Preparation method and application of surface protein imprinted polymer relying on enzyme cascade reaction

Abstract

The present invention relates to a preparation method and application of a surface protein imprinted polymer relying on an enzyme cascade reaction; the surface imprinted polymer is prepared under conditions of high monomer concentration by mediating local free radical polymerization through an enzyme cascade reaction. The local free radical polymerization reaction is initiated by an initiation system consisting of GOx / HRP, acetylacetone and glucose fixed on the surface of a nanocarrier, so that the polymerization reaction proceeds around the surface of the nanocarrier. The random freedom of free radicals in the entire solution is avoided, and even at high monomer concentrations, no agglomeration phenomenon occurs during polymerization. At the same time, the method is carried out under conditions of high monomer concentration, which is conducive to the sufficient pre-assembly of functional monomers and template proteins, thereby improving the imprinting efficiency. The imprinting capacity of the synthesized nanoimprinted microspheres for lysozyme is 145.3 mg g ‑1 , the imprinting factor is 14.2.

Description

Technical Field

[0001] The present invention belongs to the field of biological separation and purification technology. Specifically, it relates to the preparation and application of a surface protein-imprinted polymer. In particular, it relates to a method for preparing a surface protein-imprinted polymer that relies on an enzyme cascade reaction and its application in the separation and purification of proteins in complex biological samples. Background Art

[0002] Molecular imprinting techniques (MIPs) are antibody-receptor relationships, characterized by customized binding sites that complement the template molecule in shape, size, and functional groups. Due to their unique structural predictability, recognition specificity, and versatility, MIPs have been widely applied across various fields. While widely used and proven particularly effective for low-molecular-weight proteins (with molecular weights under 1500), selective recognition of macromolecules such as proteins and viruses remains a significant challenge, primarily due to difficulties in template removal and low imprinting factors.

[0003] To address these issues, researchers have proposed imprinting techniques such as epitope imprinting and surface imprinting. Epitope imprinting uses a peptide segment on the surface of a macromolecule as a template for imprinting. However, epitopes are extremely difficult to identify and synthesize. In contrast, surface imprinting uses the entire molecule as a template, forming a thin imprinted polymer layer on the surface of the nanocarrier. This reduces the mass transfer resistance of the template molecule, helping to improve elution efficiency and accessibility of the imprinted cavity. Consequently, surface imprinting has garnered significant research interest.

[0004] Methods for preparing surface imprinted polymers include sol-gel method, auto-oxidative polymerization method, surface grafting polymerization method, etc. Among them, the first two methods rely on the hydrolysis of silane monomers or the auto-oxidative polymerization of dopamine, which have the problem of poor monomer compatibility and difficulty in fully interacting with a variety of template molecules. Surface grafting polymerization is to modify vinyl groups on the surface of particles and perform free radical copolymerization with various vinyl-containing monomers. It can meet the needs of template molecules with different characteristics and is the most commonly used method for preparing surface imprinted polymers. However, this method requires a low monomer concentration, which leads to poor imprinting efficiency.

[0005] Fu et al. modified the surface of SiO2 nanoparticles with vinyl groups and carried out surface graft polymerization at a total monomer and crosslinker concentration of about 0.4 wt%. The adsorption capacity of the obtained imprinted polymer was only 11.3 mg g -1 , the imprinting factor is 1.23. Li et al. studied the adsorption capacity of surface imprinted polymers prepared by surface graft polymerization under the condition of a total monomer and cross-linker concentration of 0.6 wt% (about 78.0 mg g -1) has improved, but the imprinting factor (1.69) is still low. Increasing the monomer concentration will lead to aggregation and even gelation problems. Fu, Jing et al. prepared imprinted polymers by traditional surface grafting polymerization under high monomer concentration (about 7wt%) and found particle aggregation and even gelation problems. Therefore, when preparing imprinted polymers with high imprinting efficiency by traditional surface imprinting polymerization, there is a contradiction between the high monomer concentration requirement and the gelation problem. Summary of the Invention

