CN115254047A

CN115254047A - Molecular imprinting and coating polymer, preparation method and application thereof

Info

Publication number: CN115254047A
Application number: CN202110481022.4A
Authority: CN
Inventors: 刘震; 邢荣荣; 郭展辰; 张齐
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-11-01
Anticipated expiration: 2041-04-30
Also published as: CN115254047B

Abstract

The invention relates to a method for preparing a molecular imprinting and coating polymer by two times of molecular imprinting, the obtained molecular imprinting and coating polymer and application of the polymer. The molecular imprinting and coating polymer comprises an imprinting layer and a coating layer. The preparation method comprises the step of contacting the molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer. According to the method, a coating layer with controllable thickness is formed on the surface of the imprinting layer through a polymerization reagent, so that the coating layer can cover non-specific adsorption sites outside an imprinting cavity to improve specificity, and after a template is removed, a molecular imprinting and coating polymer capable of identifying target proteins and polypeptides with high affinity and high specificity is obtained.

Description

Molecular imprinting and coating polymer, preparation method and application thereof

Technical Field

The invention belongs to the technical field of bionic molecular recognition materials and molecular imprinting, and relates to a molecular imprinting and coating polymer capable of specifically recognizing a specific target object (such as protein or polypeptide), a controllable, universal and convenient method for preparing the molecular imprinting and coating polymer, and application of the molecular imprinting and coating polymer.

Background

The antibody is used as the last defense line in the human immune system, is an important weapon for resisting the invasion of harmful substances such as viruses and microorganisms by organisms, and is widely used for biomolecule recognition in the field of life science research. However, the antibody has obvious limitations, such as complex preparation process, high cost, long screening period, and sometimes even unavailable antibody. In addition, stability and reproducibility of antibodies are problematic. Therefore, the search for the substitute of the antibody not only has important scientific significance, but also has great economic value.

Molecularly Imprinted Polymers (MIPs) [ angelw.chem.int.ed.engl.1972, 11,341-345; nature1993,361,645-647 is a chemically synthesized receptor having antibody-binding properties synthesized by copolymerization in the presence of a template. Compared with antibodies, the molecularly imprinted polymer has the advantages of simple preparation, low cost, stable structure, tolerance to various severe environments and the like. Therefore, it has been widely used in the fields of separation, sensing, proteomics, bio-imaging, controlled drug release, and nano-medicine. Despite the significant advances made in molecular imprinting technology, there are inherent drawbacks to conventional molecular imprinting techniques. In order to obtain high specificity and strong affinity for template molecules, it is usually necessary to construct an imprinted cavity by polymerizing a plurality of functional monomers capable of interacting with the template molecules with a cross-linking agent, and the imprinted cavity is exactly complementary to the template molecules in terms of shape, size, interaction sites, and the like, so as to obtain a binding cavity capable of high specificity and strong affinity. However, the inventor finds that, because the non-imprinted surface outside the imprinted cavity is also constructed by the same type and proportion of monomers and cross-linking agents as the imprinted cavity, significant non-heterogeneous interaction sites exist, which can cause obvious cross-reactivity in the actual use process. Thus, conventional molecularly imprinted polymers do not provide both optimal affinity and optimal specificity, but rather a compromise between the two.

Disclosure of Invention

In view of the above technical problems in the existing molecular imprinting technology, the present inventors have developed a controllable, versatile, and convenient method for preparing a molecular imprinting and coating polymer through research, wherein a controllable imprinting technology is used to coat the surface of a molecular imprinting material (including a molecular imprinting polymer obtained by a conventional molecular imprinting technology) with a non-heterogeneous interaction weak polymerization reagent (e.g., tetraethylorthosilicate) as a coating reagent and participate in template imprinting, thereby forming a thin coating layer to reduce non-specific adsorption of non-imprinted regions on the surface of the imprinting material, so that the non-heterogeneous interaction sites of the obtained molecular imprinting and coating polymer are significantly reduced, and simultaneously, both specificity and affinity for a target molecule are improved.

In one aspect, the present invention provides a method for preparing a molecularly imprinted and coated polymer by two-time molecular imprinting, wherein the method comprises: and contacting the molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer.

In another aspect, the present invention provides a molecularly imprinted and coated polymer, comprising:

a blotting layer, and

a coating layer, wherein the coating layer is located on an outer surface of the imprinting layer.

In a further aspect, the present invention relates to the use of the above-described molecularly imprinted and coated polymer for the preparation of a preparation for recognizing a target molecule or a target cell.

The above preparation method of the present invention involves the following two blots: the first directional imprinting relates to imprinting treatment of template molecules by using a monomer silanization reagent and a cross-linking agent which interact with the template molecules, and is used for constructing a good imprinting cavity so as to obtain strong affinity and high specificity; the second directional imprinting (i.e., coating imprinting) involves forming a coating thin layer with controllable thickness on the surface of the molecularly imprinted layer by using a non-heterogeneous interaction weak polymerization reagent to cover non-specific interaction sites outside the imprinted layer, thereby further improving specificity (specificity), and in addition, the coating layer causes the spatially matched recognition of more amino acid residues on the template molecules by the imprinted layer, and simultaneously improves affinity. In this respect, similar documents and patents have not been reported at present.

According to the preparation method, a pure product of the target protein is not required to be provided or prepared, only the amino acid structure sequence information of the protein is required to be known, the glycosylated epitope template can be obtained by a chemical synthesis method, and the molecularly imprinted and coated polymer with higher specificity and stronger affinity can be prepared by imprinting the glycosylated epitope template twice. The method has the advantages of strong universality, easily obtained templates through solid-phase synthesis, no limitation on the types of substrate materials and the like, and is a molecularly imprinted and coated polymer preparation technology with good universality and strong applicability. The molecular imprinting and coating polymer prepared by the method can specifically identify, combine and enrich target proteins and epitopes thereof. The imprinting template is a glycated epitope, not the complete target protein. The invention adopts protein epitope saccharification treatment, breaks through the limitation of epitope selection, widens the variety of substrate materials, and adopts different types and proportions of monomer silanization reagents and cross-linking agents to carry out first directional imprinting on the epitope sequence of the target protein for the first time and then adopts nonspecific interaction weak polymerization reagents to carry out second directional imprinting (also called coating imprinting) compared with the prior protein imprinting technology, thereby not only obviously improving the specificity of the molecular imprinting material, but also enhancing the affinity. The molecular imprinting and coating polymer obtained by the invention can identify protein and polypeptide, not only has strong affinity, but also has high specificity (specificity), thereby showing good application potential in the fields of affinity separation, biochemical analysis, targeted identification, biological imaging and the like.

Drawings

FIG. 1 is a schematic diagram illustrating the principle of an exemplary method of preparing a molecularly imprinted and coated polymer according to the present invention.

FIG. 2 shows β₂-the chemical structure of the C-terminal glycated epitope of microglobulin (B2M) (a), transferrin (TRF) (B) and transferrin receptor protein (TfR) (C), the chemical structure of the N-terminal glycated epitope (e) of alpha-fetoprotein (AFP) (d) and carcinoembryonic antigen (CEA), and the chemical structure of the C-terminal glycated epitope (f) and N-terminal glycated epitope (g) of C-peptide (C-peptide).

Fig. 3 is a Transmission Electron Microscope (TEM) photograph of molecularly imprinted and coated polymers with different substrate materials. Where a is a TEM photograph of the molecularly imprinted and coated magnetic nanoparticles prepared in example 2, b is a TEM photograph of the molecularly imprinted and coated silver nanoparticles prepared in example 7, and c is a TEM photograph of the molecularly imprinted and coated FITC-doped silica nanoparticles prepared in example 9.

Figure 4 shows the selectivity of the boronic acid functionalised magnetic nanoparticles for different analytes, adenosine and deoxyadenosine (a) respectively; c-terminal epitopes of B2M, TRF and TfR, N-terminal epitopes of AFP and CEA and their corresponding glycated epitopes (B).

Fig. 5 shows the optimal imprinting factors (a) and (B) of the B2M C-terminal epitope molecular imprinting and the coated magnetic nanoparticles (B) of the B2M C-terminal epitope single-imprinted magnetic nanoparticles prepared with different ratios of the monomeric silylation agent and the cross-linking agent at the optimal imprinting time.

Fig. 6 shows the affinities of the magnetic nanoparticles (a) and the magnetic nanoparticles (B) for single imprinting of C-terminal epitope of B2M and the coated magnetic nanoparticles (B) prepared by using different ratios of the monomeric silylation agent and the cross-linking agent at the optimal imprinting time.

Fig. 7 shows the selectivity of B2M C-terminal epitope molecular imprinting and coated magnetic nanoparticles at the peptide (a) and protein (B) levels.

Fig. 8 is a result of examining the versatility of the method for preparing a molecularly imprinted and coated polymer according to the present invention with respect to C-terminal epitopes of different proteins, in which the imprinting condition optimization results (a) of the C-terminal epitope molecularly imprinted of TRF thus prepared and the coated magnetic nanoparticles and their selectivity at the peptide fragment level (C) and the protein level (e) are shown, respectively; the imprinting conditions of the TfR C-terminal epitope molecular imprinting and the coated magnetic nanoparticle optimize the result (b) and the selectivity thereof at the peptide fragment level (d) and the protein level (f).

FIG. 9 is a result of an investigation of the versatility of the method of the present invention for the preparation of molecularly imprinted and coated polymers for N-terminal epitopes of different proteins, wherein the results (a) of the optimization of the imprinting conditions of the N-terminal epitope molecularly imprinted and coated magnetic nanoparticles of AFP thus prepared and their selectivity at the peptide fragment level (c) and protein level (e) are shown, respectively; optimizing the result (b) and the selectivity of the N-terminal epitope molecular imprinting of CEA and the imprinting conditions of the coated magnetic nanoparticles at the peptide fragment level (d) and the protein level (f).

FIG. 10 shows confocal imaging after staining MCF-7, MCF-10A, hepG2 and L-02 cells with TfR C-terminal epitope molecular imprinting and coated FITC-doped silica nanoparticles and non-imprinted FITC-doped silica nanoparticles, respectively, at a nanoparticle concentration of 200 μ g/mL.

Detailed Description

The invention is described in further detail below with reference to exemplary embodiments, but the scope of the invention is not limited thereto.

In one embodiment, the present invention provides a method for preparing a molecularly imprinted and coated polymer by two-pass molecular imprinting, wherein the method comprises: and contacting the molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer.

