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

Abstract

The invention discloses a spiral multi-cell microfluidic platform and a high-throughput screening method thereof. The platform comprises four spiral flow grooves and a liquid storage tray, the spiral flow groove comprises a longitudinal partition plate, a transverse partition plate and a center partition plate, the transverse partition plate is provided with a first groove and a second groove, the center partition plate is provided with a center groove, and the longitudinal partition plate and the transverse partition plate are evenly installed in the liquid storage tray in a central symmetry mode; the height of the central groove is lower than that of the liquid storage tray, and the first grooves and the second grooves are distributed in a mirror image mode and are distributed in the spiral flow direction groove in a crossed mode. The first grooves and the second grooves are lower than the longitudinal partition plates; the liquid storage tray is provided with a liquid inlet and three liquid outlets. According to the high-throughput screening method, the change gradient of the grafting density can be obtained in a short time, the preparation efficiency of a sample is effectively improved, the workload is greatly reduced, and the problems that a traditional orthogonal experiment method is tedious in process and time-consuming are solved.

Description

Technical Field

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

[0002] Problems such as infection, incompatibility, and poor functional effects caused by poor surface characteristics of biomaterials have hindered their further development. To solve problems such as poor surface activity of titanium-based implants, the surface of the material is modified with active molecules. Due to the sequence diversity and functional controllability of polypeptides, the material surface can be precisely adjusted, and different functional polypeptides are used for surface functionalization to effectively prepare high-performance functional surfaces. However, when multiple functional polypeptides coexist, their interactions may have an important impact on surface properties, especially there is often a contradiction between the antibacterial and repair-promoting properties. In actual research, orthogonal experimental design is usually used to optimize the performance combination, but the process is cumbersome, costly, and time-consuming; and orthogonal experiments can often only deal with the interaction of two properties, and it is difficult to reveal the complex non-linear relationship and multiple interactions between multiple properties, affecting the accuracy of the conclusion.

[0003] The high-throughput experimental platform based on the gradient surface breaks through the limitations of orthogonal experiments and can test a large number of surface modification combinations in a short time to quickly and efficiently screen multifunctional surfaces. However, due to the lack of a clear boundary in the traditional high-throughput platform, the data acquisition accuracy is insufficient; on the other hand, when multiple functions are involved (especially the conflict between antibacterial and repair-promoting properties), the large amount of data makes it difficult to analyze the rules and find the optimal performance combination. In current research, if both antibacterial and repair-promoting properties are concerned, usually only two properties can be dealt with; if only the repair-promoting property is studied, it is also limited to the optimization of three functions. As an emerging method, 3D printing technology can realize the functional design of the material surface at the microscale. Because of its high printing accuracy and precise control of details, it can realize complex geometries, structures, and porosities, showing great potential in the preparation of personalized materials and being widely used in the construction of various experimental platforms. At the same time, by combining with machine learning technology, it can efficiently process massive data, mine the internal rules therein, and realize multi-dimensional data analysis, providing strong tool support for the prediction and optimization of complex properties.

[0004] The invention platform previously prepared by the research group can at most combine the properties of two polypeptides. To further improve the efficiency, the research group further developed a microfluidic platform that can test a large number of surface modification combinations in a short time to quickly and efficiently screen multifunctional surfaces.

[0005] To overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a spiral multi-cell microfluidic platform.

[0006] Another object of the present invention is to provide a high-throughput screening method for a spiral multi-cell microfluidic platform.

[0007] The primary object of the present invention can be achieved by the following technical solutions:

[0008] A spiral multi-cell microfluidic platform includes four spiral flow channels and a liquid storage tray. The spiral flow channels include a longitudinal partition, a transverse partition, and a central partition. The transverse partition is provided with a first groove and a second groove, the central partition is provided with a central groove, and the longitudinal partition and the transverse partition are symmetrically installed in the liquid storage tray in a central symmetry form; the height of the central groove is lower than the height of the liquid storage tray, the first groove and the second groove are mirror-image distributed and cross-arranged in the spiral flow channel, and the height of the first groove and the second groove is lower than the longitudinal partition; one liquid inlet and three liquid outlets are provided on the liquid storage tray.

[0009] Preferably, the height of the first groove and the second groove is 1% - 99% of the height of the longitudinal partition, and the length of the first groove and the second groove is 1% - 99% of the length of the longitudinal partition.

[0010] Another object of the present invention can be achieved by the following technical solutions:

[0011] A high-throughput screening method for a spiral multi-cell microfluidic platform includes the following steps:

[0012] S1. Design and obtain a printed part of a spiral multi-cell microfluidic platform by 3D printing. The printed part of the spiral multi-cell microfluidic platform is subjected to stress relief annealing and sandblasting treatment to obtain a spiral multi-cell microfluidic platform.

[0013] S2. Clean the spiral multi-cell microfluidic platform, and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain a surface-modified spiral multi-cell microfluidic platform.

[0014] S3. Inject a polypeptide solution into the surface-modified spiral multi-cell microfluidic platform. The polypeptide solution uniformly passes through the regional cells of the surface-modified spiral multi-cell microfluidic platform and reacts. After the polypeptide solution reacts with all the regional cells, the spiral multi-cell microfluidic platform is cleaned to obtain a polypeptide-grafted spiral multi-cell microfluidic platform.

[0015] S4. Cultivate bacteria or cells in the polypeptide-grafted spiral multi-cell microfluidic platform to determine the regional cells with the optimal bioactive polypeptide grafting density.

[0016] S5. Determine the polypeptide grafting density of the cell in the optimal bioactive polypeptide grafting density region.

[0017] More preferably, the steps for hydroxylation treatment of the helical multi-cell microfluidic platform in step S2 are as follows:

[0018] S211. Ultrasonically clean the helical multi-cell microfluidic platform successively with deionized water, acetone, absolute ethanol and deionized water for 10 - 30 minutes, and then dry the helical multi-cell microfluidic platform with a protective gas;

[0019] S212. Immerse the helical multi-cell microfluidic platform in HF acid with a mass fraction of 2% - 8% for 4 - 18 minutes;

[0020] S213. Finally, clean the helical multi-cell microfluidic platform treated with HF acid in step S212, dry the helical multi-cell microfluidic platform again with a protective gas, immerse the helical multi-cell microfluidic platform in concentrated nitric acid and let it stand for 10 - 30 minutes to obtain a surface-modified helical multi-cell microfluidic platform.

[0021] More preferably, the structural formula of the polypeptide molecule in the polypeptide solution in step S3 is HS-R-Peptide, where R includes C(PA)nP, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and Peptide includes one of KRWWKWWRR or KRWWKWWRRK-FITC or IGKYKLQYLEQWTLK or IGKYKLQYLEQWTLK-FITC or YIGSR or YIGSRK-FITC or KIPKASSVPTELSAISTLYL or KIPKASSVPTELSAISTLYLK-FITC.

[0022] More preferably, the concentration range of the polypeptide solution in step S3 is 1 μM - 5 mM.

[0023] More preferably, the reaction time of the polypeptide solution and the surface-modified helical multi-cell microfluidic platform in step S3 is 5 minutes - 24 hours.

[0024] More preferably, culturing bacteria in the polypeptide-grafted helical multi-cell microfluidic platform in step S4 includes the following steps:

[0025] S411. Prepare the required nutrient broth medium, and place the nutrient broth medium in an autoclave for sterilization at 121 °C for 2 h;

[0026] S412. Place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in the first multi-well culture plate, and sequentially add 1 μl to 100 μl of the bacterial solution with a concentration of 10 to 10 9 CFU / ml to the regional cells of the microfluidic platform with the polypeptide-grafted helical multi-cell unit, and co-incubate the bacterial solution in the microfluidic platform with the polypeptide-grafted helical multi-cell unit for 10 minutes to 24 hours;

[0027] S413. After the co-incubation is completed, place the first multi-well culture plate in an oven to evaporate the bacterial solution. After the bacterial solution has evaporated, place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in a biological safety cabinet and let it stand for 10 to 15 minutes;

[0028] S414. Sequentially add the resazurin solution with a concentration of 10 to 1000 μg / ml to the regional cells of the microfluidic platform with the polypeptide-grafted helical multi-cell unit, mix it with the nutrient broth, and co-incubate the resazurin solution in the microfluidic platform with the polypeptide-grafted helical multi-cell unit for 10 minutes to 8 hours;

[0029] S415. After the static culture of the microfluidic platform with the polypeptide-grafted helical multi-cell unit is completed, observe the colony growth of the microfluidic platform with the polypeptide-grafted helical multi-cell unit under a fluorescence microscope to determine the regional cells with the optimal bioactive polypeptide grafting density.

[0030] A more preferred option is that culturing cells in the microfluidic platform with the polypeptide-grafted helical multi-cell unit in step S4 includes the following steps:

[0031] S421. Place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in the third multi-well culture plate, sterilize the microfluidic platform with the polypeptide-grafted helical multi-cell unit, and sequentially add 1 μl to 100 μl of the cell suspension with a concentration of 10 to 10 9 cells / mL to each regional cell of the microfluidic platform with the helical multi-cell unit for static culture for 1 hour to 30 days;

[0032] S422. Take out the microfluidic platform with the polypeptide-grafted helical multi-cell unit from the third multi-well culture plate, wash the microfluidic platform with the polypeptide-grafted helical multi-cell unit with PBS, place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in the fourth multi-well culture plate, and then add paraformaldehyde to the fourth multi-well culture plate to fix the microfluidic platform with the polypeptide-grafted helical multi-cell unit in the paraformaldehyde;

[0033] S423. Add a live cell F-actin microfilament protein staining reagent into the fourth multi-well culture plate of the microfluidic platform with the polypeptide-grafted helical multi-cell unit for fluorescence labeling, and wash it with PBS. Then add DAPI dye into the fourth multi-well culture plate to perform fluorescence labeling on the microfluidic platform with the polypeptide-grafted helical multi-cell unit. Then take out the microfluidic platform with the polypeptide-grafted helical multi-cell unit and wash it with PBS.

[0034] 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 microfluidic platform with the polypeptide-grafted helical multi-cell unit in step 423 to determine the regional cells with biological activity.

[0035] The present invention has the following advantages and beneficial effects compared with the prior art:

[0036] The present invention combines 3D printing technology to design and construct a microfluidic platform with a helical multi-cell unit. The previously invented snake-shaped high-throughput experimental platform by the research group can usually only study the performance of one or two polypeptides, while the helical high-throughput experimental platform described in the present invention can construct unidirectional, bidirectional, tri-directional, and tetra-directional polypeptide surfaces. This platform breaks through the limitations of the prior art in the research of multi-functional surface modification and can study four combinations of functional characteristics simultaneously. In addition, combined with machine learning methods, the grafting behavior under different experimental conditions is predicted by means of feature screening and fitting regression; the data-driven model training method captures complex non-linear relationships, reveals the internal connection between different surface parameters (such as polypeptide grafting density) and surface functions, and conducts efficient data analysis and optimization among multiple functional indicators (such as antibacterial performance, osteogenic performance, etc.) to explore the optimal grafting parameter combination and generate multiple solutions with excellent balance among different objectives. This platform successfully reveals the mutual relationship among various functional performances, providing a theoretical basis and practical support for rational design based on performance requirements. The application of this platform provides a new tool for preparing high-quality surfaces with diverse functions, provides new ideas for the development of biomedical materials, and is of great significance for promoting the development of the biomaterials field. Description of the Drawings

[0037] Figure 1 It is a schematic diagram of the microfluidic platform (3×3 format) with a helical multi-cell unit of the present invention;

[0038] Figure 2 It is a schematic diagram of the microfluidic platform (5×5 format) with a helical multi-cell unit of the present invention;

[0039] Figure 3Schematic diagram of the spiral multi-cell microfluidic platform (7×7 format) of the present invention;

[0040] Figure 4 Schematic diagram of the usage effect of the spiral multi-cell microfluidic platform (5×5 format) of the present invention;

[0041] Figure 5 Schematic diagram of the flow direction of the polypeptide solution in the longitudinal and transverse directions (the arrow indicates the flow direction) of the spiral multi-cell microfluidic platform (5×5 format) of the present invention;

[0042] Figure 6 Schematic diagram of the structural formula of CPAPAPKRWWKWWRR in the present invention;

[0043] Figure 7 Schematic diagram of the structural formula of CPAPAPIGKYKLQYLEQWTLK in the present invention;

