Preparation method and application of patterned multifunctional bionic cilia array

Abstract

The invention relates to the technical field of bionic cilia, in particular to a preparation method and application of a patterned multifunctional bionic cilia array. The method comprises the following steps: carrying out surface modification on a patterned substrate, and then carrying out confinement assembly growth of a high-molecular polymer, so as to form a patterned bionic cilia array of nano cilia clusters or nano cilia or single cilia; the limited range assembly strategy comprises a capillary liquid bridge limited range assembly strategy, a micro-pattern limited range assembly strategy and a phase separation limited range assembly strategy; a patterned substrate is combined with a high-molecular polymer, a plurality of bionic cilia array structures with high length-diameter ratios are creatively obtained through a confinement assembly strategy, the bionic cilia array structures have large length-diameter ratios, the maximum length-diameter ratio can reach about 1200, and the bionic cilia array structures have good flexibility; in addition, after the cilia array is functionalized, the cilia array is expected to play a huge application prospect in the fields of liquid biopsy, biological separation, electronic energy, flexible sensors, microfluid and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of bionic cilia, and in particular to a preparation method and application of a patterned multifunctional bionic cilia array. Background Art

[0002] There are many kinds of ciliary structures in nature, such as respiratory cilia, digestive tract cilia, fallopian tube cilia, cerebrospinal fluid cilia and embryonic node cilia. These ciliary structures have a large aspect ratio and can maintain an upright shape, which makes the ciliary structure play an irreplaceable role in life activities. Animals such as geckos, tree frogs, and snails can crawl on walls thanks to the reversible adhesion provided by the cilia on the soles of their feet. The transportation of mucus and oocytes in animals also benefits from the coordinated swinging of the ciliary structure in the body. When immune cells clear mutated cells in the body, cilia can also provide recognition and entanglement. Inspired by the ciliary structure and its function in nature, researchers have prepared a variety of bionic ciliary arrays, which have achieved certain results in the fields of surface functional modification, chip technology, biological detection, catalysis, etc.

[0003] However, there are still great obstacles in the preparation of bionic ciliary arrays. At present, the preparation methods of bionic ciliary arrays mainly include top-down and bottom-up methods. Among them, the top-down method of preparing bionic ciliary arrays mostly focuses on a few materials such as PDMS and silicon. The preparation method is complicated, the material selection is limited, the cost is high, it is time-consuming, and the aspect ratio of the obtained cilia is not high. Polymer assembly provides a rapid and novel bottom-up strategy for the preparation of bionic ciliary arrays. However, the current polymer assembly preparation of ciliary arrays requires external force induction such as electric fields and magnetic fields. The aspect ratio of the prepared cilia is not large, it is easy to collapse, the cost is high and the yield is low, and it is difficult to maintain an upright shape like the ciliary structure in nature. These obstacles have greatly limited the application of artificial ciliary arrays.

[0004] Therefore, the present application proposes a method for preparing and applying a patterned multifunctional bionic ciliary array. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art and solve the technical problems existing in the background technology, the present invention proposes a preparation method and application of a patterned multifunctional bionic cilia array.

[0006] The present invention is achieved through the following technical solutions: A method for preparing a patterned multifunctional bionic cilia array, the method comprising the following steps: After surface modification of the patterned substrate, confined assembly growth of a polymer is performed to form a bionic ciliary array of a patterned nanociliary cluster or a nanociliary pattern or a single ciliary array; Among them, the confined assembly strategies include capillary liquid bridge confined assembly strategy, micropattern confined assembly strategy and phase separation confined assembly strategy.

[0007] Preferably, the capillary liquid bridge confinement assembly strategy is used to prepare a bionic ciliary array of nanociliary clusters, which comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer or triblock copolymer in an organic solvent to prepare a polymer oil solution with a concentration of 5-30wt%, and sealing and storing the solution for later use; S3: Surface modification of the patterned substrate in S1: A capillary bridge is constructed on the top of the micropattern to restrict polymer assembly. The top of the pattern needs to be hydrophobic and the rest of the pattern needs to be hydrophilic. The patterned substrate is first treated with piranha solution overnight to make it superhydrophilic. Then, the treated patterned substrate is contacted with fluorosilane-modified PDMS at 75-85°C for 1-5 minutes to make the top hydrophobic. S4: Constructing capillary bridges to confine the assembly of growing cilia: A 50-500 μm copper wire is placed on the hydrophilic-hydrophobic modified patterned substrate to serve as a support, and then 10-30 μl of polymer solution is dripped onto the patterned substrate, and the polymer solution is immediately pressed with a cover glass, and the entire patterned substrate is placed in a surfactant aqueous solution to volatilize the solvent; S5: Freeze-drying samples The samples with grown ciliary structures were frozen with liquid nitrogen and then placed in a freeze dryer for freeze drying, thereby avoiding the influence of capillary forces on the ciliary structure during the natural drying process and obtaining an upright ciliary array; Among them, the array pattern of the nanociliary clusters has many and dense cilia structures and can maintain an upright shape. The number of cilia in each cluster is more than 5, the diameter of the cilia is 40-400 nanometers, the length is more than 3 microns, and it has a high aspect ratio, with a maximum aspect ratio of 700-1400.

