Protein surface oriented imprinting microsphere as well as preparation method and application thereof

Abstract

The invention discloses a protein surface oriented imprinting microsphere as well as a preparation method and application thereof, and belongs to the technical field of western blot materials. According to the method provided by the invention, the protein surface imprinting material is successfully prepared by virtue of surface directional imprinting and water-phase polymerization, the stability is high, the interaction force with the protein to be detected is strong, and the target protein can be stably and specifically recognized; according to the protein surface oriented imprinting core-shell microsphere prepared by the method provided by the invention, a base sphere is of a dendritic structure, the diameter is 80nm, the surface has abundant wrinkles and high porosity, a shell layer is a polyion liquid macromonomer polymer layer, target protein is oriented and anchored, and the microsphere has abundant recognition sites, excellent dispersity and high particle size uniformity; the provided preparation method of the protein surface oriented imprinting microspheres is simple and easy to operate, the condition is mild, the used reagent is low in toxicity or non-toxic, no toxic or harmful substance is generated, and the preparation method is environment-friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of protein imprinting materials, and in particular to a protein surface directional imprinting microsphere and a preparation method and application thereof. Background Art

[0002] Surface imprinting technology can ensure that the mass transfer of the target protein is at the interface between the polymer and the liquid phase, so that the imprinted cavity is confined to the surface of the material, thereby reducing steric hindrance, making mass transfer easier, and improving accessibility to the target molecule. Nankai University used the water-soluble thermosensitive monomer di(ethylene glycol) methyl methacrylate (MEO2MA) to realize the preparation of protein surface imprinted materials. They used MEO2MA as the thermosensitive monomer, MAA and acrylamide (AAm) as functional monomers that provide Coulomb force and hydrogen bonding, respectively, and MBA as a cross-linking agent, and prepared lysozyme (Lyz) imprinted nanospheres by aqueous precipitation polymerization. Professor Liu Zhen's team at Nanjing University proposed a boric acid affinity-controlled directional surface protein imprinting strategy for the efficient separation and identification of carbohydrate biomacromolecules such as polysaccharides and glycoproteins containing cis-diols. The template glycoprotein (TRF) was fixed on the boric acid functionalized matrix, and the aqueous phase self-polymerization of dopamine and m-aminophenylboronic acid (APBA) was completed and then eluted with an acidic eluent to obtain the imprinted material. In addition, they also mimicked the unique property of lectins, which can recognize molecules containing specific sugar fragments (glycopeptides, polysaccharides, or glycoproteins). Using the polysaccharide fragment of ribonuclease B (RNase B) as a template molecule, they used surface imprinting technology to coat a lectin-like imprinted polymer on a magnetic carrier, increasing the IF to 8.40. Therefore, one of the core aspects of accurate protein recognition is to combine surface imprinting technology to design and develop "directional" protein surface imprinting materials with favorable mass transfer. Summary of the Invention

[0003] The purpose of the present invention is to provide a protein surface directional imprinting microsphere and its preparation method and application, and to provide a protein surface directional imprinting material with high stability and uniformity, good dispersibility, strong specificity, rich binding sites and simple preparation method.

[0004] To achieve the above object, the present invention provides a method for preparing protein surface directional imprinted microspheres, comprising the following steps:

[0005] S1. Synthesize an ionic liquid monomer and a modifier, wherein the modifier is 1-vinyl-3-propyltrimethoxysilane imidazole chloride, and the ionic liquid monomer includes tosylated 1-vinyl-β-cyclodextrin imidazole, 1-vinyl-3-acetamide imidazole chloride, 1-vinyl-3-propanesulfonic acid imidazole chloride, or 1-vinyl-3-styrene imidazole chloride;

[0006] S2. Prepare a polyionic liquid block copolymer macromonomer, use 2-mercapto-S-thiobenzoyl acetic acid as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and 1-vinyl-3-acetamidoimidazole chloride as a monomer, and thermally initiate to obtain Macro-CTA under N2 atmosphere, add toluenesulfonic acid 1-vinyl-β-cyclodextrin imidazole monomer and azobisisobutyronitrile to obtain a precursor Precursor-1; The precursor Precursor-2 was obtained by thermal initiation under N2 atmosphere using dimethyl sulfoxide as a chain transfer agent, azobisisobutyronitrile as an initiator, 1-vinyl-3-propanesulfonic acid imidazolium chloride and 1-vinylimidazole as monomers; the precursor Precursor-2 was reacted with 4-vinylbenzyl chloride in dimethyl sulfoxide to obtain a polyionic liquid block copolymer macromonomer p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM;

[0007] S3. Prepare a dendritic silica nanocarrier material by dissolving triethanolamine, hexadecyltrimethylammonium bromide, and sodium salicylate in deionized water. After stirring and reacting, add ethyl orthosilicate and continue the reaction. After the reaction is completed, remove the unreacted material, wash the precipitate with deionized water, dry, grind, and calcine.

[0008] S4. Double bond modification is performed on the surface of the obtained dendritic silica nanocarrier material. The dendritic silica nanocarrier material is calcined, cooled, and dispersed in anhydrous toluene. 1-vinyl-3-propyltrimethoxysilane imidazole chloride is added, stirred and refluxed, centrifuged and washed, and then solidified to obtain SiO2@VIMPS.

[0009] S5. Prepare protein surface-directed imprinted microspheres, dissolve the target protein and p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM prepared in phosphate buffer, and magnetically stir for a certain period of time to allow the target protein to be fully anchored on the surface of p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM to obtain solution I; disperse the SiO2@VIMPS obtained in S4 in phosphate buffer to obtain solution II; mix solution I and solution II and self-assemble, then add persulfate and tetramethylethylenediamine, purge with nitrogen and stir to react, completely wash the collected nanocarriers to remove the bound target protein, and freeze-dry to obtain target protein surface-directed imprinted microspheres.