[0044] Figure 8 Schematic diagram of the structural formula of CPAPAPYIGSR in the present invention;

[0045] Figure 9 Schematic diagram of the structural formula of CPAPAPKIPKASSVPTELSAISTLYL in the present invention;

[0046] Figure 10 Bar chart of the change in MFI value of the unidirectional polypeptide grafted surface grafted with CPAPAPKRWWKWWRRK-FITC in the present invention;

[0047] Figure 11 Bar chart of the change in MFI value of the bidirectional polypeptide grafted surface grafted with CPAPAPKRWWKWWRR and CPAPAPIGKYKLQYLEQWTLK-FITC in the present invention;

[0048] Figure 12 Bar chart of the change in MFI value of the tri-directional polypeptide grafted surface grafted with CPAPAPKRWWKWWRR, CPAPAPIGKYKLQYLEQWTL and CPAPAPYIGSRK-FITC in the present invention;

[0049] Figure 13 Bar chart of the change in MFI value of the four-directional polypeptide grafted surface grafted with CPAPAPKRWWKWWRR, CPAPAPIGKYKLQYLEQWTL, CPAPAPYIGSR and CPAPAPKIPKASSVPTELSAISTLYLK-FITC in the present invention;

[0050] Figure 14Bar chart of antibacterial surface results of the four-way polypeptide grafted surface in the present invention;

[0051] Figure 15 Bar chart of the surface results of promoting endothelial cell adhesion of the four-way polypeptide grafted surface in the present invention;

[0052] Figure 16 Bar chart of the surface results of promoting nerve differentiation of the four-way polypeptide grafted surface in the present invention;

[0053] Figure 17 Bar chart of the surface results of promoting osteogenic differentiation of the four-way polypeptide grafted surface in the present invention;

[0054] Markings of each component in the drawings: 1 - liquid storage tray; 101 - liquid inlet; 102 - liquid outlet; 103 - regional cell; 2 - longitudinal partition; 3 - transverse partition; 301 - first groove; 302 - second groove; 4 - central partition; 401 - central groove. Detailed implementation manners

[0055] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation manners of the present invention are not limited thereto. The materials used in the examples of the present invention can all be obtained through commercial channels.

[0056] The materials, the microfluidic platform with spiral multi-cells and the high-throughput screening method of the microfluidic platform with spiral multi-cells used in the specific embodiments are as follows:

[0057] Staphylococcus aureus (ATCC 6538P); human bone marrow mesenchymal stem cells (ATCC CRL-12424); vascular endothelial cells (CRL-2873), Schwann cells, purchase channel: VWR International, LLC, Pennsylvania, USA (VWR International Co., Ltd., Pennsylvania, USA).

[0058] The CPAPAPKRWWKWWRR, CPAPAPKRWWKWWRRK-FITC, CPAPAPIGKYKLQYLEQWTLK, CPAPAPIGKYKLQYLEQWTLK-FITC, CPAPAPYIGSR, CPAPAPYIGSRK-FITC, CPAPAPKIPKASSVPTELSAISTLYL, CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptides used in the embodiments of the present application were purchased from Shanghai Qiangyao Biotechnology Co., Ltd.

[0059] Such as Figures 1 to 3As shown in the figure, the four sides of the spiral multi-cell microfluidic platform are respectively labeled with the serial numbers "1", "2", "3" and "4". The spiral multi-cell microfluidic platform includes four spiral flow channels and a liquid storage tray 1. The spiral flow channels include longitudinal partitions 2, transverse partitions 3 and a central partition 4. The transverse partition 3 is provided with a first groove 301 and a second groove 302. The central partition 4 is provided with a central groove 401. The longitudinal partition 2 and the transverse partition 3 are uniformly installed in the liquid storage tray 1 in a centrosymmetric form; the height of the central groove is lower than the height of the liquid storage tray. The first groove and the second groove are mirror images of each other and are cross-arranged in the spiral flow channel. The height of the first groove 301 and the second groove 302 is lower than the height of the border of the longitudinal partition 2. The height of the central groove 401 is lower than the height of the first groove 301 and the second groove 302. One liquid inlet 101 and three liquid outlets 102 are arranged on the liquid storage tray.

[0060] The longitudinal partition 2 can change the flow direction of the polypeptide solution. After the liquid enters from the liquid inlet 101 and flows to the central area, it is divided into three branches and flows out to the three liquid outlets 102 at the same time, thus forming a spiral flow direction. The height of the first groove 301 and the second groove 302 is lower than the height of the liquid storage tray 1. The first groove 301 and the second groove 302 are mirror images of each other and are evenly distributed in the spiral flow channel. The height of the first groove 301 and the second groove 302 is 1% - 99% of the longitudinal partition 2. The height of the first groove 301 and the second groove 302 is 0.1 - 20 mm. The length of the first groove 301 and the second groove 302 is 1% - 99% of the length of the longitudinal partition. When the polypeptide solution is higher than the height of the first groove 301 and the second groove 302, it flows out from the second groove 302 to form a passage; the liquid storage tray 1 plays a role in storing liquid; the liquid inlet 101 is used to inject the polypeptide solution, and the liquid outlet 102 is used for the polypeptide solution that has completed the reaction to flow out.

[0061] As Figure 1 shown in the figure, the microfluidic platform includes 9 regional cells, that is, there are 9 regional cells in the spiral flow channel. The specification of the spiral multi-cell microfluidic platform is 3×3; the spiral flow channel includes 4 longitudinal partitions 2, 4 transverse partitions 3 and 4 central partitions 4. The transverse partition 3, the longitudinal partition 2 and the central partition 4 are installed in the spiral flow channel to form 9 regional cells. The side length of the regional cell is 0.05 - 100 mm.

[0062] As Figure 2 shown in the figure, the specification of the microfluidic platform is 5×5 (that is, there are a total of 25 regional cells). The spiral flow channel includes 20 transverse partitions 3, 8 longitudinal partitions 2 and 4 central partitions 4. The transverse partitions 3 are cross-arranged in the spiral flow channel to form 25 regional cells.

[0063] As shown Figure 3 in the figure, the specifications of the microfluidic platform are 7×7 (i.e., a total of 49 regional cells). The spiral flow channel includes 44 transverse partitions 3, 12 longitudinal partitions 2 and 4 central partitions 4. The transverse partitions 2 are arranged crosswise in the spiral flow channel to form 49 regional cells.

