Gradient complementary high-throughput screening method for biphase polypeptide modification

Abstract

The invention discloses a biphasic polypeptide modified gradient complementary high-throughput screening method, and aims to optimize a bifunctional surface modified by two different active molecules so as to realize optimal characterization of biological performance. According to the screening method, key parameters for preparing the functionalized polypeptide modified surface can be rapidly determined, wherein the key parameters comprise the optimal grafting density of polypeptide, the reaction time and the concentration of a polypeptide solution. The screening method effectively solves the problems that a traditional orthogonal experimental method is complex in operation and time-consuming. On the basis of a single function, a complementary gradient high-throughput screening platform is further introduced to realize visual comparison of performance differences of different bioactive polypeptides, so that the problems of multiple sample grafting times, multiple experimental groups, complex variables and the like in a traditional experiment are solved. The screening method provides guidance for functional modification of the surface of the intravascular stent, including anti-inflammatory, anti-coagulation, endothelialization promotion and other biological functions, and provides an important reference basis for parameter preparation of subsequent surface modification of the intravascular stent.

Description

Technical Field

[0001] The present invention relates to the field of surface modification of medical devices, and in particular to a gradient complementary high-throughput screening method for dual-phase polypeptide modification. Background Art

[0002] At present, with the aging of the population, the incidence of atherosclerotic stenosis is increasing year by year. Due to the advantages of small incision and short recovery period, intravascular stent intervention has gradually developed into one of the mainstream methods for treating this type of disease.

[0003] Once the vascular device is implanted, the surgical damage to the endothelium will gradually aggravate the degree of host response. Without timely and effective intervention, acute thrombosis and intimal hyperplasia may induce destructive problems such as vascular occlusion. Therefore, the surface modification strategy can effectively solve the adverse events caused by post-interventional vascular stents by giving the stent excellent anticoagulant properties and promoting endothelialization. However, the use of only a single type of biofunctional molecule has limited effect on the multifunctionalization of the vascular stent surface. Therefore, it is necessary to use two or more functional active molecules to functionalize the surface of the vascular stent.

[0004] Surface modification using bioactive peptide molecules is an effective strategy to transform traditional bioinert materials into bioactive materials. The biological properties of bioactive surfaces are related to the grafting density of bioactive peptides. Screening out the appropriate grafting density can not only effectively improve the biological properties of the material, but also reduce the biological toxicity caused by excessive grafting density. However, using traditional sample preparation strategies to modify the surface of nickel-titanium alloys requires preparing a large number of samples under the same conditions, which is time-consuming and labor-intensive, especially when the research content involves multiple factors, the preparation process will become more complicated. Therefore, multifunctional high-throughput surface design is used for construction and screening, and research related to optimizing the surface of multifunctional biomaterials has always received widespread attention. Summary of the invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the purpose of the present invention is to provide a high-throughput screening method for gradient complementation of biphasic polypeptide modification. The screening method provides guidance for the functional modification of the surface of vascular stents, including biological functions such as anti-inflammatory, anticoagulation and promotion of endothelialization, and provides an important reference for the preparation of parameters for subsequent surface modification of vascular stents.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A high-throughput screening method for gradient complementation of biphasic peptide modification comprises the following steps:

[0008] (1) designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0009] (2) ultrasonically cleaning the dual-flow nickel-titanium alloy-based high-throughput screening platform in step (1) with anhydrous ethanol and deionized water in turn, drying with N2, immersing the dual-flow nickel-titanium alloy-based high-throughput screening platform in a sodium hydroxide aqueous solution for hydroxylation treatment, introducing hydroxyl groups on the surface of the dual-flow nickel-titanium alloy-based high-throughput screening platform, and preparing a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0010] (3) dissolving alkynyl silane in ethanol to obtain an alkynyl silane solution, and immersing the hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform in the alkynyl silane ethanol solution and incubating in the dark at room temperature. After the incubation is completed, the hydroxylated nickel-titanium alloy is oven-dehydrated and fixed. After the fixation is completed, it is washed with ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0011] (4) injecting the polypeptide solution into the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform through the first liquid inlet, and after the polypeptide solution reacts with all cells of the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform, washing it to obtain a single polypeptide modified high-throughput screening platform;

[0012] (5) injecting a polypeptide solution different from that in step (4) into the single polypeptide modified high-throughput screening platform through the second liquid inlet, and after the polypeptide solution and the single polypeptide modified high-throughput screening platform have reacted, washing them to obtain a dual-phase polypeptide modified gradient complementary high-throughput screening platform;

[0013] (6) Cultivating cells on a dual-phase peptide-modified gradient complementation high-throughput screening platform to determine the optimal bioactive peptide grafting density region of cells and extracting the construction parameters of the surface of this region, namely, the peptide grafting density and the peptide reaction time;

[0014] The dual-flow nickel-titanium alloy-based high-throughput screening platform described in step (1) has multiple unit cell structures, wherein the unit cell structures include a first serpentine flow channel, a second serpentine flow channel and a liquid storage tray, wherein a first liquid inlet and a second liquid inlet are provided at both ends of the first serpentine flow channel; the first serpentine flow channel includes a first longitudinal partition and a second longitudinal partition, each of the first longitudinal partition and the second longitudinal partition being provided with a second groove lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition being installed on the liquid storage tray in a cross manner, and the second serpentine flow channel being arranged above the first serpentine flow channel.

[0015] Preferably, the second serpentine flow channel includes a first transverse baffle and a second transverse baffle, and the first transverse baffle and the second transverse baffle are both designed with a first groove lower than the height of the first longitudinal baffle and the second longitudinal baffle, and the first transverse baffle and the second transverse baffle are both installed in the first serpentine flow channel and arranged in a cross manner.