[0006] In order to avoid the occurrence of agglomeration when preparing surface-imprinted polymers using macromolecules such as proteins as templates, traditional surface grafting polymerization methods require that the process be carried out under low monomer concentration conditions. However, low monomer concentrations will limit the pre-assembly of functional monomers and template molecules, thereby affecting the imprinting efficiency of the imprinted polymer. Therefore, the present invention uses an enzyme cascade reaction to mediate local free radical polymerization to achieve the preparation of surface-imprinted polymers under high monomer concentration conditions. Specifically, the present invention utilizes an initiation system consisting of glucose oxidase / horseradish peroxidase, acetylacetone and glucose fixed on the surface of a nanocarrier to initiate the local free radical polymerization reaction, so that the polymerization reaction proceeds around the surface of the nanocarrier. The present invention avoids the random liberation of free radicals in the entire solution, and agglomeration will not occur even when polymerization is carried out at a high monomer concentration. At the same time, the method is carried out under high monomer concentration conditions, which is conducive to the sufficient pre-assembly of functional monomers and template proteins, thereby improving the imprinting efficiency.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing a surface protein imprinted polymer by enzyme cascade reaction comprises the following steps:

[0009] (1) Carboxyl modification of the nanoparticle surface: 3-aminopropyltriethoxysilane is hydrolyzed on the nanoparticle surface, and amino nanoparticles are obtained after washing;

[0010] (2) dispersing the amino-modified nanoparticles in tetrahydrofuran solution, adding succinic anhydride, stirring at room temperature for 1-8 hours, and then washing to obtain surface carboxylated nanoparticles;

[0011] (3) The washed carboxylated nanoparticles were dispersed in phosphate buffer, and glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1. Subsequently, GOx and HRP were covalently coupled to the surface of the nanoparticles under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

[0012] (4) washing the product of step (3) with deionized water to obtain a nano-ionic dispersion of the immobilized enzyme;

[0013] (5) Add the template protein, main monomer, functional monomer, and cross-linker to pH 5.0 phosphate buffer and pre-assemble for 10-60 minutes to obtain a pre-assembly solution;

[0014] (6) adding the nanoparticle dispersion of the immobilized enzyme, acetylacetone, and glucose to the pre-assembled solution of step (5) to initiate a polymerization reaction; and stopping the reaction after polymerization at room temperature for 4-8 hours to obtain a polymerization solution;

[0015] (7) The polymer solution in step (6) is centrifuged and washed 3-6 times to obtain a surface protein imprinted polymer.

[0016] The nanoparticles include SiO2, Fe3O4, Fe3O4-Cu, MOF, COF, Au, CeO2 or Ag.

[0017] The concentration of 3-aminopropyltriethoxysilane in step (1) is 0.01-0.20M.

[0018] In step (2), the amination nanoparticles are dispersed in the tetrahydrofuran solution at a particle concentration of 0.01-0.10 mg mL -1 , add succinic anhydride at a concentration of 0.01-0.20M.

[0019] In step (3), the washed carboxylated nanoparticles are dispersed in phosphate buffer at a particle concentration of 0.01-0.10 mg mL -1 Glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 0.05-1.0 M.

[0020] The nano-ion dispersion particle concentration of the immobilized enzyme obtained in step (4) is 0.01-0.10 mg mL -1 .

[0021] In the step (5), the template protein concentration is 0.01-0.10 mM, the main monomer concentration is 0.05-1.00 M, the functional monomer concentration is 1.74-34.80 mM, the methacrylic acid concentration is 1.74-34.80 mM, and the cross-linking agent concentration is 0.67-13.35 mM; the template protein in the pre-assembly solution is various acidic, neutral and alkaline proteins such as lysozyme, bovine serum albumin, cytochrome C, bovine hemoglobin, human immunoglobulin, amyloid protein, etc.; the main monomer includes N-isopropylacrylamide and acrylamide; the functional monomer includes methacrylic acid, acrylamide, styrene, N-tert-butylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and dimethylaminoethyl methacrylate; and the cross-linking agent is N,N-methylenebisacrylamide, polyethylene glycol diacrylate, and polyglutamic acid polypeptide cross-linking agent.

