Quality inspection method and preparation method for microarray chip mass production

Abstract

The present application relates to the quality inspection method of microarray chip scale production, comprising: step 1) the nanometer membrane is placed in clean environment, humidity <20%, temperature 23~25 ℃ for a period of time to make the nanometer membrane fully balanced;Step 2) preparation of sample solution, present preparation present use;Step 3) the nanometer membrane of step 1) is fully balanced, is placed in sample point instrument, is gradually sampled in the quality inspection point of nanometer membrane with the sample solution of step 2), each hole quality inspection point is not less than 2, 20nl / point;Step 4) the nanometer membrane of step 3) is sampled is loaded into the matching fixture, is placed under the lens of industrial camera, is photographed with industrial camera, is measured with software for each quality inspection point diameter and is exported;Step 5) analysis of the diameter data of each quality inspection point on the nanometer membrane, the overall standard deviation of the diameter of the quality inspection point on the nanometer membrane, the median and the range, the overall standard deviation ≤5%, the median 460 μm-480 μm, the range ≤30 μm is the nanometer membrane of quality inspection qualified.

Description

Technical Field

[0001] This invention relates to a quality inspection method and preparation method for the large-scale production of microarray chips, belonging to the field of biotechnology. Background Technology

[0002] At the beginning of the 21st century, biology underwent significant changes, posing a major challenge to traditional biological research models. With the rapid development of the Human Genome Project, the accumulation of biological data accelerated, placing higher demands on scientific analysis methods and practical analytical tools. Against this backdrop, bioinformatics, utilizing mathematical knowledge, information science, and computer science and technology, emerged. In bioinformatics, which deals with more complex gene expression data, larger data volumes, and faster data growth, microarray chips, with their unique information mining capabilities, have become a high-throughput, parallel, and miniaturized high-speed biological analysis method, a research hotspot, and another far-reaching scientific and technological revolution following large-scale integrated circuits, attracting widespread attention.

[0003] Microarray chips are designed and applied to a substrate using a spotting device. A spotting needle draws in target solutions (such as enzymes, antigens, antibodies, receptors, ligands, nucleic acids, cytokines, etc.), which are then transferred above the substrate and immobilized on the substrate surface through contact or non-contact spraying. Subsequent processing then forms the microarray chip. Microarray chips are a high-throughput analytical technique that, based on the characteristics of the target, captures analytes that specifically bind to them (found in serum, plasma, lymph, interstitial fluid, urine, exudate, cell lysate, secretions, etc.). After washing and purification, they are confirmed and subjected to biochemical analysis. This provides strong technical support for obtaining important life information (such as unknown protein components, sequences, in vivo expression levels and biological functions, their regulatory relationships with other molecules (such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc.), drug screening, and drug target selection). It is used to study the interactions between targets and has significant advantages in disease screening, early diagnosis, and screening drug targets.

[0004] However, in the fabrication of microarray chips, the accuracy of the chip results is a key concern for users. Because most microarray chips use non-contact spraying for spotting, and visualization of microarray chips outputs quantitative results through the final grayscale value of the target points, the determination of the grayscale value of the target points needs to be extremely precise. The grayscale value of the target points is highly dependent on the area of ​​the target points during the initial chip spotting process. Uniform target spotting area ensures good repeatability of subsequent experimental results; conversely, if the target spotting area is uneven, it significantly affects the repeatability and accuracy of the results.

[0005] In the fabrication of microarray chips, the uniformity of the substrate surface is crucial to the quality of the finished chip. However, due to the special characteristics of the substrate surface and the subsequent need for spotting on the surface, it is essential to ensure the surface is clean, uncontaminated, and free from mechanical damage while simultaneously inspecting the substrate surface quality.

[0006] Therefore, ultrapure water was used as the detection solution in the early stage to determine the contact angle at several points on the substrate surface. The change in contact angle was used to determine the uniformity of the substrate surface. The contact angle is the angle θ between the tangent line at the gas-liquid interface at the junction of the gas, liquid, and solid phases and the solid interface (liquid side).

[0007] So in Figure 1 As shown in the contact angle diagram, with a fixed sample volume, a constant contact angle indicates a constant contact area between the droplet and the solid surface. Therefore, the uniformity of the substrate surface can be determined by spraying the same volume of liquid onto the microarray substrate surface with ultrapure water and measuring the contact angle. Furthermore, using ultrapure water ensures no contamination or mechanical damage, while still maintaining the biocompatibility of the substrate surface.

[0008] However, when the contact angle measurement method is applied to the microarray chip manufacturing process, the following shortcomings are found:

[0009] (1) Single-point or multi-point testing with a contact angle measuring instrument still cannot meet the uniformity of the substrate at all target points of the microarray chip, and there is a certain probability of missed detection.

[0010] (2) Using ultrapure water as the spotting test solution avoids surface contamination, but the surface tension and density of ultrapure water are different from those of the actual sample dilution, so the test results cannot fully represent the quality inspection results.

[0011] (3) Contact angle measurement is a dynamic measurement, which is the angle formed at the instant when the droplet contacts the solid surface, excluding the influence of subsequent liquid penetration and diffusion. However, the target point of the microarray chip is ultimately directly related to the gray value of the sample point area, so the contact angle measurement cannot summarize the situation after subsequent liquid diffusion.

[0012] In conclusion, existing and generally accepted contact angle measurement methods are not comprehensive and effective quality control methods for microarray chips. The quality control methods employed during microarray chip fabrication play a decisive role in product production, especially large-scale production, and in the accuracy of subsequent high-precision quantitative testing results. Therefore, there is a need in the existing technology for a quality control method that is applicable to industrialization, easy to operate, and ensures the production of high-quality microarray chips. This method should guarantee good batch-to-batch and batch-to-batch repeatability of microarray chips, thereby controlling product quality and improving the accuracy of microarray chip product testing results. Summary of the Invention

[0013] To address the shortcomings of existing technologies, this invention provides a quality control method for the large-scale production of microarray chips. This method can directly and completely summarize the systematic errors of the spotting instrument and the influence of the density, viscosity, and surface tension of the spotting solution on the results. The microarray chips prepared from the nanofilm after quality control not only achieve large-scale industrial production, but also effectively test the uniformity of the microarray chip surface, so as to control the quality of the nanofilm and improve the quality of protein chip products, reduce intra-batch and inter-batch differences, and improve the accuracy of detection.

[0014] The present invention provides a quality inspection method for the mass production of microarray chips, the quality inspection method comprising:

[0015] Step 1) Place the nanofilm in a clean environment with humidity <20% and temperature 23℃~25℃ for a period of time (e.g., 20-28 hours, preferably 24 hours) to allow the nanofilm to fully equilibrate.

[0016] Step 2) Prepare the spotting solution immediately before use;

[0017] Step 3) Place the fully equilibrated nanofilm from Step 1) into a spotting apparatus. Under conditions of 45%–55% humidity and 23°C–25°C, spot the spotting solution described in Step 2) onto the quality control points of the nanofilm one by one. The quality control points of the nanofilm are the non-target areas, non-quality control areas, and non-blank control areas of the nanofilm. There are no less than 2 quality control points in each well, 20 nmol / point.

[0018] Step 4) Place each nanofilm after the sample spotting in Step 3) into the matching fixture, place it under the lens of an industrial camera, adjust the lens focus until the sample spot is clearly visible and the edge is well defined, take pictures of each hole with the industrial camera, and use software to measure the diameter of each quality inspection point and export the results.

[0019] Step 5) Analyze the diameter data of each quality inspection point on the nanofilm, and calculate the overall standard deviation, median, and range of the diameter of the quality inspection points on the nanofilm. A nanofilm with an overall standard deviation ≤ 5%, a median of 460 μm to 480 μm, and a range ≤ 30 μm is considered to have passed quality inspection. The median and range values ​​here are empirical values ​​obtained by the inventors through a large number of experiments.

[0020] The quality inspection method described in this invention directly measures the diameter of the inspection points on the nanofilm, calculates the overall standard deviation, median, and range of the diameter of the inspection points on the nanofilm, thereby determining the uniformity of the nanofilm surface. Based on the diameter statistics, the quality inspection pass rate is judged and the product qualification performance is controlled. This method can effectively control the entire nanofilm production process, improve the stability of the production process and the product stability, and achieve repeatability and accuracy of the final nanofilm-based chip in the process of detecting biological samples. The coefficient of variation of the final detection value is controlled within 15% or even within 10%, and the recovery rate is controlled between 80% and 120%, which is far superior to the actual application effect of the microarray chip prepared by the nanofilm after quality inspection by the contact angle measurement method commonly used by those skilled in the art.

[0021] In one embodiment of the present invention, the time for fully equilibrating the nanofilm in step 1) is 20 to 28 hours, preferably 24 hours.

[0022] In one embodiment of the present invention, the spotting solution in step 2) is a mixture of 5% M / V glycerol solution, 5% M / V sorbitol solution, 0.05% V / V Triton solution, dimethyl sulfoxide (DMSO) solution, and PBS (pH 6.8) solution in a volume ratio of 10:15:0.1:50:100, and is prepared and used immediately.

[0023] The spotting solution described in this invention can fully reflect the influence of the density, viscosity, and surface tension of the spotting solution on the results during the actual spotting process.

[0024] In one embodiment of the present invention, the matching fixture in step 4) includes a cover plate and a base plate. The cover plate is provided with multiple detection windows, and the base plate is provided with perforated holes at corresponding positions to the detection windows. The cover plate can be pressed and fixed on the base plate. The matching fixture in step 4) is preferably the matching fixture mentioned in Chinese Patent CN211348276U.

[0025] The present invention also relates to a method for the mass production of microarray chips, comprising: step (1) placing the qualified nanofilm in a container, adding 15ml of activation solution for surface activation, rinsing the surface activation solution with purified water after 0.5 hours, and then drying the surface of the nanofilm with clean air.

[0026] Step (2) Place the activated nanofilm from step (1) into the spotting instrument and spot the target, goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody, and blank control spotting solution onto the nanofilm according to the set program. The spotting volume is 20 nl.

[0027] Step (3) Place each nanofilm after spotting in step (2) into the matching fixture, dry it for 6 hours at a temperature of 21℃~25℃ and a humidity of <25%, and seal it with a membrane sealing solution to obtain a microarray chip.

[0028] The quality inspection method described in this invention can directly and completely reflect the systematic error of the spotting instrument and the influence of the density, viscosity and surface tension of the spotting liquid on the results. The microarray chip prepared by the nanofilm after quality inspection is not only conducive to large-scale industrial production, but also can effectively check the uniformity of the surface of the microarray chip, so as to control the quality of the nanofilm and improve the quality of protein chip products, reduce the difference between batches, and improve the accuracy of detection.

[0029] As one embodiment of the microarray chip mass production method of the present invention, the activation solution in step (1) is a solution containing 5% M / V 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and 2.5% M / V N-hydroxysuccinimide NHS.

[0030] As one embodiment of the method for large-scale production of microarray chips of the present invention, the membrane blocking solution in step (3) is a PBS solution containing 1% W / V BSA, 0.1% to 0.5% V / V glycerol, and 0.1% V / V Tween-20.

[0031] As one embodiment of the preparation method of the present invention, the target in step (2) is an animal disease virus antigen or its protein or a food safety small molecule antigen.

[0032] As one embodiment of the present invention, the target in step (2) includes porcine pseudorabies virus gD protein or gE protein, classical swine fever virus E2 protein, porcine foot-and-mouth disease virus antigen, porcine rotavirus antigen, mycotoxin antigen, and poultry antibiotic antigen.

[0033] In a preferred embodiment of the present invention, when the microarray chip is the porcine pseudorabies virus gD and gE protein antibody dual detection chip kit, the target of step (2) is the porcine pseudorabies virus gD and gE protein.

[0034] When the microarray chip is the triple detection chip for the triple detection of pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibodies in the pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip kit, the target in step (2) is the pseudorabies virus gD protein, gE protein and classical swine fever virus E2 protein.

[0035] When the microarray chip is the O-type and A-type foot-and-mouth disease virus antibody detection chip in the O-type and A-type foot-and-mouth disease virus antibody detection chip kit, the target in step (2) is the O-type foot-and-mouth disease virus antigen and the A-type foot-and-mouth disease virus antigen.

[0036] When the microarray chip is the fungal toxin quadruple detection chip in the fungal toxin quadruple detection chip kit, the target in step (2) is fumonisin antigen, aflatoxin B1 antigen, vomitoxin antigen, and zearalenone antigen.

[0037] The nanofilm prepared by the quality inspection method described in this invention can be used for the preparation of reagent kits for different scenarios, including but not limited to the detection of animal-derived antibodies and the detection of small molecules for food safety.

[0038] The term "microarray chip," also known as a biochip, refers to a biological chip that uses methods such as photoconductive in-situ synthesis or micro-spotting to orderly immobilize a large number of biological macromolecules, such as nucleic acid fragments, peptide molecules, proteins, and even tissue slices and cells, on the surface of a support (such as nanofilms, glass slides, silicon wafers, polyacrylamide, nylon membranes, etc.), forming a dense two-dimensional molecular arrangement. These molecules then react with target molecules in the labeled biological sample to be tested. The intensity of the reaction signal is rapidly, in parallel, and efficiently detected and analyzed using specific instruments, such as laser confocal scanners or charge-coupled imaging cameras, to determine the number of target molecules in the sample.

[0039] The overall standard deviation, median, and range of the diameter in this invention are as follows:

[0040] The term "population standard deviation" is the average deviation between the values ​​of all units in a population and their arithmetic mean, denoted by σ.

[0041] The term "median" is a statistical term that refers to the middle value in a set of data arranged in order. It represents a value in a sample, population, or probability distribution that divides the set of values ​​into two equal parts.

[0042] The term "range" is used to represent the difference between the maximum and minimum values ​​of the measures of variation in statistical data; that is, the data obtained by subtracting the minimum value from the maximum value.

[0043] The term "recovery rate" is calculated by adding a certain amount of a standard substance to the sample during the sample analysis, subtracting the sample's measured value from the standard substance's measured value, and obtaining the recovery rate. The formula is as follows:

[0044] Recovery rate = (Amount of sample with added standard substance - Amount of the substance in the sample) / Mass of added standard substance. Attached Figure Description

[0045] The above and other objects, features, advantages and embodiments of the present invention will be better understood with reference to the following figures:

[0046] Figure 1 This is a schematic diagram of the contact angle in the contact angle measurement method;

[0047] Figure 2 This is a schematic diagram of the spotting pattern for each well on the porcine pseudorabies virus gD and gE protein antibody dual detection chip kit (referred to as kit 1). Figure 2 In the diagram, 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J represent different spotting patterns, and the specific positions of 5 and 6 on the detection chip can be interchanged in the same diagram.

[0048] Figure 3 This is a schematic diagram of the spotting pattern for each well on the triple detection chip kit for porcine pseudorabies virus gD, gE proteins, and classical swine fever virus E2 protein antibodies (referred to as kit 2). Figure 3 In the diagram, 2A, 2B, and 2C represent different spotting patterns, and the specific positions of 5, 6, and 7 on the detection chip can be interchanged in the same diagram.

[0049] Figure 4 This is a schematic diagram of the spotting pattern for each well on the O-type and A-type foot-and-mouth disease virus antibody detection chip kit (referred to as kit 3). Figure 4 In the diagram, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, and 3J represent different spotting patterns, and the specific positions of 8 and 9 on the detection chip can be interchanged in the same diagram.

[0050] Figure 5 This is a schematic diagram of the spotting pattern for each well on the mycotoxin quad detection chip kit (referred to as kit 4). Figure 5 In the diagram, 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 4I represent different spotting patterns, and the specific positions of 10, 11, 12, and 13 on the detection chip can be interchanged within the same diagram.

[0051] Figure Labels

[0052] 1 is quality control point 1, 2 is quality control point 2, 3 is quality control point 3, 4 is blank control point, 5 is PRVgD detection point, 6 is PRVgE detection point, 7 is CSFV E2 detection point, 8 is FMDV A detection point, 9 is FMDV O detection point, 10 is fumonisin detection point, 11 is aflatoxin B1 detection point, 12 is vomitoxin detection point, 13 is zearalenone detection point, and * indicates quality inspection point. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0054] All chemical reagents used in the embodiments of this invention are of analytical grade and were purchased from Sinopharm Group.

[0055] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. Unless otherwise specified, the experimental methods described in this invention are conventional methods; and unless otherwise specified, the biological materials described are commercially available.

[0056] Example 1: Preparation of Solution

[0057] 1.1 Preparation of spotting solution

[0058] Preparation of 5% glycerol solution: Accurately weigh 5.00g of glycerol into a 100ml volumetric flask, add a small amount of purified water and gently rotate to dissolve it completely, avoiding the generation of too many bubbles. Then add purified water to the mark, invert and shake 10 times, and set aside.

[0059] Preparation of 5% sorbitol solution: Accurately weigh 5.00g of sorbitol into a 250ml beaker, add an appropriate amount of purified water and stir until completely dissolved, then transfer completely to a 100ml volumetric flask, add purified water to the mark, and shake upside down 10 times.

[0060] Preparation of 0.05% Triton solution: Pipette 50 μl of Triton into a 100 ml volumetric flask, add an appropriate amount of purified water to dissolve it completely, then add purified water to the mark, shake upside down 10 times, and set aside.

[0061] DMSO solution: DMSO reagent can be used directly;

[0062] Preparation of PBS (pH 6.8) solution: First, prepare a 0.2 mol / L disodium hydrogen phosphate solution and a 0.3 mol / L sodium dihydrogen phosphate solution, and then mix the two in a volume ratio of 49:51 to obtain a phosphate buffer solution with a pH of 6.8.

[0063] Mix the above solutions thoroughly in a volume ratio of 10:15:0.1:50:100 to prepare the spotting solution. Prepare and use immediately.

[0064] 1.2 Preparation of membrane blocking solution

[0065] The membrane blocking solution is a PBS solution containing 1% w / v BSA, 0.1%–0.5% v / v glycerol, and 0.1% v / v Tween-20, prepared according to the membrane blocking solution formula.

[0066] 1.3 Preparation of Activation Solution

[0067] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) at 5% M / V and N-hydroxysuccinimide (NHS) at 2.5% M / V were thoroughly mixed in purified water to prepare the activation solution.

[0068] Example 2: Establishment of quality control and fabrication methods for large-scale production of microarray chips

[0069] 2.1 Operational steps of quality inspection methods

[0070] A quality inspection method for the mass production of microarray chips, the quality inspection method comprising:

[0071] Step 1) Place the nanofilm in a clean environment with humidity <20% and temperature 23℃~25℃ for 24 hours to allow the nanofilm to fully equilibrate.

[0072] Step 2) Prepare the spotting solution immediately before use;

[0073] Step 3) Place the fully equilibrated nanofilm from Step 1) into a spotting apparatus. Under conditions of 45%–55% humidity and 23°C–25°C, spot the spotting solution described in Step 2) onto the quality control points of the nanofilm one by one. The quality control points of the nanofilm are the non-target areas, non-quality control areas, and non-blank control areas of the nanofilm. There are no less than 2 quality control points in each well, 20 nmol / point.

[0074] Step 4) Place each nanofilm after the sample spotting in Step 3) into the matching fixture, place it under the lens of an industrial camera, adjust the lens focus until the sample spot is clearly visible and the edge is well defined, take pictures of each hole with the industrial camera, and use software to measure the diameter of each quality inspection point and export the results.

[0075] Step 5) Analyze the diameter data of each quality inspection point on the nanofilm, and calculate the overall standard deviation, median and range of the diameter of the quality inspection points on the nanofilm. A nanofilm with an overall standard deviation ≤5%, a median of 460μm~480μm and a range ≤30μm is considered to be of qualified quality inspection.

[0076] Nanofilms that pass quality inspection can be used immediately or stored in resealable bags at room temperature for later use.

[0077] 2.2 Operational Steps of the Fabrication Method for Large-Scale Production of Microarray Chips

[0078] A method for large-scale production of microarray chips, comprising the following steps:

[0079] Step (1) Place the qualified nanofilm of Example 2.1 in a container, add an appropriate amount of activation solution for surface activation, rinse the surface activation solution with purified water after 0.5 hours, and then dry the surface of the nanofilm with clean air.

[0080] Step (2) Place the activated nanofilm from step (1) into the spotting instrument and spot the target, goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody, and blank control spotting solution onto the nanofilm according to the set program. The spotting volume is 20 nl.

[0081] Step (3) Place each nanofilm after spotting in step (2) into the matching fixture, dry it for 6 hours at a temperature of 21℃~25℃ and a humidity of <25%, and seal it with a membrane sealing solution to obtain a microarray chip.

[0082] When the microarray chip is each chip on the dual detection chip kit for porcine pseudorabies virus gD and gE protein antibodies, it is... Figure 2 As shown, the target point is Figure 2 As shown in Figures 5 and 6, 5 is the PRVgD detection point and 6 is the PRVgE detection point.

[0083] When the microarray chip is each chip on the triple detection chip kit for antibodies against porcine pseudorabies virus gD, gE protein, and classical swine fever virus E2 protein, it is... Figure 3 As shown, the target point is Figure 3 As shown in Figures 5, 6, and 7, 5 is the PRVgD detection point, 6 is the PRVgE detection point, and 7 is the CSFV E2 detection point.

[0084] When the microarray chip is each chip on the O-type and A-type foot-and-mouth disease virus antibody detection chip kit Figure 4 As shown, the target point is Figure 4 As shown in Figures 8 and 9, 8 is the FMDV A detection point and 9 is the FMDV O detection point.

[0085] When the microarray chip is each chip on the fungal toxin quadruple detection chip kit Figure 5 As shown, the target point is Figure 5 As shown in Figures 10, 11, 12, and 13, 10 is the fumonisin detection point, 11 is the aflatoxin B1 detection point, 12 is the vomitoxin detection point, and 13 is the zearalenone detection point.

[0086] 2.3 Validation of Quality Inspection Methods

[0087] To better evaluate the quality control method, this invention uses a mycotoxin quadruple detection chip kit (hereinafter referred to as kit 4) as an example to illustrate the validation of the quality control method in detail. Specifically, in kit 4, the mycotoxin quadruple detection chip is diluted with the spotting solution to apply fumonisin antigen at 0.6 ng / point to the fumonisin detection point; aflatoxin B1 antigen at 0.8 ng / point to the aflatoxin B1 detection point; vomitoxin antigen at 0.7 ng / point to the vomitoxin detection point; zearalenone antigen at 0.4 ng / point to the zearalenone detection point; goat anti-mouse polyclonal antibody at 1 ng / point to quality control point 1; goat anti-mouse polyclonal antibody at 2 ng / point to quality control point 2; goat anti-mouse polyclonal antibody at 4 ng / point to quality control point 3; and a blank control point is also sampled.

[0088] The detection steps for kit 4 are as follows:

[0089] (4-1) Sample pretreatment: Grind the grain, feed raw materials or finished feed samples to below 20 mesh (about 1 mm particle size) using a grinder. Accurately weigh 2 ± 0.02 g of sample into a 50 ml centrifuge tube, add 10 ml of sample extract, mix the sample thoroughly, vortex for 5 minutes, centrifuge at 4000 rpm for 5 minutes, take 50 μl of supernatant and add it to 950 μl of sample dilution solution and mix well. Take 50 μl for detection.

[0090] (4-2) Equilibration: Place each component of the kit at room temperature for 30 minutes to equilibrate.

[0091] (4-3) Soaking: Add 250 μl of washing solution (about 4-5 drops per well) to each well of the microarray chip, let it stand for 3 minutes, shake off and pat dry the liquid in the well;

[0092] (4-4) Add samples: Add 50 μl of sample, 25 μl of enzyme-labeled working solution, and 25 μl of antibody working solution to each well of the microarray chip.

[0093] (4-5) Incubation: Incubate at 30°C and 500 rpm in a microplate constant temperature shaker for 20 minutes;

[0094] (4-6) Wash, discard the liquid in the well, add 250μl of washing solution to each well (about 4-5 drops per well), be careful not to let the washing solution overflow the well to avoid cross-contamination and inaccurate test results. Wash 4 times, soaking for about 10 seconds each time, and pat dry on absorbent paper.

[0095] (4-7) For color development, add 100 μl of TMB substrate solution to each well and let it stand in a microplate constant temperature shaker at 30°C for 15 minutes for color development.

[0096] (4-8) Take the reading, shake off the liquid, open the matching clamp (see Chinese Patent CN211348276U), press the chip moderately with lint-free paper to absorb the liquid on the chip film surface, and use a micro-hole disk chip imager to detect it. The reading should be completed within 5 minutes, and the detection result should be checked.

[0097] Example 3: Comparison of nanofilm fabrication chip results obtained from different quality inspection methods

[0098] This invention compares the method of this invention with the contact angle measurement method.

[0099] The method and qualification criteria of this invention are as follows: the quality inspection method described in Example 2.1, and the preparation method and qualification criteria described in Example 2.2;

[0100] Contact angle measurement method and acceptance criteria: The contact angle of the nanofilm surface is measured using a contact angle meter. Ten points are randomly selected from each nanofilm for measurement, using pure water as the contact angle measurement solution. If the range of the 10 contact angle data for each nanofilm is ≤2%, the nanofilm is considered to be qualified.

[0101] 3.1 Comparison of the accuracy and repeatability of nanofilms prepared into chips after quality inspection using different methods

[0102] One batch of nanofilms (180 pieces / batch) was evenly distributed and processed according to Example 2.1 and Appendix. Figure 5 In the sampling method, any two points are selected as quality inspection points. The above two methods are used to conduct quality inspections, and nanofilms that meet the corresponding quality inspection standards are selected as backup nanofilms.

[0103] The qualified spare nanomembranes obtained from the above two methods were then used to prepare mycotoxin quadruple detection chips according to the method described in Example 2.1. Except for the different detection methods used for the nanomembranes, all other operation steps were kept consistent. The prepared chips were classified and labeled according to the "method of this invention" and the "contact angle measurement method". Three experimenters were randomly selected to conduct experiments, with one person operating two chips simultaneously (one for each of the different methods). The final results were analyzed and compared according to accuracy and repeatability.

[0104] The following methods were used to determine the following in samples: aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEN), and fumonisin (FB) using liquid chromatography-tandem mass spectrometry (LC-MS / MS) as specified in GB 5009.22-2016; and fumonisin (FB) using liquid chromatography (LC-MS / MS) as specified in GB 5009.209-2016. Samples that tested negative by all methods were pretreated with AFB1, DON, ZEN, and FB standards as follows: The samples were pulverized to below 20 mesh (approximately 1 mm particle size) using a grinder; 2 ± 0.05 g of homogenized tissue sample was accurately weighed into a 50 ml polystyrene centrifuge tube, 10 ml of sample extraction solution was added, and the sample was vortexed thoroughly for 5 minutes to disperse it; the sample was then centrifuged at 4000 rpm for 5 minutes at room temperature; 50 μL of the supernatant was taken and added to 950 μL of sample diluent and mixed thoroughly. Accuracy was measured by recovery rate, and repeatability was measured by the variability in grayscale values ​​(CV value) at the same concentration. Results are shown in Table 1.

[0105] Table 1 Summary of detection results using the two methods

[0106]

[0107] Table 1 shows that the qualified nanomembranes screened by the method of this invention have an overall standard deviation of ≤5% for the diameter of the quality inspection points, a median of 460μm to 480μm, and a range of ±30μm. The accuracy (judged by recovery rate, between 86% and 113%) and repeatability (≤6%) are significantly improved. Compared with the nanomembranes screened by the contact angle measurement method, the performance (recovery rate between 61% and 143%, repeatability >15%) is significantly improved.

[0108] 3.2 Comparison of chip fabrication results from different batches of nanofilms

[0109] Three batches of nanofilms (180 pieces / batch, a total of 540 pieces from three consecutive batches) were evenly distributed within each batch, according to Example 2.1, and according to... Figure 5 In the sampling method, any two points are selected as quality inspection points. The above two methods are used to conduct quality inspections, and nanofilms that meet the corresponding quality inspection standards are selected as backup films.

[0110] The qualified nanomembranes obtained after quality inspection using the two methods described above were used to prepare mycotoxin quadruple detection chips according to the method described in Example 2.1. Except for the different detection methods used for the nanomembranes, all other operational steps were kept consistent. The prepared chips were classified and labeled according to the "method of this invention" and the "contact angle measurement method," and three experimenters were randomly selected to conduct experiments. Each experiment involved operating two chips simultaneously (one for each method). The final results were analyzed and compared based on accuracy and repeatability. Accuracy was determined by the recovery rate, and repeatability was determined by the coefficient of variation (CV) of the grayscale values ​​at the same concentration. The results are shown in Table 2.

[0111] Table 2 Comparison of test results for chips prepared from different batches of nanofilms

[0112]

[0113] Table 2 shows that the nanomembranes that passed the quality inspection screening using the method of this invention exhibited an overall standard deviation of ≤5% for the diameter of the inspection points, a median of 460 μm to 480 μm, and a range of ±30 μm. Furthermore, the repeatability and accuracy between different batches were relatively stable, with recoveries ranging from 85% to 115% and batch-to-batch repeatability ≤8%. In contrast, the nanomembranes that passed the quality inspection screening using the contact angle measurement method showed poor repeatability (between 47% and 134%) and accuracy (>15%). Therefore, the method of this invention is superior to the traditional contact angle measurement method.

[0114] 3.3 Experimental results of nanofilms screened by different methods for different projects

[0115] One batch of nanofilms (180 pieces / batch) was evenly distributed among each batch, and quality inspection was carried out using the two methods mentioned above. Nanofilms that met the corresponding quality inspection standards were selected as spare films.

[0116] The spare membranes that passed the quality inspection of the above two methods were used to prepare a dual detection chip for porcine pseudorabies virus gD and gE protein antibodies (hereinafter referred to as the dual chip in Table 3) and a triple detection chip for porcine pseudorabies virus gD, gE protein, and classical swine fever virus E2 protein antibodies (hereinafter referred to as the triple chip in Table 3) according to Example 2.1. Except for the different detection methods used for the nanomembrane, all other operation steps were kept consistent. The prepared chips were classified and labeled according to the "method of this invention" and the "contact angle measurement method", and three experimenters were randomly selected to conduct experiments. Each experimenter operated two chips simultaneously (one for each different method), and the final results were analyzed and compared according to repeatability. Repeatability was measured by the degree of variation of gray values ​​(CV value) at the same concentration. The results are shown in Table 3:

[0117] Table 3. Experimental results of nanofilms screened using different methods for different projects.

[0118]

[0119] Table 3 shows that, in the field of animal disease chips, the performance of nanofilms screened by the method of this invention (reproducibility of 4% to 8%) is still significantly better than that of nanofilms screened by the contact angle measurement method (reproducibility ≥ 15%). Therefore, the method of this invention is superior to the traditional contact angle measurement method.

[0120] 3.4 Detection of the number of quality checkpoints in nanometer membranes

[0121] The number of quality control points for each nanofilm was measured to verify the appropriate number of sampling points.

[0122] One batch of nanofilms was randomly selected and processed according to Example 2.1 and Appendix. Figure 5 The sampling method used in the process involved selecting any 2, 3, 4, 5, 6, 7, or 8 points as quality control points. The prepared chips were classified and labeled according to the "method of this invention" and the "contact angle measurement method." Three experimenters were randomly selected to conduct the experiments, with each person operating two chips simultaneously (one for each different method) for each experiment. The final results were analyzed and compared based on accuracy and repeatability. Accuracy was measured using the recovery rate as the standard, while repeatability was measured using the variability (CV) of grayscale values ​​at the same concentration. The results are shown in Table 4.

[0123] Table 4. Detection results of the number of quality control points for nanofilms.

[0124]

[0125]

[0126] As can be seen from Table 4, the selection of the number of quality inspection points during quality inspection in the method of the present invention has no substantial impact on the recovery rate and repeatability.

[0127] Example 4: Application of different quality inspection methods in the large-scale production of microarray chips

[0128] 4.1 Application of the Mycotoxin Quadruple Detection Chip Reagent Kit

[0129] Forty clinical samples (numbered 1# to 40#) were collected and tested according to the national standard method and the method of this invention.

[0130] Ten samples, numbered 1# to 10#, were tested for aflatoxin B1 (AFB1) using liquid chromatography-tandem mass spectrometry according to the national standard method GB5009.22-2016.

[0131] Ten samples from #11 to #20 were analyzed for deoxynivalenol (DON) using liquid chromatography-tandem mass spectrometry according to the national standard method GB5009.22-2016.

[0132] Ten samples from #21 to #30 were tested for zearalenone (ZEN) in the samples using the liquid chromatography method specified in the national standard GB5009.209-2016.

[0133] Ten samples (31# to 40#) were tested for fumonisin (FB) using liquid chromatography according to the national standard method GB5009.240-2016.

[0134] Meanwhile, 40 samples were pretreated as follows: the samples were pulverized to below 20 mesh (approximately 1 mm particle size) using a grinder; 2 ± 0.05 g of homogenized tissue sample was accurately weighed into a 50 ml polystyrene centrifuge tube, 10 ml of sample extraction solution was added, and the sample was vortexed for 5 minutes to mix and disperse it; the sample was then centrifuged at 4000 rpm for 5 minutes at room temperature; 50 μL of the supernatant was taken and added to 900 μL of sample diluent and mixed well.

[0135] According to Example 2.1, according to Appendix Figure 5 Two random points were selected as quality control points for the sampling method described above. Quality control was performed using both methods, and nanofilms meeting the corresponding quality control standards were selected. Kit 4 was prepared and categorized according to the "Method of this Invention" and the "Contact Angle Measurement Method." Three experimenters were randomly selected to conduct the experiment, with each person operating two chips simultaneously (one for each method) per experiment. The processed samples were tested according to the detection steps of Kit 4 in Example 2.2. The final results were analyzed and compared based on accuracy and national standard methods. Repeatability was measured using the variation in grayscale values ​​(CV value) at the same concentration. The detection values ​​and recovery rates are shown in Table 5, and the intra-batch and inter-batch repeatability are shown in Table 6.

[0136] The results showed that the recovery rate of the method of the present invention ranged from 82.63% to 114.18%, and the intra-batch and inter-batch repeatability was less than 13%. The accuracy and repeatability of clinical samples were both good, and were superior to the results of the contact angle method.

