Iron / copper-zinc peroxide nano-enzyme with self-supply hydrogen peroxide and double-enzyme activity as well as preparation method and application of iron / copper-zinc peroxide nano-enzyme

Abstract

The invention discloses an iron / copper-zinc peroxide nano-enzyme with hydrogen peroxide self-supply and double-enzyme activity, a preparation method and application, and belongs to the field of nano-enzyme preparation, the preparation method specifically comprises the following steps: dissolving a cupric salt to obtain a mother solution; the preparation method comprises the following steps: dispersing ZnO2 and ferrous salt to obtain a mixed solution; the mother liquor and the mixed liquor are evenly dispersed, the mass ratio of the bivalent copper salt to the ZnO2 to the bivalent iron salt is (6-8): (8-12): (2-3), then the obtained sediment is washed and dried, and the iron / copper-zinc peroxide nano-enzyme with the self-supply hydrogen peroxide and double-enzyme activity is obtained. The catalyst can be used as a donor of hydrogen peroxide to continuously catalyze to generate ROS; by utilizing high-frequency ultrasonic treatment of ultrasonic waves, the hydrogen peroxide self-supply capacity, peroxidase-like activity and glutathione-like activity of the Fe / Cu-ZnO2 nano-enzyme are enhanced, a large amount of ROS (reactive oxygen species) is explosively generated, and intractable bacterial infection is efficiently treated.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme preparation, specifically an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity, its preparation method, and its application. Background Technology

[0002] Bacterial infections are a serious health problem, and antibiotics are currently the most effective treatment for them in clinical practice. However, due to the overuse of antibiotics, bacteria have developed multidrug resistance, leading to poor clinical efficacy of traditional antibiotics. Currently, drug-resistant bacterial infections have a serious negative impact on public health. The development of new antibiotics is a solution to these superbugs. However, in recent years, very few new antibiotics have been developed, and bacteria often develop new resistance to them shortly after their clinical application. Given the poor efficacy of clinical antibiotic treatment and the difficulties in developing new antibiotics, the search for new antibacterial therapies is particularly urgent.

[0003] Reactive oxygen species (ROS) act as strong oxidants, killing bacteria by disrupting their cell membranes, inactivating proteins, and breaking DNA, thus exhibiting broad-spectrum antibacterial activity. Nanozymes, as one of many ROS-generating nanomaterials, have attracted widespread attention due to their stability, low cost, good biocompatibility, and multifunctionality. However, their low catalytic activity significantly limits their antibacterial efficiency, thereby restricting their clinical antibacterial applications. The catalytic activity of nanozymes is tunable. Nanozyme materials possess unique photophysical and photochemical properties, such as tunable band gaps, diverse absorption spectra, and localized surface plasmon resonances, providing new directions for the rational regulation of nanozymes.

[0004] Photoresponsive nanozymes are photoactive nanozymes that combine the optical properties of nanozymes with enzyme-like activity to create a series of photocatalytic nanoplatforms. For example, using the optical properties of nanomaterials to enhance the catalytic activity of nanozymes can form photo-enhanced catalytic systems. Therefore, the introduction of the optical properties of nanomaterials will expand the scope of nanozymes at the optical level. However, the tissue penetration depth of light is relatively shallow (<1 cm), limiting its effectiveness for deep diseases. Compared with photoinduced therapy, which is mainly limited to skin lesions, ultrasound (US), as a mechanical wave, has superior tissue penetration mediated by ultrasound waves, penetrating to about 10 cm from the skin, thus showing great feasibility in the treatment of deep infections. Currently, there are several cases of US combined with nanozymes to enhance nanozyme activity for the treatment of bacterial infections, but its catalytic capacity needs further improvement. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity, its preparation method, and its application, in order to solve the problem that the catalytic activity of current nanozymes needs to be improved.

[0006] This invention is achieved through the following technical solution: A method for preparing Fe / Cu-ZnO2 nanozymes with self-supplying hydrogen peroxide and dual enzyme activity specifically includes the following steps: S1, dissolve the divalent copper salt to obtain the mother liquor; disperse ZnO2 and divalent iron salt to obtain the mixed liquor; S2, the mother liquor and the mixed liquor are dispersed evenly, and the mass ratio of divalent copper salt, ZnO2 and divalent iron salt is (6~8):(8~12):(2~3). The resulting precipitate is then washed and dried to obtain an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity.

