CN109959791A - It is a kind of for the multiple action imprinted material of specific recognition tumour cell and its preparation and application - Google Patents
It is a kind of for the multiple action imprinted material of specific recognition tumour cell and its preparation and application Download PDFInfo
- Publication number
- CN109959791A CN109959791A CN201711337444.4A CN201711337444A CN109959791A CN 109959791 A CN109959791 A CN 109959791A CN 201711337444 A CN201711337444 A CN 201711337444A CN 109959791 A CN109959791 A CN 109959791A
- Authority
- CN
- China
- Prior art keywords
- cell
- kinds
- modification
- tumour cell
- crosslinking agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The present invention relates to a kind of material for specific recognition tumour cell and its preparations and application.The material is a kind of multiple action imprinted material: using tumour cell as template, using mainly by disulfide bond, it can be with the crosslinking agent combination cell of the group in conjunction with tumour cell and the group composition that material polymerization can be participated in, prepolymerization system is added in the composite surface that cell and crosslinking agent are formed, it crosslinks crosslinking agent with prepolymerization system and reacts to obtain cell blots material, then the disulfide bond reduction in cell blots material is removed into tumour cell at sulfydryl, it finally utilizes sulfydryl and can be specifically bound with tumour cell, one or two or more kinds of active forces are introduced into imprinted material and are prepared.The multiple action imprinted material had both had the imprinting power of imprinted material to tumour cell, was able to achieve highly selective identification, capture and the enrichment of tumour cell.
Description
Technical field
It is specifically a kind of novel to be used for specific recognition tumour cell the invention belongs to functional biological Material Field
Multiple action imprinted material, and its preparation and application.
Background technique
In blood or lymph the efficiently concentrating of tumour cell with separate, in early diagnosis of tumor and prognostic analysis, precisely
Medicine and group are learned in analysis and research and application and have a very important role (Mol Oncol2016,10,374-394.).Currently,
Since Tumor Cell Content is extremely low in blood or lymph, and the high haemocyte of content to the enrichment of tumour cell with separate
Produce huge interference, thus realize in low cell concentration environment the enrichment of tumour cell with separate very necessary (Nature
2007,450,1235–1239)。
The technology of separation at present and enriched tumor cell mainly includes immune and physical technique.Immunological technique passes through using swollen
The antigen on oncocyte surface and its corresponding antibody combine, and realize the separation and enrichment to tumour cell.But this method due to
Many tumor cell surface antigen expression are not known and changeability, cause to be easy to produce false negative result；Physical technique mainly leads to
The difference between other interference cells such as haemocyte is crossed using tumour cell in physical property, realization divides tumour cell
From with enrichment.But this method due to the difference of tumour cell and haemocyte in terms of physical property be it is diversified, because
This, the tumour cell purity and the very poor (Nat.Rev.Cancer 2014,14,623- of sensitivity for being separated and being enriched with based on the method
631.).Therefore, antibody can either be substituted by developing one kind, and the material for capableing of specific recognition and enrichment of cell just seems very
It is important.
Molecular imprinting technology is one based on molecular recognition theory, using target molecule as template, by can and mesh
Mark interaction of molecules monomer polymerization and the subsequent elution to target molecule, obtain have on space and binding site with
The polymer technology of preparing for the imprinted sites that target molecule is mutually matched.The molecular engram material energy prepared by molecular imprinting technology
Enough specific recognitions and capture realized to target molecule achieve the purpose that separation and enrichment target molecule.The technology is in environment
High-abundance proteins removal and low-abundance protein enrichment aspect achieve a series of achievements in pollutant removal, Chinese medicine separation and blood
(Prog.Polym.Sci.2014,39,145-163.).It wherein, is a kind of using cell as the cell blots technology of template and target
The new technique of natural antibody can be substituted, not only there is to target cell separation and the accumulation ability of specificity, but also is had
Environment resistance is strong, and bio-compatibility is good, is suitble to the advantage of large-scale production.
Therefore, on the basis of cell blots technology, in order to further increase the specific recognition energy of cell blots material
Power reduces the interference of non-specific adsorption, and we have proposed a kind of cell blots materials based on multiple action power.First with to
The tumour cell of identification be template, be attached or fixed to host surface, using mainly by disulfide bond, can be thin with tumour
The group and the crosslinking agent combination cell that the group of material polymerization forms can be participated in that born of the same parents combine, after removing unbonded crosslinking agent,
Crosslinking agent and the plastidogenetic compound of template are placed in prepolymerization system, by solidifying pre-polymers in host surface
Then system is separated off host material, then the disulfide bond reduction in crosslinking agent is removed solidification post-consumer polymer surface at sulfydryl
Template cell, then using sulfydryl and can with tumour cell occur chemically specifically bind through modification nucleic acid be adapted to
It is anti-between one of body, the antibody by modification, the polypeptide by modification or ene boric acid class compound or two kinds or more
It answers, one or two or more kinds of active forces is introduced into imprinted material, obtain a kind of multiple work for specific recognition tumour cell
Use imprinted material.The material had not only had the shape recognition active force of imprinted material to tumour cell, but also had chemically special knot
With joint efforts, identification, capture, release and the enrichment to target cell are realized.
