CN115406886A

CN115406886A - Quality inspection method for large-scale production of microarray chip and preparation method

Info

Publication number: CN115406886A
Application number: CN202110579371.XA
Authority: CN
Inventors: 田克恭; 张继颖; 张许科
Original assignee: Luoyang Zhongke Biochip Technology Co ltd
Current assignee: Luoyang Zhongke Biochip Technology Co ltd
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2022-11-29

Abstract

The invention relates to a quality inspection method for the scale production of microarray chips, which comprises the following steps: step 1) placing the nano film in a clean environment, and fully balancing the nano film under the conditions that the humidity is less than 20% and the temperature is 23-25 ℃ for a period of time; step 2) preparing a sample solution, and using the sample solution as it is; step 3) placing the fully balanced nano-film obtained in the step 1) into a sample applicator, and using the sample application liquid obtained in the step 2) to sample the sample application liquid one by one on the quality detection points of the nano-film, wherein the number of the quality detection points in each hole is not less than 2 and is 20 nl/point; step 4) loading the nano film subjected to spotting in the step 3) into a matched clamp, placing the matched clamp under the lens of an industrial camera, taking pictures by holes by the industrial camera, and measuring and exporting the diameters of all quality inspection points by software; and 5) analyzing the diameter data of each quality inspection point on the nano film, and calculating the total standard deviation, median and range difference of the diameters of the quality inspection points on the nano film, wherein the total standard deviation is less than or equal to 5%, the median is 460-480 mu m, and the range difference is less than or equal to 30 mu m, and the nano film is qualified in quality inspection.

Description

Quality inspection method for large-scale production of microarray chip and preparation method

Technical Field

The invention relates to a quality inspection method for large-scale production of microarray chips and a preparation method, belonging to the technical field of biology.

Background

In the beginning of the 21 st century, the biology has changed significantly, and the traditional biological research mode has been greatly challenged. With the rapid development of the human genome project, the accumulation speed of biological data is increasing, and thus higher requirements are put forward on scientific analysis methods and practical analysis tools of biological data. Under such a background, bioinformatics using mathematical knowledge, information, and computer science and technology has come into play, and in bioinformatics dealing with more complicated gene expression data, larger data volume, and faster data growth, microarray chips have become a high-throughput, parallelized, and miniaturized high-speed bioanalysis means with their unique information mining, which is one of the hot spots of research, and is a scientific and technical revolution having a profound meaning again after large-scale integrated circuits, and thus have attracted wide attention.

The microarray chip is formed by designing and spotting on a substrate through a spotting mechanical device, sucking a target solution (such as enzyme, antigen, antibody, receptor, ligand, nucleic acid, cytokine and the like) by a spotting needle, then moving the spotting needle to the upper part of a substrate, fixing the target solution on the surface of the substrate through contact or non-contact spraying, and performing subsequent treatment. The microarray chip is a high-throughput analysis technology, and can capture objects to be detected (existing in serum, plasma, lymph, interstitial fluid, urine, exudate, cytolysis fluid, secretion fluid and the like) capable of being specifically combined with the target according to the characteristics of the target, and the objects are washed, purified, confirmed and biochemically analyzed; provides powerful technical support for obtaining important life information (such as unknown protein components, sequences, in-vivo expression level biological functions, mutual regulation and control relation with other molecules (such as enzymes, antigens, antibodies, receptors, ligands, cytokines and the like), drug screening, selection of drug target positions and the like) so as to be used for researching the interaction between targets, and has outstanding advantages in disease screening, early diagnosis and screening of targets of drug action.

However, in the production and preparation of microarray chips, the accuracy of the chip results is a major concern for users. Because the microarray chips mostly adopt non-contact type spray spotting, and the visual microarray chips output quantitative results through the final gray value of the target points, the gray value determination of the target points needs to be very accurate. The gray value of the target point has a great relationship with the area of the target point in the initial chip spotting process. In the sample application process, the sample application area of the target is uniform, so that the repeatability of the later experimental result is ensured to be good; on the contrary, if the target sample application area has the phenomenon of nonuniform size, the repeatability and accuracy of the result are obviously influenced.

In the production and preparation process of microarray chips, the uniformity of the substrate surface becomes the key to the quality of the finished chip. However, due to the special characteristics of the substrate surface and the subsequent printing characteristics on the surface, the quality of the substrate surface is tested while ensuring that the surface is clean and free from contamination and damage by mechanical force.

Therefore, ultrapure water was used as a detection solution in the previous stage to measure contact angles at several points on the surface of the substrate. The change in contact angle was used to determine the uniformity of the substrate surface. The contact angle is the tangent line to the gas-liquid interface at the intersection of the gas, liquid and solid, and the angle θ between this tangent line and the solid interface (liquid side) is assumed.

Therefore, it is understood that the contact angle shown in FIG. 1 indicates that the contact area between the droplet and the solid surface is constant when the sample application volume is fixed. The uniformity of the substrate surface can be judged as a contact angle change by spraying the same volume of liquid on the microarray substrate surface with ultrapure water and measuring the contact angle thereof. Meanwhile, the ultrapure water operation is adopted, so that the pollution and the mechanical damage are avoided, and the biological performance of the surface of the substrate can be still ensured.

However, when the contact angle measurement method is applied to the microarray chip production process, the following disadvantages are found:

(1) The single-point test or the multi-point test of the contact angle tester still can not meet the uniformity of the base material at all target points of the microarray chip, and the missing detection probability is high to a certain extent;

(2) The ultrapure water is used as the sample application quality detection test solution for sample application, so that surface pollution is avoided, but the surface tension and the density of the ultrapure water are different from those of the real sample diluent, and the test result cannot completely represent the quality detection result;

(3) The contact angle measurement belongs to dynamic measurement, is an angle formed at the moment when a liquid drop contacts a solid surface, and eliminates the influence of subsequent liquid permeation and diffusion. However, the target point of the microarray chip is directly related to the gray value through the area of the sample point, so the contact angle measurement cannot summarize the situation after subsequent liquid diffusion.

As described above, the conventional contact angle measurement method is not effective as a method for inspecting the quality of a microarray chip. The quality inspection method of the microarray chip adopted in the preparation of the microarray chip plays a decisive role in the accuracy of the quantitative detection result of the product production, particularly the large-scale production and the high precision in the later period. Therefore, there is a need in the art for a quality inspection method that can be applied to industrialization, is easy to operate, and can ensure that a high-quality microarray chip can be produced, so as to ensure good repeatability among batches of microarray chips, so as to control the product quality of microarray chips and facilitate improvement of the accuracy of microarray chip product detection results.

Disclosure of Invention

In order to solve the deficiency of the prior art, the invention provides a quality inspection method for the large-scale production of microarray chips, the quality inspection method can directly and completely summarize the system error of a sample applicator, and the influence of the density, viscosity and surface tension of a sample application liquid on the result, and the microarray chip prepared by the nano-film after quality inspection not only realizes the industrial large-scale production, but also can effectively inspect the uniformity of the surface of the microarray chip, so as to control the quality of the nano-film, be beneficial to improving the quality of protein chip products, reduce the batch-to-batch difference and improve the accuracy of detection.

