Nanometer modification and high-density coating process for surface of enzyme-linked immunosorbent assay plate

Abstract

The invention discloses a nano modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay plate. The composition comprises an interface self-assembly bottom layer solution containing tannic acid and branched polyethyleneimine, and a dynamic gating functionalized buffer solution containing sodium tetraborate, D-mannitol and trehalose. The process comprises the following steps: firstly, constructing a supramolecular bottom layer on the surface of polystyrene, and then forming a boric acid-mannitol gating interface through non-washing drying; after the antibody is added, the Fc segment sugar chain and boric acid are subjected to competitive replacement reaction, so that mannitol is replaced, and the oriented high-density fixation of the antibody is realized. According to the invention, the problems of random antibody direction and weak binding force in traditional physical adsorption are solved, the antibody immobilization amount and detection sensitivity of the elisa plate are remarkably improved, and the elisa plate has good storage stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is an analytical technique widely used in clinical diagnostics, food safety testing, and bioscience research. Polystyrene microplates have become the mainstream solid-phase carrier in ELISA technology due to their good light transmittance, low cost, and ease of molding. Traditional polystyrene ELISA plates mainly rely on the hydrophobic interactions of the material surface for the physical adsorption of proteins. While this physical adsorption mechanism is simple to operate, it has significant limitations.

[0003] During physical adsorption, the orientation of antibody molecules on the polystyrene surface is random and disordered. This results in a significant proportion of antibody antigen-binding sites (Fab ends) being sterically blocked or directly adhering to the substrate surface, failing to effectively capture target antigens in the liquid phase and thus reducing detection sensitivity. Strong interactions between hydrophobic interfaces and protein molecules can easily induce conformational changes in proteins, causing some antibodies to lose their biological activity after adsorption. Physical adsorption mainly relies on van der Waals forces and hydrophobic interactions, resulting in relatively weak binding forces. During subsequent washing and incubation steps, adsorbed antibodies are prone to desorption or displacement, leading to fluctuations in the detection signal and affecting experimental repeatability and accuracy.

[0004] To overcome the limitations of physical adsorption, existing technologies attempt to introduce active groups such as amino, carboxyl, or epoxy groups through chemical grafting, or to covalently couple using cross-linking agents such as glutaraldehyde or EDC / NHS. These chemical modification methods typically involve complex reaction steps and harsh reaction conditions, and the introduced active groups (such as succinimide esters) are easily hydrolyzed and degraded in aqueous environments, requiring strict timeliness and hindering large-scale industrial production. While indirect coating strategies using the streptavidin-biotin system or Protein A / G as an intermediate layer can improve antibody orientation, they introduce an additional protein layer, increasing preparation costs and potentially introducing new non-specific binding sites, leading to increased background noise. Therefore, developing a mild, efficient surface modification technique that enables targeted antibody immobilization is crucial for improving the performance of enzyme-linked immunosorbent assays (ELISA). Summary of the Invention

[0005] The technical problem solved by this invention is that existing enzyme-linked immunosorbent assay (ELISA) plates mainly rely on the hydrophobic interaction of polystyrene surface to physically adsorb proteins. This results in problems such as random antibody adsorption direction, easy masking of active sites, low binding strength, and large batch-to-batch differences, which leads to limited detection sensitivity and a large coefficient of variation.

[0006] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a nano-modification and high-density coating reagent composition for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, employing the following technical solution: A nano-modification and high-density coating reagent composition for the surface of enzyme-linked immunosorbent assay (ELISA) plates comprises an independent interface self-assembly bottom solution and a dynamically gated functionalized buffer. The interface self-assembly bottom solution comprises the following components at the following concentrations: tannic acid 0.2-1.0 mg / mL, branched polyethyleneimine 0.1-0.5 mg / mL, and the balance being Tris-HCl buffer at pH 7.8-8.2. The dynamically gated functionalized buffer comprises the following components at the following concentrations: sodium tetraborate 50-100 mM, D-mannitol 50-200 mM, D-(+)-trehalose 2%-10% (w / v), and the balance being carbonate buffer at pH 9.0-9.6.