[0007] A further improvement of the present invention is that: The divalent copper salt mentioned in S1 is CuCl2·2H2O, and the divalent iron salt is FeCl2·4H2O.

[0008] In S1, CuCl2·2H2O is dissolved in anhydrous ethanol to obtain the mother liquor; ZnO2 and FeCl2·4H2O are dispersed in anhydrous ethanol to obtain the mixed liquor.

[0009] S1 dissolves 0.06~0.08 g of CuCl2·2H2O in 20~30 mL of anhydrous ethanol to obtain the mother liquor.

[0010] S1 First, ZnO2 is dispersed in anhydrous ethanol, and then FeCl2·4H2O is added under stirring. The mixture is stirred until clear to obtain a mixed solution. The ratio of ZnO2 to anhydrous ethanol is 80~120 mg: 60~80 mL.

[0011] S2: The mother liquor and the mixture are stirred continuously for 1.5 to 2.5 hours, and then the resulting precipitate is washed and dried.

[0012] S2 The obtained precipitate was washed 3-5 times with anhydrous ethanol and then dried at 45-55℃ for 10-14h to obtain an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity.

[0013] An iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity, obtained by any one of the above methods, wherein the dual enzyme activity is peroxidase-like activity and glutathionease-like activity.

[0014] The application of an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity in antibacterial and antimicrobial applications was investigated. After being brought to volume with the appropriate buffer solution, high-frequency sonication was initiated, with ultrasonic power density and duration ranging from 0.5 to 1.5 W / cm². -2 5~15 min.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a method for preparing Fe / Cu-ZnO2 nanozymes with self-supplying hydrogen peroxide and dual-enzyme activity. The Fe / Cu-ZnO2 nanozymes possess both the unique properties of nanomaterials and the dual-enzyme activity of peroxidase and glutathione peroxidase. Under slightly acidic conditions (pH 5.5), the Fe / Cu-ZnO2 nanozymes can decompose to produce metal ions Fe... 3+ Cu 2+ And H2O2, producing Fe 3+ Cu 2+ Not only promote Zn + Convert to Zn 2+ And can be with Zn 2+ Together, they can consume glutathione (GSH) and be reduced to Fe. 2+ Cu + Zn + Simultaneously, a large number of metal ions act as Fenton's reagent, accelerating the decomposition of H2O2 to form a large number of cytotoxic hydroxyl radicals (•OH), thereby achieving a strong antibacterial effect. Compared with other nanozymes that rely on the addition of exogenous hydrogen peroxide, ZnO2 in Fe / Cu-ZnO2 nanozymes can act as a hydrogen peroxide donor, continuously catalyzing the generation of ROS. High-frequency ultrasonic treatment of the final volume mixture is performed, generating cavitation effect in the liquid environment. The cavitation effect generates free radicals. High temperature, high heat, and high-speed shear force enhance the self-supplying hydrogen peroxide capacity, peroxidase-like activity, and glutathionease-like activity of Fe / Cu-ZnO2 nanozymes, resulting in an explosive generation of a large amount of ROS, effectively treating stubborn bacterial infections. Attached Figure Description

[0016] Figure 1 This is the transmission image of FC-Z obtained in Example 1 of the present invention at 100 nm.

[0017] Figure 2a The image shows the energy-dispersive X-ray spectrum of FC-Z obtained in Example 1 of this invention.

[0018] Figure 2b The image shows the EDS diagram of Zn at 100 nm for FC-Z obtained in Example 1 of this invention.

[0019] Figure 2cThis is the EDS diagram of Fe at 100 nm obtained by FC-Z in Example 1 of the present invention.

[0020] Figure 2d This is the EDS image of Cu at 100 nm for FC-Z obtained in Example 1 of the present invention.

[0021] Figure 2e This is the EDS image of FC-Z at 100 nm obtained in Example 1 of the present invention.

[0022] Figure 3a The XRD patterns of FC-Z and ZnO2 obtained in Example 1 of this invention are shown.

[0023] Figure 3b The XPS spectrum of FC-Z obtained in Example 1 of this invention is shown.

