CN115254047B

CN115254047B - Molecularly imprinted and coated polymer, preparation method and application thereof

Info

Publication number: CN115254047B
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: 2024-01-02
Anticipated expiration: 2041-04-30
Also published as: CN115254047A

Abstract

The invention relates to a method for preparing a molecular imprinting and coating polymer through twice molecular imprinting, the obtained molecular imprinting and coating polymer and application of the polymer. Wherein, the molecular imprinting and coating polymer comprises an imprinting layer and a coating layer. The preparation method involves contacting a molecularly imprinted material obtained after the first imprinting with a polymerization reagent with good biocompatibility to perform a second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer. According to the method, the coating layer with controllable thickness is formed on the surface of the imprinting layer through the polymerization reagent, the nonspecific adsorption sites outside the imprinting cavity can be covered to improve the specificity, the molecular imprinting and coating polymer capable of recognizing the target protein and polypeptide with high affinity and high specificity is obtained after the template is removed, and the method has the advantages of controllability, universality, convenience and the like, and has important application prospects in the fields of separation and purification, biochemical analysis, tumor cell targeted recognition, imaging and the like.

Description

Molecularly imprinted and coated polymer, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomimetic molecular recognition materials and molecular imprinting, and relates to a molecular imprinting and coating polymer capable of specifically recognizing specific targets (such as proteins or polypeptides), 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 the organism to resist the invasion of harmful substances such as viruses and microorganisms, and is widely used for biological molecule recognition in the field of life science research. However, antibodies have significant limitations, including complex preparation processes, high cost, long screening cycles, and sometimes even inability to obtain them. In addition, stability and reproducibility of antibodies are also problematic. Therefore, finding an alternative to antibodies is not only of great scientific significance, but also of 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 the antibody, 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, bioimaging, controlled drug release, nano medicine, and the like. Although significant advances have been made in molecular imprinting techniques, conventional molecular imprinting techniques still have inherent drawbacks. In order to obtain high specificity and strong affinity for the template molecule, it is generally necessary to polymerize a plurality of functional monomers capable of interacting with the template molecule with a crosslinking agent to construct a print cavity, and the print cavity is just complementary to the template molecule in terms of shape, size, interaction site, etc., so as to obtain a binding cavity capable of high specificity and strong affinity. However, the inventors have found that since the non-imprinted surface outside the imprinted cavity is also built up from the same kind and proportion of monomers and cross-linking agents as the imprinted cavity, there are significant non-specific interaction sites which lead to significant cross-reactivity during actual use. 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 order to solve the above technical problems in the prior art, the present inventors have developed a controllable, universal and convenient preparation method of molecular imprinting and coating polymers by research, wherein the 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-specific interaction weak polymerization reagent (such as tetraethyl orthosilicate) as a coating reagent and participate in template imprinting, so that a thin coating layer is formed to reduce non-specific adsorption of a non-imprinting area on the surface of the imprinting material, thereby significantly reducing non-specific interaction sites of the obtained molecular imprinting and coating polymer, and simultaneously improving both specificity and affinity for target molecules.

In one aspect, the present invention provides a method for preparing a molecularly imprinted and coated polymer by two molecular imprinting, wherein the method comprises: and (3) contacting the molecular imprinting material obtained after the first imprinting with a polymerization reagent with good biocompatibility so as to perform second imprinting on the imprinting layer of the molecular imprinting material to form a coating layer.

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

Imprinting layer

And a coating layer, wherein the coating layer is positioned on the outer surface of the imprinting layer.

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

The preparation method of the invention relates to the following two imprinting steps: the first directional blotting involves blotting the template molecules with a monomeric silylating agent and a cross-linking agent that interact with the template molecules to build a good blotting cavity, thereby obtaining strong affinity and high specificity; the second directional blotting (i.e., coating blotting) involves forming a thin coating layer of controllable thickness on the surface of the molecular imprinting layer by using a weak polymerization reagent with non-specific interactions, which is used to cover non-specific sites outside the imprinting layer, thereby further improving specificity (specificity), and in addition, the affinity is also improved due to the spatially matched recognition of more amino acid residues on the template molecule by the imprinting layer caused by the coating layer. In this regard, similar documents and patents have not been reported.

According to the preparation method disclosed by the invention, a pure product of the target protein is not required to be provided or prepared, and only the amino acid structure sequence information of the protein is required to be known, so that the glycosylated epitope template can be obtained by utilizing a chemical synthesis method, and the molecular imprinting and coating 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 available templates through solid phase synthesis, no limitation on the types of substrate materials and the like, and is a molecular imprinting and coating polymer preparation technology with good universality and strong applicability. The molecular imprinting and coating polymer prepared by the method can specifically identify, bind and enrich target proteins and epitopes thereof. Wherein the blotting template is a glycosylated epitope, and not the whole target protein. The invention adopts protein epitope saccharification treatment, breaks through the limitation of epitope selection, widens the variety of substrate materials, and compared with the existing Western blotting technology, the invention adopts different types and proportions of monomer silanization reagents and cross-linking agents for first directional blotting aiming at the epitope sequence of target protein, and then adopts non-specific interaction weak polymerization reagents for second directional blotting (also called cladding blotting), thereby not only obviously improving the specificity of molecular blotting materials, but also enhancing the affinity. The molecular imprinting and coating polymer obtained by the invention can identify proteins and polypeptides, has strong affinity and high specificity, thereby having 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 of an exemplary method of preparing a molecularly imprinted and coated polymer according to the present invention.

FIG. 2 shows beta ₂ -chemical structures of C-terminal glycosylated epitopes of microglobulin (B2M) (a), transferrin (TRF) (B) and transferrin receptor protein (TfR) (C), chemical structures of N-terminal glycosylated epitopes (e) of Alpha Fetoprotein (AFP) (d) and carcinoembryonic antigen (CEA), and chemical structures of C-terminal glycosylated epitopes (f) and N-terminal glycosylated epitopes (g) of C-peptide (C-peptide).

Fig. 3 is a Transmission Electron Microscope (TEM) photograph of a molecular imprinting and coating polymer using different substrate materials. Wherein a is a TEM photograph of the molecularly imprinted and coated magnetic nanoparticle prepared in example 2, b is a TEM photograph of the molecularly imprinted and coated silver nanoparticle prepared in example 7, and c is a TEM photograph of the molecularly imprinted and coated FITC-doped silica nanoparticle prepared in example 9.

FIG. 4 shows the selectivity of boric acid functionalized 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 glycation epitopes (B).

Fig. 5 shows the optimal imprinting factors (a) of the B2M C-terminal epitope single-imprinted magnetic nanoparticles and the C-terminal epitope molecular imprinting of the B2M and the optimal imprinting factors of the coated magnetic nanoparticles (B) prepared with different proportions of monomer silylating agent and crosslinking agent at optimal imprinting time.

Fig. 6 shows the affinity of magnetic nanoparticles (a) single-imprinted with C-terminal epitope of B2M and C-terminal epitope molecular imprinting of B2M and coated magnetic nanoparticles (B) prepared with different ratios of monomer silylating agent and crosslinking agent at optimal imprinting time.

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

FIG. 8 is a view of the versatility of the method of preparing molecularly imprinted and coated polymers of the present invention for C-terminal epitopes of different proteins, wherein the results of optimization of imprinting conditions of the thus prepared TRF-terminal epitope molecularly imprinted and coated magnetic nanoparticles (a) and their selectivities at peptide level (C) and protein level (e), respectively, are shown; the C-terminal epitope molecular imprinting of TfR and the imprinting condition of the coated magnetic nanoparticle optimize the selectivity of the result (b) at the peptide level (d) and the protein level (f).

