A multi-cell microfluidic platform and high-throughput screening method thereof

Abstract

The present invention discloses a multi-cell microfluidic platform, including a first longitudinal partition, a second longitudinal partition and a liquid storage tray, wherein the first longitudinal partition and the second longitudinal partition are both provided with a second groove, the height of the second groove is lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition are installed on the liquid storage tray, the first longitudinal partition and the second longitudinal partition are cross-arranged to form a first serpentine flow groove, and the two ends of the first serpentine flow groove are provided with a liquid inlet and a liquid outlet. The time for sample preparation can be further reduced, and the concentration gradient of the sample can be increased to achieve the effect of improving the experimental accuracy; by preparing surfaces with different grafting densities, the gradient of grafting density changes can be obtained in a short time, which effectively improves the sample preparation efficiency and greatly reduces the workload. The present invention also discloses a high-throughput screening method for a multi-cell microfluidic platform, which solves the problem that the traditional orthogonal experimental method is cumbersome and time-consuming.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a multi-cell microfluidic platform and a high-throughput screening method thereof. Background Art

[0002] After the biomaterial is implanted in the body, all reactions occur on the surface of the material, so the properties of the material surface are crucial. Modifying the surface with various bioactive peptides can improve the biological behavior of the material surface accordingly. The surface grafting density of bioactive peptides determines the effect of the surface, so the appropriate grafting density can not only save manpower and material resources but also have a corresponding effect. It is of great significance to screen surfaces with a reasonable grafting density of bioactive peptides. Using traditional sample preparation methods to modify the surface of titanium sheets requires the preparation of many parallel samples, which greatly increases the workload of the experiment.

[0003] Peptides with antibacterial function include KRWWKWWRR, octapeptide RWWRIWKW, nonapeptide KFKWWRMLI, RGD, YIGSR, etc. Orthogonal experiments of grafting peptides of different concentrations on a uniform surface are too time-consuming and laborious. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above prior art and to provide a multi-cell microfluidic platform and a high-throughput screening method thereof.

[0005] The objective of the present invention is achieved through the following technical solutions: a multi-cell microfluidic platform, comprising a first serpentine flow trough, a second serpentine flow trough and a liquid storage tray, wherein the first serpentine flow trough is provided with a liquid inlet and a liquid outlet at both ends, the first serpentine flow trough comprises a first longitudinal partition and a second longitudinal partition, the first longitudinal partition and the second longitudinal partition are both provided with a second groove, the height of the second groove is lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition are cross-arranged and installed on the liquid storage tray, and the second serpentine flow trough is installed on the first serpentine flow trough.

[0006] A better choice is that the second serpentine flow trough includes a first transverse baffle and a second transverse baffle, the first transverse baffle and the second transverse baffle are both provided with a first groove, the height of the first groove is lower than the first longitudinal baffle and the second longitudinal baffle respectively, the first transverse baffle and the second transverse baffle are both installed in the first serpentine flow trough, and the first transverse baffle and the second transverse baffle are cross-arranged.

[0007] A better choice is that the second serpentine flow groove includes a first transverse baffle, a second transverse baffle and a third transverse baffle, the first transverse baffle and the second transverse baffle are both provided with a first groove, the height of the first groove is lower than the first longitudinal baffle and the second longitudinal baffle respectively, the height of the third transverse baffle is lower than the first longitudinal baffle and the second longitudinal baffle respectively, the third transverse baffle is installed at both ends of the first serpentine flow groove, and the first transverse baffle and the second transverse baffle are cross-arranged.

[0008] A better choice is that the second serpentine flow groove includes a first flow groove, multiple middle flow grooves and a terminal flow groove connected in sequence, the first flow groove is provided with a liquid inlet, the terminal flow groove is provided with a liquid outlet, the first flow groove includes three third transverse baffles and a first transverse baffle arranged in sequence, the multiple middle flow grooves include second transverse baffles, two third transverse baffles and a first transverse baffle arranged in sequence, the terminal flow groove includes a second transverse baffle and three third transverse baffles arranged in sequence, the first transverse baffle and the second transverse baffle are provided with a first groove, the height of the third transverse baffle is lower than that of the first longitudinal baffle and the second longitudinal baffle, and the height of the first groove is lower than that of the first longitudinal baffle and the second longitudinal baffle.

[0009] A high-throughput screening method for a multi-cell microfluidic platform comprises the following steps:

[0010] S1. Designing and obtaining a microfluidic control chip printout through 3D printing, wherein the microfluidic control chip printout is subjected to stress relief annealing treatment to obtain a microfluidic control chip;

[0011] S2, cleaning the microfluidic control chip, and performing hydroxylation treatment on the microfluidic control chip to obtain a surface-modified microfluidic control chip, wherein the surface-modified microfluidic control chip is the multi-cell microfluidic platform;

[0012] S3, injecting the polypeptide solution into the surface-modified microfluidic control chip, the polypeptide solution passes through the regional cells of the surface-modified microfluidic control chip at a uniform speed, the reaction time between the polypeptide solution and the surface-modified microfluidic control chip is 10 minutes to 20 hours, and after the polypeptide solution has reacted with all the regional cells, the microfluidic control chip is cleaned to obtain a polypeptide-grafted microfluidic control chip;

[0013] S4, culturing bacteria or cells in the polypeptide grafting microfluidic control chip to determine the optimal biologically active polypeptide grafting density area cell;

[0014] S5. Determine the polypeptide grafting density of the cells in the optimal bioactive polypeptide grafting density region.

[0015] A more preferred option is to perform hydroxylation on the microfluidic control chip in step S2, comprising the following steps:

[0016] S211, the microfluidic control chip is ultrasonically cleaned using deionized water, acetone, anhydrous ethanol and deionized water in sequence for 10 to 20 minutes, and then the microfluidic control chip is blown dry using a protective gas;

[0017] S212, immersing the microfluidic control chip in HF acid with a mass ratio of 2% to 8% for 3 to 15 minutes;

[0018] S213, finally cleaning the microfluidic control chip, drying the microfluidic control chip with protective gas again, and soaking the microfluidic control chip in concentrated nitric acid for 10 to 30 minutes to obtain a surface-modified microfluidic control chip.

[0019] A better choice is that the polypeptide molecular structure formula in the polypeptide solution in step S3 is HS-R-Peptide, wherein R includes C(PA) n P, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and the peptide includes KRWWKWWRR or KRWWKWWRRK-FITC or RWWRIWKW or RWWRIWKWK-FITC or KFKWWRMLI or KFKWWRMLIK-FITC or FKRIVQRIKDFLRNLV or FKRIVQRIKDFLRNLVK-FITC or KLLKLLKKLLKALG or KLLKLLKKLLKALGK-FITC or KRWWKWIRW or KRWWKWIRWK-FITC or RGD or RGD-FITC or YIGSR or YIGSR-FITC.

[0020] A more preferred option is that culturing bacteria in the polypeptide-grafted microfluidic control chip in step S4 comprises the following steps:

[0021] S411, preparing a certain amount of color development medium, placing the color development medium in a sterilizing pot and sterilizing it at 121° C. for 2 h;

[0022] S412, placing the polypeptide-grafted microfluidic control chip in a first multi-well culture plate, and sequentially adding 10 to 10% 9 1 μl to 100 μl of bacterial solution with a CFU / ml, incubating the bacterial solution in the polypeptide-grafted microfluidic control chip for 10 minutes to 24 hours;

[0023] S413, after the co-incubation is completed, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate, and after the bacterial solution is completely evaporated, the polypeptide-grafted microfluidic control chip is placed in a biological cabinet and allowed to stand for 10 to 15 minutes;

[0024] S414, dividing the color development medium of step S411 into two parts, adding one part of the color development medium into the polypeptide grafted microfluidic control chip and letting it stand until the color development medium solidifies, then placing the polypeptide grafted microfluidic control chip into a culture dish, using another part of the color development medium to seal the polypeptide grafted microfluidic control chip, and placing the polypeptide grafted microfluidic control chip into a constant temperature incubator for 18-48 hours after solidification;

[0025] S415. After the polypeptide grafted microfluidic control chip is statically cultured, the bacterial colony growth of the polypeptide grafted microfluidic control chip is observed under a stereo microscope to determine the regional cells with the best bioactive polypeptide grafting density.

