Long-acting antibacterial and antiviral nano ceramic coating as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibacterial and antiviral materials, and particularly relates to a long-acting antibacterial and antiviral nano ceramic coating as well as a preparation method and application thereof. The nano ceramic coating provided by the invention comprises 25-30% of methoxytrimethylsilane, 1-5% of organosilicon quaternary ammonium salt, 20-30% of nano silicon dioxide sol, 0.5-1% of a catalyst, 1-2% of a protective agent, the balance of deionized water and the like. Compared with silver, copper, titanium and the like, the modified space is large, and compared with common quaternary ammonium salt materials, bacteria are not prone to evolving drug resistance. According to the invention, O-Si-O is taken as a main body to form a 3D net-shaped structure, through an anchor gun strategy, huge flexibility and a modification space are provided for a material structure, and efficient and rapid sterilization is realized. Tests on eight moulds and two viruses confirm that the nano ceramic coating belongs to a long-acting antibacterial and antiviral self-disinfection nano ceramic coating, and the mould resistance, enterovirus resistance and novel coronavirus resistance of the material are proved.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial and antiviral materials technology, specifically relating to a long-lasting antibacterial and antiviral nano-ceramic coating, its preparation method, and its application. Background Technology

[0003] Anti-mold coatings are a type of functional coating with building protection, decoration, and mold resistance properties. Their mold resistance refers to their ability to tolerate or prevent the growth and reproduction of mold spores and mycelia. Numerous and diverse harmful bacteria and viruses exist in nature. To reduce their numbers, antibacterial and antiviral coatings containing bactericides are typically applied to object surfaces, thereby reducing the chances of cross-infection and contact infection. Studies show that applying antibacterial and antiviral functional coatings to the surfaces of commonly used household items is an effective way to block the indirect transmission of pathogenic microorganisms. Some research has been conducted on antibacterial and antiviral coatings both domestically and internationally, mainly categorized into additive antibacterial and antiviral materials and structural materials. Additive antibacterial and antiviral materials mainly include the following categories: inorganic materials, natural materials, organic materials, and other compounds. Among these, based on the composition of the antibacterial and antiviral agents, organic antibacterial and antiviral agents can be divided into low-molecular-weight and high-molecular-weight organic antibacterial and antiviral agents. Low-molecular-weight organic antibacterial and antiviral agents mainly include quaternary ammonium salts, quaternary phosphorus salts, biguanides, alcohols, and phenols. Their antibacterial and antiviral mechanisms mainly involve binding to anions on the surface of bacterial and fungal cell membranes or reacting with sulfhydryl groups to disrupt the synthesis system of proteins and cell membranes, thus exhibiting good antibacterial and antiviral effects.

[0004] Organosilicon quaternary ammonium salt antibacterial agents are characterized by their excellent water and sweat absorption and softness. Using organosilicon as a medium, they strongly adsorb bactericidal ammonium cationic groups onto the surface of bacteria, altering the permeability of the bacterial cell wall. This causes enzymes, coenzymes, and metabolic intermediates within the bacteria to leak out, leading to the cessation of respiration and death of the microorganisms, thus achieving bactericidal and bacteriostatic effects—a process known as "contact death." However, ordinary quaternary ammonium salts have low chemical activity and are generally present in a free state during application, resulting in relatively high toxicity and strong irritation. Furthermore, most antibacterial and antiviral coating materials are subject to prolonged exposure to sunlight and bacteria, leading to decomposition and loss of antibacterial and antiviral functions. This also reduces the adhesion of the antibacterial and antiviral coating material to the surface, making it easier to peel off and further weakening its antibacterial and antiviral properties.

[0005] Therefore, developing a coating that is not easily peeled off and also has long-lasting antibacterial and antiviral properties is a research hotspot in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nano-ceramic coating that significantly alters the properties of polysiloxane materials by synthesizing quaternary ammonium salts, thereby increasing their long-lasting resistance to mold and viruses, as well as their strong adhesion and resistance to peeling, thus helping to expand the application range of nano-ceramic coatings.

[0007] To achieve the above-mentioned technical effects, the technical solution adopted by the present invention is as follows:

[0008] A nano-ceramic coating comprises the following components and their mass percentages: 25-30% methoxytrimethylsilane, 1-5% organosilicon quaternary ammonium salt, 20-30% nano-silica sol, 0.5-1% catalyst, 1-2% protective agent, and deionized water to 100%.

[0009] Preferably, the nano-ceramic material comprises the following components and their mass percentages: 28% methoxytrimethylsilane, 3% organosilicon quaternary ammonium salt, 25% nano-silica sol, 0.8% catalyst, 1.5% protective agent, and deionized water to 100%.

[0010] Preferably, the catalyst is a molecular sieve catalyst, which is a crystalline aluminosilicate.

[0011] Preferably, the protective agent is composed of methacryloxysilane and epoxysilane in a mass ratio of 2-5:10-15.

