CN109959791B

CN109959791B - A multiplex imprinting material for specifically recognizing tumor cells and its preparation and application

Info

Publication number: CN109959791B
Application number: CN201711337444.4A
Authority: CN
Inventors: 张丽华; 刘路宽; 杨开广; 李潇; 高航; 张玉奎
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2021-09-24
Anticipated expiration: 2037-12-14
Also published as: CN109959791A

Abstract

The present invention relates to a material for specifically identifying tumor cells and its preparation and application. The material is a multiple-action imprinting material: using tumor cells as a template, the cells are bound by a cross-linking agent mainly composed of disulfide bonds, groups that can bind to tumor cells and groups that can participate in material polymerization. A pre-polymerization system is added to the surface of the composite formed with the cross-linking agent, so that the cross-linking agent and the pre-polymerization system undergo a cross-linking reaction to obtain a cell-imprinted material, and then the disulfide bond in the cell-imprinted material is reduced to sulfhydryl to remove tumor cells. The sulfhydryl group can be specifically combined with tumor cells, and it is prepared by introducing one or two or more forces into the imprinting material. The multiple-action imprinting material not only has the imprinting effect of the imprinting material on tumor cells, but also can realize highly selective recognition, capture and enrichment of tumor cells.

Description

Multiple-action imprinting material for specifically recognizing tumor cells and preparation and application thereof

Technical Field

The invention belongs to the field of functional biological materials, and particularly relates to a novel multi-action imprinting material for specifically identifying tumor cells, and preparation and application thereof.

Background

The high-efficiency enrichment and separation of tumor cells in blood or lymph fluid have very important functions in early diagnosis and prognosis analysis of tumors, and accurate medical and omics analysis research and application (Mol Oncol2016,10, 374-394.). At present, because the content of tumor cells in blood or lymph fluid is very low and the blood cells with very high content generate great interference to the enrichment and separation of tumor cells, it is necessary to realize the enrichment and separation of tumor cells in the environment with low cell concentration (Nature 2007,450, 1235-1239).

The current techniques for isolating and enriching tumor cells mainly include immunological and physical techniques. The immunological technology realizes the separation and enrichment of tumor cells by utilizing the combination of antigens on the surface of the tumor cells and corresponding antibodies thereof. However, this approach is prone to false negative results due to the uncertain and variable expression of many tumor cell surface antigens; the physical technology mainly realizes the separation and enrichment of tumor cells by utilizing the difference between the physical properties of the tumor cells and other interfering cells such as blood cells. However, this method is poor in purity and sensitivity of tumor cells isolated and enriched based on this method because the difference in physical properties between tumor cells and blood cells is diverse (nat. rev. cancer 2014,14, 623. 631.). Therefore, it is important to develop a material that can not only replace antibodies, but also specifically recognize and enrich cells.

The molecular imprinting technology is a polymer preparation technology which is based on a molecular recognition theory, takes target molecules as templates, and obtains imprinting sites matched with the target molecules in space and binding sites through the polymerization of monomers capable of interacting with the target molecules and the subsequent elution of the target molecules. The molecular imprinting material prepared by the molecular imprinting technology can realize the specific recognition and capture of target molecules, thereby achieving the purpose of separating and enriching the target molecules. The technology achieves a series of achievements in the aspects of environmental pollutant removal, traditional Chinese medicine separation, high-abundance protein removal and low-abundance protein enrichment in blood (prog. Polym. Sci.2014,39, 145-163). The cell imprinting technology using the cells as the template and the target is a novel technology capable of replacing natural antibodies, has specific separation and enrichment capacity on the target cells, and has the advantages of strong environmental tolerance, good biocompatibility and suitability for large-scale production.

Therefore, based on the cell imprinting technology, in order to further improve the specific recognition capability of the cell imprinting material and reduce the interference of non-specific adsorption, a cell imprinting material based on multiple acting forces is provided. Firstly, tumor cells to be identified are taken as a template and are attached or fixed on the surface of a matrix material, cells are combined by a cross-linking agent which mainly comprises disulfide bonds, groups capable of being combined with the tumor cells and groups capable of participating in material polymerization, after unbound cross-linking agent is removed, a compound formed by the cross-linking agent and the template cells is placed in a prepolymerization system, the prepolymerization system is solidified on the surface of the matrix material, then the matrix material is separated and removed, the disulfide bonds in the cross-linking agent are reduced into sulfhydryl groups to remove the template cells on the surface of the solidified polymer, and then one or more than two acting forces are introduced into the imprinting material by utilizing the reaction between the sulfhydryl groups and one or more than two of modified nucleic acid aptamers capable of chemically and specifically combining with the tumor cells, modified antibodies, modified polypeptides or alkenyl boronic acid compounds, obtaining a multiple-action imprinted material for specifically recognizing tumor cells. The material has shape recognition acting force of the imprinting material and chemical specific binding force on tumor cells, and realizes recognition, capture, release and enrichment of target cells.