[0026] More preferably, the step S4 of culturing cells in the polypeptide-grafted microfluidic control chip includes the following steps:

[0027] S421, placing the polypeptide grafted microfluidic control chip in a third multi-well culture plate, sterilizing the polypeptide grafted microfluidic control chip, and sequentially adding 10 to 10% iodine to each cell of the microfluidic control chip. 9 1 μl to 100 μl of cell suspension with a concentration of 1 cell / mL is statically cultured for 1 hour to 30 days;

[0028] S422, taking out the polypeptide-grafted microfluidic control chip from the third multi-porous culture plate, washing the polypeptide-grafted microfluidic control chip with PBS, placing the polypeptide-grafted microfluidic control chip into a fourth multi-porous culture plate, and then adding paraformaldehyde to the fourth multi-porous culture plate to fix the polypeptide-grafted microfluidic control chip in the paraformaldehyde;

[0029] S423, adding a live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate fixed with the polypeptide-grafted microfluidic control chip for fluorescent labeling and washing it with PBS, then adding DAPI dye to the fourth multi-well culture plate to fluorescently label the polypeptide-grafted microfluidic control chip, and then taking out the polypeptide-grafted microfluidic control chip and washing it with PBS;

[0030] S424. Under a laser confocal microscope, use the FITC channel to observe the cell distribution, cell adhesion number and cell differentiation on the surface of the polypeptide-grafted microfluidic control chip in step 423 to determine the regional cells with biological activity.

[0031] The concentration range of the polypeptide solution in step S3 is 1 μM to 5 mM.

[0032] The present invention has the following advantages and beneficial effects compared with the prior art:

[0033] 1. The present invention can further reduce the time for sample preparation and increase the concentration gradient of the sample through the first serpentine flow groove, the second serpentine flow groove and the liquid storage tray to achieve the effect of improving the experimental accuracy; by preparing surfaces with different grafting densities, the gradient of grafting density changes can be obtained in a short time, which effectively improves the sample preparation efficiency and greatly reduces the workload.

[0034] 2. The present invention uses a high-throughput screening method for microfluidic control chips to quickly determine the preparation parameters of polypeptide grafted microfluidic control chips, including the optimal grafting density of bioactive polypeptides, the time required for immersing titanium sheets during the preparation process, and the concentration of bioactive polypeptide solutions; it solves the problem of cumbersome and time-consuming processes in traditional orthogonal experimental methods; it can intuitively compare the differences in the performance of different bioactive polypeptides, avoiding the problems of using traditional uniform sample grafting experiments with many times, many groups, and complex variables; it guides the surface functionalization modification of titanium sheets, namely antibacterial modification, and provides preparation parameters for subsequent experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of a microfluidic control chip of the present invention (3×3 format of the first structure);

[0036] Figure 2 is a schematic diagram of a microfluidic control chip of the present invention (5×4 format of the first structure);

[0037] Figure 3 is a schematic diagram of a microfluidic control chip of the present invention (5×5 format of the first structure);

[0038] Figure 4 is a schematic diagram of a microfluidic control chip of the present invention (3×3 format of the second structure);

[0039] Figure 5 is a schematic diagram of a microfluidic control chip of the present invention (5×4 format of the second structure);

[0040] Figure 6 is a schematic diagram of a microfluidic control chip of the present invention (5×5 format of the second structure);

[0041] Figure 7 Schematic diagram of the use effect of the microfluidic control chip (second structure) of the present invention;

[0042] Figure 8It is a schematic diagram of the flow direction of the polypeptide solution in the vertical and horizontal directions (arrows indicate the flow direction) of the microfluidic control chip (5×5 format of the first structure) of the present invention;

[0043] Fig. 9 It is a schematic diagram of the flow direction of the polypeptide solution in the vertical and horizontal directions (arrows indicate the flow direction) of the microfluidic control chip (5×5 format of the second structure) of the present invention;

[0044] Fig.10 It is a schematic diagram of the flow direction of the polypeptide solution in the height direction (the arrow is the flow direction) of the microfluidic control chip (5×5 format of the first structure) of the present invention;

[0045] Fig.11 It is a schematic diagram of the structural formula of CPAPAPKFKWWRMLI in the present invention;

[0046] Fig.12 It is a schematic diagram of the structural formula of CPAPAPRWWRIWKW in the present invention;

[0047] Fig.13 It is a schematic diagram of the structure of CPAPAPKRWWKWWRR in the present invention;

[0048] Fig.14 is a schematic diagram of the structural formula of the CPAPAPRGD polypeptide of the present invention;

[0049] Fig.15 is a schematic diagram of the structural formula of the CPAPAPYIGSR polypeptide of the present invention;

[0050] Fig.16 is a bar graph showing changes in MFI values ​​of the surface grafted with CPAPAPKRWWKWWRR-FITC in the present invention;

[0051] Fig.17 is a bar graph showing changes in the MFI value of the surface grafted with CPAPAPRWWRIWKWK-FITC in the present invention;

[0052] Fig.18 is a bar graph showing changes in the MFI value of the surface grafted with CPAPAPKFKWWRMLI-FITC in the present invention;

[0053] Markings of the components in the accompanying drawings: 1-liquid storage tray; 101-liquid inlet; 102-liquid outlet; 103-first section flow trough; 104-middle section flow trough; 105-last section flow trough; 106-regional unit 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

[0054] The purpose of the present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples cannot be described one by one here, but the implementation methods of the present invention are not therefore limited to the following examples.

[0055] Example 1

[0056] Staphylococcus aureus (ATCC 6538P); Escherichia coli (ATCC 8739); Pseudomonas aeruginosa (ATCC15442) Mouse bone

[0057] Mesenchymal stem cells (ATCC CRL-12424); vascular endothelial cells ( CRL-2873), purchase channel: VWR International, LLC, Pennsylvania, USA.

[0058] The CPAPAPKRWWKWWRR, CPAPAPKRWWKWWRRK-FITC, CPAPAPRGD, and CPAPAPYIGSR polypeptides used in the examples of this application were purchased from Shanghai Qiangyao Biotechnology Co., Ltd., and CPAPAPRWWRIWKW, CPAPAPRWWRIWKWK-FITC, CPAPAPKFKWWRMLI and CPAPAPKFKWWRMLIK-FITC were purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0059] like Figure 1-6 As shown, a multi-cell microfluidic platform includes a first serpentine flow slot, a second serpentine flow slot and a liquid storage tray 1, wherein the first serpentine flow slot includes a first longitudinal partition 2 and a second longitudinal partition 3, the first longitudinal partition 2 and the second longitudinal partition 3 are both provided with a second groove 201, and the first longitudinal partition 2 and the second longitudinal partition 3 are mirror images of each other; the height of the second groove 201 is lower than the frame height of the liquid storage tray 1, the first longitudinal partition 2 and the second longitudinal partition 3 are evenly installed in the liquid storage tray 1, the first longitudinal partition 2 and the second longitudinal partition 3 are cross-arranged in the inner cavity of the liquid storage tray 1 to form a first serpentine flow slot, and the two ends of the first serpentine flow slot are respectively provided with a liquid inlet 101 and a liquid outlet 102.