[0012] Preferably, the protective agent is composed of methacryloxysilane and epoxysilane in a mass ratio of 4:13.

[0013] The present invention also provides a method for preparing the aforementioned nano-ceramic coating, comprising the following steps:

[0014] S1. Mix methoxytrimethylsilane with organosilicon quaternary ammonium salt and stir until homogeneous to obtain mixture I;

[0015] S2. Add catalyst, protective agent and deionized water to mixture I obtained in step S1, stir and mix evenly, let stand for 20-30 minutes to obtain mixture II;

[0016] S3. Treat the mixture II obtained in step S2 at a temperature of 25-40°C for 2-3 hours using a sterile metal plate, glass plate or plastic sheet carrier without antiviral activity.

[0017] Preferably, the stirring conditions in step S1 are 300-500 rpm for 20-30 min; the stirring conditions in step S2 are 800-1000 rpm for 40-50 min.

[0018] The present invention also provides the application of the aforementioned nano-ceramic coating in the preparation of antifungal and antiviral drugs.

[0019] Preferably, the molds in the antifungal and antiviral drugs include Aspergillus niger, Aspergillus flavus, Penicillium wannii, Cladosporium multiflorum, Penicillium citrinum, Trichoderma longifolium, Cladosporium buddingum, and Alternaria alternata.

[0020] Preferably, the antifungal and antiviral drugs resist viruses including enteroviruses and SARS-CoV-2.

[0021] This invention employs a specific compound of siloxane and quaternary ammonium salt: the quaternary ammonium salt contains two methoxy groups, which hydrolyze in water to form a silanized quaternary ammonium salt with two silanol groups. Even after polymerization, it maintains a water-soluble and stable linear structure. Methoxytrimethylsilane also hydrolyzes to generate hydroxyl groups. The two react in a condensation reaction under the action of a catalyst to synthesize the quaternary ammonium salt onto a polysiloxane material. Further, using a waterborne polysiloxane technology, the waterborne polysiloxane (its structure is shown in the diagram) is produced. Figure 1 As shown, the hydroxyl functional groups in the main molecular chain of the film-forming material are highly active and can anchor to the surface of various materials like a suction cup. The uniformly distributed cations can persistently and effectively destroy the cell membrane wall of the bacteria in contact with it, thereby oxidizing and inactivating the viruses parasitizing in the cell nucleus, and realizing the triggered self-disinfecting function.

[0022] Depend on Figure 1 As can be seen, compared to materials that require silver, copper, titanium, etc., the quaternary ammonium salt compound of this invention is an organic polymer, which has a large space for modification of the material structure. For example, different bacteria can be sensitive to specific structures such as branched chains. It will not be discharged into water bodies and will not be exposed over a large area, so it is not easy for bacteria to evolve drug resistance. The structure obtained in this invention is called the "anchor cannon" strategy. This anchor is also designed to strongly adhere to the surface of the substrate. This can be attributed to the flexibility of the material structure of organic polymers and the huge space for modification.

[0023] In the selection of catalysts, the inventors chose molecular sieve catalysts, which allow substances meeting the size and structural requirements to enter during the actual reaction process, effectively improving the reaction efficiency of quaternary ammonium salts and siloxanes and accelerating the formation process of the material structure. The addition of silica sol allows the reacted material to form a colloidal coating when using sterile metal plates, glass plates, or plastic sheets without antiviral properties as a carrier. Furthermore, this invention also incorporates a protective agent composed of methacryloxysilane and epoxysilane in a mass ratio of 2–5:10–15. This protective agent increases the viscosity of the reaction products, improves the adhesion of the ceramic coating, making it less prone to detachment from other materials, and, together with polysiloxane, enhances the long-lasting antibacterial and antiviral properties of the material.

[0024] Existing literature reports antibacterial and antiviral materials all physically mix various cations, achieving their antibacterial and antiviral properties through physical means. However, the material in this invention achieves its antibacterial and antiviral properties through a chemical reaction, resulting in higher bond energies and thus better persistence. Furthermore, the excellent adhesion of the antibacterial and antiviral nano-ceramic coating material of this invention ensures its longevity in antibacterial and antiviral applications. Moreover, testing has shown that the structural characteristics of the ceramic material itself also guarantee the coating's wear resistance, water resistance, and UV resistance.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention uses nano-low temperature ceramic forming technology to provide a long-lasting antibacterial and antiviral self-disinfecting nano-ceramic material that is breathable but not water-permeable. The lack of moisture and carbon-based nutrients makes it impossible for bacteria and mold to survive and reproduce.

[0027] (2) The present invention has a mold resistance level of 0 (no mold grows on the test sample) and good resistance to the mixture of 8 kinds of molds.

[0028] (3) The long-lasting antibacterial and antiviral self-disinfecting nano-ceramic materials and their aging test samples have strong antiviral effects against enterovirus and the novel coronavirus Omicron BA.5 strain. Attached Figure Description

[0029] Figure 1 This is a structural diagram of the ceramic material obtained by the present invention;

[0030] Figure 2 The image shows the control sample and the effect of the sample on inhibiting mold growth.

