CN106633167A - Blotting material used for specific recognition of cells, and preparation and applications thereof - Google Patents

Abstract

The invention relates to an imprinting material for specifically recognizing cells, as well as its preparation and application. The cell-imprinted material uses cells as templates, uses cell culture and cell fixation techniques to fix it on the surface of the matrix material, solidifies the pre-polymerization system on the surface of the matrix material, then separates and removes the matrix material, and then removes the polymer surface after solidification. Template cells are prepared. The cell imprinted material has high selectivity and high stability, and is further applied in the identification, capture and enrichment of cells.

Description

A kind of imprinting material for specific recognition of cells and its preparation and application

technical field

The invention belongs to functional biological material, its preparation technology and its application in cell identification, capture, release and enrichment, specifically a novel imprinted material which can specifically identify cells, and its preparation and application.

Background technique

High-throughput and high-sensitivity detection of cells plays a very important role in tumor diagnosis and treatment, environmental detection and biosensor research (Aherne A. et al., J.Am.Chem.Soc., 1996,118,8771- 8772). At present, the detection of cells is very difficult in an environment with low cell concentration, mainly because the cell content is small and cannot be obtained. Therefore, the premise of realizing cell detection in low cell concentration environment is to be able to identify and enrich the target from the environment.

Cell identification and enrichment Cell identification and enrichment technology is a technology that can specifically identify and capture target cells. At present, there are two main types of technologies for identifying and enriching target cells, one is based on immune principles. On the one hand, this technology uses antibodies that bind to target cells to identify and enrich cells, and on the other hand, uses antibodies that bind to interfering cells to remove non-target cells to achieve the purpose of indirect recognition and enrichment of target cells. Another technique is based on the physical properties of the target cells, including cell size, density, deformability, and chargeability. This technology utilizes the difference in the above physical properties between target cells and interfering cells to achieve the purpose of identifying and enriching target cells. However, due to the uncertainty of the expression of antigens on the cell surface, antibodies against important cells cannot be obtained, so that the technology based on the principle of immunity cannot effectively play its role. In addition, even the same cell has different physical properties under different conditions, so the purity and sensitivity of cells obtained based on physical property techniques are often poor (Catherine Alix-Panabières et al, Nat. Rev. Cancer, 2014, 14, 623- 631). Therefore, it is very important to develop a material that can not only replace antibodies, but also specifically recognize and enrich cells.

Molecular imprinting technology is a new technology based on molecular recognition theory, simulating antibody-antigen interaction, and artificially designing and synthesizing molecular recognition materials with specific structures and functions. The molecularly imprinted materials prepared by this technology realize the specific recognition and enrichment of target molecules in complex environments. Among them, cell imprinting technology is an emerging molecular imprinting technology using cells as templates. As a substitute for natural antibodies, cell imprinting materials prepared by it can not only accurately simulate the specific recognition ability of natural antibodies, but also have environmental resistance. It has the advantages of stronger acceptance, higher reuse rate and suitability for large-scale synthesis (Schirhagl, R. et al, Anal. Chem., 2014, 86, 250-261).

Therefore, on the basis of cell imprinting technology, we use cells as templates, first use cell culture and cell fixation techniques to immobilize the template cells on the matrix material, secondly initiate the curing of the pre-polymerization system on the surface of the matrix material to obtain the imprinted layer, and then The matrix material is separated from the imprinted layer, and finally the template cells on the surface of the imprinted layer are removed. When the template cells are removed, the surface of the material will form imprinted sites that have multiple interactions with the template cells. Thus, the imprinted material with the ability to specifically recognize and enrich target cells is obtained, and the recognition, capture, release and enrichment of cells are realized.