Summary of the invention
Using tumour cell as template, using mainly by disulfide bond, can with the group in conjunction with tumour cell and material can be participated in
Prepolymerization system is added in the composite surface that cell and crosslinking agent are formed in the crosslinking agent combination cell of the group composition of polymerization,
It crosslinks crosslinking agent with prepolymerization system and reacts to obtain cell blots material, then by the disulfide bond in cell blots material
Sulfydryl is reduced into remove tumour cell, finally utilize sulfydryl and can be adapted to the nucleic acid that tumour cell is specifically bound
Reaction between one of body, antibody, polypeptide or boric acid compound or two kinds or more, by one or two or more kinds of active forces
It is introduced into imprinted material and is prepared.Both there is the multiple action imprinted material shape recognition of imprinted material to make to tumour cell
Firmly, and there is chemically specific bond power, is further applied in the identification, capture and enrichment of tumour cell.To realize
Above-mentioned purpose, the technical solution adopted by the present invention are as follows:
(1) tissue culture plate, Tissue Culture Dish, the metal material of corona treatment and Mercapto-group modification glass
One of material surface or two kinds or more inoculated tumour cell are cultivated 12-72 hour in cell culture medium, are obtained surface and are pasted
Host material with tumour cell.
(2) crosslinking agent is then added to tumor cell surface to be incubated for jointly -3 hours 10 minutes；Wherein, crosslinking agent be by
Disulfide bond, can with the group in conjunction with tumour cell and can participate in material polymerization group form.It wherein, can be in conjunction with tumour cell
Chemical group mainly by one of carboxyl, maleimide and succinimide or two kinds of composition described above；It can participate in material
The chemical group of polymerization is mainly by one of alkylene, sulfydryl, maleimide and succinimide or two kinds of composition described above；
For dissolving or the solvent of decentralized crosslinking agent includes one of water, phosphate buffer and sodium chloride solution or two kinds or more；
The mass concentration range that the solution formed in solvent is dissolved or dispersed in by crosslinking agent is 1%-50%.
(3) prepolymerization system is added to the compound table that tumour cell and crosslinking agent are formed after removal cross-linking agent solution
Face is caused the reaction between prepolymerization system and crosslinking agent in a manner of light-initiated, hot initiation or free radical initiation etc., is formed by curing
Polymer.Wherein, the monomer in prepolymerization system is mainly by acrylamide, N,N-DMAA, N, N- di-2-ethylhexylphosphine oxide
One of acrylamide, n-isopropyl acrylamide, N hydroxymethyl acrylamide and N tert butyl acrylamide or two kinds with
Upper composition；For dissolving or the solvent of dispersed monomer includes water, phosphate buffer, sodium chloride solution, glucose solution and second
One of alcohol or two kinds or more；The mass concentration range that the solution formed in solvent is dissolved or dispersed in by function monomer is
10%-60%.
(4) after prepolymerization system and crosslinking agent solidify, host material is separated with imprinted layer.It then is 1mM- with concentration
One of dithiothreitol (DTT) solution, mercaptoethanol and three (2- carboxyethyl) phosphine solution of 100mM or two kinds or more are by imprinted layer
The template cell on surface completely removes, while the disulfide bond in material is reduced into sulfydryl.
(5) sulfydryl on material and aptamer, antibody, the polypeptide that can specifically bind with tumour cell are utilized
Or the reaction between one of boric acid compound or two kinds or more, one or two or more kinds of active forces are introduced into trace material
Material, obtains a kind of multiple action imprinted material for specific recognition tumour cell.Wherein, by the aptamer of modification
Specially end modified ZY sls for having one of maleimide and double bond group or two kinds or more, Anti-EGFR,
One in SYL3C, S6, A9, A10, YJ-1, APTmuc, TD05, TE02, Sgc8, Sgd5, KDED2a-3, KCHA10, KH1C12
Kind or two kinds or more；Antibody specific by modification is to be modified with one of maleimide and double bond group or two kinds or more
Anti-EGFR, Anti-EpCAM, Anti-VEGF, Anti-CTLA4, Anti-CD20, Anti-CD52, Anti-CD30,
One of Anti-CD33, Anti-HER2 or two kinds or more；Polypeptide by modification be specially be modified with maleimide and
RGD, polymyxin, bacitracin, LTX-302, Viphi A-H, Cr-ACP1 and the chest of one of double bond group or two kinds or more
One of gland pentapeptide or two kinds or more；Ene boric acid class compound is specially vinylphenylboronic acid, acrylamido phenyl boric acid
With one of the phenyl boric acid containing alkylene or two kinds or more.Aptamer of the dissolution by modification, resisting by modification
The solvent of body, the polypeptide by modification or ene boric acid class compound is mainly by water, phosphate buffer and sodium chloride solution
One or two or more kinds of compositions, the mass concentration range of the solution of formation is 5%-95%.
(6) tumour cell is not attached on host material, and non-trace is prepared according to the step of above-mentioned from (1) to (5)
Material (NIP).
