Method for making ELISA plate for paper-based ELISA

Abstract

The present invention provides a method for making an ELISA plate for paper-based enzyme-linked immunosorbent assay, comprising the following steps: preparing a paper microplate, the surface of which forms a plurality of hydrophobic regions, each of which surrounds a hydrophilic region; immersing the prepared paper microplate in a sodium periodate solution, and dropping a chitosan solution to each hydrophilic region on the paper microplate after taking it out, to form a sodium periodate-chitosan modified layer; then dropping a coated antibody solution to each hydrophilic region, and then dropping a bovine serum albumin solution to each hydrophilic region to form a closed layer, to obtain an ELISA plate. The method for making the ELISA plate enables the coated antibody solution to be connected to the sodium periodate-chitosan modified layer by chemical crosslinking, forming a coated antibody layer fixed on the surface of the hydrophilic region of the paper microplate, enhancing the stability and effectiveness of the fixed coated antibody layer, improving the sensitivity of the p-ELISA method and having good repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme-linked immunosorbent assay, and in particular to a method for preparing an ELISA plate for paper-based enzyme-linked immunosorbent assay. Background Art

[0002] The initial step of the enzyme-linked immunosorbent assay (ELISA) is to attach the coated antibody to the surface of the reaction wells of the microplate, which is called coating. The coating efficiency, that is, the density of the coated antibody finally bound to the surface of the microplate and its stability, determines the number of antigens that it subsequently binds to, that is, determines the sensitivity of the ELISA. In recent years, with the many advantages of paper microfluidics technology, in 2010, the Whitesides team first proposed the use of paper as a solid phase carrier in ELISA, and evaluated the three main advantages of paper ELISA (abbreviated as p-ELISA): small sample volume, low cost, and rapid detection, but the sensitivity of the p-ELISA method is not as good as the traditional ELISA method, which limits the scope of application of the p-ELISA method. Summary of the invention

[0003] The invention provides a method for preparing an enzyme label plate for paper-based enzyme-linked immunosorbent assay, so as to solve the technical problem that the sensitivity of the p-ELISA method in the prior art is lower than that of the traditional ELISA method.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] The invention provides a method for preparing an ELISA plate for paper-based ELISA, comprising the following steps: S1, preparing a paper microporous plate, wherein a plurality of hydrophobic areas are formed on the surface of the paper microporous plate, and each of the hydrophobic areas surrounds a hydrophilic area; S2, immersing the paper microporous plate prepared in the step S1 into a sodium periodate solution, taking it out, and then dripping a chitosan solution to each of the hydrophilic areas on the paper microporous plate to form a sodium periodate-chitosan modification layer; then dripping a coating antibody solution to each of the hydrophilic areas, and then dripping a bovine serum albumin solution to each of the hydrophilic areas to form a sealing layer, so as to obtain the ELISA plate for paper-based ELISA.

[0006] Furthermore, the step S2 further comprises: before adding the coating antibody solution to each of the hydrophilic regions, first adding a reducing agent to each of the hydrophilic regions to reduce the carbon-nitrogen double bonds in the sodium periodate-chitosan modification layer.

[0007] Furthermore, the step S2 further comprises: taking out the paper microplate from the sodium periodate solution, soaking it in a polyol solution for sufficient reaction, washing it with water and then dropping the chitosan solution.

[0008] Furthermore, in step S1, the paper microporous plate is prepared by a stamp method, and the preparation method includes the following steps: a stamp is stained with a hydrophobic agent and then pressed on the surface of a filter paper to form the hydrophobic area; after the hydrophobic agent penetrates the filter paper, it is dried and solidified to obtain the paper microporous plate.

[0009] Furthermore, the hydrophobic agent includes one or more of polydimethylsiloxane, cyclomethylsiloxane, aminosiloxane, polymethylphenylsiloxane, and polyether polysiloxane copolymer.

[0010] Furthermore, a plurality of annular protrusions are formed on the stamp, and the annular protrusions are stained with the hydrophobic agent and then pressed against the surface of the filter paper.

[0011] Furthermore, the steps of the stamp method also include: applying the hydrophobic agent on the paper surface, and allowing the annular protrusions to be stained with the hydrophobic agent on the paper surface; wherein the application thickness of the hydrophobic agent is 0.2mm to 0.6mm, and the height of the annular protrusions is 0.8mm to 1.4mm.