[0007] By adopting the above technical solution, an interface with directional capture function is constructed by utilizing the chemical synergistic effect between the components. The specific mechanism of action is as follows: The supramolecular underlying construction mechanism: Tannic acid possesses a polyphenolic hydroxyl structure, while branched polyethyleneimine has main-chain and side-chain amino groups. Under weakly alkaline conditions (pH 7.8-8.2), the two complex via hydrogen bonding and electrostatic interactions, and undergo partial Michael addition and Schiff base reactions with the assistance of tris(hydroxymethyl)aminomethane, forming a multifunctional polymer network on the polystyrene surface. This polymer network transforms the hydrophobic surface into a hydrophilic surface and provides binding sites for subsequent functionalization modifications.

[0008] Anchoring and functionalization mechanism of borate: Sodium tetraborate hydrolyzes in an alkaline carbonate buffer system to generate tetrahydroxyborate ions. Tetrahydroxyborate ions can form coordinate bonds or boron-nitrogen intracoordinate bonds with the catechol tannin structure and the amino group of polyethyleneimine in the underlying network, thereby grafting boric acid functional groups onto the polymer network surface.

[0009] Competitive substitution gating mechanism: D-mannitol, a low molecular weight polyol, acts as a competitive ligand in the system. Before antibody addition, D-mannitol forms a cyclic borate ester complex with surface-grafted borate. This complex occupies the surface active site, shielding against the non-specific positive charge adsorption of the polyethyleneimine layer. When antibody is added, the cis-diol structure on the Fc segment of the antibody has a higher binding constant to the borate, causing the antibody molecule to displace D-mannitol through a thermodynamically driven ligand exchange reaction, achieving chemical bonding at the interface. This ligand exchange mechanism filters out non-specific proteins that cannot overcome the substitution energy barrier, and because the binding site is located in the Fc segment, the antigen-binding site of the antibody is oriented towards the liquid phase, achieving directional antibody alignment.

[0010] Structure protection mechanism: During the drying process, D-(+)-trehalose forms a glassy protective layer at the interface through water substitution, which maintains the spatial conformational stability of the underlying polymer network and surface functional groups, and prevents the structure from collapsing or the active sites from being deactivated due to drying.

[0011] Preferably, in the interface self-assembly bottom solution, the concentration of tannic acid is 0.6-1.0 mg / mL, the concentration of branched polyethyleneimine is 0.3-0.5 mg / mL, and the concentration of Tris-HCl buffer is 25-50 mM; in the dynamically gated functionalized buffer, the concentration of sodium tetraborate is 75-100 mM, the concentration of D-mannitol is 125-200 mM, the mass-volume ratio of D-(+)-trehalose is 6%-10% (w / v), and the concentration of carbonate buffer is 75-100 mM.

[0012] By adopting the above technical solution, the concentration of each component is within the appropriate reaction concentration range, which ensures the film formation rate and avoids polymer agglomeration and precipitation due to excessive concentration, thus ensuring the uniformity and light transmittance of the modified layer.

[0013] Preferably, the branched polyethyleneimine has a weight-average molecular weight (Mw) of 20,000-30,000 Da; the interface self-assembly bottom layer solution is prepared by mixing component A containing tannic acid and component B containing branched polyethyleneimine in a volume ratio of 1:1 before use.

[0014] By adopting the above technical solution and selecting branched polyethyleneimine of medium molecular weight, the flowability and permeability of the solution are maintained while ensuring the density of crosslinking points; the method of dispensing and mixing avoids premature complexation failure between components.

[0015] Preferably, in the dynamically gated functionalized buffer solution, the molar ratio of sodium tetraborate to D-mannitol is 1:1 to 1:2.

[0016] By employing the above technical solution, the stoichiometric ratio of boric acid to mannitol is controlled to ensure that the surface boric acid sites are adequately shielded. If there is too little mannitol, non-specific adsorption cannot be effectively shielded; if there is too much mannitol, a high-concentration free competitive environment will be formed, hindering antibody exchange binding. This ratio range balances the shielding effect and the exchange efficiency.