[0008] Preferably, the micro-pattern confined assembly strategy is used to prepare a bionic ciliary array of a nanociliary pattern, which comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer or triblock copolymer in an organic solvent to prepare a polymer oil solution with a concentration of 5-30wt%, and sealing and storing the solution for later use; S3: Surface modification of the patterned substrate in S1: The polymer oil phase is added to the gaps of the micropattern to restrict the polymer assembly. If the top of the pattern needs to be hydrophilic and the rest of the pattern needs to be hydrophobic, the patterned substrate is first treated with piranha solution at 85-95°C overnight to make it superhydrophilic. The hydrophilized pattern is protected by microcontact imprint photoresist and then modified with fluorosilane. After removing the photoresist, the top of the pattern is made hydrophilic. S4: Micropatterned confined assembly of growing cilia: The polymer oil droplets are brushed on the micro-pattern confined space of the patterned substrate to grow cilia; when growing the nanociliary pattern, the modified patterned substrate is placed in pure water, 80-100 microliters of the polymer mixed solution is added to spread it, and then one volume of PVA aqueous solution is added, and the cilia are grown at room temperature without moving; S5: Freeze-drying samples The samples with grown ciliary structures were frozen with liquid nitrogen and then placed in a freeze dryer for freeze drying, thereby avoiding the influence of capillary forces on the ciliary structure during the natural drying process and obtaining an upright ciliary array.

[0009] Among them, the nanociliary pattern has many and dense cilia structures and can maintain an upright shape. The number of cilia in each pattern is more than 10, the diameter of the cilia is 40-400 nanometers, and the length is more than 2 microns.

[0010] Preferably, the phase separation confined assembly strategy is used to prepare a bionic ciliary array of a single ciliary array, which comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer and homopolymer or the triblock copolymer and homopolymer in an organic solvent to prepare a polymer oil solution with a concentration of 1-40wt%, wherein the content of the homopolymer is 0-90%, and sealing and storing the solution after ultrasonic dissolution. S3: Surface modification of the patterned substrate in S1: When preparing a single ciliary array by phase separation confined assembly, if the pattern needs to be made superhydrophobic, the patterned substrate is first treated with piranha solution at 80-100°C overnight, cleaned with ethanol and water, and placed in a vacuum oven, and 60-100 μL of fluorosilane liquid is added, followed by fluorosilane modification at 60-120°C for 6-24 hours to make it superhydrophobic; S4: Phase separation confined assembly of growing cilia: Place the superhydrophobic patterned substrate in pure water, add 60-200 μl of the polymer mixed solution to spread it, then slowly add one volume of PVA aqueous solution along the wall of the beaker to avoid interference with the polymer phase separation when adding PVA, and let it stand at room temperature to grow a single cilium, and remove bubbles with a pipette every 5-10 minutes; S5: Freeze-drying samples The samples with grown ciliary structures were frozen with liquid nitrogen and then placed in a freeze dryer for freeze drying, thereby avoiding the influence of capillary forces on the ciliary structure during the natural drying process and obtaining an upright ciliary array.

[0011] Among them, each cilium in a single cilium array is independent of each other and regularly distributed on the base. The cilia can maintain a vertical upward shape. Some longer cilia will bend, but they will not become entangled or collapse in the natural state. The diameter of a single cilium is 300-3000 nanometers and the length is 5-250 microns. It has a high aspect ratio of 20-200.

[0012] In the above, the patterned substrate includes a silicon wafer, a glass wafer, etc., the hydrophobic segment includes polystyrene, polylactide, etc.; the hydrophilic segment includes polyethylene glycol, polyacrylic acid, etc.; the organic solvent includes benzene, dichloromethane, etc.; the surfactant includes sodium dodecyl sulfate, polyvinyl alcohol, etc.

[0013] Preferably, the specific steps of S5 are as follows: S51: Take clean glass cups, add 100 ml of deionized water to each, and after the polymer has grown the ciliary structure, use tweezers to clamp one side of the patterned substrate, put the grown ciliary structure into the first cup of deionized water and shake it 3-5 times, then take it out and put it into the other two cups of clean deionized water to soak and wash for 2 minutes; S52: Take a clean 6-well plate and place it on the table. Add 5 ml of clean deionized water to each well in advance. Then, quickly and evenly place the soaked and cleaned ciliary structure and patterned substrate into the well plate with deionized water. S53: Take a foam box, put the well plate containing the sample into the foam box, then take 200-1000 ml of liquid nitrogen and add it into the foam box to completely freeze the sample and the water in the well plate, and immediately put it into a freeze dryer for drying for 24-48 hours after freezing; S54: After freeze drying, turn off the freeze dryer and slowly release the air to avoid damage to the ciliary structure. Finally, take out the sample and keep it in a dry environment to obtain an upright ciliary array.