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

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

Detailed Description

The invention will be described in further detail by way of exemplary embodiments, but the scope of the invention is not limited thereto.

In one embodiment, the present invention provides a method of preparing a molecularly imprinted and coated polymer by two molecular imprinting, wherein the method comprises: and (3) contacting the molecular imprinting material obtained after the first imprinting with a polymerization reagent with good biocompatibility so as to perform second imprinting on the imprinting layer of the molecular imprinting material to form a coating layer.

In some preferred embodiments, the above method of preparing a molecularly imprinted and coated polymer further comprises: anchoring a imprinting template to the surface of a substrate material, and then adding one or more monomeric silylating agents and cross-linking agents to perform said first imprinting on said substrate material, forming an imprinting layer, resulting in said molecularly imprinted material comprising said 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 the target protein as an epitope for saccharification, and obtaining a saccharified epitope as the imprinting template;

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

(3) Anchoring the imprinting template to the surface of the boric 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 template anchored substrate material to perform the first imprinting to form an imprinting layer, so as to obtain a molecular imprinting material containing the first directional imprinting of the imprinting layer;

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

(6) And eluting the molecular imprinting material of the second directional imprinting to remove the imprinting template, so as to obtain the molecular imprinting and coating polymer.

In some preferred embodiments, a polypeptide sequence of 9-15 amino acid residues at the C-terminus or N-terminus of the protein of interest is selected as the epitope. In this context, the selection of polypeptide sequences consisting of amino acid residues at the very end (C-terminal and N-terminal) of the protein as polypeptide sequences for the epitope may avoid the situation where the epitope sequence itself is glycosylated (post-translational modification).

In a further preferred embodiment, the first 6-12 amino acid residues in the epitope are first imprinted with the one or more monomeric silylating agents and cross-linking agents to obtain the first directionally imprinted molecularly imprinted material; 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 molecular imprinting 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, taking the saccharified epitope obtained after saccharification treatment of the polypeptide as a imprinting template, anchoring the imprinting template on the surface of a boric acid functionalized substrate material through boron affinity, performing first imprinting on the first nine amino acid residues of the polypeptide by using a plurality of monomer silanization reagents and a cross-linking agent, performing multiple interactions on the polypeptide by using the obtained imprinting layer to generate strong affinity, and performing second directional imprinting on the surface of the imprinting material by using a weak polymerization reagent with non-specific interactions as a coating reagent, wherein the thickness of a formed coating layer covers the last three amino acids, and removing template molecules to obtain molecular imprinting and a coating polymer with increased specificity and affinity on target molecules.

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

In some preferred embodiments, the C-terminal polypeptide sequence of the protein of interest is selected as the epitope, a residue of lysine is attached at the end of the polypeptide sequence, and the residue of lysine is then combined with a monosaccharide by a schiff base reaction for the saccharification. In some preferred embodiments, the N-terminal polypeptide sequence of the protein of interest is selected as an epitope, and the amino group of the starting amino acid of the polypeptide sequence is combined with 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 glycosylated polypeptide epitope has a stronger force against the boronic acid ligand on the substrate material, preferably fructose, glucose or a mixture thereof is selected which has a stronger affinity for the substituted boronic acid.

In some preferred embodiments, the target protein 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), and fluorescein-doped silica nanomaterials (preferably FITC-doped silica nanoparticles), among others. Preferably, the raman reporter includes, but is not limited to, p-mercaptoaniline (PATP), p-Nitrophenylthiophenol (NTP), p-mercaptophenylboronic acid (MPBA), and the like.

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

In some preferred embodiments, the substrate material is a fluorescein-doped silica nanomaterial, which is functionalized by: (i') reacting the fluorescein doped silica nanomaterial with APTES in an alcohol solution, preferably an ethanol 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, preferably methanol or ethanol solution, to obtain the boric 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), the alcohol solution contains 0.7 to 1.4vol% of the 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 substituted boric acid may include 2, 4-difluoro-3-formylphenylboric acid (DFFPBA), aldrophenylboric acid, aminophenylboric acid, carboxyphenylboric acid, mercaptophenylboric acid, or alkenylphenylboric acid, but is not limited thereto. In a further preferred embodiment, in steps (iii) and (ii'), the alcoholic solution contains 0.05 to 5w/v% of the 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 blotting template and the boric acid-functionalized substrate material are added to a buffer solution having a pH greater than 7, and after incubation (e.g., at 15-40 ℃) the template-anchored substrate material is obtained. In a further preferred embodiment, the buffer solution is selected from ammonium bicarbonate/sodium chloride buffer solution, ammonium bicarbonate buffer solution or phosphate buffer solution, but is not limited thereto.

In some preferred embodiments, the first blotting is performed by adding water and an alcohol solution, preferably an ethanol solution, of the monomeric silylating agent and the cross-linking agent to a solution of an aqueous ammonia-containing alcohol in which the template-anchored substrate material is dispersed, resulting in the first directionally-blotted molecularly-blotted material. The first blot herein is polymerized using a monomeric silylating agent and a crosslinking agent, thereby providing multiple interactions with the epitope sequence. In a more preferred embodiment, the concentration of ammonia is 25w/v% to 28w/v%. In further preferred embodiments, the monomeric silylating agent may be selected according to the amino acid type of the epitope sequence, including but not limited to APTES, UPTES, bnTES or IBTES, etc. In further preferred embodiments, the cross-linking agent may include, but is not limited to, tetraethyl orthosilicate or tetramethyl orthosilicate.

In some preferred embodiments, the second blotting is performed by adding an alcoholic solution, preferably an alcoholic solution, of the polymerization reagent to an aqueous alcoholic solution containing ammonia in which the first directionally-blotted molecularly-blotted material is dispersed, to obtain the second-blotted molecularly-blotted material. In the method, the second imprinting is polymerized by adopting a polymerization reagent, so that non-specific adsorption sites generated outside imprinting cavities in the first directional imprinting are covered, the specificity of the molecular imprinting material is remarkably improved, and meanwhile, 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 second blotting molecularly-imprinted material is eluted with an elution solution comprising acetonitrile, water, and glacial acetic acid to remove the blotting template. Preferably, the elution solution consists of acetonitrile, water and glacial acetic acid in a volume ratio of (30-70): (69-29): 1 (e.g., 50:49:1).

In the invention, a polypeptide fragment at the C end or the N end of target protein is taken as an epitope, saccharified and then taken as a imprinting template, the imprinting template is anchored on a boric acid functionalized substrate material by utilizing boron affinity, a monomer silanization reagent and a cross-linking agent with different types and proportions are adopted for carrying out first directional imprinting, and then a nonspecific interaction weak polymerization reagent is adopted for carrying out second directional imprinting, so that 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, and the epitope polypeptide fragment can meet the imprinting of any sequence after saccharification treatment, so that the technology can be applicable to various target proteins, and the prepared molecular imprinting material has higher specificity and stronger affinity. By way of example only, representative methods of preparing molecularly imprinted and coated polymers of the invention are described below:

(1) Determination of epitope sequence and saccharification

Amino acid sequence information of the target protein can be found out through protein databases (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 an epitope. Meanwhile, in order to facilitate anchoring of a polypeptide sequence as an epitope (also referred to herein as an "epitope polypeptide") to a substrate material, saccharification of the epitope polypeptide is required. The saccharification treatment is as follows: the saccharification process of the C-terminal epitope polypeptide is that the tail end of the C-terminal polypeptide is firstly connected with a lysine residue, and then the lysine residue is combined with monosaccharides such as fructose or glucose for saccharification through Schiff base reaction; the saccharification process of the N-terminal epitope polypeptide is that the amino group of the initial amino acid of the N-terminal polypeptide is saccharified by combining with monosaccharides such as fructose or glucose through a Schiff base reaction; whereby a C-terminal or N-terminal glycosylated polypeptide epitope is obtained as an imprinting template (also referred to herein as a "glycosylated epitope template").

