CN115960977A - Method for endowing substrate with function of specifically recognizing mammalian cells - Google Patents
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Abstract
The invention discloses a method for endowing a substrate with a function of specifically recognizing mammalian cells, which comprises the following steps: modifying an initiator on the surface of the substrate; constructing a specific polymer surface; and (3) regeneration treatment of the specific polymeric surface. According to the invention, mammalian cells are used for participating in polymerization reaction for the first time, the chain sequence of the polymer is regulated, the chain sequence matched with the cells is screened out by the cells, the specific polymer brush is prepared, and the mammalian cells do not need pretreatment such as fixation; compared with the antibody specific surface, the polymer brush surface has the advantages of simple preparation, low cost and high stability, and the surface can be repeatedly recycled.
Description
Technical Field
The invention belongs to the technical field of surface modification of biomedical materials, and particularly relates to a method for endowing a substrate with a function of specifically identifying mammalian cells.
Background
The treatment of cancer continues to be a significant challenge to overcome, and it is important in the process to identify target cancer cells without harming healthy cells. The identification of cancer cells and their associated markers is throughout the diagnostic and therapeutic process, meaning that this molecular recognition is involved both in vivo and in vitro. Molecular recognition is a very important process in organisms, including substance transport, signal transmission, immune regulation and the like, and has great significance for exploring and understanding the operating mechanism of human bodies, particularly for various diseases. This specific recognition is mainly based on antigen-antibody, sugar-protein, DNA-protein and other molecular interactions, some of which have been developed into commercial specific reagents such as cocktails and antibodies. However, the preparation of such widely used biological agents is complex and time consuming, often requiring stringent storage and handling conditions. Molecular Imprinting Technology (MIT) is a good chemical strategy to solve the problem of biological agents. The MIT member is a synthetic organic compound, generally insoluble in water. For polymerization, it requires elevated temperatures or exposure to ultraviolet radiation, while proteins almost lose function outside the narrow window of physiological conditions. Therefore, there is an urgent need to develop a mild chemical method for synthesizing a soluble specific reagent. Cameron Alexander and Hasan Yesikaya have obtained linear soluble polymers targeted to bacteria and proteins without the addition of cross-linking agents. These linear soluble MIT expand the range of applications. However, the biological template for linear MIT is generally a microorganism having relative rigidity like bacteria. Mammalian cells have never been used directly for linear MIT except in some instances in conventional MIT where a pre-treatment of the fixative is usually required due to the fluid film. However, pretreatment destroys the protein domain. It is clearly more desirable if living cells can be used as templates in the polymerization process. Due to the glycan effect, synthetic carbohydrate polymers have proven to be strongly selective, making them suitable for applications requiring specificity based on specific binding between the carbohydrate units and the protein. However, it is often overlooked that, despite the specific binding between carbohydrates and proteins, one type of carbohydrate can bind several specific proteins or proteins on different types of cells with far less accuracy of specificity than specific reagents such as antibodies and inducers. In previous researches, bacteria are used as a template, a saccharide polymer with strain specificity is successfully synthesized by a bacteria-saccharide monomer-adaptation-polymerization (BS-MAP) method, and the specificity accuracy can be greatly improved. It is well known that there are some specific receptor proteins that bind to carbohydrates on the surface of cells. Thus, cell-specific glycopolymers can be obtained using live mammalian cells as a template. Similarly, other than proteins, such as charge distributions, which have a differential distribution on the cell surface, other types of monomers can also act as affinity cells. Polymerization on the cell surface is difficult due to the weak membranes that lack cell wall support. At present, there are few reports on the participation of living cells in polymerization, and there is no report on the obtainment of a specific glycopolymer using living mammalian cells as a template.
Therefore, it is necessary to propose a further solution to the above situation.
Disclosure of Invention
The invention aims to provide a method for endowing a substrate with a function of specifically recognizing mammalian cells.
The technical scheme of the invention is as follows:
a method of imparting a substrate with a function of specifically recognizing mammalian cells, the method comprising the steps of:
(1) Substrate surface modification initiator: modifying the surface of a base material by using a surface initiated ATRP reagent to obtain a base material modified with an initiator;
(2) Construction process of specific polymer surface: the base material modified with the initiator is soaked in 75% ethanol and sterile water alternately for more than or equal to 3 times, then cells are planted, after 1-10 hours of adhesion, the monomer having affinity with the cells is incubated for 0.5-3 hours to obtain an incubated base material, the incubated base material is placed in a weighing bottle, two monomers, a catalytic system and a buffer solution are added, after 0.5-10 hours, the base material is taken out to stop reaction, the base material is alternately cleaned for more than 2 times by 0.5-20% SDS (sodium dodecyl sulfate) aqueous solution and competitive sugar aqueous solution, or 0.5-20% SDS aqueous solution and salt solution to remove residual cells, and the base material with the surface of a polymer brush is obtained;
(3) Regeneration treatment of specific polymeric surfaces: and (2) placing the substrate with the polymer brush surface in a sample for specific detection and capture of cells, performing cell desorption treatment after analysis is finished, and alternately washing for more than 2 times by using 0.5-20% SDS aqueous solution and competitive sugar aqueous solution or 0.5-20% SDS aqueous solution and salt solution to remove adsorbed cells so as to realize regeneration of specific surface functionalization.
Further, in the step (1), the surface of the base material is modified by a surface-initiated ATRP reagent, and the base material modified with an initiator is specifically: alternately performing ultrasonic treatment on the surfaces of silicon and silicate base materials for 5-60min by using acetone and water, cleaning for 0.5-3h by using a heated Piranha solution, washing by using a large amount of water, drying by using inert gas, then placing in an anhydrous toluene solution containing 0.1-1 mol of surface initiator for 12-36h, cleaning for 2-5 times by using an anhydrous organic solvent capable of dissolving the surface initiator to remove the non-adhered surface initiator, and drying by using the inert gas to obtain the base material modified with the initiator.
Further, in the step (1), the surface of the base material is modified by a surface-initiated ATRP reagent, and the base material modified with an initiator is specifically: alternately performing ultrasonic treatment on the surface of a gold-containing surface substrate for 5-60min by using acetone and water, exposing a clean gold-plated surface to 0.1-1mol 2-mercaptoethanol solution, forming an Au-OH surface at room temperature for 12-36h, washing the Au-OH surface by using deionized water and ethanol, drying in inert gas flow, immersing the Au-OH surface into 0.1-1 mol 3-aminopropyltrimethoxysilane solution, immersing into 95vol% ethanol solution, treating at room temperature for 12-36h, washing the surface by using ethanol, toluene and acetone in sequence, drying by using inert gas flow, keeping the obtained gold-plated surface at 52 ℃ for 2-10 h to form Au-NH 2 Surface of said Au-NH 2 The surface reacts with chloroacetyl chloride to form an Au-Cl surface, and the substrate modified with the initiator is obtained.
Further, in the step (1), the surface of the base material is modified by a surface-initiated ATRP reagent, and the base material modified with an initiator is specifically: activating the surface of a polymer substrate by formaldehyde to generate an N-hydroxymethyl polyamide membrane, and adding 2-bromoisobutyryl bromide to react to generate a 2-bromoisobutyryl immobilized membrane to obtain the substrate modified with the initiator.
Further, in the step (1), the substrate is any one of an inorganic substrate or a polymer substrate.
Further, in the step (1), the modification method includes any one of hydroxylation treatment, carboxylic acid activation, amination treatment, sulfhydrylation treatment and gold deposition.
Further, in the step (1), the surface-initiated ATRP reagent is any one of 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, 2- (2-bromoisobutyloxy) ethyl methacrylate, 2-bromo-2-methylpropanoic acid bromide, 2-bromoisobutyryl bromide (BIBB).
Further, in step (2), the cells are first cultured in RPMI-1640 medium or high-sugar DMEM medium at 37 ℃ and 5% CO 2 。
Further, in step (2), the two monomers are a cell-compatible monomer and a cell-incompatible monomer in a ratio of 1:20 to 20:1, the monomer of the compatible cell comprises any one of glucose derivatives, mannose derivatives, galactose derivatives, N- [3- (dimethylamino) propyl ] methacrylamide and charged monomers of methacryloyloxyethyl trimethyl ammonium chloride, the monomer of the incompatible cell comprises any one of poly phosphorylcholine, poly-sulfobetaine, poly-carboxylic betaine and polyethylene glycol, and the catalytic system comprises any one of cuprous ion catalyst, copper ion catalyst, iron ion catalyst and nickel catalyst.