The quality inspection method for the large-scale production of the microarray chip comprises the following steps:

step 1) placing the nano film in a clean environment, and fully balancing the nano film under the conditions that the humidity is less than 20% and the temperature is 23-25 ℃ for a period of time (for example, 20-28 hours, preferably 24 hours);

step 2) preparing a sample solution, wherein the sample solution is prepared in situ;

step 3) placing the fully balanced nano-film obtained in the step 1) into a sample application instrument, and applying the sample application liquid obtained in the step 2) to the quality detection points of the nano-film one by one under the conditions of humidity of 45% -55% and temperature of 23-25 ℃, wherein the quality detection points of the nano-film are non-target positions, non-quality control points and non-blank control positions of the nano-film, and the number of the quality detection points in each hole is not less than 2 and is 20 nl/point;

step 4) loading each nano film subjected to spotting in the step 3) into a matched clamp, placing the matched clamp under a lens of an industrial camera, adjusting the focal length of the lens until sample points are clearly presented and edges are clear, taking pictures by the industrial camera hole by hole, and measuring and exporting diameters of each quality inspection point by software;

and 5) analyzing the diameter data of each quality inspection point of the nano film, and calculating the total standard deviation, median and range of the diameters of the quality inspection points on the nano film, wherein the total standard deviation is less than or equal to 5%, the median is 460-480 mu m, and the range is less than or equal to 30 mu m, and the nano film is qualified in quality inspection. The numerical values of the median and the numerical values of the range here are empirical values obtained by the inventors through a large number of experiments.

The quality inspection method of the invention is that the diameter of the quality inspection point on the nanometer film is directly measured, and the total standard deviation, median and range of the diameter of the quality inspection point on the nanometer film are calculated, so as to judge the uniformity of the surface of the nanometer film, judge the quality inspection qualification rate and control the qualification performance of the product according to the diameter statistical data, effectively control the whole production flow of the nanometer film, and improve the stability of the production process and the stability of the product; the repeatability and the accuracy of the chip prepared by final spotting of the nano-film in the process of detecting the biological sample are achieved, the coefficient of variation of the final detection value is controlled within 15 percent or even within 10 percent, the recovery rate is controlled between 80 percent and 120 percent, and the actual application effect of the microarray chip prepared by the nano-film after the quality inspection of the contact angle measurement method universal by the technical personnel in the field is far better.

In one embodiment of the present invention, the time for allowing the nano-film to be sufficiently equilibrated in step 1) is 20 to 28 hours, preferably 24 hours.

As an embodiment of the present invention, the spotting solution in the step 2) is 5% by weight of an M/V glycerin solution, 5% by weight of an M/V sorbitol solution, 0.05% by weight of a V/V triton solution, a dimethyl sulfoxide (DMSO) solution, a PBS (pH 6.8) solution, which are mixed in turn in a volume ratio of 10: 15: 0.1: 50: 100 and ready to use.

The sample application liquid can completely reflect the influence of the density, viscosity and surface tension of the sample application liquid on the result in the real sample application process.

As an embodiment of the present invention, the matched fixture in step 4) includes a cover plate and a bottom plate, the cover plate is provided with a plurality of detection windows, the bottom plate is provided with hollow holes at positions corresponding to the detection windows, and the cover plate can be fixed on the bottom plate in a pressing manner. The matched clamp in the step 4) is preferably the matched clamp mentioned in Chinese patent CN 211348276U.

The invention also relates to a preparation method for the large-scale production of the microarray chip, which comprises the following steps: placing the qualified nano-film in a container, adding 15ml of activating solution for surface activation, washing the surface activating solution with purified water after 0.5 hour, and drying the surface of the nano-film with clean air;

step (2) the activated nano-film in the step (1) is placed in a sample application instrument, a target is diluted by using a sample application liquid according to a set program and then is used as a target point sample application liquid, a goat anti-mouse polyclonal antibody or a goat anti-mouse secondary antibody is diluted by using the sample application liquid and then is used as a quality control point sample application liquid and a blank control point sample application liquid to be respectively applied to the nano-film, and the sample application volume is 20nl;

and (3) loading each nano film subjected to point sample application in the step (2) into a matched clamp, drying for 6 hours at the temperature of 21-25 ℃ and under the humidity of less than 25%, and sealing by using a film sealing liquid to obtain the microarray chip.

The quality inspection method can directly and completely reflect the system error of the sample applicator, and the influence of the density, viscosity and surface tension of the sample application liquid on the result, and the microarray chip prepared by the nano-film after the quality inspection is not only beneficial to industrial large-scale production, but also can effectively inspect the uniformity of the surface of the microarray chip, so as to control the quality of the nano-film, improve the quality of protein chip products, reduce the difference between batches and improve the accuracy of detection.

As an embodiment of the mass production method of the microarray chip of the present invention, the activation solution in the step (1) is a solution containing 5% of M/V1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, 2.5% of M/V N-hydroxysuccinimide NHS.

As an embodiment of the mass production method of the present invention, the membrane-sealing solution in the step (3) is a PBS solution containing 1% W/V BSA, 0.1-0.5% V/V glycerol, 0.1% V/V Tween-20.

As an embodiment of the preparation method of the invention, the target in the step (2) is an animal epidemic disease virus antigen or a protein thereof or a food safety small molecule antigen.

As an embodiment of the present invention, the target in step (2) comprises 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, avian antibiotic antigen.

As a preferred embodiment of the invention, when the microarray chip is a porcine pseudorabies virus gD and gE protein antibody duplex detection chip in a porcine pseudorabies virus gD and gE protein antibody duplex detection chip kit, the targets in the step (2) are porcine pseudorabies virus gD protein and gE protein.

When the microarray chip is a porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip in the porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip kit, the targets in the step (2) are the porcine pseudorabies virus gD protein, gE protein and classical swine fever virus E2 protein.

When the microarray chip is an 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 targets of the step (2) are an O-type foot-and-mouth disease virus antigen and an A-type foot-and-mouth disease virus antigen.

When the microarray chip is a mycotoxin quadruple detection chip in the mycotoxin quadruple detection chip kit, the targets in the step (2) are fumonisin antigen, aflatoxin B1 antigen, vomitoxin antigen and zearalenone antigen.

The nano film prepared by the quality detection method can be used for the preparation of kits without scenes, including but not limited to the detection of animal source antibodies and the detection of food safety small molecules.

The term "Microarray chip" (Microarray chip) is also called biochip (biochip), and refers to a method of in-situ synthesis or micro-spotting, etc. by which a large amount of biological macromolecules such as nucleic acid fragments, polypeptide molecules, proteins, even tissue slices, cells, etc. are orderly immobilized on the surface of a support (such as a carrier such as a nano-membrane, a glass slide, a silicon wafer, polyacrylamide, nylon membrane, etc.) to form a dense two-dimensional molecular arrangement, and then react with labeled target molecules in a biological sample to be detected, and the intensity of the reaction signal is rapidly, concurrently and efficiently detected and analyzed by a specific instrument, such as a laser confocal scanner or a charge coupled camera, so as to determine the number of the target molecules in the sample.