[0014] The invention is applicable to the application of the method for preparing the patterned multifunctional biomimetic ciliary array mentioned above, wherein the method for performing cell-specific separation comprises the following steps: P1: Treat the prepared nanociliary cluster array with 2-20 mg / mL EDC / NHS solution for 0.5-2 hours to make the surface carboxyl groups active, then add 0.5-5 mg / mL streptavidin to the nanociliary cluster to make streptavidin bind to the activated carboxyl groups, and finally modify it with specific antibodies for standby use; P2: Use fluorescent dyes to stain cancer cells and leukemia cells with different colors, then add 20-1000 cancer cells and leukemia cells into 1-10 ml of culture medium respectively; P3: Place the nanociliary cluster array modified with specific antibodies into the culture medium containing cells, and perform cell capture at 20-40°C. Gently shake 3-6 times every 5-10 minutes to prevent the cells in the culture medium from sinking to the bottom. P4: After the capture is completed, the ciliary base is removed, one side of the base is clamped with forceps, and it is rinsed three times with PBS. Then the base is placed under a fluorescence microscope to take pictures and count.

[0015] The invention is applicable to the application of the method for preparing the patterned multifunctional bionic ciliary array mentioned above, wherein the method for collecting mist under high humidity comprises the following steps: Q1: Treat the prepared nanociliary strips with plasma for 8-15 minutes to make them superhydrophilic; Q2: Take 2-10 pieces of super-hydrophilic nanofibril strip substrates and glue them to the inner wall of the funnel. Use a centrifuge tube to catch the falling water drops at the mouth of the funnel. Q3: Place the mist collection device in a moisturizing box and adjust it to high humidity mode to collect water droplets; Q4: After collecting for 2-10 hours, take out the water in the centrifuge tube, weigh it with an electronic balance, and calculate the water collection efficiency.

[0016] The invention is applicable to the application of the method for preparing the patterned multifunctional bionic ciliary array mentioned above, wherein a single ciliary array is used for particle separation, wherein the method for capturing large-size particles comprises the following steps: N1: Take 10-100 mg of solid microparticle powder and add it to water, ethanol or other solutions, and ultrasonicate for 5-20 minutes to disperse it evenly to form a microparticle dispersion; N2: Place the prepared single ciliary array into the dispersion solution, allowing the microparticle dispersion solution to fully submerge the ciliary array. After standing for 1-10 minutes, take out the ciliary array base and place it flat on a clean place; N3: Evaporate the solution at a temperature of 20-70°C. After the liquid in the microparticle dispersion evaporates completely, the single ciliary array will collapse and entangle the large particle microspheres under the action of capillary force.

[0017] The beneficial effects of the present invention are: The present invention uses a patterned substrate to confine the polymer solution in a limited space for self-assembly. After the solvent evaporates, a variety of patterned bionic cilia arrays with high aspect ratios grow. The bionic cilia arrays have a large aspect ratio, with a maximum aspect ratio of about 1200, and good flexibility. Subsequently, these bionic cilia arrays can be functionalized and used in single cell analysis, mist collection, particle separation and other fields, and exhibit excellent performance. In addition, by adjusting the shape, diameter and spacing of the pattern on the substrate and the polymer parameters, the morphology and characteristics of the resulting ciliary array can be controlled. After the nanociliary cluster array is modified with specific antibodies, trace amounts of cancer cells can be efficiently and specifically captured, based on which single-cell analysis can be performed; after the nanociliary strips are superhydrophilic treated, the high aspect ratio nanociliary structure provides a larger contact area, based on which mist collection can be performed under high humidity conditions; placing a single ciliary array in a particle dispersion can tightly capture and entangle large particle microspheres, which is expected to be applied to a variety of separation fields; in addition, after the present ciliary array is functionalized, it is expected to have great application prospects in liquid biopsy, biological separation, electronic energy, flexible sensors, microfluidics and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of a patterned multifunctional biomimetic ciliary array prepared for the present invention; Figure 2 This is a schematic diagram of growing a nanociliary cluster array with a high aspect ratio on the top of a silicon pillar in Example 1 of the present invention; in, Figure 2 A in the middle is the design diagram of preparing nanociliary cluster arrays using the microfluidic bridge confined assembly strategy; Figure 2 Middle B is a diagram showing the growth principle of nanociliary clusters; Figure 2 Middle C is the SEM image at different times during the growth of the nanociliary cluster; Figure 3 This is a characteristic diagram of the nanociliary cluster array prepared on the top of the microcolumn in Example 1 of the present invention; in, Figure 3 A in the middle is the SEM image of the prepared nanociliary cluster; Figure 3 Middle B is a statistical diagram of the diameter of nanociliary clusters; Figure 3 Middle C is the length statistics of nanociliary clusters; Figure 3 Middle D is a statistical diagram of the aspect ratio of the nanociliary clusters; Figure 4This is a design diagram of growing a ciliary structure in the gap between micropatterns according to Example 2 of the present invention; in, Figure 4 A in the middle is a schematic diagram of preparing nanociliary pattern structures by micropattern confined assembly strategy; Figure 4 B is a SEM image of a nanociliary pattern grown in the gap of a patterned silicon pillar array; Figure 4 Middle C is a SEM image of a nanociliary pattern grown in the gaps between patterned long strips; Figure 4 D in the middle is a SEM image of a nanociliary pattern grown in a patterned silicon hole; Figure 5 This is a scanning electron microscope image of the nanociliary pattern array prepared in Example 3 of the present invention; Figure 6 This is a design diagram for preparing a single cilia array on a silicon pillar array according to Example 4 of the present invention; in, Figure 6 A in the middle is a schematic diagram of growing a single cilium on a patterned silicon pillar array; Figure 6 Middle B is the SEM image of the prepared single ciliary array; Figure 6 Middle C is the diameter distribution diagram of a single ciliary array; Figure 6 Middle D is the length distribution diagram of a single ciliary array; Figure 6 Middle E is the spacing distribution diagram of a single ciliary array; Figure 7 This is a characteristic diagram of a single cilium array prepared on an array of equally spaced silicon pillars in Example 5 of the present invention; in, Figure 7 A in the middle is the SEM image of the prepared single ciliary array; Figure 7 Middle B is a confocal microscopy image of a single ciliary array; Figure 7 Middle C is a three-dimensional scanning image of a single ciliary array; Figure 7 Middle D is the diameter distribution diagram of a single ciliary array; Figure 7 Middle E is the length distribution diagram of a single ciliary array; Figure 7 Middle F is the spacing distribution diagram of a single ciliary array; Figure 8 This is a design diagram of using a nanociliary cluster array for single cell analysis in Example 6 of the present invention; in, Figure 8 A in the middle is a schematic diagram of the in situ capture of cancer cells by the nanociliary cluster array; Figure 8 Middle B is the capture efficiency diagram of cells; Figure 8 Middle C is the fluorescence image of the cell after capture; Fig. 9 This is a design diagram of using a nanociliary strip pattern for mist collection in Example 7 of the present invention; in, Fig. 9 A in the middle is a schematic diagram of the nanociliary strip pattern for mist collection; Fig. 9 Middle B is the fog collection efficiency diagram; Fig. 9 Center C is the SEM image of the smooth film; Fig. 9 Middle D is the SEM image of the ciliary band membrane; Fig.10 This is a design diagram of a single ciliary array capturing polystyrene microspheres prepared in Example 8 of the present invention; in, Fig.10 A in the middle is a schematic diagram of a single ciliary array capturing polystyrene microspheres; Fig.10 Middle B is a SEM image of a single cilium erected before capture; Fig.10 Middle C is the SEM image of a polystyrene microsphere leaning against a single cilium; Fig.10 D in the middle is the SEM image of a single cilium wrapped around a polystyrene microsphere; Fig.10 Middle E is an optical microscope image of a single cilium capturing a polystyrene microsphere; Fig.10 F in the middle is a SEM image of the capture effect of a single ciliary array on polystyrene microspheres of different particle sizes. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0020] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The present invention is further described according to the accompanying drawings and specific embodiments of the present invention, and the preferred implementation methods and materials described in the present invention are only for demonstration purposes.