[0017] Secondly, the present invention provides a nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, employing the following technical solution: A nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, using the reagent composition described in the first aspect above, includes the following steps: Step S1: Tannic acid and branched polyethyleneimine are mixed and added to the wells of an enzyme-linked immunosorbent assay (ELISA) plate for incubation and deposition. After washing, a substrate with a supramolecular underlayer is obtained. Step S2: Add the dynamically gated functionalized buffer solution to the wells after step S1, incubate, remove the liquid and dry or semi-dry without washing to obtain a substrate with a thermodynamically gated interface. Step S3: Add the capture antibody solution to the wells after step S2 and incubate for coating; Step S4: Remove the antibody solution, wash, dry, and obtain the finished product.

[0018] By adopting the above technical solution, this process combines step-by-step assembly with in-situ replacement, and the specific principle is as follows: Stepwise assembly principle: Steps S1 and S2 separate the underlying construction from the functionalization construction. Step S1 removes unbound polymers through washing to prevent them from forming free complexes that interfere with the reaction in subsequent steps; Step S2 introduces functional molecules to achieve the transformation of interfacial properties.

[0019] Metastable interface retention principle: Step S2 involves direct drying without washing after liquid removal. Since the boric acid-mannitol complex is in dynamic equilibrium, washing would disrupt the surface functional molecular layer. Direct drying allows the borate, mannitol, and trehalose in the solution to concentrate in situ and deposit on the supramolecular bottom layer during solvent evaporation, forming a high-density pre-activated layer. In this state, the boric acid groups are in a metastable state protected by mannitol.

[0020] Solid-liquid interface displacement principle: In step S3, when the surface dry layer is reconstituted with antibody solution, the high-concentration local environment promotes the collision between the antibody and the surface borate ester. The antibody utilizes the multi-point synergistic binding effect of the Fc segment glycan chain, taking advantage of the binding energy difference to complete ligand displacement. This process avoids the problem of hydrolysis failure in traditional activated esters, achieving targeted antibody immobilization.

[0021] Preferably, before step S1, a pretreatment step of the enzyme-linked immunosorbent assay (ELISA) plate is included: soaking and washing with anhydrous ethanol, followed by washing with deionized water and drying.

[0022] By adopting the above technical solution, the pretreatment step removes the release agent residue and electrostatically adsorbed dust from the surface of polystyrene, exposing a clean substrate surface, which is conducive to the uniform spreading of the supramolecular bottom layer.

[0023] Preferably, in step S1, the incubation temperature for deposition is 20-25°C and the time is 15-60 minutes; in step S2, the incubation temperature is 20-25°C and the time is 30-60 minutes.

[0024] By adopting the above technical solution, interface assembly is completed at room temperature, avoiding excessive oxidation of tannic acid at high temperatures leading to agglomeration, and insufficient reaction kinetics at low temperatures, thus ensuring process reproducibility.

[0025] Preferably, in step S3, the pH value of the capture antibody solution is 7.4-8.0, and the incubation and coating conditions are: incubation at 37°C for 1-1.5 hours, or incubation at 4°C for 10-14 hours.

[0026] By adopting the above technical solution, the antibody maintains its native conformation in a neutral to weakly alkaline environment, while the formation of borate ester bonds is within a suitable pH window; the combination of temperature and time ensures that the displacement reaction proceeds fully to achieve thermodynamic equilibrium.

[0027] Preferably, in step S4, the drying method is as follows: place the washed enzyme-linked immunosorbent assay (ELISA) plate in an environment with a relative humidity of 15%-20% and dry it at 25-30°C for 2-4 hours.

[0028] By adopting the above technical solution and controlling the drying rate, trehalose forms a uniform glassy film that coats the protein surface, giving the finished plate long-term storage stability.

[0029] Thirdly, the present invention provides an enzyme-linked immunosorbent assay (ELISA) plate, which adopts the following technical solution: An enzyme-linked immunosorbent assay (ELISA) plate is prepared by the process described in the second aspect above.

[0030] By adopting the above technical solution, the enzyme-linked immunosorbent assay (ELISA) plate prepared in this application has an antibody layer arranged in an orientation on its surface. Compared with traditional physical adsorption plates, the antibody loading of the finished plate is increased and the antigen binding sites are fully exposed, thereby improving the sensitivity and signal intensity of the immunoassay and also having storage stability.