[0064] As shown Figures 1 to 3 in the figure, the above-mentioned transverse partitions 2 are all provided with a first groove 301. The second groove 302 has the same size as the first groove 301 and is lower than the heights of the longitudinal partition 3 and the liquid storage tray 1. The height of the central groove 401 is lower than the heights of the first groove 301, the second groove 302, the longitudinal partition 2 and the liquid storage tray 1.

[0065] The high-throughput screening method of the spiral multi-cell microfluidic platform in this embodiment includes the following steps:

[0066] S1. Obtain the drawing of the spiral multi-cell microfluidic platform through design. According to the drawing of the spiral multi-cell microfluidic platform, use Ti6Al4V powder as the material to 3D print the spiral multi-cell microfluidic platform printing part. The spiral multi-cell microfluidic platform printing part is subjected to stress relief annealing treatment to obtain the spiral multi-cell microfluidic platform.

[0067] The stress relief annealing treatment of the spiral multi-cell microfluidic platform printing part includes the following steps:

[0068] S101. The spiral multi-cell microfluidic platform printing part is heated to 300°C - 700°C at a heating rate of 3 - 10°C / min, then the spiral multi-cell microfluidic platform printing part is kept at this temperature for 1 - 3 hours, and after stopping heating, it is naturally cooled to room temperature in the furnace;

[0069] S102. Use a sandblaster to remove the oxide scale on the surface of the spiral multi-cell microfluidic platform printing part in step S101.

[0070] S2. Clean the spiral multi-cell microfluidic platform in step S1, and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain the surface-modified spiral multi-cell microfluidic platform;

[0071] The steps of performing hydroxylation treatment on the spiral multi-cell microfluidic platform in step S2 are as follows:

[0072] S211. The microfluidic platform with a spiral multi-cell structure is ultrasonically cleaned with deionized water, acetone, absolute ethanol, and deionized water in sequence for 10 - 30 min, and then dried with a protective gas (including nitrogen, argon, or helium).

[0073] S212. Then, the microfluidic platform with a spiral multi-cell structure is immersed in HF with a mass ratio of 2% - 8% for 4 - 18 min.

[0074] S213. Finally, the microfluidic platform with a spiral multi-cell structure that has been immersed in HF is cleaned thoroughly, dried again with a protective gas, and then immersed in concentrated nitric acid for 10 - 30 min to obtain a surface-modified microfluidic platform with a spiral multi-cell structure.

[0075] S3. Inject the polypeptide solution into the surface-modified microfluidic platform with a spiral multi-cell structure obtained in step S2. The polypeptide solution is injected into the surface-modified microfluidic platform with a spiral multi-cell structure at a uniform speed through a syringe. The polypeptide solution flows through all the regional cells 103 of the surface-modified microfluidic platform with a spiral multi-cell structure. After the polypeptide solution has flowed through all the regional cells 103 of the surface-modified microfluidic platform with a spiral multi-cell structure (the grafting reaction time is 5 min - 24 h), the microfluidic platform with a spiral multi-cell structure is cleaned with deionized water or absolute ethanol to obtain a microfluidic platform with a spiral multi-cell structure grafted with polypeptide.

[0076] The molecular structural formula of the polypeptide in the polypeptide solution used in step S3 is HS-R-Peptide, where R includes but is not limited to C(PA)nP, C represents cysteine, n is an integer from 1 to 100, P represents proline, and A represents alanine; Peptide includes but is not limited to KRWWKWWRR, or KRWWKWWRRK-FITC, or IGKYKLQYLEQWTLK, or IGKYKLQYLEQWTLK-FITC, or YIGSR, or YIGSRK-FITC, or KIPKASSVPTELSAISTLYL, or KIPKASSVPTELSAISTLYLK-FITC, etc. The concentration range of the polypeptide solution is 1 μM - 5 mM, and the reaction time is 5 min - 24 h.

[0077] S4. Cultivate bacteria or cells in the microfluidic platform with a spiral multi-cell structure grafted with polypeptide to determine the regional cells 103 with the optimal bioactive polypeptide grafting density.

[0078] The steps of cultivating bacteria in the microfluidic platform with a spiral multi-cell structure grafted with polypeptide in step S4 and then determining the regional cells 103 with qualified antibacterial performance specifically include the following steps:

[0079] S411. Prepare the required nutrient broth medium, place the nutrient broth medium in an autoclave and sterilize it at 121 °C for 2 h;

[0080] S412. Place the microfluidic platform of the polypeptide-grafted helical multi-cell unit in the first multi-well culture plate, and sequentially add 1 μl - 100 μl of the bacterial solution with a concentration of 10 - 10 9 CFU / ml to the regional cells of the microfluidic platform of the polypeptide-grafted helical multi-cell unit, and co-incubate the bacterial solution in the microfluidic platform of the polypeptide-grafted helical multi-cell unit for 10 minutes - 24 hours;

[0081] S413. After the co-incubation is completed, place the first multi-well culture plate in an oven to evaporate the bacterial solution. After the bacterial solution is completely evaporated, place the microfluidic platform of the polypeptide-grafted helical multi-cell unit in a biological safety cabinet and let it stand for 10 - 15 min;

[0082] S414. Sequentially add the resazurin solution with a concentration of 10 - 1000 μg / ml to the regional cells of the microfluidic platform of the polypeptide-grafted helical multi-cell unit, mix it with the nutrient broth, and co-incubate the resazurin solution in the microfluidic platform of the polypeptide-grafted helical multi-cell unit for 10 minutes - 8 hours;

[0083] S415. After the static culture of the microfluidic platform of the polypeptide-grafted helical multi-cell unit is completed, observe the colony growth of the microfluidic platform of the polypeptide-grafted helical multi-cell unit under a fluorescence microscope to determine the regional cells with the optimal biological activity polypeptide grafting density.