Disclosure of Invention

Tumor cells are taken as a template, cells are combined by a cross-linking agent which mainly comprises disulfide bonds, groups capable of being combined with the tumor cells and groups capable of participating in material polymerization, a prepolymerization system is added on the surface of a compound formed by the cells and the cross-linking agent, the cross-linking agent and the prepolymerization system are subjected to cross-linking reaction to obtain a cell imprinting material, then the disulfide bonds in the cell imprinting material are reduced to sulfydryl to remove the tumor cells, and finally, one or more than two acting forces are introduced into the imprinting material by utilizing the reaction between the sulfydryl and one or more than two of nucleic acid aptamers, antibodies, polypeptides or boric acid compounds capable of being specifically combined with the tumor cells to prepare the cell imprinting material. The multiple-action imprinting material has shape recognition acting force of the imprinting material and chemical specific binding force on tumor cells, and is further applied to recognition, capture and enrichment of the tumor cells. In order to achieve the purpose, the invention adopts the technical scheme that:

(1) inoculating tumor cells on one or more of the surfaces of a cell culture plate, a cell culture dish, a plasma-treated metal material and a polylysine-modified glass material, and culturing in a cell culture medium for 12-72 hours to obtain a matrix material with the tumor cells attached to the surface.

(2) Then adding the cross-linking agent to the surface of the tumor cells for incubation for 10 minutes to 3 hours; wherein the cross-linking agent is composed of disulfide bonds, groups capable of binding to tumor cells, and groups capable of participating in polymerization of the material. Wherein, the chemical group capable of combining with tumor cells mainly comprises one or more than two of carboxyl, maleimide and succinimide; the chemical group capable of participating in material polymerization mainly comprises one or more than two of alkylene, sulfydryl, maleimide and succinimide; the solvent for dissolving or dispersing the cross-linking agent comprises one or more than two of water, phosphate buffer solution and sodium chloride solution; the mass concentration range of the solution formed by dissolving or dispersing the cross-linking agent in the solvent is 1-50%.

(3) And removing the cross-linking agent solution, adding the prepolymerization system to the surface of a compound formed by the tumor cells and the cross-linking agent, initiating the reaction between the prepolymerization system and the cross-linking agent in a light initiation mode, a heat initiation mode or a free radical initiation mode, and curing to form the polymer. Wherein, the monomer in the prepolymerization system mainly comprises one or more than two of acrylamide, N-dimethylacrylamide, N-methylene bisacrylamide, N-isopropylacrylamide, N-methylolacrylamide and N-tert-butylacrylamide; the solvent for dissolving or dispersing the monomer comprises one or more than two of water, phosphate buffer solution, sodium chloride solution, glucose solution and ethanol; the mass concentration range of the solution formed by dissolving or dispersing the functional monomer in the solvent is 10-60%.

(4) After the pre-polymerization system and the cross-linking agent are cured, the matrix material is separated from the imprinting layer. Then, one or more than two of dithiothreitol solution, mercaptoethanol solution and tris (2-carboxyethyl) phosphine solution with the concentration of 1mM-100mM are used for completely removing the template cells on the surface of the imprinting layer, and simultaneously, the disulfide bonds in the material are reduced to sulfhydryl groups.