In some preferred embodiments, the above method for preparing a molecularly imprinted and coated polymer further comprises: anchoring an imprinting template on the surface of a substrate material, and then adding one or more monomer silanization reagents and a cross-linking agent to perform the first imprinting on the substrate material to form an imprinting layer, so as to obtain the molecular imprinting material containing the imprinting layer.

In some preferred embodiments, the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate.

In some preferred embodiments, the polymerization agent is tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

In some preferred embodiments, the present invention provides a method of preparing a molecularly imprinted and coated polymer, comprising:

(1) Selecting a C-terminal or N-terminal polypeptide sequence of a target protein as an epitope for saccharification to obtain a saccharification epitope as the imprinting template;

(2) Performing functional treatment on the substrate material to obtain a boric acid functionalized substrate material;

(3) Anchoring the imprinting template to the surface of the boronic acid functionalized substrate material to obtain a template anchored substrate material;

(4) Adding one or more monomer silanization reagents and a cross-linking agent into the substrate material anchored by the template to perform the first imprinting to form an imprinting layer, so as to obtain a molecularly imprinted material containing the first directional imprinting of the imprinting layer;

(5) Contacting the molecularly imprinted material subjected to the first directional imprinting with the polymerization reagent to perform second imprinting on the imprinting layer to form a coating layer, so as to obtain the molecularly imprinted material subjected to second directional imprinting and containing the coating layer, wherein the polymerization reagent is tetraethyl orthosilicate, tetramethyl orthosilicate or dopamine;

(6) And eluting the molecularly imprinted material subjected to the second directional imprinting to remove the imprinting template, so as to obtain the molecularly imprinted and coated polymer.

In some preferred embodiments, a polypeptide sequence of 9-15 amino acid residues from the C-terminus or N-terminus of the protein of interest is selected as the epitope. Herein, selecting a polypeptide sequence consisting of the amino acid residues at the extreme ends (C-and N-termini) of the protein as the polypeptide sequence of the epitope can avoid the case where the epitope sequence itself is glycosylated (post-translational modification).

In a further preferred embodiment, the one or more monomer silanization reagents and the cross-linking agent are used for carrying out first imprinting on the first 6-12 amino acid residues in the epitope to obtain the molecularly imprinted material of the first directional imprinting; and the last 3-6 amino acid residues in the epitope react with the polymerization reagent to perform second directional imprinting, so as to obtain the molecularly imprinted material of the second directional imprinting.

Taking a polypeptide consisting of 12 amino acid residues at the tail end of a target protein as a characteristic epitope as an example, a glycosylated epitope obtained after saccharification treatment is taken as an imprinting template, the imprinting template is anchored on the surface of a boric acid functionalized substrate material through boron affinity, a plurality of monomer silanization reagents and cross-linking agents are selected to perform first imprinting on the first nine amino acid residues of the epitope polypeptide, an obtained imprinting layer has multiple interactions on the epitope polypeptide to generate strong affinity, then a nonspecific heterogeneous interaction weak polymerization reagent is taken as a coating reagent to perform second directional imprinting on the surface of the imprinting material, the thickness of the formed coating layer covers the last three amino acids, and the template molecule is removed to obtain a molecular imprinting and coating polymer with increased specificity and affinity on the target molecule.

In some preferred embodiments, the epitope is obtained by solid phase synthesis as a polypeptide sequence of 9-15, preferably 12, amino acid residues from the C-terminus or N-terminus of the target protein.

In some preferred embodiments, a C-terminal polypeptide sequence of the protein of interest is selected as the epitope, a residue of lysine is attached to the end of the polypeptide sequence, and then the residue of lysine is bound to a monosaccharide by a schiff base reaction to perform the glycation. In some preferred embodiments, an N-terminal polypeptide sequence of the protein of interest is selected as the epitope, and the amino group of the starting amino acid of the polypeptide sequence is bound to a monosaccharide by a schiff base reaction for the saccharification. In a further preferred embodiment, the monosaccharide may be selected from fructose, glucose, galactose, mannose, xylose, or any mixture thereof, but is not limited thereto.

In order to ensure that the glycated polypeptide epitope has a stronger interaction with the boronic acid ligand on the substrate material, it is preferable to select fructose, glucose or a mixture thereof having a stronger affinity with the boronic acid.

In some preferred embodiments, the protein of interest may be selected from B2M, TRF, tfR, AFP, CEA, or C-peptide, but is not limited thereto.

In some preferred embodiments, the substrate material includes, but is not limited to, magnetic nanomaterials, silver nanomaterials (preferably silver nanoparticles with raman reporter molecules), fluorescein-doped silica nanomaterials (preferably FITC-doped silica nanoparticles), and the like. Preferably, the raman reporter molecule includes, but is not limited to, para-mercaptoaniline (PATP), para-Nitrobenzophenol (NTP), para-mercaptophenylboronic acid (MPBA), and the like.

In some preferred embodiments, the substrate material is a magnetic nanomaterial or a silver nanomaterial, and the substrate material is functionalized by the following steps: (i) Reacting the substrate material with ammonia water and TEOS in an alcoholic solution, preferably an ethanol solution, to obtain a substrate material with a silicon-coated surface; (ii) Reacting the substrate material with the surface coated with silicon with APTES in an alcoholic solution, preferably an alcoholic solution, to obtain an amino-functionalized substrate material; (iii) Reacting the amino-functionalized substrate material with a boronic acid and sodium cyanoborohydride in an alcoholic solution, preferably a methanol or ethanol solution, to obtain the boronic acid-functionalized substrate material.

In some preferred embodiments, the substrate material is a fluorescein-doped silica nanomaterial, and the substrate material is functionalized by the following steps: (i') reacting the fluorescein-doped silica nanomaterial with APTES in an alcoholic solution, preferably an alcoholic solution, to obtain an amino-functionalized fluorescein-doped silica nanomaterial; (ii') reacting the amino-functionalized fluorescein-doped silica nanomaterial with a boronic acid and sodium cyanoborohydride in an alcoholic solution, preferably a methanol or ethanol solution, to obtain the boronic acid-functionalized fluorescein-doped silica nanomaterial.

In a further preferred embodiment, the concentration of the aqueous ammonia is 25w/v% to 28w/v%.

In a further preferred embodiment, in step (i), said alcohol solution contains 0.7 to 1.4vol% of said TEOS.

In a further preferred embodiment, in steps (ii) and (i'), said alcoholic solution contains 0.5 to 3vol% of said APTES.

In a further preferred embodiment, the boronic acid may include 2, 4-difluoro-3-formylphenylboronic acid (DFFPBA), aldenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, or alkenylphenylboronic acid, but is not limited thereto. In a further preferred embodiment, in steps (iii) and (ii'), said alcoholic solution contains 0.05 to 5w/v% of said boronic acid.

In a further preferred embodiment, in steps (iii) and (ii'), said alcoholic solution contains 0.05 to 1w/v% of said sodium cyanoborohydride.

In some preferred embodiments, the imprinted template and the boronic acid functionalized substrate material are added to a buffer solution with a pH value above 7, and after incubation (e.g. at 15-40 ℃) a template anchored substrate material is obtained. In a further preferred embodiment, the buffer solution is selected from the group consisting of an ammonium bicarbonate/sodium chloride buffer solution, an ammonium bicarbonate buffer solution or a phosphate buffer solution, but is not limited thereto.

In some preferred embodiments, the first imprinting is performed by adding water and an alcoholic solution, preferably an alcoholic solution of the monomer silylation reagent and the cross-linking agent, to a solution of an alcohol containing ammonia, in which the template-anchored base material is dispersed, to obtain the first molecularly imprinted material. The first blot herein is polymerized using monomeric silylating and crosslinking reagents to provide multiple interactions with the epitope sequence. In a more preferred embodiment, the concentration of the aqueous ammonia is from 25w/v% to 28w/v%. In another preferred embodiment, the monomeric silylating agent is selected according to the amino acid type of the epitope sequence, including but not limited to APTES, UPTES, bnTES, IBTES, or the like. In further preferred embodiments, the crosslinking agent may include, but is not limited to, tetraethyl orthosilicate or tetramethyl orthosilicate.

In some preferred embodiments, an alcohol solution, preferably an ethanol solution, of the polymerization reagent is added to the ammonia-containing alcohol solution in which the first directionally-imprinted molecularly imprinted material is dispersed to perform the second imprinting, so as to obtain the second-imprinted molecularly imprinted material. In the method, the second imprinting is polymerized by adopting a polymerization reagent, so that nonspecific adsorption sites generated outside an imprinting cavity in the first directional imprinting are covered, the specificity of the molecular imprinting material is obviously improved, and the affinity is enhanced. In a further preferred embodiment, the concentration of the aqueous ammonia is 25w/v% to 28w/v%.

In some preferred embodiments, the imprinted template is removed by eluting the second imprinted molecularly imprinted material with an elution solution comprising acetonitrile, water and glacial acetic acid. Preferably, the elution solution consists of acetonitrile, water and glacial acetic acid in a volume ratio of (30-70): (69-29): 1 (e.g., 50.

In the invention, a polypeptide fragment at the C end or the N end of a target protein is used as an epitope, the polypeptide fragment is used as an imprinting template after saccharification treatment, the polypeptide fragment is anchored on a boric acid functionalized substrate material by utilizing boron affinity, monomer silanization reagents and cross-linking agents with different types and proportions are adopted for carrying out first directional imprinting, a non-heterogeneous interaction weak polymerization reagent is adopted for carrying out second directional imprinting, and the obtained molecular imprinting and coating polymer can specifically recognize the target protein and the epitope thereof. The technology does not need to provide or prepare a pure product of the target protein, the epitope polypeptide fragment can meet the imprinting of any sequence after saccharification treatment, the technology can be suitable for various target proteins, and the prepared molecularly imprinted material has higher specificity and stronger affinity. Representative methods of the invention for preparing molecularly imprinted and coated polymers are described herein, by way of example only, as follows:

(1) Determination of epitope sequences and saccharification

The amino acid sequence information of the target Protein can be found by a Protein database (such as UniProt, protein Date Bank, etc.) well known in the art, and the C-terminal or N-terminal polypeptide sequence of the target Protein is selected as the epitope. Meanwhile, in order to facilitate anchoring of a polypeptide sequence as an epitope (also referred to herein as "epitope polypeptide") to a substrate material, the epitope polypeptide needs to be saccharified. The saccharification treatment was as follows: the C-terminal epitope polypeptide is saccharified in such a way that the tail end of the C-terminal epitope polypeptide is firstly connected with a lysine residue, and then the lysine residue is combined with monosaccharide such as fructose or glucose through Schiff base reaction to be saccharified; the N-terminal epitope polypeptide is saccharified in such a way that amino of an initial amino acid of the N-terminal polypeptide is combined with monosaccharide such as fructose or glucose through Schiff base reaction; this resulted in a C-or N-terminal glycated polypeptide epitope as an imprinted template (also referred to herein as a "glycated epitope template").