[0010] Preferably, the preparation method of the modifier in S1 comprises the following steps:

[0011] 1-vinylimidazole and 3-chloropropyltrimethoxysilane were dissolved in a mixed solution of ethyl acetate and water, and after reflux reaction, the mixture was extracted with ethyl acetate. The aqueous solution after extraction was freeze-dried to obtain 1-vinyl-3-propyltrimethoxysilane imidazole chloride ion liquid; the molar volume ratio of 1-vinylimidazole: 3-chloropropyltrimethoxysilane: ethyl acetate: water was: 2.5 mol: 3.1 mol: 1 L: 1 L.

[0012] Preferably, the preparation method of 1-vinyl-3-acetamidoimidazole chloride in S1 comprises the following steps:

[0013] Dissolve chloroacetamide in acetone and stir until the solution is clear. Add 1-vinylimidazole dropwise under vigorous stirring to react. After vacuum distillation, wash the precipitate 3-5 times and freeze-dry the precipitate. The molar ratio of chloroacetamide to 1-vinylimidazole is 8:5. Wash the precipitate with acetone.

[0014] Preferably, the preparation method of tosylated 1-vinyl-β-cyclodextrin imidazole in S1 comprises the following steps:

[0015] β-cyclodextrin was dissolved in deionized water, and an aqueous solution of sodium hydroxide and an acetonitrile solution of toluenesulfonyl chloride were added dropwise thereto. After stirring for reaction, the precipitate was removed, and the filtrate was refrigerated overnight to recover the precipitated toluenesulfonated β-cyclodextrin. The mass volume ratio of β-cyclodextrin: deionized water: sodium hydroxide: water: toluenesulfonyl chloride: acetonitrile was 6 g: 0.05 L: 0.66 g: 0.002 L: 1 g: 0.003 L.

[0016] Toluenesulfonated β-cyclodextrin is dissolved in a mixed solution of vinylimidazole and N,N-dimethylformamide, and the reaction is carried out under a protective atmosphere. After the reaction is completed, the mixture is cooled to ambient temperature to precipitate a crude product, and the precipitate obtained by filtration is vacuum-dried to obtain a toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid. The mass volume ratio of toluenesulfonated β-cyclodextrin: 1-vinylimidazole: N,N-dimethylformamide: acetone is 6.45 g: 0.002 L: 0.015 L: 0.07 L.

[0017] Preferably, the preparation method of 1-vinyl-3-propanesulfonic acid imidazole chloride in S1 comprises the following steps:

[0018] Dissolve 1-vinylimidazole in acetonitrile, add 1,3-propane sultone dropwise under stirring, react for 24 hours, collect the precipitate, purify it, and then freeze-dry it; the molar volume ratio of 1-vinylimidazole:acetonitrile:1,3-propane sultone is 2 mol:1L:3 mol.

[0019] Preferably, the preparation method of the 1-vinyl-3-styrene imidazolium chloride ion liquid in S1 comprises the following steps:

[0020] 1-vinylimidazole and vinylbenzyl chloride are dissolved in a mixed solution of ethyl acetate and deionized water for reaction, followed by extraction with ethyl acetate, and the extracted aqueous phase is freeze-dried; the molar volume ratio of 1-vinylimidazole:vinylbenzyl chloride:ethyl acetate:deionized water is 2.5 mol:2.15 mol:1 L:1 L.

[0021] Preferably, when preparing Macro-CTA in S2, the volume ratio of transfer agent: solvent: initiator: 1-vinyl-3-acetamide imidazole chloride monomer is 1:560:0.4:20; when preparing precursor Precursor-1, the molar ratio of Macro-CTA: toluenesulfonated 1-vinyl-β-cyclodextrin imidazole monomer: azobisisobutyronitrile is 1:5:1; when preparing precursor Precursor-2, the mass volume ratio of precursor Precursor-1: dimethyl sulfoxide: azobisisobutyronitrile: 1-vinyl-3-propanesulfonic acid imidazole chloride: 1-vinyl imidazole is 500 mg: 30 mL: 33 mg: 300 mg: 800 mg, and the thermal initiation temperature is 70 ° C; preparation of p-VIMA-co-VIMCD In the case of s-co-VSPIM-co-PVIM, the mass volume ratio of Precursor-2: dimethyl sulfoxide: 4-vinylbenzyl chloride is 2 g: 25 mL: 500 mg.

[0022] Preferably, the molar ratio of triethanolamine: CTAB: sodium salicylate: ethyl orthosilicate in S3 is 7:16:4:274; the mass volume ratio of dendritic silica nanocarrier (SiO2): anhydrous toluene: 1-vinyl-3-propyltrimethoxysilane imidazole chloride in step S4 is 1.5 g: 50 mL, and the mass volume ratio of SiO2: 1-vinyl-3-propyltrimethoxysilane imidazole chloride is 150 g: 4 mL.

[0023] Preferably, the pH of the phosphate buffer in the S5 is 7.40, and the mass volume ratio of p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM: cytochrome C: SiO2@VIMPS: PBS buffer is 4g:0.1g:100mg:12.6g; the bound target protein is washed away with a methanol / acetic acid solution with a volume ratio of 9:1, a formic acid solution with a volume fraction of 20%, and ultrapure water.

[0024] A protein surface directional imprinted microsphere prepared by the method for preparing the protein surface directional imprinted microsphere as described above.

[0025] A method for preparing the above-mentioned protein surface directional imprinted microspheres and / or application of the protein surface directional imprinted microspheres in protein recognition.

[0026] Therefore, the present invention provides a protein surface directional imprinted microsphere and its preparation method and application, and its specific technical effects are as follows:

[0027] (1) The method provided by the present invention successfully prepared a protein surface imprinting material by means of surface directional imprinting and aqueous phase polymerization. The material has high stability, strong interaction with the target protein, and can stably and specifically identify the target protein accurately.

