CN113588936A - Method for manufacturing enzyme label plate for paper enzyme-linked immunosorbent assay - Google Patents
Method for manufacturing enzyme label plate for paper enzyme-linked immunosorbent assay Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention provides a method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay, which comprises the following steps: preparing a paper microporous plate, wherein a plurality of hydrophobic areas are formed on the surface of the paper microporous plate, and each hydrophobic area surrounds a hydrophilic area; immersing the prepared paper microporous plate into a sodium periodate solution, taking out the paper microporous plate, and dripping a chitosan solution into each hydrophilic area on the paper microporous plate to form a sodium periodate-chitosan modified layer; and then, dropwise adding a coating antibody solution to each hydrophilic region, and dropwise adding a bovine serum albumin solution to each hydrophilic region to form a sealing layer to obtain the ELISA plate. The enzyme label plate manufacturing method enables the coated antibody solution to be connected with the sodium periodate-chitosan modified layer in a chemical crosslinking mode to form a coated antibody layer fixed on the surface of the hydrophilic area of the paper microporous plate, so that the stability and effectiveness of the fixation of the coated antibody layer are enhanced, the sensitivity of a p-ELISA method is improved, and the repeatability is good.
Description
Technical Field
The invention relates to the technical field of enzyme-linked immunosorbent assay, in particular to a method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay.
Background
The enzyme-linked immunosorbent assay (abbreviated as ELISA) begins by attaching a coating antibody to the surface of a reaction well of a microplate, called coating, and the efficiency of coating, i.e., the density and stability of the coating antibody finally bound to the surface of the microplate, determines the amount of antigen bound thereto, i.e., the sensitivity of the enzyme-linked immunosorbent assay. In recent years, with the advantages of paper microfluidic technology, the paper used as a solid phase carrier for enzyme-linked immunosorbent assay was first proposed by the Whitesides group in 2010, and three main advantages of paper ELISA (abbreviated as p-ELISA) were evaluated: the sample amount is small, the cost is low, the detection is rapid, but the sensitivity of the p-ELISA method is not as good as that of the traditional ELISA method, and the application range of the p-ELISA method is limited.
Disclosure of Invention
The invention provides a method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay, which aims to solve the technical problem that the sensitivity of a p-ELISA method in the prior art is lower than that of the traditional ELISA method.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the invention provides a method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay, which comprises 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 hydrophobic area surrounds a hydrophilic area; s2, immersing the paper microporous plate prepared in the step S1 into a sodium periodate solution, taking out the paper microporous plate, and dripping a chitosan solution into each hydrophilic area on the paper microporous plate to form a sodium periodate-chitosan modification layer; and then dripping a coating antibody solution into each hydrophilic region, and dripping a bovine serum albumin solution into each hydrophilic region to form a sealing layer, thus obtaining the elisa plate for paper enzyme-linked immunosorbent assay.
Further, the step S2 further includes: before the coating antibody solution is dripped into each hydrophilic region, a reducing agent is firstly dripped into each hydrophilic region to reduce carbon-nitrogen double bonds in the sodium periodate-chitosan modified layer.
Further, the step S2 further includes: and taking the paper microporous plate out of the sodium periodate solution, soaking the paper microporous plate in a polyalcohol solution for full reaction, washing the paper microporous plate with water, and then dropwise adding the chitosan solution.
Further, in the step S1, the paper microporous plate is prepared by a stamp method, and the preparation method includes the following steps: and (3) the stamp is stained with a hydrophobic agent and then pressed on the surface of the filter paper to form the hydrophobic area, and the hydrophobic agent permeates the filter paper, is dried and solidified to obtain the paper microporous plate.
Further, the hydrophobic agent comprises one or more of polydimethylsiloxane, cyclomethicone, aminosiloxane, polymethylphenylsiloxane and polyether polysiloxane copolymer.
Furthermore, a plurality of annular bulges are formed on the stamp, and the annular bulges are pressed on the surface of the filter paper after being stained with the hydrophobizing agent.
Further, the step of the stamping method further comprises: coating the hydrophobic agent on the surface of paper, and enabling the annular bulge to be stained with the hydrophobic agent on the surface of the paper; wherein the coating thickness of the hydrophobic agent is 0.2 mm-0.6 mm, and the height of the annular bulge is 0.8 mm-1.4 mm.