(5) The reaction between mercapto group on the material and one or more of aptamer, antibody, polypeptide or boric acid compound capable of being specifically combined with tumor cell is utilized to introduce one or more acting forces into the blotting material, so as to obtain the multiple-acting blotting material for specifically recognizing tumor cell. Wherein the modified aptamer is specifically one or more of ZY sls, Anti-EGFR, SYL3C, S6, A9, A10, YJ-1, APTmac, TD05, TE02, Sgc8, Sgd5, KDED2a-3, KCHA10 and KH1C12, the tail end of which is modified with one or more of maleimide and double bond groups; the modified antibody is specifically one or more than two of Anti-EGFR, Anti-EpCAM, Anti-VEGF, Anti-CTLA4, Anti-CD20, Anti-CD52, Anti-CD30, Anti-CD33 and Anti-HER2 modified with one or more than two of maleimide and double bond groups; the modified polypeptide is specifically one or more of RGD modified with maleimide and double bond group, polymyxin, bacitracin, LTX-302, Viphi A-H, Cr-ACP1 and thymopentin; the alkenyl boric acid compound is one or more of vinyl benzene boric acid, acrylamide benzene boric acid and benzene boric acid containing alkylene. The solvent for dissolving the modified aptamer, the modified antibody and the modified polypeptide or the alkenyl boronic acid compound mainly comprises one or more than two of water, phosphate buffer solution and sodium chloride solution, and the mass concentration range of the formed solution is 5-95%.

(6) Non-imprinted material (NIP) was prepared by following the above-mentioned steps from (1) to (5) without attaching tumor cells to the matrix material.

(7) The cell imprinting material is used for selective recognition, capture, release and enrichment of cells in blood, body fluid and tissue samples in the fields of biological analysis, biochemical engineering, biomedicine and biotechnology.

The multiple actions mainly comprise one or more than two of shape complementary action force generated by cell imprinting, binding force of aptamer and tumor cells, binding force of antibody and tumor cells, binding force of polypeptide and tumor cells, and binding force of phenylboronic acid and tumor cells.

The invention relates to a cell imprinting material based on multiple actions between template cells and imprinting sites, which can specifically identify, capture, release and enrich target cells.

When the template cells are contacted with the prepolymerization system, action sites can be formed, and by memorizing the interaction between the template cells and the prepolymerization system in the solidification process of the prepolymerization system, after the template cells are removed, imprinting sites which interact with the template cells can be formed on the surface of the material; complementary affinity between the template cell and the imprinting site, expressed by spatial configuration and hydrogen bonding, electrostatic interaction and van der waals interaction, on spatial structure, size, shape and functional group; then introducing an aptamer, an antibody, a polypeptide and a phenylboronic acid compound which have the binding capacity to the tumor cells into the imprinted site to obtain additional chemical specific binding force.

The invention has the following advantages:

(1) the invention adopts the cell imprinting technology and the post-imprinting modification technology to prepare the cell imprinting material with multiple functions on the tumor cells, thereby improving the purity and the sensitivity of the separation and the enrichment of the tumor cells;

(2) the blotting material of the present invention has multiple functions for recognizing target cells. The cell imprinting technology provides space complementation, hydrogen bond and electrostatic acting force between tumor cells and imprinting sites; the post-imprinting modification technology provides stronger chemical affinity specificity acting force;

(3) the polymer material used in the invention has good biocompatibility and stability, is beneficial to separating and enriching living tumor cells, and provides samples for further analyzing the tumor cells;

(4) the preparation process of the imprinting material is simple, easy to operate and convenient for large-scale production.

Description of the drawings:

FIG. 1: photomicrographs of SMMC-7721 cells attached to the surface of a cell culture plate.

FIG. 2: the structural formula of the cross-linking agent.

FIG. 3: photomicrographs of complexes formed between the crosslinker and SMMC-7721 cells.

FIG. 4: blotting of SMMC-7721 cells on the surface of the layer.

FIG. 5: the resulting cell-imprinted material (MIP1) was prepared using SMMC-7721 cells as a template.

FIG. 6: bright-field and dark-field micrographs of tumor cells trapped in blood.

Detailed Description

Example 1

Preparation of a cellular blotting Material (MIP1) based on SMMC-7721 cell template and ZY sls aptamer