The overall standard deviation, median and range of diameters in the present invention are as follows:

the term "population standard deviation" is the mean deviation, expressed as σ, between the population's unit token value and its arithmetic mean.

The term "Median" (media), also known as Median, is the number of intermediate positions in a set of data in order, and a term used in statistics refers to a value in a sample, population or probability distribution, which divides the set of values into two equal parts.

The term "Range", also known as Range, is used to refer to the number of variations in the statistical data (measures of variation), the difference between the maximum and minimum, i.e., the data obtained by subtracting the maximum from the minimum.

The term "recovery rate" means that a sample is measured while adding a certain amount of a standard substance to the sample to perform measurement, and the measurement result is subtracted from the measurement value of the sample to obtain the recovery rate of the added standard substance. The calculation formula is as follows:

recovery = (measured amount of sample to which standard substance was added-measured amount of the substance in sample)/amount of standard substance added.

Drawings

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

FIG. 1 is a schematic illustration of a contact angle in a contact angle measurement method;

fig. 2 is a schematic diagram of spotting patterns of each chip well on a bigeminal detection chip kit (kit 1 for short) for gD and gE protein antibodies of porcine pseudorabies virus, wherein 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I and 1J in fig. 2 respectively represent different spotting patterns, and specific positions of 5 and 6 on the detection chip in the same figure can be interchanged;

FIG. 3 is a schematic diagram showing the spotting pattern of each chip well on a triple detection chip kit (kit 2 for short) for antibodies against porcine pseudorabies virus gD, gE protein, and classical swine fever virus E2 protein, wherein 2A, 2B, and 2C in FIG. 3 respectively show different spotting patterns, and the specific positions of 5, 6, and 7 on the detection chip can be interchanged in the same figure;

FIG. 4 is a schematic diagram showing the spotting pattern of each chip well on the O-type and A-type aftosa virus antibody detection chip kit (kit 3 for short), wherein 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I and 3J in FIG. 4 respectively show different spotting patterns, and 8 and 9 in the same figure can be interchanged in specific positions on the detection chip;

FIG. 5 is a schematic diagram showing the spotting pattern of each chip well on a mycotoxin four-joint test chip kit (abbreviated as kit 4), wherein 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I in FIG. 5 respectively show different spotting patterns, and the specific positions of 10, 11, 12, 13 on the detection chip can be interchanged in the same figure.

Reference numerals

The number 1 is that the quality control point 1, 2 is that the quality control point 2, 3 is that the quality control point 3, 4 is blank control point, 5 is that PRVgD check point, 6 is that PRVgE check point, 7 is that CSFV E2 check point, 8 is that FMDV A check point, 9 is that FMDV O check point, 10 is that fumonisin check point, 11 is that aflatoxin B1 check point, 12 is that vomitoxin check point, 13 is that zearalenone check point, the number is that the quality check point.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.

In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

EXAMPLE 1 preparation of solutions

1.1 preparing a spotting fluid

Preparation of 5% glycerol solution: precisely weighing 5.00g of glycerol into a 100ml volumetric flask, adding a small amount of purified water, slightly rotating to fully dissolve the glycerol, avoiding generating excessive bubbles, adding the purified water to a scale mark, and turning and shaking up and down for 10 times for later use;

preparation of 5% sorbitol solution: precisely weighing 5.00g of sorbitol into a 250ml beaker, adding a proper amount of purified water, stirring to completely dissolve the sorbitol, completely transferring the sorbitol into a 100ml volumetric flask, adding the purified water to a scale mark, and turning and shaking up and down for 10 times for later use;

preparation of 0.05% triton solution: measuring 50 mul of triton by using a liquid transfer gun, adding a proper amount of purified water to completely dissolve the triton into a 100ml volumetric flask, adding the purified water to a scale mark, and shaking for 10 times by turning upside down for later use;

DMSO solution: directly adopting a DMSO reagent;

PBS (pH6.8) solution preparation: firstly preparing 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution, and then mixing the two solutions according to the volume ratio of 49: 51 to obtain phosphate buffer solution with the pH value of 6.8;

the solutions are mixed evenly according to the volume ratio of 10: 15: 0.1: 50: 100 to be used as sampling solution. It is used when it is prepared.

1.2 preparation of Membrane blocking solution

The membrane blocking solution formulation was a PBS solution containing 1% W/V BSA, 0.1-0.5% V/V glycerol, and 0.1% V/V Tween-20, and was prepared according to the membrane blocking solution formulation.

1.3 preparing the activating solution

The activated solution was prepared by mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC for short) at 5% M/V and N-hydroxysuccinimide (NHS for short) at 2.5% M/V in purified water.

Example 2 quality control method for microarray chip Mass production and establishment of preparation method

2.1 operating procedure of the quality testing method

step 1) placing the nano film in a clean environment, and fully balancing the nano film under the conditions that the humidity is less than 20% and the temperature is 23-25 ℃ for 24 hours;

step 4) loading each nano film after spotting in the step 3) into a matched fixture, placing the fixture under a lens of an industrial camera, adjusting the focal length of the lens until sample points are clearly presented and the edges are clear, taking pictures by holes by the industrial camera, and measuring and leading out the diameters of all quality inspection points by software;

and 5) analyzing the diameter data of each quality inspection point of the nano film, and calculating the total standard deviation, median and range difference of the diameters of the quality inspection points on the nano film, wherein the total standard deviation is less than or equal to 5 percent, the median is 460-480 mu m, and the range difference is less than or equal to 30 mu m, and the nano film is qualified in quality inspection.

The nano-film qualified in quality inspection can be used immediately or respectively placed in self-sealing bags to be stored at room temperature for later use.

2.2 operating steps of the preparation method for the Large-Scale production of microarray chips

The preparation method for the large-scale production of the microarray chip comprises the following steps:

step (1) placing the qualified nano film of the quality inspection in the embodiment 2.1 into a container, adding a proper amount of activating solution to activate the surface, washing the surface activating solution with purified water after 0.5 hour, and drying the surface of the nano film with clean air;

When the microarray chip is each chip on the porcine pseudorabies virus gD and gE protein antibody duplex detection chip kit, the microarray chip is shown in figure 2, target points are 5 and 6 shown in figure 2, 5 is a PRVgD detection point, and 6 is a PRVgE detection point.

When the microarray chip is each chip on the porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip kit, the detection chip is shown in figure 3, the target points are 5, 6 and 7 shown in figure 3, 5 is a PRVgD detection point, 6 is a PRVgE detection point and 7 is a CSFV E2 detection point.

