A method for testing cell adhesion of biomimetic multi-scale structures

Abstract

The present invention relates to the field of biological testing technology, and in particular to a method for testing cell adhesion of a bionic multi-scale structure; the method comprises the following steps: step 1: applying glue to a cleaned substrate by a spin coater, and performing a heat baking treatment; step 2: placing the coated substrate in a photolithography machine for exposure treatment, and placing the substrate in a developer for development; step 3: performing hydrophobic modification on the substrate after photolithography; step 4: configuring a polymer solution with a block polymer, and uniformly applying the polymer solution on a substrate from which the photoresist has been washed off and reacting, to obtain a substrate with a polymer pattern; step 5: performing antibody modification on the obtained substrate with the polymer pattern; step 6: performing a cell adhesion test on the antibody-modified substrate; and the multi-scale patterned substrate is used to measure the single cell adhesion between cells and substrates, and at the same time, rapid and efficient screening of proteins can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biological testing, and in particular to a method for testing cell adhesion of a bionic multi-scale structure. Background Art

[0002] The determination of single cell adhesion has always lacked a device that can measure the overall adhesion of cells without changing the properties of cells. AFM is the most commonly used device that can accurately and quantitatively measure the adhesion between cells and substrates. This type of method mainly uses matrix protein to coat the probe and fix the cells on the cantilever. After the probe is lowered to the substrate, the cells are brought into contact with the substrate, thereby achieving mechanical measurement by measuring the degree of bending of the cantilever during the action. However, this method also has its limitations. First of all, in order to allow cells to adhere to the cantilever, they must be pretreated with lectins, streptavidin or extracellular matrix proteins. However, this will inevitably change the functional state of the cells and the distribution of surface integrins. In addition, this method can only allow cells to contact the substrate for a very short time, which means that this method can only be applied to the measurement of early adhesion. In addition, due to the limitations of materials science, strong materials suitable for fixing cells on probes without affecting cells still need to be developed.

[0003] FluidFM technology combines atomic force microscopy technology with microfluidic control systems. This technology can directly adhere cells to the probe surface through a hollow atomic force probe by negative pressure, without activating any cell pathway signals. This brings great advantages to the measurement of adhesion force. On the one hand, this method can provide much stronger adhesion than protein binding, and can firmly fix cells on the probe, so it can be used to directly separate from the matrix. On the other hand, since there is no biological treatment, this method will not change any cell surface pathways, so that the data closest to the original cell can be obtained.

[0004] Although FluidFM technology can accurately and non-destructively measure the adhesion of single cells, it has special requirements for the substrate, such as surface flatness, substrate thickness and transparency. These limitations create great difficulties for exploring cell-micro-nano surface interactions. Another inherent defect of FluidFM technology in measuring cell adhesion is its low throughput.

[0005] Therefore, the present application proposes a method for testing cell adhesion of a bionic multi-scale structure. Summary of the invention

[0006] 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 method for testing cell adhesion of a bionic multi-scale structure.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for testing cell adhesion of a bionic multi-scale structure comprises the following steps:

[0009] Step 1: Use a spin coater to apply glue to the cleaned substrate and perform heat baking;

[0010] Step 2: placing the coated substrate in a photolithography machine for exposure treatment, and placing it in a developer for development;

[0011] Step 3: hydrophobic modification of the substrate after photolithography;

[0012] Step 4: preparing a polymer solution with the block polymer, and evenly applying the polymer solution on the substrate from which the photoresist has been washed off and reacting the substrate to obtain a substrate with a polymer pattern;

[0013] Step 5: Modifying the obtained substrate with the polymer pattern with antibodies;

[0014] Step 6: Test the cell adhesion of the antibody-modified substrate.

[0015] Preferably, the cleaning of the substrate comprises: placing the substrate in a plasma cleaning apparatus for 5-10 minutes.

[0016] Preferably, the spin coater spin coats Shirply 1805 photoresist, and the rotation speed of the spin coater is 2000-3000 rpm / min, and the time is 1-2 min.