[0060] The first longitudinal partition 2 and the second longitudinal partition 3 can change the lateral flow direction of the polypeptide solution, thereby forming a serpentine flow direction. The height of the second groove 201 is lower than the height of the liquid storage tray 1, and the height of the second groove 201 is 1% to 99% of the first longitudinal partition 2 and the second longitudinal partition 3. The height of the second groove 201 is 0.1 to 20 mm, and the length of the second groove 201 is 1% to 99% of the length of the first longitudinal partition 2 and the second longitudinal partition 3. When the polypeptide solution is higher than the height of the second groove 201, it flows out from the second groove 201 to form a serpentine flow direction; the liquid storage tray 1 plays the role of storing liquid; the liquid inlet 101 is used to inject the polypeptide solution, and the liquid outlet 102 is used to flow out the polypeptide solution after the reaction is completed.

[0061] like Figure 1 As shown, the microfluidic platform of the first structure includes 9 regional cells 10, that is, the first serpentine flow groove includes 9 regional cells 106, and the specification of the microfluidic control chip is 3×3; the first serpentine flow groove includes 3 first transverse partitions 4 and 3 second transverse partitions 5, and the first transverse partitions 4 and the second transverse partitions 5 are installed in the first serpentine flow groove to form a second serpentine flow groove, forming 9 regional cells 106. The side length of the regional cell 106 is 0.05-100 mm.

[0062] like Figure 2 As shown, the specification of the microfluidic platform of the first structure is 5×4 (that is, a total of 20 regional cells 106), and the first serpentine flow groove includes 8 first transverse partitions 4 and 8 second transverse partitions 5. The first transverse partitions 4 and the second transverse partitions 5 are cross-arranged in the first serpentine flow groove to form a second serpentine flow groove, forming 20 regional cells.

[0063] like Figure 3 As shown, the specification of the microfluidic platform of the first structure is 5×5 (that is, a total of 25 regional cells 106), the first serpentine flow groove includes 10 first transverse partitions 4 and 10 second transverse partitions 5, the first transverse partitions 4 and the second transverse partitions 5 are cross-arranged in the first serpentine flow groove to form a second serpentine flow groove, forming 25 regional cells.

[0064] like Figure 1-3 As shown, the above-mentioned first transverse partition 4 and second transverse partition 5 are both provided with a first groove 401, and the first transverse partition 4 and the second transverse partition 5 are mirror images of each other; the height of the second groove 201 is the same as the size of the first groove 401, and is lower than the height of the first longitudinal partition 2, the second longitudinal partition 3 and the liquid storage tray 1.

[0065] like Figure 4As shown, the second structure of the microfluidic platform has specifications including 3×3, 5×4 and 5×5. Among them, the first serpentine flow groove of the microfluidic control chip with a specification of 3×3 includes a first section flow groove 103, a middle section flow groove 104 and a final section flow groove 105, with a total of 9 regional cells 106. The first section flow groove 103 is provided with a liquid inlet 101, which includes a third transverse partition 6 and a first transverse partition 4, and the first transverse partition 4 is provided with a first groove 401, and the height of the first groove 401 is the same as the height of the third transverse partition 6. The third transverse partition 6 and the first transverse partition 4 are installed in the first flow groove 103 according to the flow direction of the polypeptide solution; the middle flow groove 104 includes a first transverse partition 4 and a second transverse partition 5, and the second transverse partition 5 and the first transverse partition 4 are arranged in the middle flow groove 104 along the flow direction of the polypeptide solution; the last flow groove 105 includes a second transverse partition 5 and a third transverse partition 6, and the second transverse partition 5 and the third transverse partition 6 are arranged in sequence in the last flow groove according to the flow direction of the polypeptide solution. The height of the third transverse partition 6 is 1% to 99% of the height of the liquid storage tray 1.

[0066] like Figure 5 As shown, the microfluidic platform with a specification of 5×4 includes a total of 20 regional units 106, and the first serpentine flow groove includes a first flow groove, two middle flow grooves 104 and a last flow groove 105, which are connected in sequence according to the flow direction of the polypeptide solution. The first section flow trough 103 is provided with a liquid inlet 101, and the last section flow trough 105 is provided with a liquid outlet 102, and the liquid inlet 101 and the liquid outlet are both arranged on one side of the liquid storage tray 1; the first section flow trough 103 includes three third transverse baffles 6 and one first transverse baffle 4, and the three third transverse baffles 6 and the first transverse baffle 4 are arranged in the first section flow trough according to the flow direction of the polypeptide solution; the middle section flow trough 104 includes one second transverse baffle 5, two third transverse baffles 6 and one first transverse baffle 4, and the one second transverse baffle 5, two third transverse baffles 6 and the one first transverse baffle 4 are arranged in sequence in the middle section flow trough 104 according to the flow direction of the polypeptide solution; the last section flow trough 105 includes one second transverse baffle 5 and three third transverse baffles 6, and the one second transverse baffle 5 and the three third transverse baffles 6 are arranged in sequence in the last section flow trough 105 according to the flow direction of the polypeptide solution.

[0067] like Figure 6As shown, the second structure is a microfluidic platform with a specification of 5×5. The first serpentine flow groove has a total of 25 regional cells 106. The first serpentine flow groove includes a first section flow groove 103, three middle sections flow grooves 104 and a last section flow groove 105 which are sequentially connected according to the flow direction of the polypeptide solution. The first section flow groove 103 is provided with a liquid inlet 101, and the last section flow groove 105 is provided with a liquid outlet. The first section flow groove 103 includes three third transverse partitions 6 and one first transverse partition 4 which are sequentially arranged according to the flow direction of the polypeptide solution. The middle section flow groove 104 includes one second transverse partition 5, two third transverse partitions 6 and one first transverse partition 4 which are sequentially arranged according to the flow direction of the polypeptide solution; the last section flow groove 105 includes one second transverse partition 5 and three third transverse partitions 6 which are sequentially arranged according to the flow direction of the polypeptide solution.

[0068] like Figure 4-6 As shown, the height of the third transverse partition 6 is the same as the height of the first groove 401 of the first transverse partition 4, and the third transverse partition 6 is lower than the highest height of the first longitudinal partition 2, the second longitudinal partition 3 and the liquid storage tray 1. The first transverse partition 4 and the second transverse partition 5 allow the polypeptide solution to flow in a transverse direction to form a transverse serpentine flow direction; the third transverse partition 6 allows the polypeptide solution to flow in a serpentine direction in the height direction.

[0069] A high-throughput screening method for a multi-cell microfluidic platform comprises the following steps:

[0070] S1. Get the microfluidic control chip blueprint through design. According to the microfluidic control chip blueprint, use Ti 6 Al 4 The V powder is used as the material to produce a microfluidic control chip printout through 3D printing, and the microfluidic control chip printout is subjected to stress relief annealing treatment to obtain a microfluidic control chip.

[0071] The stress relief annealing treatment of the microfluidic chip printout includes the following steps:

[0072] S101, the microfluidic control chip print is heated to 300°C to 700°C at a heating rate of 3 to 10°C / min, and then the microfluidic control chip print is kept at this temperature for 1 to 3 hours, and then the heating is stopped and the print is naturally cooled to room temperature in the furnace;

[0073] S102, using a sandblasting machine to remove the oxide scale on the surface of the microfluidic control chip printed in step S101.

[0074] S2, cleaning the microfluidic control chip in step S1, and performing hydroxylation treatment on the microfluidic control chip to obtain a surface-modified microfluidic control chip;

[0075] S21, the hydroxylation treatment of the microfluidic control chip in step S2, comprises the following steps:

[0076] S211, the microfluidic control chip is ultrasonically cleaned using deionized water, acetone, anhydrous ethanol and deionized water in sequence for 10 to 20 minutes, and then the microfluidic control chip is blown dry using a protective gas (including nitrogen, argon or helium);

[0077] S212, immersing the microfluidic control chip in HF with a mass ratio of 2% to 8% for 3 to 15 minutes;

[0078] S213. Finally, the microfluidic control chip is cleaned, the microfluidic control chip is blown dry again with protective gas, and the microfluidic control chip is immersed in concentrated nitric acid for 10 to 30 minutes to obtain a surface-modified microfluidic control chip.