Further, in the step of drying and curing, the drying temperature is 50-85 ℃, and the drying time is 20-40 min.
The invention provides a method for manufacturing an enzyme label plate for paper enzyme-linked immunosorbent assay, wherein a paper micropore plate is manufactured by a stamp method, the surface of the manufactured paper micropore plate comprises a plurality of hydrophobic areas and hydrophilic areas surrounded by the hydrophobic areas, the size uniformity of the hydrophilic areas is good, no leakage phenomenon exists, and the uniformity and the tightness of the hydrophilic areas can be well kept; and forming a sodium periodate-chitosan modification layer in each hydrophilic area of the paper microporous plate, then dropwise adding a coating antibody solution on the sodium periodate-chitosan modification layer formed in the hydrophilic area, and dropwise adding a bovine serum albumin solution to form a sealing layer to prepare the enzyme label plate for paper enzyme-linked immunosorbent assay, so that the coating antibody solution is connected with the sodium periodate-chitosan modification layer in a chemical crosslinking manner to form a coating antibody layer fixed on the surface of the hydrophilic area of the paper microporous plate, thereby enhancing the stability and effectiveness of the fixation of the coating antibody layer, improving the sensitivity of the p-ELISA method and having good repeatability.
Drawings
FIG. 1 is a schematic structural diagram of an ELISA plate manufactured by the method for manufacturing the ELISA plate for paper enzyme-linked immunosorbent assay provided by the embodiment of the invention;
FIG. 2 is a scanning electron microscope image of an unmodified paper microplate (FIG. a) and a sodium periodate-chitosan modified paper microplate (FIG. b);
FIG. 3 is an infrared spectrum of a papery microporous plate, wherein line a is an unmodified papery microporous plate, line b is a papery microporous plate after oxidation of sodium periodate, and line c is a papery microporous plate modified by sodium periodate-chitosan;
FIG. 4 is a scanning electron microscope image of an unmodified paper microporous plate (FIG. a) and a paper microporous plate (FIG. b) manufactured by a stamp method;
FIG. 5 is a standard curve of detection by p-ELISA method based on sodium periodate-chitosan modified layer chemical crosslinking immobilized coated antibody;
FIG. 6 is a reaction and detection schematic diagram of the method for manufacturing an ELISA plate for paper ELISA according to the embodiment of the present invention.
Description of reference numerals:
1. a paper microporous plate; 2. a hydrophilic region.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The embodiment of the application provides a method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay, which comprises the following steps: s1, preparing a paper microporous plate 1, wherein a plurality of hydrophobic areas are formed on the surface of the paper microporous plate 1, and each hydrophobic area surrounds a hydrophilic area 2; s2, immersing the paper microporous plate 1 prepared in the step S1 into a sodium periodate solution, taking out the paper microporous plate and then dropwise adding a chitosan solution to each hydrophilic region 2 on the paper microporous plate 1 to form a sodium periodate-chitosan modification layer; and then dripping a coating antibody solution into each hydrophilic region 2, and dripping a bovine serum albumin solution into each hydrophilic region 2 to form a sealing layer, thus obtaining the elisa plate for paper enzyme-linked immunosorbent assay. Referring to fig. 1, the elisa plate manufactured in the embodiment of the present application 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.
In the conventional p-ELISA method, the coating antibody is mainly adsorbed on the surface of the paper microporous plate 1 through static electricity, the coating antibody which is not firmly fixed is easily eluted in the washing process, so that the random fixation of biomolecules is easily caused, the stability and effectiveness of the coating antibody are influenced, and the sensitivity of the p-ELISA method is reduced. The coating antibody contains a large amount of amino and carboxyl, and the research finds that the coating antibody can be chemically coupled with solid phase carriers, namely aldehyde, carboxyl, amino and other groups on the surface of the paper microporous plate 1 in a chemical crosslinking mode, so that the immunoassay performance of a p-ELISA method is enhanced.
