A method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation and its application

Abstract

The present invention relates to a method for preparing surface-imprinted microspheres by horseradish peroxidase-mediated surface initiation and its application. The method comprises the following steps: preparing and modifying nano-microspheres; dissolving a main monomer, a functional monomer, a polypeptide cross-linking agent, and a template protein in a water-soluble solvent to obtain a prepolymer solution; adding horseradish peroxidase-modified nano-microspheres and an initiator to the mixed solution to initiate free radical polymerization; and eluting the template protein to obtain polypeptide-crosslinked surface-imprinted microspheres. The nano-imprinted microspheres disclosed in the present invention are prepared by an enzyme-mediated free radical polymerization method, which can achieve sufficient pre-assembly of monomers and templates at high concentrations to improve the problem of poor imprinting effect in traditional free radical polymerization, while avoiding the agglomeration phenomenon that may occur under high-concentration reaction conditions. The introduction of the polypeptide cross-linking agent enables the imprinted microspheres to have highly specific selection and recognition capabilities for the target protein, and can significantly shorten the time for adsorbing the template protein.

Description

Technical Field

[0001] The invention belongs to the technical field of biological separation engineering, and particularly relates to a method for preparing surface-imprinted polymer microspheres by surface initiation mediated by horseradish peroxidase and application thereof. Background Art

[0002] Surface imprinting is a well-established method for preparing molecularly imprinted polymers (MIPs). Surface-imprinted nanospheres possess a larger surface area than conventional bulk-imprinted polymers. The imprinted sites are located within a thin imprinted layer on the microsphere surface, providing greater accessibility and faster mass transfer. After template removal, the three-dimensional imprinted cavities, which are complementary to the template molecule, exhibit a higher affinity for the template molecule.

[0003] Currently, surface-imprinted microspheres have been widely used in the field of protein separation and purification. To prepare surface-imprinted polymers, researchers generally use a variety of polymerization methods, including traditional free radical polymerization, precipitation polymerization, emulsion polymerization, and surface-induced atom transfer radical polymerization. However, these polymerization methods still have many problems. These polymerization methods often involve complex polymerization conditions and are difficult to control. Traditional free radical polymerization reactions usually require highly diluted monomers, resulting in low monomer and template molecule concentrations, making it difficult to fully complete preassembly, resulting in low imprinting site-specific selectivity. Furthermore, traditional polymerization methods generate free radicals in solution, and polymerization begins in solution. When the monomer concentration is too high, although the effect of monomer and template molecule preassembly can be enhanced, aggregation may occur at high monomer concentrations. For biomacromolecules, the imprinting process is more difficult due to their large molecular weight and flexible three-dimensional structure. Therefore, the surface-imprinted polymers prepared by these methods generally have poor imprinting effects on biomacromolecules. The application of surface-imprinted polymers prepared by these methods in protein separation and purification is limited. For example, Dong et al. disclosed a nanoimprinted microsphere with bovine serum albumin (BSA) as the template molecule, using N,N-methylenebisacrylamide as a crosslinker, and using ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) to initiate free radical polymerization. The polymerization process of this method is multi-step, complex and difficult to control. The reaction time is as long as 24 hours, the adsorption equilibrium time is 120 minutes, the imprinting capacity is 119.88 mg / g, and the specificity for the template protein is also poor, with an imprinting factor of only 2.25 (DONG X, MA Y, et al. Preparation of pH and temperature dual-sensitive molecularly imprinted polymers based on chitosan and N-isopropylacrylamide for recognition of bovine serum albumin [J]. Polymer International, 2019, 68 (5): 955-963.). Qian et al. prepared a nanoimprinted microsphere using Fe3O4@IL microspheres with imidazole groups and BSA through the oxidative self-polymerization of dopamine. The nanoimprinted microspheres prepared by this method have a thick imprinting layer, uncontrollable self-polymerization, poor uniformity of imprinting sites, and high mass transfer resistance. The polymerization reaction of these imprinted microparticles lasts up to 7 hours, resulting in an adsorption capacity of only 50.6 mg / mL and an imprinting factor of only 3.33, indicating low specific selectivity.(QIAN L, SUN J, et al. Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy[J]. Talanta, 2017, 168: 174-182). Wang et al. prepared BSA nanoimprinted microspheres (Fe3O4@MnO2@BSA-MIPs) by superimposing MnO2 on a Fe3O4 hollow core and using chitosan and other monomers via free radical polymerization. Although the nanoimprinted microspheres prepared by this polymerization method have a larger specific surface area, the pre-assembly of the monomer and the template is insufficient, the imprinting sites are few and the uniformity of the imprinting sites cannot be guaranteed, the integrity and utilization of the imprinting sites are low, and the specific selectivity is poor. The polymerization reaction time of this method is as long as 24 hours, and the preparation process is also complex and uncontrollable. The imprinting factor of the nanoimprinted particles is only 3.3. (WANG Y,MAY,et al.MnO2 corolla-like magneticmolecularly imprinted microspheres with enhanced adsorption capacity and specificity recognition to bovine serum albumin[J]. Chemical Engineering Journal, 2021,405:126655.). Summary of the Invention

[0004] The purpose of the present invention is to improve the limitation of existing surface imprinted polymer preparation methods that can only initiate polymerization at relatively dilute monomer concentrations. Because of this limitation, surface imprinted polymers have the problem of insufficient pre-assembly of functional monomers and template molecules, which further leads to the problem of surface imprinted polymers' specific selection ability for macromolecular templates. By adopting an enzyme-mediated in situ initiated polymerization method to generate free radicals on the surface of nanoparticles, and using nanoparticles as the reaction core, on the one hand, it can solve the problem of insufficient pre-assembly of template molecules and functional monomers in traditional free radical polymerization methods, and on the other hand, it can solve the problem of possible agglomeration of traditional polymerization methods under high monomer concentration conditions. Therefore, the method proposed by the present invention can achieve sufficient pre-assembly and avoid the occurrence of agglomeration at higher monomer concentrations, and further improve the problems of low specific recognition ability of surface imprinted polymers for template molecules and long adsorption time.

[0005] Enzyme-mediated free radical polymerization is gaining popularity in the preparation of surface microgels due to its simple and mild initiation mechanism. This method primarily uses initiation systems such as glucose oxidase and β-D-glucose or acetylacetone (ACAC)-horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) to initiate free radical polymerization. We enhance the binding capacity of the template molecule by selecting functional monomers that can form covalent or non-covalent interactions with the template molecule. Furthermore, we use peptide crosslinkers to impart a pH-responsive reversible conformational transition to the imprinted layer, enabling the imprinted sites to precisely restore a complementary three-dimensional structure to the template molecule, thereby enhancing the specificity of the imprinted sites for the template molecule. These conditions can be combined with enzyme-mediated free radical polymerization to conduct the polymerization reaction at high concentrations, thereby overcoming the challenges of traditional free radical polymerization methods, such as poor imprinting due to insufficient pre-assembly of monomers and template molecules and the potential for aggregation at high polymerization concentrations. Here, we utilize this method to prepare surface-imprinted polymers, initiating polymerization at high reaction concentrations to enhance pre-assembly of monomers and template molecules and avoid aggregation and gelation.

[0006] The present invention proposes an HRP-mediated surface initiation strategy for preparing surface-imprinted polymer microspheres and its application. The peroxidase immobilized on the surface of the nanospheres serves as an active site to catalyze the reaction of H2O2 and acetylacetone to produce free radicals that gently initiate polymerization to form a stable imprinted gel layer.

[0007] The specific technical solutions are as follows:

[0008] A method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation comprises the following steps:

[0009] 1) Horseradish peroxidase (HRP) is reacted with activated carboxyl-functionalized nanospheres to undergo amidation reaction, thereby immobilizing HRP on the surface of the microspheres as active sites to obtain HRP-modified nanoimprinted microspheres;

[0010] 2) dispersing the HRP-modified nanospheres obtained in step 1) in a phosphate buffer solution under ultrasound to obtain a dispersion of HRP nanoimprinted microspheres;

[0011] 3) adding the main monomer N-isopropylacrylamide, the functional monomers acrylamide, methacrylic acid and dimethylaminoethyl methacrylate, a polypeptide cross-linker, and the template protein bovine serum albumin (BSA) to the dispersion of HRP nanoimprinted microspheres prepared in step 2); after complete dissolution, stirring and mixing to uniformly mix, and allowing to stand to complete the pre-assembly process, thereby obtaining a pre-polymerized mixed solution;

[0012] 4) passing N2 into the prepolymerized mixed solution obtained in step 3) to remove O2 in the system;

[0013] 5) Under mechanical stirring and N2 atmosphere, the pH of the solution after excluding the system O2 in step 4) is adjusted so that the polypeptide cross-linker is in a helix conformation condition, and acetylacetone and hydrogen peroxide are added thereto to initiate a free radical polymerization reaction under HRP catalysis; after the reaction, the microspheres in the solution are magnetically separated and then washed twice with ultrapure water to obtain surface-imprinted microspheres imprinted with the template.

[0014] 6) The surface-imprinted nanospheres obtained in step 5) are placed in a phosphate buffer solution to elute the template, thereby obtaining nano-imprinted microspheres (BSA-PC-Fe3O4@MIP) with imprinted cavities.

[0015] The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation; based on the volume of the phosphate buffer solution used in step 2), the concentration of the main monomer N-isopropylacrylamide added in step 3) in the phosphate buffer solution is 5-50 mg / mL, the concentration of the functional monomer acrylamide (AAM) is 0.1-1.1 mg / mL, the concentration of methacrylic acid (MAA) is 0.001-0.011 M, and the concentration of dimethylaminoethyl methacrylate (DMAEMA) is 0.001-0.011 M.

[0016] The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation is described. The crosslinker used in step 3) is a polyglutamic acid polypeptide crosslinker with double bonds at both ends of the chain segment, a degree of polymerization ranging from 18 to 25, and a pH-responsive helix-coil conformational transition. (DONG Q, YANG M, WANG Y, et al. Peptide-crosslinked molecularly imprinted polymers for efficient separation of immunoglobulin G from human serum[J].

[0017] Biomaterials Science,2023,11(4):1398-1407)

[0018] The sum of the amounts of the monomers and functional monomers used in step 3) is taken as the total amount, and the amount of the polypeptide cross-linking agent added in step 3) is 1-5% of the total amount.

[0019] In step 5), the pH of the solution is adjusted to 4.5-5.0. The polypeptide cross-linker is in a helix conformation in this pH range, and the polypeptide cross-linker will not precipitate from the solution in this pH range.

[0020] In step 5), the HRP-mediated free radical polymerization has a reaction temperature of 20° C. to 35° C. and a reaction time of 2.5 to 6 hours;

[0021] In step 6), the template is eluted in a solution similar to the human body fluid environment, and a phosphate buffer solution with a pH of 7.4 containing 154 mM NaCl is selected as the elution medium. At this time, the polypeptide cross-linker is in a coil conformation.

[0022] The nano-imprinted microspheres of the present invention are used as adsorption materials in the high-specificity recognition and selection, rapid separation and purification of BSA.