[0079] S3. Inject the polypeptide solution into the surface modified microfluidic control chip in step S2. The polypeptide solution is injected into the surface modified microfluidic control chip at a uniform speed through a syringe. The polypeptide solution flows through all the regional cells 106 of the surface modified microfluidic control chip. After the polypeptide solution flows through all the regional cells 106 of the surface modified microfluidic control chip (the grafting reaction time is 10 minutes to 20 hours), the microfluidic control chip is cleaned with deionized water or anhydrous ethanol to obtain a polypeptide grafted microfluidic control chip.

[0080] The molecular structure of the polypeptide in the polypeptide solution used in step S3 is HS-R-Peptide, wherein R includes but is not limited to C(PA) n P, C represent cysteine, n is an integer of 1 to 100, P represents proline, and A represents alanine; peptide includes but is not limited to KRWWKWWRR or KRWWKWWRRK-FITC or RWWRIWKW or RWWRIWKWK-FITC or KFKWWRMLI or KFKWWRMLIK-FITC or FKRIVQRIKDFLRNLV or FKRIVQRIKDFLRNLVK-FITC or KLLKLLKKLLKALG or KLLKLLKKLLKALGK-FITC or KRWWKWIRW or KRWWKWIRWK-FITC or RGD or RGD-FITC or YIGSR or YIGSR-FITC, etc. The concentration range of the polypeptide solution is 1 μM to 5 mM, and the reaction time is 10 min to 20 h.

[0081] S4. Cultivate bacteria or cells in the peptide grafting microfluidic control chip and determine the optimal bioactive peptide grafting density area cell 106.

[0082] S41, in step S4, culturing bacteria in the polypeptide-grafted microfluidic control chip, and then determining the area cells 106 where the antibacterial performance reaches the standard, specifically comprises the following steps:

[0083] S411. Prepare a certain amount of colorimetric culture medium, place the colorimetric culture medium in a sterilizing pot and sterilize at 121° C. for 2 hours; the colorimetric culture medium is capable of growing red colonies after being cultured on the culture medium for 18-48 hours, which is convenient for counting and observation.

[0084] S412, placing the polypeptide grafted microfluidic control chip in the first multi-well culture plate, and sequentially adding 10 to 10% of the concentration of each cell of the polypeptide grafted microfluidic control chip. 9 1 μl to 100 μl of bacterial solution with a CFU / ml is incubated on the polypeptide-grafted microfluidic control chip for 10 min to 4 h.

[0085] S413. After the co-incubation is completed, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate. After the bacterial solution has evaporated, the polypeptide-grafted microfluidic control chip is placed in a biological cabinet and allowed to stand for 10 to 15 minutes until the surface of the polypeptide-grafted microfluidic control chip is moistened and only bacteria adhering to the polypeptide-grafted microfluidic control chip are retained;

[0086] S414, in a biological safety cabinet, divide the color development medium of step S411 into two parts, add one part of the color development medium into the polypeptide grafted microfluidic control chip and let it stand until the color development medium solidifies, then place the polypeptide grafted microfluidic control chip in a culture dish, and use another part of the color development medium to seal the polypeptide grafted microfluidic control chip. The color development medium should be 3 to 5 mm higher than the polypeptide grafted microfluidic control chip to ensure that the color development medium will not dry out. After solidification, place the polypeptide grafted microfluidic control chip in a constant temperature incubator and culture for 18 to 48 hours;

[0087] S415. After the polypeptide grafted microfluidic control chip is statically cultured, the polypeptide grafted microfluidic control chip is observed for colony growth (the number of colonies in each regional cell 106 of the polypeptide grafted microfluidic control chip) under a stereo microscope to determine the regional cell 106 with the best bioactive polypeptide grafting density.

[0088] S42, culturing cells in the polypeptide-grafted microfluidic control chip in step S4, wherein the cells include bone marrow mesenchymal stem cells, vascular endothelial cells or neural cells, using FITC fluorescent labeling, and observing the production of the cells using a laser confocal microscope, specifically includes the following steps:

[0089] S421, placing the polypeptide grafted microfluidic control chip in a third multi-porous culture plate, which is a 12-well culture plate, sterilizing the polypeptide grafted microfluidic control chip with ethanol (an ethanol solution with a volume concentration of 75%) for 2.5 hours, and then adding a concentration of 10 / mL to 10 9 The cell suspension (volume 1 μl to 100 μl) was statically cultured. The static culture conditions of the bacterial suspension were 5% CO at a temperature of 37°C. 2 Cultivate in the environment for 1 hour to 30 days;

[0090] S422, taking out the polypeptide grafted microfluidic control chip from the third multi-porous culture plate, washing the polypeptide grafted microfluidic control chip with PBS for 3 times, placing the polypeptide grafted microfluidic control chip into a fourth multi-porous culture plate, which is a 12-well culture plate, and then adding paraformaldehyde to the fourth multi-porous culture plate to fix the polypeptide grafted microfluidic control chip with paraformaldehyde, and the fixing condition of the paraformaldehyde is to fix it at a temperature of 4°C for 12 hours;

[0091] S423, adding a live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate fixed with the polypeptide-grafted microfluidic control chip for fluorescent labeling, washing with PBS (phosphate buffered saline), adding DAPI dye (DAPI, i.e. 4',6-diamidino-2-phenylindole, is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy observation) for fluorescent labeling, and then taking out the polypeptide-grafted microfluidic control chip and washing it with PBS for 3 times;

[0092] S424. Under a laser confocal microscope, use the FITC channel to observe the cell distribution, cell adhesion number and cell differentiation on the surface of the polypeptide-grafted microfluidic control chip in step 423 to determine the regional cells 106 with biological activity.

[0093] S5. Determine the polypeptide grafting density of the cell 106 in the region of optimal bioactive polypeptide grafting density.

[0094] The method for determining the polypeptide grafting density in step S5 is as follows: grafting fluorescent polypeptides such as CPAPAPKRWWKWWRR-FITC or CPAPAPRWWRIWKWK-FITC or CPAPAPKFKWWRMLIK-FITC on the surface of the polypeptide grafted microfluidic control chip, measuring the fluorescence value of the polypeptide grafted microfluidic control chip and drawing a bar graph of the MFI value changes, determining the relationship between the grafting time and the grafting density through the MFI value change bar graph, and determining the grafting density of each regional unit cell 106 of the polypeptide grafted microfluidic control chip.

[0095] The surface-modified microfluidic control chip is immersed in the HS-R-Peptide solution to prepare a surface-modified microfluidic control chip with a target bioactive peptide grafting density. The method for determining the immersion parameters, the concentration A of the bioactive peptide solution, and the immersion time B is as follows:

[0096] The surface-modified microfluidic control chip with gradient grafted density of bioactive polypeptides in step S3 is divided into n regions along the flow direction of the polypeptide solution. For an N×L chip, it needs to be divided into N×L regional cells 106, and the regional cells 106 are numbered 1 to n, then 1≤n≤N×L, the concentration of the polypeptide solution in step S3 is aμM, and the time for the polypeptide solution to flow through all regional cells 106 in step S3 is bmin; then A / μM=a, B / min=nb / N×L, N≥3, L≥3, N and L are both integers;

[0097] The target bioactive polypeptide grafting density is the same as the grafting density of the bioactive polypeptide in the optimal bioactive polypeptide grafting density region cell 106 in step S4.