It can be understood that the way of fixing the biomolecules on the paper microporous plate 1 is various, and different fixing ways have different effects on the activity of the biomolecules. The chitosan is a product of chitosan deacetylation, contains a large amount of amino, can be directly fixed on the surface of the paper microporous plate 1 through electrostatic adsorption on one hand, but is unstable in the fixing mode and easy to elute; on the other hand, the chitosan can be combined with aldehyde groups or carboxyl groups on the surface of the paper microporous plate 1 oxidized by the oxidant to firmly combine the chitosan on the surface of the paper microporous plate 1, and the mode does not need any chemical cross-linking agent and can well maintain the physical and chemical properties of the filter paper. While sodium periodate is used as an oxidizing agent for filter paper, the concentration, pH value of oxidizing solution, oxidizing time and temperature will affect the physical properties and aldehyde group content of the filter paper, so the oxidizing process must be well controlled.
In the embodiment of the application, sodium periodate and chitosan are sequentially dripped into the hydrophilic region 2 of the paper microporous plate 1, and the infrared spectrum characterization shows that the sodium periodate oxidant successfully oxidizes the surface of the paper microporous plate 1 along with the weakening of the characteristic peak of hydroxyl and the appearance of the aldehyde group peak; meanwhile, the disappearance of characteristic peak of aldehyde group and the appearance of characteristic peak of C ═ N double bond in Schiff base show that chitosan can be stably covalently bonded on the surface of the paper microporous plate 1 modified by sodium periodate, and provide good basis for stable chemical crosslinking of coating antibody.
In the embodiment of the application, the paper microporous plate 1 is provided with a plurality of hydrophobic areas, each hydrophobic area surrounds a hydrophilic area 2, and a microporous structure similar to the microporous plate is formed. The hydrophobic region is a closed structure, and the shape of the hydrophobic region is not limited in the embodiments of the present application. Immersing the paper microporous plate 1 into a sodium periodate solution, and dropwise adding a chitosan solution into each hydrophilic area 2 to form a sodium periodate-chitosan modification layer on the surface of each hydrophilic area 2; when the coated antibody solution is dripped into each hydrophilic region 2 subsequently, the coated antibody can be fixed on the surface of the hydrophilic region 2 of the paper microporous plate 1 in a chemical crosslinking mode, so that the stability and the effectiveness of the fixation of the coated antibody are enhanced, the sensitivity of a p-ELISA method is improved, and the repeatability is good. That is, the sodium periodate-chitosan modification layer formed on the surface of the hydrophilic region 2 contains amino, and the carboxyl group coated on the C end of the antibody and the amino group of the modification layer are chemically cross-linked, so that the variable region coated on the N end of the antibody is fully exposed, and the binding affinity between the antigen and the antibody is favorably improved.
Referring to the scanning electron microscope image in fig. 2, a is a paper microporous plate 1 without modified sodium periodate-chitosan, and b is a paper microporous plate 1 modified by sodium periodate-chitosan in the embodiment of the present application, the surface morphology of which is not changed, and the loose three-dimensional porous structure of the filter paper is still maintained. As can be seen from the graph 2, the original structure of the filter paper is not changed by the micromolecular chitosan through the observation of a scanning electron microscope, the specific surface area is not changed, the effective adsorption capacity of the coating antibody is ensured, the rapid and sensitive immune reaction can be presented, meanwhile, the similar porous structure of the hydrophobic area surrounding the hydrophilic area 2 is beneficial to washing substances which are not combined on the filter paper, the background influence of detection is reduced, and the good analysis performance of the paper microporous plate 1 can be kept.
Referring to fig. 3, an infrared spectrum of the sodium periodate-chitosan modified paper microporous plate 1 is shown, wherein line a is an infrared spectrum of the paper microporous plate 1 which is not modified by sodium periodate and chitosan; the line b is an infrared spectrogram of the paper microporous plate 1 modified by the sodium periodate; and the line c is an infrared spectrogram of the paper microporous plate 1 modified by the sodium periodate-chitosan.
As can be seen from the figure, the paper microporous plate 1 contains a plurality of hydroxyl groups, and only the characteristics of the hydroxyl groups can be seen on the line aSpectral peak (3300 cm)-1) (ii) a 1730cm was observed after the attachment of sodium periodate to the surface of the microwells-1The appearance of characteristic peak of aldehyde group (line b), and the characteristic spectral peak of hydroxyl group (3300 cm)-1) Weakening; after the paper microporous plate 1 is modified by chitosan, the characteristic peak of aldehyde group on the c line disappears and is 1642cm-1The characteristic peak of C ═ N double bond appears, which indicates that chitosan has been successfully bound on the surface of the micropores. The infrared spectrogram shows that the sodium periodate oxidant successfully oxidizes the paper microporous plate 1 along with the weakening of the characteristic peak of hydroxyl and the appearance of the aldehyde peak; the disappearance of characteristic peak of aldehyde group and the appearance of characteristic peak of C ═ N double bond show that chitosan can be stably covalently bonded on the surface of the hydrophilic region 2 modified by sodium periodate, and provide good basis for stable chemical crosslinking of coated antibody. The reaction scheme is shown in FIG. 6.
In the embodiment of the present application, the shape of the hydrophobic region in the paper microporous plate 1 may be annular. It can be understood that, if the hydrophobic region is configured as a ring, the hydrophilic region 2 is circular, which is the same as the shape of the liquid drop caused by the surface tension of the solution, so that the liquid drop of chitosan and other liquids can be conveniently added to the hydrophilic region 2.
In the embodiment of the application, the sodium periodate solution is formed by dissolving sodium periodate in an ammonium acetate buffer solution; the paper microplate 1 was taken out from the sodium periodate solution, washed with water and blotted dry. In addition, the coated antibody solution was prepared by dissolving EDC (i.e., 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and Sulfo-NHS (i.e., N-hydroxythiosuccinimide) with MES buffer (i.e., 2- (N-morpholino) ethanesulfonic acid), respectively, and mixing the dissolved EDC solution and the dissolved Sulfo-NHS solution to form an EDC/Sulfo-NHS mixed solution, mixing the coated antibody solution with the EDC/Sulfo-NHS mixed solution, and then dropwise adding to each hydrophilic region 2. The EDC/Sulfo-NHS solution is one of non-toxic cross-linking agents with good biocompatibility, maintains the biological activity of protein molecules, can ensure that the protein molecules are stably fixed on the surface of the paper microporous plate 1, and simultaneously does not influence the binding capacity of antigens and coating antibodies.
In some embodiments, step S2 further includes: before the antibody-coating solution is added dropwise to each hydrophilic region 2, each hydrophilic region 2 is added dropwiseReducing the carbon-nitrogen double bond in the sodium periodate-chitosan modified layer by using a reducing agent. As can be seen from the infrared spectrogram in fig. 3, in the coating layer formed on the surface of the paper microporous plate 1, a characteristic peak of N-double bond in the sodium periodate-chitosan modification layer C, which is an unstable structure, is present, and in the embodiment of the present application, a reducing agent is first dropped into each hydrophilic region 2 before a coating antibody solution is dropped, so that the C-N double bond reacts to CH2-NH2The stable state of the coating antibody layer ensures the formation of the subsequent coating antibody layer and the binding capacity of the antigen and the coating antibody. In the embodiment of the application, the reducing agent is selected from a mild reducing agent such as sodium cyanoborohydride.
In some embodiments, step S2 further includes: taking the paper microporous plate 1 out of the sodium periodate solution, soaking the paper microporous plate in a polyalcohol solution for full reaction, washing the paper microporous plate with water, and then dropwise adding a chitosan solution. Understandably, after the paper microporous plate 1 is taken out from the sodium periodate solution, the surface of the paper microporous plate 1 has the residue of the sodium periodate solution; therefore, the paper microporous plate 1 is immersed into the solution of the polyhydric alcohol, so that the polyhydric alcohol and the sodium periodate solution remained on the surface of the paper microporous plate 1 are fully reacted, and the residue of the excessive sodium periodate solution on the surface of the paper microporous plate 1 is avoided. In the examples of the present application, the polyhydric alcohol solution is ethylene glycol.
In some embodiments, in step S1, the paper microporous plate 1 is prepared by a stamp method, and the preparation method includes the following steps: and (3) the stamp is stained with a hydrophobic agent and then pressed on the surface of the filter paper to form a hydrophobic area, and the hydrophobic agent permeates the filter paper, is dried and solidified to obtain the paper microporous plate 1. In the embodiment of the application, the paper microporous plate 1 manufactured by the stamp method has the advantages that the size uniformity of the hydrophilic region 2 is good, the leakage phenomenon does not exist, and the uniformity and the impermeability of the hydrophilic region 2 can be well maintained. Referring to the scanning electron microscope image of the unmodified filter paper and the paper microporous plate 1 manufactured by the stamp method in fig. 4, it can be observed that the surface of the unmodified filter paper is rough and is interwoven to form a porous structure (fig. 4 a); the porous structure of the filter paper treated by the stamp method disappears, the smoothness is obviously improved (figure 4b), and a good hydrophobic area edge can be formed.