[0016] Preferably, the second serpentine flow channel is composed of a first transverse baffle, a second transverse baffle and a third transverse baffle, the first transverse baffle and the second transverse baffle are provided with a first groove lower than the height of the longitudinal baffle, the height of the third transverse baffle is also lower than the first longitudinal baffle and the second longitudinal baffle, and is installed at both ends of the first serpentine flow channel, and the first transverse baffle and the second transverse baffle are arranged crosswise.

[0017] Preferably, the second serpentine flow channel is composed of a first flow channel, a plurality of middle flow channels and a terminal flow channel connected in sequence; the first flow channel is provided with a first liquid inlet for the polypeptide liquid, and a second liquid inlet is provided at a position diagonally opposite to the first liquid inlet and at the same height; the first flow channel is composed of three third transverse partitions and one first transverse partition arranged in sequence, the middle flow channel is composed of two third transverse partitions and one first transverse partition arranged alternately, and the terminal flow channel is composed of three third transverse partitions and one second transverse partition arranged in sequence; the first transverse partition and the second transverse partition are both provided with a first groove, and the height of the third transverse partition is lower than the height of the first longitudinal partition and the second longitudinal partition, and the height of the first groove is also lower than the height of the first longitudinal partition and the second longitudinal partition.

[0018] Preferably, in step (2), the ultrasonic treatment time is 10-30 min, the concentration of the NaOH aqueous solution is 5M-10M, and the temperature of the hydroxylation treatment is 50-100°C.

[0019] Preferably, in step (2), the ultrasonic treatment time is 20 min, the concentration of the NaOH aqueous solution is 5 M, and the temperature of the hydroxylation treatment is 60°C.

[0020] Preferably, in step (3), the mass concentration of the alkynylsilane ethanol solution is 0.5-4 mg / mL, the light-proof incubation time is 12-48 h, the oven temperature is 70-150° C., the dehydration fixation time is 0.5-4 h, and the mass fraction of ethanol is 95%.

[0021] Preferably, in step (3), the mass concentration of the alkynylsilane ethanol solution is 5%, the light-proof incubation time is 24 hours, the oven temperature is 100° C., the dehydration fixation time is 1.5 hours, and the mass fraction of ethanol is 95%.

[0022] Preferably, the polypeptide solution in step (4) and step (5) is any one of an endothelial-promoting polypeptide REDV solution, an anticoagulant BVLD polypeptide solution or an anti-inflammatory polypeptide AP-1 solution.

[0023] Preferably, the concentration of the polypeptide solution in step (4) and step (5) is 150-400 μM, the injection speed is 80-120 μL / h, and the reaction time is 12 min-300 min.

[0024] Preferably, the cells cultured in the dual-phase polypeptide-modified gradient complementary high-throughput screening platform in step (6) are specifically located in the region where endothelial cells, smooth muscle cells, macrophages and platelets are located, and the construction parameters of the surface of the region are extracted, namely, the polypeptide grafting density, the polypeptide reaction time and the concentration of the polypeptide solution.

[0025] Preferably, the biphasic polypeptide-modified gradient complementation high-throughput screening platform culturing cells in step (6) comprises the following steps:

[0026] a. Sterilize the high-throughput screening platform with UV overnight and add 10 to 10 9 1-10 mL of cell suspension was added and incubated in a 37°C, 5% CO2 cell culture incubator for 24-72 h.

[0027] b. After the incubation, the high-throughput screening platform was taken out from the cell culture incubator, and the high-throughput screening platform was washed with PBS (pH = 7.4) to remove the non-adherent cells and cell debris, and then 2 mL of 4% paraformaldehyde was added to each high-throughput screening platform, and the cells in the model were fixed at 4°C in the dark for 12 hours;

[0028] c. After the cell fixation is completed, the high-throughput screening platform is washed with PBS for 3 times, 5 minutes each time, to remove the residual paraformaldehyde solution. Subsequently, 2 mL of 0.1% Triton-X is added to each high-throughput screening platform at room temperature for permeabilization. The permeabilization time is 5-10 minutes. After the permeabilization is completed, the high-throughput screening platform is washed with PBS for 3 times to wash away the residual Triton-X permeabilization solution;

[0029] d. After permeabilization, add 1-3 mL of PBS to each high-throughput screening platform for washing to remove the residual Triton-X permeabilization solution; after washing, add 1-3 mL of live cell F-actin microfilament protein staining reagent to each high-throughput screening platform, incubate at room temperature in the dark for 1h-24h, and wash away the residual microfilament protein fluorescent dye in the model with PBS buffer after the incubation; after washing, add 1-3 mL of cell nuclear fluorescent dye DAPI to each high-throughput screening platform, incubate at room temperature in the dark for 5-10min, and after incubation, wash with PBS buffer, and use a suction pump to remove excess PBS buffer in the high-throughput screening platform, place the treated high-throughput screening platform on the stage of a fluorescence microscope, take pictures and count the cytoskeleton and cell nucleus in the cells in the high-throughput screening platform area, and determine the regional cells with biological activity.

