CN115308992A - Protein patterning preparation method - Google Patents

Abstract

The invention belongs to the technical field of protein positioning assembly and patterning, and provides a protein patterning preparation method aiming at the problems of complex operation process and easy deformation of protein in the conventional protein patterning method. Preparing a polymer brush with enough thickness, etching the surface of the polymer brush by using an atomic force needle tip, changing the thickness of the polymer brush to obtain areas with different thicknesses, and forming the surface patterned polymer brush; and soaking the surface of the patterned polymer brush in a protein solution at room temperature, wherein the different thicknesses of the polymer brush have different adhesion forces to protein, and washing the patterned polymer brush by using ultrapure water after soaking for a period of time to obtain a protein patterned structure formed on the surface of the polymer brush. The method adopts AFM etching technology to change the thickness of the polymer brush, utilizes different thicknesses to realize protein patterning by utilizing different protein adhesion forces, has simple preparation process of the protein patterning, mild conditions and short period, and can protect the activity of protein.

Description

Protein patterning preparation method

Technical Field

The invention belongs to the technical field of protein positioning assembly and patterning, and particularly relates to a protein patterning preparation method.

Background

Proteins are known to exhibit patterned structures in vivo, but protein patterning preparation methods are lacking. Photoetching and microemulsion casting patterning technologies are the main means for controllable adsorption of protein on the surface of the current material. For example, patent application No. CN201910166615.4 discloses a method for preparing a collagen film with 3D micro-patterns, (1) forming 3D micro-patterns on a silicon wafer by using a mask with 3D micro-patterns through a photolithography technique, then casting PDMS prepolymer on the silicon wafer, and obtaining a PMDS substrate with 3D micro-patterns after cross-linking and curing; (2) Dissolving collagen in an acidic solution, adding water, uniformly stirring to obtain a collagen solution, and then adding a cross-linking agent for cross-linking reaction; (3) And (3) removing bubbles from the crosslinking reaction product obtained in the step (2), pouring the crosslinking reaction product on the PDMS substrate obtained in the step (1), air-drying to form a film, and peeling the film from the PDMS substrate to obtain the collagen film with the 3D micro-pattern. The patent application with the application number of CN201210511033.3 discloses a cell patterning chip preparation method based on an avidin-biotin system, wherein a method for preparing a biophylactic hexamethyldisilazane dot array and a biological passivated polyethylene glycol patterning area by utilizing a photoetching and chemical deposition method is adopted; then forming a sandwich structure of bovine serum albumin-biotin, avidin and biotinylated cells on the hexamethyldisilazane dot array, thereby directionally arranging the cells and realizing the separation and positioning of the cells. In addition, an electric field induction method has also emerged. The patent application with the application number of CN201911214443.X discloses a method for patterning the surface of a biological protein film, which utilizes the principle that the conformation of biological protein can be converted under the action of an external electric field, induces the conformation conversion of the biological protein film (for example, amorphous state is converted into crystalline state or carbonization) through a strong electric field in a metamaterial array gap, so that the biological protein film in an electric field action area is crosslinked, and realizes pattern direct writing on the surface of the biological protein film by moving the biological protein film and patterning the surface of the biological protein film by a developing solution. The existing protein patterning method has the problems of complex operation process, limited protein application types, protein changeability and the like when protein is subjected to additional chemical modification.

Disclosure of Invention

Aiming at the problems of complex operation process and easy deformation of protein in the existing protein patterning method, the invention provides a protein patterning preparation method.

The protein patterning preparation method comprises the following steps:

(1) Preparing a polymer brush surface;

(2) Atomic force microscope tip etching

Etching the surface of the polymer brush by using an atomic force needle tip, changing the thickness of the polymer brush to obtain areas with different thicknesses, and forming the polymer brush with the patterned surface;

(3) Protein surface adsorption to form patterned structure

And soaking the surface of the patterned polymer brush in a protein solution at room temperature, wherein the adhesion force of different thicknesses of the polymer brush to protein is different, and washing the patterned polymer brush by using ultrapure water after soaking for a period of time to obtain a protein patterned structure formed on the surface of the polymer brush.

Further, the thickness of the surface of the polymer brush prepared in the step (1) is thick enough to enable the atomic force needle tip to etch on the surface of the polymer brush, so that different thicknesses can be formed, and the different thicknesses have different adhesion forces to the protein.

As a further improvement, the step (1) of preparing the polymer brush surface comprises the following steps: heating n-tetradecene and triethoxysilane to 38-42 ℃ in an argon atmosphere, adding a karstedt catalyst, stirring in a sealed tube at 38-42 ℃ for 1.5-2.5 h, adding cyclohexane and propyl carbonate, performing phase separation, concentrating a cyclohexane phase, and purifying by using silica gel FC to obtain a functionalized silicon surface; and carrying out polymerization reaction on the functionalized silicon surface, placing the self-assembled monomolecular film of the wafer containing the initiator into a monomer, and initiating the polymerization reaction to prepare the polymer brush.