In the embodiment of the application, the filter paper is made of pure cellulose with a porous structure, the pure cellulose is a linear macromolecular chain consisting of D-glucopyranosyl, the filter paper has hydrophilicity and biocompatibility, and meanwhile, the surface of the filter paper is provided with a plurality of hydroxyl groups, so that the surface chemistry and the optical property of the filter paper have unique advantages for preparing a p-ELISA device. On one hand, the specific surface area for protein adsorption is maintained, and on the other hand, due to capillary force, liquid is easy to move on the surface. The cellulose porous structure processed by the seal method is completely sealed, and the sample and the reagent are completely confined in the hydrophilic region 2, so that the accuracy of the experiment of the hydrophilic region 2 can be ensured, and the method is a better method for manufacturing the paper microporous plate 1.
Further, the hydrophobic agent comprises one or more of polydimethylsiloxane, cyclomethicone, aminosiloxane, polymethylphenylsiloxane and polyether polysiloxane copolymer. The above hydrophobizing agents can be used in the process of manufacturing the paper microporous plate 1 by the stamp method in the embodiment of the present application, and polydimethylsiloxane is preferred.
In some embodiments, the stamp is formed with a plurality of annular protrusions, and the annular protrusions are pressed on the surface of the filter paper after being stained with the hydrophobic agent. It can be understood that when the stamp is formed with a plurality of annular protrusions, when the stamp is stained with the hydrophobizing agent and pressed on the surface of the filter paper, the hydrophobic region is formed in an annular shape, and the hydrophilic region 2 is in a circular shape, i.e. the hydrophilic region 2 and the hydrophobic region form a microporous structure similar to a microporous plate. Specifically, the seal is the rubber material, and the annular bulge on the seal forms through laser cutting technique, and annular bulge is formed with the hole on the seal, and the diameter in hole is about 6mm, and the degree of depth is 1mm, and the hole interval is 2 mm.
Further, the stamping method further comprises the following steps: coating a hydrophobic agent on the surface of the paper, and enabling the annular bulge to be stained with the hydrophobic agent on the surface of the paper; wherein the coating thickness of the hydrophobic agent is 0.2 mm-0.6 mm, and the height of the annular bulge is 0.8 mm-1.4 mm. Understandably, the contamination requirement of the seal can be met only by coating the hydrophobic agent on the surface of the paper to reach a certain thickness; in addition, the height of the annular bulge is larger than the coating thickness of the hydrophobic agent on the paper, so that the hydrophobic agent cannot enter a hole formed on the stamp by the annular bulge, and the hydrophobic agent entering the hole when the stamp is pressed on the filter paper is prevented from flowing into the hydrophilic area 2. The paper used herein may be plain paper. The thickness of the coating of the hydrophobic agent is too thick, so that the stamp is stained with too much hydrophobic agent, and the hydrophobic agent is pressed on the surface of the filter paper to pollute the hydrophilic area 2, is difficult to dry and solidify, and increases the cost; the thickness of the applied hydrophobing agent is too thin, the hydrophobing agent pressed on the filter paper by the seal is not uniform, and the uniformity and the tightness of the paper microporous plate 1 are influenced.
Further, in the step of drying and curing, the drying temperature is 50-85 ℃, and the drying time is 20-40 min. By adopting the drying temperature and the drying time, the hydrophobing agent can be completely dried, and the subsequent use is convenient.
The reagents used in the following examples are commercially available.
Example 1
A method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay comprises the following steps:
1. the paper microporous plate 1 is prepared by a stamp method.
And forming a micropore model with the diameter of 7mm, the depth of 1mm and the hole interval of 2mm on the surface of the rubber by a laser cutting technology, and thus obtaining the stamp. The PDMS hydrophobing agent is then placed on a magnetic stirrer to be slowly stirred uniformly without generating bubbles. Wherein, the PDMS hydrophobic agent is a mixture of Sylgard184 polymer and a fixing agent. PDMS is abbreviated as polydimethylsiloxane. After the hydrophobic agent is uniformly stirred, the hydrophobic agent is flatly coated on the surface of the paper, and the coating thickness is about 0.5 mm.