[0098] A high-throughput screening method of a multi-cell microfluidic platform in this embodiment includes the following steps:

[0099] S1. A square 5×5 microfluidic control chip with a size of 1.5 cm×1.5 cm is obtained through design and 3D printing. The microfluidic control chip is treated with 4% HF acid for 5 minutes, and then the microfluidic control chip is cleaned with deionized water for 5 minutes to remove the residual fluoride ions on the surface, thereby obtaining a microfluidic control chip;

[0100] S2, soaking the microfluidic control chip in concentrated nitric acid for 15 minutes to obtain sufficient hydroxyl groups for reaction, rinsing it with deionized water, and then drying it with nitrogen to obtain a surface-modified microfluidic control chip;

[0101] S3, the peptide solution is an anhydrous ethanol solution of CPAPAPKRWWKWWRRK-FITC peptide prepared with anhydrous ethanol as solvent, and its concentration is 150 μM; the volume of the peptide solution sucked by a 1 ml syringe is 400 μl, the flow rate of the peptide solution is 100 μl / h, the grafting reaction time is 4h, and the injection process is as follows Figure 7 As shown; after the reaction, the surface-modified microfluidic control chip grafted with the polypeptide is taken out, cleaned with anhydrous ethanol, and then dried with nitrogen to obtain the polypeptide-grafted microfluidic control chip;

[0102] S4, adding a water film on the surface of the polypeptide-grafted microfluidic control chip and observing it under a fluorescence microscope;

[0103] S5. Statistical fluorescence values ​​are plotted to draw a bar graph of MFI (Mean fluorescence intensity) value changes, such as Fig.16 As shown, it shows that the device can successfully construct a grafting density gradient.

[0104] Example 2

[0105] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0106] 1. In step S3 of this embodiment, the polypeptide solution uses an anhydrous ethanol solution of CPAPAPRWWRIWKWK-FITC polypeptide instead of an anhydrous ethanol solution of CPAPAPKRWWKWWRRK-FITC polypeptide. The MFI value change bar graph in this embodiment is as follows: Fig.17 shown.

[0107] Example 3

[0108] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0109] 1. The polypeptide solution in step S3 of this embodiment uses an anhydrous ethanol solution of CPAPAPKFKWWRMLIK-FITC polypeptide instead of an anhydrous ethanol solution of CPAPAPKRWWKWWRRK-FITC polypeptide. The MFI value change bar graph in this embodiment is as follows: Fig.18 shown.

[0110] Example 4

[0111] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0112] 1. In step S3 of this embodiment, the polypeptide solution uses an anhydrous ethanol solution of CPAPAPKRWWKWWRR polypeptide instead of an anhydrous ethanol solution of CPAPAPKRWWKWWRR-FITC polypeptide.

[0113] 2. In step S4 of this embodiment, the polypeptide grafted microfluidic control chip uses the agar sealing method to characterize the antibacterial performance, observe the bacterial growth of each regional cell 106, and the antibacterial effect of each regional cell 106, and determine the regional cell 106 with the best antibacterial performance. It is determined that the regional cell 106 that is effective against Staphylococcus aureus starts from the 5th regional cell 106, the regional cell 106 against Pseudomonas aeruginosa starts from the 6th regional cell 106, and the regional cell 106 against Escherichia coli starts from the 5th region to the 25th regional cell 106 with the highest concentration; therefore, the 5th region is selected as the antibacterial starting regional cell 106. The grafting density of polypeptide molecules in this region is calculated to be 28.01ng / cm 2 , the grafting density is the optimal grafting density of the polypeptide; its preparation parameters are: the concentration of the CPAPAPKRWWKWWRR polypeptide solution is 150 μM, and the immersion time is 40 minutes.

[0114] 3. Scale-up experiment: Take the 3D printed Ti with the same preparation parameters of hydroxylation treatment 6 Al 4 The Ti V sheet substrate was immersed in 1 mL of 150 μM CPAPAPKRWWKWWRR peptide solution and allowed to stand for 40 minutes. 6 Al 4 After the V sheet was taken out, it was washed three times with ethanol and deionized water respectively to obtain a TiO2 sheet uniformly grafted with CPAPAPKRWWKWWRR polypeptide. 6 Al 4 V sheet; Ti with uniform grafting of CPAPAPKRWWKWWRR polypeptide obtained by calculation 6 Al 4 The grafting density of peptide molecules on the surface of V sheet is 28.01ng / cm 2 .

[0115] 4. Uniform grafting of Ti on CPAPAPKRWWKWWRR polypeptide 6 Al 4 Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of V sheets, and the growth inhibition of bacteria on the surface was observed by agar sealing method. 6 Al 4 V sheet was used as the control group; the results showed that compared with the control group, the prepared CPAPAPKRWWKWWRR polypeptide was uniformly grafted on Ti 6 Al 4 The antibacterial rates of the V sheet surface against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were 99.36%, 98.83% and 99.68%, respectively.

[0116] Example 5

[0117] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0118] 1. In step S3 of this embodiment, the polypeptide solution uses an anhydrous ethanol solution of CPAPAPRWWRIWKW polypeptide instead of an anhydrous ethanol solution of CPAPAPKRWWKWWRRK-FITC polypeptide.

[0119] 2. In this embodiment, the antibacterial performance of the polypeptide grafted microfluidic control chip in step S4 is characterized by the agar sealing method, and the bacterial growth in each area and the antibacterial effect of each regional cell 106 are observed to determine the regional cell 106 with the best antibacterial performance. It is determined that the regional cell 106 that is effective against Staphylococcus aureus starts from the 12th regional cell 106, the anti-Pseudomonas aeruginosa region starts from the 13th regional cell 106, and the anti-Escherichia coli regional cell 106 starts from the 12th regional cell 106 to the 25th regional cell 106 with the highest concentration; therefore, the 12th regional cell 106 is selected as the antibacterial starting regional cell 106. The grafting density of the polypeptide molecule in the regional cell 106 is calculated to be 31.72ng / cm 2 , this grafting density is the optimal grafting density of the polypeptide; its preparation parameters are: the concentration of the RWWRIWKW polypeptide solution is 150μM, and the immersion time is 110 minutes.

[0120] 3. Scale-up experiment: Take the 3D printed Ti with the same preparation parameters after hydroxylation treatment 6 Al 4 The Ti substrate was immersed in 1 mL of 150 μM CPAPAPRWWRIWKW peptide solution and allowed to stand for 110 minutes. After being taken out, it was washed with ethanol and deionized water for 3 times respectively to obtain a Ti substrate uniformly grafted with CPAPAPRWWRIWKW peptide. 6 Al 4 V sheet surface; Ti CPAPAPRWWRIWKW polypeptide uniformly grafted by calculation 6 Al 4 The grafting density of peptide molecules on the surface of V sheet is 31.72ng / cm 2 ;

[0121] 4. Ti uniformly grafted with CPAPAPRWWRIWKW polypeptide 6 Al 4 Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of V sheets, and the growth inhibition of bacteria on the surface was observed by agar sealing method. 6 Al 4V sheet was used as the control group; the results showed that compared with the control group, the prepared CPAPAPRWWRIWKW polypeptide was uniformly grafted on Ti 6 Al 4 The antibacterial rates of V sheet against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli are 99.83%, 98.97% and 99.48% respectively.

[0122] Example 6

[0123] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0124] 1. In step S3 of this embodiment, the polypeptide solution uses an anhydrous ethanol solution of CPAPAPKFKWWRMLI polypeptide instead of an anhydrous ethanol solution of CPAPAPKRWWKWWRR-FITC polypeptide.