[0031] This invention provides a nano-modification and high-density coating process for the surface of enzyme-linked immunosorbent assay (ELISA) plates. It offers the following advantages: 1. This invention constructs a high-density functionalized modification layer on the surface of inert polystyrene through the self-assembly of tannic acid and branched polyethyleneimine and the cross-linking of borate. This modification layer enhances the interfacial binding force by utilizing the multi-point synergistic interaction between components, solving the problems of weak binding and large batch-to-batch variability in traditional physical adsorption. At the same time, the borate sites distributed on the interface significantly improve the antibody immobilization capacity, providing a material basis for high-sensitivity detection.

[0032] 2. This invention utilizes the competitive displacement mechanism of the boric acid-mannitol complex to achieve targeted antibody coating and shielding against non-specific adsorption. Mannitol pre-occupies the active site, and the antibody, with its Fc glycan chain, displaces the mannitol at a more favorable thermodynamic binding energy, ensuring that the antibody is immobilized with its antigen-binding end facing outwards. This mechanism avoids active site shielding caused by random adsorption, improves antigen capture efficiency, and reduces detection background noise.

[0033] 3. This invention introduces trehalose into the functionalized buffer solution and combines it with a no-wash, direct-drying process to achieve interfacial activity protection. During the drying process, trehalose forms a glassy protective layer through water substitution, maintaining the spatial structural stability of the underlying network and surface functional groups. This design enables the prepared enzyme-linked immunosorbent assay (ELISA) plate to maintain its biological activity for a long time under dry storage conditions, meeting the long-term stability requirements of commercial reagent kits. Attached Figure Description

[0034] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Reference Figure 1 The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0037] Tannic acid (purity ≥95%); branched polyethyleneimine (weight average molecular weight Mw approximately 25000 Da); sodium tetraborate decahydrate (purity ≥99.5%); D-mannitol (purity ≥98%); D-(+)-trehalose dihydrate (purity ≥99%); tris(hydroxymethyl)aminomethane (Tris); polyoxyethylene sorbitan monolaurate (Tween-20); goat anti-human IgG capture antibody (Fc fragment specific), human IgG standard, HRP-labeled goat anti-human IgG detection antibody and TMB chromogenic solution are all commercially available biotechnology grade products; sodium carbonate, sodium bicarbonate, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate and other common inorganic salts are all commercially available analytical grade reagents; the 96-well polystyrene ELISA plate is a commercially available conventional high-binding plate.

[0038] Since the core of this invention lies in a specific treatment liquid system, in order to fully support the generalization of the concentration range of each component in the claims, three preparation examples with different parameter gradients are set below, corresponding to low, medium and high value ranges respectively.

[0039] Preparation Example 1: This preparation example provides a set of low-concentration treatment reagents for modifying the surface of enzyme-linked immunosorbent assay (ELISA) plates, including an interfacial self-assembly bottom solution and a dynamically gated functionalized buffer solution, prepared as follows: Step 1: Preparation of the interface self-assembly bottom layer solution: Weigh tris(hydroxymethyl)aminomethane and dissolve it in deionized water. Add sodium chloride and adjust the pH to 7.8 to prepare a 10 mM Tris-HCl buffer solution. Dissolve tannic acid and branched polyethyleneimine separately in this buffer solution to prepare tannic acid component A1 with a concentration of 0.2 mg / mL and branched polyethyleneimine component B1 with a concentration of 0.1 mg / mL. Store the two components separately and mix them at a volume ratio of 1:1 before use.

[0040] Step 2: Preparation of dynamically gated functionalized buffer: Sodium tetraborate decahydrate, D-mannitol, and D-(+)-trehalose dihydrate were weighed and dissolved in 50 mM sodium carbonate / sodium bicarbonate buffer, and the pH was adjusted to 9.0 with sodium hydroxide or hydrochloric acid; the concentration of sodium tetraborate decahydrate was 50 mM, the concentration of D-mannitol was 50 mM, and the mass-volume ratio of D-(+)-trehalose dihydrate was 2% (w / v); after mixing evenly, the mixture was filtered through a 0.22 μm filter membrane to obtain dynamically gated functionalized buffer C1.

