Introduction and Application of Protein Microarray

In the 1990s, the concept of proteome was proposed, and proteome refers to all proteins expressed by a genome, cell or tissue in a specific environment or state. The introduction of proteomics has epoch-making significance for the study of proteins. Proteomics continues to evolve, requiring us to update our techniques to study proteins on a larger scale and efficiently. The protein microarray technology was produced under this background, thus solving this problem well. Protein microarray has high-throughput characteristics and can be used to analyze protein expression profiles. This paper summarizes protein microarray from three aspects: introduction, application, prospect and prospect of protein microarray.

1. Introduction of protein microarray

1.1 The principle of protein microarray technology

  Protein microarray is a new technology for protein research. The protein microarray consists of a carrier and immobilized proteins on the carrier. When preparing protein microarray, the solid phase carrier is often specially treated to immobilize some known protein molecular products, such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc. These molecules have different characteristics. The protein to be tested that specifically binds to it can be captured to achieve the purpose of detecting the protein. These proteins can be extracted from serum, plasma, lymph, interstitial fluid, urine, exudate, cytoplasmic fluid, and exudate. After a series of steps such as washing and purification, proteins from various sources were identified and biochemically analyzed. Protein microarray can help us detect some unknown proteins, explore the interaction between proteins, and obtain some important life information.

1.2 Construction of protein microarray

1.2.1 protein microarray carrier

  The carrier material of protein microarray should use inert material with good stability and biocompatibility. In order to couple protein molecules, some reactive groups are required on the carrier. At present, the common carriers in protein microarray include glass, silicon wafer, gold wafer, nitrocellulose membrane and nylon membrane, etc. The glass surface is smooth and the performance is stable; the background fluorescence value of nitrocellulose membrane and nylon membrane is high, and the sample droplets are easy to diffuse on the membrane, so the density of the microarray is very low; porous silicon has a larger specific surface area than the general carrier material , can bind more protein molecules, has almost no fluorescent background, is easy to make, and can be used repeatedly. Therefore, our more common carrier materials are glass or silicon.

1.2.2 Probe of protein microarray

According to different application purposes, protein microarray usually uses biologically active proteins such as specific antibodies, antigens, enzymes, water-absorbing or hydrophobic substances, chemical groups, receptors, and immune complexes as probes. Probes must be stable with high specificity and affinity. The connection methods of probes and carriers can be divided into covalent binding, surface adsorption and physical embedding. Among them, the physical embedding has good stability and is widely used. In order to avoid changes in the spatial structure of the protein, the direct spotting method is often used in the preparation process.

1.2.3 Detection of protein microarray

Samples containing protein are pretreated before spotting. In general, the sample volume is only 2~10 μL, and the sample can be tested later. The detection methods of protein microarray can be divided into labeling method and non-labeling method. The labeling method usually labels the protein to be tested or the secondary antibody with fluorescein, isotope, enzyme or gold nanometer. The labeled protein will emit a specific signal, which is then detected by a special detector such as a scanner, and further data analysis is carried out by a computer system. The labeling method has stable detection and high sensitivity, but the process of making the label is cumbersome and may destroy the activity of the analyte. Aiming at the shortcomings of the labeling method, some advanced non-labeling detection methods have been developed, such as surface plasmon resonance and protein microarray combined with surface-enhanced laser analysis/ionization mass spectrometry, etc. Surface plasmon resonance uses the change of resonance signal to detect the interaction between biomolecules in real time. It works on the principle that reflected incident light can resonate with plasmons on the metal surface. When the receptor binds or dissociates with the corresponding ligand, the corresponding resonance signal changes, thereby determining whether there is an interaction between the biomolecule and the reaction parameter. The method has the advantages of high sensitivity and good repeatability. However, the cost is high and the technical requirements of the operators are high. Surface-enhanced laser ion time-of-flight mass spectrometry (SELDI-TOF-MS) is a new technology based on mass spectrometry (MS) analysis. It can ionize the target protein adsorbed on the protein chip and calculate its mass-to-charge ratio under the action of electric field force. This method can be combined with the egg white material database to determine the molecular weight and relative content of protein fragments, and provide a basis for further research on the changes of protein mass spectra.

1.3 Classification of protein microarray

  Protein microarrays are often divided into 3 categories:

• protein microarray

• Microporin chips

• 3D Gel Chip.

1.3.1 Protein Microarray

Protein microarray Protein microarray is a library that puts different biologically active proteins into different microwell plates and screens protein functions. Immobilizing proteins on microarray surfaces is particularly difficult because they are inherently sensitive to interference.

Macbeth and Schreibel of Harvard University reported: By using a spotting mechanical device to make a protein chip, the tip is immersed in a microwell containing a purified protein solution, and then moved to a glass slide with 1nL of solution spots on the surface of the slide , and then the robot repeats this operation, pointing out different proteins. Using this device, about 10,000 kinds of proteins were immobilized, and the specific interactions between proteins and small molecules were studied. Macbeth and Schreibel first modified slides with a layer of bovine serum albumin (BSA) to prevent denaturation of proteins fixed to the surface. Since lysine widely exists in the peptide chain of proteins, lysine reacts with lysine in spotted protein samples through activators in BSA, making it bind to the surface of the substrate and expose some protein activity district. In this method, proteins are immobilized on the carrier surface by a spotting device to make a protein microarray.