[0023] The nanoimprinted microspheres of the present invention can complete pre-assembly of monomers and template molecules at high concentrations under an enzyme-mediated surface initiation system, resulting in nanoimprinted microspheres with more effective imprinting sites. At the designed concentration, the polymerization process exhibited no aggregation. The polypeptide cross-linker exists in helix and coil conformations at different pH values. These nanoimprinted microspheres exhibit highly specific selection and recognition capabilities for target proteins in the helix conformation.

[0024] The present invention's method for preparing Fe3O4 nanoimprinted microspheres via HRP-mediated free radical polymerization can be used to prepare nanoimprinted polymers based on various substrates, such as Fe3O4, SiO2, silver nanospheres, and nickel nanospheres. By modifying the Fe3O4 nanospheres with HRP as an active core, an initiation system of H2O2 and ACAC generates short-lived free radicals to rapidly initiate free radical polymerization. This polymerization method allows for the selection of higher monomer concentrations, achieving sufficient preassembly of the monomers and template molecules, improving the integrity of the imprinted sites and their specificity for the template molecule. Furthermore, in situ initiation of polymerization using HRP as the active core avoids the aggregation that can occur with traditional free radical polymerization methods. Furthermore, this method offers simple reaction conditions and easy process control. The use of a polypeptide crosslinker enables the imprinted layer to undergo a pH-responsive helix-to-coil conformational transition, enabling the imprinted cavity to precisely restore its three-dimensional structure complementary to the template protein. These advantages significantly improve the imprinting performance of the nanoimprinted microspheres, enhancing their imprinting performance and their ability to specifically select for the template protein. Protein adsorption studies have shown that the newly synthesized peptide-cross-linked nanoimprinted microspheres have an imprinting capacity of 140 mg / g for bovine serum albumin, with an imprinting factor of 10.4. The nanoimprinted microspheres can reach adsorption equilibrium within 30 minutes. Compared with results from other literature, the nanoimprinted microspheres prepared by this method show significant improvements in specific selectivity, polymerization reaction time, and time to reach adsorption equilibrium. Furthermore, this method is not limited to using bovine serum albumin as a template molecule and can be applied to enhance specific selectivity for a variety of template molecules, such as bovine hemoglobin, cytochrome C, and human serum albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Transmission electron microscopy image of polypeptide-cross-linked Fe3O4 surface-imprinted microspheres prepared by HRP-mediated free radical polymerization using BSA as the template protein, corresponding to Example 2;

[0026] Figure 2 :The effect of monomer concentration on the polydispersity of Fe3O4 nanospheres during the preparation of polypeptide-crosslinked Fe3O4 surface-imprinted microspheres;

[0027] Figure 3 :The hydrodynamic diameter of peptide-crosslinked Fe3O4 surface imprinted microspheres BSA-PC-Fe3O4@MIP changes with the reaction monomer concentration and reaction time;

[0028] Figure 4 : Adsorption kinetics and isotherms of template protein on polypeptide-cross-linked Fe3O4 surface-imprinted microspheres BSA-PC-Fe3O4@MIP and non-imprinted microspheres BSA-PC-Fe3O4@NIP, corresponding to Example 18;

[0029] Figure 5 : Selective adsorption results of peptide-cross-linked Fe3O4 nanoimprinted microspheres BSA-PC-Fe3O4@MIP and corresponding BSA-PC-Fe3O4@NIP for template protein and non-template protein;

[0030] Figure 6 : SDS-PAGE analysis of BSA extracted from fetal bovine serum, where the first lane is BSA standard sample, the second lane is untreated fetal bovine serum, the third lane is fetal bovine serum treated with imprinted microspheres, and the fourth lane is the eluate sample after fetal bovine serum treated with imprinted microspheres and eluted with phosphate buffer solution. DETAILED DESCRIPTION

[0031] In the present invention, the polymerization method can be applied to a variety of nanospheres, such as gold, silver, nickel, silica, and Fe3O4 nanospheres. Fe3O4 nanospheres are selected as the matrix due to their excellent magnetic response properties. The hydrodynamic diameter of the nanospheres is between 50 and 300 nm, with an optimal range of 100 to 200 nm. There are no specific restrictions on the modification methods of the amino and carboxyl groups and the compounds used. There are also no specific restrictions on the method used for carboxyl group activation. Enzyme modification uses peroxidase, and HRP is selected herein.

[0032] In the present invention, a prepolymer mixture is prepared by dissolving a main monomer, a functional monomer, a template protein, and a polypeptide cross-linking agent in a phosphate buffer solution. The main monomer and functional monomer used in the polymerization reaction are commonly used in the field of surface protein imprinting polymers and are not particularly limited. The template protein is a common protein such as bovine serum albumin, human serum albumin, or bovine hemoglobin, with bovine serum albumin being used herein.

[0033] In the present invention, the substrate for HRP initiation is common oxygen or a combination of H2O2 and ACAC, preferably H2O2 and ACAC. Preparation of HRP-modified Fe3O4 nanoparticles:

[0034] 1) Fe3O4 nanospheres were prepared by a solvothermal method. The washed Fe3O4 nanospheres were then ultrasonically dispersed in a mixture of 400 mL of ethanol, 12 mL of ultrapure water, and 12 mL of ammonia. After ultrasonication for 15 minutes, 0.1 mL of tetraethyl orthosilicate was added, and the mixture was mechanically stirred at 30°C for 2 hours. Next, 12 mL of APTES was added to the mixture under ultrasonication, and the mixture was stirred at 45°C for another 24 hours.