[0017] Preferably, the developer is immersed in the developer for 10-15 seconds, and the developer is rinsed with deionized water and dried with nitrogen.

[0018] Preferably, the specific steps of the hydrophobic modification are:

[0019] The photolithographic substrate was treated in a plasma cleaner for 5-10 minutes, then placed in an oven, 2 drops of fluorosilane were added, vacuum was drawn, and the substrate was modified at 100-110°C overnight.

[0020] Preferably, the specific steps of step 4 are as follows:

[0021] A1: Prepare a 10-20 wt% solution of the block polymer using dichloromethane;

[0022] A2: Use acetone to wash away the photoresist on the hydrophobic modified substrate;

[0023] A3: The polymer solution in A1 is evenly applied on the substrate treated with A2, and reacted at 35-40°C for 1-2 hours to obtain a polymer pattern on the substrate.

[0024] Preferably, the specific steps of step five are as follows:

[0025] B1: Soak the substrate in 1-2 mol L -1 Hydrolyze in hydrochloric acid for 6-12h to produce carboxyl groups, and wash with deionized water three times;

[0026] B2: 5-10mgmL -1 EDC and 5-10mgmL -1 Soak in NHS-MES buffer solution at room temperature for 1-2 h, and wash three times with PBS;

[0027] B3: 2.5-5 μg mL -1 The cells were reacted with streptavidin SA for 0.5-1 h and washed three times with PBS;

[0028] B4: 5-10 μg mL -1 The cells were incubated with biotin-anti-EpCAM for 0.5-1h and then washed with PBS solution.

[0029] Preferably, the specific steps of step six are as follows:

[0030] Place the treated substrate in a culture dish, add MCF-7 cell suspension, move the cantilever above the cells, slowly lower the cantilever to the cell surface, turn on the negative pressure system to allow the cells to adsorb on the cantilever, move the cells to just above the polymer pattern, slowly lower the cantilever to allow the cells to interact with the substrate, then lift the cantilever to complete the single cell adhesion force measurement.

[0031] Preferably, the substrate comprises a polymer region and a transparent region, and the patterned surface of the substrate comprises flat, porous, long burrs, and short burrs.

[0032] Preferably, the polymer region is a circle with a diameter of 45-55 μm and a center distance of 180-220 μm.

[0033] The beneficial effects of the present invention are:

[0034] The present invention can quickly, accurately, conveniently and with high throughput obtain the single-cell adhesion force between cells and substrates, obtain the mechanical information of the biological interface formed by cells and micro-nano topological structures, and screen proteins efficiently and quickly; and the measurement results show that the adhesion force between the polymer surface with nano-topological structure and specific antibody modification and cells is much greater than that of the surface under a single condition, and the adhesion force increases with the extension of the action time, and can effectively solve the problems of complex surface processing of traditional micro-nano structures and opaque substrates. The test process has high precision, high throughput, little damage to cells and is closer to the native state of cells.

[0035] The present invention designs a bionic micro-nano multi-scale patterned substrate through traditional photolithography technology, hydrophilic-hydrophobic patterning technology, block polymer self-assembly and other means, which is used to measure the single-cell adhesion force between cells and substrates, and can also achieve rapid and efficient screening of proteins.

[0036] The present invention divides the patterned substrate into a polymer area and a transparent area. The transparent area is convenient for observing the entire operation process, and the micro-nano multi-scale polymer area is used to measure the adhesion between cells and different surface topological structures, thereby realizing rapid high-energy flux measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a scanning electron microscope image of a polymer pattern with different surface topological structures according to Example 1 of the present invention;

[0038] Figure 2 is an optical photograph of the cell adhesion force measurement process of Example 1 of the present invention;

[0039] Figure 3 This is a typical single cell force spectrum curve diagram of Example 1 of the present invention;

[0040] Figure 4 It is a comparison diagram of cell adhesion between substrates with micro-nano burr structures and antibody modification in the present invention;

[0041] Figure 5 Microscope images of a traditional silicon substrate and a patterned polymer substrate. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] like Figure 1-Figure 5 As shown:

[0045] Embodiment 1:

[0046] (1) Glue coating: Place the clean cover glass in a plasma cleaner for 5-10 minutes, spin coat with Shirply 1805 photoresist at a speed of 2000-3000 rpm / min for 1-2 minutes, and heat bake.