[0012] Furthermore, in the drying and curing step, the drying temperature is 50° C. to 85° C., and the drying time is 20 min to 40 min.

[0013] The invention provides a method for preparing an ELISA plate for paper-based ELISA. The paper microplate is prepared by a stamp method. The surface of the prepared paper microplate includes a plurality of hydrophobic areas and a hydrophilic area surrounded by the hydrophobic areas. The size of the hydrophilic area is uniform and there is no leakage, so the uniformity and airtightness of the hydrophilic area can be well maintained. A sodium periodate-chitosan modification layer is formed in each hydrophilic area of ​​the paper microplate. Then, a coating antibody solution is dripped on the sodium periodate-chitosan modification layer formed in the hydrophilic area, and a bovine serum albumin solution is dripped to form a sealing layer to prepare the ELISA plate for paper-based ELISA. The coating antibody solution is connected to the sodium periodate-chitosan modification layer by chemical cross-linking to form a coating antibody layer fixed on the surface of the hydrophilic area of ​​the paper microplate, thereby enhancing the stability and effectiveness of the fixation of the coating antibody layer, improving the sensitivity of the p-ELISA method, and achieving good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A schematic diagram of the structure of an ELISA plate produced by the method for producing an ELISA plate for paper-based ELISA provided in an embodiment of the present invention;

[0015] Figure 2 The scanning electron micrographs are of an unmodified paper microplate (Fig. a) and a paper microplate modified with sodium periodate-chitosan (Fig. b);

[0016] Figure 3is the infrared spectrum of the paper microplate, line a is the unmodified paper microplate, line b is the paper microplate oxidized by sodium periodate, and line c is the paper microplate modified by sodium periodate-chitosan;

[0017] Figure 4 Scanning electron micrographs of an unmodified paper microplate (Fig. a) and a paper microplate made by the stamp method (Fig. b);

[0018] Figure 5 It is the standard curve of p-ELISA detection based on the chemical cross-linking and fixation of coated antibodies by sodium periodate-chitosan modification layer;

[0019] Figure 6 A reaction and detection principle diagram of a method for making an ELISA plate for paper-based ELISA provided in an embodiment of the present invention.

[0020] Description of reference numerals:

[0021] 1. Paper microporous plate; 2. Hydrophilic area. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0023] The present application embodiment provides a method for making an ELISA plate for paper-based ELISA, comprising the following steps: S1, preparing a paper microplate 1, wherein a plurality of hydrophobic areas are formed on the surface of the paper microplate 1, and each hydrophobic area surrounds a hydrophilic area 2; S2, immersing the paper microplate 1 prepared in step S1 into a sodium periodate solution, and after taking it out, adding a chitosan solution to each hydrophilic area 2 on the paper microplate 1 to form a sodium periodate-chitosan modification layer; then adding a coating antibody solution to each hydrophilic area 2, and then adding a bovine serum albumin solution to each hydrophilic area 2 to form a sealing layer, thereby obtaining an ELISA plate for paper-based ELISA. The ELISA plate made in the present application embodiment refers to Figure 1 As shown, the ELISA plate includes a paper microplate 1 provided with a plurality of hydrophobic regions and a coating layer attached to the surface of each hydrophilic region 2 .

[0024] In the conventional p-ELISA method, the coated antibody is mainly adsorbed on the surface of the paper microplate 1 by electrostatic adsorption. The coated antibody that is not firmly fixed is easily eluted during the washing process, which easily leads to random fixation of biological molecules, affecting the stability and effectiveness of the coated antibody and reducing the sensitivity of the p-ELISA method. The coated antibody contains a large number of amino and carboxyl groups. In our research, we found that it can be chemically coupled with the aldehyde, carboxyl, amino and other groups on the surface of the solid phase carrier, i.e., the paper microplate 1, by chemical cross-linking to enhance the immunoassay performance of the p-ELISA method.