[0035] 2) The amino-modified nanospheres obtained in step 1 were washed three times with a mixed solution of ethanol and ultrapure water. After washing three times with ultrapure water, they were dispersed in 400 mL of tetrahydrofuran solution by ultrasonication for 15 minutes, and then 43 g of succinic anhydride was added and the reaction was mechanically stirred for 18 hours.

[0036] 3) The carboxyl-modified nanospheres obtained in step 2 were washed three times with ultrapure water and then ultrasonically dispersed in 100 mL of a phosphate buffer solution having a pH of 5.8. 1.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was mechanically stirred at room temperature for 2 h to activate the carboxyl groups.

[0037] 4) The carboxyl-activated nanospheres obtained in step 3 were washed three times with ultrapure water, then resuspended in phosphate buffer solution (pH 5.5) containing 1.5 mg / mL horseradish peroxidase and reacted with mechanical stirring for 4 hours. The resulting HRP-modified nanospheres were washed twice with ultrapure water.

[0038] The functionalization of the nanosphere surface was accomplished by amino modification using ethyl orthosilicate and 3-aminopropyltriethoxysilane (APTES); carboxyl modification was accomplished using succinic anhydride; carboxyl activation was accomplished using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at an activator concentration of 10 mg / mL and an activation time of more than 2 h; enzyme modification was accomplished using horseradish peroxidase at a concentration of 1.5 mg / mL in the solution and a reaction time of more than 4 h (Microgel coating of magnetic nanoparticles via bienzyme-mediated free-radical polymerization for colorimetric detection of glucose[J]. Nanoscale, 2015, 7(40):16578-16582)

[0039] Example 1

[0040] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0041] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0042] 3) Add 40 mg of bovine serum albumin to the dispersion obtained in step 2 and stir for 5 minutes to mix evenly. Then add 5.7 mg of acrylamide (0.1 mg / mL), 5.1 μL of methacrylic acid (0.001 M) and 10.1 μL of dimethylaminoethyl methacrylate (0.001 M) to the solution. Then add 0.25 g of the main monomer N-isopropylacrylamide (5 mg / mL) and stir for 10 minutes to fully dissolve it. The polymerization reaction concentration is changed by controlling its concentration. Subsequently, 83 mg of a polypeptide cross-linker with a degree of polymerization of 22 is dissolved in the solution to obtain a pre-polymerization mixed solution.

[0043] 4) Pass N2 into the mixed solution of step 3 to remove O2.

[0044] 5) After removing O₂ in step 4, the pH of the mixed solution was adjusted to 5.0. Mechanically stir the mixture under an N₂ atmosphere for 15 minutes. Then, under an N₂ atmosphere, add 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide to initiate free radical polymerization on the nanosphere surface via a surface enzymatic reaction. The reaction was mechanically stirred under an N₂ atmosphere at 25°C for 4 hours. The resulting surface-imprinted microspheres were magnetically separated and washed three times with ultrapure water.

[0045] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM phosphate buffer (pH 7.4) (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant. The resulting surface-imprinted microspheres were named BSA-PC-Fe3O4@MIP. Non-imprinted microspheres, BSA-PC-Fe3O4@NIP, were prepared without the addition of template protein.

[0046] Imprinting Effect Studies:

[0047] Bovine serum albumin solutions of different concentrations were prepared using 20 mM pH 5.0 phosphate buffer solution. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer to calculate the adsorption amount. The adsorption amount was calculated using conventional calculation methods in the field, see J. Guo, et al. ANAL METHODS-UK, 7 (2015) 10018-10025. An adsorption isotherm (equilibrium protein concentration-adsorption amount curve) was drawn and the curve was fitted using the Langmuir model to obtain the imprinting capacity Q of BSA-PC-Fe3O4@MIP. max It is 26.5 mg / g and the imprinting factor is 2.1.

[0048] Example 2

[0049] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0050] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0051] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymerized mixed solution. At this point, the system has a cross-linking degree of 1%.

[0052] 4) Pass N2 into the mixed solution of step 3 to remove O2.

[0053] 5) Under an N2 atmosphere, the pH of the mixed solution obtained by removing O2 from step 4 was adjusted to 5.0. Then, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction was carried out under an N2 atmosphere at 25°C with mechanical stirring for 4 h. The resulting surface-imprinted microspheres were magnetically separated and washed three times with ultrapure water.

[0054] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0055] Imprinting Effect Research:

[0056] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor is 118 mg / g and IF is 8.25. Figure 1 As shown in FIG, the transmission electron microscopy image shows that the surface of the imprinted microspheres has an obvious imprinted layer, indicating that the method used in the present invention successfully initiates and synthesizes the imprinted polymer layer at a high monomer concentration of 40 mg / mL. Figure 2 As shown in the figure, even at a high NIPAM monomer concentration of 40 mg / mL, the polydispersity index of the imprinted microspheres is always below 0.1, indicating that the surface imprinted microspheres prepared using the enzyme-mediated surface initiation strategy have good monodispersity and will not agglomerate under high concentration reaction conditions. This method uses in situ initiation to make the polymerization more orderly, fully avoiding the agglomeration phenomenon that may occur in traditional free radical polymerization at high monomer concentrations. In addition, through the adsorption capacity Q max It can be seen from the IF that it has a significantly higher specific selection ability, indicating that under the condition of high monomer concentration, the monomer and template molecules have achieved sufficient pre-assembly and the specificity of the imprinting site has been significantly improved. Figure 3 As shown in the figure, during the preparation of BSA-PC-Fe3O4@MIP, the hydrodynamic diameter has a sudden change at 2 h and reaches growth equilibrium at 2.5 h, indicating that the reaction has been completed in 2.5 h. Therefore, the polymerization method has a fast reaction speed. The polymerization can be completed in only 4 h by this method, which can effectively shorten the time to obtain imprinted microspheres.

