. The selective fluorescence was related to the mineral phase, being absent in decalcified samples. The use of Giemsa as a fluorescent dye for mineralizing bone-like nodules presents a simple alternative method to quickly analyze biomineralization assays in vitro under fluorescence microscopy, particularly in the biological evaluation of biomaterials. (communication)

  3. X-ray fluorescence holography

    CERN Document Server

    Hayashi, K; Takahashi, Y

    2003-01-01

    X-ray fluorescence holography (XFH) is a new structural analysis method of determining a 3D atomic arrangement around fluorescing atoms. We developed an XFH apparatus using advanced X-ray techniques and succeeded in obtaining high-quality hologram data. Furthermore, we introduced applications to the structural analysis of a thin film and the environment around dopants and, discussed the quantitative analysis of local lattice distortion. (author)

  4. Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues.

    Science.gov (United States)

    Bulgari, Daniela; Casati, Paola; Faoro, Franco

    2011-11-01

    In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Micromotor-based on-off fluorescence detection of sarin and soman simulants.

    Science.gov (United States)

    Singh, Virendra V; Kaufmann, Kevin; Orozco, Jahir; Li, Jinxing; Galarnyk, Michael; Arya, Gaurav; Wang, Joseph

    2015-06-30

    Self-propelled micromotor-based fluorescent "On-Off" detection of nerve agents is described. The motion-based assay utilizes Si/Pt Janus micromotors coated with fluoresceinamine toward real-time "on-the-fly" field detection of sarin and soman simulants.

  6. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

  7. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  8. Fluorescent Nanodiamonds in Biomedical Applications.

    Science.gov (United States)

    Mitura, Katarzyna Anna; Włodarczyk, Elżbieta

    2018-04-18

    Nanoparticles have an extended surface and a large surface area, which is the ratio of the size of the surfacearea to the volume. A functionalized surface can give rise to more modifications and therefore allows this nanomaterial to have new properties. Fluorescent molecules contain fluorophore, which is capable of being excited via the absorption of light energy at a specific wavelength and subsequently emitting radiation energy of a longer wavelength. A chemically modified surface of nanodiamond (ND; by carboxylation) demonstrated biocompatibility with DNA, cytochrome C, and antigens. In turn, fluorescent nanodiamonds (FNDs) belong to a group of new nanomaterials. Their surface can be modified by joining functional groups such as carboxyl, hydroxyl, or amino, after which they can be employed as a fluorescence agent. Their fluorescent properties result from defects in the crystal lattice. FNDs reach dimensions of 4-100 nm, have attributes such as photostability, long fluorescence lifetimes (10 ns), and fluorescence emission between 600 and 700 nm. They are also nontoxic, chemically inert, biocompatible, and environmentally harmless. The main purpose of this article was to present the medical applications of various types of modified NDs.

  9. Fluorescence detection of esophageal neoplasia

    Science.gov (United States)

    Borisova, E.; Vladimirov, B.; Avramov, L.

    2008-06-01

    White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

  10. Hemoglobin detection using carbon dots as a fluorescence probe.

    Science.gov (United States)

    Barati, Ali; Shamsipur, Mojtaba; Abdollahi, Hamid

    2015-09-15

    Herein, we have described the application of high fluorescent carbon dots (CDs) without any surface modification as a simple and fast responding fluorescence probe for sensitive and selective determination of hemoglobin (Hb) in the presence of H2O2. Although Hb itself was able to quench the fluorescence of CDs, based on the inner filter effect (IFE) of the protein that affects both excitation and emission spectra of CDs, the presence of H2O2 resulted in further improvement of the sensitivity of Hb detection. The assay is based on the reaction of Hb with H2O2 that generates reactive oxygen species including hydroxyl (OH•) and superoxide (O2(•-)) radicals under heme degradation and/or iron release from Hb and the subsequent reaction of hydroxyl radicals, as strong oxidizing agents, with CDs resulting in high fluorescence quenching. The proposed probe was used for determination of Hb in concentration range of 1-100 nM with a detection limit of 0.4 nM. The method was successfully applied to the determination of Hb in human blood samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    International Nuclear Information System (INIS)

    Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

    2014-01-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

  12. Long term storage of virus templated fluorescent materials for sensing applications

    Energy Technology Data Exchange (ETDEWEB)

    Seetharam, Raviraja N; Guerra, Charles; Satir, Peter [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Blum, Amy Szuchmacher; Soto, Carissa M; Ratna, Banahalli R [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Whitley, Jessica L [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Sapsford, Kim E [George Mason University, 10910 University Boulevard, Manassas, VA 20110 (United States); Chatterji, Anju; Lin Tianwei; Johnson, John E [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)], E-mail: amy.blum@nrl.navy.mil

    2008-03-12

    Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studied for long term preservation in the presence and absence of cryoprotectants. Viral complexes were reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNase assay for structural integrity. When viruses lyophilized in the absence of cryoprotectant were rehydrated and RNase treated, UV absorption increased, indicating that the capsids were damaged. The addition of trehalose during lyophilization protected capsid integrity for at least 7 weeks. Measurements of the fluorescence peak maximum of CPMV lyophilized with trehalose and reconstituted also indicate that the virus remained intact. Microarray binding assays indicated that CPMV particles chemically modified for use as a fluorescent tracer were intact and retained binding specificity after lyophilization in the presence of trehalose. Thus, we demonstrate that functionalized CPMV nanostructures can be stored for the long term, enabling their use in practical sensing applications.

  13. Time-resolved UV-excited microarray reader for fluorescence energy transfer (FRET) measurements

    Science.gov (United States)

    Orellana, Adelina; Hokkanen, Ari P.; Pastinen, Tomi; Takkinen, Kristina; Soderlund, Hans

    2001-05-01

    Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

  14. Long term storage of virus templated fluorescent materials for sensing applications

    International Nuclear Information System (INIS)

    Seetharam, Raviraja N; Guerra, Charles; Satir, Peter; Blum, Amy Szuchmacher; Soto, Carissa M; Ratna, Banahalli R; Whitley, Jessica L; Sapsford, Kim E; Chatterji, Anju; Lin Tianwei; Johnson, John E

    2008-01-01

    Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studied for long term preservation in the presence and absence of cryoprotectants. Viral complexes were reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNase assay for structural integrity. When viruses lyophilized in the absence of cryoprotectant were rehydrated and RNase treated, UV absorption increased, indicating that the capsids were damaged. The addition of trehalose during lyophilization protected capsid integrity for at least 7 weeks. Measurements of the fluorescence peak maximum of CPMV lyophilized with trehalose and reconstituted also indicate that the virus remained intact. Microarray binding assays indicated that CPMV particles chemically modified for use as a fluorescent tracer were intact and retained binding specificity after lyophilization in the presence of trehalose. Thus, we demonstrate that functionalized CPMV nanostructures can be stored for the long term, enabling their use in practical sensing applications

  15. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

    Directory of Open Access Journals (Sweden)

    Adam R Brown

    Full Text Available Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin, were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.

  16. Automation of the dicentric chromosome assay and related assays

    International Nuclear Information System (INIS)

    Balajee, Adayabalam S.; Dainiak, Nicholas

    2016-01-01

    Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

  17. Assay strategies and methods for phospholipases

    International Nuclear Information System (INIS)

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

  18. FLUORESCENCE DIAGNOSIS FOR RECURRENT BLADDER CANCER

    Directory of Open Access Journals (Sweden)

    R. V. Ulyanov

    2017-01-01

    Full Text Available The clinical case of successful use of local fluorescence spectroscopy combined with fluorescence imaging during cystoscopy for diagnosis of recurrent bladder cancer is represented in the article. Histological study of fluorescent foci confirmed tumor growth (urothelial carcinoma in all areas with high levels of diagnostic parameter. In the fluorescent focus with low diagnostic parameter inflammation was detected.

  19. Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

    Science.gov (United States)

    Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

    2012-11-01

    Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

  20. Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

    Directory of Open Access Journals (Sweden)

    Dae Hong Jeong

    2012-03-01

    Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques.