Cascaded catalytic enzyme MOF-919-GOx as well as preparation method and application thereof

Abstract

The invention discloses a cascade catalytic enzyme MOF-919-GOx as well as a preparation method and application thereof, and relates to the technical field of nano-enzymes. According to the invention, MOF-919-GOx is synthesized by a glutaraldehyde loading method, and detection shows that the loading capacity of GOx in MOF-919 is 372.20 [mu] g / mg; when 2 mg / mL of MOF-919-GOx and 25 mM of glucose are added, the pH value of the environment can be reduced, hydroxyl radicals can be generated, and efficient peroxidase-like activity is achieved. The cascade catalytic enzyme MOF-919-GOx has a concentration-dependent inhibition effect on suspended porphyromonas gingivalis and staphylococcus aureus, and the concentration-dependent inhibition effect on the suspended porphyromonas gingivalis and staphylococcus aureus is achieved. When the concentration of the MOF-919-GOx is 0.25 mg / mL, the MOF-919-GOx can generate a remarkable removal effect on a biological membrane in a dog oral cavity artificial saliva environment, can be used for developing a dog oral cavity antibacterial material, and has application potential in the veterinary clinical field.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme technology, and in particular to a cascade catalytic enzyme MOF-919-GOx and a preparation method and application thereof. Background Art

[0002] Nanomaterials are materials with at least one nanometer-scale dimension in three-dimensional space or composed of nanoscale units. They are mesoscopic systems between macroscopic objects and microscopic atomic clusters. Nanozymes are nanomaterials with enzyme-like properties, possessing catalytic activity due to their unique structure. Compared to natural enzymes, nanozymes not only possess the same high catalytic activity but also exhibit greater stability and lower manufacturing costs. More importantly, they are resistant to bacterial resistance mechanisms, making them a promising new antimicrobial agent.

[0003] Metal-organic frameworks (MOFs) are porous polymer materials composed of metal ions or metal oxide clusters linked by organic ligands. MOF-919 (Fe-Cu-MOF), a type of MOF nanomaterial, exhibits peroxidase-like activity. Using hydrogen peroxide as a substrate, it produces •OH, a highly destructive molecule, through the Fenton reaction, promising applications in biomedicine. MOF-919 is also a bimetallic organic framework (BOF). The synergistic effect between the two metal ions within the BBOF provides a wider range of active sites and catalytic pathways, resulting in higher catalytic activity, selectivity, and stability, enabling more complex catalytic reactions.

[0004] There are various methods for loading enzymes onto metal-organic frameworks (MOFs). Cross-linking, among other methods, involves immobilizing the enzyme onto a support matrix through intermolecular reactions using a cross-linking agent. Key cross-linking agents include glutaraldehyde, dicarboxylic acids, and dimethyl adipimidate, with glutaraldehyde being the most widely used. Glutaraldehyde cross-linking offers advantages such as high stability, ease of use, versatility, and low cost, making it a widely used loading method.

[0005] There are many common bacteria in canine oral biofilm infections. Under normal physiological conditions, the types and numbers of bacteria in the oral cavity are in a dynamic balance. Once the pathogenic bacteria in the oral cavity gain the upper hand and break the balance, oral diseases will occur. In small animal clinical practice, periodontitis is very common. It is a disease caused by microbial infection that occurs widely in dogs of all ages. When the animal is found to be ill, the periodontal tissue has been irreversibly damaged, and oral ulcers, bad breath, tooth loss, and even other systemic diseases may occur. Clinically, once canine periodontitis occurs, it is difficult to cure even with long-term use of antimicrobial drugs. Bacterial control is the key to preventing and treating oral diseases such as periodontitis, and the presence of biofilms makes it difficult for traditional antibiotics and antibacterial treatments to achieve good results. There is an urgent need to find new antibacterial materials.

[0006] This technical solution aims to provide a composite cascade catalytic system, which is expected to be used for the antibacterial treatment of common bacteria in the canine oral cavity and to produce an antagonistic effect on the bacterial biofilm in the canine oral cavity. Summary of the Invention

[0007] The purpose of the present invention is to provide a cascade catalytic enzyme MOF-919-GOx and a preparation method thereof, which can be used as a new antibacterial material for the antibacterial effect of common bacteria in the canine oral cavity and produce an antagonistic effect on the bacterial biofilm in the canine oral cavity.

[0008] In order to achieve the above-mentioned object, the present invention provides a cascade catalytic enzyme MOF-919-GOx, which is obtained by loading glucose oxidase GOx onto nanozyme MOF-919 through a glutaraldehyde loading method.

[0009] The cascade catalytic enzyme MOF-919-GOx provided by the present invention can be used to inhibit Porphyromonas gingivalis P. gingivalis and Staphylococcus aureus S. aureus ; can also be used to prepare Porphyromonas gingivalis P. gingivalis and Staphylococcus aureus S. aureus antibacterial materials.

[0010] The present invention also provides a Porphyromonas gingivalis prepared by the cascade catalytic enzyme MOF-919-GOx. P. gingivalis and Staphylococcus aureus S. aureus Antibacterial materials.

[0011] The cascade catalytic enzyme MOF-919-GOx or antibacterial material provided by the present invention can be used in the field of veterinary clinical practice, and is particularly useful for inhibiting and removing canine oral bacteria and bacterial biofilm formation.

[0012] Furthermore, the above-mentioned bacteria include Porphyromonas gingivalis P. gingivalis and Staphylococcus aureusS. aureus .

[0013] The present invention also provides a method for preparing the above-mentioned cascade catalytic enzyme MOF-919-GOx, which is obtained by dispersing the nanozyme MOF-919 in deionized water, adding glucose oxidase GOx and 50% glutaraldehyde, stirring, and then centrifuging and washing to obtain MOF-919-GOx.

[0014] Preferably, the mass ratio of the above-mentioned nanozyme MOF-919 to glucose oxidase GOx is 1:1.5.

[0015] Preferably, the ratio of the above-mentioned 50% glutaraldehyde to nanozyme MOF-919 is 1 mL / 20 mg.

[0016] Preferably, the stirring in the above preparation method is stirring at room temperature in the dark for 1 hour.

[0017] The present invention has the following advantages: This application successfully synthesized MOF-919-GOx and verified that it can activate the cascade reaction in a simulated canine oral environment and has excellent peroxidase activity; at 2 mg / mL, it can activate the cascade reaction in a simulated canine oral environment and has excellent peroxidase activity. P. gingivalis and S. aureus It has a significant inhibitory effect and can significantly reduce the expression of virulence genes of the two pathogens.

[0018] As a highly effective nanozyme, MOF-919 is widely used in the antibacterial field. However, due to its optimal working environment being acidic, it is difficult to work directly in the canine oral cavity with an average pH of 8.5, and has not been used in the canine oral cavity. In order to make MOF-919 applicable to the alkaline canine oral environment, this study loaded GOx onto the surface of MOF-919 and applied MOF-919-GOx to a simulated canine oral environment for the first time, and proved that it can effectively inhibit the growth of bacteria in the simulated canine oral environment. P. gingivalis and S. aureus Has a significant inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the results of scanning electron microscopy tests of MOF-919-GOx and MOF-919 in the present invention.

[0020] Figure 2 The standard curve prepared for calculating the mass of GOx loaded on MOF-919 in the present invention.

[0021] Figure 3 The results of TMB colorimetric assay were used to detect the peroxidase activity of MOF-919 before and after loading GOx.

[0022] Figure 4The results of the methyl red test verify the effect of MOF-919 loaded with GOx on oral pH.

[0023] Figure 5 The results of the TMB colorimetric reaction to test the POD activity of MOF-919 after loading GOx.

[0024] Figure 6 The POD activity results of MOF-919 under different conditions.

[0025] Figure 7 The POD activity results of MOF-919-GOx under different action conditions.

[0026] Figure 8 The results of hemolysis tests of MOF-919 and MOF-919-GOx are shown.

[0027] Figure 9 MOF-919-GOx for suspended bacteria P. gingivalis and S. aureus Survival results of antimicrobial efficacy verification.

[0028] Figure 10 Quantitative results of crystal violet staining to verify the antibacterial effect of MOF-919-GOx on biofilm.

[0029] Figure 11 MTT test results verifying the effect of MOF-919-GOx on biofilms against P. gingivalis and S. aureus biofilms.

[0030] Figure 12 Different concentrations of MOF-919-GOx P. gingivalis Gram staining results for biofilm resistance verification.

[0031] Figure 13 Different concentrations of MOF-919-GOx S. aureus Gram staining results for biofilm resistance verification.

[0032] Figure 14 Different concentrations of MOF-919-GOx P. gingivalis Scanning electron microscopy results of biofilm resistance verification.

[0033] Figure 15 Different concentrations of MOF-919-GOx S. aureus Scanning electron microscopy results of biofilm resistance verification. DETAILED DESCRIPTION

[0034] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0035] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0036] Experimental Example 1 Synthesis and Property Verification of MOF-919-GOx 1. Synthesis of MOF-919-GOx 5 mg of nanozyme MOF-919 was dispersed in 5 mL of deionized water, and 7.5 mg of glucose oxidase GOx and 0.25 mL of 50% glutaraldehyde were added. The mixture was stirred at room temperature in the dark for 1 h, and MOF-919-GOx was obtained after centrifugation and washing with deionized water.

[0037] 2. Electron microscope scanning MOF-919-GOx and MOF-919 were gold plated using a gold plater to enhance conductivity, and the surface morphology and particle size of the samples were observed using field emission scanning electron microscopy. Figure 1 As shown in the figure, A is MOF-919 and B is MOF-919-GOx. It can be seen that both MOF-919-GOx and MOF-919 are clustered and have a petal-like surface. The loading of GOx does not affect the surface morphology of MOF-919. The particle size of MOF-919-GOx is approximately 1502.16 nm.

[0038] 3. MOF-919-GOx loading capacity detection During the centrifugation and washing step of MOF-919-GOx synthesis, the supernatant was collected to measure the loading amount of glucose oxidase.

[0039] To draw a standard curve, take 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of 100 μg / mL glucose oxidase solution, add 1, 0.8, 0.6, 0.4, 0.2, and 0 mL of 0.9% sodium chloride solution, respectively. Finally, add 3 mL of Coomassie brilliant blue dye, mix well, let stand for 5 min, and measure the absorbance at 595 nm with a microplate reader.

[0040] Calculate the enzyme loading of the sample: Take 0.1 mL of the supernatant to be tested, dilute it with 0.9 mL of 0.9% sodium chloride solution, then add 3 mL of Coomassie Brilliant Blue dye, mix well, let it stand for 5 minutes, and measure the absorbance at 595 nm using a microplate reader. Calculate the loading according to the following formula 1:

[0041] Enzyme loading (μg / mg) = Formula 1 After loading, the supernatant was collected and the protein content of the supernatant was measured by Coomassie Brilliant Blue method. The mass of GOx loaded on MOF-919 was calculated and a standard curve was prepared. The results are shown in Figure 2. Figure 2 As shown in the figure, the calculated loading amount is 372.20 μg / mg, which is within the detection range of Coomassie Brilliant Blue (0-1000 μg), and also indirectly confirms the successful loading of GOx.

[0042] 4. Detection of peroxidase activity Prepare a pH 4 buffer solution, mix 100 μL TMB (5 mM), 100 μL MOF-919-GOx or MOF-919 (2 mg / mL), 700 μL buffer solution, and 100 μL H2O2 (10 mM). Incubate the mixture at 37°C for 30 min, and measure the absorbance at 652 nm using a microplate reader.

[0043] When there is no active oxygen in the solution, TMB is colorless. When there is ·OH in the solution, TMB is oxidized to form oxTMB, the solution turns blue, and there is an absorption peak at 652 nm. By measuring the absorbance at 652 nm, the ability of the material to generate ·OH, that is, POD activity, can be indirectly reflected. The results of the TMB colorimetric test are shown in Figure 2. Figure 3 As shown, it can be seen that when MOF-919 or TMB is added alone, there is no absorption peak at 652 nm; the group with TMB and H2O2 added has a weak absorption peak at 652 nm, which is because H2O2 also has a certain oxidizing property; the group with TMB, H2O2 and MOF-919 added has a strong absorption peak at 652 nm, which is enough to show that MOF-919 converts H2O2 into ·OH with strong oxidizing ability through the Fenton reaction and has POD activity; the group with TMB, H2O2 and MOF-919-GOx added also has a strong absorption peak at 652 nm, which shows that MOF-919 still has good POD activity after loading GOx.

[0044] 5. Verification of MOF-919-GOx cascade reaction The cascade reaction consists of two parts. First, glucose oxidase catalyzes the conversion of glucose into gluconic acid, lowering the pH of the solution and producing hydrogen peroxide. Then, MOF-919-GOx exerts its POD activity, converting hydrogen peroxide into hydroxyl radicals, which plays a role in clearing biofilms.

[0045] (1) Methyl red test Glucose oxidase can convert glucose into gluconic acid, lowering the solution's pH. This was verified using a methyl red test. To a 1.5 mL centrifuge tube, 100 μL of glucose solution at varying concentrations (0.5, 1, 2, 3, 4, 6.25, 12.5, 25, 50, and 100 mM) and 100 μL of a 1 mg / mL MOF-919-GOx solution were added, along with 10 μL of methyl red indicator. The mixture was incubated at 37°C and photographed every 30 minutes.

[0046] After MOF-919 is loaded with GOx, it can lower the local pH of the oral cavity, which is verified by the methyl red test. When the pH of the solution is greater than 6.2, methyl red appears yellow. When the pH of the solution is between 4.4-6.2, methyl red appears orange. When the pH of the solution is lower than 4.4, the solution appears red.

[0047] Methyl red test results Figure 4 As shown, group A is the initial state; group B is after 30 minutes; group C is after 1 hour; group D is after 1.5 hours; and group E is after 2 hours. It can be seen that when the glucose concentration is 0.5-4 mM, the solution does not show obvious color change within two hours; after half an hour of incubation, 25 mM, 50 mM, and 100 mM all turn orange, indicating that the pH has dropped to 6.2-4.4 (see Figure 4 B); after one hour, 25 mM, 50 mM, and 100 mM all turned red, and 12.5 mM turned orange (see Figure 4 C in Figure 3); after one and a half hours, the orange color of 12.5 mM became darker (see Figure 4 D in Figure 2); after two hours, 12.5 mM turned red and 6.25 mM turned light orange (see Figure 4 E in Figure 1). This shows that GOx loaded on MOF-919 still has enzymatic activity and can activate the cascade reaction when glucose is present in the environment. The higher the glucose concentration, the faster the cascade reaction and the faster the pH change: it takes two hours for the pH to drop below 6.2 in the 6.25 mM group, while it only takes half an hour in the 25 mM group. However, when the glucose concentration is low, no obvious pH change can be observed within two hours.

[0048] (2) TMB color reaction When the pH of the solution decreases, MOF-919 can further exert its POD activity, which can be tested using the TMB colorimetric reaction: after mixing the solutions of different groups evenly (see Table 1), incubate them at 37°C for 30 minutes, and measure the absorbance at 652 nm.

[0049] Table 1 Grouping of TMB color reaction The results of TMB color reaction are as follows Figure 5 As shown in the figure, when TMB was added alone or when TMB and glucose were mixed, there was no absorption peak at 652 nm. This is because there was no oxidizing substance in the solution that could oxidize TMB. There was no absorption peak when TMB and MOF-919-GOx were mixed. Although MOF-919-GOx has catalytic activity to produce hydroxyl radicals, it lacks the substrate H2O2, so it cannot oxidize TMB. It is just that the color of MOF-919-GOx affects the absorbance, which makes the overall absorbance of this group increase. The TMB+glucose+MOF-919-GOx group showed a clear absorption peak at 652 nm.

[0050] 6. Effects of different reaction conditions on the peroxidase activity of MOF-919-GOx (1) Effect of pH on peroxidase activity of MOF-919-GOx and MOF-919 Buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, 8, and 9 were prepared. 100 μL of TMB (5 mM), 100 μL of MOF-919-GOx or MOF-919 (2 mg / mL), 700 μL of buffer solutions with different pH values, and 100 μL of H2O2 (10 mM) were added to each group. The mixture was mixed and incubated at 37°C for 30 min. The absorbance at 652 nm was measured using a microplate reader.

[0051] (2) Effect of temperature on peroxidase activity of MOF-919-GOx and MOF-919 Prepare a pH 4 buffer solution, mix 100 μL TMB (5 mM), 100 μL MOF-919-GOx or MOF-919 (2 mg / mL), 700 μL pH 4 buffer solution, and 100 μL H2O2 (10 mM). After mixing, incubate at 20, 30, 40, 50, and 60°C for 30 min, respectively, and measure the absorbance at 652 nm using a microplate reader.

[0052] (3) Effect of H2O2 concentration on peroxidase activity of MOF-919-GOx and MOF-919 H2O2 with concentrations of 1, 5, 10, 15, and 20 mM was prepared. 100 μL of TMB (5 mM), 100 μL of MOF-919-GOx or MOF-919 (2 mg / mL), 700 μL of pH 4 buffer, and 100 μL of H2O2 at different concentrations were mixed. The mixture was incubated at 37°C for 30 min, and the absorbance at 652 nm was measured every 5 min using a microplate reader.

[0053] (4) Effect of material concentration on peroxidase activity of MOF-919-GOx and MOF-919 MOF-919 solutions with concentrations of 2, 5, 10, 30, 60, and 100 μg / mL were prepared. 100 μL of TMB (5 mM), 100 μL of MOF-919-GOx or MOF-919 at different concentrations, 700 μL of pH 4 buffer, and 100 μL of H2O2 (10 mM) were mixed and incubated at 37°C for 30 min. The absorbance at 652 nm was measured every 5 min using a microplate reader.

[0054] The results of POD activity of MOF-919 under different conditions were obtained. Figure 6 As shown in the figure, A is pH, B is temperature, C is hydrogen peroxide concentration, and D is MOF-919 concentration. The optimal pH for MOF-919 to exert POD activity is usually 3-4, but the average pH of dog saliva is about 8.5. Therefore, an experiment was set up to verify the POD activity of MOF-919 under different pH environments. The results are shown in the figure. Figure 6 As shown in A in the figure, it can be seen that when the environmental pH is 3, the POD activity of MOF-919 is the strongest. As the pH increases, the POD activity gradually decreases. When the environmental pH is 8-9, MOF-919 can hardly exert its POD activity; when the environmental pH is too low, the POD activity of MOF-919 decreases. Figure 6 Figure B represents the POD activity of MOF-919 under different temperature environments. When the ambient temperature is 50°C, the POD activity of MOF-919 is the strongest. After that, the POD activity gradually decreases as the temperature increases or decreases. In addition to temperature and pH, the POD activity of MOF-919 is also affected by the concentration of hydrogen peroxide and the concentration of MOF-919 itself. Figure 6 The results in Figures C and D show that the POD activity is proportional to the concentrations of hydrogen peroxide and MOF-919, and only lower concentrations of hydrogen peroxide and MOF-919 are required to rapidly undergo the Fenton reaction to produce hydroxyl radicals.

[0055] The results of POD activity of MOF-919-GOx under different conditions were obtained. Figure 7 As shown in the figure, A represents pH, B represents temperature, C represents hydrogen peroxide concentration, and D represents MOF-919-GOx concentration. It can be seen that the optimal pH and temperature remain at 3 and 50°C, respectively. This comparison reveals that GOx-loaded MOF-919-GOx maintains excellent POD activity, rapidly generating hydroxyl radicals in the presence of low concentrations of hydrogen peroxide and MOF-919-GOx.

[0056] 7. Hemolysis test of MOF-919 and MOF-919-GOx The biosafety of MOF-919 and MOF-919-GOx was tested by hemolysis test. The test steps are as follows:

[0057] MOF-919 and MOF-919-GOx suspensions were prepared in 0.9% sterile saline at concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL, respectively. The canine red blood cell solution was diluted with sterile saline to obtain a 5% red blood cell suspension for later use.

[0058] 500 μL of each MOF-919 or MOF-919-GOx suspension at different concentrations was placed in a centrifuge tube. 500 μL of sterile saline and 500 μL of sterile PBS were then added as negative and positive controls, respectively. These solutions were incubated at 37°C for 15 minutes. After the incubation period, 500 μL of a 5% red blood cell suspension was added, mixed thoroughly, and incubated at 37°C for 1 hour. The supernatant was collected and centrifuged at 3000 rpm for 5 minutes. The absorbance at 540 nm was measured, and the hemolysis rate of MOF-919 and MOF-919-GOx was calculated according to the following formula:

[0059] 00% The results of the hemolysis test are shown in Figure 8As shown in the figure, where A is the hemolysis rate of MOF-919 and B is the hemolysis rate of MOF-919-GOx. It can be seen that the hemolysis rates of erythrocyte suspensions treated with MOF-919 suspensions at concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL were 4.33±0.26%, 2.89±0.12%, 1.13±0.11%, 1.32±0.02%, and 1.03±0.08%, respectively. The hemolysis rates of erythrocyte suspensions treated with MOF-919-GOx suspensions at concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL were 4.57±0.13%, 2.86±0.03%, 1.21±0.09%, 1.21±0.14%, and 1.00±0.04%, respectively. The results showed that the hemolysis rates of MOF-919 and MOF-919-GOx on canine red blood cells were both less than 5%, meeting the requirements of ISO international standards.

[0060] In summary, this technical solution successfully loaded grape oxidase onto MOF-919 using the glutaraldehyde loading method, with a loading capacity of 372.20 μg / mg. The cascade reaction of MOF-919-GOx was verified by methyl red and TMB colorimetric assays. The results showed that MOF-919-GOx could lower the environmental pH and generate reactive oxygen species in the presence of glucose, demonstrating its potential application in canine oral cavity. This further validated the antibacterial effect of the nanozyme.

[0061] Experimental Example 2 Verification of the Antibacterial Effect of MOF-919-GOx Based on the results of Experimental Example 1, further tests were conducted on the effects of MOF-919-GOx on P. gingivalis and S. aureus In this study, we conducted an in vitro antibacterial test to investigate the antibacterial effect of suspended bacteria and biofilms. We also prepared artificial canine saliva (pH = 8.5) to simulate the oral biofilm environment and verify whether MOF-919-GOx can function in the canine oral cavity. The details are as follows: Culture medium preparation: Preparation of BHI medium: Weigh 3.85 g of BHI powder and pour it into 100 mL of deionized water. Mix thoroughly and sterilize at 121°C for 20 min.

[0062] Preparation of 0.05% hemin: Weigh 50 mg of hemin, add 1 mL of 1 mol / L sodium hydroxide solution, and then add deionized water to make up to 100 mL. Mix well and filter sterilize with a 0.22 μm sterile syringe filter. Store at 4°C until used.

[0063] To prepare anaerobic BHI medium: Weigh 3.85 g of BHI powder into 100 mL of deionized water. Mix thoroughly. Boil the medium for 5 minutes to deoxygenate. Then, flow nitrogen through the medium for 40 minutes. Sterilize the medium by autoclaving at 121°C for 20 minutes. After cooling, add 1 mL of 0.05% hemin and 1 mL of 0.002% vitamin K1.

[0064] Preparation of artificial saliva for dogs: To 1 L of deionized water, add 1 g of beef extract powder, 2 g of yeast extract powder, 5 g of peptone, 2.5 g of porcine gastric mucin, 2.34 g of sodium chloride, 1.5 g of potassium chloride, and 0.1 g of calcium chloride. Adjust the pH to 8.5 with sodium hydroxide. Sterilize the solution in an autoclave at 120°C for 20 min. After sterilization, add 1.25 mL of 40% urea filtered through a 0.22 μm cellulose acetate membrane to obtain sterile canine artificial saliva (CAS).

[0065] Recovery and bacterial enrichment: Dissolved at room temperature P. gingivalis Use an inoculating loop to streak the dissolved bacterial solution onto BHI solid medium, then resuscitate in an anaerobic workstation at 37°C for 24 hours. After resuscitation, pick a colony and place it into BHI liquid medium, then incubate it in an anaerobic workstation at 37°C for 48 hours. S. aureus A similar method is used for resuscitation, except that an anaerobic environment is not required, and the resuscitation time is shortened to 12 h. Shaking culture is used for expansion culture.

[0066] Cultivating biofilms: 200 μL of bacterial solution was added to each well of a 96-well plate, and 3 replicate wells were set up for each group. P. gingivalis Place in an anaerobic workstation at 37°C for 48 h. S. aureus Culture in a 37°C incubator for 24 h.

[0067] 1. Verification of the antibacterial effect of MOF-919-GOx on suspended bacteria Take the bacterial solution and adjust the concentration to 10 using BHI medium. 6 CFU / mL, 50 μL of bacterial suspension, 50 μL of glucose solution of varying concentrations (25 mM, 50 mM, 75 mM, 100 mM), and 100 μL of MOF-919-GOx solution of varying concentrations (0.25, 0.5, 1, 2, and 4 mg / mL) were added to each well, resulting in a final MOF-919-GOx concentration of 0.125, 0.25, 0.5, 1, and 2 mg / mL. After incubation at 37°C for 5 min, the OD value was measured using a microplate reader, and the survival rate was calculated according to Equation 2:

[0068] Survival rate = The treated suspension P. gingivalis and S. aureus The survival rate results are shown in Figure 9 As shown, where A is P. gingivalis Survival rate; S. aureus Survival rate. It can be seen that when the concentration of MOF-919-GOx is higher, the survival rate of suspended bacteria is lower; when the concentration of glucose substrate is higher, the survival rate of bacteria is also lower. When the glucose concentration is 100 mM, the final concentration of MOF-919-GOx is 0.125 mg / mL, which can have a significant clearing effect on suspended bacteria; when the glucose concentration is 50 mM and 25 mM, the final concentration of MOF-919-GOx is 0.25 mg / mL to have a clearing effect on suspended bacteria, and the clearing effect of the 50 mM group is more obvious. In addition, by comparison, it can be found that under the same MOF-919-GOx concentration and glucose concentration conditions, MOF-919-GOx has a significant effect on suspended bacteria. P. gingivalis The antibacterial effect is stronger than S. aureus antibacterial effect.

[0069] 2. Crystal violet staining test to detect the antibacterial effect of MOF-919-GOx on biofilm MOF-919-GOx solutions with concentrations of 0.25, 0.5, 1, 2, and 4 mg / mL were prepared respectively. After confirming the formation of biofilm, they were divided into 5 groups and acted on the biofilm. Three replicate wells were set up in each group. 10 μL of artificial saliva, 10 μL of glucose solution and 20 μL of MOF-919-GOx solutions of different concentrations were added to each well to make the final concentration of the material 0.125, 0.25, 0.5, 1, and 2 mg / mL. The cells were incubated at 37°C for 5 minutes.

[0070] After incubation, remove the supernatant and rinse three times with sterile saline. Add 200 μL of 99% methanol solution and let stand at room temperature for 30 minutes. After discarding the methanol, place the biofilm in a fume hood for several minutes to dry. Then, stain with 0.1% crystal violet for 30 minutes. Rinse three times with sterile saline, dry again, and finally add 200 μL of 33% acetic acid. Measure absorbance at 490 nm.