[0084] S42. Cultivate cells in the microfluidic platform of the polypeptide-grafted helical multi-cell unit in step S4. The cells include bone marrow mesenchymal stem cells, vascular endothelial cells or nerve cells, which are labeled with FITC fluorescence and the production of the cells is observed using a laser confocal microscope. The specific steps are as follows:

[0085] S421. Place the microfluidic platform of the polypeptide-grafted helical multi-cell unit in the third multi-well culture plate, which is a 6-well culture plate. Sterilize the microfluidic platform of the polypeptide-grafted helical multi-cell unit with ethanol (ethanol solution with a volume concentration of 75%) for 2 hours, and then add a cell suspension with a concentration of 10 cells / mL - 10 9 cells / mL (volume of 1 μL - 100 μL) to the third multi-well culture plate for static culture. The static culture conditions of the cell suspension are to culture it in an environment with 5% CO2 at a temperature of 37 °C for 1 hour - 30 days;

[0086] S422. Remove the microfluidic platform with the polypeptide-grafted helical multi-cell unit from the third multi-well culture plate, wash the microfluidic platform with the polypeptide-grafted helical multi-cell unit 3 times using PBS, and place the microfluidic platform with the polypeptide-grafted helical multi-cell unit into the fourth multi-well culture plate, which is a 6-well culture plate. Then add paraformaldehyde to the fourth multi-well culture plate, and the paraformaldehyde fixes the microfluidic platform with the polypeptide-grafted helical multi-cell unit. The fixation condition of paraformaldehyde is to fix at 4°C for 12 h.

[0087] S423. Add a live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate with the fixed microfluidic platform with the polypeptide-grafted helical multi-cell unit for fluorescence labeling. After washing with PBS (phosphate buffer solution), add DAPI dye (DAPI, that is, 4',6-diamidino-2-phenylindole, which is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy observation) for fluorescence labeling. Then take out the microfluidic platform with the polypeptide-grafted helical multi-cell unit and wash it 3 times with PBS.

[0088] 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 microfluidic platform with the polypeptide-grafted helical multi-cell unit in step 423, and determine the region cell 106 with biological activity.

[0089] S5. Determine the polypeptide grafting density of the region cell 103 with the optimal biological activity polypeptide grafting density.

[0090] The method for determining the polypeptide grafting density in step S5: Graft fluorescent polypeptides such as CPAPAPKRWWKWWRR-FITC or CPAPAPIGKYKLQYLEQWTLK-FITC or CPAPAPYIGSRK-FITC or CPAPAPKIPKASSVPTELSAISTLYLK-FITC on the surface of the microfluidic platform with the polypeptide-grafted helical multi-cell unit, measure the fluorescence value of the microfluidic platform with the polypeptide-grafted helical multi-cell unit and draw a bar chart of the change in MFI value, and determine the relationship between the grafting time and the grafting density through the bar chart of the change in MFI value, and determine the grafting density of each region cell 103 of the microfluidic platform with the polypeptide-grafted helical multi-cell unit.

[0091] Immerse the surface-modified microfluidic platform with the helical multi-cell unit in the HS-R-Peptide solution to prepare a surface-modified microfluidic platform with the target biological activity polypeptide grafting density, and determine the soaking parameters. The method for the concentration A of the biological activity polypeptide solution and the soaking time B is as follows:

[0092] The surface-modified helical multi-cell microfluidic platform with gradient grafting density of bioactive polypeptide 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 103, and the regional cells 103 are labeled from 1 to n, where 1≤n≤N×L. The concentration of the polypeptide solution in step S3 is a μM, and the time for the polypeptide solution to flow through all regional cells 103 in step S3 is b min. Then A / μM = a, B / min = nb / N×L, N≥3, L≥3, and both N and L are odd numbers.

[0093] Among them, the grafting density of the target bioactive polypeptide is the same as the grafting density of the bioactive polypeptide in the regional cell 103 with the optimal bioactive polypeptide grafting density in step S4.

[0094] Example 1

[0095] The high-throughput screening method for the helical multi-cell microfluidic platform in this example includes the following steps:

[0096] S1. A square 5×5 helical multi-cell microfluidic platform print with a size of 1.62 cm×1.62 cm is obtained through design and 3D printing. The helical multi-cell microfluidic platform is treated with 4% HF acid by mass for 5 min, and then washed with deionized water for 15 min to remove the residual fluoride ions on the surface, obtaining the helical multi-cell microfluidic platform.

[0097] S2. Then, the helical multi-cell microfluidic platform is immersed in concentrated nitric acid for 15 min to obtain sufficient reactive hydroxyl groups, rinsed thoroughly with deionized water, and then dried with nitrogen to obtain the surface-modified helical multi-cell microfluidic platform.

[0098] S3. The polypeptide solution is an absolute ethanol solution of CPAPAPKRWWKWWRRK-FITC polypeptide prepared with absolute ethanol as the solvent, and its concentration is 100 μM. The volume of the polypeptide solution aspirated by a 1 ml syringe is 400 μl, the flow rate of the polypeptide solution is 100 μl / h, and the grafting reaction time is 4 h. The injection process is as Figure 7 shown (injected from the liquid inlet 101 at "1"); after the reaction, the surface-modified helical multi-cell microfluidic platform grafted with polypeptide is taken out, washed thoroughly with absolute ethanol, and then dried with nitrogen to obtain the helical multi-cell microfluidic platform grafted with polypeptide.

[0099] S4. A water film is dropped on the surface of the helical multi-cell microfluidic platform grafted with polypeptide, and it is observed under a fluorescence microscope.

[0100] S5. Statistically analyze the fluorescence values and plot a bar chart showing the changes in MFI (Mean Fluorescence Intensity) values. As shown in Figure 10 , it shows that the device can successfully construct a grafting density gradient.

[0101] Example 2

[0102] In this example, the absolute ethanol solution of CPAPAPKRWWKWWRRK-FITC polypeptide in Example 1 was replaced with the absolute ethanol solution of CPAPAPKRWWKWWRRK polypeptide. After performing the steps of Example 1, the sample was cleaned, rotated 90° clockwise, and an absolute ethanol solution of CPAPAPIGKYKLQYLEQWTLK-FITC polypeptide prepared with absolute ethanol as the solvent was introduced from the liquid outlet 102 at "2". (The subsequent steps are the same as S3 - S5. This example is mainly to prove the ability to construct a bidirectional polypeptide grafting density.) Its concentration was 100 μM; the volume of the polypeptide solution aspirated with a 1 ml syringe was 400 μl, the flow rate of the polypeptide solution was 100 μl / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified helical multi-cell microfluidic platform grafted with polypeptide was taken out, cleaned with absolute ethanol, and then dried with nitrogen to obtain a helical multi-cell microfluidic platform grafted with polypeptide; a water film was dropped on the surface of the helical multi-cell microfluidic platform grafted with polypeptide and observed under a fluorescence microscope; statistically analyze the fluorescence values and plot a bar chart showing the changes in MFI (Mean Fluorescence Intensity) values. As shown in Figure 11 , it shows that the device can successfully construct a bidirectional polypeptide grafting density gradient.

