A protein-induced self-growing transparent hydrogel antifouling coating and its preparation method

Abstract

This invention discloses a protein-induced self-growing transparent hydrogel antifouling coating and its preparation method, belonging to the field of marine antifouling gel coatings. The preparation method involves culturing bacteria that secrete large amounts of extracellular polymers and obtaining a sterile supernatant; adding a protein precipitant to obtain a precipitate; adding a protein precipitation washing reagent and mixing thoroughly to obtain the target protein, which is then freeze-dried and purified; immersing a substrate sample in a bacterial protein solution to deposit a protein coating; preparing a hydrogel precursor solution; immersing the protein coating in the hydrogel precursor solution; and allowing it to grow statically to obtain a protein-induced self-growing transparent hydrogel antifouling coating. This invention overcomes the shortcomings of current hydrogels, such as poor adhesion to the substrate and high cost, and successfully prepares a robust, low-cost, and environmentally friendly hydrogel adhesion layer with in-situ initiation and subsequent self-growth. It is not limited by traditional environmental constraints such as light and heat, thus expanding the application scenarios of hydrogels.

Description

Technical Field

[0001] This invention belongs to the field of marine antifouling gel coatings, specifically relating to a protein-induced self-growing transparent hydrogel antifouling coating and its preparation method. Background Technology

[0002] In recent years, to develop marine resources, humans have built a large number of ships and marine facilities, such as aquaculture nets and offshore drilling platforms. However, any marine facility immersed in seawater will inevitably become covered with marine organisms, leading to biofouling and adversely affecting human production activities. According to incomplete statistics, the direct economic losses caused by marine biofouling worldwide reach as high as US$150 billion annually. To more effectively reduce the enormous harm caused by marine biofouling, applying effective antifouling coatings to the surfaces of ships and marine facilities remains the most important, widely applicable, economical, and efficient way to solve the various problems caused by biofouling. Currently, antifouling coatings mainly work by creating a biotoxic environment or killing fouling organisms, but these methods also damage the marine ecosystem while preventing fouling. With further research, antifouling coating design is increasingly focusing on the interface between fouling organisms and the substrate, relying on surface modification to reduce surface free energy and decrease the adhesion of bacteria and other fouling organisms to the surface. Based on this principle, hydrogel coatings are receiving increasing attention.

[0003] Hydrogels form a hydrated layer on their surface in underwater environments, exhibiting low surface energy and ensuring a low surface adsorption tendency for fouling organisms such as bacteria. Simultaneously, the high water content and elasticity of hydrogels allow them to be easily detached from the antifouling coatings of marine facilities such as ships, even if trace amounts of fouling organisms adhere to them, due to elastic deformation and water shear forces, demonstrating good antifouling capabilities. However, in current marine antifouling applications, hydrogel coatings suffer from weak adhesion strength and poor durability, significantly limiting their practical application. Furthermore, achieving stable and convenient high-density coating of hydrogels onto substrates of various materials and shapes remains a challenge.

[0004] Fouling organisms such as bacteria can secrete a large number of extracellular polymers, including extracellular polysaccharides (RPSs), extracellular proteins (RPs), and extracellular DNA. Among them, the molecular structure of extracellular proteins contains a large number of polar groups (such as amino, carboxyl, and hydroxyl groups), which can interact with the material surface through hydrogen bonds, hydrophobic interactions, and electrostatic attraction, thereby adhering firmly to the substrate surface.

[0005] Therefore, combining bacterial proteins with hydrogel technology is an innovative direction for developing environmentally friendly coatings that combine high-efficiency antifouling, excellent durability, and good substrate compatibility. Summary of the Invention

[0006] To address the lack of existing antifouling coatings that are simple to prepare, adhere tightly to substrates, have wide application scenarios, and are environmentally friendly and pollution-free, this invention aims to provide a self-growing transparent hydrogel coating based on a bacterial protein initiation system and its preparation method. Bacteria that secrete large amounts of extracellular polymers are selected to form a dense protein adhesion layer on the substrate surface, which serves as the binding site for the hydrogel, promoting in-situ initiation and subsequent self-growth of the hydrogel. Through a simple preparation process, a transparent hydrogel antifouling coating suitable for different substrate materials, shapes, and temperature environments can be obtained, effectively reducing biofouling adhesion.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a protein-induced self-growing transparent hydrogel antifouling coating, comprising the following steps:

[0009] Extraction of bacterial supernatant: In a sterile environment, bacteria that secrete large amounts of extracellular polymers are placed in a culture medium and cultured statically. Then, sterile supernatant is obtained by high-speed centrifugation and filtration.

[0010] Extraction of bacterial extracellular proteins: After adding protein precipitant to sterile supernatant and refrigerating, the precipitate is obtained by centrifugation. Protein precipitation washing reagent is added to the precipitate, and after thorough mixing, the centrifugation process is repeated 2 or 3 times to obtain the target protein. The target protein is then lyophilized and purified, and stored frozen for later use.

[0011] Preparation of protein adhesion layer: The freeze-dried and purified target protein was prepared into a bacterial protein solution with deionized water. The substrate sample was cleaned and pretreated, and then immersed in the bacterial protein solution. After immersion, it was taken out and dried, and a protein coating was deposited on the substrate sample.

[0012] Preparation of hydrogel coating: Ammonium persulfate, acrylamide (AAm) and N,N'-methylenebisacrylamide (MBAA) were added to deionized water in sequence and mixed to prepare a hydrogel precursor solution. The substrate sample modified with protein coating was then immersed in the hydrogel precursor solution and allowed to grow statically. After rinsing, a protein-induced self-growing transparent hydrogel antifouling coating was obtained.

[0013] Furthermore, the bacteria that secrete large amounts of extracellular polymers in the extraction of bacterial supernatant are one or more of Tenacia baculum mesophilum D-6, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio sp. EF187016.

[0014] The culture medium is 2216E or LB, the culture temperature is 20℃-30℃, and the culture time is 2-5 days.