(7) the cell blots material is used for bioanalysis, biochemical industry, blood in biomedicine and field of biotechnology
The Selective recognition of cell, capture, release and enrichment in liquid, body fluid and tissue sample.
Shape complementation power, aptamer and the tumour cell that the multiple action is mainly generated by cell blots
One kind of binding force, antibody and tumour cell binding force, polypeptide and tumour cell binding force, phenyl boric acid and tumour cell binding force
Or two kinds of composition described above.
The present invention is a kind of cell blots material based on the multiple action between template cell and imprinted sites, the material
Specific recognition, capture, release and enrichment can be carried out to target cell.
It will form action site when template cell and prepolymerization system contact, pass through the solidification process in prepolymerization system
Middle memory interaction between the two, after template cell removes, material surface just will form to be occurred mutually with template cell
The imprinted sites of effect；Between template cell and imprinted sites in space structure, size, shape and functional group, pass through space
The complementary affinity that configuration and hydrogen bond action, electrostatic interaction and model ylid bloom action are shown；Then draw in imprinted sites
Enter aptamer, antibody, polypeptide and the phenylboronic acid compound that there is binding ability to tumour cell, obtains additional chemistry
Specific binding affinity.
The present invention has the advantage that
(1) present invention is prepared for having multiple work to tumour cell using modification technique after cell blots technology and trace
Cell blots material improves the purity and sensitivity of the separation and enrichment to tumour cell；
(2) imprinted material in the present invention is multiple action to the identification of target cell.Cell blots technology provides swollen
Spatial complementary, hydrogen bond and the electrostatic force formed between oncocyte and imprinted sites；Modification technique provides stronger after trace
The affine specific effect power of chemistry；
(3) present invention used in polymer material have good bio-compatibility and stability, be conducive to separation and
It is enriched to tumour cell living, provides sample for further analysis tumour cell；
(4) preparation flow of imprinted material is simple in the present invention, easily operated, is convenient for large-scale production.
Detailed description of the invention:
Fig. 1: it is attached at the microphoto of the SMMC-7721 cell of cell culture plate surface.
Fig. 2: the structural formula of crosslinking agent.
Fig. 3: the microphoto of crosslinking agent and the plastidogenetic compound of SMMC-7721.
Fig. 4: the SMMC-7721 cell of trace layer surface.
Fig. 5: the cell blots material (MIP1) being prepared using SMMC-7721 cell as template.
Fig. 6: the bright field and dark field microphoto for the tumour cell being captured in blood.
Specific embodiment
Embodiment 1
The preparation of cell blots material (MIP1) based on SMMC-7721 cell shuttering and ZY sls aptamer
By SMMC-7721 cell culture in Tissue Culture Dish (10 centimetres of diameter), when about 80% culture plate bottom quilt
When being paved with (Fig. 1), cell is cleaned with phosphate buffer (pH 7.2, PBS), the centre that mass concentration is 1%, which is then added, is
Disulfide bond, both ends are respectively the crosslinking agent (Fig. 2 a) of succinimide sodium sulfonate and double bond group, are incubated for 3 jointly with tumour cell
It after hour, is cleaned with PBS and removes extra crosslinking agent, obtain the compound (Fig. 3) that cell and crosslinking agent are formed.Then to cell
The methylene-bisacrylamide that 8 milliliters of addition in culture dish are 10% acrylamide by mass concentration and mass concentration is 1%
Then the prepolymerization aqueous solution of composition adds 1 microlitre of tetramethylethylenediamine and 10 microlitres of mass concentrations as 10% persulfuric acid
The curing reaction of aqueous ammonium initiation pre-polymer solution.After forty minutes, the imprinted layer being formed by curing is separated simultaneously with host material
Imprinted layer is overturn, in trace layer surface with the presence of SMMC-7721 cell (Fig. 4).Then three (the 2- carboxylics that concentration is 1mM are added
Ethyl) phosphine solution, 55 DEG C are incubated for 1 hour, and abundant Reduction of Disulfide is at sulfydryl and removes removing template cell, then with PBS by three (2-
Carboxyethyl) removal of phosphine solution.Finally, it is molten that the end modified ZY sls aptamer for having double bond that mass concentration is 5% is added
Liquid is reacted with the sulfydryl of material surface, obtains the cell blots material for having multiple specific active force to SMMC-7721 cell
(MIP1) (Fig. 5).
Non-imprinted material is prepared in the Tissue Culture Dish there is no SMMC-7721 cell using identical method
(NIP)。
Embodiment 2
Cell blots material (MIP1) for identification with capture SMMC-7721 cell
It is 1 × 10 by two parts of cell concentrations5The SMMC-7721 cell of cells/ml be seeded to respectively MIP1 trace face and
The surface NIP, after 3 hours, cell attachment at the imprinted sites on the surface MIP1, rather than at the imprinted sites and surface NIP without
The attaching of SMMC-7721 cell illustrates that the imprinted sites of MIP1, can be fast by the way that multiple action occurs with SMMC-7721 cell
Speed, colleges and universities and highly sensitive identification and capture target cell.SMMC-7721 cell is further added to blood of human body sample
In, according to the method described above, MIP1 also achieves identification and enrichment SMMC-7721 cell (Fig. 6) specific from blood.