Further, in the step (3), the cell desorption treatment includes a pancreatin treatment.
The invention provides a method for endowing a substrate with a function of specifically recognizing mammalian cells, which has the main advantages that:
(1) According to the invention, mammalian cells are used for participating in polymerization reaction for the first time, the chain sequence of the polymer is regulated, the chain sequence matched with the cells is screened out by the cells, the specific polymer brush is prepared, and the mammalian cells do not need pretreatment such as fixation;
(2) Compared with antibody specific surfaces, the polymer brush surface has the advantages of simple preparation, low cost and high stability, and the surface can be repeatedly recycled.
Drawings
FIG. 1 is a flow chart of example 2 of a method of imparting specific mammalian cell recognition function to a substrate according to the present invention;
FIG. 2 is a statistical thickness chart of the silicon wafer after treatment in examples 1 and 2 according to the method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention;
FIG. 3 is a statistical graph of water contact angles of treated silicon wafers in examples 1 and 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention;
FIG. 4 is a diagram showing the elemental analysis of the specific polymer surface in example 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention;
FIG. 5 is a fluorescence image and a cell morphology change image after cell desorption treatment in example 2 according to a method for imparting a substrate with a function of specifically recognizing mammalian cells of the present invention;
FIG. 6 is a statistical chart of the capture of cells by a specific functional surface in a complex mixed sample in example 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention;
FIG. 7 is a graph showing the reproducibility of the method for imparting a function of specifically recognizing mammalian cells to a substrate according to the present invention on a specific functional surface in example 3.
Detailed Description
The invention provides a method for endowing a substrate with a function of specifically recognizing mammalian cells, which selects an effective and simple polymerization method which can rapidly react in aqueous solution at room temperature without additional deoxidation in the presence of cells, utilizes surface-initiated polymerization to modify the surface of the substrate, washes off the cells, separates the cells from a polymer brush on the surface, and obtains the specific polymer brush surface of a target recognition template cell. See in particular fig. 1.
The specific affinity of the specific surface to the template cells is not dependent on the antibody, but is achieved based on the composition, proportion, arrangement and the like of the monomer of the affinity cells and the monomer of the non-affinity cells in the polymer chain thereon. The cell directly participates in polymerization, and the arrangement of monomers in a polymer chain is regulated and controlled, so that the purpose of screening is achieved.
Therefore, the invention firstly attaches the initiator to the surface of the substrate, then adds the proper monomer, and constructs the functional surface with cell selective recognition through a mild polymerization mode. The specific technical scheme is as follows:
1) Substrate surface modification initiator
In the step, a proper base material can be selected according to an application scene, and the surface of the base material is modified by using a surface-initiated ATRP reagent. The main method comprises the following steps: functionalization of the substrate surface by silane, functionalization of the substrate surface by esterification, functionalization of the substrate surface by thiol and gold, and the like, for example, adding an ATRP primer in the (co) polymerization process of a solid carrier. The substrate aiming at the silicon and silicate surface can fix the surface-initiated ATRP reagent on the surface of the substrate by means of silane coupling; the initiator can be modified on the substrate containing gold through the reaction of sulfydryl and gold; for some polymer substrates, the initiator can be modified by esterification through a large number of hydroxyl groups on the surface, and the hydroxyl groups on the surface can be self-functional groups obtained by activation and can also be obtained by deposition of exogenous sources such as dopamine. Specific examples are as follows:
silicon chip: performing ultrasonic treatment with acetone and water for 5-60min alternately, activating the surface (hydroxylation treatment is used here), washing with heated Piranha solution for 0.5-3 hr, washing with large amount of water, and blowing with inert gas. Then placing the mixture into 0.1mmol-1mol of anhydrous toluene solution of an Initiator (SI-Initiator, silicon surface Initiator) for 12-36h, cleaning the anhydrous toluene or other anhydrous organic solvents capable of dissolving the Initiator for 2-5 times to remove the non-adhered Initiator, and drying the mixture by inert gas for subsequent Atom Transfer Radical Polymerization (ATRP) initiated on the surface.