When the microarray chip is each chip on the O-type and A-type foot-and-mouth disease virus antibody detection chip kit, the microarray chip is shown in figure 4, the target points are 8, 9,8 is an FMDV A detection point and 9 is an FMDV O detection point shown in figure 4.

When the microarray chip is each chip on the mycotoxin quadruple detection chip kit, the microarray chip is shown in fig. 5, the target points are 10, 11, 12 and 13 shown in fig. 5, 10 is a fumonisin detection point, 11 is an aflatoxin B1 detection point, 12 is a vomitoxin detection point, and 13 is a zearalenone detection point.

2.3 verification of quality control method

In order to better evaluate the quality inspection method, the invention uses a mycotoxin four-joint inspection chip kit (kit 4 for short) as an example to describe the verification of the quality inspection method in detail. The mycotoxin tetrad-test chip in the kit 4 is diluted by a sample-application liquid to enable fumonisin antigens to be applied to fumonisin test points according to 0.6 ng/point, the sample-application liquid is used for diluting to enable aflatoxin B1 antigens to be applied to aflatoxin B1 test points according to 0.8 ng/point, the sample-application liquid is used for diluting to enable vomitoxin antigens to be applied to vomitoxin test points according to 0.7 ng/point, the sample-application liquid is used for diluting to enable zearalenone antigens to be applied to zearalenone test points according to 0.4 ng/point, the sample-application liquid is used for diluting to enable goat anti-mouse polyclonal antibodies to be applied to quality control points 1 according to 1 ng/point, the sample-application liquid is used for diluting to enable goat anti-mouse polyclonal antibodies to be applied to quality control points 2 according to 2 ng/point, the sample-application liquid is used for diluting to enable goat anti-mouse polyclonal antibodies to be applied to quality control points 3 according to 4 ng/point, and the sample-application liquid is applied to blank control points.

The detection steps of the kit 4 are as follows:

(4-1) pretreating a sample, namely crushing a grain, feed raw material or finished feed sample to be less than 20 meshes (about 1mm granularity) by using a crusher, accurately weighing 2 +/-0.02 g of the sample into a 50ml centrifugal tube, adding 10ml of sample extracting solution, fully and uniformly mixing the sample, then carrying out vortex for 5 minutes, centrifuging for 5 minutes at 4000 rpm, taking 50 mu l of supernate, adding the supernate into 950 mu l of sample diluent, uniformly mixing, and taking 50 mu l of supernate for detection;

(4-2) balancing, namely balancing all components of the kit at room temperature for 30 minutes;

(4-3) soaking, adding 250 mul of washing solution (about 4-5 drops per hole) into each hole of the microarray chip, standing and soaking for 3 minutes, throwing off and patting dry liquid in the hole;

(4-4) adding samples, respectively adding 50 mul of sample, 25 mul of enzyme-labeled working solution and 25 mul of antibody working solution into each hole of the microarray chip,

(4-5) incubating, namely, oscillating and incubating for 20 minutes in a microplate constant-temperature oscillator at the temperature of 30 ℃ at 500 rpm;

(4-6) washing, namely discarding liquid in holes, adding 250 mu l of washing liquid (about 4-5 drops in each hole) into each hole, paying attention to avoid the washing liquid overflowing out of the holes to avoid causing cross contamination to cause inaccurate detection results, washing for 4 times, soaking for about 10 seconds each time, and patting dry on absorbent paper;

(4-7) developing, adding 100 mul of TMB substrate solution into each hole, and standing and developing for 15 minutes in a microplate constant temperature oscillator at 30 ℃;

(4-8) reading, throwing away liquid, opening a matched clamp (see Chinese patent CN 211348276U), pressing the chip with dust-free paper appropriately, sucking the liquid on the film surface of the chip, detecting with a micropore disk chip imager, finishing reading within 5 minutes, and checking the detection result.

Example 3 comparison of the results of the chips prepared from the Nano films obtained by quality inspection with different quality inspection methods

The present invention is compared with the contact angle measurement method.

The method and the qualification standard of the invention are as follows: the quality inspection method described in example 2.1, the preparation method described in example 2.2, and the qualification standard;

contact angle determination and qualification standards: and measuring the contact angle of the surface of the nano film by using a contact angle measuring instrument. And randomly selecting 10 points from each nano film for measurement, and adopting pure water as contact angle measuring liquid. The range of 10 contact angle data of each nano film is less than or equal to 2 percent, namely the nano film is judged to be qualified.

3.1 comparison of accuracy and repeatability results of quality testing of the obtained nanometer film by different quality testing methods after preparing chip

1 batch of nano films (180 pieces/batch) are evenly distributed, any 2 points are selected as quality inspection points according to the embodiment 2.1 and the point sampling mode in the attached figure 5, quality inspection is carried out by the two methods respectively, and the nano films meeting the corresponding quality inspection standard are selected as standby nano films.

And then qualified spare nano films obtained after the quality inspection by the two methods are respectively prepared into the mycotoxin four-joint inspection chip according to the embodiment 2.1, and except that the used nano films are detected by different methods, other operation steps are ensured to be consistent. The prepared chips are classified and identified according to the method of the invention and the contact angle measuring method, three experimenters are randomly selected for experiment, one operator operates two chips (one chip in different methods) at the same time, and the final results are analyzed and compared according to the accuracy and the repeatability.

Aflatoxin B1 (AFB 1 for short) in a sample is measured by a liquid chromatography-tandem mass spectrometry method in GB5009.22-2016, deoxynivalenol (DON for short) in a sample is measured by a liquid chromatography-tandem mass spectrometry method in GB5009.22-2016, zearalenone (ZEN for short) in a sample is measured by a liquid chromatography method in GB5009.209-2016, and fumonisin (FB for short) in a sample is measured by a liquid chromatography method in GB5009.240-2016, and after the negative samples are detected by the four methods, AFB1, DON, ZEN and FB standard products are added, the treatment is carried out according to the following pretreatment methods: pulverizing the sample to below 20 mesh (about 1mm particle size) with a pulverizer; accurately weighing 2 +/-0.05 g of homogenized tissue sample into a 50ml polystyrene centrifuge tube, adding 10ml of sample extracting solution, fully whirling for 5 minutes, and mixing and dispersing the sample; centrifuging at 4000 rpm for 5 minutes at room temperature; 50 μ L of the supernatant was added to 950 μ L of the sample dilution and mixed well. The accuracy is based on the recovery rate, and the reproducibility is based on the degree of variation (CV) in the gradation value at the same concentration. The results are shown in Table 1:

TABLE 1 summary of the results of the two methods

Table 1 the results show that: the qualified nanometer film is screened by the method, wherein the overall standard deviation of the diameters of the quality inspection points on the nanometer film is less than or equal to 5 percent, the median is 460-480 mu m, and the range is +/-30 mu m, the accuracy (between 86-113 percent by taking the recovery rate as a judgment result) and the repeatability (less than or equal to 6 percent) are obviously improved, and the performance (between 61-143 percent by taking the recovery rate and more than 15 percent by comparing with the performance of the nanometer film screened by a contact angle measuring method) is obviously improved.

