Protein Microarray Chip Research Articles

Protein Microarray Characterization of the S-Nitrosoproteome

Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO's actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.

Abstract 3216: Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications.

Abstract Antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is a predictive biomarker for cisplatin based chemotherapy. High ERCC1 expression is linked to drug resistance to cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissue samples. By using high density protein microarray chip, we discovered that 8F1 not only reacts with its authentic target ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity to PCYT1A was further confirmed by other immunoassays, including Western blot, antigen absorption test, and immunohistochemistry (IHC) analyses. In this study, we also discovered that PCYT1A gene expression level is even significantly higher than ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. In summary, 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on protein microarray chip test and both worked in IHC application. Citation Format: Donghui Ma, Youmin Shu, Kehu Yuan, Wei-Wu He. Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2013-3216

Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

BackgroundAn antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein.ResultsBy using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application.ConclusionIn summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.

Humoral immune responses to Plasmodium falciparum among HIV‐1‐infected Kenyan adults

Humoral immune responses play a pivotal role in naturally acquired immunity to malaria. Understanding which humoral responses are impaired among individuals at higher risk for malaria may improve our understanding of malaria immune control and contribute to vaccine development. We compared humoral responses with 483 Plasmodium falciparum antigens between adults in, Kisumu (high, year-long malaria transmission leading to partial immunity), and adults in Kisii (low, seasonal malaria transmission). Then within each site, we compared malaria-specific humoral responses between those at higher risk for malaria (CD4(+) ≤500) and those at lower risk for malaria (CD4(+) >500). A protein microarray chip containing 483 P. falciparum antigens and 71 HIV antigens was used. Benjamini-Hochberg adjustments were made to control for multiple comparisons. Fifty-seven antigens including CSP, MSP1, LSA1 and AMA1 were identified as significantly more reactive in Kisumu than in Kisii. Ten of these antigens had been identified as protective in an earlier study. CD4(+) T-cell count did not significantly impact humoral responses. Protein microarrays are a useful method to screen multiple humoral responses simultaneously. This study provides useful clues for potential vaccine candidates. Modest decreases in CD4 counts may not significantly impact malaria-specific humoral immunity.

Protein Microarrays Based on Polymer Brushes Prepared via Surface-Initiated Atom Transfer Radical Polymerization

Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray surface coatings.

Protein microarray chip with Ni–Co alloy coated surface

Most protein microarrays based on the theory of immobilized metal affinity chromatography (IMAC) were fabricated using the chelator or compound of mono-metallic ion, such as Ni2+, to capture the histidine-tagged protein. In this study, a novel protein chip with Ni–Co alloy layer fabricated on the substrate of printed circuit board by electrodeposition was developed. It is an innovative microarray surface with bi-metallic elements, i.e. by means of adding cobalt to enhance the specific binding capability to immobilize functional proteins with His-tag attached. Taguchi method was adopted to determine the optimal electrodeposition condition using L18 orthogonal array. Then, the immunoassay was utilized to investigate the properties of the protein chip, and the signal was detected by confocal scanner. The results show that the alloy coating belongs to codeposition of Ni–Co alloy. Due to the affinity adsorption between intermediate metal and protein, this chip provides specific binding. Compared with Ni-coated chip and nitrocellulose (NC) chip, this Ni–Co alloy chip is a high sensitive protein microarray because of high detected fluorescence intensity with low fluorescent background. The proportion of Ni–Co composition will affect the affinity of electroplated layer with proteins and, consequentially, influence the results of immunoassay. Moreover, the test of long-term sensibility of Ni–Co chip showed that it had better binding capability in the first two weeks for coating buffers of pH 7.4 and pH 9 and the immobilization ability began to decay after the third week. The advantages of this novel protein chip include good performance, high repeatability, and inexpensive.