Gold sheet: performing ultrasonic treatment with acetone and water for 5-60min alternately, and hydroxylating. Exposing clean gold surfaceExposed to 0.1mmol-1mol 2-mercaptoethanol solution at room temperature for 12-36 hours to form Au-OH surface. The modified gold surface was washed with deionized water and ethanol and dried in a stream of inert gas. The freshly prepared Au-OH surface was immersed in a solution of 3-aminopropyltrimethoxysilane (0.1 mmol-1 mol) in a solution of ethanol (95 vol%). After 12-36h of treatment at room temperature, the surface was washed sequentially with ethanol, toluene and acetone and then dried with a stream of inert gas. The gold surface obtained was kept at 52 ℃ for 2-10 hours to form an amino terminated silanized surface Au-NH 2 The surface is reacted with chloroacetyl chloride to form a gold surface Au-Cl functionalized with a short chain silylation initiator for subsequent surface initiated Atom Transfer Radical Polymerization (ATRP).
Polyamide: formaldehyde activates the surface amide groups to produce an N-methylol polyamide membrane (Nylon-OH), followed by reaction of the hydroxyl groups with 2-bromoisobutyryl bromide (BIBB) to produce a 2-bromoisobutyryl fixed membrane (Nylon-Br) for subsequent surface initiated Atom Transfer Radical Polymerization (ATRP).
2) Construction process of specific polymer surface
The mammalian cells used in this step are cultured and passaged following the cell type. For example, cancer cells are cultured in RPMI-1640 medium or high-sugar DMEM medium at 37 deg.C and 5% CO before polymerization 2 。
The monomers of the compatible cells and the monomers of the incompatible cells used in this step are selected according to the cell characteristics. Depending on the cell characteristics, the cell-compatible monomers may be selected from sugar monomers that bind to sugar receptors on the cell surface or charged monomers that electrostatically interact with the cell surface. The cell-incompatible monomers may be selected from zwitterionic monomers and uncharged water-soluble neutral monomers. Alternately soaking the substrate modified with the initiator in 75% ethanol and sterile water for 3 times or more, planting cells on the substrate, adhering for 1-10h, and incubating with the monomer of the affinity cells for 0.5-3h. Placing the incubated substrate in a weighing bottle, adding two monomers (the ratio of the monomer of the compatible cell to the monomer of the incompatible cell is 1. And (3) alternately washing the reacted base material with 0.5-20% SDS aqueous solution and competitive sugar aqueous solution or salt solution for more than 2 times to remove residual cells, finishing modification of the base material, and finally obtaining the base material with the polymer brush surface.
3) Regeneration treatment of specific polymeric surfaces
The substrate with the polymer brush surface is placed in a sample (such as a blood sample, other body fluid samples and other samples needing analysis of target cells) for specific detection and capture of cells, after the analysis is finished, cell desorption treatment is carried out, and 0.5-20% SDS aqueous solution and competitive sugar water solution or salt solution are used for alternately washing for more than 2 times to remove adsorbed cells. Regeneration of specific surface functionalization is achieved.
In the above steps, the substrate can be selected from inorganic substrates such as silicon wafer, glass, gold sheet, etc., and polymer substrates such as polystyrene, polyvinyl chloride, polyurethane, nylon, acrylate, polyetheretherketone, polylactic acid, etc.
The substrate surface activation method is selected according to the substrate, and comprises hydroxylation treatment, carboxylic acid activation, amination treatment, sulfhydrylation treatment, gold deposition and the like. The hydroxylation treatment comprises endogenous hydroxylation treatment modes of converting functional groups of the base material into hydroxyl groups by plasma treatment, piranha solution treatment, formaldehyde activation and the like, and hydroxylation treatment modes introduced by exogenous modes of depositing polydopamine, polymerizing monomers with hydroxyl groups and the like.
The initiator can be any one of 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, 2- (2-bromoisobutyloxy) ethyl methacrylate, 2-bromo-2-methylpropanoic acid bromine and 2-bromoisobutyryl bromide (BIBB), and is selected according to a substrate in actual operation.