Contents of the invention

Using cells to be specifically identified as templates, cell culture and cell fixation techniques are used to immobilize them on the matrix material, and the imprinted layer is formed by solidifying the polymerization pre-polymerization system on the surface of the matrix material, separating the imprinted layer and the matrix material, and then removing the imprinted layer The template cells on the surface are obtained to obtain an imprinted material that can specifically recognize the template cells, and the material is used in the process of cell recognition, capture, release and enrichment. To achieve the above object, the technical solution adopted in the present invention is:

(1) Using physical treatment or chemical modification methods to modify the surface of the matrix material to facilitate the immobilization of cells on the surface. Wherein, the matrix material includes one or more of cell culture flasks, cell culture plates, cell culture dishes, glass slides, and cover slips, and the matrix of the matrix material is a silicone matrix, a polystyrene matrix and metal substrate. The modification method of the surface of the matrix material includes one or more of physical coating, surface heat treatment, mechanical grinding and plasma surface treatment; and antibodies, nucleic acid aptamers, polypeptides and proteins that will be able to interact with template cells One or more of them are immobilized on the surface of the matrix material.

(2) Immobilize the template cells on the surface of the untreated or treated matrix material by means of growth attachment, physical adsorption, immune function and the like. Template cells refer to animal cells, plant cells, fungi, bacteria, and spores.

(3) Subsequently, the prepolymerization system is added to the surface of the matrix material on which the cells are immobilized, and the curing of the prepolymerization system is initiated by means of photoinitiation, thermal initiation or free radical initiation. Among them, the monomers in the prepolymerization system are acrylamide, N,N-dimethylacrylamide, N,N-methylenebisacrylamide, N-isopropylacrylamide, N-methylolacrylamide, N-tert-butylacrylamide, graphene-doped acrylamide, graphene-doped N,N-dimethylacrylamide, graphene-doped N,N-methylenebisacrylamide, graphene-doped N- Isopropylacrylamide, graphene-doped N-methylolacrylamide, graphene-doped N-tert-butylacrylamide, methylchlorosilane, phenylchlorosilane, methylvinylchlorosilane, ethyl trichlorosilane Chlorosilane, Propyltrichlorosilane, Vinyltrichlorosilane, Gamma-Chloropropyltrichlorosilane and Fluorosilicone Monomer, Dimethicone, Dopamine, Allylamine, Ethylene, Propylene, Butane ethylene, styrene, 4-vinylpyridine, itaconic acid, acrylic acid, sodium acrylate, methacrylic acid, methyl methacrylate, p-styrene, ethylene glycol dimethacrylate, chlorostyrene, 2- One or more of (trifluoromethyl)acrylic acid, acrylonitrile, vinyl alcohol, vinyl acetate, acrolein, polyurethane and 2-hydroxypropionic acid; polymer materials that can be formed by self-assembly Polymers, including one or more of polysulfone, polyethersulfone and polyarylsulfone; solvents for dissolving or dispersing monomers or polymers include water, phosphate buffer, sodium chloride solution, glucose solution , methanol, ethanol, toluene, diethyl ether, petroleum ether, chloroform, dichloromethane, acetonitrile, ethyl acetate, acetone, n-hexane, cyclohexane, benzene, acetic acid, anisole, dimethyl sulfoxide, bromobenzene, One or more of carbon dichloride, carbon tetrachloride, N,N-dimethylformamide and trifluoroacetic acid; the mass of a solution formed by dissolving or dispersing functional monomers or polymers in a solvent The concentration range is 5%-90%.

(4) After the pre-polymerization system is cured, the matrix material is separated from the imprinted layer. Then, the template cells on the surface of the imprinted layer were completely removed by trypsinization or solvent extraction until no template cells were detected, and then the imprinted material (MIP) for identifying and enriching cells was obtained.

(5) No template cells are immobilized on the matrix material, and a non-imprinted material (NIP) is prepared according to the steps from (1) to (4) above.

(6) The cell imprinting material is used for selective recognition, capture, release and enrichment of cells in the fields of biological analysis, biochemical industry, biomedicine and biotechnology.

The invention is a cell imprinting material based on multiple interactions between template cells and imprinting sites, and the material can specifically recognize, capture, release and enrich target cells.

When the template cells are in contact with the pre-polymerization system, multiple interaction sites will be formed. By memorizing the multiple interactions between the two during the curing process of the pre-polymerization system, when the template cells are removed, the surface of the material will form multiple interactions with the template cells. the imprinting site.