[0022] In the step (6), the nanoparticle dispersion of immobilized enzyme is added to the pre-assembly solution in an amount of 4.0 mL, 0.01-0.10 mg mL -1 , acetylacetone concentration 0.05-0.20M, glucose concentration 0.05-0.20M.

[0023] The surface protein imprinting polymer prepared by the method of the invention by relying on enzyme cascade reaction is applied to the extraction and purification of proteins in complex biological samples.

[0024] The complex biological samples include egg white and the like.

[0025] The imprinting efficiency test of the surface protein imprinted polymer in the present invention was carried out using a common method reported in the literature, specifically as follows:

[0026] (1) The surface protein imprinted polymer (MIP) and the corresponding non-imprinted polymer (NIP) prepared by the synthesis method of the present invention are vacuum dried.

[0027] (2) Weigh approximately 10 mg of the dried MIP and corresponding NIP in step (1) into centrifuge tubes (6 each), add pH 5.0 phosphate buffer (8 mL) to fully swell, and remove the solution after the solution reaches swelling equilibrium.

[0028] (3) Prepare 1.0 mg mL -1 The lysozyme stock solution was diluted to the initial concentration (C0) of 0.1, 0.2, 0.4,

[0029] 0.6, 0.8 and 1.0 lysozyme solutions.

[0030] (4) The lysozyme solutions of different concentrations prepared in step (3) were sequentially added to the centrifuge tubes containing MIP or NIP in step (2), with 8 mL added to each centrifuge tube, and incubated at room temperature for 1 h.

[0031] (5) Determine the remaining lysozyme concentration in each centrifuge tube after incubation in step (4) by UV-visible spectrophotometer

[0032] (C).

[0033] (6) Calculate the equilibrium adsorption capacity (Q), Q = 0.8*(C0-C).

[0034] (7) The maximum saturated adsorption capacity of MIP and NIP was calculated according to the Langmuir adsorption model. The imprinting factor (IF) is the ratio of the maximum saturated adsorption capacity of MIP to the maximum saturated adsorption capacity of NIP.

[0035] (8) According to the calculation formula and method in steps (6) and (7), the maximum saturated adsorption capacity is 145.3 mg g -1 , the imprinting factor is 14.3.

[0036] Advantages and excellent effects

[0037] This enzyme-mediated localized free radical polymerization overcomes the aggregation and even gelation issues that occur when preparing surface protein-imprinted polymers using traditional surface grafting polymerization at high monomer concentrations. Furthermore, high monomer concentrations allow for sufficient pre-assembly with the template protein, increasing the number of imprinting sites and significantly improving imprinting efficiency.

[0038] The present invention uses nanoparticles as carriers, and the carrier options for the enzymatic reaction are relatively wide, and can be applied to nanoparticles such as SiO2, Fe3O4, Fe3O4-Cu, MOF, COF, Au, CeO2, and Ag. By modifying the nanoparticle surface with horseradish peroxidase and glucose oxidase as the active core, the present invention causes the polymerization reaction to proceed around the nanoparticles, avoiding the occurrence of agglomeration or even gelation under conditions of high monomer concentrations. Simultaneously, due to the immobilization of the enzyme, the enzymatic reaction activity is increased, the polymerization rate is accelerated, and the polymerization reaction can be completed rapidly in a short period of time (4-8 hours). Furthermore, the thickness of the imprinted shell can be controllably adjusted by the polymerization time and monomer concentration to meet the needs of practical applications.

[0039] In terms of monomer selection, vinyl monomers containing various functional groups can be selected to tailor artificial receptors for different proteins to meet the needs of protein purification in the biopharmaceutical field. In the present invention, main monomers include N-isopropylacrylamide and acrylamide. Functional monomers include methacrylic acid, acrylamide, styrene, N-tert-butylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and dimethylaminoethyl methacrylate.

[0040] In terms of cross-linking agent selection, N,N-methylenebisacrylamide, polyethylene glycol diacrylate, and polyglutamic acid polypeptide cross-linking agents can be selected for use in the present invention.