[0031] Figure 3 To inhibit the effects of enterovirus 71 on the sample and its aged sample;

[0032] Figure 4 To inhibit the expression of enterovirus 71 C4a gene in the sample and its aged sample;

[0033] Figure 5 To inhibit the expression of enterovirus 71 VP1 protein in the sample and its aged sample;

[0034] Figure 6 To investigate the expression of S, E, and N genes in cells of the novel coronavirus Omeprone BA.5 strain by antibacterial ceramics;

[0035] Figure 7 To inhibit the expression of S and NP proteins of the novel coronavirus Omeprón BA.5 strain in samples and samples under aging conditions. Detailed Implementation

[0036] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0037] The organosilicon quaternary ammonium salt was dimethoxymethylsilylpropyloctadecyldimethylammonium chloride, purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd., catalog number 27668-52-6. The Applied Biosystems QuantStudio 7Pro real-time fluorescence quantitative PCR system was purchased from Thermo Fisher Scientific, USA; the Axio observer 7 high-resolution fluorescence microscope was purchased from Carl Zeiss (Shanghai) Management Co., Ltd.

[0038] The inactivated fetal bovine serum, antibiotics, DMEM culture medium, trypsin-EDTA digestion solution, and phosphate buffer solution were all purchased from Thermo Fisher Scientific (China) Co., Ltd.; the PAGE purification DNA synthesis primers, AG RNA ex Pro RNA extraction reagent, Evo M-MLV reverse transcription premixed kit, and SYBR Green Pro Taq HS premixed qPCR kit were all purchased from Hunan Aikerui Biotechnology Co., Ltd.; the paraformaldehyde and Triton X-100 were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the VP1 protein and goat anti-mouse agent were purchased from Abcam, UK; bovine serum albumin (BSA) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; the potato-dextrose agar medium (PDA) was purchased from Guangdong Huankai Microbial Technology Co., Ltd.; and the Vero-E6 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0039] The *Aspergillus niger* (CGMCC 3.5487), *Aspergillus flavus* (CGMCC 3.3950), *Penicillium wansii* (CGMCC 3.4253), *Cladosporium multiflorum* (CGMCC 3.2757), *Penicillium citrinum* (CGMCC 3.2913), *Trichoderma longifolium* (CGMCC 3.2941), *Bacillus buddingus* (CGMCC 3.837), and *Alternaria alternata* (CGMCC 3.4255) were all purchased from the Guangdong Provincial Microbial Culture Collection Center.

[0040] Example 1: A nano-ceramic coating

[0041] The nano-ceramic coating comprises the following components and their mass percentages: 25% methoxytrimethylsilane, 1% organosilicon quaternary ammonium salt, 20% nano-silica sol, 0.5% crystalline aluminosilicate, 1% protective agent, and deionized water to 100%; wherein the protective agent is prepared by mixing methacryloyloxysilane and epoxysilane in a mass ratio of 2-5:10-15.

[0042] The preparation method of the nano-ceramic coating includes the following steps:

[0043] S1. Mix methoxytrimethylsilane with organosilicon quaternary ammonium salt and stir at 300 rpm for 30 min until homogeneous to obtain mixture I;

[0044] S2. Add crystalline aluminosilicate, protective agent and deionized water to mixture I obtained in step S1, stir at 800 rpm for 40 min until the mixture is uniform, let stand for 20 min to obtain mixture II.

[0045] S3. Using a sterile metal plate, glass plate, or plastic sheet without antiviral properties as a carrier, treat the mixture II obtained in step S2 at 25°C for 3 hours to obtain the final product.

[0046] Example 2: A Nano-Ceramic Coating

[0047] The nano-ceramic coating comprises the following components and their mass percentages: 30% methoxytrimethylsilane, 5% organosilicon quaternary ammonium salt, 30% nano-silica sol, 1% crystalline aluminosilicate, 2% protective agent, and deionized water to 100%; wherein the protective agent is prepared by mixing methacryloyloxysilane and epoxysilane in a mass ratio of 5:13.

[0048] The preparation method of the nano-ceramic coating includes the following steps:

[0049] S1. Mix methoxytrimethylsilane with organosilicon quaternary ammonium salt and stir at 500 rpm for 20 min until homogeneous to obtain mixture I;

[0050] S2. Add crystalline aluminosilicate, protective agent and deionized water to mixture I obtained in step S1, stir at 1000 rpm for 40 min, stir until uniform, let stand for 30 min to obtain mixture II.

[0051] S3. Using a sterile metal plate, glass plate, or plastic sheet carrier without antiviral properties, treat the mixture II obtained in step S2 at 40°C for 2 hours to obtain the final product.