As a further improvement, the monomer is a styrene monomer; the wafer self-assembly monomolecular film containing the initiator is a wafer self-assembly monomolecular film containing the alkoxy amine NMP initiator.

As a further improvement, increasing the concentration of alkoxyamine can shorten the target chain length of the silicon surface-bound polymer, thereby producing a polymer brush of lesser thickness, and conversely, a polymer brush of greater thickness.

As a further improvement, a polystyrene brush is prepared using an initiator at a concentration of 0.063 to 0.1mol% relative to styrene, giving a polystyrene brush thickness of 22 to 50 nm; polystyrene brushes were prepared using 0.2 to 0.4mol% of initiator relative to styrene, giving polystyrene brushes with a thickness of 5 to 10 nm.

As a further improvement, the step (2) comprises the following specific operation processes: after the atomic force microscope was turned on, the silicon contact mode was selected, k ≈ 42Nm-1, scan rate =0.1, set point =0.1, scan size =20m, date category off, Z range =200Nm, vertical deflection ≈ 0, horizontal deflection ≈ 0, rec3-v1.C file from Di in nanoscript, and single click "run" on the macro, repeated 3 times.

As a further improvement, the step (3) is soaked for 8 to 32 hours.

As a further improvement, the protein solution in the step (3) is a pure SDF-1 protein solution.

Has the advantages that: the invention adopts AFM etching technology to change the thickness of the polymer brush, and realizes protein patterning by utilizing different thicknesses to realize different protein adhesion forces; the protein patterning preparation process is simple, the condition is mild, the period is short, and the activity of the protein can be protected.

Drawings

FIG. 1 is a schematic view showing a process for preparing a polystyrene polymer brush of an example;

FIG. 2 is a flow chart of an example for preparing a protein patterned structure using a polymer brush;

FIG. 3 is a schematic illustration of example protein patterning preparation;

FIG. 4 is an immunofluorescence assay of the patterned structure of the SDF-1 protein of the example;

FIG. 5 is a graph showing the effect of the patterned structure of the SDF-1 protein of the example on the migration of TCam-2 cells.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Examples

(1) Preparation of Polymer Brush surfaces

N-tetradecene (0.24g, 0.43mmol, 1.0eq) and triethoxysilane (71. Mu.L, 0.39mmol, 0.90eq) were heated to 40 ℃ under an argon atmosphere, karstedt's catalyst (0.30mL, 10. Mu. Mol,1 mol%) was added, stirred in a sealed tube at 40 ℃ for 2h, and then cyclohexane and propyl carbonate were added to effect phase separation. The cyclohexane phase was concentrated and then purified with silica gel FC to give a functionalized silicon surface.

Then, the silicon surface is polymerized, the self-assembled monomolecular film (sam) containing the alkoxy amine NMP initiator is placed in a clean styrene monomer or a butyl acrylate monomer, the alkoxy amine is gradually consumed, and the alkoxy amine needs to be supplemented in the reaction process to provide enough concentration of the oxynitride, which is necessary for maintaining the polymerization mediated by the oxynitride. The reaction was carried out at 105 ℃ for 24 hours to obtain a polystyrene brush (PS brush).

In addition, unbound polymers, which are readily analyzed by Gel Permeation Chromatography (GPC), are formed during the reaction with the aid of an alkoxyamine NMP initiator, and the soluble polymers are used as an estimate of the length of the polymer chains bound to the surface.

The thickness of the polymer brush was determined by AFM experiments. PS brushes were prepared using an initiator concentration of 0.063mol% relative to styrene, giving PS brushes with a thickness of 50 nm. The target chain length of the silicon surface-bound polymer can be shortened by increasing the concentration of the alkoxyamine, and surfaces with PS thicknesses of 5nm and 10nm can be easily prepared. At an alkoxyamine concentration of 0.4mol%, a dense polystyrene polymer brush having a thickness of about 5nm was produced, an alkoxyamine concentration of 0.2mol% enabling a 10nm thick polymer brush to be produced, and an alkoxyamine concentration of 0.1mol% enabling a 22nm thick polymer brush to be produced.

(2) Atomic force microscope tip etching

And etching the surface of the polystyrene polymer brush with the thickness of 50nm by using an atomic force needle tip to change the thickness of the surface of the polystyrene polymer brush into 10nm, and preparing the surface patterned polymer brush.

The specific operation process comprises the following steps: the silicon contact mode (k ≈ 42 Nm-1) is selected after the atomic force microscope is turned on. The method specifically comprises the following steps: scan rate =0.1, set point =0.1, scan size =20m, date category select off, Z range =200nm, vertical deflection ≈ 0, horizontal deflection ≈ 0. The rec3-v1.C file was used from Di in the nanoscript and the "run" was clicked on the macro, repeating 3 times.