[0103] Example 3

[0104] In this example, the absolute ethanol solution of the CPAPAPIGKYKLQYLEQWTLK-FITC polypeptide in Example 2 was replaced with the absolute ethanol solution of the CPAPAPIGKYKLQYLEQWTLK polypeptide. After performing the steps of Example 2, the sample was cleaned, rotated 90° clockwise, and an absolute ethanol solution of the CPAPAPYIGSR-FITC polypeptide prepared with absolute ethanol as the solvent was introduced through the liquid outlet 102 at "3". (The subsequent steps are the same as S3 - S5. This example is mainly to prove that a three-way polypeptide grafting density can be constructed.) Its concentration was 100 μM; the volume of the polypeptide solution aspirated with a 1 ml syringe was 400 μl, the flow rate of the polypeptide solution was 100 μl / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified helical multi-cell microfluidic platform grafted with the polypeptide was taken out, cleaned with absolute ethanol, and then dried with nitrogen to obtain a helical multi-cell microfluidic platform grafted with the polypeptide; a water film was dropped on the surface of the helical multi-cell microfluidic platform grafted with the polypeptide and observed under a fluorescence microscope; the fluorescence values were statistically analyzed to plot a column chart of the change in MFI (Mean fluorescence intensity) values, as Figure 12 shown, indicating that the device can successfully construct a three-way polypeptide grafting density gradient.

[0105] Example 4

[0106] In this example, the absolute ethanol solution of the CPAPAPYIGSR-FITC polypeptide in Example 3 was replaced with the absolute ethanol solution of the CPAPAPYIGSR polypeptide. After performing the steps of Example 3, the sample was cleaned, rotated 90° clockwise, and an absolute ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptide prepared with absolute ethanol as the solvent was introduced through the liquid outlet 102 at "4". (The subsequent steps are the same as S3 - S5. This example is mainly to prove that a four-way polypeptide grafting density can be constructed.) Its concentration was 150 μM; the volume of the polypeptide solution aspirated with a 1 ml syringe was 400 μl, the flow rate of the polypeptide solution was 100 μl / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified helical multi-cell microfluidic platform grafted with the polypeptide was taken out, cleaned with absolute ethanol, and then dried with nitrogen to obtain a helical multi-cell microfluidic platform grafted with the polypeptide; a water film was dropped on the surface of the helical multi-cell microfluidic platform grafted with the polypeptide and observed under a fluorescence microscope; the fluorescence values were statistically analyzed to plot a column chart of the change in MFI (Mean fluorescence intensity) values, as Figure 13 shown, indicating that the device can successfully construct a four-way polypeptide grafting density gradient.

[0107] Example 5

[0108] In this example, the absolute ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL, washed, and dried with nitrogen, resulting in a gradient surface with different grafting densities of AMP-QK-YIGSR-BMP2 on the surface. After performing steps S411-S415, (I'm not sure if the specific parameters of this step need to be written again. Please help me check, teacher). Observe the growth of colonies under a fluorescence microscope. The bacterial survival rate is as Figure 14 shown, and the bacterial survival rate varies between 23.16% and 79.39%.

[0109] Example 6

[0110] In this example, the absolute ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL, washed, and dried with nitrogen, resulting in a gradient surface with different grafting densities of AMP-QK-YIGSR-BMP2 on the surface. Perform steps S421-S424. Culture vascular endothelial cells in a microfluidic platform with a polypeptide-grafted helical multi-cell unit, label them with FITC fluorescence, and observe the cell production using a laser confocal microscope. The specific steps are as follows: Place the microfluidic platform with a polypeptide-grafted helical multi-cell unit in a third multi-well culture plate, which is a 6-well culture plate. Sterilize the microfluidic platform with a polypeptide-grafted helical multi-cell unit using ethanol (an ethanol solution with a volume concentration of 75%) for 2 hours. Then, add a concentration of 8×10 4A cell suspension with a concentration of Figure 15 cells / mL (with a volume of 250 μL) was statically cultured. After one hour of static culture in an incubator, 5 mL of culture medium was added to the wells. The static culture conditions of the cell suspension were culturing for 3 days in an environment with 5% CO2 at a temperature of 37 °C. The microfluidic platform with polypeptide-grafted helical multi-unit cells was taken out from the third multi-well culture plate, and the microfluidic platform with polypeptide-grafted helical multi-unit cells was washed 3 times with PBS. The microfluidic platform with polypeptide-grafted helical multi-unit cells was placed into the fourth multi-well culture plate, and the fourth multi-well culture plate was a 6-well culture plate. Then, paraformaldehyde was added to the fourth multi-well culture plate to fix the microfluidic platform with polypeptide-grafted helical multi-unit cells. The fixation conditions of paraformaldehyde were fixing for 12 h at a temperature of 4 °C. A live cell F-actin microfilament protein staining reagent was added to the fourth multi-well culture plate with the microfluidic platform with polypeptide-grafted helical multi-unit cells fixed for fluorescence labeling. After washing with PBS (phosphate buffer solution), DAPI dye (DAPI, that is, 4',6-diamidino-2-phenylindole, is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy observation) was added for fluorescence labeling. Then, the microfluidic platform with polypeptide-grafted helical multi-unit cells was taken out and washed 3 times with PBS. Under a laser confocal microscope, the distribution of endothelial cells and the number of adhered endothelial cells on the surface of the microfluidic platform with polypeptide-grafted helical multi-unit cells in step 423 were observed using the FITC channel to determine the bioactive regional unit cells 106. The endothelial cell adhesion situation is as