3.2 comparison of results of different batches of chip preparation with Nano-film

3 batches of nano films (180/batch, total 540 continuous 3 batches are selected) are evenly distributed in each batch, any 2 points are selected as quality inspection points according to the embodiment 2.1 and the point sampling mode in the figure 5, the quality inspection is carried out by the two methods respectively, and the nano films meeting the corresponding quality inspection standard are selected as standby films.

Preparing mycotoxin quadruple detection chips by using qualified standby nano films after quality detection by the two methods according to the method in the embodiment 2.1 respectively; except that the nano-film is detected by different methods, other operation steps are consistent. The prepared chips are classified and identified according to the method of the invention and a contact angle measuring method, three experimenters are randomly selected for experiment, one person operates two chips (one chip in different methods) at the same time in each experiment, and the final results are analyzed and compared according to the accuracy and the repeatability. The accuracy was determined by the recovery rate, and the reproducibility was determined by the coefficient of variation (CV value) of the gradation value at the same concentration. The results are shown in Table 2:

TABLE 2 comparison of detection results of different batches of nano-film preparation chips

Table 2 the results show that: the qualified nano-film is screened by the quality inspection method, the diameter of the quality inspection point on the nano-film is determined to have the total standard deviation less than or equal to 5 percent, the median is 460-480 mu m and the range is +/-30 mu m, the repeatability and the accuracy among different batches are relatively stable, the recovery rate range is concentrated between 85-115 percent, the repeatability among the batches is less than or equal to 8 percent, and the qualified nano-film is screened by the quality inspection method of a contact angle, and the performances of the repeatability (between 47-134 percent) and the accuracy (more than 15 percent) are poor. Thus, the method of the present invention is superior to conventional contact angle measurements.

3.3 Experimental results of the Nano-film screened by different methods for different projects

1 batch of nanometer films (180 pieces/batch) are evenly distributed in each batch, quality inspection is carried out by the two methods respectively, and the nanometer films meeting the corresponding quality inspection standard are selected as standby films.

Preparing a porcine pseudorabies virus gD and gE protein antibody duplex detection chip (a duplex chip is abbreviated in table 3) and a porcine pseudorabies virus gD, gE protein and swine fever virus E2 protein antibody triplex detection chip (a triplex chip is abbreviated in table 3) by respectively using the qualified standby membranes after the quality inspection by the two methods according to the method in the embodiment 2.1; except that the used nano-film is detected by different methods, other operation steps are consistent. The prepared chips are classified and identified according to the method of the invention and the contact angle measuring method, three experimenters are randomly selected for experiment, one person operates two chips (one chip in different methods) at the same time in each experiment, and the final results are analyzed and compared according to repeatability. The reproducibility was measured by using the degree of variation (CV value) of the gray scale value at the same concentration as a standard. The results are shown in Table 3:

table 3 experimental results of nano-film screened by different methods for different items

Table 3 the results show: in the direction of an animal epidemic disease chip, the performance (the repeatability is 4-8%) of the nano-film screened by the method is obviously superior to the performance (the repeatability is more than or equal to 15%) of the nano-film screened by a contact angle measuring method, so the method is superior to the traditional contact angle measuring method.

3.4 detection of the number of quality detection spots of the Nanomembrane

And detecting the number of quality detection points of each nano film to verify the proper number of sample application points.

Randomly selecting 1 batch of the nano-film, and selecting any 2 points, 3 points, 4 points, 5 points, 6 points, 7 points and 8 points as quality inspection points according to the sampling mode in the embodiment 2.1 and the attached diagram 5. The prepared chips are classified and identified according to the method of the invention and a contact angle measuring method, three experimenters are randomly selected for experiment, one person operates two chips (one chip in different methods) at the same time in each experiment, and the final results are analyzed and compared according to the accuracy and the repeatability. The accuracy is based on the recovery of the results, and the reproducibility is based on the variation of the grey value (CV value) at the same concentration, and the results are shown in Table 4:

TABLE 4 detection results of quality detection number of nano-film

As can be seen from Table 4, the selection of the number of quality control spots in the quality control by the method of the present invention has no substantial effect on the recovery rate and the repetition rate.

Example 4 application of Mass production of microarray chips by heterogeneous assay methods

4.1 application of mycotoxin quadruple-joint detection chip kit

And (3) taking 40 parts of clinical samples (number 1# -40 #), and respectively detecting according to a national standard method and the method disclosed by the invention.

And (3) determining aflatoxin B1 (AFB 1 for short) in the sample by 10 parts of samples of No. 1-10 according to a liquid chromatography-tandem mass spectrometry method in a national standard method GB 5009.22-2016.

And (3) determining the deoxynivalenol (DON for short) in the sample by 10 parts of the 11# to 20# samples according to the liquid chromatography-tandem mass spectrometry in the national standard method GB 5009.22-2016.

10 parts of samples of 21# to 30# are subjected to liquid chromatography in a national standard method GB5009.209-2016 to determine zearalenone (ZEN for short) in the samples.

10 samples of 31# to 40# are subjected to liquid chromatography in a national standard method GB5009.240-2016 to measure fumonisins (FB for short) in the samples.

At the same time, 40 samples were treated according to the following pretreatment method: pulverizing the sample to below 20 mesh (about 1mm particle size) with a pulverizer; accurately weighing 2 +/-0.05 g of homogenized tissue sample into a 50ml polystyrene centrifuge tube, adding 10ml of sample extracting solution, fully whirling for 5 minutes, and mixing and dispersing the sample; centrifuging at 4000 rpm for 5 minutes at room temperature; 50 μ L of the supernatant was added to 900 μ L of the sample dilution and mixed well.

Selecting any 2 spots as quality inspection spots according to the spot-sampling method in the embodiment 2.1 and the attached figure 5, respectively performing quality inspection by the above two methods, selecting the nano-film meeting the corresponding quality inspection standard, preparing a kit 4, performing classification and identification according to the 'invention method' and the 'contact angle measurement method', randomly selecting three experimenters for experiment, and simultaneously operating two chips (one for each method) by one person for each experiment. The treated samples were tested according to the test procedure of kit 4 in example 2.2 and the final results were analyzed and compared with the national standard method for accuracy. The reproducibility was measured by using the degree of variation (CV value) of the gray scale value at the same concentration as a standard. The measurements and recovery are shown in table 5, and the batch-to-batch reproducibility is shown in table 6.

The results show that: the recovery rate result detected by the method is 82.63% -114.18%, the repeatability among batches is less than 13%, and the accuracy and the repeatability of clinical samples are better than those of the results detected by a contact angle method.