[0057] Example 3

[0058] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0059] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0060] 3) 40 mg of bovine serum albumin was added to the dispersion from step 2 and stirred for 5 minutes to mix thoroughly. Then, 53 mg of acrylamide (1.06 mg / mL), 45 μL of methacrylic acid (0.1 M), and 89 μL of dimethylaminoethyl methacrylate (0.1 M) were added to the solution. Then, 2.5 g of the main monomer, N-isopropylacrylamide (50 mg / mL), was added and stirred for 10 minutes to fully dissolve. Subsequently, 838 mg of a polypeptide cross-linker with a degree of polymerization of 22 was dissolved in the above solution to obtain a prepolymer solution.

[0061] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0062] 5) The pH of the prepolymer solution from step 4, after depleting O₂, was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction was carried out under an N₂ atmosphere at 25°C with mechanical stirring for 4 h. The resulting surface-imprinted microspheres were magnetically separated and washed three times with ultrapure water.

[0063] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0064] Imprinting Effect Research:

[0065] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max It is 109 mg / g and the imprinting factor is 8.01.

[0066] Example 4

[0067] The effect of the degree of polymerization of the peptide cross-linker on the imprinting effect at the optimal reaction monomer concentration (40 mg / mL).

[0068] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0069] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0070] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 804 mg of a polypeptide cross-linker with a degree of polymerization of 18 in the solution to obtain a prepolymer solution.

[0071] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0072] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction temperature was changed to 20°C under an N₂ atmosphere with mechanical stirring for 4 hours. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0073] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0074] Imprinting Effect Research:

[0075] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 20°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max It is 91 mg / g and the imprinting factor is 7.01.

[0076] Example 5

[0077] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0078] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0079] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 579 mg of a polypeptide cross-linker with a degree of polymerization of 25 in the above solution to obtain a prepolymer solution.

[0080] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0081] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction temperature was changed to 20°C under an N₂ atmosphere with mechanical stirring for 4 hours. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0082] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0083] Imprinting Effect Research:

[0084] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 20°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor was 7.53 and the molecular weight of the peptide cross-linker was 98 mg / g. Compared with the study in Example 2, the best imprinting effect was achieved by using a peptide cross-linker with a degree of polymerization of 22. This degree of polymerization was used in all subsequent studies.

[0085] Example 6

[0086] Effect of reaction temperature on imprinting effect at the optimal reaction monomer concentration (40 mg / mL).

[0087] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0088] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0089] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0090] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0091] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction temperature was changed to 20°C under an N₂ atmosphere with mechanical stirring for 4 hours. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0092] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0093] Imprinting Effect Research:

[0094] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 20°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The content of PEG-1 is 80 mg / g and the imprinting factor is 6.67.

[0095] Example 7

[0096] Effect of reaction temperature on imprinting effect at the optimal reaction monomer concentration (40 mg / mL).

[0097] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0098] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0099] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0100] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0101] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction temperature was changed to 35°C under an N₂ atmosphere with mechanical stirring for 4 hours. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0102] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0103] Imprinting Effect Research:

[0104] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 35°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. maxThe imprinting factor was 5.41 and the reaction temperature was 25℃, which showed the best imprinting effect and specific selectivity.

[0105] Example 8

[0106] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0107] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0108] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0109] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0110] 5) The pH of the prepolymer solution after purging O₂ from step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the nanosphere surfaces via a surface enzymatic reaction. The reaction time was changed to 2.5 h under an N₂ atmosphere at 25°C. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0111] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0112] Imprinting Effect Research:

[0113] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. maxThe concentration of 100 mg / g and the imprinting factor are 8.

[0114] Example 9

[0115] The effect of reaction time on imprinting effect under the optimal reaction monomer concentration (40 mg / mL) and reaction temperature 25℃.

[0116] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0117] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0118] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0119] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0120] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction time was changed to 6 h under an N₂ atmosphere at 25°C. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0121] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0122] Imprinting Effect Research:

[0123] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor is 7.86. The study on the effect of reaction time on the imprinting effect shows that the best imprinting effect is achieved when the reaction time is 4 hours, and the difference in imprinting effect is small when the reaction time is more than 2.5 hours. Figure 3 The reaction is basically completed after 2.5 h, so the imprinting effect is not much different.

[0124] Example 10

[0125] The effect of reaction solution pH on imprinting effect under the optimal reaction monomer concentration (40 mg / mL), reaction temperature 25℃ and reaction time 4h.

[0126] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0127] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0128] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0129] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0130] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 4.5. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The reaction was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0131] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0132] Imprinting Effect Research:

[0133] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The content of PEG-1 is 95 mg / g and the imprinting factor is 7.

[0134] Example 11

[0135] The effect of reaction solution pH on imprinting effect under the optimal reaction monomer concentration (40 mg / mL), reaction temperature 25℃ and reaction time 4h.

[0136] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0137] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0138] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg (0.84 mg / mL) of acrylamide, 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0139] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0140] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 4.8. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0141] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0142] Imprinting Effect Research:

[0143] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor was 7.51 and the pH value of the reaction solution was 101 mg / g. The best imprinting effect was found when the pH value of the reaction solution was 5.0.

[0144] Example 12

[0145] Effect of functional monomer acrylamide concentration on imprinting effect.

[0146] Effect of reaction solution pH on imprinting effect under the optimal conditions of NIPAM monomer concentration (40 mg / mL), reaction temperature 25°C and reaction time 4 h.

