Preparation method of Cu-TA-CeO2 nano-enzyme antibacterial agent

Abstract

The invention relates to a preparation method of a Cu-TA at CeO2 nano-enzyme antibacterial agent. The preparation method comprises the steps of preparation of CeO2, preparation of a Cu-TA nanosheet and preparation of the Cu-TA at CeO2 nano-enzyme antibacterial agent. Due to the fact that natural enzyme is prone to inactivation and poor in environmental adaptability, in order to solve the problem, simple hydrothermal reaction is adopted for synthesizing Cu-TA at CeO2 nano-particles, copper ions, tannic acid and cerium oxide are compounded to form artificial nano-enzyme, and the nano-enzyme is good in environmental adaptability, not prone to inactivation and good in stability. Meanwhile, under the catalytic action of hydrogen peroxide, peroxidase activity and a remarkable antibacterial effect are shown, and the compound can be better applied to the antibacterial field and the like and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme antibacterial materials technology, and relates to a method for preparing Cu-TA@CeO2 nanoenzyme antibacterial agent. Background Technology

[0002] Bacterial infections pose a serious threat to public health, causing millions of deaths each year. Antibiotic treatment remains the primary approach; however, its overuse has led to the emergence of multidrug-resistant superbugs, endangering both public health and the environment. Inspired by natural enzymes, researchers have developed nanozymes. These novel materials possess catalytic properties similar to natural enzymes, along with advantages such as low cost, high stability, durability, and tunable size. These properties make nanozymes a promising approach to combating bacterial infections, particularly in addressing the challenges posed by antibiotic resistance.

[0003] The surface and composition of nanozymes are crucial for antibacterial efficacy, and bacterial binding and antibacterial activity can be enhanced by modulating the surface and composition of nanozymes. On the one hand, surface modification of nanozymes can achieve bacterial binding and targeting, thereby improving the antibacterial properties of nanozymes, including biochemical recognition, surface charge, and surface morphology. On the other hand, enhanced antibacterial properties can be achieved by modulating the composition of nanozymes, including synergistic effects mediated by single nanozymes and cascade catalytic antibacterial applications mediated by multiple nanozymes.

[0004] Tannic acid (TA) is a natural phenolic compound extracted from plants. It possesses advantages such as non-toxicity, biocompatibility, antioxidant properties, and antibacterial activity. Tannic acid has a strong ability to complex with metal ions. Cerium oxide is a rare earth oxide; cerium ions undergo a +3 to +4 valence transition under specific conditions, thus exhibiting extremely high catalytic activity. However, tannic acid is easily oxidized in air and has weak antibacterial effects, resulting in low nanozyme activity. Therefore, complexing tannic acid with metal ions and metal oxides can enhance its nanozyme activity and antibacterial properties.

[0005] In this study, a Cu-TA@CeO2 nanozyme antibacterial agent was synthesized via a simple heating reaction. Catalyzed by hydrogen peroxide, the prepared antibacterial agent exhibited not only significantly enhanced peroxidase activity but also a remarkable antibacterial effect, thus demonstrating its potential application in the antibacterial field. Summary of the Invention

[0006] To address at least one of the aforementioned technical problems, this invention proposes a method for preparing Cu-TA@CeO2 nanozyme antibacterial agent.

[0007] One technical solution adopted by this invention to solve its technical problem is: a method for preparing Cu-TA@CeO2 nanoenzyme antibacterial agent, comprising the following steps: (1) Preparation of CeO2 powder: A certain amount of cerium nitrate hexahydrate was dissolved in deionized water, the solution was transferred to a reaction vessel and sealed, and heated to react. After the solution was cooled to room temperature, it was repeatedly centrifuged and washed with deionized water and anhydrous ethanol, and then dried to obtain CeO2 powder.

[0008] (2) Preparation of Cu-TA nanosheets: A certain amount of copper sulfate pentahydrate and tannic acid were added to deionized water and stirred evenly. After the mixture was evenly dispersed, sodium hydroxide solution was added to adjust the pH. After heating the reaction for 4 h, Cu-TA nanosheets were obtained by repeated centrifugation and washing with deionized water and anhydrous ethanol and then dried. (3) Preparation of Cu-TA@CeO2 nanoenzyme antibacterial agent: A certain amount of cerium oxide and tannic acid were dissolved in deionized water, ultrasonically dispersed evenly, a certain amount of copper sulfate pentahydrate was added, heated to react for 4 hours, centrifuged to collect the product, and dried to obtain Cu-TA@CeO2 powder.