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

Oral viral infections of adults

The field of virology has advanced greatly over the past two decades, mainly because of the introduction of sophisticated molecular tools, such as monoclonal antibodies, polymerase chain reaction (PCR)-based amplification, DNA sequencing, DNA and protein microarray chip assays, and rapid diagnostic tests. These technologies have been the driving force in the identification of viral bodies, proteins and nucleic acids in body fluids and tissue samples, and in determining the host response to viral infections. The 5th (2007) edition of Fields Virology provides an indepth description of viral methodology and medical virology, and is an important source of reference for this review (118). Viruses replicate only when present within eukaryotic (animals, plants, protists and fungi) or prokaryotic (bacteria and archaea) cells, not on their own. The extracellular virion particle ranges in size from 20 to 300 nm and consists of either DNA or RNA contained within a protective protein capsid. Some viruses have an additional envelope comprising a lipid bilayer derived from the outer cellular membrane, the internal nuclear membrane, or the endoplasmic reticulum membrane of the infected cell. Taxonomically, viruses are classified according to the presence of DNA or RNA, single-stranded or double-stranded nucleic acid, and an enveloped or nonenveloped nucleocapsid. Additional taxonomical criteria include mode of replication, type of host, capsid shape, immunological properties and disease association. The host recognizes and reacts to the infecting virus by innate and adaptive immune responses. Important cells of the innate immune system include macrophages, dendritic cells and natural killer cells. Viruses activate inflammatory cell types to release antiviral cytokines and cytotoxic agents, and to induce lymphocyte-mediated adaptive immunity. A significant cytokine release is stimulated through activation of the tumor necrosis factor-a receptor ⁄ nuclear factor-jB ⁄ extracellular signal-regulated kinase pathway (49). Virally derived proteins, which are presented by major histocompatibility complex molecules on the surface of infected cells, serve as epitopes for specific host immune cells. Nonenveloped viruses are mainly controlled by the humoral adaptive immunity. Enveloped viruses are controlled by the cellular immunity through the action of natural killer cells and cytotoxic CD8 T lymphocytes. After recognizing viral surface antigens on infected cells, cytotoxic T lymphocytes inhibit virus replication by cytolytic killing and by releasing interferons, chemokines, tumor necrosis factor-a or other pro-inflammatory mediators. Viral disease may be a direct result of cell destruction or a secondary consequence of host immune reactions against viral proteins. Proinflammatory cytokines play important roles in the antiviral immune response, but interleukin-1b, interleukin-6 and tumor necrosis factor-a cytokines can also contribute to disease manifestation. The host usually performs a delicate balancing act between promoting antiviral cytokine responses and limiting the amount of tissue damage. To counteract the immune attack, viruses employ sophisticated immunoevasive strategies to suppress antiviral host responses. For example, some viruses produce proteins that alter the major histocompatibility complex and hence the exposure of viral proteins on the surface of infected cells. Viruses may encode viral homologs of host cytokines and decoy receptors capable of binding and neutralizing host-derived cytokines. A rapid rate of mutation in critical viral genes can help viruses avoid the adaptive host defense. Other viral gene products inhibit apoptosis, which facilitates a prolonged state of replication of infected cells and spread of the virus. Viral diseases of the oral mucosa and the perioral region are often encountered in dental practice, but have received only limited research interest. Viruses are important ulcerogenic and tumorigenic agents of the human mouth. The finding of an abundance of mammalian viruses in periodontitis lesions may suggest a role for viruses in more oral diseases than

Micropatterned atmospheric pressure discharge surface modification of fluorinated polymer films for mammalian cell adhesion and protein binding

Micropatterning by an easily accessible atmospheric pressure discharge setup was performed on fluorinated polymer surfaces. Two conductively-coated glass slides are employed, together with the polymer foils to be modified and shadow masks for defining the microstructures. Surface angle measurements indicated the presence of charged groups in the surface of the fluoropolymer, enabling the growth of mammalian cells on arrays of spots with 600 μm diameter. XPS and FTIR spectra revealed the incorporation of oxygen in the surface, while the generation of aldehyde groups on the surface of fluorinated polymer films was demonstrated by selective coupling of fluorescence-labeled aminodextrane to the activated spots. The described method paves the way for producing protein microarray chips on flexible fluoropolymer substrates with standard laboratory equipment.