[0024] Figure 3c The O 1s XPS spectrum of FC-Z obtained in Example 1 of this invention is shown.

[0025] Figure 3d The image shows the Fe 2p XPS spectrum of FC-Z obtained in Example 1 of this invention.

[0026] Figure 3e The Cu 2p XPS spectrum of FC-Z obtained in Example 1 of this invention is shown.

[0027] Figure 3f The Zn 2p XPS spectrum of FC-Z obtained in Example 1 of this invention is shown.

[0028] Figure 4a This is a test diagram of hydrogen peroxide self-supply for US, FC-Z, and FC-Z+US as described in this invention.

[0029] Figure 4b This is a peroxidase-like activity test diagram of US, FC-Z, and FC-Z+US as described in this invention.

[0030] Figure 4c The graphs show the GSH-like enzyme activity assays for US, FC-Z, and FC-Z+US as described in this invention, where the lowercase letters a, b, c, and d above each data group represent significant differences.

[0031] Figure 5a This is a diagram showing the effect of ultrasonic power on hydrogen peroxide generation as described in this invention.

[0032] Figure 5b This is a graph showing the effect of ultrasonic treatment time on hydrogen peroxide generation as described in this invention.

[0033] Figure 5c This is a graph showing the effect of ultrasonic power on peroxidase-like activity as described in this invention.

[0034] Figure 5d This diagram illustrates the effect of ultrasonic treatment on peroxidase-like activity as described in this invention.

[0035] Figure 5e This is a graph showing the effect of ultrasonic power on GSH-like enzyme activity as described in this invention.

[0036] Figure 5f This is a graph showing the effect of ultrasonic treatment time on GSH-like enzyme activity as described in this invention.

[0037] Figure 6a This diagram illustrates the inhibition of Escherichia coli by different treatment groups described in this invention.

[0038] Figure 6b This diagram illustrates the inhibition of Staphylococcus aureus in different treatment groups as described in this invention.

[0039] Figure 6c The survival rate of Escherichia coli described in this invention under different treatment groups is given, where the lowercase letters a, b, c, and d above the data for each group represent significant differences.

[0040] Figure 6d The data represents the survival rate of Staphylococcus aureus as described in this invention under different treatment groups, where the lowercase letters a, b, c, and d above the data for each group represent significant differences.

[0041] Figure 7a The data represent the biofilm inhibition of Escherichia coli after FC-Z treatment according to the present invention, where the lowercase letters a, b, c, and d above each group of data represent significant differences.

[0042] Figure 7b The data above each group of data, represented by the lowercase letters a, b, c, and d, indicate significant differences in biofilm inhibition of Staphylococcus aureus after FC-Z treatment according to the present invention. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0044] This invention discloses a method for preparing Fe / Cu-ZnO2 nanozymes with ultrasound-enhanced activity and their antibacterial applications. The preparation method of the Fe / Cu-ZnO2 nanozymes specifically includes the following steps: Step 1. Preparation of ZnO2: Under stirring conditions, 0.809 g of Zn(NO3)2·6H2O and 1.4 g of polyvinylpyrrolidone (PVP) were dissolved in 60 mL of anhydrous ethanol. After stirring evenly on a magnetic stirrer, 500 μL of 15% ammonia solution was added to the above solution, followed immediately by 800 μL of H2O2 aqueous solution (1 M). After stirring for 30 min, the reaction solution was centrifuged (10000 rpm, 20 min). The precipitate was washed three times with ethanol to remove excess reactants and dried in an oven at 50 °C for later use.