[0125] 2. In this embodiment, the antibacterial performance of the polypeptide grafted microfluidic control chip in step S4 is characterized by the agar sealing method, and the bacterial growth of each regional cell 106 and the antibacterial effect of each regional cell 106 are observed to determine the regional cell 106 with the best antibacterial performance. It is determined that the regional cell 106 that is effective against Staphylococcus aureus starts from the 23rd region, the regional cell 106 against Pseudomonas aeruginosa starts from the 24th regional cell 106, and the regional cell 106 against Escherichia coli starts from the 23rd regional cell 106 to the 25th regional cell 106 with the highest concentration; therefore, the 23rd regional cell 106 is selected as the antibacterial starting regional cell 106. The grafting density of the polypeptide molecule in the regional cell 106 is calculated to be 46.93ng / cm 2 , the grafting density is the optimal grafting density of the polypeptide; its preparation parameters are: the concentration of the CPAPAPKFKWWRMLI polypeptide solution is 150μM, and the immersion time is 220min.

[0126] 3. Scale-up experiment: Take the 3D printed Ti with the same preparation parameters of hydroxylation treatment 6 Al 4 The Ti substrate was immersed in 1 mL of 150 μM CPAPAPKFKWWRMLI peptide solution and allowed to stand for 220 minutes. After being taken out, it was washed three times with ethanol and deionized water respectively to obtain a Ti substrate uniformly grafted with CPAPAPKFKWWRMLI peptide. 6 Al 4 V sheet surface; Ti is uniformly grafted with CPAPAPKFKWWRMLI polypeptide by calculation 6 Al 4 The grafting density of peptide molecules on the surface of V sheet is 46.93ng / cm 2 .

[0127] 4. Ti uniformly grafted with CPAPAPKFKWWRMLI polypeptide 6 Al 4 Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of V sheets, and the growth inhibition of bacteria on the surface was observed by agar sealing method. 6 Al 4 V sheet was used as the control group; the results showed that compared with the control group, the prepared CPAPAPKFKWWRMLI polypeptide was uniformly grafted on Ti 6 Al 4 The antibacterial rates of V sheet against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli are 99.65%, 98.26% and 99.78% respectively.

[0128] Example 7

[0129] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0130] 1. The polypeptide solution in this example uses an anhydrous ethanol solution of RGD with a concentration of 150 μM to replace an anhydrous ethanol solution of CPAPAPKRWWKWWRR-FITC polypeptide with a concentration of 150 μM.

[0131] 2. The polypeptide grafted microfluidic control chip of step S3 is divided into 25 regional cells 106, and bone marrow mesenchymal stem cells and vascular endothelial cells are cultured on the surface thereof. After 24 hours, FITC is used for fluorescence labeling. The distribution of cells on the surface of the polypeptide grafted microfluidic control chip of step S3 is observed by laser confocal microscopy to determine the optimal bioactive polypeptide grafting density regional cell 106. It has been determined that the region that effectively promotes the adhesion of bone marrow mesenchymal stem cells starts from the 7th regional cell 106 and the region cell 106 that effectively promotes the adhesion of vascular endothelial cells starts from the 7th regional cell 106 to the 25th regional cell 106 with the highest concentration; therefore, the 7th regional cell 106 is selected as the optimal cell 106 for promoting cell adhesion. After calculation, it is determined that the obtained bioactive polypeptide grafting density is 30.85ng / cm 2 The grafting density is the optimal grafting density of the polypeptide, and its preparation parameters are: the concentration of the bioactive polypeptide solution is 150 μM, and the immersion time is 60 min.

[0132] 3. Scale-up experiment: Take the 3D printed Ti with the same preparation parameters of hydroxylation treatment 6 Al 4The Ti substrate was immersed in 1 mL of anhydrous ethanol solution of 150 μM CPAPAPRGD polypeptide and allowed to stand for 60 minutes. After being taken out, it was washed three times with acetone, ethanol and deionized water respectively to obtain a Ti substrate uniformly grafted with CPAPAPRGD polypeptide. 6 Al 4 V sheet; Ti uniformly grafted with CPAPAPRGD peptide was obtained by calculation 6 Al 4 The grafting density of peptide molecules on the surface of V sheet is 30.85ng / cm 2 .

[0133] 4. Uniformly graft Ti on CPAPAPRGD polypeptide 6 Al 4 Bone marrow mesenchymal stem cells and vascular endothelial cells were cultured on the surface of the V sheet. After 24 hours, they were fluorescently labeled with FITC and observed under a laser confocal microscope. 6 Al 4 V sheet was used as the control group; the results showed that the number of bone marrow mesenchymal stem cells and vascular endothelial cells on the surface of the treated samples was 1.69 and 1.83 times that of the control group.

[0134] Example 8

[0135] Except for the following technical features, other technical features in this embodiment are the same as those in Embodiment 1:

[0136] 1. The polypeptide solution in this example uses a 150 μM CPAPAPYIGSR polypeptide anhydrous ethanol solution instead of a 150 μM CPAPAPKRWWKWWRR-FITC polypeptide anhydrous ethanol solution.

[0137] 2. The polypeptide grafted microfluidic control chip of step S3 is divided into 25 regional cells 106, and the nerve cells on its surface are fluorescently labeled with FITC after 24 hours. The differentiation of cells on the surface of the polypeptide grafted microfluidic control chip is observed by laser confocal microscopy to determine the optimal bioactive polypeptide grafting density regional cells 106. It is determined that the regional cells 106 that effectively promote nerve cell differentiation start from the 6th regional cell 106 to the 25th regional cell 106 with the highest concentration; therefore, the 6th regional cell 106 is selected as the optimal cell 106 for promoting cell adhesion. After calculation, it is determined that the obtained bioactive polypeptide grafting density is 29.84ng / cm 2 The preparation parameters are as follows: the grafting density is the optimal grafting density of the polypeptide: the concentration of the bioactive polypeptide solution is 150 μM, and the immersion time is 50 min.

[0138] 3. Scale-up experiment: Take the 3D printed Ti with the same preparation parameters of hydroxylation treatment 6 Al 4 The Ti sheet substrate was immersed in 1 mL of anhydrous ethanol solution of 150 μM CPAPAPYIGSR polypeptide and allowed to stand for 50 minutes. After being taken out, it was washed three times with acetone, ethanol and deionized water respectively to obtain a Ti substrate uniformly grafted with CPAPAPYIGSR polypeptide. 6 Al 4 V sheet; Ti uniformly grafted with CPAPAPYIGSR peptide was obtained by calculation 6 Al 4 The grafting density of peptide molecules on the surface of V sheet is 29.84ng / cm 2 .

[0139] 4. Uniform grafting of Ti on YIGSR polypeptide 6 Al 4 Neurons were cultured on the surface of the V sheet. After 24 hours, they were fluorescently labeled with FITC and observed under a laser confocal microscope. 6 Al 4 V sheets were used as the control group; the results showed that the number of nerve cells on the surface of the treated samples was 1.69 and 1.83 times that of the control group.

[0140] Comparative Example 1

[0141] The traditional method for bioactive peptide grafting density modification on titanium sheet surface is as follows:

[0142] (1) Take 75 square titanium sheets with a size of 1 cm × 1 cm, treat the titanium sheets with 4% HF acid for 5 min, wash the titanium sheets with deionized water for 5 min to remove residual fluoride ions on the surface; then ultrasonically wash the titanium sheets with deionized water for 3 min; finally, soak the titanium sheets in concentrated nitric acid for 15 min, ultrasonically wash the titanium sheets with deionized water for 3 min, and blow dry the titanium sheets with nitrogen.