[0041] The results of protein re-adsorption experiments showed that the imprinting capacity of the synthesized nanoimprinted microspheres for lysozyme was 145.3 mg g -1 , with an imprinting factor of 14.2. Protein adsorption kinetics experiments demonstrated that the nanoimprinted microspheres reached adsorption equilibrium within a relatively short period of time (30-45 minutes). Compared to other literature results, the peptide-cross-linked surface protein-imprinted polymers obtained by localized free radical polymerization mediated by a cascade reaction of horseradish peroxidase and glucose oxidase significantly improved polymerization time, specific selectivity, adsorption capacity, and elution efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 :TEM image of enzyme coated on the surface of prepared SiO2 nanoparticles

[0043] Figure 2 :TEM images of surface protein-imprinted polymers prepared by enzyme cascade-mediated localized free radical polymerization on SiO2 nanoparticles

[0044] Figure 3 :TEM images of surface protein-imprinted polymers prepared by enzyme cascade-mediated localized free radical polymerization on Fe3O4 nanoparticles

[0045] Figure 4 :Preparation of Fe3O-based localized free radical polymerization mediated by enzyme cascade reaction 4- TEM image of surface protein-imprinted polymer with Cu nanoparticles as carrier

[0046] Figure 5 :SDS-PAGE analysis of lysozyme extracted from egg white. The leftmost lane is the Mark lane, the first lane is the lysozyme standard sample lane, the second lane is the untreated egg white sample supernatant, the third lane is the egg white supernatant treated with surface protein imprinting polymer, and the fourth lane is the sample supernatant after elution. DETAILED DESCRIPTION

[0047] The present invention provides a novel method for preparing surface protein imprinted polymers, which uses localized free radical polymerization mediated by an enzyme cascade reaction to prepare surface protein imprinted polymers with high imprinting efficiency, specifically comprising the following steps:

[0048] (1) dispersing the nanoparticles in a mixture of anhydrous ethanol and deionized water, adding 3-aminopropyltriethoxysilane (0.01-0.20 M) and ammonia water (28 wt%, 0.001-0.02 M), reacting at room temperature for 10-24 h, and washing with anhydrous ethanol and water in sequence to obtain amino nanoparticles;

[0049] (2) Disperse the amino nanoparticles obtained in step (1) into tetrahydrofuran (particle concentration 0.01-0.10 mg mL -1 ), adding succinic anhydride (0.01-0.20 M), reacting at room temperature for 1-8 h, and washing with ethanol and water in sequence to obtain surface carboxyl-modified nanoparticles;

[0050] (3) Disperse the carboxyl modified nanoparticles in step (2) in phosphate buffer (particle concentration 0.01-0.10 mg / mL) -1), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and stirred at room temperature for 1-8 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.05-1.0 M);

[0051] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration 0.01-0.10 mg / mL). -1 The microscopic morphology of the particles after covalent coupling is shown in the attached manual. Figure 1 As shown, it can be clearly seen that the enzyme is successfully immobilized on the particle surface;

[0052] (5) Add a certain volume of pH 5.0 phosphate buffer solution to the template protein (0.01-0.10 mM), main monomer (N-isopropylacrylamide (0.05-1.00 M)), functional monomer (including acrylamide (1.74-34.80 mM), methacrylic acid (1.74-34.80 mM)), and cross-linker (N,N-methylenebisacrylamide or polyglutamic acid polypeptide cross-linker (0.67-13.35 mM), and pre-assemble for 10-60 min to obtain a pre-assembly solution for use;

[0053] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL,

[0054] 0.01-0.10mg mL -1 ), acetylacetone (0.05-0.20M) and glucose (0.05-0.20M) to initiate the polymerization reaction. After polymerization at room temperature for 4-8 hours, the reaction was stopped to obtain a polymer solution;

[0055] (7) The polymer solution in step (6) is centrifuged and washed 3-6 times to obtain a surface protein imprinted polymer, the microscopic morphology of which is shown in the attached manual. Figure 2 、 3 , as shown in 4.