1.3.2 Microporous protein chip

The microplate protein microarray was established by Mendoza et al. Different antigens and marker proteins were added to the microplate, and the results were measured with an immunoanalyzer. Combining the advantages of protein microarray and enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) method, the detection is sensitive, the operation is convenient and fast, and the microwell plate is relatively independent, which can avoid cross-contamination.

1.3.3 Three-dimensional gel block chip

The three-dimensional gel block chip is a chip technology jointly developed by the Argonne National Laboratory of the Russian Academy of Sciences and the Engel Institute of Molecular Biology. The three-dimensional gel block chip is mainly composed of 10,000 tiny polybenzamide gel blocks, and each gel block can be used for the analysis of target DNA, RNA and protein. The advantage of this system is that the three-dimensional structure of the gel strip can add more known samples and improve the detection sensitivity. Proteins can be analyzed in their native state and can be used in immunoassays, receptor, ligand studies, and protein composition analysis.

1.4 Features of protein microarray

• The feature of protein microarray is that it is easy to operate, and some crude products can be directly used for analysis

• Multiple proteins can be detected at the same time, reducing workload;

• High detection sensitivity, only a small amount of sample is required;

• It has high specificity and can be used for the identification of unknown proteins;

• It can be used for quantitative analysis of proteins, such as monoclonal antibody chip, because the antibody bound to the chip is quantitative, so it can be used to determine the amount of antigen; it has a wide range of applications.

2 Application of protein microarray

 

Introduction of Protein Microarray (Protein Chip) Technology and Application

2.1 Detection of biomarkers

Due to the high-throughput, high-sensitivity and easy-to-operate characteristics of protein microarray, it can be used for related research on proteins and is widely used in the screening and detection of biomarkers. Protein microarray technology also builds a bridge between drugs and proteins, greatly speeding up the drug development process.

Ge Jing et al. used proteomics technology and used 1000 protein microarrays from Raybiotech to screen molecular markers that may have clinical diagnostic value for hypertensive cerebral infarction, and found that 20 plasma proteins were up-regulated in the hypertensive cerebral infarction group. Fang Yongsheng modified the gold foil plane with bifunctional coupling agent N,N-carbonyldiimidazole and mixed sulfhydryl reagents to construct a new type of protein microarray to realize the simultaneous detection of five serum markers in patients with liver cancer, and evaluate the joint detection of these markers Clinical Value. Jiang Di obtained candidate anti-tumor-associated antigen autoantibodies with diagnostic value for lung cancer through protein microarray screening, indirect enzyme-linked immunosorbent assay, and experimental verification. Zhou Yu et al. used the high-throughput protein microarray of human recombinant proteins to discover potential autoantibody target proteins in the serum of multiple sclerosis (MS) patients. Finally, 27 proteins interacting with the serum of MS patients were screened out by protein chips, which can be used as markers for future disease diagnosis and help us understand the pathogenesis of MS.

Myoung-SchookYoou et al. used protein microarray technology to find a fragment-HIT that has inhibitory activity on B-cell lymphoma 2 (Bcelllymphoma2, Bcl-2) protein from 131 pre-selected fragment chemicals. Since protein microarray analysis is helpful for finding chemical hits of protein-protein interaction drug targets, it can be easily used as a fragment-based drug design tool to develop Bcl-2 inhibitors. YongHoLee et al. developed optimized methods for protein extraction and protein microarray, and applied low-molecular-weight proteins/peptides as potential biomarkers in three rice varieties. Yuyan Wu et al. used Renalasehuman protein microarray analysis to show that renalase is a potential biomarker of hypertrophy. ShuangWang et al. used protein microarray technology to study the neuronal factors in the serum and cerebrospinal fluid of neonates infected with human cytomegalovirus (HCMV), in order to elucidate the changes of specific neuronal factors, which can be used to develop and predict the central nervous system caused by HCMV infection. reliable indicator of damage. Yihao Zhang used protein microarray to analyze the inflammatory cytokine profile in rheumatoid arthritis (Rheumatoidarthritis, RA) synovial fibroblasts, and verified it with magnetic multi-cytokine analysis and ELISA, revealing a new mechanism of inflammation regulation in RA , to discover potential therapeutic targets.

2.2 Using protein microarray to detect allergens

  Protein microarray technology is a widely used and continuously developing technology in clinical and scientific research, which can be used for allergen detection. Accurately, quantitatively and orderly load the allergens on the specially treated carrier (such as glass sheet) that can adsorb proteins to make an allergen detection chip. The specific IgE in the serum to be tested can recognize and bind to the allergens. Fluorescently labeled antibodies are amplified for signal amplification and positive detection results can be obtained by laser scanning.