[0030] If the evaluation is for anticoagulant function, the specific steps are as follows: fresh rabbit blood containing 10% sodium citrate anticoagulant is centrifuged to obtain platelet-rich plasma (PRP). The speed used during centrifugation is 1,500-3,400 rpm, and the centrifugation time is 5-30 min. After the centrifugation, the supernatant is collected to obtain platelet-rich plasma (PRP); the high-throughput screening platform is rinsed with PBS buffer, 2-5 mL of platelet-rich plasma is added, and it is incubated in a 37°C incubator for 1-10 h. After the incubation, the non-adherent platelets are washed away with PBS buffer.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] The present invention proposes a gradient complementary high-throughput screening method for biphasic peptide modification, which aims to optimize the bifunctional surface modified by two different active molecules to achieve the best characterization of biological performance. The screening method can quickly determine the key parameters for preparing functionalized peptide-modified surfaces, including the optimal grafting density of the peptide, the reaction time, and the concentration of the peptide solution. This screening method effectively solves the problems of complex and time-consuming operation of the traditional orthogonal experimental method. On the basis of a single function, the present invention further introduces a complementary gradient high-throughput screening platform to achieve an intuitive comparison of the performance differences of different bioactive peptides, thereby avoiding the problems of multiple sample grafting times, multiple experimental groups, and complex variables in traditional experiments. This screening method provides guidance for the functional modification of the surface of vascular stents, including biological functions such as anti-inflammatory, anticoagulation, and promotion of endothelialization, and provides an important reference for the preparation of parameters for subsequent surface modification of vascular stents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (3×3 specifications of the first structure);

[0034] Figure 2 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (5×5 specifications of the first structure);

[0035] Figure 3 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (7×7 specifications of the first structure);

[0036] Figure 4 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (11×11 specifications of the first structure);

[0037] Figure 5 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (3×3 specifications of the second structure);

[0038] Figure 6 Schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform (5×5 specification of the second structure) of the present invention;

[0039] Figure 7 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (7×7 specifications of the second structure);

[0040] Figure 8 It is a schematic diagram of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (11×11 specifications of the second structure);

[0041] Fig. 9 It is a schematic diagram of the flow direction of a single polypeptide solution of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (5×5 specifications of the first structure);

[0042] Fig.10 It is a schematic diagram of the flow direction of a biphasic polypeptide solution of a dual-flow nickel-titanium alloy-based high-throughput screening platform (5×5 specifications of the first structure) of the present invention;

[0043] Fig.11 It is a schematic diagram of the flow direction of a single polypeptide solution of the dual-flow nickel-titanium alloy-based high-throughput screening platform of the present invention (5×5 specifications of the second structure);

[0044] Fig.12 It is a schematic diagram of the flow direction of a two-phase polypeptide solution in the dual-flow nickel-titanium alloy-based high-throughput screening platform (5×5 specifications of the second structure) of the present invention;

[0045] Fig.13 is a schematic diagram of the structural formula of the BVLD polypeptide of the present invention;

[0046] Fig.14 is a schematic diagram of the structural formula of the REDV polypeptide of the present invention;

[0047] Fig.15 is a schematic diagram of the structural formula of the AP1 polypeptide of the present invention;

[0048] Fig.16 is the average fluorescence intensity of the REDV-FITC gradient surface in the present invention;

[0049] Fig.17 is the average fluorescence intensity of the BVLD-FITC gradient surface in the present invention;

[0050] Fig.18 is the average fluorescence intensity of the AP1-FITC gradient surface in the present invention;

[0051] Fig.19 is the average fluorescence intensity of the BVLD-REDV complementary gradient surface in the present invention;

[0052] Fig. 20 is the average fluorescence intensity of the AP1-REDV complementary gradient surface in the present invention;

[0053] Fig.21 It is the number of platelet adhesion on the gradient surface of the BVLD single polypeptide modified high-throughput screening platform of the present invention;

[0054] Fig. 22 It is the number of endothelial cells adhering to the gradient surface of the BVLD single polypeptide modified high-throughput screening platform of the present invention;

[0055] Fig.23 is the number of endothelial cell adhesion in the REDV single polypeptide modified high-throughput screening platform of the present invention;

[0056] Fig.24 is the number of smooth muscle cells adhered in the REDV single polypeptide modified high-throughput screening platform of the present invention;

[0057] Fig.25 is the number of endothelial cell adhesion in the gradient complementation high-throughput screening platform modified with the BVLD-REDV biphasic polypeptide of the present invention;

[0058] Fig.26 is the number of smooth muscle cells adhered in the gradient complementation high-throughput screening platform modified with the BVLD-REDV biphasic polypeptide of the present invention;

[0059] Fig. 27 is the number of platelets adhered to the surface of the gradient complementary high-throughput screening platform modified with the BVLD-REDV biphasic polypeptide of the present invention;

[0060] Fig.28 It is the number of mouse mononuclear macrophages adhering to the surface of the AP1 single polypeptide modified high-throughput screening platform of the present invention;

[0061] Fig.29 is the number of endothelial cells adhering to the surface of the AP1 single polypeptide modified high-throughput screening platform of the present invention;

[0062] Fig.30 is the number of macrophage adhesion in the AP1-REDV biphasic polypeptide modified gradient complementation high-throughput screening platform of the present invention;

[0063] Fig.31 It is the number of endothelial cell adhesion in the gradient complementary high-throughput screening platform modified with the AP1-REDV biphasic polypeptide of the present invention.

[0064] Among them, 1-liquid storage tray, 101-first liquid inlet, 102-second liquid inlet, 103-first flow channel, 104-middle flow channel, 105-last flow channel, 106-region cell, 2-first longitudinal partition, 201-second groove, 3-second longitudinal partition, 4-first transverse partition, 401-first groove, 5-second transverse partition, 6-third transverse partition. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto. The materials used in the examples of the present invention can all be purchased commercially.

[0066] Example 1

[0067] Mouse leukemia mononuclear macrophage RAW 264.7 (ATCC TIB-71); human umbilical vein endothelial cells (ATCC PCS-100-010); human umbilical artery smooth muscle cells (ATCC PCS-100-012), purchased from VWR International, LLC, Pennsylvania, USA.