Complementary affinity between template cells and imprinted sites in terms of spatial structure, size, shape and functional groups, through spatial configuration and hydrogen bond interactions, electrostatic interactions, and van der Waals interactions. The present invention has the following advantages:

(1) The present invention utilizes cell imprinting technology to prepare imprinted materials that can be used to specifically identify cells. Using the imprinting sites formed on the surface of the imprinting material that have multiple interactions with cells improves the purity and sensitivity of cell recognition and enrichment, and reduces the interference of non-specific effects when enriching cells.

(2) The recognition of target cells by the imprinted material in the present invention has multiple effects, including spatial configuration and hydrogen bond interaction, electrostatic interaction and Van der Waals interaction, and shows stronger complementary affinity to target cells.

(3) The matrix material and pre-polymerization system used in the present invention have good biocompatibility and stability, which is conducive to maintaining the activity and structure of cells and repeated use, which in turn is beneficial to re-identify target cells.

(4) The preparation process of the cell-imprinted material in the present invention is simple and easy to operate. Compared with other cell identification and enrichment methods, the material can simplify the identification process, improve the identification efficiency, and have a wider range of applications.

Description of drawings

Figure 1: The attachment effect of SMMC-7721 cells on the surface of the cell culture plate;

Figure 2: SMMC-7721 cells on the surface of the imprinted layer;

Figure 3: Cell imprinted material (MIP1) prepared using SMMC-7721 cells as a template;

Figure 4: Effect diagram of recognition and capture of SMMC-7721 cells by MIP1 and NIP;

Figure 5: Cell imprinted material (MIP2) prepared using MCF-7 cells as a template.

detailed description

Example 1

Preparation of cell imprinted material (MIP1) based on SMMC-7721 cell template

SMMC-7721 cells are cultured on the surface of the twelve-well plate matrix material, and when about 80% of the bottom of the culture plate is covered (Fig. 1), the cells are washed with phosphate buffered saline (PBS) solution of PH=7.2, and then The cells were fixed by adding a paraformaldehyde solution with a mass concentration of 4%, and after 5 minutes, the paraformaldehyde solution was washed with a PBS solution of pH=7 to remove the paraformaldehyde solution. Then add 1 ml of pre-polymerized aqueous solution consisting of 30% acrylamide and 1% methylenebisacrylamide to each well of the twelve-well plate, and then add 1 microliter of tetramethylethyl ether. Diamine and 10 microliters of ammonium persulfate aqueous solution with a mass concentration of 10% initiate the curing reaction of the prepolymerization solution. After 20 minutes, the solidified imprinted layer was separated from the matrix material and the imprinted layer was turned over, and SMMC-7721 cells existed on the surface of the imprinted layer ( FIG. 2 ). The imprinted layer was repeatedly soaked in trypsin solution with a mass concentration of 0.25% until no cells were detected on the surface of the imprinted layer by the DAPI staining method, and the trypsin solution was washed with PBS solution to obtain the specific identification SMMC-7721 Cell imprinted material of cells (Figure 3). As shown in Figure 3, there are a large number of imprinted sites on the surface of imprinted materials. These imprinted sites can quickly identify and capture targets by utilizing the complementary affinity with target cells in terms of spatial structure, size, shape, and functional groups. purpose of cells.

Using the same method, a non-imprinted material (NIP) was prepared in a twelve-well plate without SMMC-7721 cells.

Example 2

Imprinted material (MIP1) for identification and capture of SMMC-7721 cells

Two copies of SMMC-7721 cells with a cell concentration of 1×10 ⁶ cells/ml were inoculated on the imprinted surface of MIP1 and the surface of NIP respectively. After 2 hours, the cells attached to the imprinted site on the surface of MIP1, but not the imprinted site There was no attachment of SMMC-7721 cells on the NIP surface (Figure 4), indicating that the imprinted site of MIP1 can identify and capture target cells quickly, efficiently and sensitively through multiple interactions with SMMC-7721 cells. SMMC-7721 cells were further added to human blood samples. According to the above method, MIP1 also realized the specific recognition and enrichment of SMMC-7721 cells from blood.

Example 3

Preparation of cell-imprinted material (MIP2) based on MCF-7 cell template

As in Example 1, the imprinted material (MIP2) that can specifically recognize and capture MCF-7 cells was prepared using MCF-7 cells as a template, as shown in FIG. 5 .

Example 4

Preparation of Escherichia coli cell-imprinted material (MIP3) based on photoinitiated graphene-doped prepolymerization system

Escherichia coli is cultured on the surface of the petri dish, and when 80% of the bottom of the petri dish is covered with Escherichia coli, a prepolymerized aqueous solution composed of graphene-doped acrylamide and methylene bisacrylamide is added, and ultraviolet light is used to trigger the prepolymerization. For the curing reaction of the polymerization system, the imprinted layer was fully washed with an aqueous solution containing acetic acid and sodium dodecyl sulfate (SDS) to remove E. coli on the surface. The prepared imprinted material (MIP3) could not only specifically identify and capture E. coli, It can also kill Escherichia coli under the irradiation of near-infrared light to achieve a sterilizing effect.

Example 5

Preparation of spore cell imprinted material (MIP4) based on urethane monomer

The spore cells of Bacillus thuringiensis were immobilized on the surface of polydimethylsiloxane (PDMS) matrix material, and mixed with 4,4'-methylenebis(phenylisocyanate), bisphenol A and m-benzene The tetrahydrofuran solution of triphenols was stirred at a temperature of 65°C to the gel point, and then the above-mentioned matrix material immobilized with spores was lightly pressed on the surface of the gel solution, and after it solidified, the matrix material and template spores were removed to prepare p-thuringiensis Bacillus spores have specific recognition and capture ability of imprinted material (MIP4).

Example 6

Preparation of yeast cell imprinted material (MIP5) based on polyethersulfone self-assembly

Fix Saccharomyces cerevisiae cells on glass slides, add 15% polyethersulfone dimethyl sulfoxide solution, then quickly add deionized water to make polyethersulfone self-assemble into an imprinted layer, and then rinse with SDS solution to remove Saccharomyces cerevisiae Yeast cells were obtained as cell imprinted material (MIP5).

Example 7

Preparation of MCF-7 cell-imprinted material (MIP6) based on aptamer-modified silicone matrix material

The aptamer that specifically recognizes MCF-7 cells was immobilized on the surface of the PDMS matrix material, and then the PBS solution of MCF-7 cells was added to the surface to allow the cells to fully interact with the aptamer, and then the cells that were not specifically bound were removed. Then, according to the method of Example 1, the imprinted material (MIP6) capable of specifically recognizing MCF-7 cells was prepared.

Example 8

Preparation of SMMC-7721 cell-imprinted material (MIP7) based on plasma-treated metal matrix material

The surface of the titanium alloy matrix material was treated with plasma, and then SMMC-7721 cells were fixed on the treated matrix material. According to the method of Example 1, an imprinted material (MIP7) capable of specifically recognizing MCF-7 cells was prepared.

Claims (10)

1. a kind of imprinted material for specific recognition cell, it is characterised in that：

It can be prepared according to the following procedure, to treat the cell of specific recognition as template cell, by mould Plate cell is attached or fixed to host surface, then is placed in prepolymerization system, by base Material surface cure prepolymerization system, is then peeled off removing host material, then removes polymerization after solidification The template cell on thing surface, obtains can be used for the imprinted material of template cell-specific identification.

2. template cell as claimed in claim 1, it is characterised in that：

Template cell is the base unit that organism is constituted in biology, including zooblast, plant be thin One or two or more kinds in born of the same parents, fungi, bacterium and spore.

3. prepolymerization system as claimed in claim 1, it is characterised in that：

Prepolymerization system is by can cause polymerization or be self-assembly of the monomer or polymer group of macromolecular material Into solution.

4. as claimed in claim 3 by causing the monomer that is polymerized or is self-assembly of macromolecular material Or the solution of polymer composition, it is characterised in that：

The monomer that polymerisation forms macromolecular material, including acrylamide, N, N- dimethyl can occur Acrylamide, N,N methylene bis acrylamide, NIPA, N- methylol acryloyls Amine, N tert butyl acrylamide, Graphene doping acrylamide, Graphene doping N, N- dimethyl propylenes Acrylamide, Graphene doping N,N methylene bis acrylamide, Graphene doping N- isopropyl acrylamides Amine, Graphene doping N hydroxymethyl acrylamide, Graphene doping N tert butyl acrylamide, methyl Chlorosilane, phenyl chlorosilane, methylvinyl-chlorosilanes, ethyl trichlorosilane, propyltrichlorosilan, Vinyl trichlorosilane, γ-chloropropyl trichloro-silane and fluoropolymer emulsion, dimethyl silicone polymer, DOPA Amine, allyl amine, ethene, propylene, butadiene, styrene, 4-vinylpridine, itaconic acid, third Olefin(e) acid, PAA, methacrylic acid, methyl methacrylate, to styrene, glycol dinitrate Base methyl acrylate, chlorostyrene, 2- (trifluoromethyl) acrylic acid, acrylonitrile, vinyl alcohol, acetic acid One or two or more kinds in ethene, methacrylaldehyde, polyurethanes and 2 hydroxy propanoic acid；Can be by certainly Assembling mode forms the polymer of macromolecular material, including the one kind in polysulfones, polyether sulfone and polyarylsulfone (PAS) Or more than two kinds；For dissolving or dispersed monomer or polymer solvent include water, phosphate buffer, Sodium chloride solution, glucose solution, methyl alcohol, ethanol, toluene, ether, petroleum ether, chloroform, two Chloromethanes, acetonitrile, ethyl acetate, acetone, n-hexane, hexamethylene, benzene, acetic acid, methyl phenyl ethers anisole, Dimethyl sulfoxide (DMSO), bromobenzene, carbon dichloride, carbon tetrachloride, N,N-dimethylformamide and trifluoroacetic acid In one or two or more kinds；Formed in solvent molten is dissolved or dispersed in by function monomer or polymer The mass concentration scope of liquid is 5%-90%.

5. the method for solidifying prepolymerization system as claimed in claim 1, it is characterised in that：Can cause Prepolymerization system solidification method, including light-initiated, thermal initiation, electric field treatment, free radical cause and One or two or more kinds in exchange of solvent；By by the polymer group of the monomer or energy self assembly that can be polymerized Into prepolymerization system be added to the host surface that is fixed with template cell, cause polymerization.

6. the method for removing removing template cell as claimed in claim 1, it is characterised in that：After solidifying Polymer surface the method that removes of template cell, it is including trypsin digestion, ultraviolet or infrared Light kills one or two or more kinds in removal method and solvent extraction.

7. host material as claimed in claim 1, it is characterised in that：

The inorganic or high-molecular organic material that cell is normally attached on its surface, including Tissue Culture Flask, One or two or more kinds in Tissue Culture Plate, Tissue Culture Dish, slide, cover glass；

In untreated or processed host surface, cell by growth attaching, physical absorption, In the fixation of hydrophobe effect, electrostatic interaction, hydrogen bond action, immunization or chemical treatments one Plant or more than two kinds.

8. inorganic or high-molecular organic material as claimed in claim 7, it is characterised in that：It is inorganic or The material of high-molecular organic material is in type siloxane matrix, polystyrene type matrix and metal matrix One or two or more kinds.

9. the host material as described in claim 1 or 7, it is characterised in that：Energy before host material application Surface modification treatment is carried out, host surface is transformed by physical treatment or chemical modification method, changed The mode of making be it is following in one or two or more kinds, including physics coating, Surface heat-treatent, machinery beat Mill and plasma surface treatment and antibody, the nucleic acid that interaction will can occur with template cell One or two or more kinds in aptamers, peptide and protein is immobilized on host surface.

10. a kind of imprinted material of the arbitrary specific recognition cell of claim 1-9 is used for cell One or two or more kinds in Selective recognition, capture, release and enrichment.