The cell-compatible monomer may be any one of a saccharide derivative such as a glucose derivative, a mannose derivative and a galactose derivative, and a charged monomer such as N- [3- (dimethylamino) propyl ] methacrylamide, methacryloyloxyethyl trimethylammonium chloride or the like, and is preferably a glucose derivative. The monomer of the incompatible cells can be selected from any one of zwitterions such as polyphosphocholine, polysulfonic acid betaine, polycarboxylic acid betaine and the like and uncharged water-soluble monomers such as polyethylene glycol and derivatives thereof, and the preferable monomer is polysulfonic acid betaine.
The catalytic system can be selected from cuprous ion catalyst, copper ion catalyst, iron ion catalyst, nickel catalyst, etc., preferably cuprous ion catalyst.
Cell desorption treatment includes pancreatin treatment, involving cell surface competition with monomers that are more cell-compatible, and influencing polymer conformation by changing light, heat, magnetism, and the like.
The inert gas is selected from any one of argon, nitrogen, helium and neon, and preferably argon.
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying the present invention are described in detail below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Chemical reagents:
d- (+) -glucosamine hydrochloride, 99%, TCI;
acryloyl chloride, 95%, aladin;
2- (N-3 sulfopropyl-N, N-dimethylammonium) ethyl methacrylate (medisa), 97%, sigma-Aldrich;
3-trimethoxysilyl propyl 2-bromo-2-methylpropionate (SI-Initiator), 95%, mackin;
cuprous bromide (CuBr) 2 )，99％，Mackin；
Tris [2- (dimethylamino) ethyl ] amine (Me 6 TREN), 98%, TCI;
30% of hydrogen peroxide and a Shanghai Linfeng chemical reagent;
concentrated sulfuric acid, AR, chinese medicine;
paraformaldehyde, 95%, sigma-Aldrich;
high purity nitrogen (N) 2 ) 99.999%, shanghai five-steel gas;
anhydrous methanol, AR, chinese medicine;
potassium carbonate, AR, chinese medicine;
dichloromethane, AR, national medicine;
anhydrous sodium sulfate, AR, chinese medicine;
penicillin streptomycin mixed solution (100 ×), biochemical reagent, solarbio;
standard Fetal Bovine Serum (FBS), biochemical, gibco;
0.25% trypsin solution (1 ×), biochemical, thermo Hyclone;
phosphate buffered saline (PBS, pH = 7.4) (10 ×), biochemical reagent, thermo Hyclone;
trypan blue solution (0.4%), biochemical, sigma-Aldrich;
calcein AM, biochemical reagents, east benevolence chemical technology ltd;
high-glucose DMEM medium, biochemical reagent, thermo Hyclone;
modified RPMI-1640 medium, biochemical reagent, thermo Hyclone;
triton X-100, biochemical reagents, shanghai-derived leaf Biotech Co., ltd.;
4', 6-diamidino-2-phenylindole (DAPI), biochemical, solarbio;
phalloidin-Fluorescein Isothiocyanate (FITC), biochemical reagent, biyuntian Biotech limited;
testing instruments and conditions:
an ellipsometer: the thickness of the polymer brush on the surface of the silicon wafer was determined by spectroscopic ellipsometry, us j.a. woollam co. The test object is the thickness of the sugar polymer brush in a dry state at room temperature. The results of the experiment were analyzed by Complete EASE software to obtain thickness values.
Water contact angle meter: the water wettability of the surfaces of the different glycomers was characterized by means of a water contact angle meter. The drop-out method was used to measure the water contact angles of different surfaces: dropping 2uL of water drops on the surfaces of the three groups of samples dried by the nitrogen, fitting the samples by using software after the samples are stable, and reading the specific numerical value of the water contact angle.
XPS: the sample is dried in a vacuum drying oven for one day, then the XPS test is carried out, the takeoff angle is 90 degrees, the excitation source is A1K alpha (1486.6 Ev), and the test data are analyzed and processed by XPS Peak 4.1.
EDS: using the sample with N 2 And after drying, placing on an SEM, selecting a proper area for element scanning, and obtaining a test result.
An Atomic Force Microscope (AFM) was used, and a Bruker Multimode 8 atomic force microscope was used, and the imaging mode was tapping.