[0028] (2) The protein surface directional imprinted microspheres prepared by the method provided by the present invention have a diameter of 80 nm, a "dendritic" structure, abundant wrinkles on the surface, high porosity, abundant recognition sites, excellent dispersibility, and high particle size uniformity;

[0029] (3) The method for preparing protein surface directional imprinted microspheres provided by the present invention is simple and easy to operate, has mild conditions, uses reagents with low or no toxicity, does not generate any toxic or harmful substances, and is environmentally friendly.

[0030] (4) Using the method provided by the present invention to prepare polyionic liquid block copolymer macromonomers, by introducing polyionic liquid block copolymer macromonomers during the imprinting process to maintain the stability of the template protein structure, combining surface imprinting and multiple anchoring protein strategies to improve the accuracy of the imprinted pores, and enhance the precise recognition of the target protein by the surface directional imprinting material;

[0031] (5) The method provided by the present invention is used to study the improvement of the selective recognition performance of the target protein by the imprinting material. The comprehensive experimental and theoretical research analyzes the stabilization rules of the protein structure by the block monomer structure ratio, monomer dosage, chain length, etc., and reveals the interaction mode and recognition mechanism between the imprinting material and the target protein.

[0032] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0034] Figure 1: This is a synthetic route for the ionic liquid monomer and modifier in Example 1 of the present invention; wherein part (A) is a schematic diagram for the synthesis of VIMPS; part (B) is a schematic diagram for the synthesis of VIMCDs ionic liquid monomer; part (C) is a schematic diagram for the synthesis of VIMA ionic liquid monomer; part (D) is a schematic diagram for the synthesis of VSPIM ionic liquid monomer; and part (E) is a schematic diagram for the synthesis of PVIM ionic liquid monomer.

[0035] Figure 2 The ionic liquid monomer and modifier synthesized in Example 1 of the present invention are 1 HNMR spectra; (A) is the H NMR spectrum of VIMPS; (B) is the H NMR spectrum of VIMCDs; (C) is the H NMR spectrum of VIMA; (D) is the H NMR spectrum of VSPIM; (E) is the H NMR spectrum of PVIM;

[0036] Figure 3 This is the infrared spectrum of the ionic liquid monomer synthesized in Example 1 of the present invention;

[0037] Figure 4 This is a flow chart for preparing the polyionic liquid block copolymer macromonomer in Example 1 of the present invention;

[0038] Figure 5 The polyionic liquid block copolymer macromonomer in Example 1 of the present invention is 1 HNMR spectrum;

[0039] Figure 6 These are the electron microscope observation results and scanning images of the dendritic silica nanocarrier material prepared in Example 1 of the present invention; wherein ac is the SEM image; df is the TEM image; gi is the EDS element scanning image, yellow represents O and red represents Si;

[0040] Figure 7 The physical and chemical properties analysis results of the dendritic silica nanocarrier material prepared in Example 1 of the present invention; wherein a is an XRD graph; b is an FT-IR graph; c is a nitrogen adsorption-desorption isotherm graph; d is a pore size distribution graph;

[0041] Figure 8 : This is an XPS spectrum of the dendritic silica nanocarrier material prepared in Example 1 of the present invention; wherein a is a full-resolution spectrum; b is a C1s high-resolution spectrum; c is an O1s high-resolution spectrum; d is a Si 2p high-resolution spectrum;

[0042] Figure 9 Schematic diagram of the preparation process of cytochrome C protein surface-directed imprinted microspheres in Example 1 of the present invention;

[0043] Figure 10This is the result of measuring the adsorption performance of Cyt-C by the surface-directed imprinted microspheres in Example 2 of the present invention;

[0044] Figure 11 These are the results of the binding specificity test of SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs with Cyt-C, OVA, and BSA proteins in Example 3 of the present invention;

[0045] Figure 12 This is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) diagram of competitive adsorption of protein surface-directed imprinted microspheres in Example 4 of the invention; wherein Lane 1 is a protein molecular weight marker; Lane 2 is a mixed solution of BSA, OVA, and Cyt-C; Lane 3 is the protein remaining after SiO2@VIMPS@MIPs adsorption; Lane 4 is the protein remaining after SiO2@VIMPS@NIPs adsorption. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0047] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0048] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0049] Example 1

[0050] Preparation of protein surface directional imprinted microspheres, the specific steps are as follows:

[0051] S1. Synthesis of ionic liquid monomer and modifier.

[0052] The 1-vinyl-3-propyltrimethoxysilane imidazole chloride (VIMPS) modifier of the ionic liquid trimethoxysilane, as well as polymerizable toluenesulfonated 1-vinyl-β-cyclodextrin imidazole (VIMCDs), 1-vinyl-3-acetamide imidazole chloride (VIMA), 1-vinyl-3-propanesulfonic acid imidazole chloride (VSPIM) and 1-vinyl-3-phenylimidazolium chloride (PVIM) containing a zwitterionic structure were prepared by alkylation substitution reaction. The synthesis processes are as follows: Figure 1 As shown in parts (A), (B), (C), (D), and (E).

[0053] 1) Synthesize VIMPS reagent, the synthesis process is as follows Figure 1 As shown in part (A) of the diagram, the specific steps are as follows:

[0054] 4.7 g of 1-vinylimidazole and 8.54 g of 3-chloropropyltrimethoxysilane were dissolved in 20 mL of ethyl acetate and refluxed at 70° C. for 24 h. After the reaction, the mixture was extracted with ethyl acetate. The aqueous solution after extraction was freeze-dried to obtain VIMPS ionic liquid.

[0055] 2) Synthesis of VIMCDs ionic liquid monomers, the synthesis process is as follows Figure 1 As shown in part (B) of the , the specific steps are as follows:

[0056] 30 g of cyclodextrin was suspended in 250 mL of water, and a solution of 3.285 g of NaOH in 10 mL of water was added. A solution of 5.021 g of p-toluenesulfonyl chloride in 15 mL of acetonitrile was added dropwise over 8 minutes. After stirring at 23°C for 2 hours, the precipitate was removed by suction filtration, and the filtrate was refrigerated at 4°C overnight. The precipitate was recovered by suction filtration to obtain tosylated β-cyclodextrin (6-TsO-β-CD).