(2) Substrate material selection and boric acid functionalization

According to the different detection methods and uses, substrate materials with different functions and types 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 into the ethanol, and stirring and reacting to obtain an amino-functionalized substrate material; and reacting the amino-functionalized substrate material, the substituted boric acid and the cyano sodium borohydride in methanol or ethanol to obtain the boric acid-functionalized substrate material.

(3) Anchoring of glycosylated epitope templates on a substrate material

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

(4) First directed blotting

And (3) carrying out first directional imprinting on the template anchored substrate material, water, ammonia water, a monomer silanization reagent and a cross-linking agent in ethanol to obtain a first directional imprinted molecular imprinting material.

(5) Second directed blotting (coated blotting)

And (3) carrying out second directional imprinting on the molecular imprinting material subjected to the first directional imprinting, ammonia water and a polymerization reagent (such as TEOS) in ethanol to obtain the molecular imprinting material subjected to the second directional imprinting.

(6) Removal of glycosylated epitope templates

And adding the molecular imprinting material subjected to the second directional imprinting into an eluting solution for eluting, and removing the glycosylated epitope template to obtain the molecular imprinting and coating polymer.

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

Magnetic nanoparticles as substrate material

Step 1), the preparation of magnetic nanoparticles can be seen 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 into the mixture, and continuing stirring for reacting for 5-60 minutes at 20-60 ℃ to obtain magnetic nanoparticles with silicon coated surfaces;

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

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

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

Step 6), dispersing the template anchored magnetic nano particles obtained in the step 5) in an ethanol solution of ammonia water, adding water into the solution, then adding an ethanol solution of a monomer silanization reagent and a cross-linking agent, performing first directional imprinting at 15 ℃ -40 ℃ (e.g. 25 ℃), and performing magnetic separation to obtain the magnetic nano particles with the first directional imprinting;

step 7), carrying out second directional imprinting on the magnetic nano particles subjected to the first directional imprinting, ammonia water and a polymerization reagent in ethanol at 15 ℃ -40 ℃ (e.g. 25 ℃) for 5-30 minutes (e.g. 10 minutes), and carrying out magnetic separation to obtain the magnetic nano particles subjected to the second directional imprinting;

step 8), adding the magnetic nano-particles obtained in the step 7) in the second imprinting to an eluting solution (for example, acetonitrile: water: glacial acetic acid=50:49:1, in v/v) to remove the template, thereby obtaining the molecular imprinting and coating polymer.

The preparation method of the non-imprinted magnetic nanoparticle-based polymer (abbreviated as "non-imprinted polymer") as a control was the same as above except that the glycosylated epitope template was not added.

Raman-responsive silver nanoparticles as substrate material

Step 1), the preparation of silver nanoparticles can be seen in the following method: phys. 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 ethanol solution, and stirring for 5-30 minutes (for example, 10 minutes); then ammonia water is added dropwise, and stirring is continued for 1-10 minutes (for example, 5 minutes); then adding an ethanol solution of TEOS, reacting at room temperature, and centrifuging to obtain Raman-responsive silver nanoparticles with silicon coated surfaces; the raman reporter used is selected according to the requirements of the assay, including but not limited to PATP, NTP, MPBA, etc.;

step 3), dispersing the Raman-responsive silver nanoparticles with silicon coated on the surface, which are obtained in the step 2), in ethanol, adding APTES into the ethanol, shaking the mixture for 0.5 to 5 hours (for example, 1 hour) at 15 to 40 ℃ (for example, 25 ℃), and centrifuging the mixture to obtain the 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 ℃ (e.g. 25 ℃), and centrifuging to obtain the 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, adding a saccharification epitope template, shaking and incubating for 0.5-5 hours at 15-40 ℃ (e.g. 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), adding an ethanol solution containing a monomer silanization reagent and a crosslinking agent, stirring at 15-40 ℃ (for example, 25 ℃) for first imprinting, and centrifuging to obtain Raman-responsive silver nanoparticles subjected to first directional imprinting;

step 7), stirring the Raman-responded silver nanoparticles subjected to the first directional imprinting, ammonia water and a polymerization reagent in an ethanol solution to perform second imprinting, and centrifuging to obtain the Raman-responded silver nanoparticles subjected to the second imprinting;

step 8), adding the raman-responsive silver nanoparticles obtained in the step 7) into an eluting solution (for example, acetonitrile: water: glacial acetic acid=50:49:1 in terms of v/v) to remove the template, and centrifuging to obtain the molecular imprinting and coating polymer.

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

FITC doped silicon dioxide nano particles as substrate material

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

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

step 3), dispersing the FITC-doped silica nanoparticles obtained in the step 2) in ethanol, then adding APTES into the ethanol, shaking for 0.5-5 hours (e.g. 2 hours) at 15-40 ℃ (e.g. 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, then adding substituted boric acid and sodium cyanoborohydride, shaking for 12-36 hours at 15-40 ℃, 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 for 0.5-5 hours at 15-40 ℃, and centrifuging to obtain template anchored FITC-doped silica nanoparticles;

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

step 7), shaking the FITC-doped silicon dioxide nano particles obtained in the step 6) with ammonia water and a polymerization reagent in ethanol at 15-40 ℃ for 5-30 minutes to perform secondary imprinting and centrifuging to obtain FITC-doped silicon dioxide nano particles with secondary directional imprinting;

Step 8), adding the FITC-doped silica nanoparticles with the second directional imprinting obtained in the step 7) into an eluting solution (for example, acetonitrile: water: glacial acetic acid=50:49:1 in terms of v/v) to remove the template, and centrifuging to obtain the molecular imprinting and coating polymer.

Preparation of non-imprinted FITC-doped silica nanoparticle-based polymer as a control (abbreviated as "non-imprinted polymer") all the steps were as above except that no glycosylated epitope template was added.

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

imprinting layer

In some preferred embodiments, the polymer further comprises a base material. In some preferred embodiments, the imprinting layer comprises a monomeric silylating agent and a crosslinking agent polymerized on the substrate 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), and fluorescein-doped silica nanomaterials (preferably FITC-doped silica nanoparticles), among others. More preferably, the raman reporter includes, but is not limited to, p-mercaptoaniline (PATP), p-Nitrophenylthiophenol (NTP), p-mercaptophenylboronic acid (MPBA), and the like.

In some preferred embodiments, the monomer silylating agent may include, but is not limited to, aminopropyl triethoxysilane (APTES), ureido triethoxysilane (upes), benzyltriethoxysilane (BnTES), and Isobutyltriethoxysilane (IBTES), among others. 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 molecularly imprinted and coated polymers described above for the preparation of a formulation for the recognition of a target molecule or target cell. In some preferred embodiments, the formulations are used in affinity purification, biochemical analysis, targeted recognition (e.g., tumor cell targeted recognition), and imaging analysis. In some preferred embodiments, the target molecules include, but are not limited to, B2M, TRF, tfR, AFP, CEA or C-peptide, and the like. In some preferred embodiments, the target cells are tumor cells or the like, including but not limited to breast, liver, lung cancer cells, and the like.

Exemplary embodiments of the present invention may be described in the following numbered paragraphs, but the scope of the present invention is not limited thereto:

1. A method of preparing a molecularly imprinted and coated polymer by two molecular imprinting, wherein the method comprises: and (3) contacting the molecular imprinting material obtained after the first imprinting with a polymerization reagent with good biocompatibility so as to perform second imprinting on the imprinting layer of the molecular imprinting material to form a coating layer.