Uniformly staining a hydrophobic agent on the surface of paper with a rubber stamp with a micropore model, then pressing the surface of the filter paper with force to form a plurality of hydrophobic areas on the surface of the filter paper, waiting for the hydrophobic agent in the hydrophobic areas to completely permeate the filter paper (about 20 s), placing the filter paper in a thermostat at 75 ℃ for heat preservation and solidification for 30min, taking out and cooling to form a paper microporous plate 1 with the diameter of the hydrophilic area 2 being 6mm and the distance between the hydrophilic areas 2 being 2 mm.
2. And forming a sodium periodate-chitosan modification layer on the surface of the hydrophilic area 2 of the paper microporous plate 1.
And (2) dissolving sodium periodate in an ammonium acetate buffer solution, completely immersing the paper microporous plate 1 prepared in the step (1) into the sodium periodate solution, shaking for 20min at room temperature in a dark place, fully washing with ultrapure water after homogenization, and sucking to dry. And then soaking the paper microporous plate 1 in an ethylene glycol solution for full reaction, fully washing with ultrapure water after 20min, and sucking to dry for later use. And (2) dropwise adding 5 mu L of chitosan solution into each hydrophilic area 2 of the paper microporous plate 1, wherein the concentration of chitosan is 0.25mg/mL, reacting at room temperature until the chitosan solution is dried, then dropwise adding 4 mu L of sodium cyanoborohydride reducing agent into each hydrophilic area 2, reacting for 20min at 37 ℃, washing for 6 times, and absorbing the redundant residual solution by absorbent paper for later use.
3. A coated antibody layer was prepared.
Respectively dissolving EDC and Sulfo-NHS by MES buffer solution (0.1mol/L, pH value 4.7), and mixing the dissolved EDC solution and Sulfo-NHS solution according to the molar ratio of 4: 5 to obtain mixed solution; and mixing the coating antibody solution with an EDC/Sulfo-NHS mixed solution, then dropwise adding the mixture to each hydrophilic region 2, activating at 37 ℃ for 15min, and then dropwise adding a bovine serum albumin solution to form a sealing layer. The concentration of the coating antibody solution was 0.2 mg/mL.
The performance of the ELISA plate prepared in example 1 was analyzed by the p-ELISA method based on the sodium periodate-chitosan chemically cross-linked immobilized antibody. Taking alpha-fetoprotein standard solutions with different concentrations as an example, 4 mu L of an enzyme-labeled antibody mixed solution is dropwise added to each hydrophilic region of an experimental group, the optimal dilution ratio of the enzyme-labeled antibody is 1: 600, 4 mu L of the enzyme-labeled antibody solution is directly added to each hydrophilic region of a control group, after reaction for 8min at room temperature, washing is carried out for 6 times by using a washing solution, and redundant residual solution is sucked; subsequently, 3. mu.L of TMB developing solution was dropped into each hydrophilic region of the experimental group and the control group to develop color, as shown in FIG. 6. The color development condition is captured by a scanner, the Image J software analyzes the Image, the RGB picture is analyzed by the Image J software, the Rn, Gn and Bn values of the experimental holes and the R0, G0 and B0 values of the comparison holes are respectively obtained, and the color development condition is obtained by a formula
The signal of the p-ELISA detection is calculated, a standard curve of the p-ELISA detection is drawn, the result is shown in figure 5, and the detection range is 0.08 ng/mL-20 ng/mL. In addition, 20 negative control Δ RGB values were measured and the average value was calculated
And standard deviation(s), and the value is substituted into the standard curve to calculate the detection limit, so that the minimum detection limit is 0.035 ng/mL.
Three alpha-fetoprotein concentrations of 20ng/mL, 10ng/mL and 1ng/mL are selected for the detection experiment to compare the repeatability of the p-ELISA detection method, and the repeatability is determined by a formula
The results are shown in table 1, and the intra-batch variation coefficients of the p-ELISA detection method based on sodium periodate-chitosan and coating antibody cross-linking are 7.66%, 8.40% and 8.68%, and the inter-batch variation coefficients are 7.78%, 9.56% and 10.49%.
TABLE 1 in-batch and inter-batch test results of the p-ELISA test method
The p-ELISA method detection and the traditional commercial 96-well plate ELISA method compare the same alpha-fetoprotein antigen, and the detection limit is obviously improved according to the result. Therefore, the sensitivity of the detection by the p-ELISA method is effectively improved. Meanwhile, the variation coefficient of the same sample in the same batch test and different batch tests can be observed through the variation coefficient between batches, which shows that the p-ELISA method has better repeatability and stability.