[0041] Preparation Example 2: This preparation example provides a set of medium-concentration treatment reagents for surface modification of enzyme-linked immunosorbent assay (ELISA) plates, including an interfacial self-assembly bottom solution and a dynamically gated functionalized buffer solution, prepared as follows: Step 1: Preparation of the interface self-assembly bottom layer solution: Weigh tris(hydroxymethyl)aminomethane and dissolve it in deionized water. Add sodium chloride and adjust the pH to 8.0 to prepare a 25mM Tris-HCl buffer solution. Dissolve tannic acid and branched polyethyleneimine separately in this buffer solution to prepare tannic acid component A2 with a concentration of 0.6 mg / mL and branched polyethyleneimine component B2 with a concentration of 0.3 mg / mL. Store the two components separately and mix them at a volume ratio of 1:1 before use.

[0042] Step 2: Preparation of dynamically gated functionalized buffer: Sodium tetraborate decahydrate, D-mannitol, and D-(+)-trehalose dihydrate were weighed and dissolved in 75 mM sodium carbonate / sodium bicarbonate buffer, and the pH was adjusted to 9.3 with sodium hydroxide or hydrochloric acid; the concentration of sodium tetraborate decahydrate was 75 mM, the concentration of D-mannitol was 125 mM, and the mass-volume ratio of D-(+)-trehalose dihydrate was 6% (w / v); after mixing evenly, the mixture was filtered through a 0.22 μm filter membrane to obtain dynamically gated functionalized buffer C2.

[0043] Preparation Example 3: This preparation example provides a set of high-concentration treatment reagents for surface modification of enzyme-linked immunosorbent assay (ELISA) plates, including an interfacial self-assembly bottom solution and a dynamically gated functionalized buffer solution, prepared as follows: Step 1: Preparation of the interface self-assembly bottom layer solution: Weigh tris(hydroxymethyl)aminomethane and dissolve it in deionized water. Add sodium chloride and adjust the pH to 8.2 to prepare a 50 mM Tris-HCl buffer solution. Dissolve tannic acid and branched polyethyleneimine separately in this buffer solution to prepare tannic acid component A3 with a concentration of 1.0 mg / mL and branched polyethyleneimine component B3 with a concentration of 0.5 mg / mL. Store the two components separately and mix them at a volume ratio of 1:1 before use.

[0044] Step 2: Preparation of dynamically gated functionalized buffer: Sodium tetraborate decahydrate, D-mannitol, and D-(+)-trehalose dihydrate were weighed and dissolved in 100mM sodium carbonate / sodium bicarbonate buffer, and the pH was adjusted to 9.6 with sodium hydroxide or hydrochloric acid; the concentration of sodium tetraborate decahydrate was 100mM, the concentration of D-mannitol was 200mM, and the mass-volume ratio of D-(+)-trehalose dihydrate was 10% (w / v); after mixing evenly, the mixture was filtered through a 0.22μm filter membrane to obtain dynamically gated functionalized buffer C3.

[0045] Example 1: This embodiment provides a nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, using the medium-concentration treatment reagent provided in Preparation Example 2, and includes the following steps: Step 1: Substrate pretreatment. Wash the 96-well polystyrene microplate three times with anhydrous ethanol for one minute each time, followed by three washes with deionized water, and air dry at room temperature.

[0046] Step 2: Supramolecular bottom layer deposition. Take the tannic acid component A2 and the branched polyethyleneimine component B2 from Preparation Example 2 and mix them evenly at a volume ratio of 1:1. Add 150 μL of the mixture to each well of the ELISA plate; incubate at 25°C for 30 minutes, aspirate the liquid from the wells, and rinse quickly 3 times with deionized water to remove unadsorbed polymers.

[0047] Step 3: Construction of thermodynamically gated interface. Add 150 μL of the dynamically gated functionalized buffer C2 provided in Preparation Example 2 to the well and incubate at 25°C for 45 minutes. After incubation, aspirate the liquid from the well and gently pat dry on absorbent paper without washing.

[0048] Step 4: Antibody-directed coating. Dilute the goat anti-human IgG capture antibody to 5 μg / mL using PBS buffer (pH 7.4), and add 100 μL of the diluted antibody solution to each well; incubate at 37°C for 1.5 hours.