[0021] like Figure 1-Figure 10 As shown: Embodiment 1:

[0022] like Figure 2 As shown: Figure 2 Figure A in the middle shows that after asymmetric modification, the top of the micropillar is grafted with fluorosilane molecules with low surface energy to make the top hydrophobic and the rest of the micropillar hydrophilic. Contact angle measurement also shows that the substrate after asymmetric modification tends to be hydrophobic as a whole. Figure 2 In the figure B, as the solvent evaporates, the liquid bridge of the oil phase at the top of the microcolumn is disconnected, and then the oil phase remaining at the top of the microcolumn will further self-assemble and grow cilia; Figure 2Middle C is the SEM image at different times during the growth of the nanociliary cluster. It can be seen from the figure that as the solvent evaporates, a narrow neck is formed on the liquid bridge, and then the oil phase is disconnected at the narrow neck. The oil phase and polymer remaining on the top of the microcolumn self-assemble and grow a nanociliary cluster structure upward.

[0023] The details are as follows: a) using photolithography and chemical etching methods to prepare a patterned array of equally spaced silicon pillars, the diameter of the silicon pillar array is 10 μm, the pitch is 10 μm, and the height is 10 μm; b) dissolving a diblock polymer having a molecular weight ratio of hydrophobic segments to hydrophilic segments of 15:1 in an organic solvent to obtain a 10 wt% block polymer solution; c) The equidistant silicon pillar array obtained by etching is used as the growth substrate of cilia, and after asymmetric hydrophilicity and hydrophobicity modification, the top is made hydrophobic and the rest of the part is super-hydrophilic; d) the polymer solution is quickly dropped onto the silicon pillar array in air and immediately covered with a hydrophobic cover glass, and then immediately placed in a 1 w / v% polyvinyl alcohol aqueous solution to evaporate the solvent at room temperature; e) After the solvent evaporates, gently peel off the coverslip, wash with deionized water and freeze-dry the sample.

[0024] The prepared nanociliary array showed a high aspect ratio of the cilia structure through SEM characterization, and the cilia grew evenly without obvious defects. Figure 3 shown.

[0025] in, Figure 3 In middle A, it can be seen that the nanocili are all grown on the top of the silicon pillar array and maintain an upright shape. Figure 3 The diameter of cilia in medium B is between 30-180 nanometers, which is not much different from the diameter of cilia in animals. Figure 3 The length of cilia in C is 5-50 μm in diameter. Figure 3 As can be seen in D, the prepared nanociliary cluster array has a large aspect ratio.