[0025] It is understandable that there are various ways to fix biomolecules on the paper microporous plate 1, and different fixation methods have different effects on the activity of biomolecules. Chitosan is the product of chitin deacetylation, and contains a large number of amino groups. On the one hand, it can be directly fixed on the surface of the paper microporous plate 1 by electrostatic adsorption, but this fixation method is unstable and easily eluted; on the other hand, it can also be combined with the aldehyde group or carboxyl group on the surface of the paper microporous plate 1 after oxidation by an oxidant, and chitosan is firmly bound to the surface of the paper microporous plate 1. This method does not require any chemical cross-linking agent and can well maintain the physical and chemical properties of the filter paper. As a filter paper oxidant, sodium periodate, its concentration, pH value of the oxidation solution, oxidation time and temperature will affect the physical properties and aldehyde group content of the filter paper, so the oxidation process must be well controlled.

[0026] In the embodiment of the present application, sodium periodate and chitosan are successively added dropwise to the hydrophilic region 2 of the paper microplate 1, and characterized by infrared spectroscopy. The results show that with the weakening of the characteristic peak of the hydroxyl group and the appearance of the aldehyde peak, the sodium periodate oxidant successfully oxidizes the surface of the paper microplate 1; at the same time, the disappearance of the characteristic peak of the aldehyde group and the appearance of the characteristic peak of the C=N double bond in the Schiff base indicate that chitosan can be stably covalently bound to the surface of the paper microplate 1 modified with sodium periodate, providing a good basis for the stable chemical cross-linking of the coated antibody.

[0027] In the embodiment of the present application, the paper microporous plate 1 is provided with a plurality of hydrophobic areas, each of which surrounds a hydrophilic area 2 to form a microporous structure similar to a microporous plate. The hydrophobic area is a closed structure, and the shape of the hydrophobic area is not limited in the embodiment of the present application. The paper microporous plate 1 is immersed in a sodium periodate solution, and a chitosan solution is added to each hydrophilic area 2 thereof, so that a sodium periodate-chitosan modification layer can be formed on the surface of each hydrophilic area 2; when a coated antibody solution is subsequently added to each hydrophilic area 2, the coated antibody can be fixed to the surface of the hydrophilic area 2 of the paper microporous plate 1 by chemical cross-linking, thereby enhancing the stability and effectiveness of the fixation of the coated antibody, improving the sensitivity of the p-ELISA method and having good repeatability. In other words, the sodium periodate-chitosan modification layer formed on the surface of the hydrophilic area 2 contains an amino group, and the carboxyl group at the C-terminus of the coated antibody is chemically cross-linked with the amino group of the modification layer, so that the N-terminal variable region of the coated antibody is fully exposed, which is beneficial to improve the affinity of the binding between the antigen and the antibody.

[0028] See also Figure 2 In the scanning electron microscope image, Figure a is a paper microporous plate 1 without sodium periodate-chitosan modification, and Figure b is a paper microporous plate 1 modified with sodium periodate-chitosan in the embodiment of the present application, whose surface morphology has not changed and still maintains the loose three-dimensional porous structure of the filter paper. Figure 2It can be seen from the scanning electron microscope observation that the small molecule chitosan did not change the original structure of the filter paper, and the specific surface area did not change, which ensured the effective adsorption amount of the coated antibody and could present a rapid and sensitive immune response. At the same time, the porous structure of the hydrophobic area surrounding the hydrophilic area 2 was conducive to washing the substances not bound to the filter paper, reducing the background effect of the detection, and maintaining the good analytical performance of the paper microplate 1.

[0029] See also Figure 3 Infrared spectrum of the paper microplate 1 modified with sodium periodate-chitosan, wherein line a is the infrared spectrum of the paper microplate 1 not modified with sodium periodate and chitosan; line b is the infrared spectrum of the paper microplate 1 after sodium periodate modification; line c is the infrared spectrum of the paper microplate 1 after sodium periodate-chitosan modification.

[0030] As can be seen from the figure, paper microplate 1 contains multiple hydroxyl groups, and only the characteristic spectral peak of hydroxyl groups (3300cm -1 ); When sodium periodate was attached to the microporous surface, 1730 cm -1 The characteristic peak of aldehyde group appears (line b), and the characteristic spectral peak of hydroxyl group (3300cm -1 ) weakened; after chitosan modified paper microplate 1, the characteristic peak of the c-line aldehyde group disappeared, and the peak at 1642cm -1 The C=N double bond characteristic peak appeared at , indicating that chitosan has been successfully bound to the microporous surface. The results of the infrared spectrum show that with the weakening of the hydroxyl characteristic peak and the appearance of the aldehyde peak, the sodium periodate oxidant successfully oxidized the paper microporous plate 1; the disappearance of the aldehyde characteristic peak and the appearance of the C=N double bond characteristic peak indicate that chitosan can be stably covalently bound to the surface of the hydrophilic region 2 modified by sodium periodate, providing a good basis for the stable chemical cross-linking of the coated antibody. Figure 6 shown.