[0057] 6.45 g of 6-TsO-β-CD was dissolved in a mixed solution of 1.8 mL of 1-vinylimidazole and 15 mL of anhydrous DMF and reacted at 75°C for 4 h under a N2 atmosphere. After cooling to ambient temperature, the crude product was precipitated with 70 mL of acetone and then filtered to obtain VIMCDs.

[0058] 3) Synthesis of VIMA ionic liquid monomer, the synthesis process is as follows Figure 1 As shown in part (C) of the diagram, the specific steps are as follows:

[0059] 2.99 g of chloroacetamide (32 mmol) was dissolved in 50 mL of acetone and stirred at 45°C for approximately 20 minutes until a clear solution was obtained. Then, 1.88 g of 1-vinylimidazole (20 mmol) was added dropwise using a constant pressure funnel with magnetic stirring at 400 rpm, followed by reaction at 50°C for 24 hours. The precipitate was then washed repeatedly with acetone and lyophilized in a freeze dryer.

[0060] 4) Synthesis of VSPIM ionic liquid monomers, the synthesis process is as follows Figure 1 As shown in part (D) of the , the specific steps are as follows:

[0061] 1-Vinyl imidazole (1.88 g, 0.02 mol) was dissolved in 10 mL of acetonitrile at 50°C. 1,3-Propane sultone (3.66 g, 0.03 mol) was slowly added dropwise to the reactor using a constant pressure funnel under magnetic stirring at 400 rpm. The mixture was stirred at 50°C for 24 h to obtain 4.53 g of a white precipitate. The precipitate was further purified by washing with acetone and then freeze-dried to obtain the VSPIM ionic liquid monomer.

[0062] 5) Synthesis of PVIM ionic liquid monomer, the synthesis process is as follows Figure 1 As shown in part (E) of the , the specific steps are as follows:

[0063] 4.6 g VIM (50 mmol) and 6.5 g vinylbenzyl chloride (43 mmol) were dissolved in 20 mL ethyl acetate and 20 mL water and reacted for 24 h. After the reaction, the mixture was extracted with ethyl acetate, and the extracted aqueous phase solution was freeze-dried to obtain PVIM.

[0064] The ionic liquid monomers obtained in steps 2)-5) and the lyophilized product of the ionic liquid modified reagent obtained in step 1) were subjected to nuclear magnetic resonance spectroscopy ( 1 HNMR) and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the chemical structure, purity and properties of the above ionic liquids.

[0065] 1 The results of HNMR and FT-IR are as follows Figure 2 and Figure 3 As shown, the scanning wavelength range is 4000~450cm -1 The spectrum shows that there are corresponding absorption peaks. In the spectrum, there is unsaturated CH stretching vibration on the imidazole ring of the imidazole ionic liquid (3200~3000cm -1 ), C=N stretching vibration (1590cm -1 ), CH out-of-plane rocking bending vibration (734cm -1 ), and C=C stretching vibration of olefins connected to imidazole rings (1651 cm -1 ). Among them, -OH stretching vibration (3341cm -1 ), ether group-COC- stretching vibration (1027cm -1 ), C=O stretching vibration in ester group (1730cm -1 )、COC stretching vibration (1280~1100cm -1 ) showed that monomer VIMCDs were successfully synthesized. C=O stretching vibration on amide (1670cm -1 ), CN stretching vibration (1404cm -1 ), NH stretching vibration (3116cm -1) showed that C.VIMA was successfully synthesized. SO stretching vibration (980cm -1 ), S=O stretching vibration (1134cm -1 ), SC stretching vibration (665cm -1 ) indicates that the monomer D.VSPIM was successfully synthesized. CH stretching vibration on the benzene ring (3056cm -1 ), CC skeleton vibration (1546cm -1 、1454cm -1 ) and the out-of-plane bending vibration of CH on the m-disubstituted benzene ring (717 cm -1 ) showed that PVIM was successfully synthesized.

[0066] S2. Prepare polyionic liquid block copolymer macromonomer.

[0067] In order to enable the polyionic liquid block copolymer macromolecular monomer to produce multiple protein anchoring effects such as electrostatic, hydrophobic, hydrogen bonding and π-π stacking with proteins in the aqueous phase, and still maintain good interactions in the aqueous medium, the imidazolyl ionic liquid structure is designed into the block copolymer macromolecular chain as a "functional unit" by utilizing the designability and protein stability of ionic liquids. The specific steps are as follows:

[0068] 1) Prepare precursor Precursor-1.

[0069] Macro-CTA was prepared by reversible addition-fragmentation chain transfer controlled radical polymerization (RAFT) using 2-mercapto-S-thiobenzoylacetic acid as a chain transfer agent (CTA), the VIMA ionic liquid prepared in step S1 as a monomer, dimethyl sulfoxide (DMSO) as a solvent, and azobisisobutyronitrile (AIBN) as an initiator under a nitrogen atmosphere. Subsequently, the VIMCDs prepared in step S1 and AIBN were added to prepare the precursor Precursor-1: p(VIMA-b-VIMCDs). The length of the p(VIMA-b-VIMCDs) was adjusted by varying the ratio of Macro-CTA to VIMCDs and VIMA monomers. The hydrophilicity of the p(VIMA-b-VIMCDs) was also controlled by varying the amounts of VIMCDs and PVIM monomers.