Embodiment 3
Preparation based on SMMC-7721 cell shuttering and SYL3C aptamer cell blots material (MIP2)
By SMMC-7721 cell culture in Tissue Culture Dish (10 centimetres of diameter), when about 80% culture plate bottom quilt
When being paved with, cell is cleaned with phosphate buffer (pH 7.2, PBS), it is two sulphur that the centre that mass concentration is 15%, which is then added,
Key, both ends are respectively the crosslinking agent (Fig. 2 b) of maleimide and double bond group, after being incubated for 1 hour jointly with tumour cell, are used
PBS cleaning removes extra crosslinking agent, obtains the compound that cell and crosslinking agent are formed.Then the addition into Tissue Culture Dish
The pre-polymerization Heshui that the methylene-bisacrylamide that 8 milliliters are 30% acrylamide by mass concentration and mass concentration is 1% forms
Then solution adds 1 microlitre of tetramethylethylenediamine and 10 microlitres of mass concentrations and causes in advance for 10% ammonium persulfate aqueous solution
The curing reaction of polymeric solution.After twenty minutes, the imprinted layer being formed by curing is separated with host material and overturns imprinted layer,
Trace layer surface is with the presence of SMMC-7721 cell.Then the dithiothreitol (DTT) solution that concentration is 20mM is added, 55 DEG C of incubations 1 are small
When, abundant Reduction of Disulfide is at sulfydryl and removes removing template cell, is then removed dithiothreitol (DTT) solution with PBS.Finally, being added
The end modified SYL3C aptamer solution for having double bond that concentration is 40% is reacted with the sulfydryl of material surface, is obtained pair
SMMC-7721 cell has the cell blots material (MIP2) of multiple specific active force.
Embodiment 4
Preparation based on Hela cell shuttering and Anti-EGFR antibody cell imprinted material (MIP3)
By Hela cell culture in Tissue Culture Dish (10 centimetres of diameter), when about 80% culture plate bottom is paved with
When, cell is cleaned with phosphate buffer (pH 7.2, PBS), it is disulfide bond, two that the centre that mass concentration is 30%, which is then added,
End is respectively the crosslinking agent (Fig. 2 c) of maleimide and mercapto groups, clear with PBS after being incubated for 30 minutes jointly with tumour cell
Wash away the compound for obtaining that cell and crosslinking agent are formed except extra crosslinking agent.Then 8 milliliters of the addition into Tissue Culture Dish
The prepolymerization aqueous solution that the methylene-bisacrylamide that by mass concentration be 60% acrylamide and mass concentration is 1% forms,
Then it is molten for 10% ammonium persulfate aqueous solution initiation prepolymerization that 1 microlitre of tetramethylethylenediamine and 10 microlitres of mass concentrations are added
The curing reaction of liquid.After twenty minutes, the imprinted layer being formed by curing is separated with host material and overturns imprinted layer, in imprinted layer
Surface is with the presence of Hela cell.Then be added concentration be 50mM three (2- carboxyethyl) phosphine solution, 55 DEG C be incubated for 1 hour, sufficiently
Reduction of Disulfide is at sulfydryl and removes removing template cell, is then removed three (2- carboxyethyl) phosphine solution with PBS.Finally, being added dense
Degree reacts for the 70% Anti-EGFR antibody for being modified with double bond with the sulfydryl of material surface, obtains having Hela cell more
The cell blots material (MIP3) of weight specific effect power.
Embodiment 5
The preparation of cell blots material (MIP4) based on the crosslinking agent containing succinimide and rgd peptide
SMMC-7721 cell is fixed on to the glass material surface of Mercapto-group modification.When about 80% culture plate bottom
When being paved with, cell is cleaned with phosphate buffer (pH 7.2, PBS), it is two that the centre that mass concentration is 50%, which is then added,
Sulfide linkage, both ends are respectively the crosslinking agent (Fig. 2 d) of succinimide and mercapto groups, after being incubated for 30 minutes jointly with tumour cell,
It is cleaned with PBS and removes extra crosslinking agent, obtain the compound that cell and crosslinking agent are formed.It is simultaneously 60% by mass concentration
Agitating solution is extremely at a temperature of 65 DEG C for the tetrahydrofuran solution of 4,4 '-di-2-ethylhexylphosphine oxides (phenyl isocyanate), bisphenol-A and phloroglucin
Then the above-mentioned host material for being fixed with cell is gently pressed on gel solution surface by gel point, after its solidification, remove matrix material
Material and template cell.Then be added concentration be 100mM mercaptoethanol solution, 55 DEG C be incubated for 1 hour, abundant Reduction of Disulfide at
Sulfydryl simultaneously removes removing template cell, is then removed mercaptoethanol solution with PBS.Finally, it is 90% to be modified with horse that concentration, which is added,
Carry out imido rgd peptide to react with the sulfydryl of material surface, obtains that there is multiple specific active force to SMMC-7721 cell
Cell blots material (MIP4).