2. The method of paragraph 1, wherein the method further comprises: anchoring a imprinting template to the surface of a substrate material, and then adding one or more monomeric silylating agents and cross-linking agents to perform said first imprinting on said substrate material, forming an imprinting layer, resulting in said molecularly imprinted material comprising said 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 polymerization agent is tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

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

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

(5) Contacting the molecularly imprinted material of the first directional imprinting with the polymerization reagent to perform the second imprinting on the imprinting layer to form a coating layer, thereby obtaining a molecularly imprinted material of the second directional imprinting including the coating layer;

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

7. The method of paragraphs 5 or 6, wherein the first blotting of the first 6-12 amino acid residues in the epitope with the one or more monomeric silylating agents and the cross-linking agent to obtain the first directionally-blotted molecularly-imprinted material; and performing the second blotting of the last 3-6 amino acid residues in the epitope by using the polymerization reagent to obtain a molecular blotting material of the second directional blotting.

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

9. The method of any one of paragraphs 5-8, wherein the C-terminal polypeptide sequence of the protein of interest is selected as the epitope, a residue of lysine is attached at the end of the polypeptide sequence, and the residue of lysine is then combined with a monosaccharide by a Schiff base reaction for the saccharification.

10. The method of any one of paragraphs 5-8, wherein the N-terminal polypeptide sequence of the target protein is selected as the epitope, and the amino group of the starting amino acid of the polypeptide sequence is combined with a monosaccharide for the saccharification by a Schiff base reaction.

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

12. The method of any one 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, or a fluorescein-doped silica nanomaterial.

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

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

16. The method of any of paragraphs 5-15, wherein the substrate material is functionalized with a substituted boric acid.

17. The method of any one 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 silicon coated surface; (ii) Reacting the substrate material with the surface coated with silicon with APTES in an alcohol solution to obtain an amino-functionalized substrate material; (iii) Reacting the amino-functionalized substrate material with a substituted boric acid and sodium cyanoborohydride in an alcohol solution to obtain the boric acid-functionalized substrate material.

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

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

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

21. The method of any one 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 substituted boronic acid comprises 2, 4-difluoro-3-formylphenylboronic acid, aldehydylphenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, or alkenylphenylboronic acid.

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

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

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

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

27. The method of any one of paragraphs 5-26, wherein the first blotting is performed by adding water and an alcohol solution of the monomer silylating agent and the crosslinking agent to a solution of an ammonia-containing alcohol dispersed with the template-anchored substrate material, resulting in the first directionally-blotted molecularly-blotted material.

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

29. The method of any of paragraphs 5-28, wherein the monomer silylating agent comprises aminopropyl triethoxysilane, ureido triethoxysilane, benzyltriethoxysilane, and isobutyltriethoxysilane.

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

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

32. The method of any of paragraphs 5-31, wherein the second directionally-blotted molecular blotting material is eluted with an elution solution comprising acetonitrile, water, and glacial acetic acid to remove the blotting template.

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:

imprinting layer

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 imprinting layer comprises a monomeric silylating agent and a crosslinking agent polymerized on the substrate material.

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

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

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

40. The molecularly imprinted and coated polymer of any of paragraphs 36-39, wherein the monomer silylating agent comprises aminopropyl triethoxysilane, ureido triethoxysilane, benzyl triethoxysilane, and isobutyl triethoxysilane.

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 layer is formed from tetraethyl orthosilicate, tetramethyl orthosilicate, 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 a target cell.

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

45. The use of paragraphs 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 the target cell is a tumor cell.

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

Examples

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto. Unless otherwise indicated, the following examples are directed to procedures known in the art (see, e.g., the following description: molecular imprinting-from basic to application, [ day ] less Gong Shanzhen et al, wu Shikang, wang Pengfei, scientific Press, month 4, molecular imprinting techniques and applications, tan Tianwei, chemical industry Press, month 7, molecular imprinting techniques, jiang Zhongyi, wu Hong, chemical industry Press, month 1, 2003, molecular imprinting techniques and pharmaceutical analysis, fu Jiang, et cetera, western An traffic university press, month 9, molecular imprinted polymer functional materials, field university hearing, scientific Press, month 3, 2017). Unless otherwise indicated, reagents, materials, and equipment used in the examples below were all commercially available.

Example 1: preparation of glycosylated polypeptide epitopes

Amino acid sequence information of the target proteins B2M, TRF, tfR, AFP, CEA and C-peptide was determined by protein databases (e.g., uniProt, protein Date Bank, etc.).

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

For TRF, the C-terminal polypeptide sequence SSLLEACTFRRP (SEQ ID NO. 3) is selected as an epitope, the 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 is combined with Fru on the connected K residue through Schiff base reaction to obtain the fructosylated epitope polypeptide SSLLEACTFRRPK-Fru.

For TfR, the C-terminal polypeptide sequence LSGDVWDIDNEF (SEQ ID NO. 5) is selected as an epitope, the polypeptide sequence LSGDVWDIDNEFK (SEQ ID NO. 6) with K connected to the tail end is directly synthesized through solid phase synthesis, and then the peptide is combined with Fru on the connected residue of K through Schiff base reaction to obtain the fructosylated epitope polypeptide LSGDVWDIDNEFK-Fru.

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

For CEA, the N-terminal polypeptide sequence KLTIESTPFNVA (SEQ ID NO. 8) is selected as an epitope, polypeptide KLTIESTPFNVA is directly synthesized through solid phase synthesis, and then the polypeptide is combined with Fru on the amino group of the initial amino acid K through Schiff base reaction, so that fructosylated epitope polypeptide Fru-KLTIESTPFNVA is obtained.

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

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

Example 2: preparation of molecular imprinting and coated magnetic nanoparticles

Step 1) preparation of magnetic nanoparticles

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

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

7.5mL of ammonia (28%, w/v) and 1.4mL of TEOS were added to 200mL of absolute ethanol and stirred at 40℃for 20 minutes. 200mg of magnetic nano particles are dispersed into 20mL of absolute ethyl alcohol by ultrasonic, then added into the solution, stirred for 20 minutes at 40 ℃, and then subjected to magnetic separation to obtain the magnetic nano particles coated with silicon dioxide, and the magnetic nano particles are respectively washed three times by water and absolute ethyl alcohol, and finally dried overnight.

The silica-coated magnetic nanoparticles were ultrasonically dispersed into 100mL of absolute ethanol, and then 3mL of APTES was added thereto, followed by stirring at 80 ℃ for 12 hours. And (3) magnetically separating to obtain amino-functionalized silica-coated magnetic nanoparticles, respectively washing the magnetic nanoparticles with water and absolute ethyl alcohol for three times, and finally drying overnight.

200mg of amino-functionalized silica-coated magnetic nanoparticles were sonicated into 80mL of methanol, then 400mg of DFFPBA and 1w/v% sodium cyanoborohydride were added and stirred at 25℃for 24 hours. And (3) magnetically separating to obtain boric acid functionalized magnetic nano particles, respectively washing the magnetic nano particles with water and absolute ethyl alcohol for three times, and then drying overnight.

Step 3), preparation of molecular imprinting and coated magnetic nano particles

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 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5), respectively, followed by addition of 20mg of boric acid-functionalized magnetic nanoparticles and ultrasonic dispersion. After 2 hours incubation at 25 ℃, magnetic separation gives glycosylated 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 sonicated into 150mL absolute ethanol containing 4.5mL aqueous ammonia (28 w/v%), then 10mL water was added and mechanically stirred for 5 minutes. 40mL of an ethanol solution of a monomer silylation agent and a crosslinking agent (the kind and mole of which are shown in FIG. 5) was added to the above solution, and the mixture was mechanically stirred at 25℃to perform a first blotting, and after magnetic separation, magnetic nanoparticles of the first directional blotting were obtained, and washed three times with absolute ethanol.