[0070] A precursor Precursor-1 was prepared by the following specific steps:

[0071] CTA (212 mg, 1 mmol) and AIBN (66 mg, 0.4 mmol) were dissolved in 40 mL of DMSO, and VIMA (3.75 g, 20 mmol) was added. A thermal reaction was initiated under a nitrogen atmosphere to prepare Macro-CTA. Subsequently, VIMCDs (6.55 g, 5 mmol), VSPIM (0.25 g, 1 mmol), and 33 mg of AIBN were added. After mixing with 30 mL of DMSO, the mixture was deoxygenated with nitrogen for 0.5 h and then placed in a 70°C oil bath for 12 h. The tube was placed in liquid nitrogen to cool the reaction and then dried in a freeze dryer to obtain Precursor-1:p(VIMA-b-VIMCDs).

[0072] 2) Preparation of polyionic liquid block copolymer macromonomers.

[0073] Using the precursor Precursor-1 as the chain transfer agent, the same RAFT polymerization method was adopted, with DMSO as the solvent, AIBN as the initiator, VSPIM zwitterionic liquid and VIM as monomers, and thermal initiation under N2 atmosphere to prepare the polyionic liquid block copolymer macromolecular precursor Precursor-2: p(VI MA)-b-(VIMCDs-co-VSPIM-co-VIM).

[0074] Subsequently, the precursor Precursor-2 was reacted with 4-vinylbenzyl chloride in DMSO at 60°C under N2 atmosphere to prepare the final product polyionic liquid block copolymer macromonomer p(VIMA-b-(VIMCDs-co-VSPIM-co-PVIM), abbreviated as p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM. The preparation process is as follows: Figure 4 shown.

[0075] During the preparation process, the length of Precursor-2 can be adjusted by changing the ratio between Macro-CTA and monomer; the composition ratio of the zwitterionic liquid structure can be adjusted by changing the amount of VSPIM and VIM.

[0076] A polyionic liquid block copolymer macromonomer is prepared by the following specific steps:

[0077] 500 mg of precursor Precursor-1, 500 mg of PVIM (4 mmol) and 0.5 mg of hydroquinone were dissolved in 25 mL of DMSO and kept at 60°C under N2 atmosphere for 24 h. The final polyionic liquid block copolymer macromonomer was obtained by freeze-drying, which was recorded as p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM.

[0078] use1 The chemical structure of the prepared p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM was characterized by H NMR. Figure 5 Shown, indicated, 1 H NMR (400MHz, DMSO-d6) δ7.40(d,J=24.8Hz,5H),7.30(s,1H),7.08(d,J=7.7Hz,2H),6.88-6.47(m,1 3H),5.77(s,44H),5.33(d,J=4.9Hz,8H),4.87(d,J=13.6Hz,71H),4.73(d,J=6.8Hz,14H),4.60(d,J =8.3Hz,2H),4.48(s,1H),4.42(s,40H),4.18(d,J=11.5Hz,20H),3.91(s,20H),3.65(s,58H),3.53( s,18H),3.34(s,149H),2.05-1.95(m,21H),1.88(s,28H),1.72(s,45H),1.45(s,1H),1.23(s,79H).

[0079] S3. Prepare dendritic silica nanocarrier materials.

[0080] In order to increase the effective imprinting area and protein imprinting amount of the surface imprinting material, a dendritic silica nanocarrier with a large specific surface area was prepared. The specific preparation steps are as follows:

[0081] Triethanolamine (TEA, 36.57 mg, 2.45 mmol), the structure-directing agent hexadecyltrimethylammonium bromide (CTAB, 2.0436 g, 5.60 mmol), and sodium salicylate (NaSaI, 22.44 mg, 1.4 mmol) were weighed and dissolved in 200 mL of ultrapure water. The mixture was stirred at 80°C and 1000 rpm for 1 hour. Then, a silicon source, tetraethyl orthosilicate (TEOS, 21.51 mL), was added and allowed to react for 8 hours. After the reaction, unreacted material was removed by filtration. After washing with ultrapure water, the resulting dendritic silica was dried in a 60°C forced air drying oven for 12 hours. After complete drying, the resulting dendritic SiO2 was ground into a powder using a quartz mortar and collected. The dendritic silica powder was placed in a porcelain boat and calcined at 600°C in air for 8 hours to remove the organic template molecules, resulting in a dendritic silica nanocarrier material.

[0082] The obtained dendritic silica nanocarrier material was analyzed by SEM, TEM, EDS, XRD, FT-IR and XPS. Figure 6-Figure 8The results show that a nanocarrier with a diameter of 80 nm and a "dendritic" structure was successfully prepared. The surface of the dendritic silica nanocarrier has a rich corrugated structure and is full of pores. The dendritic silica nanocarrier has excellent dispersibility and a relatively uniform particle size.

[0083] S4, performing surface double bond modification on the dendritic silica nanocarrier material obtained in step S3.

[0084] To successfully modify SiO2, the dendritic silica nanocarrier material must first be activated. 1.5 g of the dendritic silica nanocarrier material was calcined at 200°C for 12 hours. After cooling, it was ultrasonically dispersed in 50 mL of anhydrous toluene. 2 mL (4%, v / v) of VIMPS was added, and the mixture was stirred and refluxed at 90°C in an oil bath for 24 hours. The product was then washed by centrifugation with acetone and methanol and cured in a vacuum drying oven at 110°C for 1 hour to obtain double-bond-modified SiO2@VIMPS.

[0085] S5. Prepare cytochrome C protein surface-directed imprinted microspheres.

[0086] Cytochrome C protein (Cyt-C protein) surface imprinted microspheres were prepared by surface grafting copolymerization in an aqueous medium via redox initiation. 4.06 g p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM (2.00 mmol) and 0.1 g template protein cytochrome C (Cyt-C, 10 μmol) were dissolved in 10 mL PBS buffer solution (PBS, pH 7.40) and magnetically stirred to obtain a prepolymer solution (Solution I). At the same time, the double-bond-modified SiO2@VIMPS (100 mg) nanospheres obtained in step S4 were dispersed in 5 mL PBS solution by ultrasonic vibration (Solution II). Subsequently, Solution I and Solution II were repeatedly mixed and placed in a 50 mL round-bottom flask, and the template protein cytochrome C and p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM were allowed to self-assemble at 25°C for 3 hours.