[0052] Example 3: A Nano-Ceramic Coating

[0053] The nano-ceramic material comprises the following components and their mass percentages: 28% methoxytrimethylsilane, 3% organosilicon quaternary ammonium salt, 25% nano-silica sol, 0.8% crystalline aluminosilicate, 1.5% protective agent, and deionized water to 100%; wherein the protective agent is prepared by mixing methacryloyloxysilane and epoxysilane in a mass ratio of 4:13.

[0054] The preparation method of the nano-ceramic coating includes the following steps:

[0055] S1. Mix methoxytrimethylsilane with organosilicon quaternary ammonium salt and stir at 400 rpm for 25 min until homogeneous to obtain mixture I;

[0056] S2. Add crystalline aluminosilicate, protective agent and deionized water to mixture I obtained in step S1, stir at 900 rpm for 45 min until the mixture is uniform, let stand for 25 min to obtain mixture II.

[0057] S3. Using a sterile metal plate, glass plate, or plastic sheet carrier without antiviral properties, treat the mixture II obtained in step S2 at 35°C for 2.5 hours to obtain the final product.

[0058] Comparative Example 1: A Nano-Ceramic Coating

[0059] The composition and preparation process of the nano-ceramic coating are similar to those in Example 3;

[0060] The difference from Example 3 is that the quaternary ammonium salt in Comparative Example 1 is 80% decyl dimethyl ammonium chloride, which can be purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd., CAS No. 3401-74-9.

[0061] Comparative Example 2: A Nano-Ceramic Coating

[0062] The composition and preparation process of the nano-ceramic coating are similar to those in Example 3;

[0063] The difference from Example 3 is that in Comparative Example 2, the mass percentage of methoxytrimethylsilane is 20% and the mass percentage of organosilicon quaternary ammonium salt is 2%.

[0064] Comparative Example 3: A Nano-Ceramic Coating

[0065] The composition and preparation process of the nano-ceramic coating are similar to those in Example 3;

[0066] The difference from Example 3 is that the mass percentage of methoxytrimethylsilane in Comparative Example 3 is 35%, and the mass percentage of organosilicon quaternary ammonium salt is 7%.

[0067] Comparative Example 4: A Nano-Ceramic Coating

[0068] The composition and preparation process of the nano-ceramic coating are similar to those in Example 3;

[0069] The difference from Example 3 is that the protective agent in Comparative Example 4 is methacryloxysilane.

[0070] Comparative Example 5: A Nano-Ceramic Coating

[0071] The composition and preparation process of the nano-ceramic coating are similar to those in Example 3;

[0072] The difference from Example 3 is that the sap agent in Comparative Example 5 is an epoxy silane.

[0073] Test Example 1: Mold Resistance Test

[0074] 1. Test sample: Nano-ceramic coating prepared in Example 3;

[0075] 2. Experimental Procedure: The petri dish method was used to determine the mold resistance of the self-disinfecting nano-ceramic coating to various molds, including Aspergillus niger, Aspergillus flavus, Penicillium wannii, Cladosporium multiflorum, Penicillium citrinum, Trichoderma longifolium, Trichoderma buddingis, and Alternaria alternata. First, eight types of mold were cultured. The specific culture process was as follows: In a biosafety cabinet, mold spores were picked up with a sterile inoculation loop and inoculated onto potato dextrose agar (PDA) medium. The medium was cultured at 28°C for 10 days. When the surface of the medium was covered with mold spores, 10 mL of sterile water was added. Then, in a biosafety cabinet, under aseptic conditions, the mold spores on the surface of the mold culture were gently scraped off with a sterile inoculation loop to prepare a mold spore suspension.

[0076] Pour the mold spore suspension into a 125 mL sterile Erlenmeyer flask fitted with a stopper and pre-filled with 45 mL of sterile water and 15 sterile glass beads. Vigorously shake the flask to break up the spore clusters and release the spores from the fruiting bodies. Place a sterile glass funnel with sterile fiber filter paper over the flask and pour the shaken mold spore suspension into the funnel to filter, removing hyphae and culture medium fragments. Centrifuge the filtered mold spore suspension at 4000 rpm under aseptic conditions, discard the supernatant, add 30 mL of sterile water to the spore precipitate, mix thoroughly, and then centrifuge to obtain the mold spore precipitate. Add sterile water again, mix well, and centrifuge again to obtain the mold spore precipitate.

[0077] Accurately weigh the nutrient solution components in a beaker according to Table 1, dissolve them in water by heating, add water to 1000 mL, adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution, dispense into triangular containers, and place in an autoclave. Sterilize at (121±2)℃ for 20 min before use.

[0078] The mold spore precipitate was diluted with nutrient salt solution, and the mold spore concentration was determined using a hemocytometer and then diluted again to achieve a mold spore concentration of 1.0 × 10⁻⁶ in the suspension. 6 Each prepared mold spore suspension can be stored in a refrigerator at 4℃~10℃ for no more than 4 days. Before the experiment, equal volumes of each prepared mold spore suspension were mixed to obtain a mixed mold spore solution. The test plate loaded with the self-disinfecting nano-ceramic coating (i.e., the nano-ceramic coating of Example 3) was used as the sample group, and sterile fiber-free filter paper was used as the control group. Both were placed in the center of a culture dish containing PDA, and 0.4 mL of the mixed mold spore solution was sprayed on them. The mixture was incubated in a constant temperature and humidity incubator at 25℃ and a relative humidity of not less than 85%. The results were observed at 0d, 7d, 14d, and 28d.