[0047] (2) Photolithography and development: Expose to ultraviolet light under a photolithography machine, soak in developer for 10-15 seconds, rinse off the developer with deionized water, blow dry with nitrogen, and heat bake.

[0048] (3) Hydrophobic modification: Place the photolithographic coverslip in a plasma cleaner for 5-10 minutes and then place it in an oven. Add 2 drops of fluorosilane, evacuate the chamber, and modify it at 100-110°C overnight.

[0049] (4) Block polymer self-assembly: Use dichloromethane to prepare a 10-20wt% solution of the block polymer PLGA-PEG-PLGA with a molecular weight of 50k-1k-50k. Use acetone to wash away the photoresist on the hydrophobically modified substrate. Apply the polymer solution evenly on the substrate and react at 35-40°C for 1-2h to obtain a flat polymer pattern surface.

[0050] (5) Antibody modification: Soak the substrate in 1-2 molL -1 Hydrochloric acid was used to hydrolyze for 6-12 h to generate carboxyl groups, and then washed with deionized water three times; EDC (5-10 mg mL -1 ) and NHS (5-10 mg mL -1 ) in MES buffer solution (100-150 mM, pH 5-6) at room temperature for 1-2 h, and then washed three times with PBS; streptavidin SA (2.5-5 μg mL -1 The cells were reacted for 0.5-1 h and washed three times with PBS. Finally, biotin-anti-EpCAM (5-10 μg mL -1 ) were incubated for 0.5-1h and then washed with PBS solution.

[0051] (6) Force measurement process: Place the treated substrate in a culture dish, add MCF-7 cell suspension, move the cantilever above the cells, slowly lower the cantilever to the cell surface, turn on the negative pressure system to allow the cells to adsorb on the cantilever, move the cells to just above the polymer pattern, slowly lower the cantilever to allow the cells to interact with the substrate, then lift the cantilever to complete the single cell adhesion force measurement on the flat surface.

[0052] Embodiment 2:

[0053] (1) Glue coating: Place the clean cover glass in a plasma cleaner for 5-10 minutes, spin coat with Shirply 1805 photoresist at a speed of 2000-3000 rpm / min for 1-2 minutes, and heat bake.

[0054] (2) Photolithography and development: Expose to ultraviolet light under a photolithography machine, soak in developer for 10-15 seconds, rinse off the developer with deionized water, blow dry with nitrogen, and heat bake.

[0055] (3) Hydrophobic modification: Place the photolithographic coverslip in a plasma cleaner for 5-10 minutes and then place it in an oven. Add 2 drops of fluorosilane, evacuate the chamber, and modify it at 100-110°C overnight.

[0056] (4) Block polymer self-assembly: Use dichloromethane to prepare a 10-20wt% solution of a block polymer PLGA-PEG-PLGA with a molecular weight of 50k-4k-50k. Use acetone to wash away the photoresist on the hydrophobically modified substrate. Apply the polymer solution evenly on the substrate and react at 35-40°C for 1-2h to obtain a long-burr polymer pattern surface.

[0057] (5) Antibody modification: Soak the substrate in 1-2 molL -1 Hydrochloric acid was used to hydrolyze for 6-12 h to generate carboxyl groups, and then washed with deionized water three times; EDC (5-10 mg mL -1 ) and NHS (5-10 mg mL -1 ) in MES buffer solution (100-150 mM, pH 5-6) at room temperature for 1-2 h, and then washed three times with PBS; streptavidin SA (2.5-5 μg mL -1 The cells were reacted for 0.5-1 h and washed three times with PBS. Finally, biotin-anti-EpCAM (5-10 μg mL -1 ) were incubated for 0.5-1h and then washed with PBS solution.