SMMC-7721 cells were cultured in a cell culture dish (diameter: 10 cm), and when about 80% of the bottom of the culture plate was filled (FIG. 1), the cells were washed with phosphate buffered saline (pH 7.2, PBS), and then 1% by mass of a cross-linking agent (FIG. 2a) having a disulfide bond in the middle and sodium succinimidyl sulfonate and a double bond group at both ends, respectively, was added thereto, and after incubating with tumor cells for 3 hours, excess cross-linking agent was removed by washing with PBS, to obtain a complex of the cells and the cross-linking agent (FIG. 3). Then, 8 ml of a prepolymerization aqueous solution consisting of 10% by mass of acrylamide and 1% by mass of methylenebisacrylamide was added to the cell culture dish, and then 1. mu.l of tetramethylethylenediamine and 10. mu.l of an aqueous solution of ammonium persulfate having a mass concentration of 10% were added to initiate the solidification reaction of the prepolymerization solution. After 40 minutes, the solidified blotting layer was separated from the matrix material and turned over, with SMMC-7721 cells on the surface of the blotting layer (fig. 4). Then, a 1mM tris (2-carboxyethyl) phosphine solution was added, incubated at 55 ℃ for 1 hour to sufficiently reduce disulfide bonds to thiol groups and remove the template cells, and then the tris (2-carboxyethyl) phosphine solution was removed with PBS. And finally, adding 5% mass concentration ZY sl nucleic acid aptamer solution with double bonds modified at the tail end to react with the sulfydryl on the surface of the material to obtain the cell imprinting material (MIP1) with multiple specific acting forces on SMMC-7721 cells (figure 5).

Non-blotted material (NIP) was prepared in the same manner in a cell culture dish in the absence of SMMC-7721 cells.

Example 2

The cell blotting material (MIP1) is used for identifying and capturing SMMC-7721 cells

Two cell concentrations were 1X 10⁵cells/ml of SMMC-7721 cells are respectively inoculated on the imprinted surface of MIP1 and the surface of NIP, and after 3 hours, the cells are attached to the imprinted sites on the surface of MIP1, but not the imprinted sites and the surface of NIP are not attached with the SMMC-7721 cells, so that the imprinted sites of MIP1 can rapidly, highly-corrected and sensitively identify and capture target cells through multiple actions with the SMMC-7721 cells. Further adding SMMC-7721 cells to human blood samples, MIP1 also achieved specific recognition and enrichment of SMMC-7721 cells from blood as described above (fig. 6).

Example 3

Preparation of aptamer cellular imprinting material (MIP2) based on SMMC-7721 cell template and SYL3C

SMMC-7721 cells were cultured in a cell culture dish (diameter: 10 cm), and when about 80% of the bottom of the plate was filled, the cells were washed with phosphate buffered saline (pH 7.2, PBS), and then 15% by mass of a cross-linking agent (FIG. 2b) having a disulfide bond in the middle and maleimide and double bond groups at both ends, respectively, was added thereto, and after incubating with tumor cells for 1 hour, excess cross-linking agent was removed by washing with PBS, to obtain a complex of the cells and the cross-linking agent. Then, 8 ml of a prepolymerization aqueous solution consisting of 30% by mass of acrylamide and 1% by mass of methylenebisacrylamide was added to the cell culture dish, and then 1. mu.l of tetramethylethylenediamine and 10. mu.l of a 10% by mass aqueous solution of ammonium persulfate were added to initiate the solidification reaction of the prepolymerization solution. After 20 minutes, the solidified blotting layer was separated from the matrix material and inverted, with SMMC-7721 cells present on the surface of the blotting layer. Then, a solution of dithiothreitol having a concentration of 20mM was added, incubated at 55 ℃ for 1 hour, the disulfide bonds were sufficiently reduced to thiol groups and the template cells were removed, and then the solution of dithiothreitol was removed with PBS. And finally, adding a SYL3C aptamer solution with a double bond modified at the tail end and the concentration of 40% to react with the sulfydryl on the surface of the material to obtain the cell imprinting material (MIP2) with multiple specific acting forces on SMMC-7721 cells.

Example 4

Preparation of cell blotting material (MIP3) based on Hela cell template and Anti-EGFR antibody

Hela cells were cultured in a cell culture dish (diameter: 10 cm), and when about 80% of the bottom of the culture plate was filled, the cells were washed with phosphate buffered saline (pH 7.2, PBS), and then 30% by mass of a cross-linking agent (FIG. 2c) having a disulfide bond in the middle and maleimide and thiol groups at both ends, respectively, was added, and after incubating with tumor cells for 30 minutes, excess cross-linking agent was removed by washing with PBS, to obtain a complex of cells and cross-linking agent. Then, 8 ml of a prepolymerization aqueous solution consisting of 60% by mass of acrylamide and 1% by mass of methylenebisacrylamide was added to the cell culture dish, and then 1. mu.l of tetramethylethylenediamine and 10. mu.l of a 10% by mass aqueous solution of ammonium persulfate were added to initiate the solidification reaction of the prepolymerization solution. After 20 minutes, the solidified blotting layer was separated from the matrix material and turned over, with Hela cells present on the surface of the blotting layer. Then, a solution of tris (2-carboxyethyl) phosphine at a concentration of 50mM was added, incubated at 55 ℃ for 1 hour to sufficiently reduce disulfide bonds to thiol groups and remove the template cells, and then the solution of tris (2-carboxyethyl) phosphine was removed with PBS. And finally, adding 70% of Anti-EGFR antibody modified with double bonds to react with sulfydryl on the surface of the material to obtain the cell imprinting material (MIP3) with multiple specific acting forces on Hela cells.