(2) Selection of substrate materials and boric acid functionalization

According to different detection methods and purposes, different functions and types of substrate materials can be selected, and the boric acid functionalization process comprises the following steps: stirring and reacting a substrate material, ammonia water and TEOS in ethanol, dispersing the obtained material in the ethanol, then adding APTES, and stirring and reacting to obtain an amino functionalized substrate material; reacting the amino-functionalized substrate material, substituted boric acid and sodium cyanoborohydride in methanol or ethanol to obtain the boric acid-functionalized substrate material.

(3) Anchoring of glycated epitope templates on substrate materials

Adding the boric acid functionalized substrate material and the glycated epitope template into a buffer solution with the pH value of more than 7, and after incubation, anchoring the glycated epitope template on the surface of the boric acid functionalized substrate material to obtain the template anchored substrate material.

(4) First time orientation print

And carrying out first directional imprinting on the substrate material anchored by the template, water, ammonia water, a monomer silanization reagent and a cross-linking agent in ethanol to obtain the molecularly imprinted material for the first directional imprinting.

(5) Second orientation print (coating print)

And performing second directional imprinting on the molecularly imprinted material subjected to the first directional imprinting, ammonia water and a polymerization reagent (such as TEOS) in ethanol to obtain a molecularly imprinted material subjected to second directional imprinting.

(6) Removal of glycated epitope templates

And adding the molecularly imprinted material subjected to the second directional imprinting into an elution solution for elution, and removing the glycated epitope template to obtain the molecularly imprinted and coated polymer.

Various different base materials may be used to prepare the molecularly imprinted and coated polymer in the present invention, and for example only, the method of preparing the molecularly imprinted and coated polymer using different base materials is described below, and the preparation of the glycated epitope template is as exemplified above.

Magnetic nano-particles as substrate material

Step 1), preparation of magnetic nanoparticles can be found in the following method: chem.Sci.2013,4,4298-4303; chem.eur.j.2006,12,6341-6347;

step 2), adding ammonia water and TEOS into ethanol, stirring for 5-60 minutes at 20-60 ℃, then adding an ethanol solution of magnetic nanoparticles, and continuously stirring and reacting for 5-60 minutes at 20-60 ℃ to obtain magnetic nanoparticles with silicon-coated surfaces;

step 3), dispersing the magnetic nanoparticles with silicon coated on the surface in ethanol, then adding APTES into the ethanol, and stirring the mixture for 5 to 20 hours at the temperature of between 50 and 100 ℃ to obtain amino functionalized magnetic nanoparticles;

step 4), adding sodium cyanoborohydride into the amino functionalized magnetic nanoparticles obtained in the step 3) and the methanol solution of the substituted boric acid for reduction, and reacting at 15-40 ℃ (for example, 25 ℃) for 12-36 hours (for example, 24 hours) to obtain boric acid functionalized magnetic nanoparticles;

step 5), dispersing the boric acid functionalized magnetic nanoparticles into a buffer solution (pH is more than 7) containing a saccharification epitope template, and after incubation for 0.5-4 hours at 15-40 ℃, anchoring the saccharification epitope template on the surface of the boric acid functionalized magnetic nanoparticles to obtain template-anchored magnetic nanoparticles;

step 6), dispersing the template-anchored magnetic nanoparticles obtained in the step 5) in an ethanol solution of ammonia water, adding water, then adding an ethanol solution of a monomer silanization reagent and a cross-linking agent, performing first directional imprinting at 15-40 ℃ (for example, 25 ℃), and performing magnetic separation to obtain first directionally imprinted magnetic nanoparticles;

step 7), performing second directional imprinting on the magnetic nanoparticles subjected to the first directional imprinting, ammonia water and a polymerization reagent in ethanol at 15-40 ℃ (for example, 25 ℃) for 5-30 minutes (for example, 10 minutes), and performing magnetic separation to obtain magnetic nanoparticles subjected to second imprinting;

step 8), adding the second imprinted magnetic nanoparticles obtained in step 7) into an elution solution (e.g. acetonitrile: water: glacial acetic acid =50, in v/v) to remove the template, thereby obtaining a molecularly imprinted and coated polymer.

The preparation of a control non-imprinted magnetic nanoparticle-based polymer (abbreviated as "non-imprinted polymer") was performed in the same manner as described above except that no glycated epitope template was added.

Silver nano-particles with Raman response as substrate material

Step 1), preparation of silver nanoparticles can be found in the following method: chem,1982,86 (17), 3391-3395;

step 2), adding an ethanol solution of the Raman reporter molecule into the silver nanoparticle solution obtained in the step 1), and stirring for 20-60 minutes (for example, 40 minutes) at 15-40 ℃ (for example, 25 ℃); dispersing the obtained solution in an ethanol solution, and stirring for 5 to 30 minutes (e.g., 10 minutes); then ammonia water is dripped in, and stirring is continued for 1 to 10 minutes (for example, 5 minutes); adding a TEOS ethanol solution, reacting at room temperature, and centrifuging to obtain Raman-responsive silver nanoparticles with silicon-coated surfaces; the Raman reporter molecules used are selected according to the requirements of detection, including but not limited to PATP, NTP, MPBA and the like;

step 3), dispersing the Raman-responsive silver nanoparticles with silicon-coated surfaces obtained in the step 2) in ethanol, adding APTES into the ethanol, shaking for 0.5 to 5 hours (for example, 1 hour) at a temperature of between 15 and 40 ℃ (for example, 25 ℃), and centrifuging to obtain amino-functionalized Raman-responsive silver nanoparticles;

step 4), dispersing the amino-functionalized Raman-responsive silver nanoparticles obtained in the step 3) in ethanol, then adding substituted boric acid and sodium cyanoborohydride, reacting for 12-36 hours at 15-40 ℃ (for example, 25 ℃), and centrifuging to obtain boric acid-functionalized Raman-responsive silver nanoparticles;

step 5), dispersing the boric acid functionalized Raman-responsive silver nanoparticles obtained in the step 4) in a buffer solution, then adding a saccharification epitope template, shaking and incubating for 0.5-5 hours at 15-40 ℃ (for example, 25 ℃), and centrifuging to obtain template-anchored Raman-responsive silver nanoparticles;

step 6), dispersing the template-anchored Raman-responsive silver nanoparticles obtained in the step 5) in an ethanol solution containing ammonia water, adding water, stirring for 1-10 minutes (for example, 5 minutes), then adding an ethanol solution containing a monomer silanization reagent and a cross-linking agent, stirring at 15-40 ℃ (for example, 25 ℃) for first imprinting, and then centrifuging to obtain first directionally-imprinted Raman-responsive silver nanoparticles;

step 7), stirring the silver nanoparticles with Raman response of the first directional imprinting, ammonia water and a polymerization reagent in an ethanol solution for second imprinting, and then centrifuging to obtain silver nanoparticles with Raman response of the second imprinting;

step 8), adding the second imprinted raman-responsive silver nanoparticles obtained in step 7) into an elution solution (e.g., acetonitrile: water: glacial acetic acid =50, in v/v) to remove the template, and then centrifuging to obtain a molecularly imprinted and coated polymer.

Preparation of a control non-imprinted raman-response silver nanoparticle-based polymer (referred to as "non-imprinted polymer") all steps were the same as above except that no glycated epitope template was added.

FITC-doped silica nanoparticles as substrate material

Step 1), the preparation of FITC-APTES derivatives can be found in the following methods: J.am.chem.Soc.1978,100,8050-8055; anal, bioanal, chem, 2010,396,725-738;

step 2), uniformly mixing the ethanol solution of the FITC-APTES derivative obtained in the step 1) with the ethanol solution of TEOS, and taking the mixture as a precursor of the polycondensation reaction for later use. After absolute ethyl alcohol, water and ammonia water are mixed uniformly, the temperature is slowly raised to 30-70 ℃ under vigorous stirring (for example, 55 ℃). Then adding the precursor, continuing to react for 20-80 minutes (for example, 50 minutes) at 30-70 ℃ (for example, 55 ℃), and then centrifuging to obtain FITC-doped silica nanoparticles;

step 3), dispersing the FITC-doped silica nanoparticles obtained in the step 2) in ethanol, adding APTES into the ethanol, shaking for 0.5-5 hours (for example, 2 hours) at 15-40 ℃ (for example, 25 ℃), and centrifuging to obtain amino-functionalized FITC-doped silica nanoparticles;

step 4), dispersing the amino-functionalized FITC-doped silica nanoparticles obtained in the step 3) in methanol, adding substituted boric acid and sodium cyanoborohydride, shaking at 15-40 ℃ for 12-36 hours, and centrifuging to obtain boric acid-functionalized FITC-doped silica nanoparticles;

step 5), dispersing the boric acid functionalized FITC doped silica nanoparticles obtained in the step 4) in a buffer solution, then adding a saccharification epitope template, shaking at 15-40 ℃ for 0.5-5 hours, and centrifuging to obtain FITC doped silica nanoparticles anchored by the template;

step 6), dispersing the FITC-doped silica nanoparticles anchored by the template obtained in the step 5) into an ethanol solution containing ammonia water, adding water, stirring at 15-40 ℃ (for example, 25 ℃) for 1-10 minutes (for example, 5 minutes), then adding an ethanol solution containing a monomer silanization reagent and a cross-linking agent, stirring at 15-40 ℃ (for example, 25 ℃) for first imprinting, and centrifuging to obtain FITC-doped silica nanoparticles subjected to first directional imprinting;

step 7), shaking the FITC-doped silica nanoparticles of the first directional imprinting obtained in the step 6), ammonia water and a polymerization reagent in ethanol at 15-40 ℃ for 5-30 minutes to carry out second imprinting and then centrifuging to obtain FITC-doped silica nanoparticles of the second directional imprinting;

step 8), adding the FITC doped silica nanoparticles of the second directional imprinting obtained in step 7) into an elution solution (e.g. acetonitrile: water: glacial acetic acid =50, 1, in v/v) to remove the template, followed by centrifugation to obtain the molecular imprinting and coating polymer.

A control non-imprinted FITC-doped silica nanoparticle-based polymer (referred to simply as a "non-imprinted polymer") was prepared in the same manner as above except that no glycated epitope template was added.