Surface Chemistry for Protein Microarrays

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

From protein microarrays to diagnostic antigen discovery: a study of the pathogen Francisella tularensis

An important application of protein microarray data analysis is identifying a serodiagnostic antigen set that can reliably detect patterns and classify antigen expression profiles. This work addresses this problem using antibody responses to protein markers measured by a novel high-throughput microarray technology. The findings from this study have direct relevance to rapid, broad-based diagnostic and vaccine development. Protein microarray chips are probed with sera from individuals infected with the bacteria Francisella tularensis, a category A biodefense pathogen. A two-step approach to the diagnostic process is presented (1) feature (antigen) selection and (2) classification using antigen response measurements obtained from F.tularensis microarrays (244 antigens, 46 infected and 54 healthy human sera measurements). To select antigens, a ranking scheme based on the identification of significant immune responses and differential expression analysis is described. Classification methods including k-nearest neighbors, support vector machines (SVM) and k-Means clustering are applied to training data using selected antigen sets of various sizes. SVM based models yield prediction accuracy rates in the range of approximately 90% on validation data, when antigen set sizes are between 25 and 50. These results strongly indicate that the top-ranked antigens can be considered high-priority candidates for diagnostic development. All software programs are written in R and available at http://www.igb.uci.edu/index.php?page=tools and at http://www.r-project.org. Supplementary data are available at Bioinformatics online.

SURFACE CHEMISTRY FOR PROTEIN MICROARRAYS

Protein microarray or protein chip is an important tool in proteomics. However, duplicating the success of the DNA chip for the protein chip has been difficult. This account discusses a key issue in protein microarray development, i.e., surface chemistry. Ideally, the surface chemistry for protein microarray fabrication should satisfy the following criteria: the surface resists nonspecific adsorption; functional groups for the facile immobilization of protein molecules of interest are readily available; bonding between a protein molecule and a solid surface is balanced to provide sufficient stability but minimal disturbance on the delicate three-dimensional structure of the protein; linking chemistry allows the control of protein orientation; the local chemical environment favors the immobilized protein molecules to retain their native conformation; and finally, the specificity of linking chemistry is so high that no pre-purification of proteins is required. Strategies to achieve such an ideal situation are discussed, with successful examples from our laboratories illustrated. Finally, the need of surface technology for membrane protein microarray fabrication is addressed.

A study of APin-protein interactions using protein microarray

Protein microarray를 이용한 APin-단백질의 상호작용에 관한 연구 Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interac- tions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF- hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR. The results were as follows: 1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively. 2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation. 3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the over- expression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expres- sion of EF-hand calcium binding protein was markedly decreased by its inactivation. These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

Technologies to Shape the Future: Proteomics Applications in Anesthesiology and Critical Care Medicine

Broadly speaking, proteomics is concerned with the simultaneous characterization of the features (for example, the concentration or activity) of the many different proteins that are typically found in biological or clinical specimens. The field is being driven forward both by innovative biotechnology companies and by academicians who are introducing the technology required for the parallel identification of individual proteins. The technology currently relies heavily on two-dimensional gel electrophoresis combined with mass spectrometry, but protein microarray chips are rapidly becoming a reality. Protein biomarkers are increasingly being recognized as crucially important for the study of disease processes, both from diagnostic and prognostic points of view. Proteome level studies will therefore be used increasingly both to identify and follow the course of various pathological conditions. In the specialty of anesthesiology, this technology will allow an improved understanding of the mechanisms of action of many of the drugs that are routinely administered in the operating room and also the effects of these therapeutic drugs on protein expression. In addition, proteomic studies will increasingly be used for both diagnostic and prognostic purposes in the intensive care unit and the chronic pain clinic.