[0079] 3. Test Results: Specific test results are as follows Figure 2 As shown, it can be seen that the antifungal effect of the nano-ceramic coating group prepared in Example 3 of the present invention is significantly higher than that of the control group.

[0080] Table 1. Components of Nutrient Solution

[0081]

[0082]

[0083] Test Example 2 TCID 50 Detecting the effect of antibacterial ceramics on EV71 virus titers

[0084] 1. Test sample: Nano-ceramic coating prepared in Example 3;

[0085] 2. Experimental Procedure: Sample Preparation: The virus group was a control group containing only virus solution; the blank ceramic group was a control group (containing only aluminum plate) without antibacterial coating; the antibacterial ceramic group was an aluminum plate containing the nano-ceramic coating of this invention; the aging group was a sample irradiated with a 30W ultraviolet lamp with a wavelength of 253.7nm conforming to GB19258, with the lamp 0.8m to 1.0m away from the test plate, for 100h.

[0086] Endpoint titration dilution method (TCID) 50 The antiviral properties of the nano-ceramic coating were characterized by pipetting 0.4 mL of EV71 virus inoculum (inoculation density of 10⁻⁶). 6 TCID 500.4 mL of the virus suspension was dropped onto the surface of each sample (i.e., the aluminum plates of the blank ceramic group, antibacterial ceramic group, and aging group, with three repeated tests for each group). A 40 mm × 40 mm film was then placed over the inoculated virus suspension, and the film was gently pressed down to allow the virus suspension to spread outwards, ensuring that the virus suspension did not overflow from the edges of the film. After inoculating the samples and covering them with the film, the petri dish lid was replaced. After incubating the materials overnight, 5 mL of PBS was used for elution to obtain the virus eluent.

[0087] The virus eluent was diluted to 10 with serum-free DMEM. -1 Up to 10 -8 A series of gradients were used to infect Vero-E6 cells (Vero-E6 cells cultured to the logarithmic growth phase with 80%-90% confluence were ready for infection). After discarding the culture supernatant from the 96-well plates, 100 μL of ceramic-virus elution buffer was added to the corresponding cell culture plates. The plates were gently shaken to mix and incubated at 37°C, 5% CO2 for 1 h, gently shaking the plates every 20 min. After 2 h, the culture supernatant was aspirated, and the cells were washed twice with 200 μL of 1×PBS, replacing 200 μL of DMEM medium per well. The cells were incubated at 37°C, 5% CO2 for 72 h, with cell status observed daily and cytopathic effects recorded. Finally, the TCID of the virus was calculated using the Reed-Muench method. 50 value.

[0088] 3. Test Results: Specific test results are as follows Figure 3 As shown, this indicates that diluting the virus eluent to 10... -1 Up to 10 -8 A series of gradients were used to infect Vero-E6 cells, cytopathic effects were statistically analyzed, and the TCID of the virus was calculated. 50 value.

[0089]

[0090] The nano-ceramic coating showed an inhibition rate of 99.9% against enteroviruses, while the aged coating showed an inhibition rate of approximately 99.79% against enterovirus 71. These results suggest that both the nano-ceramic coating and its aged coating have a significant inhibitory effect on enterovirus 71. This may be because the uniformly distributed cations in the coating can persistently and effectively destroy the cell membrane walls of bacteria in contact with it, thereby oxidizing and inactivating the viruses parasitizing in the cell nucleus, thus achieving a triggered self-disinfecting function.

[0091] Experiment 3: Detection of the effect of antibacterial ceramics on the C4a gene of EV71 using qPCR

[0092] 1. Test subject: The nano-ceramic coating prepared in Example 3;

[0093] 2. Experimental Methods: Vero-E6 cells in good growth condition were digested with trypsin, centrifuged, resuspended, and seeded at 2 × 10⁶ cells per well in a 6-well plate. 4 3 mL of 500 μL DMEM medium (FBM, final FBM concentration 10%, penicillin-streptomycin concentration 1%) was used to culture cells overnight in an incubator until they were fully adhered and evenly spread. 1 mL of sample solution (i.e., the virus eluent prepared in Example 2) was added to each well to allow the virus to adsorb onto the cells. After 2 hours, the solution was removed, and the cells were washed twice with PBS. 3 mL of virus maintenance medium (DEMEM, final FBM concentration 2%, penicillin-streptomycin concentration 1%) was added to each well.