(3) Protein surface adsorption to form patterned structure

The surface of a patterned polystyrene brush (1 cm × 1 cm) was immersed in 20 μ L of pure SDF-1 protein solution at room temperature for 24 hours, and then the patterned polystyrene brush was rinsed with ultrapure water to obtain a protein patterned structure formed on the surface of the polystyrene brush.

The samples were analyzed by fluorescence microscopy. As shown in FIG. 4, immunofluorescence experiments of the SDF-1 protein patterned structure were performed using a fluorescence microscope. (a) Coating the surface of the patterned polystyrene polymer brush by using IgG antibody; (b) SDF-1 protein is adsorbed on the surface of the IgG-coated patterned polystyrene polymer brush; (c) MAB350 coats the patterned polystyrene polymer brush surface; (d) SDF-1 protein was adsorbed onto the MAB350 coated patterned polystyrene polymer brush surface. Scale 20 μm. Experiments show that the protein patterning structure is successfully prepared by the method.

(4) Functional Effect of SDF protein patterning Structure

After SDF protein patterning for 4 and 24 hours, the Tcam-2 cells in the dishes were BrdU stained and the stained cell density was analyzed using Image J. As shown in FIG. 5, the effect of the SDF-1 protein patterned structure on the migration of TCam-2 cells was observed at 4 hours (a) and 24 hours (b). The number of replicates was 4, with statistical significance of p < 0.05. It can be seen that the number of cells growing on the SDF protein patterned surface is significantly increased.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (10)

1. The protein patterning preparation method is characterized by comprising the following steps:

(1) Preparing the surface of a polymer brush;

(2) Atomic force microscope tip etching

Etching the surface of the polymer brush by using an atomic force needle tip, changing the thickness of the polymer brush to obtain areas with different thicknesses, and forming a surface patterning polymer brush;

(3) Protein surface adsorption to form patterned structure

And soaking the surface of the patterned polymer brush in a protein solution at room temperature, wherein the adhesion force of different thicknesses of the polymer brush to protein is different, and washing the patterned polymer brush by using ultrapure water after soaking for a period of time to obtain a protein patterned structure formed on the surface of the polymer brush.

2. The protein patterning preparation method according to claim 1, wherein the polymer brush prepared in step (1) has a surface thickness that is sufficiently thick to allow atomic force tips to form regions of different thicknesses when the surface of the polymer brush is etched, the regions of different thicknesses having different adhesion to the protein.

3. The method for preparing a protein pattern according to claim 1, wherein the step (1) of preparing the surface of the polymer brush is as follows: heating n-tetradecene and triethoxysilane to 38-42 ℃ in an argon atmosphere, adding a karstedt catalyst, stirring in a sealed tube at 38-42 ℃ for 1.5-2.5 h, adding cyclohexane and propyl carbonate, performing phase separation, concentrating a cyclohexane phase, and purifying by using silica gel FC to obtain a functionalized silicon surface; and carrying out polymerization reaction on the functionalized silicon surface, placing the self-assembled monomolecular film of the wafer containing the initiator into a monomer, and initiating the polymerization reaction to prepare the polymer brush.

4. The method according to claim 3, wherein the monomer is a styrene monomer; the self-assembled monomolecular film containing the initiator is a self-assembled monomolecular film containing the alkoxy amine NMP initiator.

5. A protein patterning preparation method according to claim 4, wherein increasing the concentration of the alkoxyamine enables a reduction in the target chain length of the silicon surface bound polymer, thereby producing a polymer brush of smaller thickness, and conversely, a polymer brush of greater thickness.

6. The protein patterning preparation method according to claim 5, wherein a polystyrene brush is prepared using an initiator at a concentration of 0.063 to 0.1mol% relative to styrene, resulting in a polystyrene brush having a thickness of 22 to 50 nm; polystyrene brushes were prepared using an initiator concentration of 0.2 to 0.4mol% relative to styrene, giving polystyrene brushes with a thickness of 5 to 10 nm.

7. The method for preparing protein patterns according to claim 1, wherein the step (1) of reacting n-tetradecene with triethoxysilane is performed at 40 ℃ for 2 hours.

8. The protein patterning preparation method according to claim 1, wherein the step (2) is a specific operation process: after the atomic force microscope was turned on, the silicon contact mode was selected, k ≈ 42Nm-1, scan rate =0.1, set point =0.1, scan size =20m, date category off, Z range =200Nm, vertical deflection ≈ 0, horizontal deflection ≈ 0, rec3-v1.C file from Di in nanoscript, and single click "run" on macro, repeated 3 times.

9. The protein patterning preparation method according to claim 1, wherein the step (3) is performed by soaking for 8 to 32 hours.

10. The protein patterning preparation method of claim 1, wherein the protein solution of step (3) is a pure SDF-1 protein solution.

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