In one embodiment, the present invention provides a molecularly imprinted and coated polymer comprising:

a print layer, and

In some preferred embodiments, the polymer further comprises a substrate material. In some preferred embodiments, the imprinting layer comprises a monomeric silylating agent and a cross-linking agent polymerized on the base material.

In some preferred embodiments, the substrate material may include, but is not limited to, magnetic nanomaterials, silver nanomaterials (preferably silver nanoparticles with raman reporter molecules), fluorescein-doped silica nanomaterials (preferably FITC-doped silica nanoparticles), and the like. More preferably, the raman reporter includes, but is not limited to, para-mercaptoaniline (PATP), para-Nitrobenzophenol (NTP), para-mercaptophenylboronic acid (MPBA), and the like.

In some preferred embodiments, the monomeric silylating agent may include, but is not limited to, aminopropyltriethoxysilane (APTES), ureidopropyltriethoxysilane (UPTES), benzyltriethoxysilane (BnTES), isobutyltriethoxysilane (IBTES), and the like. In some preferred embodiments, the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate. In some preferred embodiments, the coating is formed from tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

In one embodiment, the present invention relates to the use of the above-described molecularly imprinted and coated polymer for the preparation of a preparation for recognizing a target molecule or a target cell. In some preferred embodiments, the formulations are used in affinity purification, biochemical analysis, targeted recognition (e.g., targeted recognition of tumor cells), and imaging analysis. In some preferred embodiments, the target molecule includes, but is not limited to, B2M, TRF, tfR, AFP, CEA, or C-peptide, and the like. In some preferred embodiments, the target cell is a tumor cell or the like, including but not limited to breast cancer cell, liver cancer cell, lung cancer cell, and the like.

Exemplary aspects of the present invention may be illustrated by the following numbered paragraphs, but the scope of the present invention is not limited thereto:

1. a method for preparing a molecularly imprinted and coated polymer by two-pass molecular imprinting, wherein the method comprises: and contacting the molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer.

2. The method of paragraph 1, wherein the method further comprises: anchoring an imprinting template on the surface of a substrate material, and then adding one or more monomer silanization reagents and a cross-linking agent to perform the first imprinting on the substrate material to form an imprinting layer, so as to obtain the molecular imprinting material containing the imprinting layer.

3. The method of paragraph 2 wherein the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate.

4. The method of any of paragraphs 1-3, wherein the polymerizing agent is tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

5. The method of any of paragraphs 1-4, wherein the method comprises:

(1) Selecting a C-terminal or N-terminal polypeptide sequence of a target protein as an epitope for saccharification to obtain a saccharified epitope as the imprinting template;

(2) Performing functionalization treatment on the substrate material to obtain a boric acid functionalized substrate material;

(4) Adding one or more monomer silanization reagents and the cross-linking agent into the substrate material anchored by the template to perform the first imprinting to form an imprinting layer, so as to obtain a molecularly imprinted material containing the first directional imprinting of the imprinting layer;

(5) Contacting the molecularly imprinted material subjected to the first directional imprinting with the polymerization reagent to perform the second imprinting on the imprinting layer to form a coating layer, so as to obtain a molecularly imprinted material subjected to the second directional imprinting and containing the coating layer;

6. The method of paragraph 5 wherein the epitope is selected as a polypeptide sequence of 9-15 amino acid residues from the C-or N-terminus of the protein of interest.

7. The method of paragraph 5 or 6, wherein the first 6-12 amino acid residues in the epitope are subjected to the first imprinting with the one or more monomeric silylating agents and the cross-linking agent to obtain the first direct-imprinted molecularly imprinted material; and performing the second imprinting on the last 3-6 amino acid residues in the epitope by using the polymerization reagent to obtain the molecularly imprinted material of the second directional imprinting.

8. The method according to any one of paragraphs 5 to 7, wherein the epitope is a polypeptide sequence of 9 to 15 amino acid residues at the C-terminus or N-terminus of the target protein obtained by solid phase synthesis.

9. The method according to any one of paragraphs 5 to 8, wherein a C-terminal polypeptide sequence of the target protein is selected as the epitope, a residue of lysine is ligated to the end of the polypeptide sequence, and then the residue of lysine is bound to a monosaccharide by Schiff base reaction to carry out the glycation.

10. The method as described in any of paragraphs 5 to 8, wherein an N-terminal polypeptide sequence of the target protein is selected as the epitope, and an amino group of a starting amino acid of the polypeptide sequence is bound to a monosaccharide by Schiff base reaction to perform the glycation.

11. The method of paragraphs 9 or 10 wherein said monosaccharide is selected from the group consisting of fructose, glucose, galactose, mannose, xylose, and any mixture thereof.

12. The method of any of paragraphs 5-11 wherein the protein of interest is selected from B2M, TRF, tfR, AFP, CEA or C-peptide.

13. The method of any of paragraphs 5-12, wherein the substrate material is a magnetic nanomaterial, a silver nanomaterial, and a fluorescein-doped silica nanomaterial.

14. The method of any of paragraphs 5-13, wherein the substrate material is silver nanoparticles with raman reporter molecules or FITC doped silica nanoparticles.

15. The method of any of paragraphs 5-14, wherein the raman reporter molecule is p-mercaptoaniline, p-nitrobenzophenol, or p-mercaptophenylboronic acid.

16. A method as recited in any of paragraphs 5-15, wherein the base material is functionalized with a boronic acid.

17. The method according to any of paragraphs 5-16, wherein the substrate material is a magnetic nanomaterial or a silver nanomaterial, and the substrate material is functionalized by: (i) Reacting the substrate material with ammonia water and TEOS in an alcohol solution to obtain a substrate material with a silicon-coated surface; (ii) Reacting the substrate material with the surface coated with silicon with APTES in an alcoholic solution to obtain an amino functionalized substrate material; (iii) And (3) reacting the amino-functionalized substrate material with substituted boric acid and sodium cyanoborohydride in an alcoholic solution to obtain the boric acid-functionalized substrate material.

18. The method as recited in any of paragraphs 5-16, wherein the substrate material is a fluorescein-doped silica nanomaterial, and wherein the substrate material is functionalized by: (i') reacting the fluorescein-doped silica nanomaterial with APTES in an alcoholic solution to obtain an amino-functionalized fluorescein-doped silica nanomaterial; (ii') reacting the amino-functionalized fluorescein-doped silica nanomaterial with substituted boric acid and sodium cyanoborohydride in an alcoholic solution to obtain the boric acid-functionalized fluorescein-doped silica nanomaterial.

19. The method of paragraph 17 or 18 wherein the concentration of ammonia is from 25w/v% to 28w/v%.

20. The method of paragraph 17 wherein, in step (i), said alcohol solution contains 0.7 to 1.4vol% of said TEOS.

21. The process of any of paragraphs 17-20, wherein in steps (ii) and (i'), the alcoholic solution comprises 0.5 to 3vol% of the APTES.

22. The method of any of paragraphs 17-21, wherein the boronic acid comprises 2, 4-difluoro-3-formylphenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, or alkenylphenylboronic acid.

23. The method according to any of paragraphs 17 to 22, wherein in steps (iii) and (ii'), the alcoholic solution contains 0.05 to 5w/v% of the boronic acid.

24. The method according to any one of paragraphs 17-23, wherein in steps (iii) and (ii'), the alcoholic solution contains 0.05 to 1w/v% of the sodium cyanoborohydride.

25. The method of any of paragraphs 5-24, wherein the imprinted template and the boronic acid functionalized substrate material are added to a buffer solution having a pH greater than 7 and incubated to obtain a template anchored substrate material.

26. The method of paragraph 25 wherein the buffer solution is selected from ammonium bicarbonate/sodium chloride buffer solution, ammonium bicarbonate buffer solution or phosphate buffer solution.

27. The method according to any one of paragraphs 5-26, wherein the first imprinting is performed by adding water and an alcoholic solution of the monomeric silylation reagent and the cross-linking agent to a solution of an alcohol containing ammonia in which the template-anchored base material is dispersed, to obtain the first molecularly imprinted material with directional imprinting.

28. The method of paragraph 27 wherein the concentration of ammonia is from 25w/v% to 28w/v%.

29. The method of any of paragraphs 5-28, wherein the monomeric silylating agent comprises aminopropyltriethoxysilane, ureidopropyltriethoxysilane, benzyltriethoxysilane, and isobutyltriethoxysilane.

30. The method according to any one of paragraphs 5 to 29, wherein the second imprinting is performed by adding an alcoholic solution of the polymerization reagent to an aqueous ammonia-containing alcoholic solution in which the first molecularly imprinted material is dispersed, thereby obtaining the second molecularly imprinted material.

31. The method of paragraph 30 wherein the concentration of ammonia is from 25w/v% to 28w/v%.

32. The method of any of paragraphs 5-31, wherein the removal of the imprinted template is performed by eluting the molecularly imprinted material of the second directional imprinting with an elution solution comprising acetonitrile, water and glacial acetic acid.

33. The method of any of paragraphs 5-32 wherein the elution solution consists of acetonitrile, water and glacial acetic acid in a volume ratio of (30-70): (69-29): 1.

34. A molecularly imprinted and coated polymer comprising:

a print layer, and

35. The molecularly imprinted and coated polymer of paragraph 34, wherein the polymer further comprises a base material.

36. The molecularly imprinted and coated polymer of paragraph 34 or 35, wherein the imprinted layer comprises a monomeric silylation agent and a cross-linking agent polymerized on the base material.

37. The molecularly imprinted and coated polymer of paragraphs 35 or 36, wherein the base material is a magnetic nanomaterial, a silver nanomaterial, or a fluorescein-doped silica nanomaterial.

38. The molecularly imprinted and coated polymer of any one of paragraphs 35-37, wherein the base material comprises silver nanoparticles with raman reporter molecules or FITC-doped silica nanoparticles.

39. The molecularly imprinted and coated polymer of paragraph 38, wherein the Raman reporter is p-mercaptoaniline, p-nitrobenzophenol, or p-mercaptophenylboronic acid.

40. The molecularly imprinted and coated polymer of any one of paragraphs 36-39, wherein the monomeric silylating agent comprises aminopropyltriethoxysilane, ureidopropyltriethoxysilane, benzyltriethoxysilane and isobutyltriethoxysilane.

41. The molecularly imprinted and coated polymer of any one of paragraphs 36-40, wherein the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate.

42. The molecularly imprinted and coated polymer of any one of paragraphs 34-41, wherein the coating is formed from tetraethylorthosilicate, tetramethylorthosilicate, or dopamine.