[0147] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0148] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0149] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 5.7 mg of acrylamide (0.1 mg / mL), 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0150] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0151] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0152] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0153] Imprinting Effect Research:

[0154] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The content of PEG-1 is 97 mg / g and the imprinting factor is 7.1.

[0155] Example 13

[0156] Effect of functional monomer acrylamide concentration on imprinting effect.

[0157] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0158] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0159] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 55 mg of acrylamide (1.1 mg / mL), 35 μL of methacrylic acid (0.008 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0160] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0161] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0162] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0163] Imprinting Effect Research:

[0164] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor was 6.73. The effect of acrylamide on the imprinting effect showed that the best imprinting effect was obtained when the acrylamide monomer concentration was 0.84 mg / mL.

[0165] Example 14

[0166] Effect of the concentration of functional monomer methacrylic acid on the imprinting effect.

[0167] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0168] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0169] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 5.1 μL of methacrylic acid (0.001 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0170] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0171] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0172] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0173] Imprinting Effect Research:

[0174] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The content of PEG-1 is 96 mg / g and the imprinting factor is 7.1.

[0175] Example 15

[0176] Effect of the concentration of functional monomer methacrylic acid on the imprinting effect.

[0177] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0178] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0179] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 45 μL of methacrylic acid (0.011 M), and 69 μL of dimethylaminoethyl methacrylate (0.008 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the solution to obtain a prepolymer solution.

[0180] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0181] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0182] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0183] Imprinting Effect Research:

[0184] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The imprinting factor was 6.8 and the concentration of methacrylic acid was 92 mg / g. The imprinting factor was 6.8. The effect of methacrylic acid on the imprinting effect showed that the best imprinting effect was obtained when the concentration of methacrylic acid monomer was 0.008 M.

[0185] Example 16

[0186] Effect of functional monomer dimethylaminoethyl methacrylate on imprinting effect.

[0187] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0188] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0189] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 18 μL of dimethylaminoethyl methacrylate (0.001 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0190] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0191] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0192] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0193] Imprinting Effect Research:

[0194] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The content of PEG-1 is 85 mg / g and the imprinting factor is 7.08.

[0195] Example 17

[0196] Effect of functional monomer dimethylaminoethyl methacrylate on imprinting effect.

[0197] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0198] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0199] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 89 μL of dimethylaminoethyl methacrylate (0.011 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, dissolve 658 mg of a polypeptide cross-linker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0200] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0201] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0202] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0203] Imprinting Effect Research:

[0204] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max The concentration of dimethylaminoethyl methacrylate monomer was 105 mg / g, and the imprinting factor was 7.8. The effect of dimethylaminoethyl methacrylate on the imprinting effect showed that the best imprinting effect was obtained when the concentration of dimethylaminoethyl methacrylate monomer was 0.008 M.

[0205] Example 18

[0206] The effect of the amount of polyglutamic acid polypeptide cross-linker used, that is, the degree of cross-linking of the system, on the imprinting effect.

[0207] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0208] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0209] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 89 μL of dimethylaminoethyl methacrylate (0.011 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, control the system crosslinking degree to 4% and dissolve 2362 mg of a polypeptide crosslinker with a degree of polymerization of 22 in the solution to obtain a prepolymer solution.

[0210] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0211] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0212] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0213] Imprinting Effect Research:

[0214] Different concentrations of bovine serum albumin solutions were prepared using 20 mM pH 5.0 phosphate buffer solution. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated and the adsorption isotherm was plotted (see Appendix). Figure 4 B) Curve fitting using the Langmuir model to obtain the imprinted capacity Q of BSA-PC-Fe3O4@MIP max The adsorption of BSA on BSA-PC-Fe3O4@MIP was 140 mg / g, the imprinting factor was 10.4, and the adsorption equilibrium state of BSA on BSA-PC-Fe3O4@MIP was reached when the initial concentration of BSA solution was 0.5 mg / mL. At the same time, the adsorption process of BSA-PC-Fe3O4@MIP reached equilibrium in about 30 minutes. Compared with the common surface imprinted microspheres, it has the ability to reach adsorption equilibrium quickly. Figure 4A. Reference (LI X, ZHOU J, et al. Effect of crosslinking degree and thickness of thermosensitive imprinted layers on recognition and elution efficiency of protein imprinted magnetic microspheres [J]. Sensors and Actuators B: Chemical, 2016, 225: 436-445.) shows that the polymerization reaction time of the surface imprinted microspheres is 24 h, the adsorption equilibrium time is 150 min, and the imprinting effect is optimal when the system crosslinking degree is 30%, the imprinting capacity is 49.66 mg / g, and the imprinting factor is 4.56. The surface imprinted microsphere system prepared in this embodiment has the best imprinting effect with a crosslinking degree of only 4%, and has significantly improved the polymerization reaction time, the time to reach adsorption equilibrium, the specific selectivity for the template protein, and the imprinting effect.

[0215] Example 19

[0216] The effect of the amount of polypeptide cross-linker used, that is, the degree of cross-linking of the system, on the imprinting effect.

[0217] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0218] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0219] 3) Add 40 mg of bovine serum albumin to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 89 μL of dimethylaminoethyl methacrylate (0.011 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, control the system crosslinking degree to 5%, and dissolve 3290 mg of a polypeptide crosslinker with a degree of polymerization of 22 in the above solution to obtain a prepolymer solution.

[0220] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0221] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0222] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0223] Imprinting Effect Research:

[0224] Bovine serum albumin solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of the BSA-PC-Fe3O4@MIP. max It is 132 mg / g and the imprinting factor is 9.8.

[0225] Example 20

[0226] Changing the template protein used and selecting different template proteins will affect the imprinting performance of the prepared nanoimprinted microspheres.