Three alpha-fetoprotein standard substances with known concentrations of 1ng/mL, 10ng/mL and 20ng/mL are selected and added into a human serum sample of 2.01ng/mL (the human serum sample is from a traumatology hospital in Tanzhou), the recovery rates of the p-ELISA method for obtaining the sodium periodate-chitosan chemical crosslinking immobilized coating antibody through calculation are respectively 104.00%, 98.50% and 96.30%, and compared with a commercial 96-pore plate traditional ELISA method adopting the same coating antibody, the sample detection results are not obviously different, and the table 2 shows that the recovery rates of the p-ELISA method are different. The p-ELISA method proves that the result of the detection method is reliable by adding a recovery test to the sample and comparing with the traditional ELISA method.
TABLE 2 comparison of sample analysis of p-ELISA method with commercial 96-well plate traditional ELISA method
The method for applying the ELISA plate to paper enzyme-linked immunosorbent assay has good analysis performance on an object to be detected in a complex matrix environment; compared with the traditional ELISA, the method has the advantages of strong reliability of detection results and good analysis performance, and provides a low-cost, accurate, rapid and high-sensitivity method for disease detection in resource-deficient areas.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of changes or substitutions within the technical scope of the present invention, and all such changes or substitutions are included in the scope of the present invention. Moreover, the technical solutions in the embodiments of the present invention may be combined with each other, but it is necessary to be able to be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent, and is not within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (8)
1. A method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay is characterized by 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 hydrophobic area surrounds a hydrophilic area;
s2, immersing the paper microporous plate prepared in the step S1 into a sodium periodate solution, taking out the paper microporous plate, and dripping a chitosan solution into each hydrophilic area on the paper microporous plate to form a sodium periodate-chitosan modification layer; and then dripping a coating antibody solution into each hydrophilic region, and dripping a bovine serum albumin solution into each hydrophilic region to form a sealing layer, thus obtaining the elisa plate for paper enzyme-linked immunosorbent assay.
2. The method for manufacturing an elisa plate for paper elisa according to claim 1, wherein the step S2 further comprises: before the coating antibody solution is dripped into each hydrophilic region, a reducing agent is firstly dripped into each hydrophilic region to reduce carbon-nitrogen double bonds in the sodium periodate-chitosan modified layer.
3. The method for manufacturing an elisa plate for paper elisa according to claim 1, wherein the step S2 further comprises: and taking the paper microporous plate out of the sodium periodate solution, soaking the paper microporous plate in a polyalcohol solution for full reaction, washing the paper microporous plate with water, and then dropwise adding the chitosan solution.
4. The method for manufacturing an ELISA plate for paper ELISA according to any one of claims 1-3, wherein the step S1 of preparing the paper microplate by a stamp method comprises the following steps:
and (3) the stamp is stained with a hydrophobic agent and then pressed on the surface of the filter paper to form the hydrophobic area, and the hydrophobic agent permeates the filter paper, is dried and solidified to obtain the paper microporous plate.
5. The method for manufacturing the elisa plate for paper enzyme-linked immunosorbent assay according to claim 4, wherein the hydrophobic agent comprises one or more of polydimethylsiloxane, cyclomethicone, aminosiloxane, polymethylphenylsiloxane and polyether polysiloxane copolymer.
6. The method for manufacturing an ELISA plate for paper enzyme-linked immunosorbent assay according to claim 4, wherein a plurality of annular protrusions are formed on the stamp, and the annular protrusions are pressed on the surface of the filter paper after being stained with the hydrophobizing agent.
7. The method for manufacturing an elisa plate for paper elisa according to claim 6, wherein the stamping method further comprises the steps of: coating the hydrophobic agent on the surface of paper, and enabling the annular bulge to be stained with the hydrophobic agent on the surface of the paper; wherein the coating thickness of the hydrophobic agent is 0.2 mm-0.6 mm, and the height of the annular bulge is 0.8 mm-1.4 mm.
8. The method for manufacturing the elisa plate for the paper enzyme-linked immunosorbent assay according to claim 4, wherein in the step of drying and curing, the drying temperature is 50-85 ℃ and the drying time is 20-40 min.
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