Embodiment 6
The system of the cell blots material (MIP5) of metal material and acrylamido phenyl boric acid based on corona treatment
It is standby
SMMC-7721 cell is fixed on to the titanium alloy material surface of corona treatment, when about 80% cell is paved with
When, cell is cleaned with phosphate buffer (pH 7.2, PBS), it is disulfide bond, two that the centre that mass concentration is 35%, which is then added,
End is respectively the crosslinking agent (Fig. 2 e) of succinimide and maleimide base group, after being incubated for 30 minutes jointly with tumour cell,
It is cleaned with PBS and removes extra crosslinking agent, obtain the compound that cell and crosslinking agent are formed.It is simultaneously 55% by mass concentration
N-isopropylacrylamide and 1% solution of acrylamide are added to composite surface, then add 1 microlitre of tetramethyl second two
The ammonium persulfate aqueous solution that amine and 10 microlitres of mass concentrations are 10% causes the curing reaction of pre-polymer solution.After twenty minutes, will
The imprinted layer being formed by curing separates with host material and overturns imprinted layer, in trace layer surface with the presence of SMMC-7721 cell.
Then three (2- carboxyethyl) phosphine solution that concentration is 50mM are added, 55 DEG C are incubated for 1 hour, and abundant Reduction of Disulfide is at sulfydryl and goes
Then removing template cell is removed three (2- carboxyethyl) phosphine solution with PBS.Finally, the acrylamido that concentration is 95% is added
Phenyl boric acid is reacted with the sulfydryl of material surface, obtains the cell blots for having multiple specific active force to SMMC-7721 cell
Material (MIP5).
Embodiment 7
The preparation of MCF-7 cell blots material (MIP6) based on Anti-EpCAM antibody
By MCF-7 cell culture in Tissue Culture Dish (10 centimetres of diameter), when about 80% culture plate bottom is paved with
When, cell is cleaned with phosphate buffer (pH 7.2, PBS), it is disulfide bond, two that the centre that mass concentration is 20%, which is then added,
End is respectively the crosslinking agent (Fig. 2 a) of succinimide sodium sulfonate and double bond group, jointly after being incubated for 3 hours with tumour cell, is used
PBS cleaning removes extra crosslinking agent, obtains the compound that cell and crosslinking agent are formed.Then the addition into Tissue Culture Dish
The pre-polymerization Heshui that the methylene-bisacrylamide that 8 milliliters are 30% acrylamide by mass concentration and mass concentration is 1% forms
Then solution adds 1 microlitre of tetramethylethylenediamine and 10 microlitres of mass concentrations and causes in advance for 10% ammonium persulfate aqueous solution
The curing reaction of polymeric solution.After twenty minutes, the imprinted layer being formed by curing is separated with host material and overturns imprinted layer.So
Three (2- carboxyethyl) phosphine solution that concentration is 50mM are added afterwards, 56 DEG C are incubated for 1 hour, and abundant Reduction of Disulfide is at sulfydryl and removes
Then template cell is removed three (2- carboxyethyl) phosphine solution with PBS.Finally, the double bond of being modified with that concentration is 30% is added
Anti-EpCAM antibody-solutions are reacted with the sulfydryl of material surface, obtain having multiple specific active force to MCF-7 cell
Cell blots material (MIP6).
Claims (10)
1. a kind of multiple action imprinted material for specific recognition tumour cell, it is characterised in that:
It can be prepared according to the following procedure, using the tumour cell to specific recognition as template, be attached or fixed to base
Material surface, using mainly by centre be disulfide bond, one end can in conjunction with tumour cell group and the other end can participate in material
The crosslinking agent combination cell of the group composition of material polymerization, after removing unbonded crosslinking agent, crosslinking agent and template cell are formed
Compound be placed in prepolymerization system, by cause prepolymerization polymerization-filling be separated off matrix after removing extra solvent
Material, then the disulfide bond reduction in crosslinking agent is removed to the template cell for solidifying post-consumer polymer surface at sulfydryl, then utilize
Sulfydryl and can with tumour cell occur chemically specifically bind by modification aptamer, the antibody by modification,
Reaction between one of polypeptide or alkenyl benzene boric acid compound by modification or two kinds or more obtains a kind of for spy
The multiple action imprinted material of anisotropic tumor cell.
2. multiple action imprinted material as described in claim 1, it is characterised in that:
Host material refers to tissue culture plate, Tissue Culture Dish, Tissue Culture Flask, titanium alloy, the cobalt that tumour cell can attach
The one or two or more kinds of the glass material of based alloy, stainless steel material and Mercapto-group modification.