43. Use of the molecularly imprinted and coated polymer of any one of paragraphs 34-42 in the preparation of a formulation for recognizing a target molecule or target cell.

44. The use of paragraph 43 wherein the preparation is used in affinity purification, biochemical analysis, target recognition and imaging analysis.

45. The use of paragraph 43 or 44 wherein the target molecule comprises B2M, TRF, tfR, AFP, CEA or C-peptide.

46. The use of paragraphs 43 or 44 wherein said target cell is a tumor cell.

47. The use of paragraph 46 wherein said tumor cell is a lung cancer cell, a breast cancer cell or a liver cancer cell.

Examples

The invention is further illustrated by the following examples, without limiting the scope of the invention thereto. Unless otherwise stated, the operations described in the following examples can be performed according to techniques known in the art (see, for example, the following records: molecular imprinting-from the basic to the applied section, [ day ] Mingguangshan et al, wu Shikang, wang Peng Fei Ling, science publishers, 2006, 4 months; "molecular imprinting techniques and applications", tan Tianwei editors, chemical industry publishers, 2010, 7 months; "molecular imprinting techniques", jiang Zhong Yi, wu hong editors, chemical industry publishers, 2003, 1 month; "molecular imprinting techniques and pharmaceutical analyses", fu Qiang et al, xi' an university publishers, 2014, 9 months; "molecular imprinting Polymer functional materials, tianda Cai, science publishers, 2017, 3 months). Reagents, materials and equipment used in the following examples are all commercially available reagents, materials and equipment unless otherwise specified.

Example 1: preparation of glycated polypeptide epitopes

The amino acid sequence information of the target proteins B2M, TRF, tfR, AFP, CEA and C-peptide is determined by a Protein database (such as UniProt, protein Date Bank, etc.).

For B2M, a C-terminal polypeptide sequence SQPKIVKWDRDM (SEQ ID NO. 1) is selected as an epitope, a polypeptide sequence SQPKIVKWDRDMK (SEQ ID NO. 2) with lysine (K) connected to the tail end is directly synthesized through solid phase synthesis, and then the polypeptide sequence SQPKIVKWDRDMK is combined with fructose (Fru) on the connected K residue through Schiff base reaction to obtain the fructose-glycated epitope polypeptide SQPKIVKWDRDMK-Fru.

For TRF, a C-terminal polypeptide sequence SSLLEACTFRRP (SEQ ID NO. 3) is selected as an epitope, a polypeptide sequence SSLLEACTFRRPK (SEQ ID NO. 4) with K connected to the tail end is directly synthesized through solid phase synthesis, and then the peptide sequence SSLLEACTFRRPK-Fru is combined with Fru through Schiff base reaction on the connected K residue, so that the fructosylated epitope polypeptide SSLLEACTFRRPK-Fru is obtained.

For TfR, a C-terminal polypeptide sequence LSGDVWDIDDNEF (SEQ ID No. 5) is selected as an epitope, a polypeptide sequence LSGDVWDIDDNEFK (SEQ ID No. 6) with K connected to the terminal is directly synthesized by solid phase synthesis, and then the polypeptide sequence LSGDVWDIDDNEFK is combined with Fru on a residue of the connected K through Schiff base reaction to obtain the fructose-glycated epitope polypeptide LSGDVWDIDDNEFK-Fru.

For AFP, an N-terminal polypeptide sequence RTLHRNEYGIAS (SEQ ID NO. 7) is selected as an epitope, the polypeptide RTLHRNEYGIAS is directly synthesized by solid phase synthesis, and then the amino group of an initial amino acid R is combined with Fru through Schiff base reaction to obtain the fructose-glycated epitope polypeptide Fru-RTLHRNEYGIAS.

For CEA, an N-terminal polypeptide sequence KLTIESTPFNFVA (SEQ ID NO. 8) is selected as an epitope, the polypeptide KLTIESTPFNFVA is directly synthesized through solid phase synthesis, and then the polypeptide Fru-KLTIESTPFFVVA is combined with Fru through Schiff base reaction on the amino group of an initial amino acid K to obtain the fructose-glycated epitope polypeptide Fru-KLTIESTPFNVVA.

For C-peptide, selecting an N-terminal polypeptide sequence EAEDLQVGQVEL (SEQ ID NO. 9) as an epitope, directly synthesizing the polypeptide EAEDLQVGQVEL through solid phase synthesis, and then combining with Fru through Schiff base reaction on the amino group of an initial amino acid E to obtain a Fru-glycated epitope polypeptide Fru-EAEDLQVGQVEL; selecting a C-terminal polypeptide sequence SLQPLALEGSLQ (SEQ ID NO. 10) as an epitope, directly synthesizing a polypeptide sequence SLQPLALEGSLQK (SEQ ID NO. 11) connected with K at the tail end of the epitope through solid phase synthesis, and then combining the polypeptide sequence SLQPLALEGSLQK-Fru on the connected K residue through Schiff base reaction to obtain the fructose-glycated epitope polypeptide SLQPLALEGSLQK-Fru.

The chemical structures of the C-terminal glycated epitope, AFP (d) and CEA (e), and C-terminal glycated epitope (f) and N-terminal glycated epitope (g) of C-peptide are shown in FIG. 2 as B2M (a), TRF (B) and TfR (C), respectively.

Example 2: preparation of molecularly imprinted and coated magnetic nanoparticles

Step 1) preparation of magnetic nanoparticles

2.0g of ferric chloride hexahydrate, 13.0g of 1, 6-hexanediamine and 4.0g of anhydrous sodium acetate are added into 60mL of ethylene glycol, mixed uniformly and then placed into a reaction kettle lined with polytetrafluoroethylene, after reaction is carried out for 6 hours at 198 ℃, the obtained magnetic nanoparticles are respectively washed three times by water and ethanol, and finally dried overnight.

Step 2) boric acid functionalization of silica-coated magnetic nanoparticles

7.5mL of aqueous ammonia (28%, w/v) and 1.4mL of TEOS were added to 200mL of anhydrous ethanol, and stirred at 40 ℃ for 20 minutes. And ultrasonically dispersing 200mg of magnetic nanoparticles into 20mL of absolute ethyl alcohol, then adding the solution into the solution, continuously stirring the solution for 20 minutes at 40 ℃, carrying out magnetic separation to obtain the magnetic nanoparticles coated with the silicon dioxide, respectively washing the magnetic nanoparticles three times with water and absolute ethyl alcohol, and finally drying the magnetic nanoparticles overnight.

The silica-coated magnetic nanoparticles were ultrasonically dispersed in 100mL of anhydrous ethanol, and then 3mL of APTES was added thereto, followed by stirring at 80 ℃ for 12 hours. And (3) carrying out magnetic separation to obtain the magnetic nano particles coated with the silicon dioxide with the amino functionalization, respectively washing the magnetic nano particles with water and absolute ethyl alcohol for three times, and finally drying the magnetic nano particles overnight.

200mg of amino-functionalized silica-coated magnetic nanoparticles were ultrasonically dispersed in 80mL of methanol, and then 400mg of DFFPBA and 1w/v% sodium cyanoborohydride were added, followed by stirring at 25 ℃ for 24 hours. Magnetic separation is carried out to obtain boric acid functionalized magnetic nano particles, and the boric acid functionalized magnetic nano particles are respectively washed by water and absolute ethyl alcohol for three times and dried overnight.

Step 3), preparation of molecularly imprinted and coated magnetic nanoparticles

2.0mg of the C-terminal glycated epitope of B2M and the C-terminal glycated epitope of C-peptide prepared in example 1 were added to 2mL of a 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5), respectively, and then 20mg of the boronic acid-functionalized magnetic nanoparticles were added and ultrasonically dispersed. After 2 hours of incubation at 25 ℃, magnetic separation yielded glycated epitope template-anchored magnetic nanoparticles and washing three times with 50mM ammonium bicarbonate buffer solution (pH 8.5).

The template-anchored magnetic nanoparticles described above were ultrasonically dispersed in 150mL of anhydrous ethanol containing 4.5mL of ammonia (28 w/v%), followed by addition of 10mL of water and mechanical stirring for 5 minutes. 40mL of an ethanol solution of a monomer silylation reagent and a cross-linking agent (the type and molar ratio of the monomer silylation reagent and the cross-linking agent are shown in figure 5) is added into the solution, mechanical stirring is carried out at 25 ℃ to carry out first imprinting, magnetic separation is carried out to obtain magnetic nanoparticles of the first directional imprinting, and the magnetic nanoparticles are washed three times by absolute ethyl alcohol.

The magnetic nanoparticles of the first directional blotting were ultrasonically dispersed in 160mL of an absolute ethanol solution containing 2.8mL of ammonia (28 w/v%), followed by addition of 40mL of an absolute ethanol solution of 10mM TEOS and mechanical stirring at 25 ℃ for 10 minutes to perform a second blotting. And (4) obtaining magnetic nanoparticles of the second directional imprinting after magnetic separation, washing the magnetic nanoparticles for three times by using absolute ethyl alcohol, and finally drying the magnetic nanoparticles overnight.

The obtained magnetic nanoparticles of the second directional imprinting were dispersed in 2mL of an elution solution (acetonitrile: water: acetic acid 50. After removing the glycosylation epitope template, carrying out magnetic separation to obtain the molecular imprinting and the coated magnetic nanoparticles, respectively washing with water and absolute ethyl alcohol for three times, and finally drying overnight.

The preparation process of the magnetic nanoparticles of single imprinting is the same as that described above except that the template is changed from the C-terminal glycated epitope of B2M to the glycated nonapeptide epitope sqpvikvd (SEQ ID No. 12), and no second directed imprinting is performed (i.e., the resulting magnetic nanoparticles of the first directed imprinting are dispersed in 2mL of an elution solution).

The preparation of corresponding non-imprinted magnetic nanoparticles was performed in the same manner as described above except that the corresponding glycated epitope template was not added.

As shown in fig. 5, when preparing the magnetic nanoparticles (i.e., molecularly imprinted polymers) with single imprinting of C-terminal epitope of B2M described above, the optimal imprinting factor (6.2) was obtained when the species and molar ratio of the monomeric silylation reagent and the crosslinking agent were APTES/UPTES/BnTES/IBTES/TEOS = 10; in contrast, when the C-terminal epitope molecularly imprinted and coated magnetic nanoparticles (i.e., molecularly imprinted and coated polymers) of B2M are prepared, when the species and molar ratio of the monomer silylation reagent to the cross-linking agent is APTES/UPTES/BnTES/IBTES/TEOS =20, and the imprinting time is 60 minutes, an optimal imprinting factor (16.6) is obtained, which is much higher than that of the single-imprinted magnetic nanoparticles, which indicates that the non-specific adsorption sites can be significantly eliminated by using the molecular imprinting and coating strategy, thereby improving the specificity of the obtained molecularly imprinted and coated polymers.