[0056] There are no special requirements for the selection of nanoparticles in the present invention, including but not limited to SiO2, Ag, Au, Fe3O4, MOF, COF and other nanoparticles.

[0057] There is no special requirement for the selection of enzymes in the present invention, including but not limited to glucose oxidase and horseradish peroxidase.

[0058] There are no specific requirements for the template protein, main monomer and functional monomer selected for polymerization in the present invention. All proteins and monomers commonly used in this research field are used. The water-soluble solution used in this experiment is generally based on phosphate buffer, which is configured according to the pH value required by the cross-linking agent, and the ion concentration is 10mM.

[0059] In a specific implementation example, protein surface-imprinted polymers were prepared using the enzyme-mediated localized free radical polymerization method of the present invention, using different nanoparticles, different template proteins, and different types and amounts of monomers and cross-linkers. The results showed that increasing the total mass concentration of monomers and cross-linkers not only maintained the monodispersity of the surface protein-imprinted polymers but also significantly improved their adsorption efficiency.

[0060] Implementation Plan 1

[0061] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0062] (1) SiO2 nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.01 M) and ammonia water (28 wt%, 0.001 M) were added, and the mixture was reacted at room temperature for 10 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified SiO2 nanoparticles;

[0063] (2) The amination SiO2 nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.01 mg / mL) -1 ), succinic anhydride (0.01 M) was added, reacted at room temperature for 1 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified SiO2 nanoparticles;

[0064] (3) Disperse the carboxylated SiO2 nanoparticles in step (2) in phosphate buffer (particle concentration 0.01 mg / mL) -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 1 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.05 M).

[0065] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.01 mg / mL). -1 ).

[0066] (5) Lysozyme (0.01 mM), N-isopropylacrylamide (0.05 M), acrylamide (1.74 mM), methacrylic acid (1.74 mM), and polyglutamic acid polypeptide cross-linker (0.67 mM) were added to pH 5.0 phosphate buffer and preassembled for 10 minutes to obtain a preassembly solution for use.

[0067] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.01 mg mL) to the pre-assembled solution of step (1). -1), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 4 hours at room temperature to obtain a polymer solution.

[0068] (7) The polymer solution in step (6) was centrifuged and washed three times to obtain a surface protein imprinted polymer.

[0069] Implementation Plan 2

[0070] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0071] (1) SiO2 nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia water (28 wt%, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified SiO2 nanoparticles;

[0072] (2) The amination SiO2 nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified SiO2 nanoparticles;

[0073] (3) Disperse the carboxylated SiO2 nanoparticles in step (2) in phosphate buffer (particle concentration 0.05 mg / mL) -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0074] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0075] (5) Lysozyme (0.05 mM), N-isopropylacrylamide (0.50 M), acrylamide (17.40 mM), methacrylic acid (17.40 mM), and polyglutamic acid polypeptide cross-linker (6.70 mM) were added to pH 5.0 phosphate buffer and preassembled for 30 minutes to obtain a preassembly solution for use.

[0076] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0077] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0078] Implementation Plan 3

[0079] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0080] (1) SiO2 nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.20 M) and ammonia (28 wt%, 0.02 M) were added, and the mixture was reacted at room temperature for 24 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified SiO2 nanoparticles;

[0081] (2) Disperse the amination SiO2 nanoparticles obtained in step (1) into tetrahydrofuran (particle concentration of about 0.10 mg / mL) -1 ), succinic anhydride (0.20 M) was added, reacted at room temperature for 8 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified SiO2 nanoparticles;

[0082] (3) Disperse the carboxylated SiO2 nanoparticles in step (2) in phosphate buffer (particle concentration of about 0.10

[0083] mg mL -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 8 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.0 M).

[0084] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution at pH 5.0 (ion concentration of about 0.10 mg / mL). -1 ).