3. multiple action imprinted material as described in claim 1, it is characterised in that:
Crosslinking agent refer mainly to be by centre disulfide bond, one end be can be that can join with the chemical group in conjunction with tumour cell, the other end
The molecule for the chemical group composition polymerizeing with material；It can be with the chemical group in conjunction with tumour cell mainly by carboxyl, maleimide
One of amine groups and succinimide group composition；Can participate in material polymerization chemical group mainly by alkylene, sulfydryl,
One of maleimide base group and succinimide group composition；For dissolve or the solvent of decentralized crosslinking agent include water,
One of phosphate buffer, sodium chloride solution, dimethyl sulfoxide, dimethylformamide or two kinds or more；
It is dissolved in the solution formed in one of water, phosphate buffer, sodium chloride solution or two kinds or more by crosslinking agent,
The total mol concentration range of other solutes in addition to water is 50mM-200mM；In water, phosphate buffer, sodium chloride solution
One or two or more kinds in undissolved crosslinking agent be scattered in one of water, phosphate buffer, sodium chloride solution first
Or in two kinds or more, dissolution crosslinking dosage form in one of dimethyl sulfoxide, dimethylformamide or two kinds or more is then added
At solution, wherein dimethyl sulfoxide, dimethylformamide volume concentration range be 0.5%-10%.
4. multiple action imprinted material as claimed in claim 1 or 3, it is characterised in that:
Crosslinking agent is by one of following molecules or two kinds of composition described above:
Wherein, R1For-COOH,One of；R2For -
SH,One of；R3For H andOne of；N is the length of alkyl in bracket, range 2-
6；M is the length of alkyl in bracket, range 2-6.
5. multiple action imprinted material as described in claim 1, it is characterised in that:
Prepolymerization system forms the solution of the monomer composition of polymer by that can be copolymerized with crosslinking agent, wherein in prepolymerization system
Monomer mainly by acrylamide, N, N- dimethylacrylamide, N,N methylene bis acrylamide, n-isopropyl acrylamide,
One of N hydroxymethyl acrylamide and N tert butyl acrylamide or two kinds of composition described above；For dissolving or dispersed monomer
Solvent includes one of water, phosphate buffer, sodium chloride solution, glucose solution and ethyl alcohol or two kinds or more；By function
The mass concentration range that monomer is dissolved or dispersed in the solution formed in solvent is 10%-60%；
One of the cured method of prepolymerization system, including light-initiated, hot initiation, electric field treatment and free radical initiation can be caused
Or two kinds or more；Cause polymerization for template cell surface is added to by the prepolymerization system for the monomer composition that can polymerize.
6. multiple action imprinted material as described in claim 1, it is characterised in that:
The reagent of Reduction of Disulfide is one of dithiothreitol (DTT) solution, mercaptoethanol and three (2- carboxyethyl) phosphine solution or two
Kind or more.
7. multiple action imprinted material as described in claim 1, it is characterised in that:
Described can occur the aptamer by modification chemically specifically bound, by the anti-of modification with tumour cell
Body, the polypeptide by modification or ene boric acid class compound be,
Aptamer by modification refers to using tumour cell as target, sieves by the Fas lignand system evolution technology of index concentration
Obtained oligonucleotide fragment is selected, then which can modify at its end upper Malaysia acyl in conjunction with tumor cell specific
One of imines and double bond group or two kinds or more；Antibody by modification, which refers to, spy occurs with tumor cell surface antigen
The immune protein that the opposite sex combines, then modified on immune protein one of maleimide and double bond group or two kinds with
On；Polypeptide by modification refers to the protein structure and function fragment that can be specifically bound with tumor cell surface antigen,
Then maleimide and double bond group one of or two kind or more are modified it；Ene boric acid class compound refer to can with it is swollen
The polysaccharide compound on oncocyte surface forms the boric acid substance that stable compound contains alkenyl；Nucleic acid of the dissolution by modification
Aptamers, the antibody by modification, the polypeptide by modification or ene boric acid class compound solvent mainly delayed by water, phosphate
One of fliud flushing and sodium chloride solution or two kinds of composition described above, the mass concentration range of the solution of formation are 5%-95%.
8. multiple action imprinted material as claimed in claim 5, it is characterised in that: You Nengyu crosslinking agent is copolymerized to form polymer
The solution of monomer composition be,
The monomer that polymerization reaction forms polymer, including acrylamide, N,N-DMAA, N, N- methylene can occur
One of bisacrylamide, n-isopropyl acrylamide, N hydroxymethyl acrylamide, N tert butyl acrylamide or two kinds with
On；For dissolve or the solvent of dispersed monomer or polymer include one of water, phosphate buffer and sodium chloride solution or
Two kinds or more；It is 10%- by the mass concentration range that function monomer or polymer are dissolved or dispersed in the solution formed in solvent
60%.