[0031] In the embodiment of the present application, the shape of the hydrophobic area in the paper microplate 1 can be annular. It can be understood that if the hydrophobic area is set to be annular, the hydrophilic area 2 is circular, which is the same as the shape of the droplet due to the surface tension of the solution, so that it is convenient to drop liquids such as chitosan in the hydrophilic area 2.

[0032] In the embodiment of the present application, the sodium periodate solution is formed by dissolving sodium periodate in an ammonium acetate buffer solution; after the paper microplate 1 is taken out of the sodium periodate solution, it is washed with water and blotted dry. In addition, when preparing the coated antibody solution, EDC (i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and Sulfo-NHS (i.e., N-hydroxysulfosuccinimide) are dissolved in MES buffer (i.e., 2-(N-morpholinoline)ethanesulfonic acid), and the dissolved EDC solution and the dissolved Sulfo-NHS solution are mixed to form an EDC / Sulfo-NHS mixed solution, and the coated antibody solution is mixed with the EDC / Sulfo-NHS mixed solution, and then added dropwise to each hydrophilic area 2. The EDC / Sulfo-NHS solution is one of the non-toxic and biocompatible cross-linking agents, which maintains the biological activity of the protein molecules and can stably fix the protein molecules on the surface of the paper microplate 1, while not affecting the binding ability of the antigen and the coated antibody.

[0033] In some embodiments, step S2 further comprises: before adding the coating antibody solution to each hydrophilic region 2, first adding a reducing agent to each hydrophilic region 2 to reduce the carbon-nitrogen double bonds in the sodium periodate-chitosan modification layer. Figure 3 It can be seen from the infrared spectrum that in the coating layer formed on the surface of the paper microplate 1, the characteristic peak of the C=N double bond of the sodium periodate-chitosan modified layer appears, and the C=N double bond is an unstable structure. In the embodiment of the present application, before adding the coating antibody solution, a reducing agent is first added to each hydrophilic area 2, so that the C=N double bond reacts to a stable state of CH2-NH2, ensuring the subsequent formation of the coating antibody layer and the binding ability of the antigen and the coating antibody. In the embodiment of the present application, the reducing agent is a mild reducing agent such as sodium cyanoborohydride.

[0034] In some embodiments, step S2 further includes: taking out the paper microplate 1 from the sodium periodate solution, soaking it in a polyol solution for sufficient reaction, washing it with water, and then adding chitosan solution. It can be understood that after taking out the paper microplate 1 from the sodium periodate solution, there is residual sodium periodate solution on the surface of the paper microplate 1; therefore, the paper microplate 1 is immersed in a polyol solution to allow the polyol to fully react with the sodium periodate solution remaining on the surface of the paper microplate 1, thereby avoiding excessive sodium periodate solution remaining on the surface of the paper microplate 1. In the embodiment of the present application, ethylene glycol is selected as the polyol solution.

[0035] In some embodiments, the paper microporous plate 1 is prepared by a stamp method in step S1, and the preparation method comprises the following steps: a stamp is stained with a hydrophobic agent and then pressed on the surface of a filter paper to form a hydrophobic area, and after the hydrophobic agent penetrates the filter paper, it is dried and solidified to obtain the paper microporous plate 1. In the embodiment of the present application, the paper microporous plate 1 prepared by the stamp method has a good uniformity of size of the hydrophilic area 2 and no leakage, and can well maintain the uniformity and airtightness of the hydrophilic area 2. Figure 4 Scanning electron microscopy images of the unmodified filter paper and the paper microplate 1 made by the stamp method show that the surface of the unmodified filter paper is rough and intertwined to form a porous structure ( Figure 4 a); The porous structure of the filter paper treated by the stamping method disappears and the smoothness is significantly improved ( Figure 4 b), can form a good hydrophobic zone edge.