The magnetic nanoparticles of the first directional blotting were ultrasonically dispersed into 160mL of absolute ethanol solution containing 2.8mL of ammonia water (28 w/v%), then 40mL of absolute ethanol solution of 10mM TEOS was added, and mechanically stirred at 25℃for 10 minutes to perform the second blotting. And (3) obtaining magnetic nano particles with second directional imprinting after magnetic separation, washing the magnetic nano particles with absolute ethyl alcohol for three times, and finally drying the magnetic nano particles overnight.

The resulting second directionally imprinted magnetic nanoparticles were dispersed in 2mL of an elution solution (acetonitrile: water: acetic acid 50:49:1 by volume), shaken at 25 ℃ for 20 minutes, and the above procedure was repeated three times. And removing the saccharification epitope template, performing magnetic separation to obtain molecular imprinting and coated magnetic nano particles, respectively washing with water and absolute ethyl alcohol for three times, and finally drying overnight.

The preparation process of the magnetic nanoparticles of the single blotting was the same as above except that the template was changed from the C-terminal glycosylated epitope of B2M to the glycosylated nonapeptide epitope SQPKIVKWD (SEQ ID NO. 12) and the second directional blotting was not performed (i.e., the magnetic nanoparticles of the first directional blotting obtained were dispersed in 2mL of the eluting solution).

The preparation of the corresponding non-imprinted magnetic nanoparticles is the same as described above, except that no corresponding glycosylated epitope template is added.

As shown in fig. 5, when the magnetic nanoparticle with single imprinting of the C-terminal epitope of B2M (i.e., the molecularly imprinted polymer) is prepared, the optimal imprinting factor (6.2) is obtained when the types and molar ratio of the monomer silylating agent and the crosslinking agent are APTES/upes/BnTES/IBTES/teos=10:10:10:20:50 and the imprinting time is 60 minutes; in contrast, when the above-mentioned B2M C-terminal epitope molecular imprinting and coated magnetic nanoparticles (i.e., molecular imprinting and coated polymer) were prepared, when the types and molar ratios of monomer silylating agent and crosslinking agent were APTES/upes/BnTES/IBTES/teos=20:20:10:30:20, and the imprinting time was 60 minutes, an optimal imprinting factor (16.6) was obtained, which was far higher than that of the single-imprinted magnetic nanoparticles, indicating that the use of the molecular imprinting and coated strategy was able to significantly eliminate non-specific adsorption sites, thereby improving the specificity of the resulting molecular imprinting and coated polymer.

Example 3: characterization of selectivity of boric 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 boric acid functionalized magnetic nanoparticles, each was washed three times with 200 μl 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 μl of 100mM acetic acid solution and shaken for 1 hour. And magnetically separating the boric acid functionalized magnetic nano particles to obtain eluent. The absorbance of the magnetic nanoparticles functionalized with boric acid on adenosine and deoxyadenosine was obtained by uv-measuring the eluate at 260nm, as shown in fig. 4 (a), indicating that the magnetic nanoparticles functionalized with boric acid have good selectivity for adenosine containing homeopathic dihydroxy, but no boron affinity for deoxyadenosine without homeopathic dihydroxy.

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

Example 4: determination of adsorption isotherms

The fluorescein-labeled B2M C-terminal dodecapeptide (FITC-SQPKIVKWDRDM) was formulated in phosphate buffered saline (10 mM, pH 7.4) at concentrations of 10, respectively ² 、10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 200 mu L of the standard solution in pg/mL is respectively put into an ELISA plate, and the fluorescence intensity is detected by an ELISA instrument. Then 2mg of the C-terminal epitope molecular imprinting of B2M prepared in example 2 and the coated magnetic nanoparticle were added to 1mL of the standard solution of each concentration, respectively, and shaken at 25℃for 30 minutes. After magnetic separation of magnetic nanoparticles, 200 μl of the upper solution was put into an ELISA plate, and the fluorescence intensity was detected by an ELISA meter. Molecular imprinting of C-terminal epitope of B2M and coated magnetic nanoparticle before extraction The difference between the fluorescence intensity and the fluorescence intensity of the upper layer solution after extraction was fitted to the concentration logarithm of FITC-SQPKIVKWDRDM to obtain an adsorption isotherm.

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

The dissociation constants K of the single imprinting and molecular imprinting of the C-terminal epitope and the coated magnetic nano particles of the B2M prepared by the monomer silanization reagent and the cross-linking agent with different proportions under the optimal imprinting time are respectively calculated by Hill equation _d . As shown in FIG. 6, the K of the magnetic nanoparticle of B2M C-terminal epitope single imprinting under the premise of ensuring the optimal IF value _d The value is only 10 ^-7 M (a), while B2M C-terminal epitope molecular imprinting and K of coated magnetic nano particles _d A value of up to 10 ^-9 M (B), far lower than B2M C-terminal epitope single-imprinted magnetic nanoparticles. This shows that by the molecular imprinting and coating strategy of the invention, not only the specificity of the molecular imprinting material is remarkably improved, but also the affinity is greatly enhanced.

Example 5: selectivity of molecularly imprinted and coated magnetic nanoparticles

1) Selectivity at the peptide fragment level

The C-terminal epitopes of B2M, TRF and TfR obtained in example 1 and the N-terminal epitopes of AFP and CEA were dissolved in phosphate buffer solution (10 mM, pH 7.4), respectively, to prepare an epitope solution of 0.1 mg/mL. 2.0mg of the B2M C-terminal epitope molecular imprinting and coated magnetic nanoparticles and non-imprinted magnetic nanoparticles prepared in example 2 were added to 200. Mu.L of the epitope solution, respectively, and incubated at 25℃for 30 minutes. After magnetic separation of the above magnetic nanoparticles, the particles were washed three times with 200. Mu.L of phosphate buffer solution (10 mM, pH 7.4). Then, each magnetic nanoparticle was redispersed in 20. Mu.L of an elution solution (acetonitrile: water: acetic acid 50:49:1 by volume ratio) and shaken at 25℃for 10 minutes. Finally, the magnetic nanoparticles are magnetically separated and the eluate is collected.

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

2) Selectivity at the protein level

B2M, TRF, tfR, AFP and CEA were each dissolved in phosphate buffer (10 mM, pH 7.4) to prepare a protein solution of 0.1 mg/mL. 2.0mg of the B2M C-terminal epitope molecular imprinting and coated magnetic nanoparticles and non-imprinted magnetic nanoparticles prepared in example 2 were added to 200. Mu.L of a protein solution, respectively, and incubated at 25℃for 30 minutes. After magnetic separation of the above magnetic nanoparticles, the particles were washed three times with 200. Mu.L of phosphate buffer solution (10 mM, pH 7.4). Then, each magnetic nanoparticle was redispersed in 20. Mu.L of an elution solution (acetonitrile: water: acetic acid 50:49:1 by volume ratio) and shaken at 25℃for 10 minutes. Finally, the magnetic nanoparticles are 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 nanoparticle showed excellent selectivity for the target protein B2M, relative to the non-imprinted magnetic nanoparticle.

Example 6: versatility of 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 epitopes of TRF and TfR, the molar ratio of the monomer silylating reagent to the crosslinking agent APTES/UPTES/BnTES/IBTES/TEOS is 10:10:10:20:50, 10:20:10:20:40, 20:20:10:20:30, 20:20:10:30:20 and 20:30:10:30, respectively, and the optimal blotting time is 70min, 60min and 50min, respectively, and the ratio of the 5 reagents and the optimal blotting time can be used as the universal blotting conditions of the target protein C-terminal epitope. All preparation procedures were as in example 2 except that the template was changed to the corresponding C-terminal glycated epitope of TRF and TfR, and the above-described ratios of monomeric silylating agent and crosslinker were used with the corresponding optimal blotting times.