[0143] (2) The titanium sheet of step (1) was laid flat and fixed in a container, and 300 μl of anhydrous ethanol solution of the bioactive polypeptide CPAPAPKRWWKWWRR-FITC was added, with a concentration of 150 μM, so that it covered the bottom of the titanium sheet. The 75 titanium sheets were divided into 25 groups, each group had 3 titanium sheets, and the immersion time of the 25 groups of titanium sheets in the bioactive polypeptide CPAPAPKRWWKWWRR-FITC solution was 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, 130 min, 140 min, 150 min, 160 min, 170 min, 180 min, 190 min, 200 min, 210 min, 220 min, 230 min and 240 min, respectively.

[0144] (3) Taking out the titanium sheet from step (2), washing the titanium sheet three times with anhydrous ethanol and deionized water respectively, to obtain titanium sheets with different bioactive polypeptide grafting densities.

[0145] (4) Use the FITC channel of a fluorescence microscope to characterize changes in surface fluorescence values.

[0146] Comparative Example 2

[0147] In this comparative example, except for the following technical features, other technical features are the same as those of comparative example 1:

[0148] The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPRWWRIWKW-FITC instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR-FITC.

[0149] Comparative Example 3

[0150] In this comparative example, except for the following technical features, other technical features are the same as those of comparative example 1:

[0151] The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKFKWWRMLI-FITC instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR-FITC.

[0152] Comparative Example 4

[0153] In this comparative example, except for the following technical features, other technical features are the same as those of comparative example 1:

[0154] 1. The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR-FITC.

[0155] 2. In this comparative example, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of the titanium sheet in step (3). The distribution of bacteria on the surface of the titanium biomaterial and the antibacterial effect under each condition were observed by using the agar sealing method. The titanium substrate was determined to promote the best antibacterial effect through the above experiments. The preparation process conditions under the optimal grafting density were determined to be an immersion concentration of 150 μM and an immersion time of 40 min. The grafting density of polypeptide molecules in this area was calculated to be 28.01 ng / cm2; at the same time, the untreated titanium substrate was used as the control group; the results showed that the surface resistance of the treated sample to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was 99.43%, 98.82% and 99.59% respectively. Therefore, the optimal preparation parameters are: the concentration of the bioactive polypeptide solution is 150 μM, and the immersion time is 40 minutes.

[0156] Comparative Example 5

[0157] In this comparative example, except for the following technical features, other technical features are the same as those of comparative example 1:

[0158] 1. The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPRWWRIWKW instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR-FITC.

[0159] 2. In this comparative example, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of the titanium sheet in step (3), and the distribution of bacteria on the surface of the titanium sheet and the antibacterial effect under each condition were observed by using the agar sealing method. The titanium sheet was determined to promote the best antibacterial effect through the above experiments. The preparation process conditions under the optimal grafting density were determined to be an immersion concentration of 150 μM and an immersion time of 110 min. The grafting density of the polypeptide molecules in this area was calculated to be 31.72 ng / cm 2 ; At the same time, the untreated titanium sheet was used as the control group; the results showed that the surface of the treated sample was 99.43%, 98.82% and 99.59% anti-Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli respectively. Therefore, the optimal preparation parameters are: the concentration of the bioactive peptide solution is 150μM, and the immersion time is 110 minutes.

[0160] Comparative Example 6

[0161] In this comparative example, except for the following technical features, other technical features are the same as those of comparative example 1:

[0162] 1. The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKFKWWRMLI instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR-FITC.

[0163] 2. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on the surface of the titanium sheet in step (3). The distribution of bacteria on the surface of the titanium sheet and the antibacterial effect under each condition were observed by using the agar sealing method. The titanium sheet was determined to promote the best antibacterial effect through the above experiments. The preparation process conditions under the optimal grafting density were determined to be an immersion concentration of 150 μM and an immersion time of 220 min. The grafting density of the polypeptide molecules in this area was calculated to be 46.93 ng / cm 2 ; At the same time, the untreated titanium sheet was used as the control group; the results showed that the surface of the treated sample was 99.37%, 98.72% and 99.38% anti-Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli respectively. Therefore, the optimal preparation parameters are: the concentration of the bioactive peptide solution is 150μM, and the immersion time is 220 minutes.

[0164] Comparative Example 7

[0165] Except for the following technical features, the other technical features of this comparative example are the same as those of comparative example 1:

[0166] 1. The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPRGD instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR.

[0167] 2. Bone marrow mesenchymal stem cells and vascular endothelial cells were cultured on the titanium sheet of step (3). After 24 hours, FITC was used for fluorescence labeling. The distribution of cells on the surface of the titanium sheet was observed by laser confocal microscopy to determine the optimal area of ​​cell distribution. It was determined that the preparation process conditions under the optimal grafting density were an immersion concentration of 150 μM and an immersion time of 60 min. The grafting density of the polypeptide molecules in this area was calculated to be 30.85 ng / cm 2 ; At the same time, the untreated titanium substrate was used as the control group; the results showed that the number of bone marrow mesenchymal stem cells and vascular endothelial cells on the surface of the treated samples was 1.69 and 1.83 times that of the control group.

[0168] Comparative Example 8

[0169] Except for the following technical features, the other technical features of this comparative example are the same as those of comparative example 1:

[0170] 1. The polypeptide solution in this comparative example uses an anhydrous ethanol solution of the biologically active polypeptide CPAPAPYIGSR instead of an anhydrous ethanol solution of the biologically active polypeptide CPAPAPKRWWKWWRR.

[0171] 2. Cultivate neural cells on the surface of the titanium sheet from step (3), and use FITC for fluorescence labeling after 24 hours. Observe the differentiation of cells on the surface through laser confocal microscopy to determine the optimal area for cell differentiation, measure the degree of cell differentiation based on the aspect ratio of the surface cells, and statistically compare the biological properties of promoting cell differentiation at different locations on the surface. It has been determined that the preparation process conditions under the optimal grafting density are an immersion concentration of 150 μM and an immersion time of 50 min. The grafting density of polypeptide molecules in this area is calculated to be 29.84 ng / cm 2 ; At the same time, the untreated titanium sheet was used as the control group; the results showed that the differentiation of neural cells on the surface of the treated samples was 1.38 and 1.49 times that of the control group.

[0172] From the above examples and comparative 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 of the prepared active polypeptide grafted modification, including the optimal grafting density of the biologically active polypeptide, the time required for the material in the preparation process, and the concentration of the biologically active polypeptide solution.

[0173] The above specific implementation modes are preferred embodiments of the present invention and cannot be used to limit the present invention. Any other changes or other equivalent replacement methods that do not deviate from the technical solution of the present invention are included in the protection scope of the present invention.

Claims

1. A multi-cell microfluidic platform, characterized in that: The invention comprises a first serpentine flow groove, a second serpentine flow groove and a liquid storage tray, wherein the first serpentine flow groove is provided with a liquid inlet and a liquid outlet at both ends, the first serpentine flow groove comprises a first longitudinal partition and a second longitudinal partition, the first longitudinal partition and the second longitudinal partition are both provided with a second groove, the height of the second groove is lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition are cross-arranged and installed on the liquid storage tray, and the second serpentine flow groove is installed on the first serpentine flow groove; The second serpentine flow groove includes a first transverse baffle and a second transverse baffle, the first transverse baffle and the second transverse baffle are both provided with a first groove, the height of the first groove is lower than the first longitudinal baffle and the second longitudinal baffle respectively, the first transverse baffle and the second transverse baffle are both installed in the first serpentine flow groove, and the first transverse baffle and the second transverse baffle are cross-arranged.