[0058] (6) Force measurement process: Place the treated substrate in a culture dish, add MCF-7 cell suspension, move the cantilever above the cells, slowly lower the cantilever to the cell surface, turn on the negative pressure system to allow the cells to adsorb on the cantilever, move the cells to just above the polymer pattern, slowly lower the cantilever to allow the cells to interact with the substrate, then lift the cantilever, and the single cell adhesion force measurement on the burr surface is completed.

[0059] Comparative Example 1

[0060] A silicon wafer substrate was selected and placed on a fluid force microscope. It was found that the needle tip could not be observed due to the opacity of the silicon wafer, so the next adhesion test process could not be carried out.

[0061] Any matters not described in the present invention are applicable to the prior art.

Claims

1. A method for testing cell adhesion of a bionic multi-scale structure, characterized in that: The following steps are involved: Step 1: Use a spin coater to apply glue to the cleaned substrate and perform heat baking; Step 2: placing the coated substrate in a photolithography machine for exposure treatment, and placing it in a developer for development; Step 3: hydrophobic modification of the substrate after photolithography; Step 4: preparing a polymer solution with the block polymer, and evenly applying the polymer solution on the substrate from which the photoresist has been washed off and reacting the substrate to obtain a substrate with a polymer pattern; Step 5: Modifying the obtained substrate with the polymer pattern with antibodies; Step 6: Test the cell adhesion of the antibody-modified substrate.

2. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The cleaning of the substrate comprises: placing the substrate in a plasma cleaning apparatus for 5-10 minutes.

3. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The spin coater spin coats Shirply 1805 photoresist at a rotation speed of 2000-3000 rpm / min for 1-2 min.

4. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The developer is immersed in the developer for 10-15 seconds, and the developer is rinsed with deionized water and dried with nitrogen.

5. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The specific steps of the hydrophobic modification are: The photolithographic substrate was treated in a plasma cleaner for 5-10 minutes, then placed in an oven, 2 drops of fluorosilane were added, vacuum was drawn, and the substrate was modified at 100-110°C overnight.

6. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The specific steps of step 4 are as follows: A1: Prepare a 10-20 wt% solution of the block polymer using dichloromethane; A2: Use acetone to wash away the photoresist on the hydrophobic modified substrate; A3: The polymer solution in A1 is evenly applied on the substrate treated with A2, and reacted at 35-40°C for 1-2 hours to obtain a polymer pattern on the substrate.

7. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The specific steps of step five are as follows: B1: Soak the substrate in 1-2 mol L -1 Hydrolyze in hydrochloric acid for 6-12h to produce carboxyl groups, and wash with deionized water three times; B2: 5-10mgmL -1 EDC and 5-10mgmL -1 Soak in NHS-MES buffer solution at room temperature for 1-2 h, and wash three times with PBS; B3: 2.5-5 μg mL -1 The cells were reacted with streptavidin SA for 0.5-1 h and washed three times with PBS; B4: 5-10 μg mL -1 The cells were incubated with biotin-anti-EpCAM for 0.5-1h and then washed with PBS solution.

8. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The specific steps of step six are as follows: Place the treated substrate in a culture dish, add MCF-7 cell suspension, move the cantilever above the cells, slowly lower the cantilever to the cell surface, turn on the negative pressure system to allow the cells to adsorb on the cantilever, move the cells to just above the polymer pattern, slowly lower the cantilever to allow the cells to interact with the substrate, then lift the cantilever to complete the single cell adhesion force measurement.

9. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 1, characterized in that: The substrate includes a polymer region and a transparent region, and the patterned surface of the substrate includes flat, porous, long burrs, and short burrs.

10. The method for testing cell adhesion of a biomimetic multi-scale structure according to claim 9, characterized in that: The polymer region is a circle with a diameter of 45-55 μm and a center distance of 180-220 μm.

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