The eluate was measured by UV analysis at 214nm and the results are shown in fig. 8, with the molar ratio of monomer silylating reagent and crosslinker APTES/upes/BnTES/IBTES/TEOS being 10:20:10:20:40, C-terminal epitope molecular imprinting of TRF prepared at 60min imprinting time and coated magnetic nanoparticles yielding the highest IF value (a of fig. 8); the molar ratio of monomer silylating reagent and crosslinker APTES/upes/BnTES/IBTES/TEOS was 20:20:10:30:20, C-terminal epitope molecular imprinting of TfR prepared at 60min imprinting time and coated magnetic nanoparticles gave the highest IF value (fig. 8 b).

The characterization of the selectivity of the magnetic nanoparticles described above was performed as described in example 5. The results show that the C-terminal epitope molecular imprinting and coated magnetic nanoparticles of the TRF (C, e of fig. 8) and the C-terminal epitope molecular imprinting and coated magnetic nanoparticles of TfR (d, f of fig. 8) show excellent specificity at both peptide and protein levels.

For N-terminal epitopes of AFP and CEA, the molar ratio of monomer silylating reagent to cross-linking agent APTES/UPTES/BnTES/IBTES/TEOS is 10:10:10:20:50, 10:20:10:20:40, 20:20:10:20:30, 20:20:10:30:20 and 20:30:10:30:10, and the corresponding optimal blotting time is 60min, 50min and 40min respectively. Thus, the ratio of these 5 reagents and the corresponding optimal blotting time can be used as universal blotting conditions for blotting the N-terminal epitope of the protein. All preparation procedures were as in example 2 except that the template was changed to the corresponding N-terminal epitope of AFP and CEA and the above proportions of monomeric silylating agent and crosslinker were used with the corresponding optimal blotting time.

The eluate was measured by UV analysis at 214nm and the results are shown in figure 9, the molar ratio of monomeric silylating agent and cross-linker APTES/upes/BnTES/IBTES/TEOS being 20:20:10:20:30, N-terminal epitope molecular imprinting of AFP prepared at 50min imprinting time and coated magnetic nanoparticles yielding the highest IF value (a of figure 9); the molar ratio of monomer silylating reagent and crosslinker APTES/upes/BnTES/IBTES/TEOS was 10:20:10:20:40, N-terminal epitope molecular imprinting of CEA prepared at 50min imprinting time and coated magnetic nanoparticles gave the highest IF value (fig. 9 b).

The characterization of the selectivity of the magnetic nanoparticles described above was performed as described in example 5. The results show that the N-terminal epitope molecular imprinting and coating magnetic nanoparticles (c and e of fig. 9) of the AFP and the N-terminal epitope molecular imprinting and coating magnetic nanoparticles (d and f of fig. 9) of the CEA show excellent specificity at peptide segment and protein level.

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

From the results of this example, it can be seen that the method of preparing the molecularly imprinted and coated polymer of the invention is simple, has strong versatility, and can be easily extended to imprinting of other proteins.

Example 7: preparation of molecular imprinting and coated Raman-responsive silver nanoparticles

Step 1) preparation of Raman-responsive silver nanoparticles

36mg of silver nitrate was dissolved in 200mL of ultrapure water, heated to boiling with continuous stirring, 4mL of a freshly prepared trisodium citrate solution (1%, w/v) was rapidly added, stirring was continued and kept at boiling for about 40 minutes, and then naturally cooled to room temperature to give a silver sol having a particle size of about 55nm, and stored at 4℃for further use.

Using PATP as a Raman reporter, 10mL of the silver sol solution was added with 20. Mu.L of 1mM PATP in ethanol, and the mixture was stirred at room temperature for 40 minutes. The resulting solution was dispersed in 40mL of ethanol solution, and after stirring for 10 minutes to mix the solution uniformly, 0.7mL of aqueous ammonia (28 w/v%) was added dropwise and stirred for 5 minutes. Then, 10mL of an ethanol solution of 10mM TEOS was added thereto, and the reaction was magnetically stirred at room temperature for 50 minutes, 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 raman-responsive silica-coated silver nanoparticles were dispersed in 10mL of absolute ethanol. To modify boric acid on raman-responsive silica-coated silver nanoparticles, 100 μl 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 3 times with absolute ethanol to give amino-functionalized raman-responsive silver nanoparticles. The amino-functionalized raman-responsive silver nanoparticles were dispersed in 30mL ethanol. mu.L of 5mg/mL DFFPBA and 300. Mu.L of 5mg/mL sodium cyanoborohydride were added to 30mL of the ethanol suspension of the silver nanoparticles obtained above. After 24 hours of reaction, boric acid functionalized raman-responsive silver nanoparticles were obtained by centrifugation at 8000rpm for 10 minutes, and then washed 3 times with absolute ethanol and water, respectively.

Step 3), preparation of molecular imprinting and coated Raman-responsive silver nanoparticles

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

The template-anchored raman-responsive silver nanoparticles were dispersed into 15mL of absolute 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. Then 4mL of an absolute ethanol solution containing a mixture of monomeric silylating agent and crosslinking agent (molar ratio of APTES, UPTES, IBTES to TEOS 30:20:30:20) was added to the above suspension and stirred at room temperature for a first blotting. Finally, the resulting raman-responsive silver nanoparticles of the first directional blotting were collected by centrifugation at 8000rpm for 10 minutes and washed 3 times with absolute ethanol.

The raman-responsive silver nanoparticles of the first directional blotting described above were redispersed in 16mL of absolute ethanol containing 0.45mL of aqueous ammonia (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 blotting. Finally, the resulting second directionally imprinted raman-responsive silver nanoparticles were collected by centrifugation at 8000rpm for 10 minutes and washed 3 times with absolute ethanol.

The second directionally-imprinted raman-responsive silver nanoparticles were dispersed into 10mL of elution solution (acetonitrile: water: acetic acid=50:49:1, in v/v) and stirred at room temperature for 20 minutes, and this step was repeated 3 times. After removing the glycosylated epitope template, the prepared molecular imprinting and coated raman-responsive silver nanoparticles with imprinting cavities were centrifuged at 8000rpm for 10 minutes and washed with absolute ethanol and water, respectively, 3 times. Finally, the molecularly imprinted and coated raman-responsive silver nanoparticles were dispersed in 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 above except that no glycosylated epitope template is added.

Example 8: dual epitope specific molecular imprinting and polymer coated plasmonic immune sandwich method (duMIP-PISA)

For the applicability of each molecular imprinting and coated Raman-responsive silver nanoparticle prepared in example 7, we propose a dual epitope-specific molecular imprinting 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 enrichment and detection process of C-peptide, the specificity is further improved by simultaneously grabbing the C-peptide by utilizing the MIPs which specifically recognize the N-terminal epitope and have the Raman labeling function and the MIPs which specifically recognize the C-terminal epitope and have the rapid magnetic separation function and are prepared in the embodiment 7. After a sandwich structure of the C-terminal epitope MIP-C-peptide-N-terminal epitope MIP is formed, the sandwich structure is subjected to high-sensitivity detection by a portable Raman spectrometer.