[0085] (5) Lysozyme (0.10 mM), N-isopropylacrylamide (1.00 M), acrylamide (34.80 mM), methacrylic acid (34.80 mM), and polyglutamic acid polypeptide cross-linker (13.40 mM) were added to pH 5.0 phosphate buffer and preassembled for 60 minutes to obtain a preassembly solution for use.

[0086] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 8 h at room temperature to obtain a polymer solution.

[0087] (7) The polymer solution in step (6) was centrifuged and washed 6 times to obtain a surface protein imprinted polymer.

[0088] Implementation Plan 4

[0089] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0090] (1) Ag nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia (28 wt %, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The resultant mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified Ag nanoparticles.

[0091] (2) The amination-modified Ag nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified Ag nanoparticles;

[0092] (3) The carboxylated Ag nanoparticles in step (2) were dispersed in phosphate buffer (particle concentration 0.05 mg / mL) -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0093] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0094] (5) Add bovine serum albumin (0.05 mM), N-isopropylacrylamide (0.50 M), acrylamide (17.40 mM), methacrylic acid (17.40 mM), and polyethylene glycol diacrylamide (6.70 mM) to pH 5.0 phosphate buffer and pre-assemble for 30 minutes to obtain a pre-assembly solution for use.

[0095] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0096] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0097] Implementation Plan 5

[0098] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0099] (1) CeO2 nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia water (28 wt%, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The resultant ammoniated Ag nanoparticles were washed with anhydrous ethanol and water in sequence.

[0100] (2) The amination-modified CeO2 nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified Ag nanoparticles;

[0101] (3) Disperse the carboxylated CeO2 nanoparticles in step (2) in phosphate buffer (particle concentration 0.05 mg / mL) -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0102] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0103] (5) Human immunoglobulin (0.05 mM), N-isopropylacrylamide (0.50 M), acrylamide (17.40 mM), methacrylic acid (17.40 mM), and N,N-methylenebisacrylamide (6.70 mM) were added to a pH 5.0 phosphate buffer solution and preassembled for 30 minutes to obtain a preassembly solution for use.

[0104] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0105] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0106] Implementation Plan 6

[0107] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0108] (1) Au nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia (28 wt %, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The resulting mixture was washed with anhydrous ethanol and water in turn to obtain amino-modified Ag nanoparticles.

[0109] (2) The amino Au nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified Ag nanoparticles;

[0110] (3) The carboxylated Au nanoparticles in step (2) were dispersed in phosphate buffer (particle concentration 0.05 mg mL -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0111] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0112] (5) Human immunoglobulin (0.05 mM), N-isopropylacrylamide (0.50 M), N-tert-butylacrylamide (17.40

[0113] mM), 2-acrylamido-2-methylpropanesulfonic acid (17.40 mM), N,N-methylenebisacrylamide (6.70 mM)

[0114] A pH 5.0 phosphate buffer solution was added and pre-assembled for 30 minutes to obtain a pre-assembly solution for use.

[0115] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0116] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0117] Implementation Plan 7

[0118] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0119] (1) MOF nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia (28 wt %, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified MOF nanoparticles;

[0120] (2) The amination-modified MOF nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified MOF nanoparticles;

[0121] (3) Disperse the carboxyl modified MOF nanoparticles in step (2) in phosphate buffer (particle concentration 0.05

[0122] mg mL -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0123] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0124] (5) Cytochrome C (0.05 mM), N-isopropylacrylamide (0.50 M), styrene (17.40 mM), dimethylaminoethyl methacrylate (17.40 mM), and N,N-methylenebisacrylamide (6.70 mM) were added to a pH 5.0 phosphate buffer solution and preassembled for 30 minutes to obtain a preassembly solution for use.

[0125] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0126] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0127] Implementation Plan 8

[0128] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0129] (1) COF nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia (28 wt %, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amino-modified MOF nanoparticles;

[0130] (2) The amino COF nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified COF nanoparticles;

[0131] (3) Disperse the carboxyl modified COF nanoparticles in step (2) in phosphate buffer (particle concentration 0.05 mg mL -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0132] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0133] (5) Add bovine hemoglobin (0.05 mM), N-isopropylacrylamide (0.50 M), acrylamide (17.40 mM), dimethylaminoethyl methacrylate (17.40 mM), N,N-methylenebisacrylamide (6.70 mM) to pH

[0134] 5.0 phosphate buffer, pre-assemble for 30 min to obtain the pre-assembly solution for use.