[0094] After 72 hours, nucleic acid was extracted and purified from each group of cells using AG RNAex Pro RNA extraction reagent (AG 21102). The nucleic acid was diluted with enzyme-free water to approximately 400 ng / μL. Reverse transcription was performed using the Evo M-MLV reverse transcription premixed kit (AG 11728). Detection was then performed using the SYBR Green Pro TaqHS premixed qPCR kit (AG 11718), with the following procedure: denaturation at 90℃ for 30 s, followed by reactions at 90℃ for 5 s and 60℃ for 30 s, repeated for 40 cycles. The primers used are as follows.

[0095] GAPDH-F:5'-GCACCGTCAAGGCTGAGAAC-3';

[0096] GAPDH-R:5'-TGGTGAAAGACGCCAGTGGA-3';

[0097] C4a-F:5'-ACAAGCAACAGGTGGTTACAGTA-3';

[0098] C4a-R:5'-GGTGGAATGAAGTCTACAGTGGAT-3'.

[0099] Then, the expression level of the virus's C4a gene after sample treatment was calculated using GraphPad Prism software.

[0100] 3. Test Results: Specific test results are as follows Figure 4As shown, the expression levels of enterovirus EV71 C4a after treatment with the self-cleaning nano-ceramic coating and its aged coating were 0.291 and 0.378, respectively, demonstrating that both the self-cleaning nano-ceramic coating and its aged coating effectively inhibited the expression of enterovirus EV71 C4a. Furthermore, compared to the nano-ceramic coating of this invention, although the ability of the aged coating to inhibit enterovirus EV71 C4a expression was slightly weaker, it still significantly inhibited C4a expression, proving its ability to persistently inhibit C4a expression.

[0101] Experiment 4: Detection of the effect of antibacterial ceramics on VP1 protein of EV71 using immunofluorescence assay

[0102] 1. Test sample: Nano-ceramic coating prepared in Example 3

[0103] 2. Experimental Methods: Vero-E6 cells in good growth condition were digested with trypsin, centrifuged, resuspended, and seeded at 2×10⁶ cells per well in a 24-well plate. 4 Cells / 500 μL of culture medium (same as in Example 3) were cultured overnight in an incubator until they were fully adhered and evenly spread. 200 μL of sample solution (i.e., the virus eluent prepared in Example 2) was added to each well to allow the virus to adsorb onto the cells. After 2 hours, the solution was removed, and the cells were washed twice with PBS. Virus maintenance medium (same as in Example 3) was added, and the cells were incubated in a CO2 incubator for 24 hours. After 24 hours, the culture in the 24-well plate was terminated, the supernatant was discarded, and the cells were washed three times with PBS. After washing each well, the 24-well plate was fixed in 4% paraformaldehyde solution for 1 hour. Permeabilization was performed with 0.5% Triton X-100 in PBS for 30 minutes. Blocking was performed in 5% BSA for 30 minutes. Primary antibody was used to stain for VP1 protein (Anti-Enterovirus 71 antibody (ab36367), 1:500) and incubated overnight at 4°C. Secondary antibody was used for goat anti-mouse (Goat Anti-Mouse Alexa). 488 (ab150113), 1:600) was incubated at room temperature for 1 h; DAPI was incubated at room temperature for 0.5 h. Fluorescence signals were detected using an inverted microscope and observed with an Axio Observer 7 inverted fluorescence microscope in conjunction with ZEN software. Three non-overlapping fields were randomly selected from each specimen.

[0104] 3. Test Results: Specific test results are as follows Figure 5 As shown, it can be seen that 10 6 TCID 50After 24 hours of contact between the virus and antibacterial ceramics, and between the virus group and the blank sample, the expression of VP1 protein was significantly reduced. The results indicate that antibacterial ceramics and aged antibacterial ceramics have a significant inhibitory effect on the VP1 protein of EV71. VP1 plays an important role in the adsorption and penetration of EV71, enhancing viral stability and protecting the virus from other substances. These experimental results demonstrate that antibacterial ceramics may interfere with viral particle assembly and viral entry into cells by inhibiting the expression of the VP1 protein in EV71.

[0105] Experimental Example 5: Detection of the effect of antibacterial ceramics on the novel coronavirus using the ELISPOT method.

[0106] 1. Test sample: Nano-ceramic coating prepared in Example 3;

[0107] 2. Experimental Methods: Vero-E6 cells were cultured in triplicate. Cell culture plates were washed twice with PBS buffer. 100 μL of virus eluent (prepared as in Example 2, but with the virus replaced by SARS-CoV-2 Omeprone BA.5 strain) was added to each well. The plates were gently shaken to mix and incubated at 37°C with 5% CO2 for 2 hours, gently shaking every 20 minutes. After 1 hour, the plates were washed twice with PBS, and DMEM medium was added. The plates were incubated at 37°C with 5% CO2 for 72 hours. After PBS washing, 4% PFA (paraformaldehyde) was added for fixation overnight. The plates were then transferred to another laboratory, washed twice with PBS, permeabilized with 0.2% Triton X-100 for 10 minutes, and washed twice with PBS. 50 μL of primary antibody was added to each well, and the plates were incubated at room temperature for 2 hours. After removing the primary antibody, the plates were washed twice with 1×PBS. 50 μL of secondary antibody per well, incubate at room temperature for 0.5 h, remove the antibody and wash twice with PBS. 50 μL of peroxidase base per well, incubate at room temperature for 10 min, remove the peroxidase and wash twice with PBS. Wrap the 96-well plate with a soft cotton pad and absorbent paper to thoroughly dry any remaining 1×PBS, then scan and count the cells.

