CN106633167A - Blotting material used for specific recognition of cells, and preparation and applications thereof - Google Patents
Blotting material used for specific recognition of cells, and preparation and applications thereof Download PDFInfo
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Abstract
The invention relates to a blotting material used for specific recognition of cells, and preparation and applications thereof. According to the blotting material, cells are taken as a template, cell culturing and cell immobilization technology are adopted to immobilize the cells on the surface of a substrate material, curing of a pre-polymerization system is carried out on the surface of the substrate material, the substrate material is removed via separation, and the template cells on the surface of an obtained cured polymer are removed so as to obtain a finished product. The blotting material is high in selectivity and stability, and can be used in identification, capturing, and enriching of cells.
Description
Technical field
The invention belongs to functional biological materials, and its technology of preparing and its cell recognition, capture,
Release and the application in being enriched with, specifically it is a kind of it is new can be with the trace of specific recognition cell
Material, and its prepare and apply.
Background technology
The high flux and high-sensitivity detection of cell is in diagnosing tumor and treatment, environment measuring and biological biography
Have a very important role in sensor research (Aherne A.et al., J.Am.Chem.Soc., 1996,
118,8771-8772).At present, in the relatively low environment of cell concentration, the detection of cell is very difficult,
Its main cause is that cell content is less, it is impossible to obtained.Therefore, realize thin in low cell concentration environment
Target is recognized and is enriched with out by premise is that for born of the same parents' detection from environment.
Cell recognition is that one kind can be with specific recognition and capture mesh with enrichment of cell identification and beneficiation technologies
The technology of mark cell.At present, identification mainly has two types with the technology of enrichment target cell, a kind of
It is based on the technology of immunity principle.The technology on the one hand using the antibody recognition combined with target cell and
Enrichment of cell, on the other hand utilizes the antibody combined with interference cell to remove untargeted cells, between reaching
Connect identification and be enriched with the purpose of target cell.Another kind of technology is based on the skill of target cell physical property
Art, including the size of cell, density, deformability and charging property etc..The technology utilizes target cell
Identification is reached with difference of the interference cell in terms of above-mentioned physical property and be enriched with the purpose of target cell.
However, due to the uncertainty of cell surface antigen expression, it is impossible to obtain the antibody for important cells,
So as to cause the technology based on immunity principle effectively to play its effect.Even if in addition, same thin
At different conditions its physical property is also not quite similar born of the same parents, so based on the thin of physical property technology acquisition
Born of the same parents often purity and sensitivity it is poor (Catherine Alix-Panabieres et al, Nat.Rev.Cancer,
2014,14,623-631). therefore, exploitation one kind can either substitute antibody, again can specific recognition and
The material of enrichment of cell just seems particularly significant.
Molecular imprinting be one based on molecular recognition theory, analog antibody-AI
Mode, the new skill of the molecular recognition material with ad hoc structure and function by engineer and synthesis
Art.The molecular engram material prepared using the technology is realized to the special of the target molecule in complex environment
Property identification and be enriched with.Wherein, cell blots technology is a kind of emerging molecular engram with cell as template
Technology, cell blots material prepared therefrom, not only can essence used as a kind of substitute of natural antibody
The really specific recognition capability of simulation natural antibody, and, recycling higher with environmental resistance
The advantage such as rate is higher and suitable scale is combined to (Schirhagl, R.et al, Anal.Chem., 2014,86,
250-261)。
Therefore, on the basis of cell blots technology, we with cell as template, initially with cell
Culture is fixed on template cell on host material with cell technique for fixing, secondly causes prepolymerization system
Imprinted layer is obtained in the solidification of host surface, host material and imprinted layer is then peeled off, most
The template cell of trace layer surface is removed afterwards.After template cell is removed, material surface will be formed with
There are the imprinted sites of multiple action in template cell.So as to obtain with specific recognition and enrichment mesh
The imprinted material of mark cell ability, and realize identification, capture, release and the enrichment of cell.
The content of the invention
To treat that the cell of specific recognition, as template, is consolidated using cell culture with cell technique for fixing
On host material, by solidifying polymerization prepolymerization System forming imprinted layer in host surface,
Imprinted layer and host material are separated, the template cell of trace layer surface is then removed, obtaining one kind can
The imprinted material of specific recognition template cell, and by the materials application in the identification of cell, capture,
In release and enrichment process.For achieving the above object, the technical solution used in the present invention is：
(1) using physical treatment or chemical modification method transformation host surface so as to beneficial to cell
Fix on its surface.Wherein, host material includes Tissue Culture Flask, Tissue Culture Plate, cell culture
One or two or more kinds in ware, slide, cover glass, the matrix of host material is type siloxane base
Matter, polystyrene type matrix and metal matrix.The method of modifying of host surface include physics coating,
One or two or more kinds of Surface heat-treatent, mechanical grinding and plasma surface treatment；And by energy
With template cell occur interact antibody, aptamer, peptide and protein in one kind or
It is immobilized on host surface for more than two kinds.
(2) be not required to process or processed host surface using growth attaching, physical absorption,
The mode fixed form cell such as immunization.Template cell refer to zooblast, plant cell, fungi,
Bacterium and spore.
(3) subsequently prepolymerization system is added to the host surface for being fixed with cell, with it is light-initiated,
The mode such as thermal initiation or free radical initiation causes prepolymerization system to solidify.Wherein, in prepolymerization system
Monomer is acrylamide, N, N- DMAAs, N,N methylene bis acrylamide, N- isopropyls
Base acrylamide, N hydroxymethyl acrylamide, N tert butyl acrylamide, Graphene doping acryloyl
Amine, Graphene doping N, N- DMAAs, Graphene doping N,N methylene bis acrylamide,
Graphene doping NIPA, Graphene doping N hydroxymethyl acrylamide, Graphene are mixed
Miscellaneous N tert butyl acrylamide, methylchlorosilane, phenyl chlorosilane, methylvinyl-chlorosilanes, second
Base trichlorosilane, propyltrichlorosilan, vinyl trichlorosilane, γ-chloropropyl trichloro-silane and fluorine silicon
Monomer, dimethyl silicone polymer, dopamine, allyl amine, ethene, propylene, butadiene, benzene second
Alkene, 4-vinylpridine, itaconic acid, acrylic acid, PAA, methacrylic acid, methacrylic acid
Methyl esters, to styrene, ethyleneglycol dimethacrylate methyl esters, chlorostyrene, 2- (trifluoromethyl) third
In olefin(e) acid, acrylonitrile, vinyl alcohol, vinyl acetate, methacrylaldehyde, polyurethanes and 2 hydroxy propanoic acid
One or two or more kinds；The polymer of macromolecular material can be formed by self assembly mode, including poly-
One or two or more kinds in sulfone, polyether sulfone and polyarylsulfone (PAS)；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, dichloromethane, acetonitrile, ethyl acetate, acetone, n-hexane,
Hexamethylene, benzene, acetic acid, methyl phenyl ethers anisole, dimethyl sulfoxide (DMSO), bromobenzene, carbon dichloride, carbon tetrachloride,
One or two or more kinds in N,N-dimethylformamide and trifluoroacetic acid；By function monomer or polymer
The mass concentration scope for being dissolved or dispersed in the solution formed in solvent is 5%-90%.
(4) after the solidification of prepolymerization system, host material is separated with imprinted layer.Then digested with pancreatin
Method or solvent extraction etc. thoroughly remove the template cell of trace layer surface, until can't detect template
The presence of cell, and then it is identified the imprinted material (MIP) with enrichment of cell.
(5) the not immobilized template cell on host material, the step of according to above-mentioned from (1) to (4)
Prepare non-imprinted material (NIP).
(6) the cell blots material is used for into bioanalysis, biochemical industry, biomedicine and biological skill
The Selective recognition of art field inner cell, capture, release and it is enriched with.
The present invention is a kind of cell blots material of the multiple action based between template cell and imprinted sites
Material, the material can carry out specific recognition, capture, release to target cell and be enriched with.
Multiple action position can be formed when template cell is contacted with prepolymerization system, by prepolymerization
Multiple action therebetween is remembered in the solidification process of system, after template cell is removed, material list
Face will form the imprinted sites that multiple action occurs with template cell.
Between template cell and imprinted sites in space structure, size, shape and functional group, pass through
The complementary affinity that steric configuration and hydrogen bond action, electrostatic interaction and model ylid bloom action show.
The invention has the advantages that：
(1) present invention utilizes cell blots technology to prepare the print that can be used for specific recognition cell
Mark material.There are the imprinted sites of multiple action with cell in what is formed using imprinted material surface, improve
The purity and sensitivity of cell recognition and enrichment, nonspecific action is dry when reducing enrichment of cell
Disturb.
(2) identification of the imprinted material to target cell in the present invention is multiple action, including space structure
Type and hydrogen bond action, electrostatic interaction and model ylid bloom action, to target cell performance it is higher go out complementation
Affinity.
(3) in the present invention host material used and prepolymerization system have good bio-compatibility and
Stability, beneficial to the activity and structure and reuse that keep cell, and then is conducive to recognizing mesh again
Mark cell.
(4) preparation flow of cell blots material is simple in the present invention, it is easy to operate.Compared to other
Cell recognition and enrichment method, using the material identification process can be simplified, and improve recognition efficiency, application
Scope is wider.
Description of the drawings
Fig. 1：Attaching design sketch of the SMMC-7721 cells in cell culture plate surface；
Fig. 2：The SMMC-7721 cells of trace layer surface；
Fig. 3：With the cell blots material (MIP1) that SMMC-7721 cells are prepared as template；
Fig. 4：MIP1 and NIP identifications and the design sketch for capturing SMMC-7721 cells；
Fig. 5：With the cell blots material (MIP2) that MCF-7 cells are prepared as template.
Specific embodiment
Embodiment 1
Preparation based on the cell blots material (MIP1) of SMMC-7721 cell shutterings
By SMMC-7721 cell culture in 12 orifice plate host surfaces, when about 80% training
When foster plate bottom is paved with (Fig. 1), cleaned carefully with phosphate buffer (PBS) solution of PH=7.2
Born of the same parents, are subsequently adding the paraformaldehyde solution that mass concentration is 4% and fix cell, after 5 minutes, use PH=7
PBS solution cleaning remove paraformaldehyde solution.Then 1 milliliter is added to the every hole in 12 orifice plates
The methylene-bisacrylamide that by mass concentration be 30% acrylamide and mass concentration is 1% is constituted
The prepolymerization aqueous solution, it is 10% then to add 1 microlitre of tetramethylethylenediamine and 10 microlitres of mass concentrations
Ammonium persulfate aqueous solution cause pre-polymer solution curing reaction.After 20 minutes, by what is solidify to form
Imprinted layer is separated with host material and overturns imprinted layer, has SMMC-7721 cells in trace layer surface
Exist (Fig. 2).Imprinted layer is soaked in the trypsin solution of mass concentration 0.25% repeatedly, until adopting
Exist less than cell in imprinted layer Surface testing with DAPI decoration methods, with PBS solution by trypsin solution
The cell blots material (Fig. 3) for specific recognition SMMC-7721 cell is obtained after cleaning.
As shown in figure 3, on imprinted material surface with the presence of abundant traces site, these imprinted sites are by profit
With the complementary affinity interaction shown in space structure, size, shape and functional group with target cell,
Reach quick identification and capture the purpose of target cell.
Using identical method, in 12 orifice plates that there is no SMMC-7721 cells, prepare
Non-imprinted material (NIP).
Embodiment 2
Cell blots material (MIP1) is used to recognizing and capturing SMMC-7721 cells
It is 1 × 10 by two parts of cell concentrations6The SMMC-7721 cells of cells/ml are seeded to respectively
MIP1 traces face and NIP surfaces, after 2 hours, imprinted sites of the cell attachment on MIP1 surfaces
Place, rather than attaching (Fig. 4) of at the imprinted sites and NIP surfaces without SMMC-7721 cells, explanation
The imprinted sites of MIP1 by with SMMC-7721 cells occur multiple action, can quickly, colleges and universities
With high-sensitive identification and capture target cell.Further SMMC-7721 cells are added into human body blood
In liquid sample, according to the method described above, MIP1 also achieves the identification and enrichment of the specificity from blood
SMMC-7721 cells.
Embodiment 3
Preparation based on the cell blots material (MIP2) of MCF-7 cell shutterings
With embodiment 1, having been prepared as template with MCF-7 cells can be with specific recognition and capture
The imprinted material (MIP2) of MCF-7 cells, as shown in Figure 5.
Embodiment 4
The Bacillus coli cells imprinted material (MIP3) of prepolymerization system of being adulterated based on light-initiated Graphene
Preparation
Escherichia coli are cultivated on culture dish surface, when 80% culture dish bottom is paved with by Escherichia coli
Afterwards, addition is water-soluble doped with the prepolymerization that the acrylamide and methylene-bisacrylamide of Graphene are constituted
Liquid, using the curing reaction of ultraviolet light-initiated pre-polymers system, with containing acetic acid and lauryl sodium sulfate
(SDS) the aqueous solution fully rinses imprinted layer to remove the Escherichia coli on surface, the trace for preparing
Material (MIP3) not only can specific recognition and capture Escherichia coli, can be near infrared light
Irradiation is lower to kill Escherichia coli, plays the effect of sterilizing.
Embodiment 5
Preparation based on the spore cell imprinted material (MIP4) of urethane monomer
The spore cell of thuringiensis is fixed on into dimethyl silicone polymer (PDMS) matrix material
Material surface, while 4, the four of 4 '-di-2-ethylhexylphosphine oxide (phenyl isocyanate), bisphenol-A and phloroglucin will be mixed with
Hydrogen tetrahydrofuran solution at a temperature of 65 DEG C agitating solution to gel point, then by the above-mentioned base for being fixed with spore
Material is gently pressed on gel solution surface, after its solidification, removes host material and template spore, system
The standby imprinted material for obtaining there is the spore of thuringiensis specific recognition and capture ability
(MIP4)。
Embodiment 6
Preparation based on the yeast cells imprinted material (MIP5) of polyether sulfone self assembly
Brewing yeast cell is fixed on slide, the dimethyl of the polyether sulfone that concentration is 15% is added
Sulfoxide solution, being then rapidly added deionized water makes polyether sulfone be self-assembled into imprinted layer, then uses SDS
Solution washes off brewing yeast cell and obtains cell blots material (MIP5).
Embodiment 7
MCF-7's cell blots materials (MIP6) of the silicone matrix material modified based on aptamers
Prepare
The aptamers of specific recognition MCF-7 cell are fixed on into PDMS host surfaces, then
After its surface adds the PBS solution of MCF-7 cells cell is fully acted on aptamers, remove not
The cell for being specifically bound.Then according to the method preparation of embodiment 1 being capable of specific recognition MCF-7
The imprinted material (MIP6) of cell.
Embodiment 8
SMMC-7721 cell blots materials (MIP7) based on the metal matrix material of plasma treatment
Preparation
Titanium alloy host surface is done into corona treatment, then fixes in SMMC-7721 cells
Host material after process, according to the method for embodiment 1, preparation being capable of specific recognition MCF-7
The imprinted material (MIP7) of cell.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510726565.2A CN106633167B (en) | 2015-10-30 | 2015-10-30 | It is a kind of for the imprinted material of specific recognition cell and its preparation and application |
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| CN107245143A (en) * | 2017-01-15 | 2017-10-13 | 北京林业大学 | A kind of preparation method of porous polyaniline material applied to hexavalent chromium removal |
| CN109959791A (en) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | It is a kind of for the multiple action imprinted material of specific recognition tumour cell and its preparation and application |
| CN110655634A (en) * | 2019-11-13 | 2020-01-07 | 万华化学集团股份有限公司 | High flame-retardant polyurethane foam composite material and high flame-retardant polyurethane foam prepared from same |
| CN111085118A (en) * | 2019-12-09 | 2020-05-01 | 太原理工大学 | Preparation method of polydopamine modified polyether sulfone imprinted composite membrane |
| CN111909407A (en) * | 2020-07-31 | 2020-11-10 | 华中科技大学 | Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof |
| KR20210055019A (en) * | 2019-11-06 | 2021-05-14 | 재단법인 아산사회복지재단 | Surface modification method for cell sheet generation with spontaneous chemical reaction |
| CN112898624A (en) * | 2019-12-04 | 2021-06-04 | 中国科学院大连化学物理研究所 | Substitutional template imprinted polymer for specifically recognizing exosome and application thereof |
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| CN107245143A (en) * | 2017-01-15 | 2017-10-13 | 北京林业大学 | A kind of preparation method of porous polyaniline material applied to hexavalent chromium removal |
| CN109959791A (en) * | 2017-12-14 | 2019-07-02 | 中国科学院大连化学物理研究所 | It is a kind of for the multiple action imprinted material of specific recognition tumour cell and its preparation and application |
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| CN110655634B (en) * | 2019-11-13 | 2022-04-19 | 万华化学集团股份有限公司 | High flame-retardant polyurethane foam composite material and high flame-retardant polyurethane foam prepared from same |
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| CN112898624A (en) * | 2019-12-04 | 2021-06-04 | 中国科学院大连化学物理研究所 | Substitutional template imprinted polymer for specifically recognizing exosome and application thereof |
| CN112898624B (en) * | 2019-12-04 | 2022-05-31 | 中国科学院大连化学物理研究所 | Alternative template imprinted polymer for specifically recognizing exosomes and application thereof |
| CN111085118A (en) * | 2019-12-09 | 2020-05-01 | 太原理工大学 | Preparation method of polydopamine modified polyether sulfone imprinted composite membrane |
| CN111085118B (en) * | 2019-12-09 | 2022-02-25 | 太原理工大学 | Preparation method of polydopamine modified polyether sulfone imprinted composite membrane |
| CN111909407B (en) * | 2020-07-31 | 2021-10-08 | 华中科技大学 | Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof |
| CN111909407A (en) * | 2020-07-31 | 2020-11-10 | 华中科技大学 | Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof |
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