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Improving the sensitivity of protein microarray by evanescent-field-induced fluorescence

Journal of Zhejiang University-Science A(Applied Physics & Engineering) 2005年第7期6卷 623-626页

作者：王立强陆祖康 State Key Lab of Modern Optical Instrumentation Zhejiang University

To improve the sensitivity of protein microarray, a prism surface replaces the surface of the common microscope *** protein targets arrayed on the surface are hybridized and labelled by fluorescent probes. Evanescent excitation occurs when the convergent laser reaches the surface, and a photomultiplier tube detects the emitted fluorescent signal. A two-dimensional actuator scans the whole surface to achieve planar laser excitation and fluorescence collection. The penetration depth of the evanescent field into the protein targets is only some hundred nanometers and can be controlled by different incident angle of the laser beam, so the undesired background signals are reduced dramatically and the detection sensitivity is improved by a factor of 50 to 100 comparing to confocal excitation. This approach can detect low abundance analytes without signal amplification.

关键词： protein microarray Evanescent excitation Sensitivity Total reflection

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Non-contact protein microarray fabrication using a procedure based on liquid bridge formation

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ANALYTICAL AND BIOANALYTICAL CHEMISTRY 2009年第2期393卷 591-598页

作者： Hartmann, Michael Sjodahl, Johan Stjernstrom, Mayenrten Redeby, Johan Joos, Thomas Roeraade, Johan Royal Inst Technol Dept Analyt Chem Sch Chem Sci & Engn S-10044 Stockholm Sweden Univ Tubingen NMI Nat & Med Sci Inst Reutlingen Germany

Contemporary microarrayers of contact or noncontact format used in protein microarray fabrication still suffer from a number of problems, e. g. generation of satellite spots, inhomogeneous spots, misplaced or even absent spots, and sample carryover. In this paper, a new concept of non-contact sample deposition that reduces such problems is introduced. To show the potential and robustness of this pressure-assisted deposition technique, different sample solutions known to cause severe problems or to be even impossible to print with conventional microarrayers were accurately printed. The samples included 200 mg mL(-1) human serum albumin, highly concentrated sticky cell adhesion proteins, pure high-salt cell-lysis buffer, pure DMSO, and a suspension of 5-mu m polystyrene beads. Additionally, a water-immiscible liquid fluorocarbon, which was shown not to affect the functionality of the capture molecules, was employed as a lid to reduce evaporation during microarray printing. The fluorocarbon liquid lid was shown to circumvent hydrolysis of water-sensitive activated surfaces during long-term deposition procedures.

关键词： Non-contact microarrayer Liquid lid Fluorocarbon Capillary microarrayer protein microarray

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Label-Free, Single protein Detection on a Near-Infrared Fluorescent Single-Walled Carbon Nanotube/protein microarray Fabricated by Cell-Free Synthesis

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NANO LETTERS 2011年第7期11卷 2743-2752页

作者： Ahn, Jin-Ho Kim, Jong-Ho Reuel, Nigel F. Barone, Paul W. Boghossian, Ardemis A. Zhang, Jingqing Yoon, Hyeonseok Chang, Alice C. Hilmer, Andrew J. Strano, Michael S. MIT Dept Chem Engn Cambridge MA 02139 USA

Excessive sample volumes continue to be a major limitation in the analysis of protein protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni2+ chelated by N alpha,N alpha-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni2+ acts as a proximity quencher with the Ni2+/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein protein interactions in the staurosporine-induced apoptosis of SH-SYSY cells, confirming literature predictions.

关键词： protein microarray nanotube protein-protein interaction single-walled carbon nanotubes label-free detection

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Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

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ANALYTICA CHIMICA ACTA 2006年第2期570卷 137-143页

作者： Varnum, Susan M. Warner, Marvin G. Dockendorff, Brian Anheier, Norman C., Jr. Lou, Jianlong Marks, James D. Smith, Leonard A. Feldhaus, Michael J. Grate, Jay W. Bruckner-Lea, Cynthia J. Pacific NW Natl Lab Fundamental Sci Directorate Richland WA 99352 USA Univ Calif San Francisco Dept Anesthesia San Francisco CA 94110 USA USA Med Res Inst Infect Dis Ft Detrick MD 21702 USA

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL(-1)). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min. (c) 2006 Elsevier B.V. All rights reserved.

关键词： immunoassay protein microarray renewable microcolumn sensor monoclonal antibody botulinum neurotoxin A

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Use of protein microarray to identify gene expression changes of Yersinia pestis at different temperatures

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CANADIAN JOURNAL OF MICROBIOLOGY 2011年第4期57卷 287-294页

作者： Li, Bei Tan, Yafang Guo, Jingyu Cui, Baizhong Wang, Zuyun Wang, Hu Zhou, Lei Guo, Zhaobiao Zhu, Ziwen Du, Zongmin Yang, Ruifu Inst Microbiol & Epidemiol State Key Lab Pathogen & Biosecur Lab Analyt Microbiol Beijing 100071 Peoples R China Hubei Med Univ Taihe Hosp Shiyan 442000 Hubei Province Peoples R China Qinghai Inst Endem Dis Prevent & Control Xining 811602 Qinghai Prov Peoples R China

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 degrees C, and mammalian hosts, which have a body temperature of 37 degrees C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague scrum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 degrees C, or adsorbed by Y pestis cultivated at 26 and 37 degrees C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 degrees C but not at 26 degrees C, or expressed at significantly higher levels at 37 degrees C than at 26 degrees C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 degrees C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.

关键词： Yersinia pestis protein microarray antibody profiling temperature

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A polypyrrole protein microarray for antibody-antigen interaction studies using a label-free detection process

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ANALYTICAL BIOCHEMISTRY 2005年第2期347卷 193-200页

作者： Grosjean, L Cherif, B Mercey, E Roget, A Levy, Y Marche, PN Villiers, MB Livache, T Univ Grenoble 1 INSERM U548 Lab Immunochim CEA GDRDC F-38054 Grenoble France UJF CNRS CEADRFMC CREBUMR 5819 F-38054 Grenoble France Inst Opt Orsay LCFIO F-91043 Orsay France

protein microarray is a promising technology that should combine rapidity and easy use with high throughput and versatility. This article describes a method in which an electrocopolymerization process is employed to graft biological molecules on to a chip so that surface plasmon resonance imaging may be used to detect molecular interactions. Copolymerization of pyrrole-modified protein and pyrrole is an efficient grafting process which immobilizes molecules at defined positions on a gold surface. Surface plasmon resonance imaging is an optical technique that allows real-time simultaneous detection of molecular interactions on a large number of spots without labeling. This method was successfully used to analyze antibody-antigen interactions. This illustrates its high specificity and good sensitivity and demonstrates its suitability for biological studies. (c) 2005 Elsevier Inc. All rights reserved.

关键词： protein microarray polypyrrole surface plasmon resonance imaging antigen antibody

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protein Structural Denaturation Evaluated by MCR-ALS of protein microarray FTIR Spectra

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ANALYTICAL CHEMISTRY 2021年第40期93卷 13441-13449页

作者： De Meutter, Joelle Goormaghtigh, Erik Univ Libre Bruxelles Ctr Struct Biol & Bioinformat Lab Struct & Funct Biol Membranes Campus PlaineCP206-2 B-1050 Brussels Belgium

The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the efficiency and safety of biotherapeutic proteins. Yet, recognizing the structural features associated with intermolecular bridging in a high-throughput manner remains a challenge. We combined here the use of protein microarrays spotted at a density of ca 2500 samples per cm(2) and Fourier transform infrared (FTIR) imaging to analyze structural modifications in a set of 85 proteins characterized by widely different secondary structure contents, submitted or not to mild denaturing conditions. Multivariate curve resolution alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subject to denaturing conditions. In the native protein set, 6 components were found to be sufficient to obtain good modeling of the spectra. Furthermore, their shape allowed them to be assigned to alpha-helix, beta-sheet, and other structures. Their content in each protein was correlated with the known secondary structure, confirming these assignments. In the denatured proteins, a new component was necessary and modeled by MCR-ALS. This new component could be assigned to the intermolecular beta-sheet, bridging protein molecules. MCR-ALS, therefore, unveiled a potential spectroscopic marker of protein aggregation and allowed a semiquantitative evaluation of its content. Insight into other structural rearrangements was also obtained.

关键词： Fourier-transform infrared spectroscopy protein protein aggregation protein structure protein microarray

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LABEL-FREE DETECTION OF protein microarray WITH HIGH THROUGHPUT SURFACE PLASMON RESONANCE IMAGING(SPRI)

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Journal of Innovative Optical Health Sciences 2008年第1期1卷 107-114页

作者： SHUKUAN XU GUOLIANG HUANG CHENG DENG JIANG ZHU CHAO HAN XIAOYONG YANG Medical Systems Biology Research Center Tsinghua University School of Medicine Beijing China National Engineering Research Center for Beijing Biochip Technology BeijingChina

A surface plasmon resonance imaging(SPRI)system was developed for the discrimination of proteins on a gold *** a label-free and high-throughput technique,SPRI enables simultaneously monitoring of the biomolecular interactions at low *** used SPRI as a label-free and parallel method to detect different proteins based on protein *** Serum Albumin(BSA),Casein and Immunoglobulin G(IgG)were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6×6 *** proteins can be discriminated directly by changing the incident angle of *** reproducibility for label-free detection of protein molecules was *** SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.

关键词： Surface plasmon resonance imaging protein microarray label-free highthroughput

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A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood

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ANALYTICAL BIOCHEMISTRY 2008年第1期382卷 9-15页

作者： Burgess, Stewart T. G. Kenyon, Fiona O'Looney, Nichola Ross, Alan J. Kwan, Marisa Chong Beattie, John S. Petrik, Juraj Ghazal, Peter Campbell, Colin J. Univ Edinburgh Div Pathway Med Coll Med & Vet Med NRIE Edinburgh EH16 4SB Midlothian Scotland Royal Dick Vet Coll Transfus Transmitted Infect Dept SNBTS Edinburgh EH9 1QH Midlothian Scotland

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format. (C) 2008 Elsevier Inc. All rights reserved.

关键词： protein microarray Human immunodeficiency virus Hepatitis C virus Blood typing Serodiagnosis

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Simultaneous Determination of Malachite Green, Diethylstilbestrol, Medroxyprogesterone, and 3-Amino-2-Oxazolidone in Synbranchoid Eels by a protein microarray Method

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FOOD ANALYTICAL METHODS 2015年第4期8卷 1058-1066页

作者： Liu, Ying-Chun Wang, Quan Jiang, Wei Chen, Yong-Jun Zhao, Xin-Yu Jing, Zhen-Yu Zhang, Meng Chinese Acad Agr Sci Shanghai Vet Res Inst Shanghai 200241 Peoples R China

protein chips have emerged as a useful approach for a wide variety of applications, including the identification of small molecule targets. In this study, a protein microarray method was developed for the simultaneous detection of the following four veterinary drug residues in synbranchoid eels: leucomalachite green (LMG), diethylstilbestrol (DES), medroxyprogesterone (MPA), and 3-amino-2-oxazolidone (AOZ). The analytes were coupled to ovalbumin (OVA) before immobilization on the surface of modified glass slides. After immobilization, a mixture of antibodies to the four analytes and either standard solutions containing the analytes or samples were added to the array reaction area. Then, Cy3-labeled goat anti-rabbit IgG was added and analyte residues were detected quantitatively. The results indicated that the 50 % inhibitory concentrations of DES, MPA, LMG, and AOZ were 2.0, 1.98, 1.65, and 0.355 mu g/L, respectively. The limit of detection of DES, MPA, LMG, and AOZ in synbranchoid eels were 0.325, 0.37, 0.271, and 0.103 mu g/kg, respectively, and recovery rates ranged from 60.7 to 98.6 % for fortified samples at levels of 2, 5, and 10 mu g/kg with coefficient of variation values < 15 %. This protein microarray method was compared with commercially available kits for these four analytes;both methods yielded similar results. These results indicate that the protein microarray method is suitable for the detection of DES, MPA, LMG, and AOZ, as well as other potential drug residues in animal tissues.

关键词： protein microarray Drug residue Malachite green Diethylstilbestrol Medroxyprogesterone 3-amino-2-oxazolidone

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