Protein patterning preparation method

Abstract

The present invention belongs to the technical field of protein positioning assembly and patterning. In view of the problems of complex operation process and easy deformation of proteins in existing protein patterning methods, the present invention provides a protein patterning preparation method. Prepare a polymer brush with sufficient thickness, etch the surface of the polymer brush with an atomic force needle tip, change the thickness of the polymer brush, obtain areas of different thicknesses, and form a surface patterned polymer brush; soak the patterned polymer brush surface in a protein solution at room temperature, and the different thicknesses of the polymer brush have different adhesion to the protein. After soaking for a period of time, rinse the patterned polymer brush with ultrapure water to obtain a protein patterned structure formed on the surface of the polymer brush. The present invention uses AFM etching technology to change the thickness of the polymer brush, and uses different thicknesses to achieve protein patterning. The protein patterning preparation process is simple, the conditions are mild, the cycle is short, and the activity of the protein can be protected.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein localization assembly and patterning, and in particular relates to a protein patterning preparation method. Background Art

[0002] It is known that proteins present patterned structures in vivo, but there is a lack of protein patterning preparation methods. Photolithography and microemulsion casting patterning technology are currently the main means of controllable adsorption of proteins on material surfaces. For example, a patent application with application number CN201910166615.4 discloses a method for preparing a collagen film with a 3D micropattern, (1) using a mask with a 3D micropattern, a 3D micropattern is formed on a silicon wafer by photolithography, and then a PDMS prepolymer is cast on the silicon wafer, and a PMDS substrate with a 3D micropattern is obtained after cross-linking and curing; (2) collagen is dissolved in an acidic solution, water is added and stirred evenly to obtain a collagen solution, and then a cross-linking agent is added for cross-linking reaction; (3) the cross-linking reaction product of step (2) is degassed and cast on the PDMS substrate of step (1), and after air drying to form a film, the film is peeled off from the PDMS substrate to obtain a collagen film with a 3D micropattern. The patent application with application number CN201210511033.3 discloses a method for preparing a cell patterned chip based on an avidin-biotin system. This method uses photolithography and chemical deposition methods to prepare a biophilic hexamethyldisilazide dot array and a bio-passivated polyethylene glycol patterned area; then a sandwich structure of bovine serum albumin-biotin, avidin and biotinylated cells is formed on the hexamethyldisilazide dot array, thereby directional arrangement of cells to achieve cell separation and positioning. In addition, an electric field induction method has also appeared. The patent application with application number CN201911214443.X discloses a method for patterning the surface of a biological protein membrane. This method uses the principle that the conformation of biological proteins will change under the action of an external electric field, and induces the conformational change of the biological protein membrane (for example, from an amorphous state to a crystalline state or carbonization) through a strong electric field in the gap of a metamaterial array, thereby cross-linking the biological protein membrane in the electric field action area, and by moving the biological protein membrane, a pattern is directly written on the surface of the biological protein membrane, and the surface patterning of the biological protein membrane is achieved through a developer. Existing protein patterning methods require additional chemical modifications to proteins, but have many problems, such as complex operating procedures, limited protein application types, and protein variability. Summary of the invention

[0003] In view of the above problems of the existing protein patterning methods, such as the complicated operation process and the easy deformation of proteins, the present invention provides a protein patterning preparation method.

[0004] The protein patterning preparation method comprises the following steps:

[0005] (1) Preparation of polymer brush surfaces;

[0006] (2) Atomic force microscope tip etching

[0007] The surface of the polymer brush is etched with an atomic force needle tip to change the thickness of the polymer brush to obtain areas of different thicknesses, thereby forming a surface patterned polymer brush;

[0008] (3) Protein surface adsorption to form patterned structures

[0009] The patterned polymer brush surface is immersed in a protein solution at room temperature. The different thicknesses of the polymer brushes have different adhesion to the protein. After soaking for a period of time, the patterned polymer brush is rinsed with ultrapure water to obtain a protein patterned structure formed on the surface of the polymer brush.

[0010] Furthermore, the surface thickness of the polymer brush prepared in step (1) is thick enough so that when the atomic force needle tip etches on the polymer brush surface, different thicknesses can be formed, and the different thicknesses have different adhesion forces to the protein.

[0011] As a further improvement, the preparation method of the polymer brush surface in step (1) is as follows: heating n-tetradecene and triethoxysilane to 38-42° C. under an argon atmosphere, adding a karstedt catalyst, stirring at 38-42° C. in a sealed tube for 1.5-2.5 hours, then adding cyclohexane and propyl carbonate, performing phase separation, concentrating the cyclohexane phase, and then purifying it with silica gel FC to obtain a functionalized silicon surface; performing a polymerization reaction on the functionalized silicon surface, placing a wafer self-assembled monomolecular film containing an initiator in a monomer, initiating a polymerization reaction, and obtaining a polymer brush.