[0068] The flow direction of the peptide solution in the dual-flow nickel-titanium alloy-based high-throughput screening platform is as follows Figure 1 As shown, the screening platform has a total of 25 regional cells. The first polypeptide solution is pumped into the screening platform from the first inlet through a fully automatic constant flow injection pump, and flows from region 1 to region 25 along the first serpentine flow channel. The second polypeptide solution is pumped into the screening platform from the second inlet through a fully automatic constant flow injection pump, and flows from region 25 to region 1 along the second serpentine flow channel. That is, in the dual-flow nickel-titanium alloy-based high-throughput screening platform, the two polypeptides present a single peptide gradient distribution in region 1-region 25 and region 25-region 1, respectively. At the same time, the two polypeptides also present a complementary gradient distribution in region 1-region 25 of the dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0069] Method for determining peptide grafting density:

[0070] Designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0071] The dual-flow nickel-titanium alloy-based high-throughput screening platform was ultrasonically cleaned for 10 minutes using water and anhydrous ethanol respectively, and after drying with nitrogen, the dual-flow nickel-titanium alloy-based high-throughput screening platform was hydroxylated using a 5M NaOH aqueous solution, and hydroxyl groups were introduced on the surface of the dual-flow nickel-titanium alloy-based high-throughput screening platform to prepare a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0072] The hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the dual-flow nickel-titanium alloy-based high-throughput screening platform was placed in a 100° C. oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput model was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0073] In the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform, a constant-flow injection pump was used to introduce N3-REDV-FITC peptide solution with a fluorescent group FITC from the inlet 1 at a rate of 120 μL / h. The working solution concentration was 150 μM, the working solution volume was 600 μL, and the flow time was 5 hours. The injection process is shown in the figure. After the reaction, the fluorescent peptide-modified single polypeptide-modified high-throughput screening platform was cleaned with deionized water to remove the unreacted fluorescent peptide, and dried with nitrogen to obtain the fluorescent peptide-modified single polypeptide-modified high-throughput screening platform. Select platform; add a deionized water film to the above-mentioned single polypeptide modified high-throughput screening platform modified with fluorescent polypeptide, and place it under a fluorescence microscope for observation, take pictures and record the average fluorescence intensity of each regional cell, and statistically analyze the average fluorescence intensity of the regional cells of the single polypeptide modified high-throughput screening platform modified with fluorescent polypeptide, and draw the statistical results into a bar graph, as shown in Figure 16. The results show that the high-throughput screening platform can successfully construct a gradient surface of a single polypeptide, and realize the grafting density gradient surface of a single polypeptide in this high-throughput screening platform.

[0074] Same as the above steps, the N3-REDV-FITC fluorescent peptide solution was replaced with the N3-BVLD-FITC peptide solution to obtain the fluorescent BVLD single peptide modification high-throughput screening platform, and its regional unit cell average fluorescence intensity change bar graph is shown in Figure 17;

[0075] Same as the above steps, the N3-REDV-FITC fluorescent peptide solution was replaced with the N3-AP1-FITC peptide solution to obtain a fluorescent AP-1 single peptide modification high-throughput screening platform, and its regional cell average fluorescence intensity change bar graph is shown in Figure 18;

[0076] For the determination of the polypeptide grafting density in the regional unit cells of the gradient complementary high-throughput screening platform modified with BVLD and REDV dual-phase polypeptides, a constant-current injection pump was used to inject the N3-BVLD polypeptide solution without fluorescent groups from the first liquid inlet of the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform at an injection rate of 120 μL / h, wherein the working concentration of the BVLD polypeptide solution was 150 μL, and the reaction time after injection was 5 h. After the reaction, the high-throughput screening platform was washed with deionized water to remove the unreacted polypeptide solution to obtain the BVLD single polypeptide modified high-throughput screening platform; then, a constant-current injection pump was used to inject the N3-REDV-FITC containing fluorescent groups from the second liquid inlet into the BVLD gradient high-throughput screening platform at a flow rate of 120 μL / h, wherein N3-REDV-FITC The working concentration is 150 μL, and the reaction time after introduction is 5 hours. After the reaction, the high-throughput screening platform is cleaned with deionized water to remove the unreacted N3-REDV-FITC solution, and blown dry with nitrogen to obtain a gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic polypeptides; a deionized water film is added to the above-mentioned gradient high-throughput screening platform modified with fluorescent polypeptides, and placed under a fluorescence microscope for observation, photographing and recording the average fluorescence intensity of each regional cell, and the average fluorescence intensity of the regional cells of the gradient high-throughput screening platform modified with fluorescent polypeptides is statistically analyzed, and the statistical results are plotted into a bar graph, as shown in Figure 19. The results show that the high-throughput screening platform can successfully construct a complementary gradient surface of biphasic polypeptides, and realize the grafting density complementary gradient surface of biphasic polypeptides in this high-throughput screening platform.

[0077] For the determination of the peptide grafting density in the regional cells of the AP1 and REDV dual-phase peptide-modified gradient complementary high-throughput screening platform, a constant-current injection pump was used to introduce the N3-AP1 peptide solution without fluorescent groups from the first liquid inlet of the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform at an injection rate of 100 μL / h, wherein the working concentration of the AP1 peptide solution was 200 μL, and the reaction time after introduction was 6 hours. After the reaction, the high-throughput screening platform was washed with deionized water to remove the unreacted peptide solution to obtain the AP1 single peptide-modified high-throughput screening platform; then, a constant-current injection pump was used to introduce the fluorescent group-containing N3-REDV-FITC into the AP1 gradient high-throughput screening platform from the second liquid inlet at a flow rate of 120 μL / h, wherein the working concentration of N3-REDV-FITC was 200 μL. The concentration was 150 μL, and the reaction time after introduction was 5 hours. After the reaction, the high-throughput screening platform was cleaned with deionized water to remove the unreacted N3-REDV-FITC solution, and dried with nitrogen to obtain a gradient complementary high-throughput screening platform modified with AP1 and REDV biphasic polypeptides; a deionized water film was added to the above-mentioned gradient high-throughput screening platform modified with fluorescent polypeptides, and the platform was placed under a fluorescence microscope for observation, photographed, and the average fluorescence intensity of each regional cell was recorded. The average fluorescence intensity of the regional cells of the gradient high-throughput screening platform modified with fluorescent polypeptides was statistically analyzed, and the statistical results were plotted into a bar graph, as shown in Figure 20. The results show that the high-throughput screening platform can successfully construct a complementary gradient surface of biphasic polypeptides, and realize the grafting density complementary gradient surface of biphasic polypeptides in the high-throughput screening platform.