[0049] Step 5: Curing and Drying. Discard the reaction solution in the wells and wash twice with PBS containing 0.05% Tween-20; then place the ELISA plate in an environment with a relative humidity of 20% and dry it at 25°C for 3 hours, then seal and store.

[0050] Example 2: This embodiment provides a nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate. It uses the low-concentration treatment reagent provided in Preparation Example 1, and the process parameters are biased towards rapid processing. The process includes the following steps: Step 1: Substrate pretreatment. Same as Example 1.

[0051] Step 2: Supramolecular bottom layer deposition. Take the tannic acid component A1 and branched polyethyleneimine component B1 from Preparation Example 1 and mix them evenly at a volume ratio of 1:1. Add 100 μL of the mixture to each well of the ELISA plate; incubate at 20°C for 15 minutes, aspirate the liquid from the wells, and rinse quickly 3 times with deionized water.

[0052] Step 3: Construction of thermodynamically gated interface. Add 100 μL of the dynamically gated functionalized buffer C1 provided in Preparation Example 1 to the well and incubate at 20°C for 30 minutes. After incubation, aspirate the liquid from the well, gently pat dry, and do not perform a washing step.

[0053] Step 4: Antibody-directed coating. Dilute the goat anti-human IgG capture antibody to 1 μg / mL using PBS buffer (pH 7.4), and add 100 μL of the diluted antibody solution to each well; incubate at 37°C for 1 hour.

[0054] Step 5: Curing and Drying. Discard the reaction solution in the wells and wash once with PBS containing 0.05% Tween-20; then place the ELISA plate in an environment with a relative humidity of 15% and dry it at 30°C for 2 hours, then seal and store.

[0055] Example 3: This embodiment provides a nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate. It utilizes the high-concentration treatment reagent provided in Preparation Example 3, and the process parameters are biased towards high loading and low-temperature protection. The process includes the following steps: Step 1: Substrate pretreatment. Same as Example 1.

[0056] Step 2: Supramolecular bottom layer deposition. Take the tannic acid component A3 and branched polyethyleneimine component B3 from Preparation Example 3 and mix them evenly at a volume ratio of 1:1. Add 200 μL of the mixture to each well of the ELISA plate; incubate at 25°C for 60 minutes, aspirate the liquid from the wells, and rinse rapidly 5 times with deionized water.

[0057] Step 3: Construction of thermodynamically gated interface. Add 150 μL of the dynamically gated functionalized buffer C3 provided in Preparation Example 3 to the well and incubate at 25°C for 60 minutes. After incubation, aspirate the liquid from the well, gently pat dry, and do not perform a washing step.

[0058] Step 4: Antibody directional coating. Dilute the goat anti-human IgG capture antibody to 10 μg / mL using PBS buffer (pH 8.0), and add 100 μL of the diluted antibody solution to each well; incubate at 4°C for 12 hours (overnight).

[0059] Step 5: Curing and Drying. Discard the reaction solution in the wells and wash twice with PBS containing 0.1% Tween-20; then place the ELISA plate in a vacuum drying oven at 20% relative humidity and dry at 25°C for 4 hours, then seal and store.

[0060] Comparative Example 1: This comparative example provides a traditional physical adsorption coating method. The difference from Example 1 is that steps two (supramolecular bottom layer deposition) and three (thermodynamically gated interface construction) are omitted; in step four, goat anti-human IgG capture antibody is dissolved in 50mM sodium carbonate / sodium bicarbonate buffer (pH 9.6) and directly added to the wells of the ELISA plate treated in step one for coating; the remaining steps (such as washing and drying) are the same.

[0061] Comparative Example 2: This comparative example provides a modification method without introducing a competing gating agent. The difference from Example 1 is that the dynamically gating functionalized buffer used in step three does not contain D-mannitol, but only 75 mM sodium tetraborate and 6% trehalose; the concentrations of other components, preparation methods, and subsequent process steps are the same.