TABLE 5 test values and recovery results of the method of the invention

TABLE 6 results of batch-to-batch repeatability

4.2 application of porcine pseudorabies virus gD and gE protein antibody bigeminy detection chip kit

Selecting 3 spots as quality inspection spots according to the sampling method in the embodiment 2.1 and the figure 2, respectively performing quality inspection by the two methods, selecting a nano-film meeting the specification of the corresponding quality inspection standard, preparing a porcine pseudorabies virus gD and gE protein antibody duplex detection chip (kit 1 for short), wherein the porcine pseudorabies virus gD and gE protein duplex detection chip is diluted by a sampling solution to sample the porcine pseudorabies virus gD and gE protein duplex detection chip at a PRVgD detection spot according to 4.0 ng/spot, the porcine pseudorabies virus gE protein is diluted by the sampling solution to sample at a PRVgE detection spot according to 1.0 ng/spot, the goat anti-mouse polyclonal antibody is diluted by the sampling solution to sample at a PRVgE detection spot 1 ng/spot, the goat anti-mouse polyclonal antibody is sampled at a quality control spot 1 ng/spot, the goat anti-mouse polyclonal antibody is diluted by the sampling solution to sample at a 2 ng/spot, and the goat anti-mouse polyclonal antibody is sampled at a 3 ng/spot for a control contrast sample.

The detection method of the porcine pseudorabies virus gD and gE protein antibody bigeminy detection chip kit (kit 1 for short) is as follows:

(1-1) balancing, namely balancing all components of the kit at room temperature for 30 minutes;

(1-2) soaking, namely adding 300 mu l/hole of washing solution into each hole of the microarray chip, and after 3 minutes of soaking, discarding the solution and patting dry;

(1-3) adding sample, namely adding 50 mu l/hole of sample to be detected after 50 mu l/hole of sample diluent, and incubating for 30 minutes at 37 ℃ and 500 r/min in a constant-temperature shaking incubator; adding 300 mul/hole of washing liquid into the waste liquid, and soaking for 30-60 seconds to remove the liquid; repeatedly washing for 5 times, and drying in the last time;

(1-4) adding an enzyme standard reagent, adding 100 mu l/hole of the enzyme standard reagent, and placing the mixture in a constant-temperature shaking incubator at 37 ℃ and incubating for 30 minutes at 500 r/min; discarding the solution, adding 300 mu l/hole of washing solution, soaking for 30-60 seconds, and discarding the solution; repeatedly washing for 5 times, and drying for the last time;

(1-5) developing, adding 100 mu l/hole of substrate solution, incubating at 37 ℃ for 15 minutes, discarding the solution, and patting dry;

(1-6) after the upper cover of the chip is removed, the dust-free paper is placed on the detection chip, the detection chip is lightly pressed, and the result is measured by a micropore disk chip imager within 10 minutes, so that an S/N value (sample S value/quality control point N value) is derived.

And (3) testing validity judgment: the S value of the quality control point is not less than 6000, the S value of the blank control point is not more than 3000, otherwise, the test is invalid, and the judgment is automatically completed by an internal data processing and analyzing system. The criteria for result determination (which is also done by the internal one-touch intelligent data processing and analyzing system, and does not require the technician to perform data calculation and statistical analysis) are as follows:

calculation of the S/N ratio: S/N = sample A value/negative quality control article A value (wherein the sample A value is a sample detection gray value; the negative quality control article A value is a detection gray value of a certain quality control point, and the quality control point is preferably selected according to the condition that the analysis value of the calibrated known negative and positive sample is in accordance with the standard value when the batch is debugged, and the preferred standard is a quality control article point corresponding to the condition that the gray value ratio of the calibrated known negative and positive sample to the three quality control article points 1, 2 and 3 is calculated, and the coincidence degree of the standard value and the judgment result is the highest).

When the S/N is judged to be less than or equal to 0.600 positively, the PRV gD and gE antibodies are positive;

when the S/N is more than 0.700, the PRV gD and gE antibodies are negative;

when S/N is more than 0.600 and less than or equal to 0.700, PRV gD and gE antibodies are suspicious, and re-sampling is needed for detection, and if the detection is still suspicious, the result is negative.

10 positive samples (the number is 41# to 50#, PRVgD antibodies and PRVgE antibodies are positive, wherein 41# to 45# CSFV antibodies are also positive), 10 negative samples (the number is 51# to 60#, PRVgD antibodies and PRVgE antibodies are negative) of clinical samples detected by a porcine pseudorabies virus neutralization test and a commercial porcine pseudorabies virus ELISA antibody kit (the detection result of the Biochek PRVgB kit and the IDEXX PRVgE kit is negative), a classical swine fever virus ELISA antibody kit (the detection result of the IDEXX CSFV antibody kit is positive).

Optionally, 3 batches (batch numbers marked as A, B and C) of nano films are respectively prepared into a kit 1, classification identification is carried out according to the method and the contact angle measurement method, three experimenters are randomly selected for carrying out experiments, one person simultaneously operates any one chip in 3 batches for carrying out batch-to-batch repeatability measurement in each experiment, any 3 chips in 1 batch are randomly selected for carrying out batch-to-batch repeatability measurement, and meanwhile, the kits prepared by the contact angle measurement method are compared. 20 clinical samples were tested according to the test procedure of kit 1 in example 4.2, and the final results were analyzed and compared according to the repeatability and the contact angle measurement, the results of the repeatability between batches in the two methods are shown in Table 7, especially the consistency of the test results of the kit prepared from the nano-membrane of the quality test method of the invention and the test results of the commercial kit is better than that of the contact angle measurement. Moreover, the kit 1 prepared by the nano-membrane for quality inspection of the method provided by the invention has the batch-to-batch and in-batch variation coefficients of PRVgD antibody and PRVgE antibody within 5%, and the kit 1 prepared by the nano-membrane for quality inspection of the contact angle measurement method has the batch-to-batch and in-batch variation coefficients of PRVgD antibody and PRVgE antibody within 8-15%, so that the quality inspection effect of the method provided by the invention is better than that of the contact angle measurement method.

TABLE 7 comparison of test results of kits prepared from nano-films for quality testing by different methods

4.3 application of triple detection chip kit for antibodies of gD, gE proteins and E2 proteins of porcine pseudorabies viruses

Selecting any 2 spots as quality inspection spots according to the spot-printing mode in the embodiment 2.1 and the figure 3, respectively performing quality inspection by the two methods, selecting a nano-film meeting the specification of the corresponding quality inspection standard, and then preparing a porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip in the porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein triple detection chip kit (kit 2 for short), wherein the porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein triple detection chip is diluted by a spot-printing liquid to spot the porcine pseudorabies virus gD protein at a PRVgD detection spot according to 4.0 ng/spot, the porcine pseudorabies virus gE protein is diluted by the spot-printing liquid to spot at a PRVgE detection spot according to 1.0 ng/spot, the porcine pseudorabies virus gE protein is spotted at a PRVgE detection spot according to 6.4 ng/spot, the porcine pseudorabies virus gE protein is spotted at a PRVgE detection spot according to 1.0 ng/spot, the anti-rabies virus gE 2 protein is cloned at a multi-spot-printing liquid for a control, and the anti-pestivirus monoclonal antibody is cloned and diluted by a sample-spotting liquid for a control sample-control point.