[0026] Embodiment 2:

[0027] like Figure 4 As shown in A: a) Different patterned silicon substrates were prepared by photolithography and chemical etching, wherein the diameter of the silicon pillar array was 12 μm, the pitch was 8 μm, and the height was 10 μm; the width of the silicon strip was 5 μm, the pitch was 5 μm, and the height was 5 μm; the diameter of the silicon hole array was 10 μm, the pitch was 10 μm, and the depth was 5 μm; b) dissolving a diblock polymer having a molecular weight ratio of hydrophobic segments to hydrophilic segments of 15:1 in an organic solvent to obtain a 10 wt% block polymer solution; c) The patterned silicon template is used as the growth substrate of cilia, and after hydrophilic and hydrophobic modification, it has different wettability, among which the top of the silicon column, the top of the long strip and the silicon pore are hydrophobic, and the rest of the parts are superhydrophilic; d) In pure water, the polymer solution is brushed onto the confined space of the micropattern, and then a 4 w / v% polyvinyl alcohol aqueous solution is immediately added to evaporate the solvent at room temperature; e) After the solvent evaporates, wash the sample with deionized water and freeze-dry it.

[0028] The prepared nanociliary structures formed by SEM characterization were dense and uniform in the gaps between the silicon pillars, with no obvious defects. Figure 4 shown.

[0029] in, Figure 4 In middle B, we can see that dense nanociliary structures have grown in the gaps between the silicon pillars, forming a suction cup-like pattern; Figure 4 In the middle C, we can see that dense nanociliary structures have also grown in the gaps between the long strips. Figure 4 Middle D shows an upright array of nanociliary clusters with a high aspect ratio grown in a silicon hole.

[0030] Embodiment 3:

[0031] like Figure 5 As shown: a) using photolithography and chemical etching methods to prepare different patterned silicon substrates, which mainly include triangular silicon pillar arrays, square silicon pillar arrays, rectangular silicon pillar arrays, pentagonal silicon pillar arrays, hexagonal silicon pillar arrays, isosceles trapezoidal silicon pillar arrays, circular silicon pillar arrays and long strip arrays; b) dissolving a triblock polymer having a molecular weight ratio of hydrophobic segments to hydrophilic segments of 9:1 in an organic solvent to obtain a 20 wt% block polymer solution; c) Silicon pillar arrays with different patterns are used as the growth substrate of cilia. After hydrophilic-hydrophobic asymmetric modification, the top is hydrophilic and the rest is hydrophobic. The main structures of the silicon pillar patterns are triangle, square, rectangle, pentagon, hexagon, isosceles trapezoid, ring and long strip; d) In pure water, the polymer solution is brushed onto different pattern confined spaces, and then a 4 w / v% polyvinyl alcohol aqueous solution is immediately added with one volume to evaporate the solvent at room temperature; e) After the solvent evaporates, wash the sample with deionized water and freeze-dry it.

[0032] SEM characterization showed that the prepared nanocilia formed dense cilia structures in the gaps between different silicon pillar patterns, grew evenly, and had no obvious defects. Figure 5As shown, the main structures formed include triangular cilia pattern, square cilia pattern, rectangular cilia pattern, pentagonal cilia pattern, hexagonal cilia pattern, isosceles trapezoidal cilia pattern, circular cilia pattern and long strip cilia pattern.

[0033] Embodiment 4:

[0034] like Figure 6 As shown in A: a) using photolithography and chemical etching to prepare a patterned non-pitch silicon pillar array, the silicon pillar array has a diameter of 8 μm, a pitch of 5 μm, and a height of 20 μm; b) blending the triblock copolymer and the polystyrene homopolymer and dissolving them in an organic solvent to obtain a 20 wt % polymer oil solution, wherein the content of polystyrene is 5%; c) Using a non-uniformly spaced silicon column array as the growth substrate for cilia. The silicon column substrate was first treated with piranha solution at 95°C overnight, cleaned with ethanol and water, and then plasma cleaned to make it super hydrophilic. Then, it was placed in a vacuum oven, 100 microliters of fluorosilane liquid was added, and fluorosilane modification was performed at 60°C for 18 hours to make it super hydrophobic. d) Next, the modified substrate is placed in pure water, 70 microliters of the polymer mixture solution is added to spread it out, and then one volume of 2 wt% polyvinyl alcohol aqueous solution is added to allow the solvent to develop at room temperature. As the polymer undergoes phase separation and self-assembly, a single cilium grows on the silicon pillar array; e) After the solvent evaporates, wash the sample with deionized water and freeze-dry it.

[0035] The single cilia array prepared by SEM characterization showed a high aspect ratio of cilia structure, cilia growth was uniform, and there were no obvious defects. Figure 6 shown.

[0036] in, Figure 6 In B, we can see that individual cilia grow on the silicon column array and maintain an upright shape. Figure 6 In middle C, it can be seen that the diameter of the cilia is between 400-2400 nanometers. Figure 6 In middle D, we can see that the length of cilia ranges from 50 to 140 microns, with an average length of about 100 microns. Figure 6 As can be seen in Figure E, the prepared single cilia are independent of each other, with a spacing between 11-18 microns and an average spacing of about 15 microns.