[0108] 3. Experimental results: The specific experimental results are shown in Table 2.

[0109] Table 2. Effects of different test samples on the novel coronavirus.

[0110]

[0111] Therefore, the results of ELISPOT testing on the novel coronavirus Omeprón BA.5 strain (Table 2) show that the antiviral rate of the sample against the novel coronavirus Omeprón BA.5 strain was 99.35%, and the antiviral efficiency of the aged sample against the novel coronavirus Omeprón BA.5 strain was 98.03%. The results suggest that the sample has a significant anti-COVID-19 effect.

[0112] Experiment 6: Detection of the effect of antibacterial ceramics on the S, E, and N genes of SARS-CoV-2 using qPCR.

[0113] 1. Test sample: Nano-ceramic coating prepared in Example 3;

[0114] 2. Experimental Procedure: Vero-E6 cells in good growth condition were digested with trypsin, centrifuged, resuspended, and seeded at 2 × 10⁶ cells per well in a 6-well plate. 4 3 mL of 500 μL DMEM medium (final concentration of fetal bovine serum 10%, penicillin-streptomycin concentration 1%) was used to culture cells overnight in an incubator until they were fully adhered and evenly spread. 1 mL of sample solution was added to each well to allow the virus to adsorb onto the cells. After 2 hours, the solution was removed, and the cells were washed twice with PBS. Virus maintenance medium (same as in Example 3) was added, and after 72 hours, viral nucleic acid extraction, reverse transcription, and qPCR detection were performed (the procedure was the same as in Example 3, with primers replaced as follows). The expression levels of the S, E, and N genes of the virus after sample treatment were calculated using GraphPad Prism software.

[0115] SF:5'-ACTTGCAGATGCTGGCTTCA-3';

[0116] SR:5'-TCTGTGAGCAAAGGTGGCAA';

[0117] EF:5'-TCGGAAGAGACAGGTACGTTAAT-3';

[0118] ER:5'-GCGCAGTAAGGATGGCTAGT-3';

[0119] NF:5'-GCAGTCAAGCCTCTTCTCGT-3';

[0120] NR:5'-CAAGCAGCAGCAAAGCAAGA-3'.

[0121] 3. Test Results: Specific test results are as follows Figure 6 As shown, after treatment with the self-cleaning nano-ceramic coating and its aged coating, the expression of the S, E, and N genes of the novel coronavirus Omeprone BA.5 strain was almost zero, proving that both the self-cleaning nano-ceramic coating and its aged coating can effectively inhibit the expression of the S, E, and N genes. Furthermore, compared with conventional coatings, although the ability of the coating to inhibit the expression of the S, E, and N genes did not decrease after aging treatment, it demonstrates its ability to persistently inhibit the expression of the S, E, and N genes.

[0122] Experimental Example 7: Detection of the effect of antibacterial ceramics on the S and NP proteins of the novel coronavirus using immunofluorescence assay.

[0123] 1. Test sample: Nano-ceramic coating prepared in Example 3;

[0124] 2. Experimental Procedure: Vero-E6 cells in good growth condition were digested with trypsin, centrifuged, resuspended, and seeded at 2×10⁶ cells per well in a 24-well plate. 4 Cells / 500 μL of culture medium were incubated overnight in an incubator until they were fully adhered and evenly spread. 200 μL of sample solution was added to each well to allow the virus to adsorb onto the cells. After 2 hours, the solution was removed, and the cells were washed twice with PBS. Virus maintenance medium was added, and the cells were incubated in a CO2 incubator for 72 hours. After 72 hours, the culture in the 24-well plate was terminated, the supernatant was discarded, and the cells were washed three times with PBS. After washing each well, the 24-well plate was immersed in 4% paraformaldehyde solution and incubated overnight at 4°C. The cells were then permeated with 0.5% Triton X-100 in PBS for 30 minutes, blocked with 5% BSA for 1 hour, and stained overnight with primary antibodies for SARS-CoV-2 spike protein (SARS-CoV-2(COVID-19) Spike antibody, (GTX635693) 1:400) and nucleoprotein (SARS / SARS-CoV-2 Nucleocapsid Monoclonal Antibody (MA5-29981), 1:400). Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor Plus 555 donkey anti-rabbit IgG (Invitrogen, 1:500) secondary antibodies were incubated at room temperature for 1 h, followed by DAPI incubation at room temperature for 0.5 h. All antibodies were diluted in blocking buffer (5% BSA solution). Fluorescence signals were detected using an inverted microscope and observed with an Axio Observer 7 inverted fluorescence microscope in conjunction with ZEN software.