[0036] In the embodiment of the present application, the filter paper is composed of pure cellulose with a porous structure. Pure cellulose is a linear polymer chain composed of D-pyranose glucopyranose, which is hydrophilic and biocompatible. At the same time, the surface has several hydroxyl groups, so that its surface chemical and optical properties also have unique advantages for the preparation of p-ELISA devices. On the one hand, it maintains the specific surface area for protein adsorption, and on the other hand, due to capillary force, liquid is easy to move on its surface. The porous structure of cellulose treated by the stamp method is completely closed, and the samples and reagents are all confined in the hydrophilic area 2, which can ensure the accuracy of the experiment in the hydrophilic area 2, and is a better method for making paper microplates 1.

[0037] Furthermore, the hydrophobic agent includes one or more of polydimethylsiloxane, cyclomethicone, aminosiloxane, polymethylphenylsiloxane, and polyether polysiloxane copolymer. The above hydrophobic agents can be used in the process of making the paper microplate 1 by the stamp method of the embodiment of the present application, and polydimethylsiloxane is preferred.

[0038] In some embodiments, a plurality of annular protrusions are formed on the seal, and the annular protrusions are stained with a hydrophobic agent and pressed on the surface of the filter paper. It can be understood that when a plurality of annular protrusions are formed on the seal, when the seal is stained with a hydrophobic agent and pressed on the surface of the filter paper, the hydrophobic area formed is annular, and the hydrophilic area 2 is circular, that is, the hydrophilic area 2 and the hydrophobic area form a microporous structure similar to a microporous plate. Specifically, the seal is made of rubber, and the annular protrusions on the seal are formed by laser cutting technology. The annular protrusions form holes on the seal, and the diameter of the holes is about 6 mm, the depth is 1 mm, and the hole spacing is 2 mm.

[0039] Furthermore, the steps of the stamp method also include: applying a hydrophobic agent to the surface of the paper, and contaminating the annular protrusion with the hydrophobic agent on the surface of the paper; wherein the thickness of the hydrophobic agent applied is 0.2mm-0.6mm, and the height of the annular protrusion is 0.8mm-1.4mm. Understandably, it is necessary to apply a hydrophobic agent to a certain thickness on the surface of the paper to meet the contamination requirements of the seal; in addition, the height of the annular protrusion is greater than the thickness of the hydrophobic agent applied on the paper, so that the hydrophobic agent will not enter the hole formed by the annular protrusion on the seal, and the hydrophobic agent entering the hole when the seal is pressed on the filter paper will not flow into the hydrophilic area 2. The paper here can be ordinary paper. If the thickness of the hydrophobic agent applied is too thick, the seal will be contaminated with too much hydrophobic agent, and after being pressed on the surface of the filter paper, the hydrophilic area 2 will be polluted, and it will be difficult to dry and solidify, which will increase the cost; if the thickness of the hydrophobic agent applied is too thin, the hydrophobic agent pressed on the filter paper by the seal will be uneven, affecting the uniformity and airtightness of the paper microporous plate 1.

[0040] Furthermore, in the drying and curing step, the drying temperature is 50° C. to 85° C. and the drying time is 20 min to 40 min. The above drying temperature and drying time can ensure that the hydrophobic agent is completely dried for subsequent use.

[0041] The reagents used in the following examples are all commercially available.

[0042] Example 1

[0043] A method for preparing an ELISA plate for paper-based ELISA comprises the following steps:

[0044] 1. Prepare paper microplate 1 by stamp method.

[0045] A microporous model with a hole diameter of 7mm, a depth of 1mm and a hole spacing of 2mm is formed on the rubber surface by laser cutting technology to make a stamp. Then, the PDMS hydrophobic agent is placed on a magnetic stirrer and slowly stirred evenly without generating bubbles. Among them, the PDMS hydrophobic agent is a mixture of Sylgard184 polymer and a fixing agent. PDMS is the abbreviation of polydimethylsiloxane. After the hydrophobic agent is stirred evenly, it is evenly applied to the surface of the paper, and the thickness of the application is about 0.5mm.