Example 3: characterization of selectivity of boronic acid functionalized magnetic nanoparticles

1.0mg/mL of adenosine and deoxyadenosine were dissolved in 200. Mu.L of 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5), respectively, and then 2.0mg of the boric acid functionalized magnetic nanoparticles obtained in example 2 were added, respectively, and incubated at 25 ℃ for 2 hours. After magnetic separation of the boronic acid functionalized magnetic nanoparticles, they were washed three times with 200. Mu.L of 50mM ammonium bicarbonate/500 mM sodium chloride buffer (pH 8.5) and 50mM ammonium bicarbonate buffer (pH 8.5), respectively, and then redispersed in 20. Mu.L of 100mM acetic acid solution with shaking for 1 hour. And magnetically separating the boric acid functionalized magnetic nanoparticles to obtain an eluent. The absorbance of the boronic acid functionalized magnetic nanoparticles to adenosine and deoxyadenosine was obtained by measuring the eluate at 260nm with ultraviolet light, as shown in fig. 4 (a), indicating that the boronic acid functionalized magnetic nanoparticles have good selectivity to adenosine containing a homeotic dihydroxy, but no boron affinity to deoxyadenosine containing no homeotic dihydroxy.

To further demonstrate the selectivity of the boronic acid functionalized magnetic nanoparticles, the C-terminal epitopes of B2M, TRF and TfR and the N-terminal epitopes of AFP and CEA and their corresponding glycated epitopes obtained in example 1 were used as analytes, and the experimental procedure was the same as described above except that adenosine and deoxyadenosine were replaced with the above-described epitopes and glycated epitopes and the eluate was changed to measure the ultraviolet absorbance at 214nm, and as a result, the boronic acid functionalized magnetic nanoparticles showed good selectivity for cis-dihydroxy compound-containing compounds (C-terminal glycated epitopes of B2M, TRF and TfR, N-terminal glycated epitopes of AFP and CEA) relative to the corresponding epitopes, as shown in fig. 4 (B).

Example 4: determination of adsorption isotherms

Fluorescein-labeled C-terminal dodecapeptide (FITC-SQPKIVKWDRDM) of B2M was prepared at concentrations of 10 and 10 using phosphate buffer (10 mM, pH 7.4), respectively²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰And (3) taking 200 mu L of pg/mL standard solution, putting the solution into an enzyme label plate, and detecting the fluorescence intensity of the solution by an enzyme label instrument. Then, 2mg of the molecular imprinting of the C-terminal epitope of B2M prepared in example 2 and the coated magnetic nanoparticles were added to 1mL of the standard solutions of the above-mentioned respective concentrations, respectively, and shaken at 25 ℃ for 30 minutes. After magnetic separation of magnetic nanoparticles, 200. Mu.L of the upper solution was placed in an ELISA plate, and the fluorescence intensity was detected by an ELISA reader. And fitting the logarithm of the concentration of the FITC-SQPKIVKWDRDM by using the difference between the fluorescence intensity of the standard solution before the C-terminal epitope molecular imprinting of the B2M and the fluorescence intensity of the upper solution after extraction to obtain an adsorption isotherm.

For the adsorption isotherm measurement of the magnetic nanoparticles of the single imprinting of the C-terminal epitope of B2M, all the experimental steps were the same as above except that the molecular imprinting of the C-terminal epitope of B2M and the coated magnetic nanoparticles were changed to the magnetic nanoparticles of the single imprinting, and FITC-SQPKIVKWDRDM was changed to FITC-KIVWDRDM.

Respectively calculating dissociation constants K of single imprinting and molecular imprinting of C-terminal epitope of B2M and coated magnetic nanoparticles prepared by using monomer silanization reagents and cross-linking agents in different proportions under optimal imprinting time through Hill equation_d. As shown in FIG. 6, the K of the magnetic nanoparticle with single imprinting of the C-terminal epitope of B2M is shown on the premise of ensuring the optimal IF value_dValue of only 10^-7M (a), and B2M C-terminal epitope molecular imprinting and K of coated magnetic nanoparticles_dCan reach a value of 10^-9M (B), is far lower than the magnetic nanoparticles of single imprinting of C-terminal epitope of B2M. This shows that the molecular imprinting and coating strategy of the invention not only significantly improves the specificity of the molecular imprinting material, but also greatly enhances the affinity.

Example 5: selectivity of molecularly imprinted and coated magnetic nanoparticles

1) Selectivity at the level of peptide fragments

The C-terminal epitopes of B2M, TRF and TfR and the N-terminal epitopes of AFP and CEA obtained in example 1 were dissolved in a phosphate buffer solution (10 mM, pH 7.4), respectively, to prepare an epitope solution of 0.1 mg/mL. 2.0mg of the C-terminal epitope molecular blot of B2M prepared in example 2 and the coated and non-imprinted magnetic nanoparticles were added to 200. Mu.L of the epitope solution, respectively, and incubated at 25 ℃ for 30 minutes. After magnetically separating each of the above magnetic nanoparticles, it was washed three times with 200. Mu.L of a phosphate buffer solution (10mM, pH 7.4). Then, each magnetic nanoparticle was redispersed in 20 μ L of an elution solution (acetonitrile: water: acetic acid 50, in volume ratio). Finally, each magnetic nanoparticle is magnetically separated and the eluate is collected.

The eluate was measured at 214nm by UV analysis, and as a result, as shown in fig. 7 (a), the C-terminal epitope of B2M molecularly imprinted and the coated magnetic nanoparticles showed excellent selectivity for the C-terminal epitope of B2M relative to non-imprinted magnetic nanoparticles.

2) Selectivity at the protein level

B2M, TRF, tfR, AFP and CEA were dissolved in a phosphate buffer solution (10 mM, pH 7.4) to prepare a 0.1mg/mL protein solution, respectively. 2.0mg of the C-terminal epitope molecular blot of B2M prepared in example 2 and the coated and non-imprinted magnetic nanoparticles were added to 200. Mu.L of the protein solution, respectively, and incubated at 25 ℃ for 30 minutes. After magnetically separating each of the above magnetic nanoparticles, it was washed three times with 200. Mu.L of a phosphate buffer solution (10mM, pH 7.4). Then, each magnetic nanoparticle was redispersed in 20 μ L of an elution solution (acetonitrile: water: acetic acid 50, in volume ratio). Finally, each magnetic nanoparticle is magnetically separated and the eluate is collected.

The eluate was measured at 214nm by UV analysis, and as a result, as shown in (B) of fig. 7, the C-terminal epitope molecular imprinting of B2M and the coated magnetic nanoparticles showed excellent selectivity for the target protein B2M, relative to non-imprinted magnetic nanoparticles.

Example 6: versatility of the molecular imprinting and coating methods of the invention

Next, the C-terminal epitopes of TRF and TfR and the N-terminal epitopes of AFP and CEA were blotted and coated. For the C-terminal epitope of TRF and TfR, the molar ratio of monomeric silylating agent and cross-linking agent APTES/UPTES/BnTES/IBTES/TEOS is 10. All preparation procedures were the same as in example 2 except that the template was changed to the C-terminal glycated epitope of TRF and TfR, and the monomer silylation reagent and the cross-linking agent in the above-mentioned proportions and the corresponding optimal blotting time were used.

The eluates were measured by UV analysis at 214nm and the results are shown in fig. 8, the molar ratio of monomeric silylating agent and cross-linker APTES/UPTES/BnTES/IBTES/TEOS was 10; the highest IF values were obtained for the C-terminal epitope molecular imprinting of TfR and coated magnetic nanoparticles prepared at 60min imprinting time with a molar ratio of monomer silylating reagent and cross-linker APTES/UPTES/BnTES/IBTES/TEOS of 20.

The selective characterization of the magnetic nanoparticles described above was performed as described in example 5. The results show that the C-terminal epitope molecular engram and the coated magnetic nanoparticles (C and e in fig. 8) of the TRF and the C-terminal epitope molecular engram and the coated magnetic nanoparticles (d and f in fig. 8) of the TfR show excellent specificity in peptide fragments and protein water average.

For the N-terminal epitope of AFP and CEA, the molar ratio of the monomers silylating agent and the cross-linking agent APTES/UPTES/BnTES/IBTES/TEOS is 10. Therefore, the ratio of these 5 reagents and the corresponding optimal blotting time can be used as a universal blotting condition for blotting an epitope on the N-terminal side of a protein. All preparation procedures were the same as in example 2 except that the template was changed to the corresponding AFP and CEA N-terminal epitope and the monomer silylation reagent and cross-linking agent were used in the above proportions and the corresponding optimal imprinting time was used.

The eluates were measured by UV analysis at 214nm and the results are shown in fig. 9, the molar ratio of monomeric silylating agent and cross-linker APTES/UPTES/BnTES/IBTES/TEOS was 20; the molar ratio of monomeric silylating reagent and cross-linking agent APTES/UPTES/BnTES/IBTES/TEOS was 10.

The selective characterization of the magnetic nanoparticles described above was performed as described in example 5. The results show that the AFP N-terminal epitope molecular engram and the coated magnetic nanoparticles (c and e in figure 9) and the CEA N-terminal epitope molecular engram and the coated magnetic nanoparticles (d and f in figure 9) show excellent specificity in peptide fragment and protein level.

Non-imprinted magnetic nanoparticles corresponding to those described above were prepared as described in example 2.

From the results of this example, it can be seen that the method of the present invention for preparing molecularly imprinted and coated polymers is simple, versatile, and easily extendable to imprinting of other proteins.

Example 7: preparation of molecularly imprinted and coated Raman-responsive silver nanoparticles

Step 1), preparation of Raman-responsive silver nanoparticles

Dissolving 36mg of silver nitrate in 200mL of ultrapure water, heating to boiling under continuous stirring, rapidly adding 4mL of newly prepared trisodium citrate solution (1%, w/v), continuing stirring and maintaining the boiling state for about 40 minutes, then naturally cooling to room temperature to obtain silver sol with the particle size of about 55nm, and storing at 4 ℃ for later use.