[0009] Furthermore, the mass of cerium nitrate hexahydrate used in step (1) above is 1.085 g.

[0010] Furthermore, in step (1) above, the heating temperature is 220℃ and the reaction time is 12 h.

[0011] Furthermore, in step (2) above, the amounts of copper sulfate pentahydrate and tannic acid used are 0.875 g and 0.027 g, respectively.

[0012] Furthermore, the concentration of the sodium hydroxide solution in step (2) above is 1 M.

[0013] Furthermore, in step (2) above, the solution pH is adjusted to 7.4.

[0014] Furthermore, in step (3) above, the amounts of cerium oxide, copper sulfate pentahydrate and tannic acid are 0.5 g, 0.875 g and 0.027 g, respectively.

[0015] Furthermore, the drying temperature in steps (1) and (2) above is 60°C.

[0016] Furthermore, in steps (1), (2) and (3) above, the centrifugation speed is 8000 rpm and the centrifugation time is 5 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This preparation method yields Cu-TA@CeO2 nanozyme antibacterial agent through a simple heating reaction, which is easy to control. Compared to natural enzymes, this nanozyme is less prone to inactivation and possesses peroxidase activity, exhibiting excellent antibacterial effects under the catalysis of hydrogen peroxide. For future large-scale applications, it has lower production requirements, lower costs, and is more economical and controllable. Attached Figure Description

[0018] Figure 1 The X-ray diffraction patterns of CeO2 powder, Cu-TA nanosheets, and Cu-TA@CeO2 nanozymes prepared in this invention are shown below. Figure 2 These are peroxidase performance test graphs of CeO2 powder, Cu-TA nanosheets, and Cu-TA@CeO2 nanozymes prepared in this invention; Figure 3 This is a graph showing the effect of the concentration of Cu-TA@CeO2 nanozyme prepared in this invention on peroxidase activity. Figure 4 This is a graph showing the effect of pH on peroxidase activity of the Cu-TA@CeO2 nanozyme prepared in this invention. Figure 5 This is a diagram showing the antibacterial effect of the Cu-TA@CeO2 nanozyme antibacterial agent prepared according to the present invention. Detailed Implementation

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0020] The following is in conjunction with the attached diagram. Figures 1-5 The present invention will be described in detail with reference to specific embodiments. However, the embodiments of the present invention are not limited to the following embodiments. Example 1

[0021] This example provides a method for preparing Cu-TA@CeO2 nanozyme antibacterial agent. Specifically, it includes the following steps: (1) Preparation of CeO2 powder: 1.085 g of cerium nitrate hexahydrate was dissolved in 80 mL of deionized water and stirred for 30 min to disperse evenly. The solution was transferred to a reaction vessel and sealed. The reaction was heated at 220℃ for 12 h. After the solution cooled to room temperature, it was repeatedly washed by centrifugation with deionized water and anhydrous ethanol. The centrifugation speed was 8000 rpm and the centrifugation time was 5 min. After drying in a vacuum oven at 60℃, CeO2 powder was obtained.

[0022] (2) Preparation of Cu-TA nanosheets: 0.027 g of tannic acid was added to 100 mL of deionized water and sonicated for 10 min to disperse evenly. After even dispersion, 0.875 g of copper sulfate pentahydrate was added and sonicated for another 10 min. Then, the pH was adjusted to 7.4 with 1 M sodium hydroxide solution and reacted in an oil bath at 70 °C for 4 h. The nanosheets were washed repeatedly by centrifugation with deionized water and anhydrous ethanol at a speed of 8000 rpm for 5 min. After drying in a vacuum oven at 60 °C, Cu-TA nanosheets were obtained.

[0023] (3) Preparation of Cu-TA@CeO2 nanoenzyme antibacterial agent: 0.5 g of cerium oxide and 0.027 g of tannic acid were dissolved in 100 mL of deionized water and ultrasonically dispersed for 10 min. After uniform dispersion, 0.875 g of copper sulfate pentahydrate was added and ultrasonically dispersed for another 10 min. Then, the pH was adjusted to 7.4 with 1 M sodium hydroxide solution and reacted in an oil bath at 70 °C for 4 h. The mixture was washed repeatedly by centrifugation with deionized water and anhydrous ethanol at a speed of 8000 rpm for 5 min. After drying in a vacuum oven at 60 °C, Cu-TA@CeO2 powder was obtained.