[0111] Example 7

[0112] In this example, the absolute ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL, washed, and dried with nitrogen to obtain a gradient surface with a four-way polypeptide grafting density of AMP-QK-YIGSR-BMP2 with different grafting densities on the surface. Steps S421 - S424 were carried out. Schwann cells were cultured in the microfluidic platform with polypeptide-grafted helical multi-unit cells, fluorescence-labeled with FITC, and the production of cells was observed using a laser confocal microscope, specifically including the following steps: The microfluidic platform with polypeptide-grafted helical multi-unit cells was placed in the third multi-well culture plate, and the third multi-well culture plate was a 6-well culture plate. The microfluidic platform with polypeptide-grafted helical multi-unit cells was sterilized with ethanol (an ethanol solution with a volume concentration of 75%) for 2 hours. Then, a cell suspension with a concentration of 8×10 3A cell suspension with a concentration of Figure 16 per mL (with a volume of 250 μL) was statically cultured. After statically culturing in an incubator for one hour, 5 mL of medium was added to the wells. The static culture conditions of the cell suspension were culturing for 3 days in an environment with 5% CO2 at a temperature of 37 °C; the microfluidic platform with polypeptide-grafted helical multi-cells was taken out from the third multi-well culture plate, and the microfluidic platform with polypeptide-grafted helical multi-cells was washed 3 times with PBS cleaning, and the microfluidic platform with polypeptide-grafted helical multi-cells was placed into the fourth multi-well culture plate. This fourth multi-well culture plate was a 6-well culture plate. Then, paraformaldehyde was added to the fourth multi-well culture plate, and the microfluidic platform with polypeptide-grafted helical multi-cells was fixed. The fixation conditions of paraformaldehyde were fixing at 4 °C for 12 h; a live cell F-actin microfilament protein staining reagent was added to the fourth multi-well culture plate with the microfluidic platform with the polypeptide-grafted helical multi-cells fixed for fluorescence labeling. After washing with PBS (phosphate buffer solution), DAPI dye (DAPI, that is, 4',6-diamidino-2-phenylindole, is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy observation) was added for fluorescence labeling. Then, the microfluidic platform with the polypeptide-grafted helical multi-cells was taken out and washed 3 times with PBS; under a laser confocal microscope, the Schwann cell differentiation on the surface of the microfluidic platform with the polypeptide-grafted helical multi-cells in step 423 was observed using the FITC channel. The Schwann cell differentiation was as Figure 16 shown. The L / W value showed a trend of first increasing and then decreasing from the c1 region to the c6 region. The high-concentration regions were inhibited. In the c4 region, the value of L / W reached the highest, which was 9.91 (Figure 3c). At this time, the grafting densities of AMP, QK, YIGSR, and BMP-2 were 0.80 per nm 2 , 0.44 per nm 2 , 2.21 per nm 2 , 0.41 per nm 2 .

[0113] Example 8

[0114] In this example, the absolute ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC polypeptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL, washed, and dried with nitrogen to obtain a gradient surface with different grafting densities of AMP-QK-YIGSR-BMP2 in four directions on the surface. The prepared platform was placed in a 6-well plate, and 4×10 4 / mL of human bone marrow mesenchymal stem cells. After 3 days of culture, 50μM ascorbic acid, 10mM β-glycerophosphate and 100nM dexamethasone were added to the hBMSCs complete medium, and the hBMSCs complete medium was replaced with osteogenic induction medium. After another 7 days of culture, the substrate was washed with PBS and fixed with 4% paraformaldehyde (4°C, overnight). Then it was stained with ALP working solution for 15 minutes and then washed with PBS. The samples were then characterized using a digital fluorescence microscope (DM6M, Lecia, Germany) under the bright field channel, and the indicators were quantified using Lecia's own software. The degree of osteogenic differentiation is quantified using the ALP staining intensity. The higher the degree of differentiation, the greater the ALP staining intensity and the smaller the value. Figure 17 As shown, the ALP staining intensity varied from 4320.19 au to 6195.34 au.

[0115] Comparative Example 1

[0116] The conventional method for modifying the surface of titanium sheets by bioactive polypeptide grafting density comprises the following steps: (1) taking 33 square titanium sheets with a size of 1 cm×1 cm, treating the titanium sheets with 4% HF acid for 5 min, washing the titanium sheets with deionized water for 5 min to remove residual fluoride ions on the surface; then ultrasonically washing the titanium sheets with deionized water for 3 min; finally, soaking the titanium sheets with concentrated nitric acid for 15 min, ultrasonically washing the titanium sheets with deionized water for 3 min, and drying the titanium sheets with nitrogen;

[0117] (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 100 μM, so that it covered the bottom of the titanium sheet. The 33 titanium sheets were divided into 11 groups, each group had 3 titanium sheets, and the immersion time of the 11 groups of titanium sheets in the bioactive polypeptide CPAPAPKRWWKWWRR-FITC solution was 0 min, 18.5 min, 55.5 min, 74 min, 92.5 min, 111 min, 129.5 min, 148 min, 166.5 min, 203.5 min, and 240.5 min, respectively;

[0118] (3) taking out the titanium sheet from step (2), and washing the titanium sheet three times with anhydrous ethanol and deionized water respectively, to obtain titanium sheets with different bioactive polypeptide grafting densities;

[0119] (4) Use the FITC channel of a fluorescence microscope to characterize changes in surface fluorescence values.

[0120] Comparative Example 2

[0121] Staphylococcus aureus was cultured on the surface of Ti 6Al 4V sheets uniformly grafted with CPAPAPKRWWKWWRR polypeptide, and the agar sealing method was used to observe the growth inhibition of bacteria on its surface. At the same time, the Ti 6Al 4V sheet only subjected to hydroxylation treatment was used as a control group; the results showed that compared with the control group, the surface of the Ti 6Al 4V sheet uniformly grafted with CPAPAPKRWWKWWRR polypeptide prepared had an 80.36% inhibition rate against Staphylococcus aureus.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. A spiral multi-cell microfluidic platform, characterized in that, It includes four spiral flow channels and a liquid storage tray. The spiral flow channels include longitudinal partitions, transverse partitions, and a central partition. The transverse partition is provided with a first groove and a second groove, and the central partition is provided with a central groove. The longitudinal partitions and the transverse partitions are evenly installed in the liquid storage tray in a centrosymmetric form; the height of the central groove is lower than the height of the liquid storage tray. The first groove and the second groove are mirror-symmetrically distributed and cross-arranged in the spiral flow channel, and the height of the first groove and the second groove is lower than that of the longitudinal partition; one liquid inlet and three liquid outlets are provided on the liquid storage tray.