At present, the main method for detecting allergen components is the ImmunoCAP method. The reagents used in this method are expensive and the inspection cost is high. In view of this, Zhai Kangle et al expressed and purified several common allergen components of peaches using yeast expression system. Combined with protein microarray technology, a protein microarray that can be used to detect various components of peach allergens in serum was prepared; protein chips were used to detect antibodies against peach allergen component proteins in clinical serum samples, and compared with the ImmunoCAP method, the protein Sensitivity and specificity of microarray detection.

2.3 Using protein microarray for disease monitoring

  Protein microarray can be detected in real time and online, and does not require labeling and purification, and consumes very little sample. It is often used in disease diagnosis and disease status monitoring.

Yue Wu et al. established a visible protein microarray and a cyanine dye 3 (Cy3)-labeled protein microarray to detect porcine parvovirus antibodies in clinical serum of pigs, with good sensitivity and specificity. Chen Fan and others tried to use the specific adsorption surface treated with polyethylene glycol polymer, and used the specific antigen of falciparum histidine-rich protein II (Histidine-rich protein II, HRP2) as the capture probe to construct the surface plasmon resonance technology protein. Microarray for rapid detection of falciparum malaria. GuiyingSun et al. identified new anti-tumor-associated antigenautoantibodies (TAAbs) autoantibodies, and explored the best diagnostic model for detecting esophageal squamous cell carcinoma based on protein microarray. The identification of 12 candidate autoantibodies based on protein microarray can be used as an effective method to identify novel TAAbs.

2.4 Using protein chips to detect pesticide and veterinary drug residues

At present, the methods for detecting pesticide and veterinary drug residues using protein microarray mainly include biochemical technology, gas chromatography, high performance liquid chromatography and ELISA. As a traditional method, biochemical method has always played a huge role, but due to the rapid development of food production and processing technology and biotechnology, this technology has exposed obvious shortcomings such as complex process, slow speed, and low precision; gas chromatography and high-efficiency Liquid chromatography has high sensitivity, but the pretreatment process is complicated, slow, and the degree of instrumentation is high; the microbiological method is simple to operate, but the sensitivity and specificity are low; ELISA has high specificity and low detection limit, but the detection of components Single, not suitable for multi-component detection at the same time. Compared with the above methods, the protein microarray based on competitive immune response has the advantages of high throughput, parallelism, simultaneous determination of multiple components, high sensitivity, and strong specificity. Using protein microarray to detect pesticide and veterinary drug residues has become a new detection tool.

Based on the protein microarray protein chip method has the advantages of high throughput, short detection time, high specificity, simple operation, low cost, etc., Zhong Wenying et al. made sulfa-bovine serum albumin (Bovineserumalbumin, BSA) and quinolones-BSA conjugates As an antigen, use a biochip spotter to spot antigens of different mass concentrations, fix the artificial antigen on the bottom of the petri dish, prepare in each well of the petri dish, add sulfadiazine and enrofloxacin mixed standard and sulfaquinolones mixed single After cloning the antibody, goat anti-mouse IgG was added and labeled with nanosilver. The results were processed and analyzed by chip analysis software. A protein microarray method for the simultaneous detection of sulfonamides and quinolones residues in milk was established.

2.5 Using biochips to detect microorganisms in food

Pathogens detected by biochips have always been the focus of health inspections. Pathogenic microorganisms in food are too complex and diverse, which brings great challenges to detection. The previous biochemical detection methods were single, sample processing was complicated and the operation was cumbersome. When some sudden rare microbial contamination occurred, the shortcomings of these traditional detection methods would be highlighted. Protein microarray has the characteristics of high precision, high throughput, and rapidity, and can detect common microorganisms in a short period of time.

Cai Tingting et al. used TiO2-porous silicon as a carrier and based on immunoassay, established a protein microarray technology for simultaneous detection of multiple mycotoxins. This technology can be used for the simultaneous detection of mycotoxins, with a wide detection range, fast linearity, and simple sample pretreatment, which provides greater selectivity for the detection of mycotoxins.

3 Application prospect of protein microarray

Antibody Chip Carrier Display

  The protein microarray technology has powerful detection ability, high sensitivity and less sample usage. at present. Protein microarray technology is maturing and can be used in disease diagnosis, new drug development, biotoxin detection, proteomics and other aspects. However, the development speed of protein microarray is still very slow compared with gene chip. The main reason is that the protein is relatively unstable, and it is difficult to maintain the integrity and biological activity of the protein during protein purification, sample collection and chip storage. Second, it is easier to obtain the target gene than to obtain the protein. For example, obtaining monoclonal antibodies often requires culturing hybridoma cells. This process is time-consuming, laborious and costly. In addition, the protein may bind to the carrier non-specifically, resulting in inaccurate determination. Detection technology is also one of the reasons for the limited development of protein microarray.

However, with the rapid development of science and technology, these problems have been gradually solved. The emergence of antibody phage display technology and ribosome display technology can greatly shorten the preparation process of monoclonal antibody. In addition, when preparing protein microarray, we increase the lattice speed of chip production, provide appropriate temperature and humidity, and add a stabilizing solution.

Improving surface treatment techniques for matrix materials can reduce nonspecific binding of proteins. Therefore, we can expect that in the near future, protein microarray technology will become the main body of high-throughput detection and will be widely used in various fields.