[0135] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0136] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0137] Implementation Plan 9

[0138] Preparation of surface protein-imprinted polymers by localized free radical polymerization mediated by enzyme cascade reaction:

[0139] (1) Fe3O4 nanoparticles were dispersed in a mixture of anhydrous ethanol and deionized water, 3-aminopropyltriethoxysilane (0.10 M) and ammonia (28 wt%, 0.01 M) were added, and the mixture was reacted at room temperature for 15 h. The mixture was washed with anhydrous ethanol and water in sequence to obtain amination-modified Fe3O4 nanoparticles;

[0140] (2) The amination-treated Fe3O4 nanoparticles obtained in step (1) were dispersed in tetrahydrofuran (particle concentration 0.05 mg / mL) -1 ), succinic anhydride (0.10 M) was added, reacted at room temperature for 4 h, and washed with ethanol and water in sequence to obtain surface carboxyl-modified Fe3O4 nanoparticles;

[0141] (3) Disperse the carboxylated Fe3O4 nanoparticles in step (2) in phosphate buffer (particle concentration 0.05

[0142] mg mL -1 ), glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the mixture was stirred at room temperature for 4 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.50 M).

[0143] (4) The reaction product was washed with deionized water and dispersed in a phosphate buffer solution with a pH of 5.0 (ion concentration of 0.05 mg / mL). -1 ).

[0144] (5) Add amyloid protein (0.05 mM), N-isopropylacrylamide (0.50 M), styrene (17.40 mM), dimethylaminoethyl methacrylate (17.40 mM), and polyglutamic acid peptide crosslinker (6.70 mM) to pH 5.0 phosphate buffer and pre-assemble for 30 minutes to obtain a pre-assembly solution for use.

[0145] (6) Add the nanoparticle dispersion of immobilized enzyme obtained in step (4) (4.0 mL, 0.10 mg mL) to the pre-assembled solution of step (1). -1 ), acetylacetone (0.05 M) and glucose (0.05 M) were added to initiate the polymerization reaction. The polymerization was stopped after 6 h at room temperature to obtain a polymer solution.

[0146] (7) The polymer solution in step (6) was centrifuged and washed 4 times to obtain a surface protein imprinted polymer.

[0147] Implementation Plan 10

[0148] The surface protein imprinted polymer prepared by the synthesis method of the present invention was applied to the separation and purification of lysozyme from egg white:

[0149] (1) vacuum drying the surface protein imprinted polymer (MIP) prepared by the synthesis method of the present invention;

[0150] (2) Weigh about 10 mg of the dried MIP in step (2) into a centrifuge tube and add pH 5.0 phosphate buffer (8 mL)

[0151] Allow it to swell fully and reach swelling equilibrium before removing the solution;

[0152] (3) Dilute the egg white of fresh eggs 50 times with pH 5.0 phosphate buffer;

[0153] (4) Take 5 mL of the egg white solution diluted 50% in step (3) and add it to the centrifuge tube containing MIP in step (2) and incubate at room temperature for 1 hour to extract lysozyme from the egg white.

[0154] (5) The solution in step (4) was completely removed, and 5 mL of pH 7.4 phosphate buffer was added to elute the lysozyme adsorbed by the MIP.

[0155] (6) The extraction effect was tested by gel electrophoresis. Lysozyme standard solution, 50-fold diluted egg white solution, the remaining solution after MIP adsorbed lysozyme, and the eluate were used as four sample solutions for gel electrophoresis. Figure 5 ), the lysozyme in the egg white was almost completely adsorbed by the MIP, while other proteins were well retained (lane 3). After the MIP was eluted, the lysozyme was well eluted with almost no other proteins (lane 4).