[0227] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0228] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0229] 3) Add 40 mg of another protein, such as bovine hemoglobin (BHb), to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 89 μL of dimethylaminoethyl methacrylate (0.011 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, control the system's crosslinking degree to 4% and dissolve 2362 mg of a polypeptide crosslinker with a degree of polymerization of 22 in the solution to obtain a prepolymer solution.

[0230] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0231] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0232] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0233] Imprinting Effect Research:

[0234] BHb solutions of varying concentrations were prepared in 20 mM pH 5.0 phosphate buffer. BHb-PC-Fe3O4@MIP or BHb-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of BHb-PC-Fe3O4@MIP. max It is 129 mg / g and the imprinting factor is 9.58.

[0235] Example 21

[0236] Changing the template protein used and selecting different template proteins will affect the imprinting performance of the prepared nanoimprinted microspheres.

[0237] 1) HRP and carboxyl-functionalized Fe3O4 nanospheres are covalently fixed on the surface of the microspheres to obtain HRP-modified nanospheres.

[0238] 2) The HRP-modified nanospheres prepared in step 1 were dispersed into 50 mL of phosphate buffer solution by ultrasonication to obtain a dispersion.

[0239] 3) Add 40 mg of another protein, such as human serum albumin (HSA), to the dispersion from step 2 and stir for 5 minutes to mix thoroughly. Then, add 42 mg of acrylamide (0.84 mg / mL), 35 μL of methacrylic acid (0.008 M), and 89 μL of dimethylaminoethyl methacrylate (0.011 M) to the solution. Then, add 2 g of the main monomer, N-isopropylacrylamide (40 mg / mL), and stir for 10 minutes to fully dissolve. Subsequently, control the system's crosslinking degree to 4% and dissolve 2362 mg of a polypeptide crosslinker with a degree of polymerization of 22 in the solution to obtain a prepolymer solution.

[0240] 4) Pass N2 into the prepolymer solution of step 3 to remove O2.

[0241] 5) The pH of the prepolymer solution after purging O₂ in step 4 was adjusted to 5.0. Under an N₂ atmosphere, 2.04 mL of acetylacetone and 0.75 mL of hydrogen peroxide were added to initiate free radical polymerization on the surface of the nanospheres via a surface enzymatic reaction. The mixture was mechanically stirred at 25°C under an N₂ atmosphere for 4 h. The resulting surface-imprinted microspheres were washed three times with ultrapure water.

[0242] 6) The template protein was eluted from the surface-imprinted microspheres in step 5 with 40 mL of 20 mM pH 7.4 phosphate buffer (containing 154 mM NaCl). The template protein in the supernatant of the eluate was measured using a UV spectrophotometer. After two elutions, no template protein was detected in the supernatant.

[0243] Imprinting Effect Research:

[0244] HSA solutions of varying concentrations were prepared using 20 mM pH 5.0 phosphate buffer. HSA-PC-Fe3O4@MIP or HSA-PC-Fe3O4@NIP was added at 25°C. After reaching adsorption equilibrium, the concentration of bovine serum albumin in the supernatant was measured using a UV spectrophotometer. The adsorption amount was calculated, and adsorption isotherms were plotted. The Langmuir model was used for curve fitting to obtain the imprinting capacity, Q, of HSA-PC-Fe3O4@MIP. max The adsorption capacity was 134 mg / g, and the imprinting factor was 9.9. The results showed that the method used in the present invention can be used to prepare surface-imprinted microspheres of various template proteins, and still have excellent specific adsorption effects.

[0245] Specific adsorption studies:

[0246] Various protein solutions, including bovine serum albumin, lactate dehydrogenase (LDH), glucose oxidase (GOX), trypsin inhibitor (Try), myoglobin (Mb), and cytochrome C (Cyt C), were prepared at a concentration of 0.5 mg / mL using 20 mM pH 5.0 phosphate buffer. BSA-PC-Fe3O4@MIP or BSA-PC-Fe3O4@NIP was added at 25°C. After sufficient adsorption, the absorbance of the supernatant was detected by UV light, and the adsorption amount was calculated. The imprinting factors of the various proteins were 10.4 (BSA), 1.9 (LDH), 1.7 (GOX), 1.53 (Try), 1.3 (Mb), and 1.26 (Cyt C), respectively. See the attached instructions for the instructions. Figure 5 This result shows that the BSA-PC-Fe3O4@MIP surface imprinted microspheres prepared by the present invention can selectively adsorb BSA with high specificity and have a high affinity for BSA, while the specific selectivity for non-template proteins is much lower.

[0247] Extraction of BSA from fetal bovine serum:

[0248] Fetal bovine serum was diluted 50 times with 20mM pH 5.0 phosphate buffer solution. 20mg BSA-PC-Fe3O4@MIP surface imprinted microspheres were swollen in pH 5.0 phosphate buffer solution (20mM) at 25°C for 1h. The imprinted microspheres were magnetically separated and added to 5mL of 50-fold diluted fetal bovine serum and adsorbed at 25°C for 1h. After adsorption, the imprinted microspheres were magnetically separated and eluted with 20mM pH 7.4 phosphate buffer solution containing 154mMNaCl to recover the adsorbed BSA. The extraction results were analyzed by SDS-PAGE. Figure 6 In the figure, the first lane is natural BSA, the second lane is untreated fetal bovine serum sample, the third lane is egg white sample treated with nano-imprinted microspheres, and the fourth lane is the sample of surface imprinted microsphere eluate. Figure 6 The results in the Figure 15 demonstrate that the BSA surface-imprinted microspheres synthesized in Example 18 can highly specifically extract BSA from fetal bovine serum. In lane 3, BSA from the fetal bovine serum was almost completely extracted, while in lane 4, only a BSA band was present in the eluate. HPLC analysis of the sample extraction results revealed a purity of over 95%.