[0087] Afterwards, VIMPS (10 mg, 0.04 mmol) and TEMED (15 μL, 0.1 mmol) were added to the reaction solution, purged with nitrogen for 30 minutes, and then magnetically stirred for 24 hours. The nanospheres were collected, filtered, and washed with methanol / acetic acid (9 / 1, v / v), 20% formic acid (v / v), and water in sequence until the template molecules could no longer be detected by UV spectroscopy in the extract. The product was then freeze-dried and named cytochrome C protein surface-directed imprinted microspheres (SiO2@VIMPS@MIPs). The preparation process is shown in the following figure. Figure 9The synthesis conditions of the corresponding non-imprinted nanospheres (SiO2@VIMPS@NIPs) were the same as those of the cytochrome C protein surface-directed imprinted microspheres, except that the template protein cytochrome C was not added during the preparation process.

[0088] Example 2

[0089] The adsorption performance of the SiO2@VIMPS@MIPs prepared in Example 1 was tested using Cyt-C protein as the test protein, as follows:

[0090] 5.0 mg of SiO2@VIMPS@MIPs (NIPs) prepared in Example 1 was dispersed in a centrifuge tube containing 6.0 mL of a 0.5 mg / mL Cyt-C protein solution. After constant temperature adsorption at 37°C for 1 hour, the solution was centrifuged at 3000 rpm for 15 minutes to obtain a protein supernatant. The absorbance at a wavelength of 410 nm was detected using a UV-visible spectrophotometer to determine the Cyt-C protein concentration in the supernatant.

[0091] The adsorption capacity (Q) of SiO2@VIMPS@MIPs to proteins was calculated using the following formula (1):

[0092] Q=(C0-C)V / M (1)

[0093] Where: C0 is the initial concentration of the protein solution (mg / mL); C is the protein concentration of the supernatant after adsorption (mg / mL); V is the volume of the protein solution (mL); M is the mass of SiO2@VIMPS@MIPs (g).

[0094] Typically, the imprinting factor (IF) and selectivity factor (β) are used to evaluate the selective recognition performance of SiO2@VIMPS@MIPs, which are defined as follows:

[0095] IF=Q MIPs / Q NIPs (2)

[0096] Where: Q MIPs is the adsorption capacity of SiO2@VIMPS@MIPs on protein (mg / g); Q NIPs is the adsorption amount of protein by SiO2@VIMPS@NIPs (mg / g).

[0097] β=IF / IF ana (3)

[0098] Where: IF is the imprinting factor of SiO2@VIMPS@MIPs on template protein Cyt-C; IF ana It is the imprinting factor of SiO2@VIMPS@MIPs on competing proteins.

[0099] The adsorption performance of SiO2@VIMPS@MIPs on Cyt-C protein is shown in the following figure: Figure 10 As shown in the figure, the adsorption capacity of Cyt-C by SiO2@VIMPS@MIPs (Q = 56 mg / g) was significantly higher than that of SiO2@VIMPS@NIPs (Q = 23 mg / g), and the IF value was 2.43, indicating that the cavities of SiO2@VIMPS@MIPs have a highly specific recognition ability for Cyt-C. This indicates that the surface-imprinted microspheres prepared in Example 1 have potential application value in the field of protein separation and purification.

[0100] Example 3

[0101] The selective adsorption capacity of SiO2@VIMPS@MIPs prepared in Example 1 was investigated as follows:

[0102] Three 20mg portions of SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs were weighed and placed in 10mL sample vials. 5.0mL of 0.8mg / mL Cyt-C, BSA, and OVA solutions were then added to the vials. After mixing, the solutions were placed in a constant temperature water bath shaker at 28°C for 2h. The mixtures were then centrifuged at 10,000rpm for 10min, and the supernatants were aspirated. The concentrations of BSA and OVA were measured by UV-Vis at a wavelength of 280nm, and the concentration of Cyt-C in the solution was measured at 410nm. This yielded the adsorption capacity of the target proteins and analogs onto SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs.

[0103] Considering the molecular weight of Cyt-C (MW 12kDa, pI 9.6), several proteins were selected as analogs, including BSA (MW 68kDa, pI 4.9) and OVA (MW 43kDa, pI 4.7). The specific recognition performance of SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs for the above proteins is shown in Figure 2. Figure 11 As shown in Figure 2, SiO2@VIMPS@MIPs has the strongest specific recognition ability for Cyt-C (IF=2.43). BSA and OVA have larger molecular weights, which increase the mass transfer resistance in the imprinted cavity on HMS@MIP, and the IF is lower (IF BSA =1.36,IF OVA=1.29), indicating poor steric complementarity with the imprinted site. Furthermore, the adsorption capacities of BSA and OVA by SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs were similar to the nonspecific adsorption of Cyt-C by SiO2@VIMPS@MIPs. Physical interactions such as hydrophobic interactions and hydrogen bonding play a significant role in the adsorption process. Based on these analyses, differences in electrical properties and spatial structure preclude the effective binding of these analogs to the imprinted cavity of Cyt-C.