[0062] (Note: This comparative example is used to verify the core role of D-mannitol in shielding non-specific adsorption of PEI and constructing substitution sites.) Comparative Example 3: This comparative example provides a modification method relying solely on the electrostatic adsorption of polyelectrolytes. The difference from Example 1 is that the solution used in step three does not contain sodium tetraborate and D-mannitol, but only a carbonate buffer solution (pH 9.3) containing 6% trehalose; the remaining steps are the same.

[0063] (Note: This comparative example is used to verify the necessity of borate groups for antibody targeting.) Comparative Example 4: This comparative example provides a modification method for lacking the supramolecular underlying framework. Compared with Example 1, the difference is that step two (supramolecular underlying deposition) is omitted, and the ELISA plate treated in step one is directly added to the dynamically gated functionalized buffer described in Example 1 for step three. All other steps are the same.

[0064] (Note: This comparative example is used to verify the contribution of the tannic acid / PEI substrate to the stability of antibody fixation.) Comparative Example 5: This comparative example provides a modification method that uses a conventional monosaccharide to replace a specific sugar alcohol. The difference from Example 1 is that, in preparing the dynamically gated functionalized buffer, an equimolar concentration of D-glucose is used instead of D-mannitol; all other components and steps remain the same.

[0065] (Note: This comparative example is used to verify the necessity of a polyol (mannitol) with a specific binding constant for achieving effective competitive substitution. Glucose has a weaker binding affinity to boric acid and is difficult to form an effective shielding layer.) Comparative Example 6: This comparative example provides a chemical cross-linking method (glutaraldehyde method). Compared with Example 1, the difference is that in step two, 1% glutaraldehyde solution is used instead of TA / PEI solution for 1 hour of treatment; in step three, after washing with PBS, boric acid / mannitol treatment is not performed, and antibody is directly added for coating; the remaining steps are the same.

[0066] Test Example 1: Validation of the effectiveness of surface chemical component modification and antibody immobilization This test example uses the curcumin colorimetric method to detect the binding of borate ions on the surface and the Coomassie Brilliant Blue staining method to characterize the total protein adsorption on the surface. Take the semi-finished ELISA plates from Examples 1-3 and Comparative Examples 1-4 before the antibody coating step (Comparative Example 1 used an untreated light plate), and add 100 μL of a 0.5 mg / mL curcumin ethanol solution containing 0.5 mg / mL oxalic acid to each well. After standing at room temperature for 15 minutes, discard the liquid in the wells and wash three times with anhydrous ethanol. After drying, add 100 μL of isopropanol to each well to dissolve the product generated on the surface, and measure the absorbance at 540 nm. Separately, take the finished ELISA plates from Examples 1-3 and Comparative Examples 1-4 after completing all preparation steps, and add 100 μL of Coomassie Brilliant Blue G-250 staining solution to each well. Incubate at room temperature for 30 minutes, and rinse with deionized water until the washings are colorless. 100 μL of an aqueous solution containing 50% methanol and 10% acetic acid was added to each well for desorption. After shaking, the absorbance at 595 nm was measured. The test results are shown in Table 1.

[0067] Table 1: Surface boron element response value and antibody immobilization data for each experimental group

[0068] Table 1 shows that the OD540 values ​​of Examples 1 to 3 ranged from 0.655 to 0.919, while the OD540 values ​​of Comparative Examples 1 and 4 were 0.045 and 0.126, respectively. The OD540 values ​​of Examples 1 to 3 were higher than those of Comparative Examples 1 and 4, indicating that the underlying network formed by tannic acid and branched polyethyleneimine allowed borate ions to remain on the polystyrene surface. Comparative Example 4 omitted the underlying assembly step, resulting in a low amount of borate adhering to the hydrophobic polystyrene surface.

[0069] Regarding protein immobilization, the OD595 value of Comparative Example 2 was 1.452, higher than that of Example 1 (1.229). The OD595 value of Comparative Example 3 was 0.892. Comparative Example 3 contained only a polyethyleneimine layer, exhibiting electrostatic adsorption capacity. Comparative Example 2 did not contain mannitol; the antibody and impurity proteins accumulated at the interface through electrostatic interactions and boric acid binding, resulting in the highest total protein adsorption. The system of Example 1 contained mannitol, which occupied some binding sites and shielded some non-specific electrostatic adsorption. The antibody molecules underwent a displacement reaction with boric acid through the Fc-terminal glycan chain, displacing mannitol and binding it at the interface. The total protein adsorption of Example 1 was lower than that of Comparative Example 2, but higher than the physical adsorption of Comparative Example 1 (0.433), indicating that the proposed method achieved borate modification and antibody binding on the polystyrene surface.

[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A nano-modification and high-density coating reagent composition for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, characterized in that, The composition comprises an independent interface self-assembly substrate solution and a dynamically gated functionalized buffer solution; The interface self-assembly substrate solution comprises the following components at the following concentrations: Tannic acid: 0.2-1.0 mg / mL; Branched polyethyleneimine: 0.1-0.5 mg / mL; Tris-HCl buffer solution at pH 7.8-8.2: balance; The dynamically gated functionalized buffer solution comprises the following components at the following concentrations: Sodium tetraborate: 50-100 mM; D-Mannitol: 50-200 mM; D-(+)-trehalose: 2%-10% (w / v); Carbonate buffer solution with pH 9.0-9.6: balance.

2. The nano-modification and high-density coating reagent composition for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 1, characterized in that, In the interface self-assembly bottom solution, the concentration of tannic acid is 0.6-1.0 mg / mL, the concentration of branched polyethyleneimine is 0.3-0.5 mg / mL, and the concentration of Tris-HCl buffer is 25-50 mM. In the dynamically gated functionalized buffer solution, the concentration of sodium tetraborate is 75-100 mM, the concentration of D-mannitol is 125-200 mM, the mass-volume ratio of D-(+)-trehalose is 6%-10% (w / v), and the concentration of carbonate buffer solution is 75-100 mM.

3. The nano-modification and high-density coating reagent composition for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 1, characterized in that, The branched polyethyleneimine has a weight-average molecular weight (Mw) of 20,000-30,000 Da; the interface self-assembly bottom layer solution is prepared by mixing component A containing tannic acid and component B containing branched polyethyleneimine in a volume ratio of 1:1 before use.

4. The nano-modification and high-density coating reagent composition for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 1, characterized in that, In the dynamically gated functionalized buffer solution, the molar ratio of sodium tetraborate to D-mannitol is 1:1 to 1:

2.

5. A nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate, using the reagent composition according to any one of claims 1-4, characterized in that, Includes the following steps: Step S1: Tannic acid and branched polyethyleneimine are mixed and added to the wells of an enzyme-linked immunosorbent assay (ELISA) plate for incubation and deposition. After washing, a substrate with a supramolecular underlayer is obtained. Step S2: Add dynamic gated functionalized buffer to the wells after step S1, incubate, remove the liquid and dry or semi-dry without washing to obtain a substrate with a thermodynamically gated interface. Step S3: Add the capture antibody solution to the wells after step S2 and incubate for coating; Step S4: Remove the antibody solution, wash, dry, and obtain the finished product.

6. The nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 5, characterized in that, Before step S1, the enzyme-linked immunosorbent assay (ELISA) plate is pretreated by immersion in anhydrous ethanol, followed by washing with deionized water and drying.

7. The nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 5, characterized in that, In step S1, the incubation temperature for deposition is 20-25℃ and the time is 15-60 minutes; in step S2, the incubation temperature is 20-25℃ and the time is 30-60 minutes.

8. The nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 5, characterized in that, In step S3, the pH of the capture antibody solution is 7.4-8.0, and the incubation and coating conditions are: incubation at 37°C for 1-1.5 hours, or incubation at 4°C for 10-14 hours.

9. The nano-modification and high-density coating process for the surface of an enzyme-linked immunosorbent assay (ELISA) plate according to claim 5, characterized in that, In step S4, the specific drying method is as follows: place the washed enzyme-linked immunosorbent assay (ELISA) plate in an environment with a relative humidity of 15%-20% and dry it at 25-30℃ for 2-4 hours.

10. An enzyme-linked immunosorbent assay (ELISA) plate, characterized in that, The enzyme-linked immunosorbent assay (ELISA) plate is prepared by a nano-modification and high-density coating process on the surface of an ELISA plate as described in any one of claims 5-9.

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