[0024] The crystal structure of the CeO2, Cu-TA, and Cu-TA@CeO2 powders obtained in Example 1 was analyzed using a Rigaku D / max2500PC rotating X-ray diffractometer (Japan). The X-ray target was Cu Kα (λ = 1.54056 Å), voltage was 40 kV, current was 100 mA, step size was 0.02°, and scanning range was 5°–80°. The X-ray diffraction patterns are shown below. Figure 1 As shown, the peaks of the prepared CeO2 conform to the standard peaks, and the XRD pattern of Cu-TA nanosheets shows many sharp peaks, indicating that Cu-TA nanosheets have a crystalline structure. In the Cu-TA@CeO2 powder obtained by combining the two, peaks of CeO2 and peaks of Cu-TA nanosheets appear, indicating that Cu-TA@CeO2 powder was successfully prepared. Example 2

[0025] This example provides a peroxidase performance test of the prepared Cu-TA@CeO2 nanozyme.

[0026] In this embodiment, the peroxidase performance of CeO2, Cu-TA, and Cu-TA@CeO2 powders prepared in Example 1 was mainly assessed by measuring their colorimetric reaction with tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) and their ultraviolet absorption at 652 nm. The effects of Cu-TA@CeO2 composite sample concentration and solution pH on their peroxidase activity were also investigated.

[0027] (1) Prepare a 40 mM tetramethylbenzidine (TMB) solution by dissolving 48.1 mg of TMB in 5 mL of dimethyl sulfoxide (DMSO) and dispersing it evenly by ultrasonication. (2) Prepare a 1 M hydrogen peroxide (H2O2) solution by adding 511 μL of 30% hydrogen peroxide to 4.489 mL of deionized water and mixing thoroughly. (3) Prepare an acetate-sodium acetate buffer solution with pH 4. Dissolve 0.123 g of anhydrous sodium acetate and 0.49 mL of glacial acetic acid in 80 mL of deionized water, mix well, and then bring the volume to 100 mL.

[0028] (4) Take 50 μL of 40 mM TMB solution, 50 μL of 1 M hydrogen peroxide solution, and 200 μL of 2 mg / mL CeO2, Cu-TA, and Cu-TA@CeO2 powder sample solutions respectively. After mixing, add 3 ml of acetate-sodium acetate buffer solution with pH 4. React in a water bath at 37℃ for 30 min. Set up four experimental groups: H2O2+TMB, H2O2+TMB+CeO2, H2O2+TMB+Cu-TA, and H2O2+TMB+Cu-TA@CeO2.

[0029] (5) Take 50 μL of 40 mM TMB solution, 50 μL of 1 M hydrogen peroxide solution, and different amounts of 2 mg / mL Cu-TA@CeO2 powder sample solution, mix them, and then add 3 ml of acetate-sodium acetate buffer solution with pH 4. React in a water bath at 37℃ for 30 min to obtain five experimental groups of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL and 250 μg / mL.

[0030] (6) Take 50 μL of 40 mM TMB solution, 50 μL of 1 M hydrogen peroxide solution, and 200 μL of 2 mg / mL Cu-TA@CeO2 powder sample solution, and then add 3 ml of acetate-sodium acetate buffer solution with pH 3, 4, 6, 8 and 10 respectively. React in a water bath at 37℃ for 30 min to obtain five experimental groups.

[0031] The test results of step (4) in Example 3 are as follows: Figure 2 As shown, compared with H2O2+TMB and H2O2+TMB+CeO2, the UV absorption peak of H2O2+TMB is relatively weak, indicating that its peroxidase activity is relatively low. Compared with H2O2+TMB and H2O2+TMB+TA-Cu, the absorption peak of H2O2+TMB+Cu-TA@CeO2 is also relatively weak. Compared with other groups, the absorption peak of H2O2+TMB+Cu-TA@CeO2 is significantly enhanced, indicating that the peroxidase activity of Cu-TA@CeO2 formed by the two is significantly improved. This is because Cu-TA@CeO2 can generate hydroxyl radicals under the catalysis of hydrogen peroxide, which oxidizes TMB and results in a higher UV absorption peak at 652 nm.