[0037] Embodiment 5:

[0038] like Figure 7 As shown: a) using photolithography and chemical etching methods to prepare a patterned array of equally spaced silicon pillars, the diameter of the silicon pillar array is 10 μm, the pitch is 12 μm, and the height is 12 μm; b) blending the triblock copolymer and the polystyrene homopolymer and dissolving them in an organic solvent to obtain a 10 wt % block polymer oil solution, wherein the content of polystyrene is 5%; c) An array of equally spaced silicon pillars was used as the growth substrate for cilia. The silicon pillar substrate was first treated with piranha solution at 95°C overnight, cleaned with ethanol and water, and then plasma cleaned to make it super hydrophilic. Then, it was placed in a vacuum oven, 100 microliters of fluorosilane liquid was added, and fluorosilane modification was performed at 60°C for 18 hours to make it super hydrophobic. d) Next, the modified substrate is placed in pure water, 70 microliters of the polymer mixture solution is added to spread it out, and then one volume of 2 wt% polyvinyl alcohol aqueous solution is added to allow the solvent to develop at room temperature. As the polymer undergoes phase separation and self-assembly, a single cilium grows on the silicon pillar array; e) After the solvent evaporates, wash the sample with deionized water and freeze-dry it.

[0039] The single cilia array prepared by SEM and confocal microscopy showed a high aspect ratio of cilia structure, cilia growth was uniform, and there were no obvious defects. Figure 7 shown.

[0040] in, Figure 7 In middle A, we can see that individual cilia also grow on the silicon column array and maintain an upright shape. Figure 7 In B, confocal microscopy also shows that a single ciliary array grows from the silicon pillar and maintains an upright morphology. Figure 7 The three-dimensional scanning image in center C shows that the length of a single ciliary array is relatively large, with a maximum height of about 120 microns. Figure 7 In middle D, we can see that the diameter of a single cilium ranges from 400 to 1700 nanometers, with an average diameter of about 1100 nanometers. Figure 7 The length of a single cilium in center E ranges from 20 to 130 microns, with an average length of about 65 microns. Figure 7 In the middle F, it can be seen that the prepared single cilia are independent of each other, with a spacing between 6-26 microns and an average spacing of about 15 microns.

[0041] Embodiment 6:

[0042] like Figure 8 As shown: a) treating the nanociliary cluster array prepared in Example 1 with 10 mg / mL EDC / NHS reagent for 1 hour to fully activate the carboxyl groups on the surface, then combining streptavidin with the activated carboxyl groups and modifying the nanociliary clusters, and finally modifying the nanociliary clusters with specific antibodies for standby use; b) Use red fluorescent DID to stain cancer cells and green fluorescent DIO to stain leukocytes. Add 25, 50, 100, 200, and 500 stained cancer cells and leukocytes to 2 ml of culture medium respectively; c) Next, the nanociliary cluster array modified with specific antibodies is placed flatly in a culture medium containing cells and placed at 37°C for 45 minutes; d) After capturing the cells, they were washed three times with PBS and then photographed and counted under a fluorescence microscope.

[0043] Fluorescence microscopy showed that the prepared nanociliary cluster array could efficiently capture trace amounts of cancer cells and had a lower capture efficiency for cancer cells, showing high specificity. Figure 8 shown.

[0044] in, Figure 8 In B, we can see that the nanociliary cluster array is able to efficiently and specifically capture cancer cells. It has high capture efficiency among 25, 50, 100, 200, and 500 spiked cells and has the ability to perform single-cell analysis. Figure 8 In the C fluorescence microscope, it can be seen that more than 90% of the captured cells are cancer cells, which has a strong specificity.

[0045] Embodiment 7:

[0046] like Fig. 9 As shown: a) treating the nanociliary strips prepared in Example 2 with plasma for 10 minutes to make them superhydrophilic; b) Next, glue 4 super-hydrophilic nanofibril strips to the inner wall of the funnel, and use a centrifuge tube to catch the falling water drops at the mouth of the funnel; c) Then, the device is placed in a high humidity environment to collect mist; d) After 4 hours, take out the water in the centrifuge tube, weigh it and calculate the water collection efficiency.

[0047] After weighing with an electronic balance, it can be seen that the nanociliary strip membrane has a stronger mist collection ability than the smooth membrane, and the mist collection membrane is characterized using SEM.

[0048] in, Fig. 9As can be seen in A, the ciliary strip membrane has a larger specific surface area, which can increase the contact area with the mist and improve the mist collection efficiency. The collected water droplets will flow into the centrifuge tube along the gaps between the strips. Fig. 9 In Figure B, we can see that the water collection efficiency of the nanociliary strip membrane can reach 10 mg / cm 3 / h, which is more than 2 times higher than that of smooth film. Fig. 9 In the middle C, it can be seen that there is no ciliary structure on the smooth membrane, and the specific surface area is small. Fig. 9 The nanociliary strip membrane shown in D has a high specific surface area, which plays a vital role in improving the mist collection efficiency.

[0049] Embodiment 8: a) freeze-drying the single ciliary array prepared on the non-equidistant silicon pillar array in Example 4, and storing it under dry conditions for later use; b) dispersing 20 mg of polystyrene microspheres of different particle sizes in 10 mL of water to form a 2 mg / mL aqueous dispersion. The particle size of the polystyrene microspheres can be between 5 and 100 μm; c) Take 10 ml of polystyrene microsphere aqueous dispersion in a culture dish, place the single ciliary array prepared in implementation 4 in a culture dish, then take 5 ml of polystyrene microsphere aqueous dispersion and add it to the culture dish to submerge the ciliary array, let it stand for 2 minutes, then take out the ciliary base and place it flat on a clean place; d) Wait for the aqueous dispersion to evaporate completely at room temperature, and the single ciliary array will collapse and entangle the large particle microspheres under the action of capillary force; e) The process of a single ciliary array capturing polystyrene microspheres was observed under an optical microscope. Then, samples were taken for freeze drying at different times, and the capture process of a single ciliary array on polystyrene microspheres was observed using SEM.