[0071] The quantitative results after crystal violet staining test are shown in Figure 10 As shown, A: absorbance of P. gingivalis; B: absorbance of S. aureus; (*P < 0.05, **P < 0.01, ***P < 0.001). It can be seen that compared with the control group, the cells treated with the materials at the final concentrations of 0.25, 0.5, 1, and 2 mg / mL P. gingivalisThe biofilm was significantly reduced in all samples (P<0.001), and the absorbance decreased by 49.97±2.65%, 76.72±0.99%, 92.11±0.22%, and 96.73±0.29%, respectively. P. gingivalis Biofilm was significantly (P<0.05) reduced by 18.89±2.41%.

[0072] Compared with the control group, the cells treated with materials at final concentrations of 0.125, 0.25, 0.5, 1, and 2 mg / mL S. aureus biofilm, the survival rates were extremely significantly reduced (P<0.01), and the survival rates decreased by 17.75±4.40%, 38.25±2.80%, 71.16±0.61%, 86.81±1.15%, and 95.00±1.07%, respectively.

[0073] The results showed that MOF-919-GOx P. gingivalis and S. aureus Biofilm has obvious clearing effect and P. gingivalis The removal effect is better than S. aureus The cleaning effect.

[0074] 3. MTT test to detect the antibacterial effect of MOF-919-GOx on biofilm MOF-919-GOx solutions with concentrations of 0.25, 0.5, 1, 2, and 4 mg / mL were prepared respectively. After confirming the formation of biofilm, they were divided into 5 groups and acted on the biofilm. Three replicate wells were set up in each group. 10 μL of artificial saliva, 10 μL of glucose solution and 20 μL of MOF-919-GOx solutions of different concentrations were added to each well to make the final concentration of the material 0.125, 0.25, 0.5, 1, and 2 mg / mL. The cells were incubated at 37°C for 5 minutes.

[0075] After the incubation period, remove the 96-well plate, remove the liquid from the plate, and dry it in a fume hood. Add 100 μL of PBS and 5 μL of MTT solution to each well and incubate at 37°C for 3 h. After the incubation period, remove the plate, remove the supernatant, then add 100 μL of the lysis buffer and shake at low speed for 10 min. Measure the absorbance at 570 nm to calculate the bacterial survival rate.

[0076] The MTT test results of the two strains are shown in Figure 11 As shown, A: P. gingivalis survival rate; B: S. aureus survival rate; (*P < 0.05, **P < 0.01, ***P < 0.001). It can be seen that compared with the control group, the cells treated with the materials at the final concentrations of 0.25, 0.5, 1, and 2 mg / mL P. gingivalis The survival rates of biofilms were significantly reduced (P<0.001), with the survival rates reduced by 37.99±8.14%, 68.51±10.03%, 92.07±1.63%, and 96.86±1.25%, respectively. P. gingivalis No significant effect, see Figure 11 A in.

[0077] See also Figure 11 Compared with the control group, the material B at a final concentration of 0.125 mg / mL had a significant S. aureus No significant effect was observed; the material at a concentration of 0.25 mg / mL S. aureus The survival rate of the mice treated with the materials at the final concentrations of 0.5, 1, and 2 mg / mL decreased significantly (P<0.05) by 26.90±7.17%. S. aureus biofilm, the survival rates were extremely significantly reduced (P<0.001), with the survival rates reduced by 62.68±1.71%, 88.41±0.69%, and 95.23±0.97%, respectively.

[0078] 4. Gram staining microscopy to detect the antibacterial effect of MOF-919-GOx on biofilm Add bacterial solution and cell culture slides to the 12-well plate. P. gingivalis Anaerobic culture at 37°C for 48 h. S. aureus Incubate in a 37°C incubator for 24 hours. Remove the supernatant and wash with sterile PBS. Then, add 100 μL of artificial saliva, 100 μL of glucose solution, and 200 μL of MOF-919-GOx solutions of varying concentrations (0.5 and 4 mg / mL) to each well, giving final concentrations of 0.25 mg / mL and 2 mg / mL, respectively. Incubate at 37°C for 5 minutes. Remove the slides, dry them in a fume hood, and observe them under a microscope after Gram staining.

[0079] get P. gingivalis and S. aureus Gram staining results (100 times) are shown in Figure 12 、 13 As shown, A, B, and C are 0, 0.25, and 2 mg / mL MOF-919-GOx, respectively. It can be seen that after Gram staining, P. gingivalis Dyed red, S. aureusWhen the biofilm was treated with MOF-919-GOx at a final concentration of 0.25 mg / mL, the coverage of the biofilm was significantly reduced, and microscopic examination showed that most of the biofilm had been removed. When the biofilm was treated with MOF-919-GOx at a final concentration of 2 mg / mL, the stained part of the slide was even smaller, and microscopic observation showed that only a small amount of biofilm remained on the slide. It can be seen that low concentrations of MOF-919-GOx have an effect on the biofilm. P. gingivalis and S. aureus The biofilm with a lower concentration had a better removal effect, while higher concentrations of MOF-919-GOx removed it more thoroughly.