The procedure for detecting C-peptide in human serum and urine (from 2 healthy subjects (abbreviated as "normal"), 2 type I diabetics and 2 type II diabetics, respectively) based on dual epitope-specific molecular imprinting and polymer-coated plasmonic immune sandwich method is as follows: (1) enrichment and labelling. 50 μl of the solution of the molecularly imprinted and coated magnetic nanoparticles prepared in example 2 (dispersed in 10mM phosphate buffer solution, pH 7.4) was taken. Then 50. Mu.L of a C-peptide standard solution or a sample to be tested (human serum and urine) and 30. Mu.L of a solution of molecularly imprinted and coated Raman-responsive silver nanoparticles prepared in example 7 (dispersed in 10mM phosphate buffer solution, pH 7.4) were simultaneously added thereto, and the mixture was shaken at room temperature for 20 minutes; (2) cleaning. Removing substances not bound to the magnetic nanoparticles and raman-responsive silver nanoparticles by magnetic separation, and washing the obtained magnetic nanoparticles with 100 μl of phosphate buffer (10 mm, ph 7.4) 3 times; (3) detection. The resulting magnetic nanoparticles were dispersed in 10 μl of phosphate buffer solution (10 mm, ph 7.4) to give a sandwich-structured nanocomposite solution. And (3) taking 1 mu L of the nano-composite solution with the sandwich structure to be spotted on a smooth glass sheet coated with aluminum foil paper, 5 liquid drops are sprayed on each sample spot, and collecting Raman spectra of the center position of each liquid drop for 3 times after the sample spots are dried at room temperature.

Meanwhile, ELISA kits purchased from Shanghai enzyme-linked biotechnology limited company (China) are used as controls to detect corresponding human serum and urine according to the product specification. The results are shown in table 1 below.

Table 1 comparison of double epitope specific molecular imprinting and polymer coated plasmonic immune sandwich method with commercial ELISA kit

* The content of C-peptide in serum of normal human is usually 0.78-3.1ng/mL. The content of C-peptide in urine of normal people is 45-117 mug/24 h.

Example 9: preparation of molecular imprinting and coated FITC-doped silica nanoparticles

Step 1) preparation of FITC-doped silica nanoparticles

50. Mu.L of APTES was added to 10mL of absolute ethanol, and after mixing well, 10mg of FITC was added thereto, and the mixture was shaken overnight at 25℃in the absence of light to obtain an ethanol solution of the FITC-APTES derivative.

The prepared 6.25mL of ethanol solution of FITC-APTES derivative is uniformly mixed with 1.5mL of TEOS and 20mL of absolute ethanol to serve as a precursor of polycondensation reaction for standby. 200mL of absolute ethanol, 12.125mL of water and 9.0mL of ammonia water (28 w/v%) were added to a 250mL round-bottomed flask, and the mixture was placed in an oil bath after uniform mixing, and the temperature was slowly raised to 55℃with vigorous stirring. The precursor was then added and the reaction 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 absolute ethanol and water, respectively. Finally, the FITC-doped silica nanoparticles were redispersed in 20mL of absolute ethanol and stored at 25 ℃ protected from light.

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

100. Mu.L of APTES was added to 20mL of absolute ethanol containing the above FITC-doped silica nanoparticles, and the mixture was shaken at 25℃for 2 hours. The resulting solution was centrifuged at 11000rpm for 15min to obtain amino-functionalized FITC-doped silica nanoparticles, which were then washed twice with absolute ethanol and water, respectively. Finally, the amino-functionalized FITC-doped silica nanoparticles were dispersed in 20mL of 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 absolute ethanol and water, respectively. Finally, boric acid functionalized FITC doped silica nanoparticles were dispersed in 50mM ammonium bicarbonate/500 mM sodium chloride buffer (pH 8.5) and stored at 25 ℃ protected from light.

Step 3), preparation of molecular imprinting and coated FITC-doped silica nanoparticles

4mg of the C-terminal glycosylated epitope template of TfR prepared in example 1 was added to 4mL of 50mM ammonium bicarbonate/500 mM sodium chloride buffer solution (pH 8.5) containing 1mg/mL of boric acid functionalized FITC-doped silica nanoparticles, and shaken at 25℃for 2 hours. The resulting solution was centrifuged at 8000rpm for 10 minutes to obtain template-anchored FITC-doped silica nanoparticles, which were then washed three times with 50mM ammonium bicarbonate buffer solution (pH 8.5). Finally, 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 of absolute ethanol solution containing 0.45mL of aqueous ammonia (28 w/v%), 1mL of water was added, and stirred at 25 ℃ for 5 minutes. 4mL of an absolute ethanol solution containing a mixture of monomeric silylating agent and crosslinking agent (molar ratio of APTES, UPTES, bnTES, IBTES to TEOS 20:20:10:30:20) was then added to the above suspension and stirred at 25℃for a first blotting. Finally, centrifugation was performed at 8000rpm for 10 minutes to collect the resulting FITC-doped silica nanoparticles of the first directional imprinting.

The FITC-doped silica nanoparticles of the first directional blotting were dispersed into 16mL of an absolute ethanol solution containing 0.28mL of aqueous ammonia (28 w/v%), then 4mL of an absolute ethanol solution containing 10mM TEOS was added, and shaken at 25℃for 10 minutes to perform the second blotting. Finally, centrifugation was performed at 8000rpm for 10 minutes to collect the resulting FITC-doped silica nanoparticles of the second directional imprinting.

The second directionally imprinted FITC-doped silica nanoparticles were dispersed into 20mL of an elution solution (acetonitrile: water: acetic acid 50:49:1 by volume ratio), stirred at 25 ℃ for 20 minutes, and the above-described washing procedure was repeated three times. After removing the glycosylated epitope template, the mixture was centrifuged at 8000rpm for 10 minutes to obtain molecular imprinting and coated FITC-doped silica nanoparticles, which were then washed three times with absolute ethanol and water, respectively. Finally, the molecular imprinting and the coated FITC-doped silica nanoparticles are redispersed in a 1 XPBS buffer solution.

The preparation process of the non-imprinted FITC-doped silica nanoparticles is the same as above except that no glycosylated epitope template is 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 ℃ C., 5% CO) ₂ ) HepG-2 cells (purchased from ATCC) were cultured in DMEM medium containing 10% FBS for 2-3 days (37 ℃,5% CO) ₂ ) L-02 cells (purchased from ATCC) were cultured in DMEM medium containing 10% FBS for 2-3 days (37 ℃ C., 5% CO) ₂ ) Two aliquots of each cell were cultured. After removal of the medium, the cells were washed twice with 1 XPBS buffer. 200. Mu.L of a 1 XPBS buffer solution containing 200. Mu.g/mL of the C-terminal epitope molecular imprinting of TfR prepared in example 9 and coated FITC-doped silica nanoparticles and non-imprinted FITC-doped silica nanoparticles were added, respectively. The dishes treated as described above were placed in a cell incubator (37 ℃,5% co) ₂ ) After 30 minutes of incubation, 1 XPBS buffer was added to wash three times to remove unbound nanoparticles. Subsequently, 100. Mu.L of DAPI was added to the petri dish, and after staining for 10 minutes, the dish was washed twice with 1 XPBS buffer. Finally, 1mL of 1 x PBS buffer solution was added to the dish and cell imaging was performed under a laser confocal fluorescence microscope.

Through the above cell imaging analysis, as shown in fig. 10, the TfR C-terminal epitope molecular imprinting 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); whereas there is little fluorescence signal to normal cells (normal mammary epithelial cells MCF-10A and normal hepatocytes L-02). Meanwhile, the non-imprinted FITC-doped silica nanoparticles have no obvious fluorescence signals on tumor cells and normal cells. The result shows that the TfR C-terminal epitope molecular imprinting and the coated FITC-doped silica nanoparticle prepared by the method can be used for distinguishing tumor cells from normal cells obviously by selectively combining TfR which is highly expressed on the surface of the tumor cells.