[0125] 3. Test Results: Specific test results are as follows Figure 7 As shown, after 24 hours of contact with antibacterial ceramics and aged antibacterial ceramics, the expression of SARS-CoV-2 S and NP proteins was completely inhibited compared to the virus group in the blank sample. Since the S protein can recognize and bind to receptors on the surface of host cells, and the N protein plays an important role in viral replication, we hypothesize that antibacterial ceramics and aged antibacterial ceramics may exert their antiviral effect by disrupting the surface structure of SARS-CoV-2 and interfering with its adsorption to host cells.

[0126] Test Example 8: Durability Test of Antiviral and Antibacterial Properties

[0127] 1. Test samples: Nano-ceramic coatings prepared in Examples 1-3 and Comparative Examples 1-5;

[0128] 2. Test methods: The testing standards are as follows: antiviral performance and antiviral durability testing shall be conducted in accordance with T / CNCIA03002-202 for testing EV71 virus;

[0129] For testing of antibacterial properties and antibacterial durability, refer to T / CNCIA 01014-2020 for testing Aspergillus niger and Paecilomyces wanyi.

[0130] 3. Experimental results: The specific experimental results are shown in Table 3.

[0131] Table 3 Comparison of antibacterial properties of different test samples of the present invention

[0132]

[0133]

[0134] Therefore, the nano-ceramic coatings prepared in Examples 1-3 of this invention exhibit excellent antiviral and antibacterial durability. After 100 hours, their resistance to viruses remains above 99%, and no mold growth is observed. In contrast, the antibacterial and antiviral effects of Comparative Examples 1-5 are reduced to varying degrees and lack long-lasting efficacy.

[0135] In summary, the self-disinfecting nano-ceramic coating provided by this invention has a mold resistance rating of 0, demonstrating significant mold resistance. It also exhibits significant inhibitory effects against enteroviruses and the novel coronavirus, with inhibition rates of 99.90% and 99.35%, respectively. The aged samples showed inhibition rates of 99.79% against enterovirus EV71 and 98.03% against the novel coronavirus. Mechanistic studies revealed that this coating effectively inhibits the expression of enterovirus VP1 protein and the expression of S and NP proteins of the novel coronavirus. Furthermore, it effectively inhibits the expression of the enterovirus C4a gene and the expression of the S, E, and N genes of the novel coronavirus. This long-lasting antibacterial and antiviral self-disinfecting nano-ceramic coating demonstrates significant antiviral and anti-COVID-19 properties.

[0136] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A nano-ceramic coating, characterized in that, It includes the following components and their mass percentages: 25-30% methoxytrimethylsilane, 1-5% organosilicon quaternary ammonium salt, 20-30% nano-silica sol, 0.5-1% catalyst, 1-2% protective agent, and deionized water to 100%.

2. The nano-ceramic material as described in claim 1, characterized in that, It includes the following components and their mass percentages: 28% methoxytrimethylsilane, 3% organosilicon quaternary ammonium salt, 25% nano-silica sol, 0.8% catalyst, 1.5% protective agent, and deionized water to 100%.

3. The nano-ceramic coating as described in claim 1 or 2, characterized in that, The catalyst is a molecular sieve catalyst, which is a crystalline aluminosilicate.

4. The nano-ceramic coating as described in claim 1 or 2, characterized in that, The protective agent is composed of methacryloxysilane and epoxysilane in a mass ratio of 2-5:10-15.

5. The nano-ceramic coating as described in claim 4, characterized in that, The protective agent is composed of methacryloxysilane and epoxysilane in a mass ratio of 4:

13.

6. A method for preparing a nano-ceramic coating as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Mix methoxytrimethylsilane with organosilicon quaternary ammonium salt and stir until homogeneous to obtain mixture I; S2. Add catalyst, protective agent and deionized water to mixture I obtained in step S1, stir and mix evenly, let stand for 20-30 minutes to obtain mixture II; S3. Treat the mixture II obtained in step S2 at a temperature of 25-40°C for 2-3 hours using a sterile metal plate, glass plate or plastic sheet carrier without antiviral activity.

7. The preparation method according to claim 6, characterized in that, The stirring conditions described in step S1 are: processing at a speed of 300-500 rpm for 20-30 minutes; the stirring and mixing conditions described in step S2 are: processing at a speed of 800-1000 rpm for 40-50 minutes.

8. The application of the nano-ceramic coating as described in claim 1 in the preparation of antifungal and antiviral drugs.

9. The application as described in claim 8, characterized in that, The molds in the mold-resistant and antiviral drugs include Aspergillus niger, Aspergillus flavus, Penicillium wannii, Cladosporium multiflorum, Penicillium citrinum, Trichoderma longiflorum, Cladosporium buddingum, and Alternaria alternata.

10. The application as described in claim 8, characterized in that, The viruses resisted by the antifungal and antiviral drugs include enteroviruses and SARS-CoV-2.

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