[0156] The technical solutions disclosed and proposed by the present invention can be implemented by those skilled in the art by drawing on the content herein and appropriately changing the conditions, routes, and other aspects. Although the methods and preparation techniques of the present invention have been described through preferred embodiments, it is obvious that those skilled in the art can modify or recombine the methods and technical routes described herein without departing from the content, spirit, and scope of the present invention to achieve the ultimate preparation technology. It is particularly important to point out that all similar substitutions and modifications that are obvious to those skilled in the art are considered to be included in the spirit, scope, and content of the present invention.

Claims

1. A method for preparing a surface protein imprinted polymer by enzyme cascade reaction, characterized in that: The steps include: (1) Carboxyl modification of the nanoparticle surface: 3-aminopropyltriethoxysilane is used to hydrolyze the nanoparticle surface, and amino nanoparticles are obtained after washing; (2) Dispersing the amino-modified nanoparticles in tetrahydrofuran solution, adding succinic anhydride, stirring at room temperature for 1-8 h, and then washing to obtain surface carboxylated nanoparticles; (3) The washed carboxylated nanoparticles were dispersed in phosphate buffer, and glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:

1. Subsequently, GOx and HRP were covalently coupled to the surface of the nanoparticles under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide; (4) washing the product of step (3) with deionized water to obtain a dispersion of immobilized enzyme nanoparticles; (5) Add the template protein, main monomer, functional monomer, and cross-linker to pH 5.0 phosphate buffer and pre-assemble for 10-60 minutes to obtain a pre-assembly solution; the main monomer is N -isopropylacrylamide, with a concentration range of 0.05-1.00 M; (6) Adding the nanoparticle dispersion of the immobilized enzyme, acetylacetone, and glucose to the pre-assembled solution of step (5) to initiate a polymerization reaction; stopping the reaction after polymerization for 4-8 hours at room temperature to obtain a polymerization solution; (7) The polymer solution in step (6) is centrifuged and washed 3-6 times to obtain a surface protein imprinted polymer.

2. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, characterized in that: Nanoparticles include SiO2, Fe3O4, Fe3O4-Cu, MOF, COF, Au, CeO2 or Ag.

3. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, characterized in that: The concentration of 3-aminopropyltriethoxysilane in step (1) is 0.01-0.20 M.

4. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, wherein: In step (2), the amino nanoparticles are dispersed in tetrahydrofuran solution at a particle concentration of 0.01-0.10 mg·mL -1 , add succinic anhydride at a concentration of 0.01-0.20 M.

5. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, wherein: In step (3), the washed carboxylated nanoparticles are dispersed in phosphate buffer at a particle concentration of 0.01-0.10 mg·mL -1 Glucose oxidase (GOx) and horseradish peroxidase (HRP) were added at a molar ratio of 1.5:1, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 0.05-1.0 M.

6. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, wherein: The particle concentration of the nanoparticle dispersion of immobilized enzyme obtained in step (4) is 0.01-0.10 mg·mL -1 .

7. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, wherein: In step (5), the template protein concentration is 0.01-0.10 mM, the functional monomer concentration is 1.74-34.80 mM, and the cross-linker concentration is 0.67-13.35 mM; the template proteins in the pre-assembly solution are lysozyme, bovine serum albumin, cytochrome C, bovine hemoglobin, human immunoglobulin, and amyloid protein; the functional monomers include methacrylic acid, acrylamide, styrene, N-tert-butylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and dimethylaminoethyl methacrylate; the cross-linker is N,N -Methylene bisacrylamide, polyethylene glycol diacrylate, polyglutamic acid polypeptide cross-linking agent.

8. The method for preparing a surface protein imprinted polymer by enzyme cascade reaction according to claim 1, wherein: Step (6) Add 4.0 mL of the immobilized enzyme nanoparticle dispersion to the pre-assembly solution, 0.01-0.10 mg mL -1 , acetylacetone concentration 0.05-0.20 M, glucose concentration 0.05-0.20 M.

9. The surface protein imprinted polymer prepared by the method of claim 1 and relying on enzyme cascade reaction is used for extracting and purifying proteins in complex biological samples.

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