Example 1
This embodiment demonstrates a method for imparting specific mammalian cell recognition functionality to a substrate by:
priming agent is connected to surface of base material
Silicon wafers for silicon and silicate based substrates are exemplified, gold flakes for gold containing surfaces are exemplified, and polyamide for polymer surfaces are exemplified.
Silicon chip: subjecting to ultrasonic treatment with acetone and water for 15min, and adding Piranha solution (instant fish solution H) at 90 deg.C 2 SO 4 :H 2 O 2 = 7) washing for 2h, and after washing with a large amount of water, N 2 And (5) drying. And then placing the mixture in an anhydrous toluene solution of 4Mm Initiator (SI-Initiator, surface Initiator) for 24h, cleaning the mixture with anhydrous toluene for 5 times to remove the Initiator which is not adhered, and drying the mixture with nitrogen for later use. Referring to FIGS. 2 and 3, FIG. 2 is a thickness statistical chart of a silicon wafer after processing according to an embodiment 1 and 2 of a method for imparting a function of specifically recognizing mammalian cells to a substrate according to the present invention; FIG. 3 is a statistical graph of water contact angles of treated silicon wafers in examples 1 and 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention. Successful modification of the initiator to the silicon surface was confirmed by the changes in thickness and water contact angle in fig. 2 and 3. Referring to FIG. 4, FIG. 4 is a diagram showing the elemental analysis of the specific polymer surface in example 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate of the present invention. The success of the initiator was also confirmed by the presence of bromine signal peaks as shown in FIG. 4And (5) modifying.
Gold sheet: carrying out ultrasonic treatment with acetone and water for 5-60min alternately, and hydroxylating. The clean gold surface was exposed to a 0.1mmol-1mol solution of 2-mercaptoethanol for 24 hours at room temperature to form an Au-OH surface. The modified gold surface was washed with deionized water and ethanol and dried in a stream of inert gas. The freshly prepared Au-OH surface was immersed in a solution of 3-aminopropyltrimethoxysilane (0.1 mmol-1 mol) in a solution of ethanol (95 vol%). After 24h of treatment at room temperature, the surface was washed successively with ethanol, toluene and acetone and then dried with a stream of inert gas. The gold surface obtained was kept at 52 ℃ for 5 hours to form an amino-terminated silanized surface Au-NH 2 The surface is reacted with chloroacetyl chloride to form a gold surface Au-Cl functionalized with a short chain silylation initiator for subsequent surface initiated Atom Transfer Radical Polymerization (ATRP).
Polyamide: formaldehyde activates the surface amide groups to produce an N-methylol polyamide membrane (Nylon-OH), followed by reaction of the hydroxyl groups with 2-bromoisobutyryl bromide (BIBB) to produce a 2-bromoisobutyryl-fixed membrane (Nylon-Br) for subsequent surface-initiated Atom Transfer Radical Polymerization (ATRP).
Example 2
This embodiment demonstrates a method of imparting specific recognition of mammalian cell function to a substrate by:
construction process of specific polymer surface
L929 and Hela cells are exemplified. L929 cells were cultured in RPMI-1640 medium containing 10% FBS,100U/mL penicillin and 100ug/mL streptomycin, and Hela was cultured in high-sugar DMEM medium containing 10% FBS,100U/mL penicillin and 100ug/mL streptomycin, at 37 ℃ for each culture environment, and 5% CO 2 . When the cell density in the culture flask reaches about 90%, the cells are subjected to digestion treatment: after the cells are rinsed by 1mL sterile PBS, 1mL pancreatin is added, when the cells become round and bright under a microscope, 2mL culture medium is added to stop digestion, the cells are centrifuged at 1200rpm/min for 5min, then the supernatant is discarded, and the cell precipitates are uniformly blown by the culture medium and can be directly used or passaged.
75% ethanol and none for base material modified with initiatorThe bacteria and water are alternately soaked for 3 times, wherein the time is 15min,10min and 5min. 5 ten thousand cells (Hela/L929) were plated on the plates and incubated for 2h with 1mg/mL AGA for 0.5h. The incubated pieces were placed in a weighing flask, and 279.4mg MEDSA,233.1mg AGA,27.5uL ME added 6 TREN，14.3mg CuBr，6mL H 2 And O, taking out the silicon wafer after 0.5h to stop the reaction. The reaction-completed sheet was washed 6 times with 2% SDS aqueous solution and 1mg/mL mannose aqueous solution alternately to remove residual cells. Samples were retained at each step and stained with DAPI: fixing with paraformaldehyde for 10min, washing with PBS for 3 times, adding appropriate amount of DAPI staining solution, spreading on sample surface, sucking off dye after 5min, washing with PBS for 3 times, sucking dry with filter paper, and observing under microscope. The cell state of each step was observed by SEM.