[0080] 5. Scanning electron microscopy detection of the antibacterial effect of MOF-919-GOx on biofilm Add bacterial solution and cell culture slides to the 12-well plate. P. gingivalis Anaerobic culture at 37°C for 48 h. S. aureus Incubate in a 37°C incubator for 24 h. Discard the supernatant, wash two to three times with sterile PBS, and add 100 μL of artificial saliva, 100 μL of glucose solution, and 200 μL of different concentrations of MOF-919-GOx (0.5 and 4 mg / mL) to each well, giving final concentrations of 0.25 and 2 mg / mL, respectively. Incubate at 37°C for 5 min. Wash with sterile PBS and fix with 2 mL of 2.5% glutaraldehyde for 12 h. Wash twice with sterile PBS and sterile deionized water, respectively. Dehydrate in ethanol for 10 min in 30%, 50%, and 70% ethanol, followed by 15 min each in 80% and 90% ethanol, and finally with anhydrous ethanol for 20 min. After air drying at room temperature, the biofilms were treated with a gold plater to enhance conductivity. Scanning electron microscopy was used to observe the biofilms.

[0081] get P. gingivalis and S. aureus The scanning electron microscopy results are shown in Figure 14 、 15 As shown, A (2000 times), D (5000 times): 0 mg / mL MOF-919-GOx; B (2000 times), E (5000 times): 0.25 mg / mL MOF-919-GOx; C (2000 times), F (5000 times): 2.0 mg / mL MOF-919-GOx. P. gingivalisThe cells appeared as short rods of varying lengths. Compared to the control group, the bacterial count in the low-concentration group (0.25 mg / mL) decreased by 46.09±4.94%, while the high-concentration group decreased by 98.17±0.85%. Furthermore, the bacteria were spherical. In the untreated control group, the biofilm densely covered the slide, with many overlapping bacteria. However, the cells on the slide treated with 0.25 mg / mL MOF-919-GOx decreased by 55.78±8.57%, leaving only a single layer of biofilm, no longer overlapping, and with individual cells visible. In the high-concentration group, the cell count decreased by 88.77±2.27%, with more individual, scattered cells.

[0082] The above results show that MOF-919-GOx has a good ability to remove biofilms. The effect can be seen at low concentrations, and the effect is better after treatment with higher concentrations of MOF-919-GOx. Through qualitative and quantitative tests, it is proved that MOF-919-GOx can remove biofilms. P. gingivalis and S. aureus It has antibacterial and biofilm-removing effects. When the concentration is 0.25 mg / mL and 25 mM glucose is added exogenously, it can exert a significant antibacterial effect, and this effect is concentration-dependent.

[0083] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A cascade catalytic enzyme MOF-919-GOx, characterized in that Glucose oxidase GOx was loaded onto the nanozyme MOF-919 by glutaraldehyde loading method.

2. The cascade catalytic enzyme MOF-919-GOx according to claim 1 is effective in inhibiting Porphyromonas gingivalis P. gingivalis and Staphylococcus aureus S. aureus Application in.

3. Porphyromonas gingivalis prepared by the cascade catalytic enzyme MOF-919-GOx according to claim 1 P. gingivalis and Staphylococcus aureus S. aureus Antibacterial materials.

4. Use of the cascade catalytic enzyme MOF-919-GOx according to claim 1 or the antibacterial material according to claim 3 in the field of veterinary clinical practice.

5. The use according to claim 4, characterized in that Contains products for inhibiting and eliminating oral bacteria and bacterial biofilm formation in dogs.

6. The use according to claim 5, characterized in that The bacteria include Porphyromonas gingivalis P. gingivalis and Staphylococcus aureus S. aureus .

7. The method for preparing the cascade catalytic enzyme MOF-919-GOx according to claim 1, wherein: Nanozyme MOF-919 was dispersed in deionized water, glucose oxidase GOx and 50% glutaraldehyde were added, stirred and centrifuged to obtain MOF-919-GOx.

8. The preparation method according to claim 7, characterized in that The mass ratio of the nanozyme MOF-919 to glucose oxidase GOx is 1:1.

5.

9. The preparation method according to claim 7, characterized in that The ratio of the amount of 50% glutaraldehyde to the nanozyme MOF-919 is 1 mL / 20 mg.

10. The preparation method according to claim 7, characterized in that The stirring is carried out at room temperature in the dark for 1 hour.

Images