[0032] The test results of step (5) in Example 4 are as follows: Figure 3 As shown, the absorbance at 652 nm increases with the increase of Cu-TA@CeO2 sample concentration, indicating that the peroxidase activity of Cu-TA@CeO2 increases with the increase of sample concentration.

[0033] The test results of step (6) in Example 4 are as follows: Figure 3 As shown, the activity of Cu-TA@CeO2 peroxidase is pH dependent, with the highest activity at pH 4. Example 3

[0034] This example provides an in vitro antibacterial test of the prepared Cu-TA@CeO2 nanozyme antibacterial agent.

[0035] (1) Prepare culture medium. Prepare liquid culture medium and solid plate culture medium according to GB / T20944.1-2007 standard. Weigh 3.6 g of nutrient broth and 3.0 g of agar powder and dissolve them in 200 ml of distilled water to prepare solid culture medium. The liquid culture medium is the same as the above formula, but without adding agar powder. Autoclave at 121 ℃ for 30 min to form the original sterile culture medium.

[0036] (2) Preparation of bacterial suspension: Take out the frozen solution of Staphylococcus aureus and Escherichia coli, and use an inoculation loop to streak rice-grain-sized amounts into the prepared solid culture medium. Incubate at 37 ℃ for 24 h. Take the bacteria from the culture medium and inoculate them into liquid culture medium. Dilute the bacterial strain to 10⁻⁶ oz. 8 10 7 10 6 In liquid culture medium, the bacterial concentration was calculated using absorbance (OD 600 nm) to prepare a culture containing 1.0 × 10⁻⁶ bacteria. 6 CFU / ml bacterial suspension.

[0037] (3) The culture medium and bacterial suspension prepared in steps (1) and (2) were mixed with the Cu-TA@CeO2 prepared in Example 1. Blank groups, H2O2 groups, Cu-TA@CeO2 groups, and Cu-TA@CeO2 + H2O2 groups for Escherichia coli and Staphylococcus aureus were set up so that the concentration of the sample solution was 500 μg / ml and the bacterial concentration was 1.0 × 10⁻⁶. 6 The concentration of CFU / ml and H2O2 was 100mM. Then, 20 μL of the solution was taken and spread on a solid plate culture medium and incubated at 37°C for 24 h.

[0038] The in vitro antibacterial test results of Example 3 are as follows: Figure 4 As shown, the H2O2 group had no antibacterial effect compared to the blank group; the Cu-TA@CeO2 group showed a relatively good antibacterial effect, and after the addition of H2O2, it showed a significant antibacterial effect with an antibacterial rate of over 99%.

[0039] The above provides a detailed description of the purification method for eplerenone methyl ester provided by the present invention. Specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a Cu-TA@CeO2 nanozyme antibacterial agent, characterized in that: Comprising the following steps: Step (1) Preparation of CeO2 powder: A certain amount of cerium nitrate hexahydrate is dissolved in deionized water, sealed in a reaction kettle, heated to react at 220°C, repeatedly centrifuged and washed, and dried to obtain CeO2 powder; Step (2) Preparation of Cu-TA nanosheet: Copper sulfate pentahydrate and tannic acid are added to deionized water and stirred uniformly, then 1 M sodium hydroxide solution is added to adjust the pH, heated to react for 4 h, repeatedly centrifuged and washed with deionized water and anhydrous ethanol, and dried to obtain Cu-TA nanosheet; Step (3) Preparation of Cu-TA@CeO2 nanoscale enzyme antibacterial agent: A certain amount of cerium oxide and tannic acid are dissolved in deionized water, ultrasonically dispersed uniformly, then a certain amount of copper sulfate pentahydrate is added, heated to react for 4 h, the product is collected by centrifugation, and dried to obtain Cu-TA@CeO2 powder.

2. The preparation method of the Cu-TA@CeO2 nanoscale enzyme antibacterial agent according to claim 1, characterized in that: The concentration of cerium nitrate hexahydrate in step (1) is 31.2 mmol / L.

3. The preparation method of the Cu-TA@CeO2 nanoscale enzyme antibacterial agent according to claim 1, characterized in that: The amounts of copper sulfate pentahydrate and tannic acid used in step (2) are 0.875 g and 0.027 g, respectively.

4. The preparation method of the Cu-TA@CeO2 nanoscale enzyme antibacterial agent according to claim 1, characterized by: The amounts of cerium oxide, copper sulfate pentahydrate and tannic acid used in step (3) are 0.5 g, 0.875 g and 0.027 g, respectively.

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