Non-template control polymerization with control (Non-T): the polymerization was carried out in the absence of cells, the initiator-modified substrate was placed in a weighing flask, 279.4mg MEDSA,233.1mg AGA, 27.5. Mu. LME were added 6 TREN，14.3mg CuBr，6mL H 2 And O, taking out the sheet after 0.5h, and stopping the reaction.
Referring to fig. 1, fig. 1 is a flow chart of an embodiment 2 of the method for providing a substrate with a function of specifically recognizing mammalian cells according to the present invention, as shown in fig. 1, the whole process includes cell culture, in situ surface-induced polymerization, and removal of template cells after polymerization. Continuing with fig. 2 and 3, the successful modification of the polymer to the substrate surface is evidenced by the changes in thickness and water contact angle in fig. 2 and 3. Continuing with FIG. 4, the occurrence of the monomer signal peak also confirms successful modification of the polymer. Referring to fig. 5, fig. 5 is a fluorescence image and a cell morphology change image after cell desorption treatment in example 2 according to a method for imparting a substrate with a function of specifically recognizing mammalian cells of the present invention. As shown in FIG. 5, it can be seen from the fluorescence image and the change of the surface morphology before and after, the cells can be well removed from the surface of the polymer brush after the desorption treatment.
Example 3
Regeneration treatment of specific polymeric surfaces
The cells are desorbed by a pancreatic method. Washing the functional surface used in step 2) with water to remove serum and prevent serum from inhibiting the cell desorption process by pancreatin, immersing with pancreatin, standing for 15min, and washing with 2% SDS aqueous solution and 1mg/mL mannose aqueous solution alternately for 6 times to remove residual cells. And putting the test piece into use again to evaluate the specific performance. Referring to FIG. 6, FIG. 6 is a statistical chart showing the capture of cells by specific functional surfaces in a complex mixed sample in example 2 according to a method for imparting a function of specifically recognizing mammalian cells to a substrate according to the present invention. As shown in FIG. 6, the capture capacity of the specific polymer surface on the template cells is stronger than that of the non-template cells, such as L929-T on L929 and the number of L929 captured in the mixed sample of Hela is more than that of Hela. Referring to FIG. 7, FIG. 7 is a graph showing the reproducibility of a specific functional surface in example 3 according to a method of the present invention for imparting a function of specifically recognizing mammalian cells to a substrate. As shown in FIG. 7, the cells adhered to the surface were removed cleanly by treatment with pancreatin and SDS, and the specific capture efficiency remained above 90% in many cycles, indicating a good regeneration effect.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A method of imparting a substrate with a function of specifically recognizing mammalian cells, the method comprising the steps of:
(1) Substrate surface modification initiator: modifying the surface of a base material by using a surface initiated ATRP reagent to obtain the base material modified with an initiator;
(2) Construction process of specific polymer surface: the base material modified with the initiator is soaked in 75% ethanol and sterile water alternately for more than or equal to 3 times, then cells are planted, after 1-10 hours of adhesion, the monomer having affinity with the cells is incubated for 0.5-3 hours to obtain an incubated base material, the incubated base material is placed in a weighing bottle, two monomers, a catalytic system and a buffer solution are added, after 0.5-10 hours, the base material is taken out to stop reaction, the base material is alternately cleaned for more than 2 times by 0.5-20% SDS (sodium dodecyl sulfate) aqueous solution and competitive sugar aqueous solution, or 0.5-20% SDS aqueous solution and salt solution to remove residual cells, and the base material with the surface of a polymer brush is obtained;
(3) Regeneration treatment of specific polymeric surfaces: and (2) placing the substrate with the polymer brush surface in a sample for specific detection and capture of cells, performing cell desorption treatment after analysis is finished, and alternately washing for more than 2 times by using 0.5-20% SDS aqueous solution and competitive sugar aqueous solution or 0.5-20% SDS aqueous solution and salt solution to remove adsorbed cells so as to realize regeneration of specific surface functionalization.
2. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in the step (1), the modifying the surface of the base material with the surface-initiated ATRP reagent to obtain the base material modified with the initiator specifically comprises: the surface of a silicon and silicate base material is subjected to alternate ultrasonic treatment for 5-60min by using acetone and water, the silicon and silicate base material is cleaned by using a heated Piranha solution for 0.5-3h, the silicon and silicate base material is subjected to massive water washing and then dried by inert gas, the silicon and silicate base material is placed in an anhydrous toluene solution containing 0.1mmol-1mol of surface initiator for 12-36h, the silicon and silicate base material is cleaned for 2-5 times by using an anhydrous organic solvent capable of dissolving the surface initiator to remove the non-adhered surface initiator, and the base material modified with the initiator is obtained after the inert gas is dried by blowing.
3. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in the step (1), the modifying the surface of the base material with the surface-initiated ATRP reagent to obtain the base material modified with the initiator specifically comprises: alternately performing ultrasonic treatment on the surface of the gold-containing surface substrate for 5-60min by using acetone and water, exposing the clean gold-plated surface to 0.1-1mol 2-mercaptoethanol solution at room temperature for 1 min2-36 hours to form Au-OH surface, washing the Au-OH surface with deionized water and ethanol, and drying in inert gas stream, immersing the Au-OH surface in a solution of 0.1mmol-1mol of 3-aminopropyltrimethoxysilane, further in a 95vol% ethanol solution, treating at room temperature for 12-36 hours, washing the surface with ethanol, toluene and acetone in order, then drying with inert gas stream, maintaining the obtained gold-plated surface at 52 ℃ for 2-10 hours to form Au-NH 2 Surface of said Au-NH 2 The surface reacts with chloroacetyl chloride to form an Au-Cl surface, and the substrate modified with the initiator is obtained.
4. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in the step (1), the modifying the surface of the base material with the surface-initiated ATRP reagent to obtain the base material modified with the initiator specifically comprises: activating the surface of a polymer substrate by formaldehyde to generate an N-hydroxymethyl polyamide membrane, and adding 2-bromoisobutyryl bromide to react to generate a 2-bromoisobutyryl fixed membrane to obtain the substrate modified with the initiator.
5. The method of claim 1, wherein the step of providing the substrate with a function of specifically recognizing mammalian cells comprises: in the step (1), the substrate is any one of an inorganic substrate or a polymer substrate.
6. The method of claim 1, wherein the step of providing the substrate with a function of specifically recognizing mammalian cells comprises: in the step (1), the modification method comprises any one of hydroxylation treatment, carboxylic acid activation, amination treatment, sulfhydrylation treatment and gold deposition.
7. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in step (1), the surface-initiated ATRP reagent is any one of 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, 2- (2-bromoisobutyloxy) ethyl methacrylate, 2-bromo-2-methylpropanoic acid bromide, 2-bromoisobutyryl bromide (BIBB).
8. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in step (2), the cells are first cultured in RPMI-1640 medium or high-glucose DMEM medium in each case at 37 ℃ and 5% CO 2 。
9. The method of claim 1, wherein the step of providing the substrate with a specific recognition of mammalian cell function comprises: in step (2), the two monomers are a monomer with affinity to cells and a monomer without affinity to cells, and the ratio is 1:20 to 20:1, the monomer of the compatible cell comprises any one of glucose derivatives, mannose derivatives, galactose derivatives, N- [3- (dimethylamino) propyl ] methacrylamide and charged monomers of methacryloyloxyethyl trimethyl ammonium chloride, the monomer of the incompatible cell comprises any one of poly phosphorylcholine, poly-sulfobetaine, poly-carboxylic betaine and polyethylene glycol, and the catalytic system comprises any one of cuprous ion catalyst, copper ion catalyst, iron ion catalyst and nickel catalyst.
10. The method of claim 1, wherein the step of providing the substrate with a function of specifically recognizing mammalian cells comprises: in step (3), the cell desorption treatment comprises pancreatin treatment.
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