[0137] Table 5. Detection values ​​and recovery rates of the method of the present invention.

[0138]

[0139]

[0140] Table 6. Results of intra-batch and inter-batch repeatability

[0141]

[0142]

[0143] 4.2 Application of the dual detection chip kit for porcine pseudorabies virus gD and gE protein antibodies

[0144] According to Example 2.1, according to Figure 2 In the sampling method, any three points are selected as quality inspection points. Quality inspection is performed using the two methods described above, and nanomembranes meeting the corresponding quality inspection standards are selected. Then, the porcine pseudorabies virus gD and gE protein dual-detection antibody chip kit (referred to as kit 1) is prepared. The porcine pseudorabies virus gD and gE protein dual-detection antibody chip is diluted with the sampling solution to make the porcine pseudorabies virus gD protein at a ratio of 4... Spot 0.0 ng / point at the PRVgD detection point. Dilute with spotting solution to spot 1.0 ng / point of porcine pseudorabies virus gE protein at the PRVgE detection point. Dilute with spotting solution to spot 1 ng / point of goat anti-mouse polyclonal antibody at the quality control point. Dilute with spotting solution to spot 2 ng / point of goat anti-mouse polyclonal antibody at the quality control point. Dilute with spotting solution to spot 4 ng / point of goat anti-mouse polyclonal antibody at the quality control point. Spot with spotting solution at the blank control point.

[0145] The detection method for the porcine pseudorabies virus gD and gE protein antibody dual detection chip kit (referred to as kit 1) is as follows:

[0146] (1-1) Equilibration: Place each component of the kit at room temperature for 30 minutes to equilibrate.

[0147] (1-2) Soaking: Add 300 μl of washing solution to each well of the microarray chip, soak for 3 minutes, then discard the solution and pat dry.

[0148] (1-3) Add sample: Add 50 μl of sample diluent per well, then add 50 μl of the sample to be tested per well. Incubate in a constant temperature shaking incubator at 37°C and 500 rpm for 30 minutes. Discard the liquid: Add 300 μl of washing solution per well, soak for 30-60 seconds, and discard the liquid. Wash repeatedly 5 times, and pat dry for the last time.

[0149] (1-4) Add enzyme-labeled reagent, add 100 μl of enzyme-labeled reagent per well, and incubate in a constant temperature shaking incubator at 37°C and 500 rpm for 30 minutes; discard the liquid, add 300 μl of washing buffer per well, soak for 30-60 seconds, and discard the liquid; wash repeatedly 5 times, and pat dry for the last time;

[0150] (1-5) For color development, add 100 μl of substrate solution per well, incubate at 37°C for 15 minutes, then discard the solution and pat dry.

[0151] (1-6) After removing the chip cover, place the lint-free paper on the detection chip, press it gently, and measure the result using a micro-disk chip imager within 10 minutes, and export the S / N value (sample S value / quality control point N value).

[0152] Determination of experimental validity: The S-value of the quality control point should be ≥6000, and the S-value of the blank control point should be ≤3000; otherwise, the experiment is invalid. This determination is automatically completed by the internal data processing and analysis system. Result determination (this determination is also automatically completed by the internal one-click intelligent data processing and analysis system, eliminating the need for technical personnel to perform data calculations and statistical analysis) standards are as follows:

[0153] Calculation of S / N ratio: S / N = Sample A value / Negative control A value (where sample A value is the gray value of the sample; negative control A value is the gray value of a certain quality control point, which is selected based on the conformity of the analytical values ​​of known negative and positive samples calibrated during the batch commissioning with the standard values. The selection criterion is the result calculated by the ratio of the gray values ​​of the calibrated known negative and positive samples to the gray values ​​of three quality control points 1, 2, and 3, and the quality control point corresponding to the condition with the highest conformity to the standard value and judgment result).

[0154] When the positive criterion S / N ≤ 0.600, PRV gD and gE antibodies are considered positive.

[0155] When the negative determination S / N > 0.700, PRV gD and gE antibodies are negative;

[0156] If the PRV gD and gE antibodies are suspected (0.600 < S / N ≤ 0.700), resampling and testing are required. If the test is still suspected, the result is considered negative.

[0157] Ten positive clinical samples (numbered 41# to 50#, both PRVgD and PRVgE antibodies were positive, and CSFV antibodies were also positive in samples 41# to 45#) and ten negative samples (numbered 51# to 60#, both PRVgD and PRVgE antibodies were negative) were collected after testing with porcine pseudorabies virus neutralization test and commercial porcine pseudorabies virus ELISA antibody kits (including Biochek PRVgB kit and IDEXX PRVgE kit) and classical swine fever virus ELISA antibody kit (detected by IDEXX CSFV antibody detection kit).

[0158] Three batches (batch numbers A, B, and C) of nanofilm were randomly selected to prepare reagent kit 1. These kits were categorized and labeled according to the "method of this invention" and the "contact angle measurement method." Three experimenters were randomly selected to conduct the experiments. Each experiment involved operating one chip from any of the three batches simultaneously for inter-batch repeatability testing, and randomly selecting three chips from one batch for intra-batch repeatability testing. The reagent kits prepared using the contact angle measurement method were compared with the results. Twenty clinical samples were tested according to the detection steps of reagent kit 1 in Example 4.2. The final results were analyzed and compared based on repeatability and the contact angle measurement method. The intra-batch and inter-batch repeatability results of the two methods are shown in Table 7. In particular, the test results of the reagent kit prepared using the nanofilm method of this invention showed better consistency with the test results of the commercially available reagent kit than those of the contact angle measurement method. Furthermore, the batch-to-batch and intra-batch coefficients of variation for PRVgD and PRVgE antibodies detected by the nanofilm preparation kit 1 prepared by the method of the present invention are both within 5%, while the batch-to-batch and intra-batch coefficients of variation for PRVgD and PRVgE antibodies detected by the contact angle measurement method are both between 8% and 15%. Therefore, the quality inspection effect of the method of the present invention is better than that of the contact angle measurement method.

[0159] Table 7 Comparison of test results for nanofilm preparation kits using different quality control methods

[0160]

[0161]

[0162] 4.3 Application of the triple detection chip kit for antibodies against porcine pseudorabies virus gD, gE proteins, and classical swine fever virus E2 protein

[0163] According to Example 2.1, according to Figure 3 In the sampling method described above, any two points are selected as quality control points. Quality control is performed using both methods described above, and nanomembranes meeting the corresponding quality control standards are selected. Then, the pseudorabies virus (PRV) gD, gE protein, and classical swine fever virus (CSFV) E2 protein antibody triple detection chip kit (referred to as Kit 2) is prepared. Specifically, the PRV gD protein is diluted with the sampling solution and sampled at 4.0 ng / point at the PRV gD detection point; the PRV gE protein is diluted with the sampling solution and sampled at 1.0 ng / point at the PRV gE detection point; and the CSFV E2 protein is diluted with the sampling solution and sampled at 6.4 ng / point at the CSFV detection point. At the E2 test point, dilute the sample solution and apply 1 ng / point of goat anti-mouse polyclonal antibody to the quality control point 1. Dilute the sample solution and apply 2 ng / point of goat anti-mouse polyclonal antibody to the quality control point 2. Apply the sample solution to the blank control point.