Example 5

Preparation of a cell-imprinted material (MIP4) based on a crosslinker comprising succinimide and RGD polypeptide

SMMC-7721 cells were immobilized on the surface of polylysine-modified glass material. When about 80% of the bottom of the plate was filled, the cells were washed with phosphate buffered saline (pH 7.2, PBS), and then 50% by mass of a cross-linking agent having a disulfide bond in the middle and succinimide and thiol groups at both ends (FIG. 2d) was added, and after incubating with the tumor cells for 30 minutes, excess cross-linking agent was removed by washing with PBS, to obtain a complex of the cells and the cross-linking agent. And simultaneously stirring the tetrahydrofuran solution of 4, 4' -methylene bis (phenyl isocyanate), bisphenol A and phloroglucinol with the mass concentration of 60% at the temperature of 65 ℃ until the solution reaches a gel point, lightly pressing the matrix material fixed with the cells on the surface of the gel solution, and removing the matrix material and the template cells after the matrix material is solidified. Then, a mercaptoethanol solution having a concentration of 100mM was added, incubated at 55 ℃ for 1 hour, the disulfide bonds were sufficiently reduced to the thiol groups and the template cells were removed, and then the mercaptoethanol solution was removed with PBS. And finally, adding 90% of RGD polypeptide modified with maleimide to react with sulfydryl on the surface of the material to obtain the cell imprinting material (MIP4) with multiple specific acting forces on SMMC-7721 cells.

Example 6

Preparation of a plasma-treated Metal Material and acrylamidophenylboronic acid-based cytoblot Material (MIP5)

SMMC-7721 cells are fixed on the surface of the titanium alloy material treated by the plasma, when about 80% of the cells are fully paved, the cells are washed by phosphate buffer (pH 7.2, PBS), then 35% of cross-linking agent with disulfide bond in the middle and succinimide group and maleimide group at two ends is added by mass concentration (figure 2e), and after incubation for 30 minutes with tumor cells, excess cross-linking agent is removed by washing with PBS, and a compound formed by the cells and the cross-linking agent is obtained. Simultaneously adding a solution of isopropyl acrylamide with the mass concentration of 55% and acrylamide with the mass concentration of 1% to the surface of the compound, and then adding 1 microliter of tetramethyl ethylene diamine and 10 microliter of ammonium persulfate aqueous solution with the mass concentration of 10% to initiate the curing reaction of the prepolymerization solution. After 20 minutes, the solidified blotting layer was separated from the matrix material and inverted, with SMMC-7721 cells present on the surface of the blotting layer. Then, a solution of tris (2-carboxyethyl) phosphine at a concentration of 50mM was added, incubated at 55 ℃ for 1 hour to sufficiently reduce disulfide bonds to thiol groups and remove the template cells, and then the solution of tris (2-carboxyethyl) phosphine was removed with PBS. And finally, adding acrylamide phenylboronic acid with the concentration of 95% to react with sulfydryl on the surface of the material to obtain the cell imprinting material (MIP5) with multiple specific acting forces on SMMC-7721 cells.