The detection method of the porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip kit (kit 2 for short) is carried out according to the detection method of the kit 1, and a determination method of the swine fever antibody is added on the basis of the determination method of the kit 1, wherein the determination method of the swine fever antibody is as follows:

PI = (1-sample A value/negative quality control A value) × 100%.

Judging that the antibody of CSFV is positive when PI is more than or equal to 40 percent;

judging the CSFV antibody to be suspicious when PI is more than 30% and less than 40%;

and judging that the antibody is CSFV antibody negative when the PI is less than or equal to 30 percent.

According to the embodiment 2.1 and the spotting mode shown in the attached figure 3, any 2 spots are selected as quality testing spots, the two methods are respectively used for quality testing, nano films meeting the corresponding quality testing standards are selected, 3 batches (batch numbers marked as A, B and C) of nano films are selected to be respectively prepared into a kit 2, classification identification is carried out according to the method for the invention and a contact angle measuring method, three experimenters are randomly selected for experiment, one person operates any chip in 3 batches simultaneously in each experiment to carry out batch-to-batch repeatability measurement, any 3 chips in 1 batch are randomly selected to carry out batch-to-batch repeatability measurement, and the kits prepared by the contact angle measuring method are compared. 20 clinical samples are detected according to the detection procedure of the reagent kit 2 in the example 4.3, the final results are analyzed and compared according to repeatability and comparison with a contact angle measurement method, the repeatability results between batches in the two methods are shown in tables 8 to 9, and particularly, the consistency of the detection result of the reagent kit prepared by the nano membrane for quality detection of the method of the invention and the detection result of a commercial reagent kit is superior to that of the contact angle measurement method. Moreover, the kit 2 prepared by the nano-membrane for quality inspection of the method of the invention has the batch-to-batch and in-batch variation coefficients of PRVgD antibody, PRVgE antibody and CSFV antibody within 5%, and the kit 2 prepared by the nano-membrane for quality inspection of the contact angle measurement method has the batch-to-batch and in-batch variation coefficients of PRVgD antibody, PRVgE antibody and CSFV antibody within 8-15%, so the quality inspection effect of the method of the invention is better than that of the contact angle measurement method.

TABLE 8 comparison of the test results of the kit prepared from the nano-film for quality testing by the method of the present invention

TABLE 9 comparison of detection results of kit prepared from nano-film for quality control by contact angle measurement method

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

According to the embodiment 2.1, selecting any 2 spots as quality detection spots according to the spot-casting mode in fig. 4, after the quality detection is qualified, preparing O-type and A-type foot-and-mouth disease virus antibody detection chips in an O-type and A-type foot-and-mouth disease virus antibody detection chip kit (kit 3 for short), wherein the O-type and A-type foot-and-mouth disease virus antibody detection chips are diluted by using a spot-casting liquid to spot O-type and A-type foot-and-mouth disease virus antigens at 16 ng/spot on FMDV O detection spots, diluted by using the spot-casting liquid to spot the A-type and A-type foot-and-mouth disease virus antigens at 32 ng/spot on FMDV detection spots, diluted by using the spot-casting liquid to spot the goat anti-mouse polyclonal antibody at 2 ng/spot on quality control spots 1, diluted by using the spot-casting liquid to spot the goat anti-mouse polyclonal antibody at 4 ng/spot on quality control spots 2, diluted by using the spot casting liquid to spot the goat anti-mouse polyclonal antibody at 6 ng/spot on quality control spots 3, and spot on blank control spots by using the spot casting liquid.

The detection method of the O-type and A-type foot-and-mouth disease virus antibody detection chip kit (kit 3 for short) is as follows:

(3-1) pre-diluting the sample, and diluting the serum sample by 10 times with a sample diluent;

(3-2) numbering, namely numbering the chip holes according to the sequence of the samples;

(3-3) soaking, adding 300 mul/hole of washing solution, and after 3 minutes of soaking, discarding the solution and patting dry;

(3-4) adding samples, adding 50-100 mul/hole of the diluted sample, and placing the sample in a constant temperature oscillator at 37 ℃ and incubating for 30 minutes at 500 r/min;

(3-5) adding an enzyme labeling reagent, adding 50-100 mu l/hole of the enzyme labeling reagent, and placing the mixture in a constant temperature oscillator at 37 ℃ and incubating the mixture for 30 minutes at 500 r/min; discarding the liquid, adding 300 mul/hole of washing liquid, soaking for 10-20 seconds, discarding the liquid, repeatedly washing for 5 times, and finally drying by beating for the last time;

(3-6) developing, adding 100 mu l/hole of substrate solution, and incubating for 15 minutes at 37 ℃;

and (3-7) measuring, vertically discarding liquid, throwing off, removing the upper cover of the chip, reversely buckling the chip on a piece of dust-free paper, slightly pressing, measuring the result by using a micropore disk chip imager within 10 minutes, and deriving the blocking rate (1-sample gray value/quality control point gray value).

And (3) testing validity judgment: the gray value of the quality control point is more than or equal to 10000, the gray value of the blank control point is less than or equal to 3000, otherwise, the test is invalid, and the judgment is automatically completed through an internal data processing and analyzing system. The criteria for result determination (which is also done by the internal one-touch intelligent data processing and analyzing system, and does not require the technician to perform data calculation and statistical analysis) are as follows:

calculation of blocking rate: blocking ratio = (1-sample gray value/quality control point gray value) × 100%

And (3) positive judgment: when the blocking rate is more than or equal to 50 percent, the FMDV O and A antibodies are positive;

and (4) negative judgment: FMDV O and A antibodies are negative when the blocking rate is less than 50 percent.

10 parts of positive samples (number: 61# -70 #, FMDV O type and A type antibodies are positive) and 10 parts of negative samples (number: 71# -80 #, FMDV O type and A type antibodies are negative) detected by commercial foot and mouth disease virus ELISA antibody kits (including Lanshou foot and mouth disease virus O type antibody liquid blocking ELISA detection kit and Lanshou foot and mouth disease virus A type antibody liquid blocking ELISA detection kit) are collected.

Selecting 3 random spots as quality inspection spots according to the spotting mode in the embodiment 2.1 and the figure 4, respectively performing quality inspection by the two methods, selecting the nano-film meeting the corresponding quality inspection standard, optionally selecting 3 batches (batch numbers marked as A, B and C) of nano-films to respectively prepare a kit 3, performing classification identification according to the method of the invention and the contact angle measurement method, randomly extracting three experimenters for experiment, simultaneously operating any chip in 3 batches by one person for each experiment to perform batch-to-batch repeatability measurement, randomly selecting any 3 chips in 1 batch for batch-to-batch repeatability measurement, and simultaneously comparing the kits prepared by the contact angle measurement method. 20 clinical samples were tested according to the test procedure of kit 3 in example 4.4, and the final results were compared and analyzed according to the repeatability and the contact angle measurement, the results of the repeatability between the two methods are shown in Table 10, especially the test results of the kit prepared from the nanomembrane of the quality test of the method of the present invention are better consistent with the test results of the commercialized foot-and-mouth disease virus ELISA antibody kit than those of the contact comparison method. The kit 3 prepared by the nano-film for quality inspection of the method detects FMDV O-type antibodies and A-type antibodies with the inter-batch and intra-batch variation coefficients within 5 percent, and the kit 3 prepared by the nano-film for quality inspection of the contact angle measurement method detects FMDV O-type antibodies and A-type antibodies with the inter-batch and intra-batch variation coefficients within 7 to 15 percent, so the quality inspection effect of the method of the invention is better than the quality inspection effect of the contact angle measurement method.