[0104] Example 4

[0105] The competitive adsorption performance of SiO2@VIMPS@MIPs prepared in Example 1 was investigated as follows:

[0106] Two 20mg portions of SiO2@VIMPS@MIPs and two 20mg portions of SiO2@VIMPS@NIPs were weighed and placed in 10mL sample vials. The samples were then divided into two groups, each containing one portion of SiO2@VIMPS@MIPs and one portion of SiO2@VIMPS@NIPs. A mixed protein solution of Cyt-C was added to one group at a final concentration of 0.8mg / mL. The mixture was placed in a constant temperature water bath shaker at 28°C for 2 hours and then centrifuged at 10,000 rpm for 10 minutes. The Cyt-C concentration in the supernatant was measured by UV-Vis at 410nm and 280nm, respectively. In another group, a ternary protein mixture of BSA, OVA, and Cyt-C (the three proteins were mixed in a 1:1:1 volume ratio) was added to a final concentration of 0.8 mg / mL. The mixture was shaken and adsorbed at 28°C for 2 hours. The solid particles were separated by centrifugation at 10,000 rpm for 10 minutes. The proteins adhering to the surfaces of SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs were removed with PBS (0.02 M, pH 7.4) buffer, and the supernatant was separated and decanted. The proteins adsorbed to SiO2@VIMPS@MIPs and SiO2@VIMPS@NIPs were then eluted with 0.5 M NaCl solution until the characteristic protein absorption peaks were no longer detectable by UV spectroscopy. The eluate was collected and desalted using a dialysis bag. The solution in the dialysis bag was then freeze-dried. The obtained solid was dissolved in 3 mL of phosphate buffer. 10 μL of the solution was taken and separated and detected using SDS-PAGE. The electrophoresis test conditions were: 12.5% ​​polyacrylamide as the separation gel and 5% polyacrylamide as the stacking gel.

[0107] The results of competitive adsorption SDS-PAGE are as follows Figure 12As shown. Lane 1 provides a molecular weight standard used to determine the molecular weight of the proteins in the other lanes. Lane 2 shows the initial state of the three proteins in the mixed solution. From top to bottom, bands corresponding to BSA, OVA, and Cyt-C can be observed. Lane 3 shows the remaining proteins after adsorption by SiO2@VIMPS@MIPs. Compared with lane 2, the intensities of the three protein bands are significantly weakened, especially for Cyt-C, indicating that SiO2@VIMPS@MIPs has a high adsorption capacity for Cyt-C. Compared with lane 2, the change in band intensity in lane 4 is smaller, indicating that the adsorption capacity of NIPs for proteins is weaker. This indicates that compared with SiO2@VIMPS@NIPs, SiO2@VIMPS@MIPs has higher selectivity and can effectively recognize and adsorb the target protein. By comparing lanes 3 and 4, it can be determined that SiO2@VIMPS@MIPs has a higher affinity and specific recognition ability for Cyt-C. SiO2@VIMPS@MIPs can preferentially adsorb the target protein Cyt-C in a competitive environment, which is of great significance for protein separation and purification in practical applications.

[0108] Therefore, the method provided by the present invention is used to successfully prepare a protein surface imprinted material with the help of surface directional imprinting and aqueous phase polymerization. The protein surface imprinted material has high stability, strong interaction with the protein to be tested, and can stably and specifically identify the target protein. The protein surface directional imprinted core-shell microspheres prepared by the method provided by the present invention have a base ball with a "dendritic" structure, a diameter of 80 nm, abundant wrinkles on the surface, high porosity, and a shell layer of a polyionic liquid macromolecular monomer polymer layer, which directionally anchors the target protein, has abundant recognition sites, and has excellent dispersibility and high particle size uniformity. The preparation method of the provided protein surface directional imprinted microspheres is simple and easy to operate, has mild conditions, uses low-toxic or non-toxic reagents, does not generate toxic or harmful substances, and is environmentally friendly.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing protein surface directional imprinted microspheres, characterized in that: Here are the steps: S1. Synthesize an ionic liquid monomer and a modifier, wherein the modifier is 1-vinyl-3-propyltrimethoxysilane imidazole chloride, and the ionic liquid monomer includes tosylated 1-vinyl-β-cyclodextrin imidazole, 1-vinyl-3-acetamide imidazole chloride, 1-vinyl-3-propanesulfonic acid imidazole chloride, or 1-vinyl-3-styrene imidazole chloride; S2. Prepare a polyionic liquid block copolymer macromonomer, using 2-mercapto-S-thiobenzoyl acetic acid as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, 1-vinyl-3-acetamidoimidazole chloride as a monomer, and thermal initiation under N2 atmosphere to obtain Macro-CTA, and add toluenesulfonated 1-vinyl-β-cyclodextrin imidazole monomer and azobisisobutyronitrile to obtain a precursor Precursor-1; use the precursor Precursor-1 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, 1-vinyl-3-propanesulfonic acid imidazole chloride and 1-vinylimidazole as monomers, and thermal initiation under N2 atmosphere to obtain a precursor Precursor-2; react the precursor Precursor-2 with 4-vinylbenzyl chloride in dimethyl sulfoxide to obtain a polyionic liquid block copolymer macromonomer p-VIMA-co-VIMCD s-co-VSPIM-co-PVIM; S3. Prepare a dendritic silica nanocarrier material by dissolving triethanolamine, hexadecyltrimethylammonium bromide, and sodium salicylate in deionized water. After stirring and reacting, add ethyl orthosilicate and continue the reaction. After the reaction is completed, remove the unreacted material, wash the precipitate with deionized water, dry, grind, and calcine. S4. Double bond modification is performed on the surface of the obtained dendritic silica nanocarrier, the dendritic silica nanocarrier material is calcined, and after cooling, it is dispersed in anhydrous toluene, 1-vinyl-3-propyltrimethoxysilane imidazole chloride is added, and after stirring and refluxing, the reaction is centrifuged and washed, and then solidified to obtain SiO2@VIMPS; S5. Prepare protein surface-directed imprinted microspheres, dissolve the target protein and p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM prepared in phosphate buffer, and magnetically stir for a certain period of time to allow the target protein to be fully anchored on the surface of p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM to obtain solution I; disperse the SiO2@VIMPS obtained in S4 in phosphate buffer to obtain solution II; mix solution I and solution II and self-assemble, then add persulfate and tetramethylethylenediamine, purge with nitrogen and stir to react, completely wash the collected nanocarriers to remove the bound target protein, and freeze-dry to obtain target protein surface-directed imprinted microspheres.