[0046] The rubber stamp with the microporous model is evenly stained with the hydrophobic agent on the paper surface, and then pressed against the surface of the filter paper to form multiple hydrophobic areas on the filter paper surface. The hydrophobic agent in the hydrophobic area is allowed to completely penetrate the filter paper (about 20 seconds), and the filter paper is placed in a constant temperature box at 75°C for 30 minutes of curing. It is then taken out and cooled to form a paper microporous plate 1 with a diameter of 6 mm in the hydrophilic area 2 and a spacing of 2 mm between the hydrophilic areas 2.

[0047] 2. A sodium periodate-chitosan modification layer is formed on the surface of the hydrophilic region 2 of the paper microplate 1.

[0048] Dissolve sodium periodate in ammonium acetate buffer, immerse the paper microplate 1 prepared in step 1 completely in the sodium periodate solution, shake in the dark at room temperature for 20 minutes, and wash it thoroughly with ultrapure water and dry it after it is evenly mixed. Then immerse the paper microplate 1 in ethylene glycol solution for full reaction, wash it thoroughly with ultrapure water after 20 minutes and dry it for standby use. Add 5 μL of chitosan solution to each hydrophilic area 2 of the paper microplate 1, the concentration of chitosan is 0.25 mg / mL, react at room temperature until dry, then add 4 μL of sodium cyanoborohydride reducing agent to each hydrophilic area 2, react at 37°C for 20 minutes, wash with water 6 times, absorb excess residual liquid with absorbent paper, and standby use.

[0049] 3. Prepare the coating antibody layer.

[0050] Dissolve EDC and Sulfo-NHS in MES buffer (0.1 mol / L, pH 4.7) respectively, then mix the dissolved EDC solution with the Sulfo-NHS solution at a molar ratio of 4:5 to obtain a mixed solution; mix the coating antibody solution with the EDC / Sulfo-NHS mixed solution, then drop it into each hydrophilic area 2, activate it at 37°C for 15 minutes, and then drop bovine serum albumin solution to form a blocking layer. The concentration of the coating antibody solution is 0.2 mg / mL.

[0051] The p-ELISA detection performance of the antibody fixed by sodium periodate-chitosan chemical cross-linking was analyzed by using the ELISA plate prepared in Example 1. Taking the alpha-fetoprotein standard solution of different concentrations as an example, 4 μL of the alpha-fetoprotein standard solution and the enzyme-labeled antibody mixture was dripped into each hydrophilic area of ​​the experimental group, and the optimal dilution ratio of the enzyme-labeled antibody was 1:600. 4 μL of the enzyme-labeled antibody solution was directly added to each hydrophilic area of ​​the control group. After reacting at room temperature for 8 minutes, the plate was washed 6 times with a washing solution and the excess residual liquid was dried; then 3 μL of TMB color developing solution was dripped into each hydrophilic area of ​​the experimental group and the control group for color development, and the color was obtained by referring to the results of the experiment. Figure 6 The color development was captured by a scanner, and the image was analyzed by Image J software. The RGB images were analyzed by Image J software to obtain the Rn, Gn, and Bn values ​​of the experimental wells and the R0, G0, and B0 values ​​of the control wells. The formula Calculate the signal of p-ELISA detection and draw the standard curve of p-ELISA detection. The results are as follows: Figure 5 As shown in the figure, the detection range is 0.08ng / mL to 20ng / mL. In addition, the ΔRGB values ​​of 20 negative controls were measured and the average value was calculated. The detection limit was calculated by substituting the value and standard deviation (s) into the standard curve, and the minimum detection limit was 0.035 ng / mL.

[0052] The test experiment used three AFP concentrations of 20 ng / mL, 10 ng / mL and 1 ng / mL to compare the repeatability of the p-ELISA test method. The results are shown in Table 1. The intra-batch coefficients of variation of the p-ELISA detection method based on sodium periodate-chitosan and coated antibody cross-linking were 7.66%, 8.40% and 8.68%, and the inter-batch coefficients of variation were 7.78%, 9.56% and 10.49%.

[0053] Table 1 Intra-batch and inter-batch test results of p-ELISA test method

[0054]

[0055] The above p-ELISA method was compared with the traditional commercial 96-well plate traditional ELISA method for the same alpha-fetoprotein antigen. The results showed that the detection limit was significantly increased. Therefore, the sensitivity of the p-ELISA method has been effectively improved. At the same time, through the intra-batch and inter-batch coefficients of variation, it can be observed that the coefficient of variation of the same sample in the same batch test and different batches of tests is small, indicating that the p-ELISA method has good repeatability and stability.