[0078] Example 2

[0079] Designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0080] The high-throughput screening platform was ultrasonically cleaned for 10 minutes using water and anhydrous ethanol respectively, and after drying with nitrogen, the high-throughput screening platform was hydroxylated using a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the high-throughput screening platform to prepare a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0081] The hydroxylated high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the high-throughput screening platform was placed in a 100° C. oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput screening platform was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0082] The 150 μM reaction concentration of BVLD peptide was pumped into the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform through a constant flow injection pump at 120 μL / h, flowing from area 1 of the screening platform to area 25 in a serpentine flow (the reaction time of BVLD peptide with each cell in the screening platform is 0-300 min). After the reaction is completed, the high-throughput screening platform is cleaned with deionized water or ultrapure water to obtain the BVLD single polypeptide modified high-throughput screening platform.

[0083] Platelet-rich plasma (PRP) was added to the high-throughput screening platform modified with the BVLD polypeptide, and incubated in a shaker at 37°C and 80 rpm for 2 hours. After the incubation, the high-throughput screening platform was washed 3 times with PBS. After the washing, the platelets in the BVLD high-throughput screening platform were stained with F-actin at room temperature for 1 hour. After the staining, the residual fluorescent staining liquid was washed away with PBS, and the number of platelet adhesion in the regional cells of the high-throughput screening platform was observed and counted with a fluorescence microscope to determine the BVLD regional cells with the best anti-platelet adhesion. It was determined by statistics that the cell regions 1 to 15 that effectively inhibited platelet adhesion could effectively inhibit platelet adhesion, and the grafting density of the region range was 557.53-804.07 ng / cm according to the surface average fluorescence intensity result. 2 , taking the silanized surface as the control group, as shown in the figure, the number of platelet adhesion on the surface after BVLD modification decreased by more than 40% compared with the control group surface.

[0084] Human umbilical vein endothelial cells (HUVEC) were cultured in the regional cells of the BVLD single polypeptide modified high-throughput screening platform for 24 hours and 72 hours, and the cytoskeleton was stained for 1 hour at room temperature using the live cell F-actin microfilament staining reagent and the cell nucleus was stained for 8 minutes at room temperature using the DAPI cell nucleus staining reagent, and then washed three times with PBS. Under a laser confocal microscope, the number of cell adhesions in each regional cell of the high-throughput screening platform was observed and counted to determine the regional cell range with the best bioactive polypeptide grafting density; it was determined statistically that the endothelial cells in the regional cells of regions 15-20 had the most adhesion, and the grafting density of this regional range was converted to 557.53-661.51 ng / cm based on the surface average fluorescence intensity results. 2

[0085] Example 3

[0086] A dual-flow nickel-titanium alloy-based high-throughput screening platform was designed and obtained through 3D printing, and the printed dual-flow nickel-titanium alloy-based high-throughput screening platform was subjected to stress annealing treatment in a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform; the high-throughput screening platform was ultrasonically cleaned for 10 minutes using water and anhydrous ethanol respectively, and after drying with nitrogen, the high-throughput screening platform was hydroxylated using a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the high-throughput screening platform to prepare a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0087] The hydroxylated high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the high-throughput model was placed in a 100° C. oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput screening platform was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0088] 150-300 μM REDV peptide solution was injected into the silanized high-throughput screening platform from the first inlet at a liquid inlet rate of 120 μL / h using an electric liquid inlet pump, and reacted at room temperature for 5 hours. After the peptide solution and the cells of the high-throughput screening platform were completely reacted, the high-throughput screening platform was washed three times with deionized water and dried with nitrogen to obtain a REDV single peptide modified high-throughput screening platform;

[0089] Human umbilical vein endothelial cells (HUVEC), human umbilical artery smooth muscle cells (HUASMC) and mouse mononuclear macrophage leukemia cells (Raw264.7) were cultured for 24 h and 72 h, respectively, and the cytoskeleton was stained for 1 h at room temperature using a live cell F-actin microfilament protein staining reagent and the cell nucleus was stained for 8 min at room temperature using a DAPI cell nucleus staining reagent, and the cells were washed three times with PBS after staining. Under a laser confocal microscope, the number of cell adhesion in each high-throughput screening platform area was observed and counted to determine the area range of cells with the best bioactive peptide grafting density. The number of cells in each cell was counted and plotted into a bar graph. The results showed that in the REDV single peptide modified high-throughput screening platform, endothelial cells in the cells of regions 18 to 20 had the most adhesion, and smooth muscle cells in this range had less adhesion. Mouse mononuclear macrophage leukemia cells in the cells of regions 16 to 19 had the least adhesion. The average fluorescence intensity in the REDV gradient high-throughput screening platform showed that the grafting density range of REDV in the above-mentioned area cells was 168.07-191.70 ng / cm 2Taking the silanized surface as the control group, the number of cells adhering within the REDV grafting density range was 8.0 times that of the control group, the number of smooth muscle cells adhering was 0.4 times that of the control group, and the number of mouse mononuclear macrophages adhering was 0.45 times that of the control group.