[0012] As a further improvement, the monomer is a styrene monomer; the initiator-containing wafer self-assembled monomolecular film is a wafer self-assembled monomolecular film containing an alkoxyamine NMP initiator.

[0013] As a further improvement, increasing the concentration of alkoxyamine can shorten the target chain length of the polymer bound to the silicon surface, thereby producing a polymer brush with a smaller thickness. Conversely, a polymer brush with a larger thickness can be produced.

[0014] As a further improvement, a polystyrene brush is prepared using an initiator with a concentration of 0.063 to 0.1 mol% relative to styrene, and a polystyrene brush with a thickness of 22 to 50 nm is obtained; a polystyrene brush is prepared using an initiator with a concentration of 0.2 to 0.4 mol% relative to styrene, and a polystyrene brush with a thickness of 5 to 10 nm is obtained.

[0015] As a further improvement, the specific operation process of step (2) is as follows: after the atomic force microscope is turned on, the silicon contact mode is selected, k≈42Nm-1, the scanning rate = 0.1, the set point = 0.1, the scanning size = 20m, the date category is selected off, the Z range = 200nm, the vertical deflection ≈0, the horizontal deflection ≈0, the rec3-v1.c file is used on Di from the nanoscript, and "run" is clicked on the macro to run, and repeated 3 times.

[0016] As a further improvement, the soaking in step (3) is performed for 8-32 hours.

[0017] As a further improvement, the protein solution in step (3) is a pure SDF-1 protein solution.

[0018] Beneficial effects: The present invention adopts AFM etching technology to change the thickness of the polymer brush, and utilizes the different adhesion of different thicknesses to proteins to achieve protein patterning; the protein patterning preparation process is simple, the conditions are mild, the cycle is short, and the activity of the protein can be protected. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation process of polystyrene polymer brushes in Example;

[0020] Figure 2 A flowchart of preparing a protein patterned structure using a polymer brush is shown in the following example;

[0021] Figure 3 Schematic diagram of protein patterning preparation in Example;

[0022] Figure 4 This is an immunofluorescence experiment of the patterned structure of SDF-1 protein in Example;

[0023] Figure 5 This is an example of the effect of SDF-1 protein patterned structure on the migration of TCam-2 cells. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to specific embodiments.

[0025] Example

[0026] (1) Preparation of polymer brush surfaces

[0027] n-Tetradecene (0.24 g, 0.43 mmol, 1.0 eq) and triethoxysilane (71 μL, 0.39 mmol, 0.90 eq) were heated to 40 °C under argon atmosphere, and Karstedt catalyst (0.30 mL, 10 μmol, 1 mol%) was added. In a sealed tube, the mixture was stirred at 40 °C for 2 h, and then cyclohexane and propyl carbonate were added for phase separation. The cyclohexane phase was concentrated and then purified by silica gel FC to obtain a functionalized silicon surface.

[0028] Next, the polymerization reaction is carried out on the functionalized silicon surface. The wafer self-assembled monolayer (SAM) containing alkoxyamine NMP initiator is placed in clean styrene monomer or butyl acrylate monomer. The alkoxyamine is gradually consumed. During the reaction, alkoxyamine needs to be added to provide sufficient concentration of nitroxide, which is necessary to maintain nitroxide-mediated polymerization. The reaction is carried out at 105°C for 24 hours to obtain a polystyrene brush (PS brush).

[0029] In addition, with the assistance of the alkoxyamine NMP initiator, unbound polymer was also formed during the reaction, which was easily analyzed by gel permeation chromatography (GPC), and the soluble polymer was used to estimate the length of the polymer chain bound to the surface.

[0030] The thickness of the polymer brushes was determined by AFM experiments. PS brushes with a thickness of 50 nm were prepared using an initiator concentration of 0.063 mol% relative to styrene. The target chain length of the silicon surface-bound polymer can be shortened by increasing the concentration of alkoxyamines, and PS surfaces with thicknesses of 5 nm and 10 nm can be easily prepared. At an alkoxyamine concentration of 0.4 mol%, a dense polystyrene polymer brush with a thickness of approximately 5 nm was prepared, an alkoxyamine concentration of 0.2 mol% enabled the preparation of a 10 nm thick polymer brush, and an alkoxyamine concentration of 0.1 mol% enabled the preparation of a 22 nm thick polymer brush.

[0031] (2) Atomic force microscope tip etching

[0032] The surface of a 50 nm thick polystyrene polymer brush was etched with an atomic force needle tip to make the surface thickness become 10 nm, thereby preparing a surface patterned polymer brush.

[0033] Specific operation process: After the atomic force microscope is turned on, select the silicon contact mode (k≈42Nm-1). The specific settings are: scan rate = 0.1, set point = 0.1, scan size = 20m, date category select off, Z range = 200nm, vertical deflection ≈ 0, horizontal deflection ≈ 0. Use the rec3-v1.c file on Di from nanoscript and click "run" on the macro to run it, repeat 3 times.