[0164] The detection method for the triple detection chip kit for porcine pseudorabies virus gD, gE protein, and classical swine fever virus E2 protein antibodies (hereinafter referred to as Kit 2) is the same as that for Kit 1, with the addition of a method for determining classical swine fever antibodies. The method for determining classical swine fever antibodies is as follows:

[0165] PI = (1 - Sample A value / Negative control A value) × 100%.

[0166] A pI ≥ 40% is considered a positive CSFV antibody test.

[0167] A CSFV antibody level of 30% < PI < 40% is considered suspicious.

[0168] A PI ≤ 30% is considered a negative CSFV antibody result.

[0169] According to Example 2.1, according to Appendix Figure 3 In the sampling method described in Example 4.3, any two points were selected as quality control points. Quality control was performed using both methods, and nanofilms meeting the corresponding quality control standards were selected. Three batches (batch numbers A, B, and C) of nanofilms were randomly selected to prepare reagent kit 2. These kits were classified and labeled according to the "method of this invention" and the "contact angle measurement method." Three experimenters were randomly selected to conduct the experiments. Each experiment involved operating one chip from any of the three batches simultaneously for inter-batch repeatability testing, and randomly selecting three chips from one batch for intra-batch repeatability testing. The reagent kits prepared using the contact angle measurement method were compared with those prepared using the same method. Twenty clinical samples were tested according to the detection steps of reagent kit 2 in Example 4.3. The final results were analyzed and compared based on repeatability and the contact angle measurement method. The intra-batch and inter-batch repeatability results of the two methods are shown in Tables 8 and 9. In particular, the test results of the reagent kit prepared using the nanofilms tested by the method of this invention showed better consistency with the test results of the commercially available reagent kit than those prepared using the contact angle measurement method. Furthermore, the inter-batch and intra-batch coefficients of variation for PRVgD antibody, PRVgE antibody, and CSFV antibody detected by the nanomembrane preparation kit 2 of the present invention are all within 5%, while the inter-batch and intra-batch coefficients of variation for PRVgD antibody, PRVgE antibody, and CSFV antibody detected by the contact angle measurement method are all between 8% and 15%. Therefore, the quality inspection effect of the present invention method is better than that of the contact angle measurement method.

[0170] Table 8 Comparison of test results for the kit used in the quality control of nanofilm preparation according to the method of the present invention.

[0171]

[0172]

[0173] Table 9 Comparison of test results for nanofilm preparation kits using the contact angle measurement method.

[0174]

[0175] 4.4 Application of the O-type and A-type foot-and-mouth disease virus antibody detection chip kit

[0176] According to Example 2.1, according to Figure 4 For the sampling method, any two points are selected as quality control points. After passing the quality control, the O-type and A-type foot-and-mouth disease virus antibody detection chip kit (referred to as kit 3) is prepared. In the O-type and A-type foot-and-mouth disease virus antibody detection chip, the O-type foot-and-mouth disease virus antigen is diluted with the sampling solution and sampled at 16 ng / point at the FMDV O detection point. The A-type foot-and-mouth disease virus antigen is diluted with the sampling solution and sampled at 32 ng / point at the FMDV A detection point. The sheep anti-mouse polyclonal antibody is diluted with the sampling solution and sampled at 2 ng / point at quality control point 1. The sheep anti-mouse polyclonal antibody is diluted with the sampling solution and sampled at 4 ng / point at quality control point 2. The sheep anti-mouse polyclonal antibody is diluted with the sampling solution and sampled at 6 ng / point at quality control point 3. The blank control point is sampled with the sampling solution.

[0177] The detection method for the O-type and A-type foot-and-mouth disease virus antibody detection chip kit (referred to as kit 3) is as follows:

[0178] (3-1) Sample pre-dilution: Dilute the serum sample 10 times with sample diluent;

[0179] (3-2) Numbering: Number the chip holes according to the sample sequence;

[0180] (3-3) Soak, add 300 μl of washing solution per well, soak for 3 minutes, then discard the solution and pat dry;

[0181] (3-4) Add 50-100 μl of diluted sample to each well and incubate at 37°C and 500 rpm for 30 minutes in a constant temperature shaker.

[0182] (3-5) Add enzyme-labeled reagent, add 50-100 μl of enzyme-labeled reagent per well, incubate in a constant temperature shaker at 37℃ and 500 rpm for 30 minutes; discard the liquid, add 300 μl of washing buffer per well, soak for 10-20 seconds, discard the liquid, wash repeatedly 5 times, and finally pat dry;

[0183] (3-6) For color development, add 100 μl of substrate solution per well and incubate at 37°C for 15 minutes;

[0184] (3-7) Determination: Vertically discard the liquid, shake clean, remove the chip cover, invert onto a lint-free paper, press gently, and measure the result within 10 minutes using a micro-disk chip imager. Export the blocking rate (1 - sample gray value / quality control point gray value).

[0185] Determination of test validity: The gray value of the quality control point should be ≥10000, and the gray value of the blank control point should be ≤3000; otherwise, the test is invalid. This determination is automatically completed by the internal data processing and analysis system. Result determination (this determination is also automatically completed by the internal one-click intelligent data processing and analysis system, eliminating the need for technical personnel to perform data calculations and statistical analysis) standards are as follows:

[0186] Calculation of blocking rate: Blocking rate = (1 - Sample gray value / Quality control point gray value) × 100%

[0187] Positive criteria: FMDV O and A antibodies are positive when the blocking rate is ≥50%;

[0188] Negative determination: When the blocking rate is <50%, FMDV O and A antibodies are negative.

[0189] Ten positive clinical samples (numbered 61# to 70#, both FMDV type O and type A antibodies were positive) and ten negative samples (numbered 71# to 80#, both FMDV type O and type A antibodies were negative) were collected using commercially available foot-and-mouth disease virus ELISA antibody kits (including the Lanzhou Veterinary Research Institute Foot-and-Mouth Disease Virus Type O Antibody Liquid Phase Blocking ELISA Detection Kit and the Lanzhou Veterinary Research Institute Foot-and-Mouth Disease Virus Type A Antibody Liquid Phase Blocking ELISA Detection Kit).

