Preparation method of hydrophobic cell culture plate

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a preparation method of a hydrophobic cell culture plate, which comprises the following steps: cleaning the surface of a culture plate; carrying out plasma etching treatment on the cleaned culture plate; (3-chloropropyl) trichlorosilane coating is carried out on the etched culture plate; the hydrophobic cell culture plate solves the problem that an existing polystyrene culture plate is poor in weather resistance, a silicon-oxygen hydrophobic layer and a hydrolytic polycondensation layer of the (3-chloropropyl) trichlorosilane are utilized to form a double-protection barrier structure, and the hydrophobic cell culture plate has the advantages that the hydrophobic cell culture plate is high in weather resistance, high in weather resistance and good in weather resistance, and the service life of the hydrophobic cell culture plate is prolonged. The polystyrene is prevented from being oxidized, and the stability of the culture plate is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a method for preparing a hydrophobic cell culture plate. Background Art

[0002] Cell culture refers to a method of simulating the in vivo environment in vitro to enable cells to survive, grow, reproduce, and maintain their main structures and functions. In cell culture, cell suspension culture is currently a more commonly used laboratory cell culture method. This cell suspension culture refers to a method of suspending free single cells or cell clusters in a liquid culture medium at a certain cell density for culture. It can be applied to various primary cells or cell lines, and can also be used for various cell clusters in the process of primary or stem cell differentiation.

[0003] As disposable plastic culture plates are widely used in many fields such as biology, medicine, and chemical engineering, plastic products have become the preferred polymer materials for experimental analysis in the biology, medicine and other industries due to their low prices. Among them, polystyrene culture plates have become a more commonly used material for cell suspension culture due to their non-hydrophilic surface. However, polystyrene culture plates are mainly made of polystyrene materials. Under storage conditions such as light, the surface of polystyrene culture plates will undergo oxidation and deterioration, resulting in a decrease in hydrophobicity, and even partial hydrophilicity, which cannot meet the requirements. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a method for preparing a hydrophobic cell culture plate, which solves the problem of poor weather resistance of existing polystyrene culture plates. A double protective barrier structure is formed by using a silicone hydrophobic layer and a hydrolysis condensation layer of (3-chloropropyl)trichlorosilane to prevent polystyrene from oxidizing and ensure the stability of the culture plate.

[0005] In order to achieve the above technical objectives, the technical solution of the present invention is: A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 20-30 minutes, ultrasonically treating for 1-2 hours, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixed solution of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 2-4:1-3:2, the soaking temperature is 20-30°C, the ultrasonic treatment temperature is 5-10°C, the ultrasonic frequency is 70-90kHz, and the nitrogen purging adopts dry nitrogen at 40-60°C, and the speed after purging is 10-20mL / min; this step utilizes the mutual solubility of ethanol, acetone and water to form a homogeneous mixed solution, and during the soaking and ultrasonic treatment, the mixed solution directly acts on the surface of the culture plate, not only removing the surface dust and granular impurities, but also dissolving and peeling off the sticky impurities deposited on the surface, thereby cleaning the surface of the culture plate; Step 2, subjecting the culture plate to plasma etching treatment to obtain a preliminarily modified culture plate, wherein the gas for the plasma etching treatment is a mixed gas of oxygen and argon, and the volume ratio of oxygen to argon is 1:2-3, the vacuum degree is 100-150mT, the temperature is 25-35°C, the gas flow rate is 30-50sccm, the power is 100-300W, and the processing time is 200-600s; this step utilizes the mixed gas formed by oxygen and argon to etch the surface of the polystyrene culture plate to increase the roughness of the surface of the polystyrene culture plate, and at the same time, the presence of oxygen can form surface oxidation on the polystyrene, thereby increasing the activity of the surface of the polystyrene culture plate; Step 3: Add (3-chloropropyl)trichlorosilane to ether and stir evenly to form a coating solution, and then spray it onto the surface of the preliminarily modified culture plate, and dry it after standing to obtain a coated culture plate. The concentration of (3-chloropropyl)trichlorosilane in ether is 300-500g / L, the stirring speed for uniform stirring is 300-500r / min, and the spraying amount is 0.9-1.3mL / cm 2 The static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10-13:1, the static temperature is 5-10°C, and the drying temperature is 20-30°C; this step utilizes (3-chloropropyl)trichlorosilane to form a stable dissolution dispersion in ether, and then forms an ether liquid film on the surface of the preliminarily modified culture plate in a spraying manner, and in an atmosphere containing water vapor, (3-chloropropyl)trichlorosilane forms in-situ hydrolysis to convert the chlorine group into a hydroxyl group, and the ether itself is slightly soluble in distilled water, which effectively controls the contact between water vapor and (3-chloropropyl)trichlorosilane, and the volatility of ether is utilized during drying to form rapid volatilization, thereby forming a surface film of (3-hydroxypropyl)trihydroxysilane; Step 4, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 20-30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 20-30 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:1-2:1-2:1-2, and the concentration of trichloromethylsilane in ether is 5-10g / L, the ultrasonic frequency of the ultrasonic treatment is 70-90kHz, the temperature is 5-10°C, the amount of distilled water added is 3-8% of the mass of trichloromethylsilane, and the dropping is accelerated The temperature of the low-temperature ultrasonic treatment is 0-5°C, and the ultrasonic frequency is 80-100kHz; this step utilizes the solubility of trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane in diethyl ether to form a homogeneous mixed dispersion, and also ensures that trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane are fully mixed; distilled water is added dropwise to diethyl ether, and the slight solubility of diethyl ether in distilled water is utilized to slowly penetrate, and trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane are hydrolyzed to obtain a hydrolyzed silane liquid; Step 5: spray the coating liquid on the surface of the coating culture plate, dry it and let it stand for 20-30 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 5-10°C and the spray volume is 0.3-0.6mL / cm 2The drying temperature is 10-20°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10-12:1, the temperature is 30-40°C, and the temperature of the heating treatment is 60-70°C; this step uses a spray method to deposit the coating liquid on the surface of the coating culture plate, and removes ether during drying to form a silicon oxide precursor film. During the static process, water vapor in the atmosphere is used to form an in-situ hydrolysis to achieve a hydroxyl-rich silane hydrolyzate; at this time, the coating layer on the surface of the coating culture plate is (3-hydroxypropyl) trihydroxysilane, which contains a 3-hydroxypropyl group, and the hydroxyl group is located at The propyl end can penetrate into the hydroxyl-rich silane hydrolyzate and form a hydroxyl polycondensation with the hydroxyl in the silane hydrolyzate during the heating treatment, thereby achieving the effect of penetration and fixation, effectively improving the connection stability between the silane hydrolyzate film and the (3-hydroxypropyl) trihydroxysilane film. It is particularly important to note that the silicon element of (3-hydroxypropyl) trihydroxysilane is also connected to a hydroxyl group, which can also form a hydroxyl polycondensation with the silane hydrolyzate film, thereby forming an integrated film composite structure; the silane hydrolyzate film forms an in-situ polycondensation after the heating treatment, forming a siloxane hydrophobic layer containing ethyl, methyl, isopropyl and propyl short-chain branches. Therefore, the siloxane hydrophobic layer contains a large number of alkyl short-chain branches, which reduces the adhesion of the siloxane surface, thereby achieving an excellent hydrophobic effect. At the same time, the self-condensation of (3-hydroxypropyl) trihydroxysilane can form an internal dense hydrophobic layer and barrier properties, effectively improving the protectiveness of the polystyrene surface. It can be seen from the above description that the present invention has the following advantages: 1. The present invention solves the problem of poor weather resistance of existing polystyrene culture plates, and utilizes a silicon-oxygen hydrophobic layer and a hydrolysis-condensation layer of (3-chloropropyl)trichlorosilane to form a double protective barrier structure to prevent polystyrene from being oxidized, thereby ensuring the stability of the culture plate.

[0006] 2. The present invention utilizes trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane as silane raw materials to form short hydrophobic silane branches on the surface of the silicon-oxygen hydrophobic layer, which not only improves the regional hydrophobicity of the silicon-oxygen material surface, but also reduces the surface physical stability of the silicon-oxygen hydrophobic layer.

[0007] 3. The present invention utilizes the chlorine-terminated propyl group of (3-chloropropyl)trichlorosilane to penetrate the siloxane hydrophobic layer, and utilizes the hydroxyl group after the hydrolysis of the chlorine-terminated group to form a penetrating polycondensation reaction with the siloxane hydrophobic layer, thereby improving the connection stability between the siloxane hydrophobic layer and the hydrolysis polycondensation layer of (3-chloropropyl)trichlorosilane.

[0008] 4. The present invention utilizes plasma etching to improve the activity of the surface of the polystyrene culture plate, increases the oxygen-containing groups on the surface, and can react with the hydrolyzed hydroxyl groups of the silicon-oxygen hydrophobic layer and (3-chloropropyl)trichlorosilane, and forms a stable chemical connection during the temperature increase treatment, thereby ensuring the stability of the hydrophobic layer and reducing the bubbles in the hydrophobic layer. At the same time, the silicon-oxygen hydrophobic layer and the hydrolyzed condensation layer of (3-chloropropyl)trichlorosilane are both made of silicon-oxygen materials, which have good transparency and do not affect experimental observation. DETAILED DESCRIPTION

[0009] The present invention is described in detail with reference to the embodiments, but no limitation is imposed on the claims of the present invention.

[0010] Example 1 A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 20 minutes, ultrasonically treating for 1 hour, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 2:1:2, the soaking temperature is 20°C, the ultrasonic treatment temperature is 5°C, the ultrasonic frequency is 70kHz, the nitrogen purging adopts 40°C dry nitrogen, and the speed after purging is 10mL / min; Step 2, the culture plate is subjected to plasma etching to obtain a preliminarily modified culture plate, wherein the plasma etching gas is a mixed gas of oxygen and argon, and the volume ratio of oxygen to argon is 1:2, the vacuum degree is 100mT, the temperature is 25°C, the gas flow rate is 30sccm, the power is 100W, and the processing time is 200s; Step 3: Add (3-chloropropyl)trichlorosilane to ether and stir evenly to form a coating solution, and then spray it onto the surface of the preliminarily modified culture plate. After standing and drying, a coated culture plate is obtained. The concentration of (3-chloropropyl)trichlorosilane in ether is 300 g / L, the stirring speed is 300 r / min, and the spraying amount is 0.9 mL / cm 2 The static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10:1, the static temperature is 5°C, and the drying temperature is 20°C; Step 4, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 20 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 20 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:1:1:1, and the concentration of trichloromethylsilane in ether is 5g / L, the ultrasonic frequency of the ultrasonic treatment is 70kHz, the temperature is 5°C, the amount of distilled water added is 3% of the mass of trichloromethylsilane, and the dropping speed is 0.2mL / min; the temperature of the low-temperature ultrasonic treatment is 0°C, and the ultrasonic frequency is 80kHz; Step 5: spray the coating liquid on the surface of the coating culture plate, dry it and let it stand for 20 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 5°C and the spray volume is 0.3 mL / cm 2 The drying temperature is 10°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10:1, the temperature is 30°C, and the temperature of the heating treatment is 60°C.

[0011] Example 2 A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 30 minutes, ultrasonically treating for 1-2 hours, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 4:3:2, the soaking temperature is 30°C, the ultrasonic treatment temperature is 10°C, the ultrasonic frequency is 90kHz, the nitrogen purging adopts 60°C dry nitrogen, and the speed after purging is 20mL / min; Step 2, the culture plate is subjected to plasma etching to obtain a preliminarily modified culture plate, wherein the plasma etching gas is a mixed gas of oxygen and argon, and the volume ratio of oxygen to argon is 1:3, the vacuum degree is 150mT, the temperature is 35°C, the gas flow rate is 50sccm, the power is 300W, and the processing time is 600s; Step 3: Add (3-chloropropyl)trichlorosilane to ether and stir evenly to form a coating solution, and then spray it onto the surface of the preliminarily modified culture plate. After standing and drying, a coated culture plate is obtained. The concentration of (3-chloropropyl)trichlorosilane in ether is 500 g / L, the stirring speed is 500 r / min, and the spraying amount is 1.3 mL / cm 2 The static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 13:1, the static temperature is 10°C, and the drying temperature is 30°C; Step 4, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 30 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:2:2:2, and the concentration of trichloromethylsilane in ether is 10g / L, the ultrasonic frequency of the ultrasonic treatment is 90kHz, the temperature is 10°C, the amount of distilled water added is 8% of the mass of trichloromethylsilane, and the dropping speed is 0.4mL / min; the temperature of the low-temperature ultrasonic treatment is 5°C, and the ultrasonic frequency is 100kHz; Step 5: spray the coating liquid on the surface of the coating culture plate, dry it and let it stand for 30 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 10°C and the spray volume is 0.6 mL / cm 2 The drying temperature is 20°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 12:1, the temperature is 40°C, and the temperature of the heating treatment is 70°C.

[0012] Example 3 A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 25 minutes, ultrasonically treating for 2 hours, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 3:2:2, the soaking temperature is 25°C, the ultrasonic treatment temperature is 8°C, the ultrasonic frequency is 80kHz, the nitrogen purging adopts 50°C dry nitrogen, and the speed after purging is 15mL / min; Step 2, subjecting the culture plate to plasma etching to obtain a preliminarily modified culture plate, wherein the plasma etching gas is a mixed gas of oxygen and argon, and the volume ratio of oxygen to argon is 1:3, the vacuum degree is 130mT, the temperature is 30°C, the gas flow rate is 40sccm, the power is 200W, and the processing time is 400s; Step 3: Add (3-chloropropyl)trichlorosilane to ether and stir evenly to form a coating solution, and then spray it onto the surface of the preliminarily modified culture plate. After standing and drying, a coated culture plate is obtained. The concentration of (3-chloropropyl)trichlorosilane in ether is 400 g / L, the stirring speed is 400 r / min, and the spraying amount is 1.1 mL / cm 2The static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 12:1, the static temperature is 8°C, and the drying temperature is 25°C; Step 4, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 20-30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 25 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:2:1:2, and the concentration of trichloromethylsilane in ether is 8g / L, the ultrasonic frequency of the ultrasonic treatment is 80kHz, the temperature is 8°C, the amount of distilled water added is 6% of the mass of trichloromethylsilane, and the dropping speed is 0.3mL / min; the temperature of the low-temperature ultrasonic treatment is 4°C, and the ultrasonic frequency is 90kHz; Step 5: spray the coating liquid on the surface of the coating culture plate, dry it and let it stand for 25 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 8°C and the spray volume is 0.5 mL / cm 2 The drying temperature is 15°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 11:1, the temperature is 35°C, and the temperature of the heating treatment is 65°C.

[0013] Example 4 A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 25 minutes, ultrasonically treating for 2 hours, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 3:2:2, the soaking temperature is 25°C, the ultrasonic treatment temperature is 8°C, the ultrasonic frequency is 80kHz, the nitrogen purging adopts 50°C dry nitrogen, and the speed after purging is 15mL / min; Step 2: Add (3-chloropropyl)trichlorosilane to ether and stir evenly to form a coating solution, then spray it onto the surface of the culture plate, and dry it after standing to obtain a coated culture plate. The concentration of (3-chloropropyl)trichlorosilane in ether is 400 g / L, the stirring speed is 400 r / min, and the spraying amount is 1.1 mL / cm 2 The static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 12:1, the static temperature is 8°C, and the drying temperature is 25°C; Step 3, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 20-30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 25 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:2:1:2, and the concentration of trichloromethylsilane in ether is 8g / L, the ultrasonic frequency of the ultrasonic treatment is 80kHz, the temperature is 8°C, the amount of distilled water added is 6% of the mass of trichloromethylsilane, and the dropping speed is 0.3mL / min; the temperature of the low-temperature ultrasonic treatment is 4°C, and the ultrasonic frequency is 90kHz; Step 4: spray the coating liquid on the surface of the coating culture plate, dry it and let it stand for 25 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 8°C and the spray volume is 0.5 mL / cm 2 The drying temperature is 15°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 11:1, the temperature is 35°C, and the temperature of the heating treatment is 65°C.

[0014] Example 5 A method for preparing a hydrophobic cell culture plate comprises the following steps: Step 1, soaking the culture plate in a ternary mixed solution for 25 minutes, ultrasonically treating for 2 hours, taking it out and purging it with nitrogen to obtain a cleaned culture plate, wherein the culture plate adopts a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 3:2:2, the soaking temperature is 25°C, the ultrasonic treatment temperature is 8°C, the ultrasonic frequency is 80kHz, the nitrogen purging adopts 50°C dry nitrogen, and the speed after purging is 15mL / min; Step 2, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane, and trichloroisopropylsilane to ether and ultrasonically treating for 20-30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 25 minutes to obtain a coating solution; the molar ratio of the trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane is 1:2:1:2, and the concentration of trichloromethylsilane in ether is 8g / L, the ultrasonic frequency of the ultrasonic treatment is 80kHz, the temperature is 8°C, the amount of distilled water added is 6% of the mass of trichloromethylsilane, and the dropping speed is 0.3mL / min; the temperature of the low-temperature ultrasonic treatment is 4°C, and the ultrasonic frequency is 90kHz; Step 3: spray the coating solution on the surface of the culture plate, dry it and let it stand for 25 minutes to obtain a prefabricated culture plate, and then heat it to obtain a hydrophobic cell culture plate. The spray temperature is 8°C and the spray volume is 0.5 mL / cm2 The drying temperature is 15°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 11:1, the temperature is 35°C, and the temperature of the heating treatment is 65°C.

[0015] Comparative Example The comparative example used an untreated polystyrene culture plate. Performance Testing The culture plates of Examples 1-3 and Comparative Examples 1-3 were used as test samples. Water drops were added to the culture plates to detect the contact angle between the water drops and the culture plates. The hydrophilicity of the culture plates was represented by the water contact angle. The results are as follows:

[0016] After the culture plates of the above-mentioned Examples 1-3 and the comparative example were cleaned, they were irradiated with 100W ultraviolet light for 12 hours, and then the hydrophilicity test was performed. The results are as follows:

[0017] Comparison of the above data shows that the culture plate provided by the present technical solution has good hydrophobicity, and the short branched chain structure on the surface can effectively improve the surface hydrophobicity; after ultraviolet light irradiation, the culture plate generally maintains good hydrophobicity. Compared with Example 5 and Comparative Example 1, the stability of the present technical solution is better. It is understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are within the protection scope of the present invention.

Claims

1. A method for preparing a hydrophobic cell culture plate, characterized in that: The steps include: Step 1: Soak the culture plate in the ternary mixed solution for 20-30 minutes, perform ultrasonic treatment for 1-2 hours, take it out and purge it with nitrogen to obtain a cleaned culture plate. Step 2, subjecting the culture plate to plasma etching to obtain a preliminarily modified culture plate; Step 3, adding (3-chloropropyl)trichlorosilane to ether and stirring evenly to form a coating solution, and then spraying it onto the surface of the preliminarily modified culture plate, and drying it after standing to obtain a coated culture plate; Step 4, adding trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane to ether and ultrasonically treating for 20-30 minutes to obtain a mixed solution, then dropping distilled water and ultrasonically treating at low temperature for 20-30 minutes to obtain a coating solution; Step 5, spraying the coating liquid on the surface of the coating culture plate, drying and standing for 20-30 minutes to obtain a prefabricated culture plate, and heating the plate to obtain a hydrophobic cell culture plate.

2. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The culture plate in step 1 is a polystyrene culture plate, the ternary mixed solution is a mixture of ethanol, acetone and water, and the volume ratio of ethanol, acetone and water is 2-4:1-3:2, and the immersion temperature is 20-30°C.

3. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The temperature of the ultrasonic treatment in step 1 is 5-10° C., the ultrasonic frequency is 70-90 kHz, the nitrogen purging uses dry nitrogen at 40-60° C., and the speed after purging is 10-20 mL / min.

4. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The plasma etching gas in step 2 is a mixed gas of oxygen and argon, and the volume ratio of oxygen to argon is 1:2-3, the vacuum degree is 100-150mT, the temperature is 25-35°C, the gas flow rate is 30-50sccm, the power is 100-300W, and the processing time is 200-600s.

5. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The concentration of (3-chloropropyl)trichlorosilane in diethyl ether in step 3 is 300-500 g / L, the stirring speed is 300-500 r / min, and the spray volume is 0.9-1.3 mL / cm 2 .

6. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The standing atmosphere in step 3 is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10-13:1, the standing temperature is 5-10°C, and the drying temperature is 20-30°C.

7. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The molar ratio of trichloromethylsilane, trichloroethylsilane, trichloropropylsilane and trichloroisopropylsilane in step 4 is 1:1-2:1-2:1-2, and the concentration of trichloromethylsilane in diethyl ether is 5-10 g / L. The ultrasonic frequency of the ultrasonic treatment is 70-90 kHz and the temperature is 5-10°C.

8. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The amount of distilled water added in step 4 is 3-8% of the mass of trichloromethylsilane, and the dropping speed is 0.2-0.4 mL / min; the temperature of the low-temperature ultrasonic treatment is 0-5° C., and the ultrasonic frequency is 80-100 kHz.

9. The method for preparing a hydrophobic cell culture plate according to claim 1, characterized in that: The spray temperature in step 5 is 5-10°C, and the spray volume is 0.3-0.6 mL / cm 2 The drying temperature is 10-20°C, the static atmosphere is a mixed atmosphere of nitrogen and water vapor, and the volume ratio of nitrogen to water vapor is 10-12:1, the temperature is 30-40°C, and the temperature of the heating treatment is 60-70°C.

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