TABLE 10 comparison of test results of kits prepared from nano-film for quality testing by different methods

In conclusion, the quality inspection method can reproduce the sample application process in the quality inspection process, avoid the system error brought in the sample application process and really embody the stability and the uniformity of the preparation process of the nano-film. The method for measuring the diameters of the sample points reflects the surface uniformity of the nano film to the greatest extent, and the nano film prepared by the quality inspection method shows excellent experimental results in the subsequent chip preparation process. The quality inspection method is characterized in that the sample application solution fixed on the surface of the nano-film can be thoroughly cleaned in the process of preparing the chip through the nano-film, no residue is left, and no influence is caused on the surface of the nano-film.

The high-flux microarray chip project is characterized in that targets such as protein and the like are fixed on the surface of a nano-film through spotting, finally, blue precipitates are generated on corresponding sample spots through an enzyme-linked immunosorbent assay and a color reaction, and the final result is judged by reading a gray value. Therefore, the size uniformity of the sample spot becomes a quality key of the microarray chip, particularly, the microarray chip for quantitative determination.

The nano film is used as a main substrate of a micro array chip and a carrier of targets such as protein, and the surface uniformity of the nano film plays a decisive role in the quality of the chip. Due to the characteristic that the main component is silica gel, the quality inspection method of the nano film per se becomes a difficult point of quality control. At present, no instrument quality inspection method which is suitable, effective and meets the requirement of industrial production can be used for quality inspection of the nano-film, so that the invention adds the whole spotting process into the quality inspection process of the nano-film from spotting, comprehensively controls the uniformity of the surface of the nano-film and provides powerful guarantee for the quality control of the microarray chip.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A quality inspection method for the mass production of microarray chips comprises the following steps: step 1) placing the nano film in a clean environment, wherein the humidity is less than 20%, and the temperature is 23-25 ℃ for a period of time to fully balance the nano film;

step 3) placing the nano-film fully balanced in the step 1) into a sample application instrument, and applying the sample application liquid in the step 2) to the quality inspection points of the nano-film one by one under the conditions of humidity of 45-55% and temperature of 23-25 ℃, wherein the quality inspection points of the nano-film are non-target positions, non-quality control points and non-blank control positions of the nano-film, and the number of the quality inspection points in each hole is not less than 2 and is 20 nl/point;

step 4) loading the nano film subjected to the spotting in the step 3) into a matched fixture, placing the fixture under a lens of an industrial camera, adjusting the focal length of the lens until sample points are clearly presented and the edges are clear, taking pictures by holes by the industrial camera, and measuring and leading out the diameters of all quality inspection points by software;

and 5) analyzing the diameter data of each quality inspection point on the nano film, and calculating the total standard deviation, median and range difference of the diameters of the quality inspection points on the nano film, wherein the total standard deviation is less than or equal to 5%, the median is 460-480 mu m, and the range difference is less than or equal to 30 mu m, and the nano film is qualified in quality inspection.

2. The method for quality inspection in mass production of microarray chips according to claim 1, wherein the time for fully equilibrating the nanomembrane in step 1) is 20 to 28 hours, preferably 24 hours.

3. The method for quality control in mass production of microarray chips of claim 1, wherein the spotting solution of step 2) is a 5% M/V glycerol solution, a 5% M/V sorbitol solution, a 0.05% V/V triton solution, a dimethyl sulfoxide (DMSO) solution, and a PBS (pH 6.8) solution, which are mixed in the order of 10: 15: 0.1: 50: 100 by volume.

4. The quality inspection method for the large-scale production of the microarray chips according to claim 1, wherein the matched clamp in the step 4) comprises a cover plate and a bottom plate, the cover plate is provided with a plurality of detection windows, the bottom plate is provided with hollowed-out holes at positions corresponding to the detection windows, and the cover plate can be fixed on the bottom plate in a pressing mode.

5. A preparation method for the mass production of microarray chips comprises the following steps: placing the nano film qualified in quality inspection according to claims 1-4 in a container, adding 15ml of activating solution for surface activation, washing the activating solution with purified water after 0.5 hour, and drying the surface of the nano film with clean air;

step (2) placing the activated nano-film in the step (1) into a sample application instrument, diluting a target with a sample application solution according to a set program to serve as a target point sample application solution, diluting a goat anti-mouse polyclonal antibody or a goat anti-mouse secondary antibody with the sample application solution to serve as a quality control point sample application solution and a blank control point sample application solution, and respectively applying the sample application volumes to the nano-film, wherein the sample application volumes are 20nl;

and (3) loading the nano-film subjected to the spotting in the step (2) into the matched clamp, drying for 6 hours under the conditions that the temperature is 21-25 ℃ and the humidity is less than 25%, and sealing by using a film sealing liquid to obtain the microarray chip.

6. The method of mass-producing microarray chips according to claim 5, wherein the activating solution in the step (1) is a solution containing 5% of M/V1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, 2.5% of M/V N-hydroxysuccinimide NHS.

7. The method of claim 5, wherein the membrane blocking solution in step (3) is a PBS solution containing 1% W/V BSA, 0.1-0.5% V/V glycerol, 0.1-V Tween-20.

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

9. The method for preparing microarray chips of claim 5, wherein the targets of step (2) comprise porcine pseudorabies virus gD or gE protein, classical swine fever virus E2 protein, swine foot and mouth disease virus antigen, porcine rotavirus antigen, mycotoxin antigen, and avian antibiotic antigen.

10. The preparation method for the large-scale production of the microarray chip according to claim 5, wherein when the microarray chip is a porcine pseudorabies virus gD and gE protein antibody duplex detection chip, the targets in the step (2) are porcine pseudorabies virus gD protein and gE protein;

when the microarray chip is a porcine pseudorabies virus gD, gE protein and classical swine fever virus E2 protein antibody triple detection chip, the target in the step (2) is the porcine pseudorabies virus gD protein, gE protein and classical swine fever virus E2 protein;

when the microarray chip is an O-type and A-type foot-and-mouth disease virus antibody detection chip, the targets in the step (2) are O-type foot-and-mouth disease virus antigens and A-type foot-and-mouth disease virus antigens; when the microarray chip is a mycotoxin quadruple detection chip, the targets in the step (2) are fumonisin antigen, aflatoxin B1 antigen, vomitoxin antigen and zearalenone antigen.