INTEGRATION OF NANOPARTICLES WITH PROTEIN MICROARRAYS

A variety of DNA, protein or cell microarray devices and systems have been developed and commercialized. In addition to the biomolecule related analysis, they are also being used for pharmacogenomic research, infectious and genetic disease and cancer diagnostics, and proteomic and cellular analysis.1 Currently, microarray is fabricated on a planar surface; this limits the amount of biomolecules that can be bounded on the surface. In this work, a planar protein microarray chip with nonplanar spot surface was fabricated to enhance the chip performance. A nonplanar spot surface was created by first coating the silica nanoparticles with albumin and depositing them into the patterned microwells. The curve surfaces of the nanoparticles increase the surface area for immobilization of proteins, which helps to enhance the detection sensitivity of the chip. Using this technique, proteins are immobilized onto the nanoparticles before they are deposited onto the chip, and therefore the method of protein immobilization can be customized at each spot. Furthermore, a nonplanar surface promotes the retention of native protein structure better than planar surface.2 The technique developed can be used to produce different types of microarrays, such as DNA, protein and antibody microarrays.

Optimal design of nickel-coated protein chips using Taguchi approach

For the traditional ion-metal affinity chromatography, the nickel is used to capture the histidines(His)-tagged protein to make up a rapid purification system from the organism lysate. In this paper, a nickel-coated substrate was fabricated by electroplating technology in order to design a planar protein microarray chip. The Taguchi method was adopted to determine the optimal process of the nickel-coated chip using L18 orthogonal array for eight electroplating parameters, such as electroplating buffer, four kinds of electroplating additives, current density, and temperature of electroplating buffer, with three levels for each factor. Then, we utilized the immunoassay to investigate the properties of the protein chip, and the signal was detected by confocal scanner. The result shows the Ni-coated slide has better specific binding ability than nitrocellulose coated slide and the S/N ratio of the immune assay can be improved from 34.12 to 83.42 after optimizing eight electroplating parameters.

High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.

Reversible Hydrophobic Barriers Introduced by Microcontact Printing: Application to Protein Microarrays

Microcontact printing (µCP) has been used to introduce temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels on gold. Among the investigated types of inks, tetraoctadecylammonium bromide (TOAB), electrostatically bound to the CMD layer, provided the most well-defined features both with respect to pattern-definition and reversibility upon exposure to a regeneration solution. The printed patterns were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), microscopic wetting and imaging null ellipsometry to explore the influence of concentration of ink solution and contact time on the appearance of the printed layer. AFM revealed that the printed TOAB molecules aggregated into clusters rather than into a homogeneous mono- or multilayer on the CMD hydrogel. It was also observed that printed areas of TOAB that are larger than 25?µm are inhomogeneous most likely because of an edge transfer lithography (ETL) mechanism. A protein model system based on Protein A-rabbit antimouse Fc ? was used to evaluate the potential of the patterned surface as a protein microarray chip by means of surface plasmon microscopy (SPM). Moreover, non-specific adsorption of several proteins onto TOAB barriers was also studied using surface plasmon resonance (SPR), and it is evident that undesired adsorption can be eliminated by removing barriers after ligand immobilization, but prior to analyte exposure, by treating the patterned surface with a simple salt regeneration solution. © Springer-Verlag/Wien 2004.

95. Formation of germanium films by thermal evaporation of a vacuum with application of a gas purge: L N Butorina and V A Tolomasov,Kristallografiya, 10 (4), 1965, 547, (in Russian)

For the traditional ion-metal affinity chromatography, the nickel is used to capture the histidines(His)-tagged protein to make up a rapid purification system from the organism lysate. In this paper, a nickel-coated substrate was fabricated by electroplating technology in order to design a planar protein microarray chip. The Taguchi method was adopted to determine the optimal process of the nickel-coated chip using L18 orthogonal array for eight electroplating parameters, such as electroplating buffer, four kinds of electroplating additives, current density, and temperature of electroplating buffer, with three levels for each factor. Then, we utilized the immunoassay to investigate the properties of the protein chip, and the signal was detected by confocal scanner. The result shows the Ni-coated slide has better specific binding ability than nitrocellulose coated slide and the S/N ratio of the immune assay can be improved from 34.12 to 83.42 after optimizing eight electroplating parameters.