2. A multi-cell microfluidic platform, characterized in that: The invention comprises a first serpentine flow groove, a second serpentine flow groove and a liquid storage tray, wherein the first serpentine flow groove is provided with a liquid inlet and a liquid outlet at both ends, the first serpentine flow groove comprises a first longitudinal partition and a second longitudinal partition, the first longitudinal partition and the second longitudinal partition are both provided with a second groove, the height of the second groove is lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition are cross-arranged and installed on the liquid storage tray, and the second serpentine flow groove is installed on the first serpentine flow groove; The second serpentine flow groove includes a first transverse baffle, a second transverse baffle and a third transverse baffle. The first transverse baffle and the second transverse baffle are both provided with a first groove. The height of the first groove is lower than that of the first longitudinal baffle and the second longitudinal baffle, respectively. The height of the third transverse baffle is lower than that of the first longitudinal baffle and the second longitudinal baffle, respectively. The third transverse baffle is installed at both ends of the first serpentine flow groove. The first transverse baffle and the second transverse baffle are arranged crosswise.

3. A multi-cell microfluidic platform, characterized in that: The invention comprises a first serpentine flow groove, a second serpentine flow groove and a liquid storage tray, wherein the first serpentine flow groove is provided with a liquid inlet and a liquid outlet at both ends, the first serpentine flow groove comprises a first longitudinal partition and a second longitudinal partition, the first longitudinal partition and the second longitudinal partition are both provided with a second groove, the height of the second groove is lower than the height of the liquid storage tray, the first longitudinal partition and the second longitudinal partition are cross-arranged and installed on the liquid storage tray, and the second serpentine flow groove is installed on the first serpentine flow groove; The second serpentine flow groove includes a first flow groove, multiple middle flow grooves and a terminal flow groove connected in sequence, the first flow groove is provided with a liquid inlet, the terminal flow groove is provided with a liquid outlet, the first flow groove includes three third transverse baffles and a first transverse baffle arranged in sequence, the multiple middle flow grooves include second transverse baffles, two third transverse baffles and a first transverse baffle arranged in sequence, the terminal flow groove includes a second transverse baffle and three third transverse baffles arranged in sequence, the first transverse baffle and the second transverse baffle are provided with a first groove, the height of the third transverse baffle is lower than that of the first longitudinal baffle and the second longitudinal baffle, and the height of the first groove is lower than that of the first longitudinal baffle and the second longitudinal baffle.

4. A high-throughput screening method for a multi-cell microfluidic platform, characterized in that: The following steps are involved: S1. Designing and obtaining a microfluidic control chip printout through 3D printing, wherein the microfluidic control chip printout is subjected to stress relief annealing treatment to obtain a microfluidic control chip; S2, cleaning the microfluidic control chip, and performing hydroxylation treatment on the microfluidic control chip to obtain a surface-modified microfluidic control chip, wherein the surface-modified microfluidic control chip is a multi-cell microfluidic platform according to any one of claims 1 to 3; S3, injecting the polypeptide solution into the surface-modified microfluidic control chip, the polypeptide solution passes through the regional cells of the surface-modified microfluidic control chip at a uniform speed, the reaction time of the polypeptide solution and the surface-modified microfluidic control chip is 10 minutes to 20 hours, and after the polypeptide solution has reacted with all the regional cells, the microfluidic control chip is cleaned to obtain a polypeptide-grafted microfluidic control chip; S4, culturing bacteria or cells in the polypeptide grafting microfluidic control chip to determine the optimal biologically active polypeptide grafting density area cell; S5. Determine the polypeptide grafting density of the cells in the optimal bioactive polypeptide grafting density region.

5. The high-throughput screening method of a multi-cell microfluidic platform according to claim 4, characterized in that: The step S2 of performing hydroxylation on the microfluidic control chip comprises the following steps: S211, the microfluidic control chip is ultrasonically cleaned using deionized water, acetone, anhydrous ethanol and deionized water in sequence for 10 to 20 minutes, and then the microfluidic control chip is blown dry using a protective gas; S212, immersing the microfluidic control chip in HF acid with a mass ratio of 2% to 8% for 3 to 15 minutes; S213, finally cleaning the microfluidic control chip, drying the microfluidic control chip with protective gas again, and soaking the microfluidic control chip in concentrated nitric acid for 10 to 30 minutes to obtain a surface-modified microfluidic control chip.

6. The high-throughput screening method of a microfluidic control chip according to claim 4, characterized in that: The polypeptide molecular structure in the polypeptide solution in step S3 is HS-R-Peptide, wherein R includes C(PA) n P, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and the peptide includes KRWWKWWRR or KRWWKWWRRK-FITC or RWWRIWKW or RWWRIWKWK-FITC or KFKWWRMLI or KFKWWRMLIK-FITC or FKRIVQRIKDFLRNLV or FKRIVQRIKDFLRNLVK-FITC or KLLKLLKKLLKALG or KLLKLLKKLLKALGK-FITC or KRWWKWIRW or KRWWKWIRWK-FITC or RGD or RGD-FITC or YIGSR or YIGSR-FITC.

7. The high-throughput screening method of a multi-cell microfluidic platform according to claim 4, characterized in that: Cultivating bacteria in the polypeptide-grafted microfluidic control chip in step S4 comprises the following steps: S411, preparing a certain amount of color development medium, and placing the color development medium in a sterilizing pot and sterilizing it at 121° C. for 2 hours; S412, placing the polypeptide-grafted microfluidic control chip in a first multi-well culture plate, and sequentially adding 10 to 10% 9 1 μl to 100 μl of bacterial solution with a CFU / ml, incubating the bacterial solution in the polypeptide-grafted microfluidic control chip for 10 minutes to 24 hours; S413, after the co-incubation is completed, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate, and after the bacterial solution is completely evaporated, the polypeptide-grafted microfluidic control chip is placed in a biological cabinet and allowed to stand for 10 to 15 minutes; S414, dividing the color development medium of step S411 into two parts, adding one part of the color development medium into the polypeptide grafted microfluidic control chip and letting it stand until the color development medium solidifies, then placing the polypeptide grafted microfluidic control chip into a culture dish, using another part of the color development medium to seal the polypeptide grafted microfluidic control chip, and placing the polypeptide grafted microfluidic control chip into a constant temperature incubator for 18-48 hours after solidification; S415. After the polypeptide grafted microfluidic control chip is statically cultured, the bacterial colony growth of the polypeptide grafted microfluidic control chip is observed under a stereo microscope to determine the regional cells with the best bioactive polypeptide grafting density.

8. The high-throughput screening method of a multi-cell microfluidic platform according to claim 4, characterized in that: Cultivating cells in the polypeptide-grafted microfluidic control chip in step S4 comprises the following steps: S421, placing the polypeptide grafted microfluidic control chip in a third multi-well culture plate, sterilizing the polypeptide grafted microfluidic control chip, and sequentially adding 10 to 10% iodine to each cell of the microfluidic control chip. 9 1 μl to 100 μl of cell suspension with a concentration of 1 cell / mL is statically cultured for 1 hour to 30 days; S422, taking out the polypeptide-grafted microfluidic control chip from the third multi-porous culture plate, washing the polypeptide-grafted microfluidic control chip with PBS, placing the polypeptide-grafted microfluidic control chip into a fourth multi-porous culture plate, and then adding paraformaldehyde to the fourth multi-porous culture plate to fix the polypeptide-grafted microfluidic control chip in the paraformaldehyde; S423, adding a live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate fixed with the polypeptide-grafted microfluidic control chip for fluorescent labeling and washing it with PBS, then adding DAPI dye to the fourth multi-well culture plate to fluorescently label the polypeptide-grafted microfluidic control chip, and then taking out the polypeptide-grafted microfluidic control chip and washing it with PBS; S424. Under a laser confocal microscope, use the FITC channel to observe the cell distribution, cell adhesion number and cell differentiation on the surface of the polypeptide-grafted microfluidic control chip in step 423 to determine the regional cells with biological activity.

9. The high-throughput screening method of a multi-cell microfluidic platform according to claim 4, characterized in that: The concentration range of the polypeptide solution in step S3 is 1 μM to 5 mM.

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