9. multiple action imprinted material as claimed in claim 1 or 7, it is characterised in that: aptamer, warp by modification
The antibody, the polypeptide by modification and ene boric acid class compound, the aptamer by modification for crossing modification are specially end
Be modified with ZY sls of one of maleimide and double bond group or two kinds or more, Anti-EGFR, SYL3C, S6, A9,
One of A10, YJ-1, APTmuc, TD05, TE02, Sgc8, Sgd5, KDED2a-3, KCHA10, KH1C12 or two kinds or more；
The tumour cell that can accordingly capture refers to liver cancer cells, colorectal cancer cell, glioblastoma cells, adenocarcinoma cell, leaching
One of bar oncocyte, leukaemia cell, lung carcinoma cell or two kinds or more；
Antibody specific by modification is the Anti- for being modified with one of maleimide and double bond group or two kinds or more
EGFR、Anti-EpCAM、Anti-VEGF、Anti-CTLA4、Anti-CD20、Anti-CD52、Anti-CD30、Anti-CD33、
One of Anti-HER2 or two kinds or more；The tumour cell that can accordingly capture refer to liver cancer cells, colorectal cancer cell,
One of glioblastoma cells, adenocarcinoma cell, lymphoma cell, leukaemia cell, lung carcinoma cell or two kinds or more；
Polypeptide by modification is specially the RGD for being modified with one of maleimide and double bond group or two kinds or more, more
One of colistin, bacitracin, LTX-302, Viphi A-H, Cr-ACP1 and thymopeptide-5 or two kinds or more；It accordingly can be with
The tumour cell of capture refer to liver cancer cells, colorectal cancer cell, glioblastoma cells, adenocarcinoma cell, lymphoma cell,
One of leukaemia cell, lung carcinoma cell or two kinds or more；
Ene boric acid class compound is specially in vinylphenylboronic acid, acrylamido phenyl boric acid and phenyl boric acid containing alkylene
One or two or more kinds.The tumour cell that can accordingly capture refers to liver cancer cells, colorectal cancer cell, glioblastoma
One of cell, adenocarcinoma cell, lymphoma cell, leukaemia cell, lung carcinoma cell or two kinds or more.
10. any multiple action imprinted material for specific recognition tumour cell of claim 1-9, for biology
Analysis, biochemical industry, one of in biomedicine and field of biotechnology or two kinds or more blood, body fluid and tissue sample
One of or two kinds or more cells one of Selective recognition, capture, release and enrichment or two kinds or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711337444.4A CN109959791B (en) | 2017-12-14 | 2017-12-14 | Multiple-action imprinting material for specifically recognizing tumor cells and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711337444.4A CN109959791B (en) | 2017-12-14 | 2017-12-14 | Multiple-action imprinting material for specifically recognizing tumor cells and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109959791A true CN109959791A (en) | 2019-07-02 |
| CN109959791B CN109959791B (en) | 2021-09-24 |
Family
ID=67017793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711337444.4A Active CN109959791B (en) | 2017-12-14 | 2017-12-14 | Multiple-action imprinting material for specifically recognizing tumor cells and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109959791B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003031591A2 (en) * | 2001-10-10 | 2003-04-17 | Superarray Bioscience Corporation | Detecting targets by unique identifier nucleotide tags |
| WO2008083323A1 (en) * | 2006-12-29 | 2008-07-10 | Invitrogen Corporation | Detection apparatus |
| US20100203422A1 (en) * | 2009-02-11 | 2010-08-12 | Yanxiu Zhou | Proton selective membrane for solid polymer fuel cells |
| CN102268119A (en) * | 2010-11-01 | 2011-12-07 | 中南大学肝胆肠外科研究中心 | Preparation method of molecular imprinted polymer for detecting lung cancer tumor markers |
| CN103275273A (en) * | 2013-06-08 | 2013-09-04 | 南开大学 | Preparation method of core-shell molecular imprinting nano-material, and application of nano-material |
| CN103408697A (en) * | 2013-07-10 | 2013-11-27 | 江苏大学 | Method for preparing thermosensitive molecularly-imprinted polymers (T-MIPs) through surface initiation |
| US20140120534A1 (en) * | 2011-02-15 | 2014-05-01 | Mats Nilsson Bernitz | Methods for identifying nucleic acid sequences |
| CN104525131A (en) * | 2015-01-05 | 2015-04-22 | 福州大学 | CDTA modified magnetic nano-adsorbent and preparation method and application thereof |
| CN103406109B (en) * | 2013-08-06 | 2016-08-10 | 南京大学 | A kind of controlled, general orientation surface immunoblot method and the application of gained molecularly imprinted polymer |
| CN106596918A (en) * | 2017-01-10 | 2017-04-26 | 河南省人口和计划生育科学技术研究院 | Preparation method of nanometer materials for capturing circulating tumor cells |
| CN106633167A (en) * | 2015-10-30 | 2017-05-10 | 中国科学院大连化学物理研究所 | Blotting material used for specific recognition of cells, and preparation and applications thereof |
| KR101847893B1 (en) * | 2016-12-08 | 2018-04-11 | 주식회사 피엔에스테크놀로지 | Sensor electrode for measuring 1-OHP, preparation method of the same and electrochemical sensor for measuring 1-OHP comprising the same |
-
2017
- 2017-12-14 CN CN201711337444.4A patent/CN109959791B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003031591A2 (en) * | 2001-10-10 | 2003-04-17 | Superarray Bioscience Corporation | Detecting targets by unique identifier nucleotide tags |