[0050] The single ciliary array obtained by optical microscopy and SEM characterization can capture and entangle the polystyrene microspheres well. The results are shown in the attached Fig.10 shown.

[0051] in, Fig.10 After the ciliary base in Figure A is placed in the microsphere dispersion, the polystyrene microsphere will contact and lean against the single cilium. As the dispersion evaporates, the flexible single cilium will collapse under the action of capillary force and entangle the polystyrene microsphere. Fig.10 Middle B is a SEM image of a single ciliary array used to capture polystyrene microspheres. It can be seen that the ciliary array is able to maintain an upright morphology without collapsing. Fig.10 Middle C is the SEM image of the polystyrene microsphere leaning against a single cilium. It can be seen that the polystyrene microsphere begins to lean against the single cilium and the cilium begins to collapse completely. Fig.10D in the middle is a SEM picture of a single cilium wrapped around a polystyrene microsphere. It can be seen that under the action of capillary force, a single cilium tightly wraps around the polystyrene microsphere. Fig.10 Figure E is an optical microscope process diagram of a single cilium capturing polystyrene microspheres. It can be seen that as the water evaporates, under the action of capillary force, the cilium will gradually fall towards the polystyrene microspheres, and finally collapse completely and tightly entangle the microspheres. From Figure F, it can be seen that the particle sizes of the captured polystyrene microspheres include 15 microns, 20 microns, 40 microns, and 60 microns. It can be seen that a single cilium array can well capture and entangle polystyrene microspheres of different particle sizes. This method of capturing microparticles is expected to be applied to a variety of separation fields.

[0052] Any matters not described in the present invention are applicable to the prior art.

Claims

1. A method for preparing a patterned multifunctional bionic cilia array, characterized in that: The method comprises the following steps: After surface modification of the patterned substrate, confined assembly growth of a polymer is performed to form a bionic ciliary array of a patterned nanociliary cluster or a nanociliary pattern or a single ciliary array; Among them, the confined assembly strategies include capillary liquid bridge confined assembly strategy, micropattern confined assembly strategy and phase separation confined assembly strategy.

2. The method for preparing a patterned multifunctional bionic cilia array according to claim 1, characterized in that: The capillary liquid bridge confinement assembly strategy is used to prepare a bionic ciliary array of nanociliary clusters, which comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer or triblock copolymer in an organic solvent to prepare a polymer oil solution with a concentration of 5-30wt%, and sealing and storing the solution for later use; S3: Surface modification of the patterned substrate in S1: A capillary bridge is constructed on the top of the micropattern to restrict polymer assembly. The top of the pattern needs to be hydrophobic and the rest of the pattern needs to be hydrophilic. The patterned substrate is first treated with piranha solution overnight to make it superhydrophilic. Then, the treated patterned substrate is contacted with fluorosilane-modified PDMS at 75-85°C for 1-5 minutes to make the top hydrophobic. S4: Constructing capillary bridges to confine the assembly of growing cilia: A 50-500 micron copper wire is placed on the hydrophilic-hydrophobic modified patterned substrate to serve as a support, and then 10-30 microliters of polymer solution is dripped on the patterned substrate. The polymer solution is immediately pressed with a coverslip, and the entire patterned substrate is placed in a surfactant aqueous solution to evaporate the solvent, thereby achieving the growth of the ciliary structure.

3. The method for preparing a patterned multifunctional bionic cilia array according to claim 1, characterized in that: The micro-pattern confined assembly strategy is used to prepare a bionic ciliary array of a nano-ciliary pattern, and comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer or triblock copolymer in an organic solvent to prepare a polymer oil solution with a concentration of 5-30wt%, and sealing and storing the solution for later use; S3: Surface modification of the patterned substrate in S1: The polymer oil phase is added to the gaps of the micropattern to restrict the polymer assembly. If the top of the pattern needs to be hydrophilic and the rest of the pattern needs to be hydrophobic, the patterned substrate is first treated with piranha solution at 85-95°C overnight to make it superhydrophilic. The hydrophilized pattern is protected by microcontact imprint photoresist and then modified with fluorosilane. After removing the photoresist, the top of the pattern is made hydrophilic. S4: Micropatterned confined assembly of growing cilia: Polymer oil droplets are brushed on the confined space of the micro-pattern of the patterned substrate to grow cilia; when growing the nanociliary pattern, the modified patterned substrate is placed in pure water, 80-100 microliters of the polymer mixed solution is added to spread it out, and then one volume of PVA aqueous solution is added, and the cilia are grown while standing at room temperature to achieve the growth of the ciliary structure.