[0190] According to Example 2.1, according to Figure 4 Three random points were selected as quality control points for the sampling method described above. Quality control was performed using both methods, and nanomembranes meeting the corresponding quality control standards were selected. Three batches (batch numbers A, B, and C) of nanomembranes were randomly selected to prepare reagent kit 3. These kits were classified and labeled according to the "method of this invention" and the "contact angle measurement method." Three experimenters were randomly selected to conduct the experiments. Each experiment involved operating one chip from any of the three batches simultaneously for inter-batch repeatability testing, and randomly selecting three chips from one batch for intra-batch repeatability testing. The reagent kits prepared using the contact angle measurement method were compared. Twenty clinical samples were tested according to the detection steps of reagent kit 3 in Example 4.4. The final results were analyzed and compared based on repeatability and the contact angle measurement method. The intra-batch and inter-batch repeatability results of the two methods are shown in Table 10. In particular, the test results of the reagent kit prepared using the nanomembrane quality control method of this invention showed better consistency with the test results of the commercially available foot-and-mouth disease virus ELISA antibody kit than those of the contact angle measurement method. Furthermore, the batch-to-batch and intra-batch coefficients of variation for FMDV type O and type A antibodies detected by the nanomembrane preparation kit 3 using the method of the present invention are all within 5%, while the batch-to-batch and intra-batch coefficients of variation for FMDV type O and type A antibodies detected by the contact angle measurement method are all between 7% and 15%. Therefore, the quality inspection effect of the method of the present invention is better than that of the contact angle measurement method.

[0191] Table 10 Comparison of test results for nanofilm preparation kits using different quality control methods

[0192]

[0193] In summary, the quality inspection method of this invention can reproduce the spotting process during quality inspection, avoiding systematic errors introduced during spotting and truly reflecting the stability and uniformity of the nanofilm preparation process. The method of measuring the sample spot diameter reflects the uniformity of the nanofilm surface to the greatest extent. Nanofilms obtained through the above quality inspection method have shown excellent experimental results in subsequent chip fabrication processes. Furthermore, the spotting solution fixed on the nanofilm surface by this quality inspection method can be thoroughly washed away during the chip fabrication process, leaving no residue and having no impact on the nanofilm surface.

[0194] High-throughput microarray chip projects involve spotting samples to immobilize targets such as proteins onto the surface of a nanomembrane. Finally, enzyme-linked immunosorbent assay (ELISA) and a colorimetric reaction are used to produce a blue precipitate at the corresponding sample spot, and the final result is determined by reading the grayscale value. Therefore, the uniformity of sample spot size is crucial to the quality of microarray chips, especially those used for quantitative analysis.

[0195] As the primary substrate for microarray chips and a carrier for targets such as proteins, the surface uniformity of nanofilms plays a decisive role in chip quality. However, due to their primary component being silica gel, the quality control of nanofilms themselves presents a significant challenge. Currently, there are no suitable, effective, and industrially viable instrument-based quality control methods for nanofilms. Therefore, this invention, starting with the spotting process, incorporates the entire spotting process into the nanofilm quality control process, comprehensively controlling the surface uniformity of the nanofilm and providing strong assurance for the quality control of microarray chips.

[0196] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A quality inspection method for the mass production of microarray chips, the quality inspection method comprising: Step 1) Place the nanofilm in a clean environment with humidity <20% and temperature 23℃~25℃ for a period of time to allow the nanofilm to fully equilibrate. Step 2) Prepare the spotting solution, and prepare it fresh for each use; the spotting solution is obtained by mixing 5% M / V glycerol solution, 5% M / V sorbitol solution, 0.05% V / V Triton solution, dimethyl sulfoxide solution, and pH 6.8 PBS solution in a volume ratio of 10:15:0.1:50:100 in sequence. Step 3) Place the fully balanced nanofilm from Step 1) into a spotting apparatus and spot it one by one at the quality control points of the nanofilm using the spotting solution from Step 2) under conditions of 45%–55% humidity and 23°C–25°C. The quality control points of the nanofilm are the non-target areas, non-quality control areas, and non-blank control areas of the nanofilm. There are no less than 2 quality control points in each well, 20 nmol / point. Step 4) Place the nanofilm obtained after spotting in Step 3) into the matching fixture, place it under the lens of an industrial camera, adjust the lens focus until the quality inspection points are clearly visible and the edges are well defined, take pictures of each hole with the industrial camera, and use software to measure the diameter of each quality inspection point and export the results. Step 5) Analyze the diameter data of each quality inspection point on the nanofilm, and calculate the overall standard deviation, median and range of the diameter of the quality inspection points on the nanofilm. A nanofilm with an overall standard deviation ≤5%, a median of 460μm~480μm and a range ≤30μm is considered to be of qualified quality inspection.

2. The quality inspection method for large-scale production of microarray chips according to claim 1, wherein, The time for fully equilibrating the nanofilm in step 1) is 20 to 28 hours.

3. The quality inspection method for large-scale production of microarray chips according to claim 2, wherein, The time for fully equilibrating the nanofilm in step 1) is 24 hours.

4. The quality inspection method for large-scale production of microarray chips according to claim 1, wherein, The matching fixture in step 4) includes a cover plate and a base plate. The cover plate is provided with multiple detection windows, and the base plate is provided with perforated holes at corresponding positions to the detection windows. The cover plate can be pressed and fixed on the base plate.

5. A method for the large-scale production of a microarray chip, the method comprising: Step (1) Place the qualified nanofilm of any one of claims 1 to 4 into a container, add 15 ml of activation solution for surface activation, rinse the activation solution with purified water after 0.5 hours, and then dry the surface of the nanofilm with clean air. Step (2) Place the activated nanofilm from step (1) into a spotting instrument and spot the target, goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody, and blank control spotting solution onto the nanofilm according to the set program. The spotting volume is 20 nl. Step (3) The nanofilm after spotting in step (2) is loaded into the matching fixture and dried for 6 hours under the conditions of temperature 21℃~25℃ and humidity <25%. After sealing with membrane sealing liquid, it becomes a microarray chip.

6. The method for large-scale production of microarray chips according to claim 5, wherein, The activation solution in step (1) is a solution containing 5% M / V 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and 2.5% M / V N-hydroxysuccinimide NHS.

7. The method for large-scale production of microarray chips according to claim 5, wherein, The membrane blocking solution in step (3) is a PBS solution containing 1% W / V BSA, 0.1% to 0.5% V / V glycerol, and 0.1% V / V Tween-20.

8. The method for large-scale production of microarray chips according to claim 5, wherein, The target in step (2) is an animal disease virus antigen or its protein or a food safety small molecule antigen.

9. The method for large-scale production of microarray chips according to claim 5, wherein, The targets in step (2) include one or more of the following: porcine pseudorabies virus gD protein or gE protein, classical swine fever virus E2 protein, porcine foot-and-mouth disease virus antigen, porcine rotavirus antigen, mycotoxin antigen, and poultry antibiotic antigen.

10. The method for large-scale production of microarray chips according to claim 5, wherein, When the microarray chip is a dual detection chip for antibodies against porcine pseudorabies virus gD and gE proteins, the target of step (2) is porcine pseudorabies virus gD and gE proteins. When the microarray chip is a triple detection chip for antibodies against porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein, the target of step (2) is porcine pseudorabies virus gD protein, gE protein and classical swine fever virus E2 protein. When the microarray chip is a type O and type A foot-and-mouth disease virus antibody detection chip, the target in step (2) is type O foot-and-mouth disease virus antigen and type A foot-and-mouth disease virus antigen. When the microarray chip is a fungal toxin quadruple detection chip, the targets in step (2) are fumonisin antigen, aflatoxin B1 antigen, vomitoxin antigen and zearalenone antigen.

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