[0015] Furthermore, in the extraction of bacterial supernatant, the high-speed centrifugation speed was 12000rpm-15000rpm, the time was 10min-20min, and the temperature was 2℃-8℃.

[0016] The filtration was performed using membrane filtration, with the filter membrane having a pore size of 0.22 μm for filtering the supernatant.

[0017] Furthermore, in the extraction of bacterial extracellular proteins, the protein precipitant is 100% trichloroacetic acid, isopropanol, or polyethylene glycol, and the protein precipitation washing reagent is 100% pure acetone, ether, or a mixture of methanol and ethanol.

[0018] Furthermore, in the extraction of bacterial extracellular proteins, the volume ratio of protein precipitant to sterile supernatant is 1:(8-12); the centrifugation speed to obtain the precipitate is 12000rpm-15000rpm, and the time is 10min-20min.

[0019] The volume ratio of protein precipitation washing reagent to precipitate is (10-20):1; the centrifugation speed for each acquisition of target protein is 8000rpm-10000rpm, and the time is 5min-10min;

[0020] The temperature during the acquisition of the target protein is 2℃-8℃;

[0021] The freeze-drying time for the target protein is 24h-72h;

[0022] Frozen storage temperature is below -60℃.

[0023] Furthermore, in the preparation of the protein adhesion layer, the substrate sample materials include glass, PVC, PMMA, silicon wafers, rubber, 316L stainless steel, titanium alloy, etc., and the substrate shape includes two-dimensional plane, three-dimensional cube, three-dimensional hemisphere, tube structure, etc.

[0024] The substrate sample cleaning process involves ultrasonically cleaning the substrate sample in deionized water and ethanol solution for 10-20 minutes in sequence, drying it, and then cleaning it with an oxygen plasma cleaner at 200W-300W for 3-5 minutes.

[0025] Furthermore, in the preparation of the protein adhesion layer, the concentration of the bacterial protein solution was 6 mg / mL-10 mg / mL, the soaking time was 24 h-72 h, and the temperature was 2 ℃-8 ℃.

[0026] Furthermore, in the preparation of the hydrogel coating, the amount of ammonium persulfate added is 1 mg / mL-5 mg / mL, the amount of acrylamide added is 1 M-4 M, and the amount of N,N'-methylenebisacrylamide added is 1 mg / mL-5 mg / mL.

[0027] Furthermore, in the preparation of the hydrogel coating, the static growth time is 3-5 minutes, and the temperature is 15℃-25℃.

[0028] Secondly, the present invention provides a protein-induced self-growing transparent hydrogel antifouling coating, which is prepared by the above-described preparation method.

[0029] Advantages and effects of the present invention:

[0030] 1. This invention utilizes the characteristic that the extracellular polymers secreted by bacteria bind tightly to the substrate to extract extracellular proteins that play an important role, and enables the proteins to adhere in large quantities to the substrate surface. This overcomes the shortcomings of current hydrogels, such as poor adhesion to the substrate, high requirements for preparation conditions, high preparation costs, and easy environmental pollution, and successfully prepares a low-cost, non-toxic and environmentally friendly transparent hydrogel adhesion layer.

[0031] 2. The bacterial protein provided by this invention can serve as a binding site for hydrogels, adsorbing ammonium persulfate and generating persulfate free radicals, thereby realizing in-situ initiation and subsequent self-growth of hydrogels, without being limited by traditional environmental constraints such as light and heat, thus increasing the application scenarios of hydrogels.

[0032] 3. The self-growing hydrogel prepared by this invention has transparency, stability, durability and high-efficiency antifouling properties, providing a way for the development of high-efficiency and pollution-free antifouling coatings in marine engineering. Attached Figure Description

[0033] Figure 1 The image shows a laser confocal microscopy (CLSM) image of the growth process of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 1.

[0034] Figure 2 The graph shows the changes of the protein-induced self-growing transparent hydrogel antifouling coating obtained in Example 1 after immersion in ultrapure water for one month. In the graph, (a) is the coating thickness before immersion and (b) is the coating thickness after immersion for one month.

[0035] Figure 3 The image shows the effect of the protein-induced self-growing transparent hydrogel antifouling coating obtained in Example 1 on Escherichia coli and Staphylococcus aureus.

[0036] Figure 4The image shows a scanning electron microscope (SEM) image of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 1, which resists Escherichia coli and Staphylococcus aureus.

[0037] Figure 5 This is a laser confocal microscopy (CLSM) image of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 1, which resists Escherichia coli and Staphylococcus aureus.

[0038] Figure 6 The full-spectral transmittance curve of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 1 is shown.

[0039] Figure 7 The image shows the actual product of the hydrogel antifouling coating grown on a cube substrate in Example 2, where (a) is the blank substrate and (b) is the stained hydrogel antifouling coating.

[0040] Figure 8 The image shows the actual product of the hydrogel antifouling coating grown on a hemispherical substrate in Example 4, where (a) is the blank substrate and (b) is the stained hydrogel antifouling coating.

[0041] Figure 9 The image shows the actual hydrogel antifouling coating grown on the tube structure in Example 5, where (a) is a blank substrate and (b) is the stained hydrogel antifouling coating.

[0042] Figure 10 This is a statistical chart showing the growth thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared under bacterial protein solutions of different concentrations according to the present invention.

[0043] Figure 11 This is a statistical chart showing the growth thickness of the protein-induced self-growing transparent hydrogel antifouling coatings prepared at different growth temperatures according to the present invention. Detailed Implementation

[0044] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects according to the present invention application is provided in conjunction with the accompanying drawings and preferred embodiments.

[0045] Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in textbooks and experimental guides, or those recommended by the manufacturer, which are well known or readily available to those skilled in the art. The following embodiments are merely preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0046] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0047] Extraction of bacterial supernatant: One or more of the bacteria that secrete large amounts of extracellular polymers, such as Tenacia baculum mesophilum D-6, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio sp. EF187016, are placed in 2216E or LB medium and cultured at 20℃-30℃ for 2-5 days. Then, they are centrifuged at 12000rpm-15000rpm and 2℃-8℃ for 10-20 minutes. Finally, the supernatant is filtered using a membrane filtration method with a pore size of 0.22μm to obtain sterile supernatant.

[0048] Extraction of bacterial extracellular proteins: Add 100% concentration of trichloroacetic acid, isopropanol, or polyethylene glycol as a protein precipitant to sterile supernatant and allow to stand under cold conditions. The volume ratio of protein precipitant to sterile supernatant is 1:(8-12). Centrifuge at 12000-15000 rpm and 2℃-8℃ for 10-20 min to obtain a precipitate. Add 100% purity ice-cold acetone, ice-cold diethyl ether, or ice-cold methanol-ethanol mixture as a protein precipitation washing reagent. The volume ratio of protein precipitation washing reagent to precipitate is (10-20):1. After thorough mixing, centrifuge at 8000-10000 rpm and 2℃-8℃ for 5-10 min. Repeat centrifugation 2 or 3 times to obtain the target protein. Freeze-dry the target protein for 24-72 h to purify it, and store it frozen below -60℃ for later use.

[0049] Preparation of the protein adhesion layer: The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 6 mg / mL-10 mg / mL using deionized water. The substrate sample was ultrasonically cleaned in deionized water and ethanol solution for 10 min-20 min in sequence. After drying, it was cleaned with an oxygen plasma cleaner at 200W-300W for 3 min-5 min. Then, the substrate sample was immersed in the bacterial protein solution at 2℃-8℃ for 24 h-72 h. After that, it was taken out and dried. A protein coating was deposited on the substrate sample. The materials of the substrate sample are glass, PVC, PMMA, silicon wafer, rubber, 316 L stainless steel, titanium alloy, etc. The shape of the substrate is a two-dimensional plane, a three-dimensional cube, a three-dimensional hemisphere, or an internal structure of a tube.

[0050] Preparation of hydrogel coating: Take appropriate amounts of ammonium persulfate, AAM and MBAA and add them to deionized water in sequence to prepare hydrogel precursor solution. The concentration of ammonium persulfate in the hydrogel precursor solution is 1 mg / mL-5 mg / mL, the concentration of acrylamide is 1M-4M, and the concentration of N,N'-methylenebisacrylamide is 1 mg / mL-5 mg / mL. Then immerse the substrate sample modified with protein coating in the hydrogel precursor solution and let it stand at 15℃-25℃ for 3 min-5 min. Then take it out and rinse it to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0051] A protein-induced self-growing transparent hydrogel antifouling coating was prepared using the above-described preparation method.

[0052] Bacteria capable of producing large amounts of extracellular polymers were selected. Bacterial extracellular proteins are an important component of extracellular polymers, containing abundant amino acid residues, which are key to strong adhesion to the surface. At the same time, bacterial extracellular proteins also have catalytic activity, which can catalyze ammonium persulfate to generate active atoms or atomic groups, initiating monomer polymerization. Thanks to the strong adhesion and catalytic activity of bacterial proteins, they are used as the protein initiation layer in this invention, which is firmly adhered to the surface of the material substrate.

[0053] Hydrogels are a class of three-dimensional cross-linked polymer materials with hydrophilic surface properties, low surface energy, and anti-adhesion properties. They can inhibit the adsorption and colonization of biofouling substances such as proteins and bacteria on the material surface through physical or chemical actions, and are widely used in the field of antifouling coatings for ships. Due to their low surface energy and hydration layer, they isolate pollutants from the material surface, reduce interfacial adhesion, and make pollutants easily washed away by water flow. Thanks to the excellent antifouling properties of hydrogels, they are used as the main material in the antifouling coating of this invention.

[0054] This invention uses extracellular proteins extracted from bacterial extracellular polymers as initiators for hydrogel coatings, overcoming the shortcomings of traditional hydrogel coatings that rely on additional conditions such as light, heat, and transition elements for polymerization. The tight binding ability of bacterial proteins to the substrate surface allows the hydrogel coating to adhere firmly to the substrate, improving its stability. The broad-spectrum adhesion of bacterial proteins also allows the growth of the hydrogel coating to be extended to different substrates and carriers of different shapes. The protein hydrogels in the various embodiments of this invention can grow on the surfaces of metals, polymers, inorganic materials, and organic materials, enriching the application scenarios of hydrogels. At the same time, the growth of protein hydrogels on curved surfaces, cubic edges, cubic cusps, and inside tubes is realized, greatly compensating for the current problem of strict requirements for substrate flatness in hydrogel formation. The hydration layer on the surface of the hydrogel coating gives the modified substrate low surface energy, further improving the material's resistance to biofouling adhesion.

[0055] Example 1

[0056] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0057] Extraction of bacterial supernatant:

[0058] In a sterile environment, add one Tenacibaculum mesophilum D-6 colony cultured on solid medium to 1L of 2216E medium, place it in a 30℃ incubator, and incubate statically for 3 days.

[0059] The 2216E medium consists of: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0060] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 12,000 rpm and 4°C for 10 minutes.

[0061] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0062] Extraction of bacterial extracellular proteins:

[0063] Add 100% trichloroacetic acid to the sterile supernatant at a volume ratio of 1:10 and let it stand in a refrigerator at 4°C for 12 hours.

[0064] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 15,000 rpm and 4°C for 10 minutes.

[0065] Retain the precipitate after centrifugation, add ice-cold acetone and mix thoroughly to disperse the protein. The volume ratio of ice-cold acetone to the retained precipitate is 10:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 10,000 rpm and 4°C for 5 min. Repeat the centrifugation twice to obtain the target protein.

[0066] The target protein was freeze-dried for 24 hours to purify it, and then stored in a -80°C freezer for later use.

[0067] Preparation of protein adhesion layer:

[0068] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 10 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0069] A 1cm×1cm square glass slide was ultrasonically cleaned in ethanol solution for 10 minutes and ultrasonically cleaned in deionized water for 10 minutes, then dried. It was then cleaned with an oxygen plasma cleaner at 300W for 5 minutes to obtain a cleaned glass slide substrate.

[0070] The cleaned glass substrate was placed in a bacterial protein solution and soaked at 4°C for 24 hours. After soaking, it was removed, dried with nitrogen, and a protein coating was deposited on the glass substrate.

[0071] Preparation of hydrogel coating:

[0072] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 1 mg / mL, the concentration of AAM 2 M, and the concentration of MBAA 3 mg / mL, thus preparing the hydrogel precursor solution.

[0073] The glass slide substrate modified with the protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 25°C for 5 minutes. Afterward, it was removed, rinsed, and gently washed three times with deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating. The self-growth process of this transparent hydrogel antifouling coating was detected using rhodamine staining and observed under a laser confocal microscope. Figure 1 As shown, the hydrogel grows very quickly, forming a thin layer on the surface in 20 seconds. The coating thickness increases over time. The hydrogel coating grows very quickly in the first 180 seconds, and then the growth gradually slows down in the last 120 seconds.

[0074] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 1 is 313 μm.

[0075] Performance Analysis:

[0076] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, observation with a macro camera showed that... Figure 2 As shown, the gel remained transparent before and after soaking, with no significant change in gel thickness and no swelling.

[0077] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0078] Antibacterial rate: After co-culturing the hydrogel antifouling coating and a blank substrate in a bacterial solution for 24 hours, bacteria were collected from the surfaces of the coating and the substrate for a plate coating experiment. Figure 3 The CPU coating data shown indicates an antibacterial rate of 98.03% against Escherichia coli and 98.57% against Staphylococcus aureus, demonstrating that the protein-induced self-growing transparent hydrogel antifouling coating effectively resists the adhesion of Escherichia coli and Staphylococcus aureus to the substrate; Figure 4 The scanning microscopy results shown more clearly demonstrate that, compared to the blank substrate surface, the surface of the protein-induced self-growing transparent hydrogel antifouling coating is virtually free of bacterial adhesion; such as Figure 5 As shown, after fluorescent staining, the protein-induced self-growing transparent hydrogel antifouling coating showed virtually no green live bacteria or red dead bacteria stained with fluorescent dye, further demonstrating its excellent antifouling effect.

[0079] Transmittance: Compared with blank glass, a full-spectrum scan was performed using an ultraviolet spectrophotometer, such as... Figure 6 As shown, the hydrogel coating has a transmittance of 98%, indicating that a transparent hydrogel antifouling coating has been obtained.

[0080] Example 2

[0081] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0082] Extraction of bacterial supernatant:

[0083] In a sterile environment, add one Vibrioparahaemolyticus colony cultured on solid medium to 1L of LB medium, place it in a 20℃ incubator, and incubate statically for 2 days.

[0084] LB medium composition: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride;

[0085] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 13,000 rpm and 2°C for 12 minutes.

[0086] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0087] Extraction of bacterial extracellular proteins:

[0088] Add 100% isopropanol to the sterile supernatant at a volume ratio of 1:8 and let it stand in a refrigerator at 2°C for 12 hours.

[0089] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 12,000 rpm and 2°C for 12 minutes.

[0090] Retain the precipitate after centrifugation, add ice-cold ether and mix thoroughly to disperse the protein. The volume ratio of ice-cold ether to the retained precipitate is 12:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 8000 rpm and 2°C for 6 min. Repeat the centrifugation twice to obtain the target protein.

[0091] The target protein was freeze-dried for 36 hours to purify it, and then stored in a -65°C freezer for later use.

[0092] Preparation of protein adhesion layer:

[0093] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 7 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0094] The three-dimensional PVC cube was ultrasonically cleaned in ethanol solution for 12 minutes and ultrasonically cleaned in deionized water for 12 minutes, then dried, and then cleaned with an oxygen plasma cleaner at 200W for 3 minutes to obtain the cleaned PVC cube substrate.

[0095] The cleaned PVC cube substrate was placed in a bacterial protein solution and soaked at 2°C for 36 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the PVC cube substrate.

[0096] Preparation of hydrogel coating:

[0097] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 2 mg / mL, the concentration of AAM 1 M, and the concentration of MBAA 1 mg / mL, thus preparing the hydrogel precursor solution.

[0098] The PVC cube substrate with modified protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 20°C for 4 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0099] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 2 is 253 μm. Figure 7 As shown in (a), this is the surface morphology of the PVC cube substrate in its unmodified state, as follows: Figure 7(b) shows the transparent hydrogel antifouling coating on the PVC cube substrate. To make it more visible, the coating was dyed with methylene blue dye.

[0100] Performance Analysis:

[0101] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0102] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0103] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 96.38%, and the antibacterial rate of Staphylococcus aureus was 98.73%.

[0104] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0105] Example 3

[0106] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0107] Extraction of bacterial supernatant:

[0108] In a sterile environment, add one Vibrioalginolyticus colony cultured on solid medium to 1L of 2216E medium, place it in a 22℃ incubator, and incubate statically for 4 days.

[0109] The 2216E medium consists of: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0110] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 14,000 rpm and 3°C for 11 minutes.

[0111] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0112] Extraction of bacterial extracellular proteins:

[0113] Add 100% polyethylene glycol to the sterile supernatant at a volume ratio of 1:9 and let it stand in a refrigerator at 3°C ​​for 12 hours.

[0114] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 13,000 rpm and 3°C for 14 minutes.

[0115] Retain the precipitate after centrifugation, add ice-cold methanol-ethanol mixture and mix thoroughly to disperse the protein. The volume ratio of ice-cold methanol-ethanol mixture to the retained precipitate is 14:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 9000 rpm and 3°C for 7 min. Repeat the centrifugation 3 times to obtain the target protein.

[0116] The target protein was freeze-dried for 72 hours to purify it, and then stored in a -70°C freezer for later use.

[0117] Preparation of protein adhesion layer:

[0118] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 8 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0119] The three-dimensional PMMA cube was ultrasonically cleaned in ethanol solution for 14 min and ultrasonically cleaned in deionized water for 14 min in sequence, then dried, and then cleaned with an oxygen plasma cleaner at 220W for 4 min to obtain the cleaned PMMA cube substrate.

[0120] The cleaned PMMA cube substrate was placed in a bacterial protein solution and soaked at 3°C ​​for 72 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the PMMA cube substrate.

[0121] Preparation of hydrogel coating:

[0122] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 3 mg / mL, the concentration of AAM 3 M, and the concentration of MBAA 2 mg / mL, thus preparing the hydrogel precursor solution.

[0123] The PMMA cube substrate with modified protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 15°C for 5 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0124] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 3 is 215 μm.

[0125] Performance Analysis:

[0126] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0127] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0128] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 95.37%, and the antibacterial rate of Staphylococcus aureus was 96.75%.

[0129] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 98%, indicating that a transparent hydrogel antifouling coating was obtained.

[0130] Example 4

[0131] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0132] Extraction of bacterial supernatant:

[0133] In a sterile environment, add one Tenacibaculum mesophilum D-6 colony cultured on solid medium to 1L of 2216E medium, place it in a 30℃ incubator, and incubate statically for 3 days.

[0134] The 2216E medium consists of: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0135] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 12,000 rpm and 4°C for 10 minutes.

[0136] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0137] Extraction of bacterial extracellular proteins:

[0138] Add 100% trichloroacetic acid to the sterile supernatant at a volume ratio of 1:10 and let it stand in a refrigerator at 4°C for 12 hours.

[0139] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 15,000 rpm and 4°C for 10 minutes.

[0140] Retain the precipitate after centrifugation, add ice-cold acetone and mix thoroughly to disperse the protein. The volume ratio of ice-cold acetone to the retained precipitate is 10:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 10,000 rpm and 4°C for 5 min. Repeat the centrifugation twice to obtain the target protein.

[0141] The target protein was freeze-dried for 24 hours to purify it, and then stored in a -80°C freezer for later use.

[0142] Preparation of protein adhesion layer:

[0143] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 6 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0144] The three-dimensional glass hemisphere was ultrasonically cleaned in ethanol solution for 10 min and ultrasonically cleaned in deionized water for 10 min in sequence, then dried, and then cleaned with an oxygen plasma cleaner at 300W for 5 min to obtain the cleaned glass hemisphere substrate.

[0145] The cleaned glass hemispherical substrate was placed in a bacterial protein solution and soaked at 4°C for 24 hours. After soaking, it was removed, dried with nitrogen, and a protein coating was deposited on the glass hemispherical substrate.

[0146] Preparation of hydrogel coating:

[0147] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 1 mg / mL, the concentration of AAM 2 M, and the concentration of MBAA 3 mg / mL, thus preparing the hydrogel precursor solution.

[0148] The glass hemispherical substrate modified with protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 25°C for 3 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0149] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 4 is 211 μm. Figure 8 As shown in (a), this is the surface morphology of the glass hemispherical substrate in its unmodified state, as follows: Figure 8 (b) shows a transparent hydrogel antifouling coating on the glass hemispherical substrate. The coating was dyed with methylene blue dye to make it more visible.

[0150] Performance Analysis:

[0151] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0152] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0153] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 95.48%, and the antibacterial rate of Staphylococcus aureus was 97.43%.

[0154] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0155] Example 5

[0156] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0157] Extraction of bacterial supernatant:

[0158] In a sterile environment, add one Vibrio sp. EF187016 colony cultured on solid medium to 1L of LB medium, place it in a 24℃ incubator, and incubate statically for 5 days.

[0159] LB medium composition: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride;

[0160] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 15,000 rpm and 5°C for 14 minutes.

[0161] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0162] Extraction of bacterial extracellular proteins:

[0163] Add 100% trichloroacetic acid to the sterile supernatant at a volume ratio of 1:11 and let it stand in a refrigerator at 5°C for 12 hours.

[0164] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 14,000 rpm and 5°C for 16 minutes.

[0165] Retain the precipitate after centrifugation, add ice-cold acetone and mix thoroughly to disperse the protein. The volume ratio of ice-cold acetone to the retained precipitate is 16:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 10,000 rpm and 5°C for 8 minutes. Repeat the centrifugation 3 times to obtain the target protein.

[0166] The target protein was freeze-dried for 30 hours to purify it, and then stored in a -75°C freezer for later use.

[0167] Preparation of protein adhesion layer:

[0168] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 9 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0169] The silicone tube was ultrasonically cleaned in ethanol solution for 16 min and ultrasonically cleaned in deionized water for 16 min in sequence, then dried, and then cleaned with an oxygen plasma cleaner at 230W for 5 min to obtain the cleaned silicone tube internal structure substrate.

[0170] The cleaned silicone tube inner structure substrate was placed in a bacterial protein solution and soaked at 5°C for 30 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the silicone tube inner structure substrate.

[0171] Preparation of hydrogel coating:

[0172] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 4 mg / mL, the concentration of AAM 4 M, and the concentration of MBAA 4 mg / mL, thus preparing the hydrogel precursor solution.

[0173] The silicone tube substrate with modified protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 20°C for 3 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0174] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 5 is 226 μm. Figure 9 As shown in (a), this is the surface morphology of the unmodified substrate within the silicone tube. Figure 9 (b) shows the transparent hydrogel antifouling coating on the inner structure substrate of the silicone tube. To make it more visible, the coating was stained with methylene blue dye.

[0175] Performance Analysis:

[0176] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0177] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0178] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 96.41%, and the antibacterial rate of Staphylococcus aureus was 97.33%.

[0179] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0180] Example 6

[0181] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0182] Extraction of bacterial supernatant:

[0183] In a sterile environment, add one Vibrioparahaemolyticus and one Vibrio alginolyticus colony cultured on solid medium to 1L of 2216E medium, place in a 26℃ incubator, and incubate statically for 3 days.

[0184] The 2216E medium consists of: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0185] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 12,000 rpm and 6°C for 15 minutes.

[0186] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0187] Extraction of bacterial extracellular proteins:

[0188] Add 100% isopropanol to the sterile supernatant at a volume ratio of 1:12 and let stand in a refrigerator at 6°C for 12 hours.

[0189] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 15,000 rpm and 6°C for 18 minutes.

[0190] Retain the precipitate after centrifugation, add ice-cold ether and mix thoroughly to disperse the protein. The volume ratio of ice-cold ether to the retained precipitate is 18:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 8000 rpm and 6°C for 9 min. Repeat the centrifugation 3 times to obtain the target protein.

[0191] The target protein was freeze-dried for 48 hours to purify it, and then stored in a -60°C freezer for later use.

[0192] Preparation of protein adhesion layer:

[0193] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 10 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0194] A 1cm×1cm square rubber substrate was ultrasonically cleaned in ethanol solution for 18 minutes and ultrasonically cleaned in deionized water for 18 minutes, then dried. It was then cleaned with an oxygen plasma cleaner at 250W for 3 minutes to obtain a cleaned square rubber substrate.

[0195] The cleaned square rubber substrate was placed in a bacterial protein solution and soaked at 6°C for 48 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the square rubber substrate.

[0196] Preparation of hydrogel coating:

[0197] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 5 mg / mL, the concentration of AAM 2 M, and the concentration of MBAA 5 mg / mL, thus preparing the hydrogel precursor solution.

[0198] The square rubber substrate with modified protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 21°C for 4 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0199] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 6 is 273 μm.

[0200] Performance Analysis:

[0201] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0202] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0203] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 95.17%, and the antibacterial rate of Staphylococcus aureus was 96.97%.

[0204] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0205] Example 7

[0206] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0207] Extraction of bacterial supernatant:

[0208] In a sterile environment, add one Vibrio parahaemolyticus and Vibrio sp. EF187016 colony cultured on solid medium to 1L of LB medium, place it in an incubator at 27℃ and incubate statically for 2 days;

[0209] LB medium composition: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride;

[0210] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 13,000 rpm and 7°C for 16 minutes.

[0211] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0212] Extraction of bacterial extracellular proteins:

[0213] Add 100% polyethylene glycol to the sterile supernatant at a volume ratio of 1:10 and let it stand in a refrigerator at 7°C for 12 hours.

[0214] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 12,000 rpm and 7°C for 20 minutes.

[0215] Retain the precipitate after centrifugation, add ice-cold methanol-ethanol mixture and mix thoroughly to disperse the protein. The volume ratio of ice-cold methanol-ethanol mixture to the retained precipitate is 20:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 9000 rpm and 7°C for 10 min. Repeat the centrifugation 3 times to obtain the target protein.

[0216] The target protein was freeze-dried for 40 hours to purify it, and then stored in a -80°C freezer for later use.

[0217] Preparation of protein adhesion layer:

[0218] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 6 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0219] The three-dimensional 316L stainless steel cube was ultrasonically cleaned in ethanol solution for 20 minutes and ultrasonically cleaned in deionized water for 20 minutes, then dried, and then cleaned with an oxygen plasma cleaner at 270W for 4 minutes to obtain the cleaned 316L stainless steel cube substrate.

[0220] The cleaned 316L stainless steel cube substrate was placed in a bacterial protein solution and soaked at 7°C for 40 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the 316L stainless steel cube substrate.

[0221] Preparation of hydrogel coating:

[0222] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 2 mg / mL, the concentration of AAM 1 M and the concentration of MBAA 3 mg / mL, and thus prepare the hydrogel precursor solution.

[0223] The 316L stainless steel cube substrate with modified protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 22°C for 5 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0224] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 7 is 297 μm.

[0225] Performance Analysis:

[0226] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0227] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0228] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 97.72%, and the antibacterial rate of Staphylococcus aureus was 98.21%.

[0229] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 96%, indicating that a transparent hydrogel antifouling coating was obtained.

[0230] Example 8

[0231] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0232] Extraction of bacterial supernatant:

[0233] In a sterile environment, add one Vibrio alginolyticus and Vibrio sp. EF187016 colony cultured on solid medium to 1L of 2216E medium, place it in a 28℃ incubator, and incubate statically for 4 days.

[0234] The 2216E medium consists of: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0235] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 14,000 rpm and 8°C for 18 minutes.

[0236] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0237] Extraction of bacterial extracellular proteins:

[0238] Add 100% isopropanol to the sterile supernatant at a volume ratio of 1:9 and let it stand in a refrigerator at 8°C for 12 hours.

[0239] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 13,000 rpm and 8°C for 14 minutes.

[0240] Retain the precipitate after centrifugation, add ice-cold acetone and mix thoroughly to disperse the protein. The volume ratio of ice-cold acetone to the retained precipitate is 13:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 10,000 rpm and 8°C for 6 min. Repeat the centrifugation 3 times to obtain the target protein.

[0241] The target protein was freeze-dried for 50 hours to purify it, and then stored in a -70°C freezer for later use.

[0242] Preparation of protein adhesion layer:

[0243] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 8 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0244] The three-dimensional titanium alloy hemisphere was ultrasonically cleaned in ethanol solution for 13 min and ultrasonically cleaned in deionized water for 13 min in sequence, then dried, and then cleaned with an oxygen plasma cleaner at 280W for 4 min to obtain the cleaned titanium alloy hemisphere substrate.

[0245] The cleaned titanium alloy hemispherical substrate was placed in a bacterial protein solution and soaked at 8°C for 50 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the titanium alloy hemispherical substrate.

[0246] Preparation of hydrogel coating:

[0247] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 3 mg / mL, the concentration of AAM 3 M, and the concentration of MBAA 1 mg / mL, thus preparing the hydrogel precursor solution.

[0248] The titanium alloy hemispherical substrate modified with protein coating was then immersed in the hydrogel precursor solution and allowed to grow at 23°C for 3 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0249] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 8 is 241 μm.

[0250] Performance Analysis:

[0251] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0252] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0253] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 96.09%, and the antibacterial rate of Staphylococcus aureus was 97.21%.

[0254] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0255] Example 9

[0256] A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating includes the following steps:

[0257] Extraction of bacterial supernatant:

[0258] In a sterile environment, add one colony of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio sp. EF187016 cultured on solid medium to 1L of LB medium, place it in an incubator at 29℃ and incubate statically for 5 days;

[0259] LB medium composition: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride;

[0260] The cultured bacterial solution was dispensed into centrifuge tubes, balanced, and then placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 15,000 rpm and 4°C for 20 minutes.

[0261] The supernatant after centrifugation was retained and filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0262] Extraction of bacterial extracellular proteins:

[0263] Add 100% polyethylene glycol to the sterile supernatant at a volume ratio of 1:11 and let it stand in a refrigerator at 4°C for 12 hours.

[0264] After standing, the solution was dispensed into centrifuge tubes, balanced, and placed in a pre-cooled high-speed centrifuge. The tubes were then centrifuged at 14,000 rpm and 3°C for 16 minutes.

[0265] Retain the precipitate after centrifugation, add ice-cold ether and mix thoroughly to disperse the protein. The volume ratio of ice-cold ether to the retained precipitate is 15:1. Aliquot the mixture into centrifuge tubes, balance them, and place them in a pre-cooled high-speed centrifuge. Centrifuge at 8000 rpm and 6°C for 8 min. Repeat the centrifugation 3 times to obtain the target protein.

[0266] The target protein was freeze-dried for 60 hours to purify it, and then stored in a -60°C freezer for later use.

[0267] Preparation of protein adhesion layer:

[0268] The freeze-dried and purified target protein was prepared into a bacterial protein solution with a concentration of 10 mg / mL using deionized water. After stirring for 10 min, a stable protein aqueous solution was obtained.

[0269] The PVC pipe was ultrasonically cleaned in ethanol solution for 15 minutes and ultrasonically cleaned in deionized water for 15 minutes in sequence, then dried, and then cleaned with an oxygen plasma cleaner at 290W for 5 minutes to obtain the cleaned PVC pipe internal structure substrate.

[0270] The cleaned PVC pipe inner structural substrate was placed in a bacterial protein solution and soaked at 5°C for 60 hours. After soaking, it was removed and dried with nitrogen gas, and a protein coating was deposited on the PVC pipe inner structural substrate.

[0271] Preparation of hydrogel coating:

[0272] Take appropriate amounts of ammonium persulfate, AAM and MBAA, and add them in sequence to 30 mL of deionized water to make the concentration of ammonium persulfate in the hydrogel precursor solution 4 mg / mL, the concentration of AAM 4 M, and the concentration of MBAA 2 mg / mL, thus preparing the hydrogel precursor solution.

[0273] The PVC pipe inner structure substrate modified with protein coating was then immersed in hydrogel precursor solution and allowed to grow at 24°C for 4 minutes. After that, it was taken out and rinsed, and then gently rinsed 3 times in deionized water to obtain a protein-induced self-growing transparent hydrogel antifouling coating.

[0274] The thickness of the protein-induced self-growing transparent hydrogel antifouling coating prepared in Example 9 is 255 μm.

[0275] Performance Analysis:

[0276] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0277] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0278] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 96.15%, and the antibacterial rate of Staphylococcus aureus was 97.07%.

[0279] Transmittance: Compared with blank glass, the full spectrum was scanned using an ultraviolet spectrophotometer. The transmittance of the hydrogel coating was 97%, indicating that a transparent hydrogel antifouling coating was obtained.

[0280] Comparative Example 1

[0281] A method for preparing a protein-induced self-growing transparent hydrogel coating differs from Example 1 only in that the concentration of the bacterial protein solution in the preparation of the protein adhesion layer is 2 mg / mL, and the thickness of the protein-induced self-growing transparent hydrogel coating prepared in Comparative Example 1 is 12 μm.

[0282] Performance Analysis:

[0283] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0284] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0285] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria were taken from the surface of the coating and the substrate for a plate coating experiment. The antibacterial rate of Escherichia coli was 0%, and the antibacterial rate of Staphylococcus aureus was 0%, so the antibacterial function could not be achieved.

[0286] Transmittance: Compared with blank glass, the transmittance of the hydrogel coating was 97% when the full spectrum was scanned using a UV spectrophotometer, indicating that a transparent hydrogel coating can be obtained.

[0287] Comparative Example 2

[0288] A method for preparing a protein-induced self-growing transparent hydrogel coating differs from Example 2 only in that the concentration of the bacterial protein solution in the preparation of the protein adhesion layer is 4 mg / mL, and the thickness of the protein-induced self-growing transparent hydrogel coating prepared in Comparative Example 2 is 148 μm.

[0289] Performance Analysis:

[0290] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0291] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0292] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 83.68%, and the antibacterial rate of Staphylococcus aureus was 81.42%, indicating limited antibacterial function.

[0293] Transmittance: Compared with blank glass, the transmittance of the hydrogel coating was 97% when the full spectrum was scanned using a UV spectrophotometer, indicating that a transparent hydrogel coating can be obtained.

[0294] Comparative Example 3

[0295] A method for preparing a protein-induced self-growing transparent hydrogel coating differs from Example 3 only in that the temperature during the preparation of the hydrogel coating is 10°C. The thickness of the protein-induced self-growing transparent hydrogel coating prepared in Comparative Example 3 is 133 μm.

[0296] Performance Analysis:

[0297] Durability: After immersing the hydrogel antifouling coating in ultrapure water for one month, macro photography showed that it remained transparent before and after immersion, with no significant change in gel thickness and no swelling.

[0298] Stability: After being immersed in ultrapure water for one month, the hydrogel antifouling coating showed no damage to the gel surface and no change in morphology.

[0299] Antibacterial rate: After co-culturing the hydrogel antifouling coating and the blank substrate in a bacterial solution for 24 hours, bacteria on the surface of the coating and the substrate were taken for plate coating experiments. The antibacterial rate of Escherichia coli was 77.42%, and the antibacterial rate of Staphylococcus aureus was 73.99%, indicating limited antibacterial function.

[0300] Transmittance: Compared with blank glass, the transmittance of the hydrogel coating was 97% when the full spectrum was scanned using a UV spectrophotometer, indicating that a transparent hydrogel coating can be obtained.

[0301] To clarify the relationship between the thickness of the hydrogel antifouling coating and the concentration of the bacterial protein solution and the hydrogel growth temperature, Example 1 was used as a baseline, such as Figure 10 As shown, by changing only the concentration of the bacterial protein solution in the preparation of the protein adhesion layer, the thickness of the hydrogel antifouling coating was tested over time at concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL. Except for the 2 mg / mL concentration where the trend was not obvious, the thickness of the hydrogel antifouling coating increased with increasing protein solution concentration. Figure 11 As shown, by simply changing the hydrogel growth temperature in the preparation of the hydrogel coating, the thickness of the hydrogel antifouling coating gradually increases with the increase of temperature.

[0302] In summary, this invention utilizes bacterial proteins as both the binding and initiation layer of the hydrogel, enabling the preparation of a stable, swelling-resistant, and environmentally friendly antifouling hydrogel coating. Bacterial proteins endow the hydrogel with initiation capabilities; the concentration of the bacterial protein solution and the temperature of the hydrogel growth environment determine the final growth thickness of the coating. The protein-initiated hydrogel coating allows monomers to polymerize normally at different temperatures, forming a hydrogel. The stable binding ability of bacterial proteins to the substrate not only enables the hydrogel coating to adhere to the material surface but also endows the hydrogel with swelling resistance, ensuring its stability in practical applications. This significantly enhances the coating's environmental adaptability and greatly improves its antifouling durability.

Claims

1. A method for preparing a protein-induced self-growing transparent hydrogel antifouling coating, characterized in that, Includes the following steps: Extraction of bacterial supernatant: Under sterile conditions, bacteria secreting large amounts of extracellular polymers were placed in a culture medium and incubated statically. The sterile supernatant was then obtained by high-speed centrifugation and filtration. The bacteria secreting large amounts of extracellular polymers in the extraction of the bacterial supernatant are... Tenacibaculum mesophilum D-6 Vibrio parahaemolyticus , Vibrio alginolyticus , Vibrio One or more of sp. EF187016; Extraction of bacterial extracellular proteins: After adding protein precipitant to sterile supernatant and refrigerating, the precipitate is obtained by centrifugation. Protein precipitation washing reagent is added to the precipitate, and after thorough mixing, the centrifugation process is repeated 2 or 3 times to obtain the target protein. The target protein was freeze-dried and purified, then frozen and stored for later use. Preparation of protein adhesion layer: The freeze-dried and purified target protein was prepared into a bacterial protein solution with deionized water. The substrate sample was cleaned and pretreated, and then immersed in the bacterial protein solution. After immersion, it was taken out and dried, and a protein coating was deposited on the substrate sample. Preparation of hydrogel coating: Ammonium persulfate, acrylamide and N,N'-methylenebisacrylamide were added to deionized water in sequence and mixed to prepare hydrogel precursor solution. The substrate sample modified with protein coating was then immersed in the hydrogel precursor solution and allowed to grow statically. After rinsing, a protein-induced self-growing transparent hydrogel antifouling coating was obtained.

2. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, The culture medium for extracting bacterial supernatant is 2216E or LB, the culture temperature is 20℃-30℃, and the culture time is 2-5 days.

3. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, The extraction of bacterial supernatant was carried out by high-speed centrifugation at 12,000 rpm to 15,000 rpm for 10 to 20 minutes at a temperature of 2°C to 8°C. The filtration was performed using membrane filtration, with the filter membrane having a pore size of 0.22 μm for filtering the supernatant.

4. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the extraction of bacterial extracellular proteins, the protein precipitant is 100% trichloroacetic acid, isopropanol, or polyethylene glycol.

5. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the extraction of bacterial extracellular proteins, the volume ratio of protein precipitant to sterile supernatant is 1:(8-12); the centrifugation speed to obtain the precipitate is 12000rpm-15000rpm, and the time is 10min-20min. The volume ratio of protein precipitation washing reagent to precipitate is (10-20):1; the centrifugation speed for each acquisition of target protein is 8000rpm-10000rpm, and the time is 5min-10min; The temperature during the acquisition of the target protein is 2℃-8℃; The freeze-drying time for the target protein is 24h-72h; Frozen storage temperature is below -60℃.

6. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the preparation of the protein adhesion layer, the substrate sample materials include glass, PVC, PMMA, silicon wafer, rubber, 316L stainless steel, and titanium alloy, and the substrate shape includes two-dimensional plane, three-dimensional cube, three-dimensional hemisphere, and tube structure. The substrate sample cleaning process involves ultrasonically cleaning the substrate sample in deionized water and ethanol solution for 10-20 minutes in sequence, drying it, and then cleaning it with an oxygen plasma cleaner at 200W-300W for 3-5 minutes.

7. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the preparation of the protein adhesion layer, the concentration of the bacterial protein solution was 6 mg / mL-10 mg / mL, the soaking time was 24 h-72 h, and the temperature was 2 ℃-8 ℃.

8. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the preparation of the hydrogel coating, the amount of ammonium persulfate added is 1 mg / mL-5 mg / mL, the amount of acrylamide added is 1 M-4 M, and the amount of N,N'-methylenebisacrylamide added is 1 mg / mL-5 mg / mL.

9. The method for preparing a protein-induced self-growing transparent hydrogel antifouling coating as described in claim 1, characterized in that, In the preparation of the hydrogel coating, the static growth time is 3-5 minutes and the temperature is 15℃-25℃.

10. A protein-induced self-growing transparent hydrogel antifouling coating, characterized in that, The antifouling coating was prepared using the method described in any one of claims 1-9 for the preparation of a protein-induced self-growing transparent hydrogel.

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