Using PATP as a Raman reporter, 10mL of the above silver sol solution was added to 20. Mu.L of a 1mM ethanol solution of PATP, and the mixture was stirred at room temperature for 40 minutes. The resulting solution was dispersed in 40mL of an ethanol solution, stirred for 10 minutes to mix the solution uniformly, and then 0.7mL of aqueous ammonia (28 w/v%) was added dropwise and stirred for 5 minutes. Then, 10mL of 10mM TEOS ethanol solution was added, the reaction was magnetically stirred at room temperature for 50 minutes, and the mixture was centrifuged at 8000rpm for 10 minutes and washed with absolute ethanol 3 times to obtain Raman-responsive silica-coated silver nanoparticles (Ag/PATP @ SiO2 NPs).

Step 2) preparation of boric acid functionalized Raman responsive silver nanoparticles

The above raman-responsive silica-coated silver nanoparticles were dispersed in 10mL of anhydrous ethanol. In order to modify boric acid on the raman-responsive silica-coated silver nanoparticles, 100 μ L of APTES was added dropwise to 10mL of the above nanoparticle solution, stirred at room temperature for 1 hour, centrifuged at 8000rpm for 10 minutes and the precipitate was washed with absolute ethanol 3 times to obtain amino-functionalized raman-responsive silver nanoparticles. The amino functionalized raman responsive silver nanoparticles were dispersed in 30mL ethanol. 300. Mu.L of 5mg/mL DFFPBA and 300. Mu.L of 5mg/mL sodium cyanoborohydride were added to 30mL of the above-obtained ethanol suspension of silver nanoparticles. After 24 hours of reaction, the silver nanoparticles were centrifuged at 8000rpm for 10 minutes to obtain boric acid-functionalized raman response, and then washed with anhydrous ethanol and water 3 times, respectively.

Step 3), preparation of molecularly imprinted and coated Raman-responsive silver nanoparticles

The boric acid functionalized raman-responsive silver nanoparticles were dispersed in 9mL of phosphate buffer solution (10 mm, ph 7.4). 2mL of 1.0mg/mL of the N-terminal glycated epitope of C-peptide prepared in example 1 dissolved in a phosphate buffer solution (10mM, pH 7.4) was added to 9mL of the above-described boric acid-functionalized Raman-responsive silver nanoparticle solution. After 2 hours of incubation at room temperature, the corresponding glycated epitope template-anchored raman-responsive silver nanoparticles were obtained by centrifugation at 8000rpm for 10 minutes and washed 3 times with phosphate buffer solution (10mm, ph 7.4).

The template-anchored raman-responsive silver nanoparticles were dispersed in 15mL of anhydrous ethanol containing 0.45mL of aqueous ammonia (28 w/v%), and then 1mL of water was added to the resulting suspension and stirred for 5 minutes. Next, 4mL of an absolute ethanol solution containing a mixture of monomeric silylating agent and cross-linking agent (APTES, UPTES, IBTES and TEOS at a molar ratio of 30. Finally, the resulting raman-responsive silver nanoparticles of the first directional blot were collected by centrifugation at 8000rpm for 10 minutes and washed 3 times with anhydrous ethanol.

The silver nanoparticles of the raman response of the first directional blotting were redispersed in 16mL of anhydrous ethanol containing 0.45mL of ammonia water (28 w/v%), and stirred for 5 minutes. Then 4mL of 10mM TEOS in ethanol was added and stirred at room temperature for 10 minutes for a second blot. Finally, the obtained silver nano particles with the Raman response of the second directional imprinting are collected by centrifugation at 8000rpm for 10 minutes and washed 3 times by absolute ethyl alcohol.

The raman-responsive silver nanoparticles of the second directional blot were dispersed into 10mL of an elution solution (acetonitrile: water: acetic acid =50, in v/v). After removing the glycosylation epitope template, the prepared molecular imprinting with imprinting cavity and the coated Raman-responsive silver nanoparticles were centrifuged at 8000rpm for 10 minutes, and washed 3 times with anhydrous ethanol and water, respectively. Finally, the molecularly imprinted and coated raman-responsive silver nanoparticles were dispersed in a phosphate buffer solution (10 mm, ph 7.4) for use.

The preparation process of the non-imprinted Raman-responsive silver nanoparticles is the same as the preparation process of the non-imprinted Raman-responsive silver nanoparticles except that the glycated epitope template is not added.

Example 8: double epitope specificity molecular engram and polymer coating plasmon immune sandwich method (DuMIP-PISA)

For the applicability of each molecularly imprinted and coated raman-responsive silver nanoparticle prepared in example 7, we propose a dual epitope specific molecularly imprinted and polymer-coated plasmon immune sandwich method (duMIP-PISA) combined with a portable raman spectrometer to rapidly and highly sensitively detect C-peptide in human body fluid. In the process of enriching and detecting the C-peptide, the MIPs which are used for specifically recognizing the N-terminal epitope and have the Raman labeling function and the MIPs which are used for specifically recognizing the C-terminal epitope and have the rapid magnetic separation function and are prepared in the embodiment 7 are used for grabbing the C-peptide simultaneously, so that the specificity is further improved. After a sandwich structure of C-terminal epitope MIP-C-peptide-N-terminal epitope MIP is formed, the high-sensitivity detection is carried out on the sandwich structure by a portable Raman spectrometer.

The steps for detecting C-peptide in human serum and urine (from 2 healthy subjects (short for normal people), 2 patients with type I diabetes and 2 patients with type II diabetes) based on the dual epitope specific molecular imprinting and polymer-coated plasmon immune sandwich method are as follows: (1) enrichment and labeling. 50 μ L of 10mg/mL solution of the molecularly imprinted and coated magnetic nanoparticles prepared in example 2 (dispersed in 10mM phosphate buffer, pH 7.4) was taken. Then, 50. Mu.L of C-peptide standard solution or sample to be tested (human serum and urine) and 30. Mu.L of the solution of the molecularly imprinted and coated Raman-responsive silver nanoparticles prepared in example 7 (dispersed in 10mM phosphate buffer, pH 7.4) were added thereto at the same time, and shaken at room temperature for 20 minutes; and (2) cleaning. Removing the substance not bound to the magnetic nanoparticles and the raman-responsive silver nanoparticles by magnetic separation, and washing the resulting magnetic nanoparticles 3 times with 100 μ L of a phosphate buffer (10 mm, ph 7.4); and (3) detecting. The obtained magnetic nanoparticles were dispersed in 10. Mu.L of a phosphate buffer solution (10 mM, pH 7.4) to obtain a nanocomposite solution of a sandwich structure. A1 mu L of nano compound solution with a sandwich structure is spotted on a smooth glass sheet coated by aluminum foil paper, 5 liquid drops are spotted on each sample, and after the sample is aired at room temperature, the Raman spectrum of the central position of each liquid drop is collected for 3 times.

Meanwhile, ELISA kits purchased from Shanghai enzyme-linked bioscience, inc. (China) are used as a reference to detect corresponding human serum and urine according to product specifications. The results are shown in table 1 below.

Table 1 comparison of dual epitope specific molecular imprinting and polymer-coated plasmon immunization sandwich method with commercial ELISA kit

* The content of C-peptide in the serum of a normal person is 0.78-3.1ng/mL. The content of C-peptide in normal human urine is 45-117 mu g/24h.

Example 9: preparation of molecularly imprinted and coated FITC (FITC-doped silica) nanoparticles

Step 1), preparation of FITC-doped silica nanoparticles

Adding 50 mu L of APTES into 10mL of absolute ethanol, uniformly mixing, then adding 10mg of FITC, and shaking overnight at 25 ℃ in the dark to obtain an ethanol solution of the FITC-APTES derivative.

The prepared ethanol solution of FITC-APTES derivative of 6.25mL is uniformly mixed with TEOS of 1.5mL and absolute ethyl alcohol of 20mL to be used as a precursor of the polycondensation reaction for later use. 200mL of absolute ethanol, 12.125mL of water and 9.0mL of aqueous ammonia (28 w/v%) were added to a 250mL round bottom flask, mixed well, placed in an oil bath, and slowly warmed to 55 ℃ with vigorous stirring. The precursor was then added and the reaction was continued for 50 minutes at 55 ℃. The resulting solution was centrifuged at 11000rpm for 15min to obtain FITC-doped silica nanoparticles, which were then washed twice with anhydrous ethanol and water, respectively. Finally, the FITC-doped silica nanoparticles were redispersed in 20mL absolute ethanol and stored at 25 ℃ in the dark.

Step 2) preparation of boric acid functionalized FITC doped silica nanoparticles

100 μ L of APTES was added to 20mL of anhydrous ethanol containing the FITC-doped silica nanoparticles described above, and shaken for 2 hours at 25 ℃. And centrifuging the obtained solution at 11000rpm for 15min to obtain amino-functionalized FITC-doped silica nanoparticles, and then respectively washing with absolute ethyl alcohol and water twice. Finally, amino functionalized FITC doped silica nanoparticles were dispersed in 20mL methanol.

DFFPBA and sodium cyanoborohydride were added to a methanol solution of the amino-functionalized FITC-doped silica nanoparticles at final concentrations of 5mg/mL and 1mg/mL, and shaken at 25 ℃ for 24 hours. The resulting solution was centrifuged at 8000rpm for 10 minutes to obtain boric acid functionalized FITC doped silica nanoparticles, which were then washed three times with anhydrous ethanol and water, respectively. Finally, the boronic acid functionalized FITC doped silica nanoparticles were dispersed in 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5) and stored at 25 ℃ protected from light.

Step 3), preparation of molecularly imprinted and coated FITC-doped silica nanoparticles

4mg of the TfR C-terminal glycated epitope template prepared in example 1 was added to 4mL of a 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5) containing 1mg/mL of boronic acid-functionalized FITC-doped silica nanoparticles and shaken at 25 ℃ for 2 hours. The resulting solution was centrifuged at 8000rpm for 10 minutes to give template-anchored FITC-doped silica nanoparticles, which were then washed three times with 50mM ammonium bicarbonate buffer solution (ph 8.5). Finally, the template-anchored FITC-doped silica nanoparticles were collected by centrifugation at 8000rpm for 10 minutes.

Template-anchored FITC-doped silica nanoparticles were dispersed in 15mL absolute ethanol solution containing 0.45mL ammonia (28 w/v%), followed by addition of 1mL water and stirring at 25 ℃ for 5 minutes. Then 4mL of an absolute ethanol solution containing a mixture of monomeric silylating agent and crosslinker (APTES, UPTES, bnTES, IBTES and TEOS at a 20. Finally, the resulting first directional imprinted FITC-doped silica nanoparticles were collected by centrifugation at 8000rpm for 10 minutes.

The FITC-doped silica nanoparticles of the first directed blotting were dispersed in 16mL of an absolute ethanol solution containing 0.28mL of ammonia (28 w/v%), followed by addition of 4mL of an absolute ethanol solution containing 10mM TEOS, and shaking at 25 ℃ for 10 minutes to perform a second blotting. Finally, the resulting second directionally imprinted FITC-doped silica nanoparticles were collected by centrifugation at 8000rpm for 10 minutes.

The second directional imprinted FITC-doped silica nanoparticles were dispersed in 20mL of an elution solution (acetonitrile: water: acetic acid 50, by volume ratio, 49) and stirred at 25 ℃ for 20 minutes, and the above washing process was repeated three times. After removing the glycated epitope template, centrifuging at 8000rpm for 10 minutes to obtain the molecularly imprinted and coated FITC-doped silica nanoparticles, and then washing with absolute ethanol and water respectively three times. Finally, the molecularly imprinted and coated FITC-doped silica nanoparticles were redispersed in 1 × PBS buffer solution.

The preparation of non-imprinted FITC-doped silica nanoparticles was performed in the same manner as above except that no glycated epitope template was added.

Example 10: cell culture and imaging

MCF-7 cells (purchased from ATCC) were cultured in RPMI-1640 medium containing 10% FBS for 2-3 days (37 ℃,5% CO)₂) MCF-10A cells were cultured in RPMI-1640 medium containing 10% FBS for 2-3 days (37 ℃,5% CO)₂) Culturing HepG-2 cells (purchased from ATCC) in DMEM medium containing 10% FBS for 2-3 days (37 ℃,5%₂) Culturing L-02 cells (purchased from ATCC) in DMEM medium containing 10% FBS for 2-3 days (37 ℃,5%₂) Each cell was cultured in duplicate. After removal of the medium, the cells were washed twice with 1 × PBS buffer. 200 μ L of 1 XPBS buffer solution containing 200 μ g/mL of the molecular imprint of the C-terminal epitope of TfR prepared in example 9 and the coated FITC-doped silica nanoparticles and non-imprinted FITC-doped silica nanoparticles were added, respectively. The culture dish thus treated was placed in a cell culture incubator (37 ℃,5% CO)₂) After 30 minutes of incubation, the cells were washed three times by adding 1 × PBS buffer solution to remove unbound nanoparticles. Subsequently, 100. Mu.L of DAPI was added to the culture dish, and after staining for 10 minutes, it was washed twice with 1 XPBS buffer solution. Finally, the culture dish was supplemented with 1mL of 1 XPBS buffer solution and cell imaging was performed under a laser confocal fluorescence microscope.

Through the above cell imaging analysis, as shown in fig. 10, the molecular imprinting of the C-terminal epitope of TfR and the coated FITC-doped silica nanoparticles showed very strong fluorescence signals to tumor cells (breast cancer cell MCF-7 and liver cancer cell HepG-2); and has almost no fluorescence signal to normal cells (normal mammary epithelial cells MCF-10A and normal liver cells L-02). Meanwhile, the non-imprinted FITC-doped silica nanoparticles have no obvious fluorescent signal on tumor cells and normal cells. The results show that the TfR C-terminal epitope molecular imprinting prepared by the method and the coated FITC-doped silica nanoparticles can obviously distinguish tumor cells from normal cells by selectively combining the TfR with high expression on the surface of the tumor cells.

Sequence listing

<110> Nanjing university

<120> molecular imprinting and coating polymer, preparation method and application thereof

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Ser Ser Leu Leu Glu Ala Cys Thr Phe Arg Arg Pro Lys

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<213> Artificial Sequence (Artificial Sequence)

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<223> C-terminal polypeptide sequence of TfR

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Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe

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<213> Artificial Sequence (Artificial Sequence)

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<223> C-terminal polypeptide sequence of C-peptide having lysine linked to terminal thereof

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1 5

Claims

1. A method for preparing a molecularly imprinted and coated polymer by two-time molecular imprinting, wherein the method comprises: and contacting the molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer.

2. The method of claim 1, further comprising: anchoring an imprinting template on the surface of a substrate material, and then adding one or more monomer silanization reagents and a cross-linking agent to perform the first imprinting on the substrate material to form an imprinting layer, so as to obtain the molecular imprinting material containing the imprinting layer;

preferably, the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate;

preferably, the polymerization agent is tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine;

preferably, the method comprises:

3. The method according to claim 2, wherein a polypeptide sequence of 9-15 amino acid residues from the C-terminus or N-terminus of the target protein is selected as the epitope;

preferably, the first 6-12 amino acid residues in the epitope are subjected to the first imprinting by using the one or more monomeric silylation reagents and the cross-linking reagent to obtain the molecularly imprinted material of the first directional imprinting; and performing the second imprinting on the last 3-6 amino acid residues in the epitope by using the polymerization reagent to obtain a molecularly imprinted material for the second directional imprinting;

preferably, a polypeptide sequence of 9-15 amino acid residues at the C terminal or N terminal of the target protein is obtained as the epitope through solid phase synthesis;

preferably, a C-terminal polypeptide sequence of the target protein is selected as the epitope, a residue of lysine is linked to the end of the polypeptide sequence, and then the residue of lysine is bound to monosaccharide by schiff base reaction to perform the glycation;

or, preferably, an N-terminal polypeptide sequence of the target protein is selected as the epitope, and an amino group of an initial amino acid of the polypeptide sequence is bound to a monosaccharide through a schiff base reaction to perform the glycation;

preferably, the monosaccharide is selected from fructose, glucose, galactose, mannose, xylose, or any mixture thereof;

preferably, the protein of interest is selected from B2M, TRF, tfR, AFP, CEA or C-peptide.

4. The method of claim 2 or 3, wherein the substrate material is a magnetic nanomaterial, a silver nanomaterial, and a fluorescein-doped silica nanomaterial;

preferably, the substrate material is silver nanoparticles with raman reporter molecules or FITC doped silica nanoparticles;

preferably, the raman reporter molecule is p-mercaptoaniline, p-nitrobenzophenol or p-mercaptophenylboronic acid;

preferably, the base material is functionalized with boronic acid.

5. The method according to any one of claims 2 to 4, wherein the base material is a magnetic nanomaterial or a silver nanomaterial, the base material being functionalized by: (i) Reacting the substrate material with ammonia water and TEOS in an alcohol solution to obtain a substrate material with a silicon-coated surface; (ii) Reacting the substrate material with the surface coated with silicon with APTES in an alcoholic solution to obtain an amino functionalized substrate material; (iii) Reacting the amino-functionalized substrate material with substituted boric acid and sodium cyanoborohydride in an alcoholic solution to obtain the boric acid-functionalized substrate material;

or preferably, the substrate material is a fluorescein-doped silica nanomaterial, and the substrate material is functionalized by the following steps: (i') reacting the fluorescein-doped silica nanomaterial with APTES in an alcoholic solution to obtain an amino-functionalized fluorescein-doped silica nanomaterial; (ii') reacting the amino-functionalized fluorescein-doped silica nanomaterial with substituted boric acid and sodium cyanoborohydride in an alcoholic solution to obtain the boronic acid-functionalized fluorescein-doped silica nanomaterial;

preferably, the concentration of the ammonia water is 25w/v% -28 w/v%;

preferably, in step (i), said alcoholic solution contains 0.7 to 1.4vol% of said TEOS;

preferably, in steps (ii) and (i'), said alcoholic solution contains 0.5 to 3vol% of said APTES;

preferably, the boronic acid comprises 2, 4-difluoro-3-formylphenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, or alkenylphenylboronic acid;

preferably, in steps (iii) and (ii'), said alcoholic solution contains 0.05 to 5w/v% of said boronic acid;

preferably, in steps (iii) and (ii'), said alcoholic solution contains 0.05 to 1w/v% of said sodium cyanoborohydride;

preferably, the imprinting template and the boric acid functionalized substrate material are added into a buffer solution with the pH value of more than 7, and after incubation, a template anchoring substrate material is obtained;

preferably, the buffer solution is selected from an ammonium bicarbonate/sodium chloride buffer solution, an ammonium bicarbonate buffer solution or a phosphate buffer solution.

6. The method of any one of claims 2-5, wherein the first imprinting is performed by adding water and an alcohol solution of the monomeric silylation reagent and the cross-linking agent to a solution of ammonia-containing alcohol in which the template-anchored substrate material is dispersed, resulting in the first directionally-imprinted molecularly imprinted material;

preferably, the concentration of the ammonia water is 25w/v% -28 w/v%;

preferably, the monomeric silylating agent comprises aminopropyltriethoxysilane, ureidopropyltriethoxysilane, benzyltriethoxysilane, and isobutyltriethoxysilane.

7. The method of any one of claims 2 to 6, wherein the second imprinting is carried out by adding an alcoholic solution of the polymerization reagent to an ammonia-containing alcoholic solution in which the molecularly imprinted material of the first directional imprinting is dispersed, so as to obtain the molecularly imprinted material of the second directional imprinting;

preferably, the concentration of the ammonia water is 25w/v% -28 w/v%;

preferably, the molecularly imprinted material of the second directional imprinting is eluted by an elution solution containing acetonitrile, water and glacial acetic acid to remove the imprinted template;

preferably, the elution solution consists of acetonitrile, water and glacial acetic acid in a volume ratio of (30-70): (69-29): 1.

8. A molecularly imprinted and coated polymer comprising:

a print layer, and

9. The molecularly imprinted and coated polymer of claim 1, wherein the polymer further comprises a base material;

preferably, the blotting layer comprises a monomeric silylating agent and a cross-linking agent polymerized on the base material;

preferably, the substrate material is a magnetic nano material, a silver nano material or a fluorescein-doped silicon dioxide nano material;

preferably, the substrate material comprises silver nanoparticles with raman reporter molecules or FITC doped silica nanoparticles;

preferably, the monomeric silylating agent comprises aminopropyltriethoxysilane, ureidopropyltriethoxysilane, benzyltriethoxysilane, and isobutyltriethoxysilane;

preferably, the coating layer is formed from tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

10. Use of the molecularly imprinted and coated polymer of claim 8 or 9 for the preparation of a preparation for recognizing a target molecule or a target cell;

preferably, the preparation is used in affinity purification, biochemical analysis, target recognition and imaging analysis;

preferably, the target molecule comprises B2M, TRF, tfR, AFP, CEA, or C-peptide;

preferably, the target cell is a tumor cell; preferably, the tumor cell is a lung cancer cell, a breast cancer cell or a liver cancer cell.