| WO2008083323A1 (en) * | 2006-12-29 | 2008-07-10 | Invitrogen Corporation | Detection apparatus |
| US20100203422A1 (en) * | 2009-02-11 | 2010-08-12 | Yanxiu Zhou | Proton selective membrane for solid polymer fuel cells |
| CN102268119A (en) * | 2010-11-01 | 2011-12-07 | 中南大学肝胆肠外科研究中心 | Preparation method of molecular imprinted polymer for detecting lung cancer tumor markers |
| US20140120534A1 (en) * | 2011-02-15 | 2014-05-01 | Mats Nilsson Bernitz | Methods for identifying nucleic acid sequences |
| CN103275273A (en) * | 2013-06-08 | 2013-09-04 | 南开大学 | Preparation method of core-shell molecular imprinting nano-material, and application of nano-material |
| CN103408697A (en) * | 2013-07-10 | 2013-11-27 | 江苏大学 | Method for preparing thermosensitive molecularly-imprinted polymers (T-MIPs) through surface initiation |
| CN103406109B (en) * | 2013-08-06 | 2016-08-10 | 南京大学 | A kind of controlled, general orientation surface immunoblot method and the application of gained molecularly imprinted polymer |
| CN104525131A (en) * | 2015-01-05 | 2015-04-22 | 福州大学 | CDTA modified magnetic nano-adsorbent and preparation method and application thereof |
| CN106633167A (en) * | 2015-10-30 | 2017-05-10 | 中国科学院大连化学物理研究所 | Blotting material used for specific recognition of cells, and preparation and applications thereof |
| KR101847893B1 (en) * | 2016-12-08 | 2018-04-11 | 주식회사 피엔에스테크놀로지 | Sensor electrode for measuring 1-OHP, preparation method of the same and electrochemical sensor for measuring 1-OHP comprising the same |
| CN106596918A (en) * | 2017-01-10 | 2017-04-26 | 河南省人口和计划生育科学技术研究院 | Preparation method of nanometer materials for capturing circulating tumor cells |
Non-Patent Citations (6)
| Title |
|---|
| HORIKAWA R , SUNAYAMA H , KITAYAMA Y , ET AL.: "A Programmable Signaling Molecular Recognition Nanocavity Prepared by Molecular Imprinting and Post-imprinting Modifications", 《ANGEWANDTE CHEMIE》 * |
| LIU H , LI Y , SUN K , ET AL.: "Dual-Responsive Surfaces Modified with Phenylboronic Acid-Containing Polymer Brush To Reversibly Capture and Release Cancer Cells", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 * |
| SCHIRHAGL ROMANA等: "表面印迹聚合物及其在微流控器件中的应用", 《中国科学:化学》 * |
| ZHANG Y, DENG C, LIU S, WU J, CHEN Z, LI C, LU W: "Active targeting of tumors through conformational epitope imprinting", 《ANGEW CHEM INT ED ENGL》 * |
| 余元勋 等: "《中国分子肝癌学》", 30 April 2016 * |
| 周雪, 韩俊, 吴一凡,等: "纳米技术在肿瘤靶向诊疗中的应用研究进展", 《转化医学电子杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109959791B (en) | 2021-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ansari et al. | Molecularly imprinted polymers for capturing and sensing proteins: Current progress and future implications | |
| Yang et al. | Protein-imprinted materials: rational design, application and challenges | |
| Yang et al. | Design and preparation of self-driven BSA surface imprinted tubular carbon nanofibers and their specific adsorption performance | |
| Li et al. | Size matters: Challenges in imprinting macromolecules | |
| Yang et al. | Molecularly imprinted polymers for the identification and separation of chiral drugs and biomolecules | |
| Moczko et al. | Epitope approach in molecular imprinting of antibodies | |
| Li et al. | Photolithographic boronate affinity molecular imprinting: a general and facile approach for glycoprotein imprinting. | |
| EP2417249B1 (en) | Dna-cell conjugates | |
| Khumsap et al. | Epitope-imprinted polymers: applications in protein recognition and separation | |
| Pang et al. | Bovine serum albumin-imprinted polyacrylamide gel beads prepared via inverse-phase seed suspension polymerization | |
| Sergeyeva et al. | In situ formation of porous molecularly imprinted polymer membranes | |
| Liu et al. | Core–shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein | |
| Hillberg et al. | Biomolecule imprinting: Developments in mimicking dynamic natural recognition systems | |
| Li et al. | Smart bio-separation materials | |
| CN106633167B (en) | It is a kind of for the imprinted material of specific recognition cell and its preparation and application | |
| JP2008540723A (en) | Synthetic receptor | |
| Nagase et al. | Temperature-responsive chromatography for bioseparations: A review | |
| Yemiş et al. | Molecularly imprinted polymers and their synthesis by different methods | |
| Nguyen et al. | An aptamer-based microfluidic device for thermally controlled affinity extraction | |
| CN104530314A (en) | Preparation method and application of bionic temperature-sensitive molecular engram composite film | |
| JP2008543980A (en) | Synthetic receptor | |
| CN108456280A (en) | A kind of phospholipid organic polymer integral material and its preparation method and application | |
| CN108003287A (en) | A kind of preparation method of albumen based on acrylamide race metal-chelating monomer is affine trace aquogel polymer | |
| Koda et al. | One-shot preparation of polyacrylamide/poly (sodium styrenesulfonate) double-network hydrogels for rapid optical tissue clearing | |
| CN109959791A (en) | It is a kind of for the multiple action imprinted material of specific recognition tumour cell and its preparation and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |