An immunoassay method based on amplification of high molecular signal

Abstract

The application discloses an immunological analysis method based on high-molecular amplification signal amplification and belongs to the technical field of biological medicines. The application combines protein-high-molecular conjugates with biological analysis together, and successfully develops a method for protein quantitative analysis. On the basis of taking silica microspheres as a solid-phase carrier material, target analysis antigen protein molecules are coated in advance, specific recognition reaction of antigen and antibody is utilized, antibody IgG-I macromolecules grafted with a photo initiator in advance are indirectly fixed on the surface of the microspheres, and a "competition" immunological detection method is constructed. The method is a rapid, simple, inexpensive and enzyme-free cascade amplification detection technology, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an immunoassay method based on polymer amplification signal amplification. Background Technology

[0002] Protein drugs have several advantages over other drugs, such as high specificity and good compatibility. Nevertheless, protein or peptide drugs still have some limitations, such as low cellular uptake, low bioavailability, susceptibility to environmental interference, and easy inactivation. These limitations are hindering the development and utilization of protein drugs.

[0003] Proteins play a vital role in various physiological activities within the human body, and the sensitive and specific detection of protein molecules is of great significance in molecular diagnostics research and the biomedical field. To improve the sensitivity of protein detection methods and lower the detection limit, developing a suitable signal amplification method is essential. In recent years, the technology of polymer-polymer protein conjugation has advanced rapidly. By conjugating polymers, proteins can be endowed with new functions and properties, showing broad application prospects in drug delivery, medical diagnostics, and many other fields. Among these applications, the aggregation of multiple polymeric repeating units can amplify signals, possessing potential value in bioanalysis. This invention develops a novel method for synthesizing protein-polymer conjugates and, based on this, develops a convenient and rapid signal amplification method for application in protein immunoassay.

[0004] Bovine serum albumin (BSA) is a globular protein that is widely recognized as the most popular template in various biochemical analyses due to its low cost, easy availability, and high structural homology with human serum albumin (HSA). Similarly, BSA plays a crucial role in the pathology of various human-related diseases, such as diabetes, mad cow disease, and kidney disease. Therefore, this invention develops a method for the rapid and accurate detection of BSA. Summary of the Invention

[0005] The purpose of this invention is to address the problem of low sensitivity in protein molecule detection in existing technologies by providing an immunoassay method based on polymer amplification signal amplification. This method realizes the application of signal amplification strategies based on Photo ATRP technology in bioanalysis and develops a rapid, simple, inexpensive, and enzyme-free cascade amplification detection technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An immunoassay method based on polymer amplification signal amplification includes the following steps:

[0008] (1) Surface modification of silica microspheres: APTMS and glutaraldehyde were used to chemically modify the surface of silica microspheres to obtain an aldehyde-amino-functionalized silica microsphere solution;

[0009] (2) Preparation of silica microspheres loaded with antigen protein molecules: The amino group of the antigen protein molecule is used to form a Schiff base by aldehyde-amine condensation with the aldehyde group on the surface of the aldehyde-amino-functionalized silica microspheres, thereby fixing the antigen protein molecule on the surface of the silica microspheres in the form of covalent crosslinking.

[0010] (3) Blocking non-specific binding sites on the surface of silica microspheres; using a sealing agent to block non-specific binding sites on the surface of microspheres;

[0011] (4) Preparation of antibody IgG-photoinitiator macromolecule: The strong oxidizing property of sodium periodate is used to open the six-membered ring of antibody IgG glycosylation site to form aldehyde group. Then, the photoinitiator is subjected to site-specific oxime reaction with aldehyde group to synthesize antibody IgG-photoinitiator macromolecule.

[0012] (5) Competitive immunoassay of antigen protein molecule content by photo-mediated ATRP reaction: The antigen protein molecule sample solution, the silica microsphere solution loaded with antigen protein molecules blocking non-specific sites, and the antibody IgG-photoinitiator macromolecule solution are added to the polymerization solution; under the action of a catalyst, the antibody IgG-photoinitiator macromolecule and the ligand and signal molecule monomer in the polymerization solution are copolymerized through Photo ATRP reaction to form antibody IgG-polymer conjugate with photoinitiator tag; using the specific recognition ability between antigen and antibody, the antigen protein molecules to be detected in the sample compete with the antigen protein molecules fixed on the surface of silica microspheres for the antibody IgG-polymer conjugate with photoinitiator tag, thereby indirectly fixing the antibody IgG-polymer conjugate on the surface of silica microspheres, and detecting the antigen protein molecule content by the change of fluorescence intensity.

[0013] Furthermore, the antigen protein molecules mentioned in the above methods include, but are not limited to, bovine serum albumin (BSA), ovalbumin (OVA), and human serum albumin (HSA).

[0014] Furthermore, the surface modification of the silica microspheres in step (1) of the above method specifically includes the following steps:

[0015] 1) Surface modification of silica microspheres: 1 mg / ml of silica microsphere suspension was mixed with 3-aminopropyltrimethoxysilane at a molar ratio of 1:10. The mixture was stirred continuously at 4 °C for 18 h. The precipitate was collected by centrifugation, washed three times with anhydrous ethanol and ultrapure water respectively, and then dispersed with ultrapure water to obtain an amino-functionalized silica microsphere solution for later use.

[0016] 2) Add 2.5 wt% glutaraldehyde solution to the amino-functionalized silica microsphere solution and stir rapidly at room temperature for 2 h. After the reaction is completed, centrifuge the resulting aldehyde-amino-functionalized silica microsphere mixture to obtain a precipitate, wash it three times with ultrapure water, and redisperse it in PBS to obtain the aldehyde-amino-functionalized silica microsphere solution.

[0017] Furthermore, the preparation of silica microspheres loaded with antigen protein molecules in step (2) of the above method specifically includes: mixing the aldehyde-amino-functionalized silica microsphere solution with the antigen protein molecule solution, stirring at 4°C for 24 h, washing and centrifuging to collect the precipitate and dispersing it in PBS solution to obtain silica microspheres loaded with antigen protein molecules.

[0018] The above step (3) blocks the non-specific binding sites on the surface of silica microspheres, specifically including: taking the prepared silica microspheres loaded with antigen protein molecules, adding serum diluted with PBS solution at a volume ratio of 1:20, mixing well and placing at 37℃ for continuous stirring reaction for 2 h to block the non-specific sites on the surface of silica microspheres that have not been bound; washing three times with PBST buffer, centrifuging to collect the precipitate and dispersing it in PBS solution to obtain a silica microsphere solution loaded with antigen protein molecules that blocks non-specific sites.

[0019] Furthermore, step (4) of the above method, the preparation of antibody IgG-photoinitiator macromolecules, specifically includes the following steps:

[0020] 1) Oxidation of IgG: Sodium periodate solution was added to an IgG solution with a concentration of 1 mg / mL, mixed quickly, and placed on crushed ice to react in the dark for 30 min; the strong oxidizing property of sodium periodate was used to open the six-membered ring of the glycosylation site of BSA antibody IgG to form an aldehyde group; after the oxidation reaction was completed, sodium metabisulfite solution was added to the oxidation reaction solution to stop the reaction for 5 min. After the reaction was completed, the solution was placed in PBS buffer and dialyzed at 4℃ for 24 h with stirring to obtain the IgG oxidation reaction solution; the molar ratio of IgG:sodium periodate:sodium metabisulfite was 1:10:10.

[0021] 2) Synthesis of IgG-photoinitiator macromolecules: In step 1), 2-aminooxy-3-bromo-2-methylbutyrate photoinitiator solution was added to the IgG-I oxidation reaction solution. After rapid mixing, the mixture was placed in a water bath at 37°C to carry out a site-specific oxime reaction between the photoinitiator and the aldehyde group for 1 h. After the reaction was completed, the mixture was placed in PBS buffer and dialyzed at 4°C for 24 h with stirring to obtain IgG-photoinitiator macromolecule solution; wherein the molar ratio of IgG to photoinitiator was 1:200.

[0022] Furthermore, step (5) of the above method, the light-mediated ATRP reaction competitively detecting BSA content, specifically includes the following steps:

[0023] 1) The silica microsphere solution blocking non-specific sites, the antigen protein molecule sample solution, and the antibody IgG-photoinitiator macromolecule solution were added to the polymerization solution and mixed to form a mixed system. The mixture was sealed with a rubber stopper to create a closed space and protected from light. After deoxygenation under nitrogen atmosphere for 20 min, the UV lamp was turned on to initiate the Photo ATRP reaction. The Photo ATRP reaction copolymerized to form an antibody IgG-polymer conjugate IgG-NIPAM-FOA with a photoinitiator tag. The target antigen protein molecules to be detected in the sample compete with the antigen protein molecules immobilized on the surface of the silica microspheres for the antibody IgG-polymer conjugate with the photoinitiator tag, indirectly immobilizing the antibody IgG-polymer conjugate on the surface of the microspheres. After UV irradiation for 30 min, the mixed system was exposed to air to terminate the reaction.

[0024] The concentration of the silica microsphere solution containing the antigen protein molecules blocking non-specific sites is 1 mg / ml, and the concentration of the antibody IgG-photoinitiator macromolecule solution is 50 ug / ml; the concentration of the antigen protein molecule sample solution is 0.1 ng / ml to 5000 ng / ml; the volume ratio of the silica microsphere solution containing the antigen protein molecules blocking non-specific sites, the antigen protein molecule sample solution, and the antibody IgG-photoinitiator macromolecule solution is 100 μL: 50 μL: 100 μL.

[0025] 2) After the reaction was terminated, the fluorescence intensity of the solution at an excitation wavelength of 468 nm and an emission wavelength of 512 nm was measured using a fluorescence spectrometer. The concentration of antigen protein molecules in the sample solution was obtained by comparing it with the standard curve.

[0026] Furthermore, the above-mentioned immunoassay method based on polymer amplification signal amplification is applied in immunoassay.

[0027] The principle of the method of this invention:

[0028] To apply signal amplification strategies based on Atom Transfer Radical Polymerization (ATRP) technology to bioanalysis, a competitive immunoassay for the quantitative analysis of BSA will be constructed, drawing upon the mechanisms and methods for constructing protein-polymer conjugates. The surface of silica microspheres will be chemically modified with 3-aminopropyltrimethoxysilane (APTMS) and glutaraldehyde (GA). A Schiff base will be formed by aldehyde-amine condensation between the amino groups on BSA and the aldehyde groups on the microsphere surface, thus covalently immobilizing BSA on the surface. A sealing agent (serum) will be used to block non-specific binding sites on the microsphere surface to prevent false positives. Then, utilizing the specific recognition ability between antigen and antibody, the target analyte BSA in the sample and the BSA immobilized on the microsphere surface will compete for quantification of BSA IgG-photoinitiator macromolecules tagged with a photoinitiator. After a simple centrifugation and washing step, the IgG-photoinitiator macromolecules will be indirectly and selectively immobilized on the microsphere surface (see principle below). Figure 1 Finally, under ultraviolet light irradiation, fluorescent polymers were directionally grafted onto the immobilized photoinitiator macromolecules using a signal amplification strategy based on Photo ATRP technology. As the BSA concentration in the solution increased, the content of photoinitiator macromolecules on the surface of the silica microspheres decreased, and the overall fluorescence intensity of the solution also decreased. Within a certain range, a negative linear correlation between BSA concentration and fluorescence intensity was established, and actual samples were analyzed.

[0029] The significant advantages of this invention are:

[0030] (1) According to the method of the present invention, a method for the quantitative analysis of BSA is successfully developed by combining protein-polymer conjugates with bioanalysis through photocatalytic surface-initiated polymerization technology. Based on silica microspheres as solid-phase carrier material, the target analyte BSA is pre-coated, and the anti-BSA IgG-I macromolecules grafted with photoinitiators are indirectly immobilized on the surface of the microspheres by utilizing the specific recognition reaction of antigen and antibody, thus constructing a "competitive" immunoassay method.

[0031] (2) The sensing method of the present invention combines the advantages of silica material and photocatalytic surface-initiated ATRP polymerization. Compared with traditional signal amplification methods, it is a novel, convenient, simple and enzyme-free cascade amplification method. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of an immunoassay method based on polymer amplification signal amplification.

[0033] Figure 2 Electrophoresis diagram (left) and fluorescence spectrum (right) of anti-BSA IgG-NIPAM-FOA protein.

[0034] Figure 3 The change in Zeta potential before and after modification with silica microspheres.

[0035] Figure 4 The particle size changes after modification with silica microspheres are shown.

[0036] Figure 5 Standard curve of BSA concentration versus fluorescence intensity.

[0037] Figure 6 This is to ensure the specificity of the BSA detection method. Detailed Implementation

[0038] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0039] Example 1

[0040] An immunoassay method based on polymer amplification signal amplification includes the following steps:

[0041] (1) Surface modification of silica microspheres: The surface of silica microspheres was chemically modified using APTMS and glutaraldehyde to obtain an aldehyde-amino-functionalized silica microsphere (SiO2-NH2-CHO) solution. The specific steps included:

[0042] 1) Surface modification of silica microspheres: The silica microspheres (SiO2) used in this experiment were obtained from Suzhou Nanomicro Technology Co., Ltd. A suspension of silica microspheres with a concentration of 1 mg / ml was prepared and then ultrasonically dispersed for 30 min. 600 μL of the suspension was added to a reaction vessel. Then, 3-aminopropyltrimethoxysilane (APTMS) was added to the vessel at a molar ratio of 1:10 (silica microspheres to 3-aminopropyltrimethoxysilane). The mixture was continuously stirred at 4 °C for 18 h. The resulting amino-functionalized silica microspheres (SiO2-NH2) were collected by centrifugation at 5000 rpm for 10 min, washed three times with anhydrous ethanol and ultrapure water respectively, and then dispersed with ultrapure water to obtain a 1 mg / ml amino-functionalized silica microsphere (SiO2-NH2) solution for later use.

[0043] 2) Add 2 mL of 2.5 wt% glutaraldehyde solution to the amino-functionalized silica microspheres (SiO2-NH2) solution and stir rapidly at room temperature for 2 h. After the reaction is completed, the resulting aldehyde-amino-functionalized silica microspheres (SiO2-NH2-CHO) mixture is centrifuged at 5000 rpm for 10 min to obtain a precipitate, which is washed three times with ultrapure water and redispersed in PBS to obtain a 1 mg / mL aldehyde-amino-functionalized silica microspheres (SiO2-NH2-CHO) solution.

[0044] (2) Preparation of silica microspheres loaded with BSA (SiO2-BSA): The amino groups on BSA are used to form Schiff bases by condensation of aldehyde groups on the surface of aldehyde-amino-functionalized silica microspheres with aldehyde groups, thereby fixing BSA on the surface of silica microspheres in the form of covalent crosslinking.

[0045] Accurately measure 200 μL of 1 mg / mL aldehyde-amino-functionalized silica microspheres (SiO2-NH2-CHO) solution, add 1 mL of BSA solution (2 mg / mL), and stir at 200 rpm for 24 h at 4 °C. Wash three times with PBS, centrifuge at 3000 rpm for 10 min, collect the precipitate and disperse it in PBS solution to obtain 1 mg / mL BSA-loaded silica microspheres (SiO2-BSA), which are stored at 4 °C for later use. Collect the supernatant after centrifugation for protein content determination.

[0046] (3) Blocking non-specific binding sites on the surface of silica microspheres: Take 1 mL of the prepared 1 mg / mL silica microspheres loaded with BSA (SiO2-BSA), add 1 mL of serum (PBS solution diluted 1:20), mix well and place at 37℃ for 2 h with continuous stirring to block the non-specific binding sites on the surface of silica microspheres. Wash three times with PBST buffer, centrifuge at 3000 rpm for 10 min, collect the precipitate and disperse it in PBS solution to obtain a 1 mg / mL solution of silica microspheres loaded with BSA blocking non-specific sites;

[0047] (4) Preparation of BSA antibody IgG-photoinitiator macromolecules: The strong oxidizing property of sodium periodate is used to open the six-membered ring at the glycosylation site of BSA antibody IgG, forming an aldehyde group. Subsequently, the photoinitiator is subjected to a site-specific oxime reaction with the aldehyde group to synthesize BSA antibody IgG-photoinitiator macromolecules. Specifically, the following steps are included:

[0048] 1) Oxidation of BSA antibody IgG: Add sodium periodate solution to a 1 mg / mL BSA antibody IgG solution, mix quickly, and place on crushed ice to react in the dark for 30 min; after the oxidation reaction is complete, add sodium metabisulfite solution to the oxidation reaction solution, stop the reaction for 5 min, and after the reaction is complete, place in PBS buffer, stir and dialyze at 4℃ for 24 h to obtain IgG oxidation reaction solution; wherein the molar ratio of IgG: sodium periodate: sodium metabisulfite is 1:10:10;

[0049] 2) Synthesis of BSA antibody IgG - photoinitiator macromolecule: In step 1), a photoinitiator (2-aminooxy-3-bromo-2-methylbutyrate) solution was added to the IgG-I oxidation reaction solution. After rapid mixing, the mixture was placed in a water bath at 37°C for 1 hour. After the reaction was complete, the mixture was placed in PBS buffer and dialyzed at 4°C for 24 hours with stirring to obtain a BSA antibody IgG - photoinitiator macromolecule solution; wherein the molar ratio of BSA antibody IgG to photoinitiator was 1:200. The photoinitiator solid was dissolved in PBS solution to prepare a photoinitiator solution.

[0050] (5) Photo-mediated ATRP (Photo ATRP) reaction for BSA content detection: A BSA sample solution, a solution of BSA-loaded silica microspheres blocking non-specific sites, and a BSA antibody-IgG photoinitiator macromolecule solution are added to the polymerization solution. Under the action of a catalyst, the BSA antibody-IgG photoinitiator macromolecules copolymerize with ligands and signal molecule monomers in the polymerization solution via Photo ATRP reaction to form BSA antibody-IgG-polymer conjugates. Utilizing the specific recognition ability between antigen and antibody, the target analyte BSA in the sample competes with the BSA immobilized on the surface of the silica microspheres for the photoinitiator-tagged IgG-polymer conjugates, indirectly immobilizing the IgG-polymer conjugates on the microsphere surface. Specifically, the following steps are included:

[0051] 1) The BSA-loaded silica microsphere solution, BSA sample solution, and BSA antibody IgG-photoinitiator macromolecule solution were added to the polymerization solution and mixed to form a mixed system. The mixture was sealed with a rubber stopper and protected from light. After deoxygenation under nitrogen atmosphere for 20 min, the UV lamp was turned on to start the Photo ATRP reaction. After UV irradiation for 30 min, the mixed system was exposed to air to terminate the reaction.

[0052] The polymerization solution consisted of 800 ml of solvent water and 20 mg of catalyst CuBr, mixed evenly at room temperature, followed by the addition of 15 ml of tris(2-dimethylaminoethyl)amine (Me6TERN), 10 mg of signal molecule monomer N-isopropylacrylamide (NIPAM), and 10 mg of signal molecule fluorescent monomer fluorescein-o-acrylate (FOA).

[0053] The concentration of the silica microsphere solution loaded with BSA that blocks non-specific sites is 1 mg / ml, and the concentration of the BSA antibody IgG-photoinitiator macromolecule solution is 50 ug / ml; the concentration of the BSA sample solution is 0.1 ng / ml to 5000 ng / ml.

[0054] The volume ratio of the BSA-loaded silica microsphere solution, the BSA sample solution, and the BSA antibody IgG-photoinitiator macromolecule solution that blocks non-specific sites is 100 μL: 50 μL: 100 μL.

[0055] The copolymerization forms a BSA antibody IgG-polymer conjugate, which is an IgG-NIPAM-FOA conjugate.

[0056] 2) The fluorescence intensity of the solution after the reaction was terminated was measured using a fluorescence spectrometer at an excitation wavelength of 468 nm and an emission wavelength of 512 nm. The concentration of BSA in the sample solution was obtained by comparing it with the standard curve.

[0057] Electrophoresis image of BSA antibody IgG macromolecule grafted with NIPAM (N-isopropylacrylamide) protein, results are as follows: Figure 2 As shown on the left, the original IgG band is clearly visible, and it can be clearly observed that the treatment with sodium periodate and photoinitiator has no effect on the molecular weight of IgG protein. However, after the Photo ATRP reaction, the original protein band almost disappears, and a high molecular weight, broadly distributed band appears instead, proving that NIPAM monomers are grafted onto the IgG-I macromolecule.

[0058] The fluorescence spectrum of IgG-NIPAM-FOA is shown in the image. Figure 2 (Right). The original IgG band, as shown in the image, remained clear even after oxidation initiation, indicating that the band was unaffected. After polymerization of NIPAM and FOA fluorescent monomers, the original protein band almost disappeared, replaced by a blurred fluorescent band, demonstrating that NIPAM and FOA monomers were grafted onto the IgG macromolecule. Spectral scanning revealed a fluorescent sheen on the band, further confirming the successful polymerization of NIPAM and FOA monomers.

[0059] The potentials before and after modification with silica microspheres are shown in the following results. Figure 3As shown, the zeta potential of the original SiO2 sample was -38.56 mV, indicating good stability of the solution. After APTMS modification and glutaraldehyde treatment, the zeta potential of the solution system changed to a negatively charged -3.57 mV, and the absolute value remained basically unchanged, indicating that the system was still in an unstable state prone to aggregation. This is because the aldehyde group (-CHO) itself is hydrophilic, and the electronegativity of O and C is greater than that of H (O is greater than that of C). The electrons of H will be biased, resulting in H being electronegative and O being negatively charged, thus making the aldehyde group as a whole negatively charged. After the Photo ATRP reaction, the zeta potential changed from negative to positive, and the absolute value increased, which may be caused by the polymer chains grafted on the surface of the microspheres. The control group was SiO2 without any modification. Compared with the original sample, the absolute value of the zeta potential increased, which may be because the monomers were non-specifically adsorbed on the surface of the silica microspheres, increasing the repulsive force between particles.

[0060] The particle size after silica microsphere modification is as follows: Figure 4 As shown. Figure 4 As can be seen, the particle size of silica microspheres (SiO2) changed after a series of modifications. The original SiO2 particles had a particle size of approximately 1 µm. Then, the particles modified with APTMS were treated with glutaraldehyde, coating their surface with an aldehyde group to form SiO2-NH-CHO particles. The particle size increased to 1.433 µm, and the dispersibility improved. Finally, after polymerization, polymer chains (SiO2-I-pNIPAM-o-FOA) were grafted in situ at the initiator sites on the SiO2-I surface. The particle size increased significantly to 4.316 µm, and the dispersibility further decreased. These particle size changes demonstrate that each step of the modification of the silica microspheres was successful, and that polymer chains were ultimately grafted in situ onto the surface of the anti-BSA IgG-I macromolecule using its initiator sites.

[0061] Example 2: Construction of the Standard Curve

[0062] BSA standard solutions of different concentrations were prepared (concentrations of 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.6 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, 32 ng / mL, 64 ng / mL, 128 ng / mL, 512 ng / mL, 1024 ng / mL, 2048 ng / mL, 4096 ng / mL, 5000 ng / mL, and 10000 ng / mL), with three parallel samples for each concentration. The blank control was the original silica microsphere solution (SiO2). A solution of BSA-loaded silica microspheres with 1 mg / ml blocking nonspecific sites, BSA standard solutions of different concentrations, and a 50 μg / ml BSA antibody IgG-photoinitiator macromolecule solution were added to a polymerization solution [800 ml of solvent water and 20 mg of catalyst CuBr, mixed thoroughly at room temperature, followed by 15 ml of tris(2-dimethylaminoethyl)amine (Me6TERN), 10 mg of signal molecule monomer N-isopropylacrylamide (NIPAM), and 10 mg of signal molecule fluorescent monomer fluorescein-o-acrylate (FOA)] at a volume ratio of 100 μL: 50 μL: 100 μL. The mixture was then sealed with a rubber stopper to create a closed system and protected from light. After deoxygenation under nitrogen atmosphere for 20 min, the UV lamp was turned on to initiate the Photo ATRP reaction. After UV irradiation for 30 min, the mixture was exposed to air to terminate the reaction. The solution after the reaction was terminated was measured using a fluorescence spectrometer with an excitation wavelength of 468 nm and an emission wavelength of 512 nm. The fluorescence intensity at the location was measured. A standard curve was plotted with BSA concentration on the x-axis and fluorescence intensity on the y-axis.

[0063] Standard curve see Figure 5 The fluorescence intensity and Log CBSA showed a good linear relationship in the range of 0.1 ng / mL to 5000 ng / mL, with the linear equation being y = 2.91 × 10⁷ - 4318363 * Log CBSA, R² = 0.9921.

[0064] Example 3: Method for detecting BSA.

[0065] A. Specificity of BSA detection methods.

[0066] This invention selected three groups of snail enzyme (SA), bovine serum albumin (BSA), and ovalbumin (OVA) at a uniform concentration of 1 mg / ml as experimental subjects for specificity testing, and also set up a PBS buffer solution group and a blank control group as references. Figure 6As shown, when the fluorescence intensity of the solution was measured at an excitation wavelength of 468 nm and an emission wavelength of 512 nm, only the target analyte BSA showed the highest fluorescence signal value. The signal values ​​of other substances were all low, indicating that the detection method can specifically detect BSA without interference from other substances, and that this method has good specificity for detecting BSA.

Claims

1. An immunoassay method based on polymer amplification signal amplification, characterized in that, Includes the following steps: (1) Surface modification of silica microspheres: APTMS and glutaraldehyde were used to chemically modify the surface of silica microspheres to obtain an aldehyde-amino-functionalized silica microsphere solution; (2) Preparation of silica microspheres loaded with antigen protein molecules: The amino group of the antigen protein molecule is used to form a Schiff base by aldehyde-amine condensation with the aldehyde group on the surface of the aldehyde-amino-functionalized silica microsphere, thereby fixing the antigen protein molecule on the surface of the silica microsphere in the form of covalent crosslinking, and obtaining silica microspheres loaded with antigen protein molecules. (3) Blocking non-specific binding sites on the surface of silica microspheres; using a sealing agent to block non-specific binding sites on the surface of microspheres; (4) Preparation of antibody IgG-photoinitiator macromolecule: The strong oxidizing property of sodium periodate is used to open the six-membered ring of antibody IgG glycosylation site to form aldehyde group. Then, the photoinitiator is subjected to site-specific oxime reaction with aldehyde group to synthesize antibody IgG-photoinitiator macromolecule. (5) Competitive immunoassay of antigen protein molecule content by photo-mediated ATRP reaction: An antigen protein molecule sample solution, a silica microsphere solution containing antigen protein molecules loaded with non-specific sites, and an antibody IgG-photoinitiator macromolecule solution are added to a polymerization solution; under the action of a catalyst, the antibody IgG-photoinitiator macromolecule and the ligand and signal molecule monomer in the polymerization solution copolymerize through a Photo ATRP reaction to form an antibody IgG-polymer conjugate tagged with a photoinitiator; utilizing the specific recognition ability between antigen and antibody, the antigen protein molecules to be detected in the sample compete with the antigen protein molecules immobilized on the surface of silica microspheres for the antibody IgG-polymer conjugate tagged with a photoinitiator, indirectly immobilizing the antibody IgG-polymer conjugate on the surface of silica microspheres, and detecting the antigen protein molecule content by changes in fluorescence intensity; the antigen protein molecule is bovine serum albumin (BSA); Step (4) specifically includes the following steps: 1) Oxidation of IgG: Add sodium periodate solution to IgG solution with a concentration of 1 mg / mL, mix quickly, and place on crushed ice to react in the dark for 30 min; utilize the strong oxidizing property of sodium periodate to open the six-membered ring of BSA antibody IgG glycosylation site to form aldehyde group; after the oxidation reaction is complete, add sodium metabisulfite solution to the oxidation reaction solution, stop the reaction for 5 min, after the reaction is complete, place in PBS buffer, stir and dialyze for 24 h to obtain IgG oxidation reaction solution; wherein the molar ratio of IgG:sodium periodate:sodium metabisulfite is 1:10:10; 2) Synthesis of IgG-photoinitiator macromolecules: In step 1), a photoinitiator solution of 2-aminooxy-3-bromo-2-methylbutyrate was added to the IgG oxidation reaction solution. After rapid mixing, the mixture was placed in a water bath at 37°C to allow the photoinitiator to undergo a site-specific oxime reaction with the aldehyde group for 1 hour. After the reaction was completed, the mixture was placed in PBS buffer and dialyzed at 4°C for 24 hours with stirring to obtain an IgG-photoinitiator macromolecule solution; wherein the molar ratio of IgG to photoinitiator was 1:

200. Step (5) specifically includes the following steps: 1) A mixture of silica microsphere solution containing antigen protein molecules with blocked non-specific sites, antigen protein molecule sample solution, and antibody IgG-photoinitiator macromolecule solution was added to the polymerization solution and mixed thoroughly. The mixture was then sealed with a rubber stopper to create a closed space and protected from light. After deoxygenation under nitrogen for 20 minutes, the UV lamp was turned on to initiate the Photo ATRP reaction. The Photo ATRP reaction copolymerized to form an antibody IgG-polymer conjugate IgG-NIPAM-FOA tagged with the photoinitiator. The target antigen protein molecules to be detected in the sample compete with the antigen protein molecules immobilized on the surface of the silica microspheres for the antibody IgG-polymer conjugate tagged with the photoinitiator, indirectly immobilizing the antibody IgG-polymer conjugate on the surface of the microspheres. After UV irradiation for 30 minutes, the mixture was exposed to air to terminate the reaction. 2) After the reaction was terminated, the fluorescence intensity of the solution was measured using a fluorescence spectrometer at an excitation wavelength of 468 nm and an emission wavelength of 512 nm. The concentration of antigen protein molecules in the sample solution was obtained by comparing it with the standard curve.

2. The method according to claim 1, characterized in that: The surface modification of the silica microspheres in step (1) specifically includes the following steps: 1) Surface modification of silica microspheres: 1 mg / ml silica microsphere suspension was mixed with 3-aminopropyltrimethoxysilane at a molar ratio of 1:

10. The mixture was stirred continuously at 4 °C for 18 h. The precipitate was collected by centrifugation, washed three times with anhydrous ethanol and ultrapure water respectively, and then dispersed with ultrapure water to obtain an amino-functionalized silica microsphere solution for later use. 2) Add 2.5 wt% glutaraldehyde solution to the amino-functionalized silica microsphere solution and stir rapidly at room temperature for 2 h. After the reaction is completed, centrifuge the resulting aldehyde-amino-functionalized silica microsphere mixture to obtain a precipitate, wash it three times with ultrapure water, and redisperse it in PBS to obtain the aldehyde-amino-functionalized silica microsphere solution.

3. The method according to claim 1, characterized in that: The preparation of silica microspheres loaded with antigen protein molecules in step (2) specifically includes: mixing aldehyde-amino-functionalized silica microsphere solution with antigen protein molecule solution, stirring at 4°C for 24 h, washing and centrifuging to collect the precipitate and dispersing it in PBS solution to obtain silica microspheres loaded with antigen protein molecules.

4. The method according to claim 1, characterized in that: Step (3) involves blocking the non-specific binding sites on the surface of silica microspheres. Specifically, this includes: taking the prepared silica microspheres loaded with antigen protein molecules, adding serum diluted in PBS solution at a volume ratio of 1:20, mixing well, and stirring continuously at 37°C for 2 hours to block the non-specific binding sites on the surface of the silica microspheres; washing three times with PBST buffer, centrifuging to collect the precipitate and dispersing it in PBS solution to obtain a silica microsphere solution loaded with antigen protein molecules that has blocked the non-specific sites.

5. The method according to claim 1, characterized in that: In step (5), step 1), the concentration of the silica microsphere solution containing the antigen protein molecules blocking non-specific sites is 1 mg / ml, and the concentration of the antibody IgG-photoinitiator macromolecule solution is 50 ug / ml; the concentration of the antigen protein molecule sample solution is 0.1 ng / ml to 5000 ng / ml; the volume ratio of the silica microsphere solution containing the antigen protein molecules blocking non-specific sites, the antigen protein molecule sample solution, and the antibody IgG-photoinitiator macromolecule solution is 100 μL: 50 μL: 100 μL.

6. The application of the method as described in claim 1 in immune detection.

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