2. The method for preparing protein surface directional imprinted microspheres according to claim 1, characterized in that: The preparation method of the modifier in S1 comprises the following steps: 1-vinylimidazole and 3-chloropropyltrimethoxysilane were dissolved in a mixed solution of ethyl acetate and water, and after reflux reaction, the mixture was extracted with ethyl acetate. The aqueous solution after extraction was freeze-dried to obtain 1-vinyl-3-propyltrimethoxysilane imidazole chloride ion liquid; the molar volume ratio of 1-vinylimidazole: 3-chloropropyltrimethoxysilane: ethyl acetate: water was: 2.5 mol: 3.1 mol: 1 L: 1 L.

3. The method for preparing protein surface directional imprinted microspheres according to claim 1, characterized in that: The preparation method of 1-vinyl-3-acetamidoimidazole chloride in S1 comprises the following steps: Dissolve chloroacetamide in acetone and stir until the solution is clear. Add 1-vinylimidazole dropwise under vigorous stirring to react. After vacuum distillation, wash the precipitate 3-5 times and freeze-dry the precipitate. The molar ratio of chloroacetamide to 1-vinylimidazole is 8:

5. Wash the precipitate with acetone.

4. The method for preparing protein surface-directed imprinted microspheres according to claim 1, characterized in that: The preparation method of tosylated 1-vinyl-β-cyclodextrin imidazole in S1 comprises the following steps: β-cyclodextrin was dissolved in deionized water, and an aqueous solution of sodium hydroxide and an acetonitrile solution of toluenesulfonyl chloride were added dropwise thereto. After stirring for reaction, the precipitate was removed, and the filtrate was refrigerated overnight to recover the precipitated toluenesulfonated β-cyclodextrin. The mass volume ratio of β-cyclodextrin: deionized water: sodium hydroxide: water: toluenesulfonyl chloride: acetonitrile was 6 g: 0.05 L: 0.66 g: 0.002 L: 1 g: 0.003 L. Toluenesulfonated β-cyclodextrin is dissolved in a mixed solution of vinylimidazole and N,N-dimethylformamide, and the reaction is carried out under a protective atmosphere. After the reaction is completed, the mixture is cooled to ambient temperature to precipitate a crude product, and the precipitate obtained by filtration is vacuum-dried to obtain toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid. The mass volume ratio of toluenesulfonated β-cyclodextrin: 1-vinylimidazole: N,N-dimethylformamide: acetone is 6.45 g: 0.002 L: 0.015 L: 0.07 L.

5. The method for preparing protein surface-directed imprinted microspheres according to claim 1, characterized in that: The preparation method of 1-vinyl-3-propanesulfonic acid imidazole chloride in S1 comprises the following steps: Dissolve 1-vinylimidazole in acetonitrile and add 1,3-propane sultone dropwise under stirring to react. Collect the precipitate, purify it, and freeze-dry it. The molar volume ratio of 1-vinylimidazole:acetonitrile:1,3-propane sultone is 2 mol:1 L:3 mol. The preparation method of 1-vinyl-3-styrene imidazolium chloride ion liquid comprises the following steps: 1-vinylimidazole and vinylbenzyl chloride are dissolved in a mixed solution of ethyl acetate and deionized water for reaction, followed by extraction with ethyl acetate, and the extracted aqueous phase is freeze-dried; the molar volume ratio of 1-vinylimidazole:vinylbenzyl chloride:ethyl acetate:deionized water is 2.5 mol:2.15 mol:1 L:1 L.

6. The method for preparing protein surface-directed imprinted microspheres according to claim 1, characterized in that: When Macro-CTA is prepared in S2, the volume ratio of transfer agent: solvent: initiator: 1-vinyl-3-acetamido imidazole chloride monomer is 1:560:0.4:20; when Precursor-1 is prepared, Macro The molar ratio of o-CTA: toluenesulfonated 1-vinyl-β-cyclodextrin imidazole monomer: azobisisobutyronitrile is 1:5:1; when preparing precursor Precursor-2, the mass volume ratio of precursor Precursor-1: dimethyl sulfoxide: azobisisobutyronitrile: 1-vinyl-3-propanesulfonic acid imidazole chloride: 1-vinylimidazole is 500 mg:30 mL:33 mg:300 mg:800 mg, and the thermal initiation temperature is 70°C; when preparing p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM, the mass volume ratio of Precursor-2: dimethyl sulfoxide: 4-vinylbenzyl chloride is 2 g:25 mL:500 mg.

7. The method for preparing protein surface-directed imprinted microspheres according to claim 1, characterized in that: The molar ratio of triethanolamine: CTAB: sodium salicylate: ethyl orthosilicate in S3 is 7:16:4:274; the mass volume ratio of dendritic silica nanocarrier (SiO2): anhydrous toluene: 1-vinyl-3-propyltrimethoxysilane imidazole chloride in step S4 is 1.5 g:50 mL, and the mass volume ratio of SiO2: 1-vinyl-3-propyltrimethoxysilane imidazole chloride is 150 g:4 mL.

8. The method for preparing protein surface-directed imprinted microspheres according to claim 1, characterized in that: The pH of the phosphate buffer in the S5 is 7.40, and the mass volume ratio of p-VIMA-co-VIMCDs-co-VSPIM-co-PVIM: cytochrome C: SiO2@VIMPS: PBS buffer is 4g:0.1g:100mg:12.6g; the bound target protein is washed away with a methanol / acetic acid solution with a volume ratio of 9:1, a formic acid solution with a volume fraction of 20%, and ultrapure water.

9. Protein surface directional imprinted microspheres prepared by the method for preparing protein surface directional imprinted microspheres according to any one of claims 1 to 8.

10. A method for preparing the protein surface directional imprinted microspheres according to any one of claims 1 to 8 and / or use of the protein surface directional imprinted microspheres according to claim 9 in protein recognition.

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