Example 7

Preparation of Anti-EpCAM antibody-based MCF-7 cell blotting Material (MIP6)

MCF-7 cells are cultured in a cell culture dish (diameter is 10 cm), when about 80% of the bottom of the culture plate is fully paved, the cells are washed by phosphate buffer (pH 7.2, PBS), then 20% of cross-linking agent (figure 2a) with disulfide bond in the middle and sodium succinimide sulfonate and double bond groups at two ends is added, after 3 hours of incubation with tumor cells, excess cross-linking agent is removed by PBS washing, and a compound formed by the cells and the cross-linking agent is obtained. Then, 8 ml of a prepolymerization aqueous solution consisting of 30% by mass of acrylamide and 1% by mass of methylenebisacrylamide was added to the cell culture dish, and then 1. mu.l of tetramethylethylenediamine and 10. mu.l of a 10% by mass aqueous solution of ammonium persulfate were added to initiate the solidification reaction of the prepolymerization solution. After 20 minutes, the print layer formed by curing was separated from the matrix material and the print layer was inverted. Then, a solution of tris (2-carboxyethyl) phosphine at a concentration of 50mM was added, incubated at 56 ℃ for 1 hour to sufficiently reduce disulfide bonds to thiol groups and remove the template cells, and then the solution of tris (2-carboxyethyl) phosphine was removed with PBS. And finally, adding 30% double-bond-modified Anti-EpCAM antibody solution to react with sulfydryl on the surface of the material to obtain the cell imprinting material (MIP6) with multiple specific acting forces on the MCF-7 cells.

Claims

1. A multiplex imprinting material for specifically identifying tumor cells, characterized in that:

It can be prepared according to the following process. The tumor cell to be specifically recognized is used as a template, and it is attached or fixed on the surface of the matrix material. The cross-linking agent composed of groups that can participate in the polymerization of the material binds the cells. After removing the unbound cross-linking agent, the complex formed by the cross-linking agent and the template cells is placed in the pre-polymerization system. By initiating the polymerization of the pre-polymerization system, the excess is removed. After the solvent, the matrix material is separated and removed, and the disulfide bond in the cross-linking agent is reduced to sulfhydryl to remove the template cells on the surface of the polymer after curing. The reaction between one or more of the nucleic acid aptamers, modified antibodies, modified polypeptides or alkenyl phenylboronic acid compounds to obtain a multiple-action imprinting material for specific recognition of tumor cells ;Crosslinking agent mainly refers to a molecule composed of a disulfide bond in the middle, one end is a chemical group that can be combined with tumor cells, and the other end is a chemical group that can participate in the polymerization of materials; The chemical group that can be combined with tumor cells mainly It is composed of one of carboxyl group, maleimide group and succinimide group; the chemical groups that can participate in the polymerization of materials are mainly composed of olefin group, sulfhydryl group, maleimide group and succinimide group A composition in the group; the solvent for dissolving or dispersing the cross-linking agent includes one or more of water, phosphate buffer, sodium chloride solution, dimethyl sulfoxide, and dimethyl formamide ;

In a solution formed by dissolving the cross-linking agent in one or more of water, phosphate buffer, and sodium chloride solution, the total molar concentration of other solutes other than water ranges from 50 mM to 200 mM; The crosslinking agent insoluble in one or more of water, phosphate buffer, and sodium chloride solution is first dispersed in one or more of water, phosphate buffer, and sodium chloride solution , and then add one or more of dimethyl sulfoxide and dimethyl formamide to dissolve the cross-linking agent to form a solution, wherein the volume concentration range of dimethyl sulfoxide and dimethyl formamide is 0.5% -10%;

The cross-linking agent is composed of one or more of the following molecules:

, where _R1 is

,

,

one of ; R ₂ is

,

,

,

,

,

one of; R ₃ is H and

One of; n is the length of the alkane group in brackets, ranging from 2-6; m is the length of the alkane group in brackets, ranging from 2-6.

2. The multiplexed imprinted material of claim 1, wherein:

The matrix material refers to one or more of cell culture plates, cell culture dishes, cell culture flasks, titanium alloys, cobalt-based alloys, stainless steel materials and polylysine-modified glass materials to which tumor cells can adhere.

3. The multiplexed imprinted material of claim 1, wherein:

The prepolymerization system is a solution composed of monomers that can be copolymerized with a crosslinking agent to form a polymer, wherein the monomers in the prepolymerization system are mainly composed of acrylamide, N,N-dimethylacrylamide, N,N-methylene One or two or more of bisacrylamide, N-isopropylacrylamide, N-methylolacrylamide and N-tert-butylacrylamide; solvents for dissolving or dispersing monomers include water, One or more of phosphate buffer, sodium chloride solution, glucose solution and ethanol; the mass concentration range of the solution formed by dissolving or dispersing the functional monomer in the solvent is 10%-60%;

The method for initiating the curing of the prepolymerization system includes one or more of photoinitiation, thermal initiation, electric field treatment and free radical initiation; the prepolymerization system composed of the polymerizable monomer is added to the surface of the template cell to initiate the polymerization.