[0056] Three known concentrations of alpha-fetoprotein standards of 1 ng / mL, 10 ng / mL and 20 ng / mL were selected and added to 2.01 ng / mL human serum samples (human serum samples were obtained from Zhuzhou Traditional Chinese Medicine Traumatology Hospital). The recovery rates of the p-ELISA method with sodium periodate-chitosan chemical cross-linking fixed coated antibodies were calculated to be 104.00%, 98.50% and 96.30%, respectively. Compared with the commercial 96-well plate traditional ELISA method using the same coated antibodies, there was no significant difference in the sample detection results, see Table 2. The p-ELISA method was tested for sample addition recovery and compared with the traditional ELISA method, indicating that the results of the detection method are reliable.

[0057] Table 2 Comparison of sample analysis between p-ELISA and traditional ELISA in commercial 96-well plates

[0058]

[0059] The ELISA plate of the embodiment of the present application is used for the method of paper-based enzyme-linked immunosorbent assay, which has good analytical performance for the analyte in a complex matrix environment; compared with traditional ELISA, its detection results are highly reliable and have good analytical performance, providing a low-cost, accurate, rapid and highly sensitive method for disease detection in resource-poor areas.

[0060] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be covered within the protection scope of the present invention. In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the ability of ordinary technicians in the field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing an ELISA plate for paper-based ELISA, characterized in that: The following steps are involved: S1. preparing a paper microporous plate, wherein a plurality of hydrophobic regions are formed on the surface of the paper microporous plate, each of the hydrophobic regions being surrounded by a hydrophilic region; S2, immersing the paper microplate prepared in step S1 into a sodium periodate solution, taking it out, and then dropping a chitosan solution into each of the hydrophilic areas on the paper microplate to form a sodium periodate-chitosan modification layer; then dropping a coating antibody solution into each of the hydrophilic areas, and then dropping a bovine serum albumin solution into each of the hydrophilic areas to form a sealing layer, so as to obtain the ELISA plate for paper-based ELISA; step S2 also includes: before dropping the coating antibody solution into each of the hydrophilic areas, first dropping a reducing agent into each of the hydrophilic areas to reduce the carbon-nitrogen double bonds in the sodium periodate-chitosan modification layer.

2. The method for preparing a paper-based ELISA plate according to claim 1, characterized in that: The step S2 further comprises: taking out the paper microporous plate from the sodium periodate solution, soaking it in a polyol solution for sufficient reaction, washing it with water and then dripping the chitosan solution.

3. The method for preparing an ELISA plate for paper-based ELISA according to claim 1 or 2, characterized in that: In step S1, the paper microplate is prepared by a stamp method, and the preparation method comprises the following steps: The seal is stained with a hydrophobic agent and then pressed on the surface of the filter paper to form the hydrophobic area. After the hydrophobic agent penetrates the filter paper, it is dried and solidified to obtain the paper microporous plate.

4. The method for preparing an ELISA plate for paper-based ELISA according to claim 3, characterized in that: The hydrophobic agent includes one or more of polydimethylsiloxane, cyclomethylsiloxane, aminosiloxane, polymethylphenylsiloxane, and polyether polysiloxane copolymer.

5. The method for preparing an ELISA plate for paper-based ELISA according to claim 3, characterized in that: A plurality of annular protrusions are formed on the stamp, and the annular protrusions are stained with the hydrophobic agent and then pressed on the surface of the filter paper.

6. The method for preparing an ELISA plate for paper-based ELISA according to claim 5, characterized in that: The steps of the stamp method also include: applying the hydrophobic agent on the paper surface, and making the annular protrusions on the paper surface contaminate the hydrophobic agent; wherein the application thickness of the hydrophobic agent is 0.2mm-0.6mm, and the height of the annular protrusions is 0.8mm-1.4mm.

7. The method for preparing an ELISA plate for paper-based ELISA according to claim 3, characterized in that: In the drying and curing step, the drying temperature is 50° C. to 85° C., and the drying time is 20 min to 40 min.

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