Sequence listing

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Claims

1. A method of preparing a molecularly imprinted and coated polymer by two molecular imprinting, wherein the method comprises:

selecting a C-terminal or N-terminal polypeptide sequence of a target protein as an epitope for saccharification to obtain a saccharified epitope as a imprinting template, anchoring the imprinting template to the surface of a substrate material, and then adding one or more monomer silylation reagents and a cross-linking agent to perform first imprinting on the substrate material to form an imprinting layer, so as to obtain a molecular imprinting material containing the imprinting layer;

and (3) enabling the molecularly imprinted material to be in contact with a polymerization reagent with weak nonspecific interaction and good biocompatibility so as to perform second imprinting on an imprinting layer of the molecularly imprinted material to form a coating layer, and covering nonspecific adsorption sites generated outside an imprinting cavity.

2. The method of claim 1, wherein the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate.

3. The method of claim 1 or 2, wherein the polymerization agent is tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

4. The method of claim 1, wherein the method comprises:

5. The method of claim 4, wherein a polypeptide sequence of 9-15 amino acid residues at the C-terminus or N-terminus of the target protein is selected as the epitope.

6. The method of claim 4 or 5, wherein the first blotting of the first 6-12 amino acid residues in the epitope with the one or more monomeric silylating agents and the cross-linking agent to obtain the first directionally-blotted molecularly-imprinted material; and performing the second blotting of the last 3-6 amino acid residues in the epitope by using the polymerization reagent to obtain a molecular blotting material of the second directional blotting.

7. The method according to claim 4 or 5, wherein 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 by solid phase synthesis.

8. The method according to claim 4, wherein a C-terminal polypeptide sequence of the target protein 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 combined with a monosaccharide by a schiff base reaction to carry out the saccharification.

9. The method of claim 4, wherein the N-terminal polypeptide sequence of the target protein is selected as the epitope, and the saccharification is performed by combining an amino group of a starting amino acid of the polypeptide sequence with a monosaccharide through a schiff base reaction.

10. The method of claim 8 or 9, wherein the monosaccharide is selected from fructose, glucose, galactose, mannose, xylose, or any mixture thereof.

11. The method of claim 4 or 5, wherein the target protein is selected from B2M, TRF, tfR, AFP, CEA or C-peptide.

12. The method of claim 4 or 5, wherein the substrate material is a magnetic nanomaterial, a silver nanomaterial, and a fluorescein doped silica nanomaterial.

13. The method of claim 4 or 5, wherein the substrate material is silver nanoparticles or FITC-doped silica nanoparticles with raman reporter molecules.

14. The method of claim 13, wherein the raman reporter is p-mercaptoaniline, p-nitrophenylthiophenol, or p-mercaptophenylboronic acid.

15. The method of claim 4 or 5, wherein the substrate material is functionalized with a substituted boric acid.

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

17. The method of claim 4, wherein the substrate material is a fluorescein doped silica nanomaterial, and the substrate material is functionalized by: (i') reacting the fluorescein-doped silica nanomaterial with APTES in an alcohol solution to obtain an amino-functionalized fluorescein-doped silica nanomaterial; (ii') reacting the amino-functionalized fluorescein-doped silica nanomaterial with a substituted boric acid and sodium cyanoborohydride in an alcohol solution to obtain the boric acid-functionalized fluorescein-doped silica nanomaterial.

18. The method of claim 16, wherein the concentration of the aqueous ammonia is 25 wt% -28 wt%.

19. The method of claim 16, wherein in step (i), the alcohol solution contains 0.7 to 1.4 vol% of TEOS.

20. The method according to any one of claims 16 to 19, wherein in steps (ii) and (i'), the alcohol solution contains 0.5 to 3vol% of APTES.

21. The method of any one of claims 16-19, wherein the substituted boronic acid comprises 2, 4-difluoro-3-formylphenylboronic acid, aldehydylphenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, or alkenylphenylboronic acid.

22. The method of any one of claims 16-19, wherein in steps (iii) and (ii'), the alcoholic solution contains 0.05-5 w/v% of the substituted boronic acid in mg/mL.

23. The method according to any one of claims 16 to 19, wherein in steps (iii) and (ii'), the alcoholic solution contains 0.05 to 1 w/v% of the sodium cyanoborohydride in mg/mL.

24. The method of claim 4 or 5, wherein the blotting template and the boric acid functionalized substrate material are added to a buffer solution having a pH above 7, and after incubation, a template anchored substrate material is obtained.

25. The method of claim 24, wherein the buffer solution is selected from ammonium bicarbonate/sodium chloride buffer, ammonium bicarbonate buffer, or phosphate buffer.

26. The method according to claim 4 or 5, wherein the first blotting is performed by adding water and an alcohol solution of the monomer silylating agent and the crosslinking agent to a solution of an ammonia-containing alcohol dispersed with the template-anchored base material, to obtain the first directionally-imprinted molecularly-imprinted material.

27. The method of claim 26, wherein the concentration of the aqueous ammonia is 25wt% to 28wt%.

28. The method of claim 4 or 5, wherein the monomer silylating agent comprises aminopropyl triethoxysilane, ureido triethoxysilane, benzyl triethoxysilane, and isobutyl triethoxysilane.

29. The method according to claim 4 or 5, wherein the second blotting is performed by adding an alcohol solution of the polymerization reagent to an aqueous alcohol solution containing ammonia in which the molecularly imprinted material of the first directional blotting is dispersed, to obtain the molecularly imprinted material of the second directional blotting.

30. The method of claim 29, wherein the concentration of the aqueous ammonia is 25wt% to 28wt%.

31. The method of claim 4 or 5, wherein the second directionally-blotted molecular engram material is eluted with an elution solution comprising acetonitrile, water and glacial acetic acid to remove the engram template.

32. The method of claim 31, wherein the eluting solution is comprised of acetonitrile, water and glacial acetic acid in a volume ratio of (30-70): (69-29): 1.

33. A molecularly imprinted and coated polymer prepared by the method of any one of claims 1-32, comprising:

a print layer comprising a monomeric silylating agent and a cross-linking agent polymerized on a substrate material, an

34. The molecularly imprinted and coated polymer of claim 33, wherein the substrate material is a magnetic nanomaterial, a silver nanomaterial, or a fluorescein-doped silica nanomaterial.

35. A molecularly imprinted and encapsulated polymer according to claim 33 or 34, wherein the substrate material comprises silver nanoparticles or FITC-doped silica nanoparticles with raman reporter molecules.

36. The molecularly imprinted and coated polymer of claim 35, wherein the raman reporter is p-mercaptoaniline, p-nitrophenylthiophenol, or p-mercaptophenylboronic acid.

37. A molecularly imprinted and coated polymer according to claim 33 or 34, wherein the monomer silylating agent comprises aminopropyl triethoxysilane, ureido triethoxysilane, benzyl triethoxysilane, and isobutyl triethoxysilane.

38. A molecularly imprinted and encapsulation polymer according to claim 33 or 34, wherein the cross-linking agent is tetraethyl orthosilicate or tetramethyl orthosilicate.

39. A molecularly imprinted and encapsulation polymer according to claim 33 or 34, wherein the encapsulation layer is formed by tetraethyl orthosilicate, tetramethyl orthosilicate, or dopamine.

40. Use of a molecularly imprinted polymer according to any one of claims 33-39 for preparing a formulation for recognizing a target molecule or a target cell.

41. The use according to claim 40, wherein the formulation is used in affinity purification, biochemical analysis, target recognition and imaging analysis.

42. The use according to claim 40 or 41, wherein the target molecule comprises B2M, TRF, tfR, AFP, CEA or C-peptide.

43. The use according to claim 40 or 41, wherein the target cell is a tumor cell.

44. The method of claim 43, wherein the tumor cell is a lung cancer cell, a breast cancer cell or a liver cancer cell.