[0090] Example 4

[0091] (1) designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0092] (2) The high-throughput model was ultrasonically cleaned for 10 min using water and anhydrous ethanol respectively, and after drying with nitrogen, the high-throughput screening platform was hydroxylated using a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the high-throughput screening platform to prepare a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0093] (3) The prepared hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the high-throughput screening platform was placed in a 100° C. oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput screening platform was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0094] (4) A 100-400 μM AP-1 peptide solution was injected from the first inlet into the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform using an electric inlet pump at an inlet rate of 100 μL / h, and reacted at room temperature for 6 hours. After the peptide solution reacted with the cells of the high-throughput screening platform, the high-throughput model was washed three times with deionized water and dried with nitrogen to obtain an AP-1 single peptide modified high-throughput screening platform.

[0095] Human umbilical vein endothelial cells (HUVEC) and human aortic artery smooth muscle cells (HUASMC) were cultured for 24 hours and 72 hours respectively in the AP-1 single polypeptide modified high-throughput screening platform, and the cytoskeleton was stained for 1 hour at room temperature using a live cell F-actin microfilament staining reagent and the cell nucleus was stained for 8 minutes at room temperature using a DAPI cell nucleus staining reagent, and the cells were washed three times with PBS after staining. Under a laser confocal microscope, the number of cell adhesions in each high-throughput screening platform area cell was observed and counted to determine the area cell range of the optimal bioactive polypeptide grafting density;

[0096] Mouse mononuclear macrophage leukemia cells (Raw264.7) were cultured in the AP-1 single polypeptide modified high-throughput screening platform for 24 hours and 72 hours, and the cytoskeleton was stained for 1 hour at room temperature using the live cell F-actin microfilament staining reagent and the cell nucleus was stained for 8 minutes at room temperature using the DAPI cell nucleus staining reagent, and then washed 3 times with PBS. Under a laser confocal microscope, the number of cell adhesions in each high-throughput screening platform area was observed and counted to determine the area cell range with the best bioactive polypeptide grafting density; the number of cells in each cell was counted and plotted into a bar graph to obtain a graph, and the results showed that in the AP1 single polypeptide modified high-throughput screening platform, the mouse mononuclear macrophage leukemia cells in the area 19-area 25 area cells had the least adhesion; the AP1 grafting density of the above-mentioned area cells was calculated by the average fluorescence intensity in the AP1 single polypeptide modified high-throughput screening platform, and the range was 233.68-305.02ng / cm 2 Taking the silanized surface as the control group, the cell density of mouse mononuclear macrophages within this AP1 grafting density range was 0.44 times that of the control group.

[0097] Example 5

[0098] Designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0099] The high-throughput screening platform was ultrasonically cleaned for 10 min using water and anhydrous ethanol respectively, and after drying with nitrogen, the high-throughput screening platform was hydroxylated using a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the high-throughput model, thereby preparing a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0100] The hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the high-throughput screening platform was placed in a 100° C. oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput model was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0101] The BVLD polypeptide solution was injected into the first liquid inlet of the silanized high-throughput screening platform at a liquid inlet rate of 120 μL / h using an electric liquid inlet pump, and reacted at room temperature for 5 hours. After the polypeptide solution and the high-throughput model unit cell were reacted, the high-throughput screening platform was washed three times with deionized water and dried with nitrogen to obtain a BVLD single polypeptide modified high-throughput screening platform;

[0102] The REDV polypeptide solution was injected into the second inlet of the BVLD single polypeptide modified high-throughput screening platform at a liquid inlet rate of 120 μL / h using an electric inlet pump, and reacted at room temperature for 5 hours. After the polypeptide solution and the high-throughput screening platform unit cell reacted, the high-throughput screening platform was washed three times with deionized water and dried with nitrogen to obtain a gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic polypeptides;

[0103] Human umbilical vein endothelial cells (HUVEC) and human aortic artery smooth muscle cells (HUASMC) were cultured in the regional cells of the BVLD and REDV biphasic peptide modified high-throughput screening platform for 24 hours and 72 hours, respectively, and the cytoskeleton was stained for 1 hour at room temperature using a live cell F-actin microfilament protein staining reagent and the cell nucleus was stained for 8 minutes at room temperature using a DAPI cell nucleus staining reagent, and the cells were washed three times with PBS after staining. Under a laser confocal microscope, the number of cell adhesion in each high-throughput screening platform area was observed and counted to determine the area cell range with the optimal bioactive peptide grafting density. The number of cells in each cell was counted and plotted into a bar graph. The results showed that in the gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic peptides, the endothelial cells in the cells of area 14 to area 20 had the most adhesion, and the smooth muscle cells in this range had less adhesion. The average fluorescence intensity in the gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic peptides was calculated to show that the grafting density of REDV in the above-mentioned area cells ranged from 112.92 to 189.07 ng / cm 2 The grafting density of BVLD ranges from 377.22 to 565.83 ng / cm 2 Taking the silanized surface as the control group, the number of cell adhesion within this REDV grafting density range was 1.46 times that of the control group, and the number of smooth muscle cell adhesion was 0.45 times that of the control group.

[0104] Determine the peptide grafting density range corresponding to the cell range of the optimal bioactive peptide grafting density region.

[0105] Platelet-rich plasma (PRP) was added to the gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic polypeptides, and incubated at 37°C and 80rpm in a shaker for 2 hours. After the incubation, the high-throughput screening platform was washed 3 times with PBS. After the washing, the platelets in the BVLD high-throughput screening platform were stained with F-actin at room temperature for 1 hour. After the staining, the residual fluorescent staining solution was washed away with PBS, and the number of platelet adhesion in the regional cells of the high-throughput screening platform was observed and counted with a fluorescence microscope to determine the best anti-platelet adhesion regional cells of the gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic polypeptides; it was determined by statistics that the cell regions 1 to 18 of the gradient complementary high-throughput screening platform modified with BVLD and REDV biphasic polypeptides can effectively inhibit platelet adhesion, and the BVLD grafting density range of this region was converted to 467.23-801.59 ng / cm according to the surface average fluorescence intensity result. 2 REDV grafting density ranges from 47.27 to 178.56 ng / cm 2 , the silanized surface was used as the control group. As shown in the figure, compared with the control group surface, the number of platelet adhesion on the surface after BVLD modification decreased by more than 40%.