[0249] As can be seen from the above examples, the surface imprinted microspheres prepared by HRP-mediated free radical polymerization provided by the present invention effectively avoid the agglomeration problem that may occur in traditional free radical polymerization by fixing HRP on the surface of the imprinted microspheres as the active core and using acetylacetone and hydrogen peroxide as the initiation system to initiate polymerization on the surface of the microspheres in situ. In addition, this polymerization method confirms the feasibility of the method for preparing surface imprinted microspheres by enzyme-mediated free radical polymerization under conditions of higher monomer concentration (40 mg / mL). This method promotes the full preassembly of monomers and template molecules, improves the integrity and specificity of the imprinted sites, and the obtained BSA-PC-Fe3O4@MIP has a higher adsorption capacity (Q max =140mg / g) and imprinting factor (IF=10.4). In addition, this polymerization method is simpler, the polymerization process is easier to control, and the polymerization reaction can be completed within 4h. Regarding the introduction of the polypeptide cross-linker, because it has a pH-responsive helix-coil conformational transition, when the pH is adjusted to 4.5-5.0, the polypeptide cross-linker exists in the helix conformation and will not precipitate from the reaction solution. The imprinted cavity can accurately restore the shape that is complementary to the size and spatial configuration of the template protein under the condition of changing pH, and can accurately recognize the template protein, thereby further improving the specific selection ability for the template protein. At the same time, due to the presence of the polypeptide cross-linker, the cross-linking degree of the imprinted shell is reduced and the swelling degree is increased. The combined effect of the above conditions makes the BSA-PC-Fe3O4@MIP surface imprinted microspheres have a high specific selection ability for the template protein.

Claims

1. A method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation; characterized in that: The following steps are involved: 1) Horseradish peroxidase (HRP) is reacted with activated carboxyl-functionalized Fe3O4 nanoparticles to undergo amidation reaction, and HRP is immobilized on the surface of the microspheres as active sites to obtain HRP-modified nanoimprinted microspheres; 2) dispersing the HRP-modified nanoimprinted microspheres obtained in step 1) in a phosphate buffer solution under ultrasound to obtain a dispersion of HRP-modified nanoimprinted microspheres; 3) adding the main monomer N-isopropylacrylamide, the functional monomers acrylamide, methacrylic acid and dimethylaminoethyl methacrylate, a polypeptide cross-linker, and the template protein bovine serum albumin (BSA) to the dispersion of the HRP-modified nanoimprinted microspheres prepared in step 2); after complete dissolution, stirring and mixing uniformly, and allowing to stand to complete the pre-assembly process, thereby obtaining a pre-polymerized mixed solution; the cross-linker is a polyglutamic acid polypeptide cross-linker having double bonds at both ends of the chain segment; 4) passing N2 into the prepolymerized mixed solution obtained in step 3) to remove O2 in the system; 5) Under mechanical stirring and an N2 atmosphere, the pH of the solution after purging the system O2 from step 4) is adjusted to maintain the polypeptide cross-linker in a helix conformation, and acetylacetone and hydrogen peroxide are added thereto to initiate a free radical polymerization reaction catalyzed by HRP; after completion of the reaction, the microspheres in the solution are magnetically separated and then washed twice with ultrapure water to obtain surface-imprinted nanospheres imprinted with the template; 6) The surface-imprinted nanospheres obtained in step 5) are placed in a phosphate buffer solution to elute the template, thereby obtaining nano-imprinted microspheres with imprinted cavities.

2. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation according to claim 1, wherein: Based on the volume of the phosphate buffer solution used in step 2), the concentration of the main monomer N-isopropylacrylamide added in step 3) in the phosphate buffer solution is 5 to 50 mg / mL, the concentration of the functional monomer acrylamide (AAM) is 0.1 to 1.1 mg / mL, the concentration of methacrylic acid (MAA) is 0.001 to 0.011 M, and the concentration of dimethylaminoethyl methacrylate (DMAEMA) is 0.001 to 0.011 M.

3. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation according to claim 1, wherein: The cross-linking agent used in step 3) has a degree of polymerization ranging from 18 to 25 and has a pH-responsive helix-coil conformational transition.

4. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation according to claim 1, wherein: The sum of the amounts of the monomers and functional monomers used in step 3) is taken as the total amount, and the amount of the polypeptide cross-linking agent added in step 3) is 1-5% of the total amount.

5. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface initiation according to claim 1, characterized in that: In step 5), the pH of the solution is adjusted to 4.5-5.

0. The polypeptide cross-linker is in a helix conformation in this pH range, and the polypeptide cross-linker will not precipitate from the solution in this pH range.

6. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface priming according to claim 1, wherein: In step 5), the HRP-mediated free radical polymerization has a reaction temperature of 20° C. to 35° C. and a reaction time of 2.5 to 6 h.

7. The method for preparing surface-imprinted polymer microspheres by horseradish peroxidase-mediated surface priming according to claim 1, wherein: In step 6), the template is eluted in a solution similar to the human body fluid environment, and a phosphate buffer solution with a pH of 7.4 containing 154 mM NaCl is selected as the elution medium. At this time, the polypeptide cross-linker is in a coil conformation.

8. The surface-imprinted polymer microspheres prepared by horseradish peroxidase-mediated surface initiation according to claim 1 are used as adsorption materials in the high-specificity recognition and selection, rapid separation and purification of BSA.

Images