4. The multiplexed imprinted material of claim 1, wherein:

The reagent for reducing the disulfide bond is one or more of dithiothreitol solution, mercaptoethanol and tris(2-carboxyethyl)phosphine solution.

5. The multiplexed imprinted material of claim 1, wherein:

The modified nucleic acid aptamer, modified antibody, modified polypeptide or alkenyl boronic acid compound that can chemically and specifically bind to tumor cells are,

Modified nucleic acid aptamers refer to oligonucleotide fragments that target tumor cells and are screened by exponentially enriched ligand system evolution technology. The fragments can specifically bind to tumor cells, and then modified at their ends. One or more of leimide and double bond groups; modified antibody refers to an immune protein that can specifically bind to tumor cell surface antigens, and then maleimide and maleimide are modified on the immune protein. One or more than two double bond groups; modified polypeptides refer to protein structure and functional fragments that can specifically bind to tumor cell surface antigens, and then modify maleimide and double bond groups on them. One or more of these; alkenyl boronic acid compounds refer to boronic acids containing alkenyl groups that can form stable complexes with polysaccharide compounds on the surface of tumor cells; dissolve modified nucleic acid aptamers, modified The solvent of the antibody, modified polypeptide or alkenyl boronic acid compound is mainly composed of one or more of water, phosphate buffer and sodium chloride solution, and the mass concentration range of the formed solution is 5%-95% .

6. The multiple-action imprinting material according to claim 3, wherein the solution consisting of monomers capable of copolymerizing with a crosslinking agent to form a polymer is,

Monomers that can polymerize to form polymers, including acrylamide, N,N-dimethylacrylamide, N,N-methylenebisacrylamide, N-isopropylacrylamide, N-methylol acrylamide One or more of N-tert-butylacrylamide; solvents used to dissolve or disperse monomers or polymers include one or more of water, phosphate buffer and sodium chloride solution ; The mass concentration range of the solution formed by dissolving or dispersing the functional monomer or polymer in the solvent is 10%-60%.

7. The multiplexed imprinted material of claim 1, wherein:

The modified nucleic acid aptamer is specifically ZY sls, Anti-EGFR, SYL3C, S6, A9, A10, YJ-1, ZY sls, Anti-EGFR, SYL3C, S6, A9, A10, YJ-1, One or more of APTmuc, TD05, TE02, Sgc8, Sgd5, KDED2a-3, KCHA10, KH1C12; corresponding tumor cells that can be captured refer to liver cancer cells, colorectal cancer cells, glioblastoma cells, adenocarcinoma cells One or more of cancer cells, lymphoma cells, leukemia cells, and lung cancer cells;

The modified antibody is specifically Anti-EGFR, Anti-EpCAM, Anti-VEGF, Anti-CTLA4, Anti-CD20, Anti-CD52 modified with one or more of maleimide and double bond groups One or more of Anti-CD30, Anti-CD33, Anti-HER2; the corresponding tumor cells that can be captured refer to liver cancer cells, colorectal cancer cells, glioblastoma cells, adenocarcinoma cells, lymphoma cells One or more of cells, leukemia cells and lung cancer cells;

The modified polypeptide is specifically RGD, polymyxin, bacitracin, LTX-302, Viphi A-H, Cr-ACP1 and thymopenta modified with one or more of maleimide and double bond groups One or more of the peptides; the corresponding tumor cells that can be captured refer to one of liver cancer cells, colorectal cancer cells, glioblastoma cells, adenocarcinoma cells, lymphoma cells, leukemia cells, and lung cancer cells or two or more;

The alkenyl boronic acid compound is specifically one or more than two of vinylbenzene boronic acid, acrylamido phenyl boronic acid and phenyl boronic acid containing an alkene group;

Correspondingly captured tumor cells refer to one or more of liver cancer cells, colorectal cancer cells, glioblastoma cells, adenocarcinoma cells, lymphoma cells, leukemia cells, and lung cancer cells.

8. The multiple-action imprinting material for specifically recognizing tumor cells according to any one of claims 1-7, which is used for one or more than two kinds of blood in the fields of bioanalysis, biochemical engineering, biomedicine and biotechnology. One or more of selective recognition, capture, release and enrichment of one or more cells in body fluids and tissue samples.