[0106] Example 6

[0107] Designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0108] The high-throughput screening platform was ultrasonically cleaned for 10 min using water and anhydrous ethanol respectively, and after drying with nitrogen, the high-throughput model was hydroxylated using a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the high-throughput screening platform to prepare a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform.

[0109] 2. The hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform was completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 hours. After the incubation, the high-throughput screening platform was placed in a 100°C oven for dehydration and fixation for 1 hour. After the fixation, the high-throughput model was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform;

[0110] The AP-1 polypeptide solution was injected into the first inlet of the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform using an electric inlet pump at an inlet rate of 100 μL / h, and reacted at room temperature for 6 hours. After the polypeptide solution and the high-throughput model unit cell were reacted, the high-throughput screening platform was washed three times with deionized water and dried with nitrogen to obtain a high-throughput screening platform modified with the BVLD polypeptide;

[0111] The REDV polypeptide solution was injected into the second inlet of the high-throughput model modified with the BVLD polypeptide at an inlet rate of 120 μL / h using an electric inlet pump, and reacted at room temperature for 5 hours. After the polypeptide solution and the high-throughput screening platform unit cell were reacted, the high-throughput screening platform was washed three times with deionized water and dried with nitrogen to obtain a gradient complementary high-throughput screening platform modified with AP-1 and REDV biphasic polypeptides;

[0112] Human umbilical vein endothelial cells (HUVEC) and mouse mononuclear macrophage leukemia cells (Raw264.7) were cultured in the regional cells of the AP-1 and REDV biphasic polypeptide modified gradient complementary high-throughput screening platform for 24 hours and 72 hours, respectively, and the cytoskeleton was stained for 1 hour at room temperature using a live cell F-actin microfilament protein staining reagent and the cell nucleus was stained for 8 minutes using a DAPI cell nucleus staining reagent at room temperature, and the cells were washed three times with PBS after staining. Under a laser confocal microscope, the number of cell adhesions in each high-throughput screening platform area was observed and counted to determine the area range of cells with the best bioactive peptide grafting density. In the gradient complementary high-throughput screening platform modified with AP1 and REDV biphasic peptides, endothelial cells in the cells of areas 1-7 and 13-18 had the most adhesion, and smooth muscle cells in this range had less adhesion. Mouse mononuclear macrophage leukemia cells in the cells of areas 17-25 had the least adhesion. The average fluorescence intensity in the REDV gradient high-throughput screening platform showed that the grafting density of REDV in the cells of the above areas ranged from 183.82 to 210.08 ng / cm 2 、77.47-129.99ng / cm 2 and 56.46-85.35ng / cm 2 The grafting density of AP1 ranges from 2.95 to 24.59 ng / cm 2 、66.38-221.27ng / cm 2 and 142.60-299.95ng / cm 2 Taking the silanized surface as the control group, the REDV grafting density ranged from 56.46 to 85.35 ng / cm 2 The grafting density of AP1 is 142.60-299.95 ng / cm2 In the area, the number of mouse monocytes and macrophages adhered was 0.41 times that of the control group, and the number of endothelial cells was 1.48 times that of the control group.

[0113] From the above examples, it can be seen that compared with the traditional orthogonal experimental method, the method of the present invention can quickly determine the preparation parameters of the functionalized surface modified by grafting active polypeptides, including the optimal grafting density of the biologically active polypeptides, the time required for the material in the preparation process, and the concentration of the biologically active polypeptide solution.

[0114] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A high-throughput screening method for gradient complementation of biphasic peptide modifications, characterized in that: The following steps are included: (1) designing and obtaining a dual-flow nickel-titanium alloy-based high-throughput screening platform through 3D printing, and performing stress annealing treatment on the printed dual-flow nickel-titanium alloy-based high-throughput screening platform through a high-temperature furnace to obtain a dual-flow nickel-titanium alloy-based high-throughput screening platform; (2) ultrasonically cleaning the dual-flow nickel-titanium alloy-based high-throughput screening platform in step (1) with anhydrous ethanol and deionized water in turn, drying with N2, immersing the dual-flow nickel-titanium alloy-based high-throughput screening platform in a sodium hydroxide aqueous solution for hydroxylation treatment, introducing hydroxyl groups on the surface of the dual-flow nickel-titanium alloy-based high-throughput screening platform, and preparing a hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform; (3) dissolving alkynyl silane in ethanol to obtain an alkynyl silane solution, and immersing the hydroxylated dual-flow nickel-titanium alloy-based high-throughput screening platform in the alkynyl silane ethanol solution and incubating in the dark at room temperature. After the incubation is completed, the hydroxylated nickel-titanium alloy is oven-dehydrated and fixed. After the fixation is completed, it is washed with ethanol and dried with nitrogen to obtain a silanized dual-flow nickel-titanium alloy-based high-throughput screening platform; (4) injecting the polypeptide solution into the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform through the first liquid inlet, and after the polypeptide solution reacts with all cells of the silanized dual-flow nickel-titanium alloy-based high-throughput screening platform, washing it to obtain a single polypeptide modified high-throughput screening platform; (5) injecting a polypeptide solution different from that in step (4) into the single polypeptide modified high-throughput screening platform through the second liquid inlet, and after the polypeptide solution and the single polypeptide modified high-throughput screening platform have reacted, washing them to obtain a dual-phase polypeptide modified gradient complementary high-throughput screening platform; (6) Cultivating cells on a dual-phase peptide-modified gradient complementation high-throughput screening platform to determine the optimal bioactive peptide grafting density region of cells and extracting the construction parameters of the surface of this region, namely, the peptide grafting density and the peptide reaction time; The dual-flow nickel-titanium alloy-based high-throughput screening platform described in step (1) has multiple unit cell structures, wherein the unit cell structures include a first serpentine flow channel, a second serpentine flow channel and a liquid storage tray, wherein a first liquid inlet and a second liquid inlet are provided at both ends of the first serpentine flow channel; the first serpentine flow channel includes a first longitudinal partition and a second longitudinal partition, each of the first longitudinal partition and the second longitudinal partition being provided with a second groove lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition being installed on the liquid storage tray in a cross manner, and the second serpentine flow channel being arranged above the first serpentine flow channel.

2. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The second serpentine flow channel includes a first transverse baffle and a second transverse baffle, and the first transverse baffle and the second transverse baffle are both designed with a first groove lower than the height of the first longitudinal baffle and the second longitudinal baffle. The first transverse baffle and the second transverse baffle are both installed in the first serpentine flow channel and arranged in a cross manner.

3. The gradient complementation high-throughput screening method for biphasic polypeptide modification according to claim 1, characterized in that: The second serpentine flow channel is composed of a first transverse baffle, a second transverse baffle and a third transverse baffle. The first transverse baffle and the second transverse baffle are provided with a first groove which is lower than the height of the longitudinal baffle. The height of the third transverse baffle is also lower than the first longitudinal baffle and the second longitudinal baffle, and it is installed at both ends of the first serpentine flow channel. The first transverse baffle and the second transverse baffle are arranged crosswise.

4. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The second serpentine flow channel is composed of a first flow channel, a plurality of middle flow channels and a terminal flow channel connected in sequence; the first flow channel is provided with a first liquid inlet for the polypeptide liquid, and a second liquid inlet is provided at a position diagonally opposite to the first liquid inlet and at the same height; the first flow channel is composed of three third transverse partitions and one first transverse partition arranged in sequence, the middle flow channel is composed of two third transverse partitions and one first transverse partition arranged alternately, and the terminal flow channel is composed of three third transverse partitions and one second transverse partition arranged in sequence; the first transverse partition and the second transverse partition are both provided with a first groove, and the height of the third transverse partition is lower than that of the first longitudinal partition and the second longitudinal partition, and the height of the first groove is also lower than that of the first longitudinal partition and the second longitudinal partition.

5. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that Preferably, the ultrasonic treatment time in step (2) is 10-30 min, the concentration of the NaOH aqueous solution is 5M-10M, and the temperature of the hydroxylation treatment is 50-100°C.

6. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The mass concentration of the alkynylsilane ethanol solution in step (3) is 0.5-4 mg / mL, the light-proof incubation time is 12-48 h, the oven temperature is 70-150° C., the dehydration fixation time is 0.5-4 h, and the mass fraction of ethanol is 95%.

7. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The polypeptide solution in step (4) and step (5) is any one of the endothelial polypeptide REDV solution, the anticoagulant BVLD polypeptide solution or the anti-inflammatory polypeptide AP-1 solution.

8. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The concentration of the polypeptide solution in step (4) and step (5) is 150-400 μM, the injection speed is 80-120 μL / h, and the reaction time is 12 min-300 min.

9. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The cells cultured in the dual-phase polypeptide-modified gradient complementary high-throughput screening platform in step (6) are specifically the regions where endothelial cells, smooth muscle cells, macrophages and platelets are located, and the construction parameters of the surface of the region are extracted, namely the polypeptide grafting density, the polypeptide reaction time and the concentration of the polypeptide solution.

10. The method for high-throughput screening of gradient complementation of biphasic polypeptide modification according to claim 1, characterized in that: The step (6) of culturing cells on the dual-phase polypeptide modified gradient complementation high-throughput screening platform comprises the following steps: a. Sterilize the high-throughput screening platform with UV overnight and add 10 to 10 9 pc / mL cell suspension, add 1-10mL volume, incubate in a 37℃, 5% CO2 cell culture incubator for 24-72h; b. After the incubation, the high-throughput screening platform was taken out from the cell culture incubator, and the high-throughput screening platform was washed with PBS (pH = 7.4) to remove the non-adherent cells and cell debris, and then 2 mL of 4% paraformaldehyde was added to each high-throughput screening platform, and the cells in the model were fixed at 4°C in the dark for 12 hours; c. After the cell fixation is completed, the high-throughput screening platform is washed with PBS for 3 times, 5 minutes each time, to remove the residual paraformaldehyde solution. Subsequently, 2 mL of 0.1% Triton-X is added to each high-throughput screening platform at room temperature for permeabilization. The permeabilization time is 5-10 minutes. After the permeabilization is completed, the high-throughput screening platform is washed with PBS for 3 times to wash away the residual Triton-X permeabilization solution; d. After permeabilization, add 1-3 mL of PBS to each high-throughput screening platform for washing to remove the residual Triton-X permeabilization solution; after washing, add 1-3 mL of live cell F-actin microfilament protein staining reagent to each high-throughput screening platform, incubate at room temperature in the dark for 1h-24h, and wash away the residual microfilament protein fluorescent dye in the model with PBS buffer after the incubation; after washing, add 1-3 mL of cell nuclear fluorescent dye DAPI to each high-throughput screening platform, incubate at room temperature in the dark for 5-10min, and after incubation, wash with PBS buffer, and use a suction pump to remove excess PBS buffer in the high-throughput screening platform, place the treated high-throughput screening platform on the stage of a fluorescence microscope, take pictures and count the cytoskeleton and cell nucleus in the cells in the high-throughput screening platform area, and determine the regional cells with biological activity.

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