[0034] (3) Protein surface adsorption to form patterned structures

[0035] The surface of the patterned polystyrene brush (1 cm×1 cm) was immersed in 20 μL of pure SDF-1 protein solution at room temperature. After immersion for 24 hours, the patterned polystyrene brush was rinsed with ultrapure water to obtain a protein patterned structure formed on the surface of the polystyrene brush.

[0036] The above samples were analyzed using a fluorescence microscope. Figure 4 As shown, the immunofluorescence experiment of the SDF-1 protein patterned structure was carried out using a fluorescence microscope. (a) The surface of the patterned polystyrene polymer brush was coated with IgG antibody; (b) SDF-1 protein was adsorbed on the surface of the patterned polystyrene polymer brush coated with IgG; (c) MAB350 was coated on the surface of the patterned polystyrene polymer brush; (d) SDF-1 protein was adsorbed on the surface of the patterned polystyrene polymer brush coated with MAB350. Scale bar 20μm. The experiment shows that the protein patterned structure was successfully prepared by the present invention.

[0037] (4) Functional effects of SDF protein patterning

[0038] After 4 and 24 hours of SDF protein patterning, Tcam-2 cells in the culture dish were stained with BrdU, and the density of stained cells was analyzed using Image J. Figure 5 As shown, the effect of SDF-1 protein patterning structure on the migration of TCam-2 cells, the time points are 4 hours (a) and 24 hours (b). The parallel samples were 4 times, and the statistical significance was p < 0.05. It can be seen that the number of cells growing on the SDF protein patterned surface increased significantly.

[0039] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A protein patterning preparation method, characterized in that: The steps include: (1) Preparation of a polymer brush surface, the method is as follows: heating n-tetradecene and triethoxysilane to 38-42° C. under an argon atmosphere, adding a karstedt catalyst, stirring at 38-42° C. in a sealed tube for 1.5-2.5 hours, then adding cyclohexane and propyl carbonate, performing phase separation, concentrating the cyclohexane phase, and then purifying it with silica gel FC to obtain a functionalized silicon surface; performing a polymerization reaction on the functionalized silicon surface, placing a wafer self-assembled monolayer containing an initiator in a monomer, initiating a polymerization reaction, and obtaining a polymer brush; (2) Atomic force microscope tip etching The surface of the polymer brush is etched with an atomic force needle tip to change the thickness of the polymer brush to obtain areas of different thicknesses, thereby forming a surface patterned polymer brush; (3) Protein surface adsorption to form patterned structures The patterned polymer brush surface is immersed in a protein solution at room temperature. The different thicknesses of the polymer brushes have different adhesion to the protein. After soaking for a period of time, the patterned polymer brush is rinsed with ultrapure water to obtain a protein patterned structure formed on the surface of the polymer brush.

2. The protein patterning preparation method according to claim 1, characterized in that: The surface thickness of the polymer brush prepared in step (1) is thick enough so that when the atomic force needle tip etches on the surface of the polymer brush, different thickness regions can be formed, and the different thickness regions have different adhesion forces to proteins.

3. The protein patterning preparation method according to claim 1, characterized in that: The monomer is a styrene monomer; the wafer self-assembled monomolecular film containing an initiator is a wafer self-assembled monomolecular film containing an alkoxyamine NMP initiator.

4. The protein patterning preparation method according to claim 3, characterized in that: Increasing the concentration of alkoxyamine can shorten the target chain length of the polymer bound to the silicon surface, thereby producing a polymer brush with a smaller thickness. Conversely, a polymer brush with a larger thickness can be produced.

5. The protein patterning preparation method according to claim 4, characterized in that: The polystyrene brush is prepared by using an initiator with a concentration of 0.063-0.1 mol% relative to styrene to obtain a polystyrene brush with a thickness of 22-50 nm; the polystyrene brush is prepared by using an initiator with a concentration of 0.2-0.4 mol% relative to styrene to obtain a polystyrene brush with a thickness of 5-10 nm.

6. The protein patterning preparation method according to claim 1, characterized in that: In the step (1), n-tetradecene and triethoxysilane are reacted at 40° C. for 2 hours.

7. The protein patterning preparation method according to claim 1, characterized in that: The specific operation process of step (2) is as follows: after the atomic force microscope is turned on, the silicon contact mode is selected, k≈42Nm-1, the scanning rate = 0.1, the set point = 0.1, the scanning size = 20m, the date category is selected as off, the Z range = 200nm, the vertical deflection ≈0, the horizontal deflection ≈0, the rec3-v1.c file is used on Di in nanoscript, and "run" is clicked on the macro to run, and repeated 3 times.

8. The protein patterning preparation method according to claim 1, characterized in that: The step (3) is performed by soaking for 8-32 hours.

9. The protein patterning preparation method according to claim 1, characterized in that: The protein solution in step (3) is a pure SDF-1 protein solution.

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