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protein microarray spots are modulated by patterning method, surface chemistry and processing conditions

BIOSENSORS & BIOELECTRONICS 2019年 130卷 397-407页

作者： Clancy, Kathryn F. A. Dery, Sebastien Laforte, Veronique Shetty, Prasad Juncker, David Nicolau, Dan V. McGill Univ Dept Bioengn Montreal PQ Canada McGill Univ Dept Biomed Engn Montreal PQ Canada McGill Univ Dept Neurol & Neurosurg Montreal PQ Canada McGill Univ Montreal PQ Canada Genome Quebec Innovat Ctr Montreal PQ Canada

The uniformity of the protein patterns, their shape, and the contrast between the fluorescence signal of the pattern and the background, critically modulate the quantitative accuracy of the microarray-derived data. While significant research focused of the identification of the factors that impact the protein microarray patterns, these studies usually have focused on the optimization of one set of these factors, e.g., how the spot uniformity is affected by different additives, or by different surfaces. However, the complex interaction between proteins, carrier fluids, surfaces, and patterning methodologies used would suggest a systematic and more comprehensive study that considers all these parameters, as well as their inter-relationship. The present work compared the patterning of two fluorescently-tagged proteins, i.e., IgG, BSA, on surfaces with different hydrophobicity and chemistry, and printed by inkjet, pin, and microcontact printing (mu CP). The quantification of the spot size regularity, its morphology, the signal intensity and its distribution within spots were used to assess the quality of a specific printing method, on a specific surface, with a specific solute of the printed protein. It was found that the optimal uniformity for both droplet-based methods depend on surface chemistry, with glass slides modified with 3-Glycidoxypropyl-dimethoxymethyl silane (GPS) and 3-(Aminopropyl)-triethoxy silane (APTES) exhibiting the greatest uniformity, while uniformity of the mu CP patterns was relatively independent of the surface chemistry. For the inkjet and pin printing, the largest fluorescence signal and contrast with the background was found on APTES modified glass slides, whereas for the mu CP the fluorescence signal increased with increasing hydrophilicity.

关键词： protein microarray Inkjet printing Pin printing Micro-contact printing microarray spot

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protein microarray analysis of apoptosis-related protein expression following heat shock in human tongue squamous cell carcinomas containing different p53 phenotypes

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INTERNATIONAL JOURNAL OF HYPERTHERMIA 2008年第8期24卷 605-612页

作者： Kajihara, Atsuhisa Takahashi, Akihisa Ohnishi, Ken Imai, Yuichiro Yamakawa, Nobuhiro Yasumoto, Jun-ichi Ohnishi, Takeo Kirita, Tadaaki Nara Med Univ Sch Med Dept Biol Nara 6348521 Japan Nara Med Univ Sch Med Dept Oral & Maxillofacial Surg Nara 6348521 Japan

Purpose: Hyperthermia is useful in the treatment of human head and neck cancers, because it is relatively easy to regulate temperatures when compared to tumors located in deep organs. In this study, attention was focused on p53 as a possible predictive indicator for the efficacy of hyperthermic cancer therapy. Methods: Two kinds of cell lines were used. These were derived from a human squamous cell carcinoma (SAS) and had identical genetic backgrounds except for their p53 gene status. It was previously reported that the heat sensitivity and frequency of apoptosis in wild-type p53 cells (SAS/neo) were clearly elevated when compared with mutated p53 cells (SAS/mp53). In order to study the expression of apoptosis related proteins after heat treatment, protein microarray analysis was used. Results: The expression of apoptosis inhibitory proteins such as Bcl-2, Bcl-xL, NF-B, COX2, STAT3, IL-6, and IKK/1 was seen to increase after heat treatment in SAS/mp53 cells, but not in SAS/neo cells. Conclusion: The result of these observations indicates that apoptosis inhibitory proteins (such as Bcl-2, Bcl-xL, IL-6, etc.) were highly induced in SAS/mp53 cells after heat treatment when compared to control SAS/neo cells.

关键词： Apoptosis protein microarray heat p53 human tongue carcinoma

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protein microarray biosensors based on imaging ellipsometry techniques and their applications

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protein & Cell 2011年第6期2卷 445-455页

作者： Yu Niu Gang Jin NML Institute of MechanicsChinese Academy of SciencesBeijing 100190China Graduate School of the Chinese Academy of Sciences Beijing 100049China

After years of development,biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering *** experimental set... 详细信息

关键词： biosensor protein microarray imaging ellipsometry total internal reflection imaging ellipsometry applications

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protein microarray with horseradish peroxidase chemiluminescence for quantification of serum -fetoprotein

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JOURNAL OF INTERNATIONAL MEDICAL RESEARCH 2015年第5期43卷 639-647页

作者： Zhao, Yuanshun Zhang, Yonghong Lin, Dongdong Li, Kang Yin, Chengzeng Liu, Xiuhong Jin, Boxun Sun, Libo Liu, Jinhua Zhang, Aiying Li, Ning Capital Med Univ Beijing Youan Hosp Beijing 100069 Peoples R China Capital Med Univ Beijing Youan Hosp Beijing Inst Hepatol Beijing 100069 Peoples R China

Objectives To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of -fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). Methods A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Results Serum AFP concentrations determined via protein microarray were positively correlated (r=0.973) with those determined via ELISA in patients with HCC (n=60) and healthy control subjects (n=30). protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974;95% CI 0.000, 1.000). Conclusion protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum.

关键词： protein microarray -fetoprotein horseradish peroxidase hepatocellular carcinoma

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protein microarray detection strategies: focus on direct detection technologies

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JOURNAL OF IMMUNOLOGICAL METHODS 2004年第1-2期290卷 121-133页

作者： Espina, V Woodhouse, EC Wulfkuhle, J Asmussen, HD Petricoin, EF Liotta, LA US FDA NCI Clin Proteom Program Pathol LabCtr Canc Res Bethesda MD 20892 USA Univ Colorado Boulder CO 80309 USA US FDA Ctr Biol Evaluat & Res NCI Clin Proteom Program Bethesda MD 20892 USA

protein microarrays are being utilized for functional proteomic analysis, providing information not obtainable by gene arrays. microarray technology is applicable for studying protein-protein, protein-ligand, kinase activity and posuranslational modifications of proteins. A precise and sensitive protein microarray, the direct detection or reverse-phase microarray, has been applied to ongoing clinical trials at the National Cancer Institute for studying phosphorylation events in EGF-receptor-mediated cell signaling pathways. The variety of microarray applications allows for multiple, creative microarray designs and detection strategies. Herein, we discuss detection strategies and challenges for protein microarray technology, focusing on direct detection of protein microarrays. Published by Elsevier B.V.

关键词： protein microarray catalyzed reporter deposition molecular profiling fluorescence chromogens

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protein microarray analysis identifies human cellular prion protein interactors

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NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY 2009年第1期35卷 16-35页

作者： Satoh, J. Obayashi, S. Misawa, T. Sumiyoshi, K. Oosumi, K. Tabunoki, H. Meiji Pharmaceut Univ Dept Bioinformat & Mol Neuropathol Tokyo 2048588 Japan NCNP Natl Inst Neurosci Dept Immunol Tokyo Japan

Aims: To obtain an insight into the function of cellular prion protein (PrPC), we studied PrPC-interacting proteins (PrPIPs) by analysing a protein microarray. Methods: We identified 47 novel PrPIPs by probing an array of 5000 human proteins with recombinant human PrPC spanning amino acid residues 23-231 named PR209. Results: The great majority of 47 PrPIPs were annotated as proteins involved in the recognition of nucleic acids. Coimmunoprecipitation and cell imaging in a transient expression system validated the interaction of PR209 with neuronal PrPIPs, such as FAM64A, HOXA1, PLK3 and MPG. However, the interaction did not generate proteinase K-resistant proteins. KeyMolnet, a bioinformatics tool for analysing molecular interaction on the curated knowledge database, revealed that the complex molecular network of PrPC and PrPIPs has a significant relationship with AKT, JNK and MAPK signalling pathways. Conclusions: protein microarray is a useful tool for systematic screening and comprehensive profiling of the human PrPC interactome. Because the network of PrPC and interactors involves signalling pathways essential for regulation of cell survival, differentiation, proliferation and apoptosis, these observations suggest a logical hypothesis that dysregulation of the PrPC interactome might induce extensive neurodegeneration in prion diseases.

关键词： cellular prion protein KeyMolnet protein microarray protein-protein interaction

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protein microarray assay for the screening of SH3 domain interactions

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ANALYTICAL AND BIOANALYTICAL CHEMISTRY 2010年第5期398卷 1937-1946页

作者： Asbach, Benedikt Kolb, Michaela Liss, Michael Wagner, Ralf Schaeferling, Michael Univ Regensburg Inst Analyt Chem Chemo & Biosensors D-93053 Regensburg Germany Univ Regensburg Inst Med Microbiol & Hyg D-93053 Regensburg Germany Geneart AG D-93053 Regensburg Germany

Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. protein interactions are often mediated by conserved modular domains, e.g., SH3-domains, which recognize proline-rich sequences in their cognate ligands. In the course of this study, different microarray formats (reactive silane monolayers and nitrocellulose on glass slides) and assay work flows were evaluated to develop a microarray based screening assay that permits the reliable identification of interactions between certain target proteins with a set of SH3 domains. Nine representative SH3 domains which were produced and purified as GST-fusion proteins were spotted on the microarray substrates and probed with two well-characterized ligands, the Nef protein from HIV-1 and the human protein Sam68. The best results from these low-density model arrays were obtained with nitrocellulose slides. We show that a straightforward and highly robust detection of ligand binding is achieved by staining with a fluorescently labeled antibody directed against the N-terminal His-tag attached to these proteins. The optimized assay protocol reported here allows for the identification of SH3-interactions with high reproducibility and adequate signal-to-background and signal-to-noise ratios, as well as the quantitative determination of relative binding affinities.

关键词： protein microarray Biochip analysis SH3 domains Signal transduction Proteomics

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protein microarray-mediated detection of antienterovirus antibodies in serum

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JOURNAL OF INTERNATIONAL MEDICAL RESEARCH 2016年第2期44卷 287-296页

作者： Zhang, Aiying Xiu, Bingshui Zhang, Heqiu Li, Ning Capital Med Univ Beijing YouAn Hosp Beijing Inst Hepatol B Bldg8 Xitoutiao Beijing 100069 Peoples R China Inst Basic Med Sci Dept Vaccine Engn East Bldg27 Taiping Rd Beijing 100850 Peoples R China

Objective To utilize prokaryotic gene expression and protein microarray to develop and evaluate a sensitive, accurate protein microarray assay for detecting antienterovirus antibodies in serum samples from patients with hand, foot and mouth disease (HFMD). Enterovirus 71 (EV71) and coxsackievirus A16 (CA16), two common causative agents for HFMD, were used for assay development. Methods Serum was collected from patients with HFMD and healthy controls. EV71 and CA16 VP1 and VP3 genes were expressed in transfected Escherichia coli;the resultant VP1 and 3 proteins were used in a microarray assay for human serum EV71 and CA16 immunoglobulin (Ig) M and IgG. To validate the microarray assay, serum samples were tested for EV71 IgM using enzyme-linked immunosorbent assay (ELISA). Results Out of 50 patients with HFMD, EV71 IgM and CA16 IgM was detected in 80% and 44% of serum samples, respectively, using protein microarray, and EV71 IgM was detected in 78% of samples using ELISA. protein microarray and ELISA showed 100% specificity for EV71-IgM detection. Conclusion The protein microarray assay developed in the present study shows potential as a sensitive technique for detecting EV71 IgM in serum samples from patients with HFMD.

关键词： Hand foot and mouth disease EV71 CA16 protein microarray

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protein microarray platform for the multiplex analysis of biomarkers in human sera

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JOURNAL OF IMMUNOLOGICAL METHODS 2006年第1-2期316卷 1-7页

作者： Urbanowska, Teresa Mangialaio, Sara Zickler, Christophe Cheevapruk, Sirinda Hasler, Paul Regenass, Stephan Legay, Francois Novartis Pharma AG Werk Klybeck CH-4002 Basel Switzerland Kantonsspital Aarau Rheumaklin Aarau Switzerland Univ Basel Hosp Dept Rheumatol CH-4031 Basel Switzerland

Many biomarkers are currently used to monitor patients in clinical studies. Technologies which evaluate, validate and monitor biomarkers in a cost effective and efficient manner are a necessity. Here we describe the development, validation and implementation of a protein microarray platform for the quantitative and simultaneous analysis of six proteins: IL-1 beta, IL-1ra, IL-6, IL-8, MCP-1 and TNF alpha. The platform utilizes a 96-well plate as a solid support on which antibodies are immobilized using non-contact piezoelectric printing. The reaction is based on a sandwich ELISA and the signal is quantified by chemiluminescence with a CCD camera. The robustness and reproducibility of the methodology was investigated using the Food and Drug Administration (FDA.) regulatory guidelines for pharmacokinetic assay validation, in which a spike-recovery validation test was elaborated and run over 3 days. The method was shown to be both quantitative and reproducible, with assay accuracy between 70% and 130%, and an assay precision of less than 30%. In addition, protein microarray performance was compared with the classical ELISA approach. Sera collected from a total of 78 individuals were assayed using both approaches. Correlation coefficients (R-2) between the two technologies were calculated for each of the analytes: 0.90 for IL-1 beta, 0.60 for IL-1ra, 0.93 for IL-6, 0.96 for IL-8, 0.94 for MCP-1 and 0.95 for TNF alpha. The results obtained demonstrate the applicability of this protein microarray for quantitative and simultaneous analysis of IL-1 beta, IL-1ra, IL-6, IL-8, MCP-1 and TNF alpha in clinical samples. (c) 2006 Published by Elsevier B.V.

关键词： biomarker protein microarray immunoassay

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protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives

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PEDIATRIC DIABETES 2007年第5期8卷 252-260页

作者： Vrabelova, Zuzana Kolouskova, Stanislava Boehmova, Kristyna Faresjoe, Maria Karlsson Sumnik, Zdenek Pechova, Marta Kverka, Miloslav Chudoba, Daniel Zacharovova, Klara Stadlerova, Gabriela Pithova, Pavlina Hladikova, Marie Stechova, Katerina Charles Univ Prague Fac Med 2 Dept Pediat Prague Czech Republic Linkoping Univ Diabet Res Ctr Fac Hlth Sci Div Pediat Linkoping Sweden Charles Univ Prague Fac Med 2 Dept Chem & Biochem Prague Czech Republic Acad Sci Czech Republic Inst Microbiol Prague Czech Republic Charles Univ Prague Fac Med 2 Dept Biol & Genet Prague Czech Republic Inst Clin & Expt Med Dept Diabetol Prague Czech Republic Charles Univ Prague Fac Med 2 Dept Internal Med Prague Czech Republic Charles Univ Prague Fac Med 2 Dept Med Informat & Stat Prague Czech Republic

Background Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis. Objectives: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. Methods: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543;proinsulin a.a. 9-23;and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-gamma, tumor necrosis factor beta, transforming growth factor beta 1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. Results: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-gamma (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. Conclusion: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.

关键词： autoantigen autoreactive T cells cytokine protein microarray T1D

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