2. The spiral multi-cell microfluidic platform according to claim 1, characterized in that The height of the first groove and the second groove is 1% - 99% of the height of the longitudinal partition, and the length of the first groove and the second groove is 1% - 99% of the length of the longitudinal partition.

3. A high-throughput screening method for a spiral multi-cell microfluidic platform according to any one of claims 1 or 2, characterized in that, It includes the following steps: S1. Design and obtain a printed part of a spiral multi-cell microfluidic platform through 3D printing. The printed part of the spiral multi-cell microfluidic platform is subjected to stress relief annealing and sandblasting treatment to obtain a spiral multi-cell microfluidic platform. S2. Clean the spiral multi-cell microfluidic platform, and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain a surface-modified spiral multi-cell microfluidic platform. S3. Inject a polypeptide solution into the surface-modified spiral multi-cell microfluidic platform. The polypeptide solution uniformly passes through the regional cells of the surface-modified spiral multi-cell microfluidic platform and reacts. After the polypeptide solution reacts with all the regional cells, clean the spiral multi-cell microfluidic platform to obtain a polypeptide-grafted spiral multi-cell microfluidic platform. S4. Culture bacteria or cells in the polypeptide-grafted spiral multi-cell microfluidic platform to determine the regional cells with the optimal bioactive polypeptide grafting density. S5. Determine the polypeptide grafting density of the regional cells with the optimal bioactive polypeptide grafting density.

4. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The steps for performing hydroxylation treatment on the spiral multi-cell microfluidic platform in step S2 are as follows: S211. Ultrasonically clean the spiral multi-cell microfluidic platform successively with deionized water, acetone, absolute ethanol, and deionized water for 10 - 30 minutes, and then blow-dry the spiral multi-cell microfluidic platform with a protective gas. S212. Immerse the spiral multi-cell microfluidic platform in HF acid with a mass fraction of 2% - 8% for 4 - 18 minutes. S213. Finally, clean the spiral multi-cell microfluidic platform treated with HF acid in step S212, blow-dry the spiral multi-cell microfluidic platform again with a protective gas, and immerse the spiral multi-cell microfluidic platform in concentrated nitric acid and let it stand for 10 - 30 minutes to obtain a surface-modified spiral multi-cell microfluidic platform.

5. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The structural formula of the polypeptide molecule in the polypeptide solution described in step S3 is HS-R-Peptide, where R includes C(PA)nP, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and Peptide includes one of KRWWKWWRR or KRWWKWWRRK-FITC or IGKYKLQYLEQWTLK or IGKYKLQYLEQWTLK-FITC or YIGSR or YIGSRK-FITC or KIPKASSVPTELSAISTLYL or KIPKASSVPTELSAISTLYLK-FITC.

6. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The concentration range of the polypeptide solution described in step S3 is 1 μM to 5 mM.

7. The spiral multi-cell microfluidic platform according to claim 3, characterized in that The reaction time of the polypeptide solution described in step S3 and the surface-modified helical multi-cell microfluidic platform is 5 minutes to 24 hours.

8. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, Culturing bacteria in the polypeptide-grafted helical multi-cell microfluidic platform in step S4 includes the following steps: S411. Prepare the required nutrient broth medium, and place the nutrient broth medium in an autoclave for sterilization at 121 °C for 2 h; S412. Place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in the first multi-well culture plate, and sequentially add 1 μl to 100 μl of the bacterial solution with a concentration of 10 - 10 9 CFU / ml into the regional cells of the microfluidic platform with the polypeptide-grafted helical multi-cell unit, and co-incubate the bacterial solution with the microfluidic platform with the polypeptide-grafted helical multi-cell unit for 10 minutes to 24 hours; S413. After the co-incubation is completed, place the first multi-well culture plate in an oven to let the bacterial solution evaporate. After the bacterial solution has evaporated, place the polypeptide-grafted helical multi-cell microfluidic platform in a biological safety cabinet and let it stand for 10 - 15 min; S414. Sequentially add a resazurin solution with a concentration of 10 - 1000 μg / ml to the regional cells of the polypeptide-grafted helical multi-cell microfluidic platform and mix it with the nutrient broth. The resazurin solution is co-incubated with the polypeptide-grafted helical multi-cell microfluidic platform for 10 minutes to 8 hours; S415. After the static culture of the polypeptide-grafted helical multi-cell microfluidic platform is completed, observe the colony growth of the polypeptide-grafted helical multi-cell microfluidic platform under a fluorescence microscope to determine the regional cells with the optimal bioactive polypeptide grafting density.

9. The spiral multi-cell microfluidic platform according to claim 3, characterized in that Culturing cells in the polypeptide-grafted helical multi-cell microfluidic platform in step S4 includes the following steps: S421. Place the microfluidic platform with the polypeptide-grafted helical multi-cell unit in a third multi-well culture plate, sterilize the microfluidic platform with the polypeptide-grafted helical multi-cell unit, and sequentially add 1 μl to 100 μl of cell suspension with a concentration of 10 to 10 9 cells / mL to each regional cell unit of the microfluidic platform with the helical multi-cell unit, and let it stand for culture for 1 hour to 30 days; S422. Take out the polypeptide-grafted helical multi-cell microfluidic platform from the third multi-well culture plate, wash the polypeptide-grafted helical multi-cell microfluidic platform with PBS, place the polypeptide-grafted helical multi-cell microfluidic platform in a fourth multi-well culture plate, and then add paraformaldehyde to the fourth multi-well culture plate to fix the polypeptide-grafted helical multi-cell microfluidic platform in the paraformaldehyde. S423. Add a live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate of the microfluidic platform fixed with the polypeptide-grafted helical multi-cell unit for fluorescence labeling, and wash it with PBS. Then add DAPI dye to the fourth multi-well culture plate to perform fluorescence labeling on the microfluidic platform of the polypeptide-grafted helical multi-cell unit. Then take out the microfluidic platform of the polypeptide-grafted helical multi-cell unit and wash 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 microfluidic platform of the polypeptide-grafted helical multi-cell unit in step 423 to determine the regional cells with biological activity.

Images