4. The method for preparing a patterned multifunctional bionic cilia array according to claim 1, characterized in that: The phase separation confined assembly strategy is used to prepare a bionic ciliary array of a single ciliary array, which comprises the following steps: S1: Preparation of patterned substrate: The smooth substrate is subjected to surface micro-nano processing by photolithography and chemical etching methods to form a substrate with different pattern arrays; S2: Prepare polymer solution: Dissolving the diblock copolymer and homopolymer or the triblock copolymer and homopolymer in an organic solvent to prepare a polymer oil solution with a concentration of 1-40wt%, wherein the content of the homopolymer is 0-90%, and sealing and storing the solution after ultrasonic dissolution to make it uniform; S3: Surface modification of the patterned substrate in S1: When preparing a single ciliary array by phase separation confined assembly, if the pattern needs to be made superhydrophobic, the patterned substrate is first treated with piranha solution at 80-100°C overnight, cleaned with ethanol and water, and placed in a vacuum oven, and 60-100 μL of fluorosilane liquid is added, followed by fluorosilane modification at 60-120°C for 6-24 hours to make it superhydrophobic; S4: Phase separation confined assembly of growing cilia: Place the superhydrophobic modified patterned substrate in pure water, add 60-200 μl of polymer mixed solution to spread it out, then slowly add one volume of PVA aqueous solution along the wall of the beaker to avoid interfering with the polymer phase separation when adding PVA. Let it stand at room temperature to grow a single cilium, and remove bubbles with a pipette every 5-10 minutes to achieve the growth of the ciliary structure.

5. A method for preparing a patterned multifunctional bionic cilia array according to claim 2, 3 or 4, characterized in that: After the growth of the ciliary structure is completed, the sample is freeze-dried, and the specific steps are as follows: S51: Take clean glass cups, add 100 ml of deionized water to each, and after the polymer has grown the ciliary structure, use tweezers to clamp one side of the patterned substrate, put the grown ciliary structure into the first cup of deionized water and shake it 3-5 times, then take it out and put it into the other two cups of clean deionized water to soak and wash for 2 minutes; S52: Take a clean 6-well plate and place it on the table. Add 5 ml of clean deionized water to each well in advance. Then, quickly and evenly place the soaked and cleaned ciliary structure and patterned substrate into the well plate with deionized water. S53: Take a foam box, put the well plate containing the sample into the foam box, then take 200-1000 ml of liquid nitrogen and add it into the foam box to completely freeze the sample and the water in the well plate, and immediately put it into a freeze dryer for drying for 24-48 hours after freezing; S54: After freeze drying, turn off the freeze dryer and slowly release the air to avoid damage to the ciliary structure. Finally, take out the sample and keep it in a dry environment to obtain an upright ciliary array.

6. An application of the method for preparing a patterned multifunctional bionic ciliary array according to any one of claims 1 to 5, characterized in that: Single cell analysis is performed using a nanociliary cluster array, wherein the method for performing cell-specific separation comprises the following steps: P1: Treat the prepared nanociliary cluster array with 2-20 mg / mL EDC / NHS solution for 0.5-2 hours to make the surface carboxyl groups active, then add 0.5-5 mg / mL streptavidin to the nanociliary cluster to make streptavidin bind to the activated carboxyl groups, and finally modify it with specific antibodies for standby use; P2: Use fluorescent dyes to stain cancer cells and leukemia cells with different colors, then add 20-1000 cancer cells and leukemia cells into 1-10 ml of culture medium respectively; P3: Place the nanociliary cluster array modified with specific antibodies into the culture medium containing cells, and perform cell capture at 20-40°C. Gently shake 3-6 times every 5-10 minutes to prevent the cells in the culture medium from sinking to the bottom. P4: After the capture is completed, the ciliary base is removed, one side of the base is clamped with forceps, and it is rinsed three times with PBS. Then the base is placed under a fluorescence microscope to take pictures and count.

7. An application of the method for preparing a patterned multifunctional bionic ciliary array according to any one of claims 1 to 5, characterized in that: The method of collecting mist using a nanofibril strip pattern under high humidity comprises the following steps: Q1: Treat the prepared nanociliary strips with plasma for 8-15 minutes to make them superhydrophilic; Q2: Take 2-10 pieces of super-hydrophilic nanofibril strip substrates and glue them to the inner wall of the funnel. Use a centrifuge tube to catch the falling water drops at the mouth of the funnel. Q3: Place the mist collection device in a moisturizing box and adjust it to high humidity mode to collect water droplets; Q4: After collecting for 2-10 hours, take out the water in the centrifuge tube, weigh it with an electronic balance, and calculate the water collection efficiency.

8. An application of the method for preparing a patterned multifunctional bionic ciliary array according to any one of claims 1 to 5, characterized in that: A method for particle separation using a single ciliary array, wherein capturing large-size particles comprises the following steps: N1: Take 10-100 mg of solid microparticle powder and add it to water, ethanol or other solutions, and ultrasonicate for 5-20 minutes to disperse it evenly to form a microparticle dispersion; N2: Place the prepared single ciliary array into the dispersion solution, allowing the microparticle dispersion solution to fully submerge the ciliary array. After standing for 1-10 minutes, take out the ciliary array base and place it flat on a clean place; N3: Evaporate the solution at a temperature of 20-70°C. After the liquid in the microparticle dispersion evaporates completely, the single ciliary array will collapse and entangle the large particle microspheres under the action of capillary force.

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