[0045] Step 2. Preparation of Fe / Cu-ZnO2: First, dissolve 0.06–0.08 g of CuCl₂·2H₂O in 20–30 mL of anhydrous ethanol to prepare a mother liquor for later use. Then, disperse 80–120 mg of ZnO₂ in 60–80 mL of deionized water, and add 0.02–0.03 g of FeCl₂·4H₂O under magnetic stirring until the mixture becomes clear. Next, add the prepared mother liquor to the mixture and continue stirring for 1.5–2.5 h. Collect the precipitate by centrifugation, wash 3–5 times with anhydrous ethanol, and dry in an oven at 45–55 °C for 10–14 h for later use. Example 1 A method for preparing Fe / Cu-ZnO2 nanozymes with self-supplying hydrogen peroxide and dual enzyme activity specifically includes the following steps: Step 1. Preparation of ZnO2: Under stirring conditions, 0.809 g of Zn(NO3)2·6H2O and 1.4 g of polyvinylpyrrolidone (PVP) were dissolved in 60 mL of anhydrous ethanol. After stirring evenly on a magnetic stirrer, 500 μL of 15% ammonia solution was added to the above solution, followed immediately by 800 μL of H2O2 aqueous solution (1 M) to the above mixture. After stirring for 30 min, the reaction solution was centrifuged (10000 rpm, 20 min), and the precipitate was washed three times with ethanol and dried in an oven at 50 °C for later use.

[0046] Step 2. Preparation of Fe / Cu-ZnO2: First, 0.075 g of CuCl2·2H2O was dissolved in 25 mL of anhydrous ethanol to prepare a mother liquor for later use. Then, 100 mg of ZnO2 was dispersed in 75 mL of deionized water, and 0.025 g of FeCl2·4H2O was added under magnetic stirring until the mixture became clear. The prepared mother liquor was then added to the mixture, and stirring was continued for 2 h. The mixture was collected by centrifugation, washed three times with anhydrous ethanol, and dried in a 50°C oven for 12 h for later use. Step 3. The morphology of the prepared Fe / Cu-ZnO2 (FC-Z) was characterized using TEM. For example... Figure 1 As shown, FC-Z exhibits a regular spherical particle structure with a diameter of approximately 25 nm. Elemental mapping analysis ( Figure 2b , Figure 2c , Figure 2d and Figure 2e It can be seen that the synthesized nanomaterials contain four elements: Zn, Fe, Cu, and O, which corresponds to energy-dispersive X-ray spectroscopy (EDS). Figure 2a The results are consistent with those in ().

[0047] By comparing and analyzing XRD patterns ( Figure 3a ) and using XPS to analyze the composition and valence state of the synthesized material ( Figure 3b , Figure 3c Analysis showed that Fe / Cu-ZnO2 was successfully prepared, and based on... Figure 3d , Figure 3e and Figure 3f The material contains mixed valence states (Cu) + / Cu 2 + Fe 2+ / Fe 3+ Zn 2+ ).

[0048] Step 4. Hydrogen peroxide self-supply and dual-enzyme activity of Fe / Cu-ZnO2 nanozymes ADHP, as a specific fluorescent probe for hydrogen peroxide, reacts with H2O2 to generate a substance with a fluorescent signal (excitation wavelength: 571 nm, emission wavelength: 585 nm). The H2O2 generated by FC-Z was measured using this fluorescent probe. Experimental procedure: Take a 5 mL plastic centrifuge tube, then add 1 mL of NaAc-HAc buffer (pH 5.5) and FC-Z (250 μg / mL) sequentially, and finally adjust the volume to 2 mL with buffer. Incubate at 37℃ for 30 min. Take 100 μL and 100 μL of ADHP (4 mM) of the reaction solution after different treatments, incubate at room temperature for 5 min, and measure the emission wavelength of the projection solution at 585 nm. Figure 4aAs shown, compared with the blank control group (C), the composite material FC-Z can react with ADHP (10-acetyl-3,7-dihydroxyphenazine) under slightly acidic conditions (pH 5.5). FC-Z has a fluorescence signal at 585 nm, indicating that the reaction system produces hydrogen peroxide, which proves that FC-Z has the ability to supply hydrogen peroxide on its own.

[0049] To determine the peroxidase-like activity characteristics of FC-Z, this experiment used a TMB (3,3',5,5'-tetramethylbenzidine) colorimetric assay (which also involved buffer dilution). Figure 4b As shown, at pH 5.5, FC-Z catalyzes the TMB reaction, resulting in a blue solution with an absorption peak at 652 nm, indicating that FC-Z possesses peroxidase-like activity. DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) reacts with the thiol group of GSH to generate yellow 5-thio-2-nitrobenzoic acid (also after buffer reconstitution). This product has a maximum absorption peak at 405 nm; therefore, the glutathione content can be indirectly determined by measuring the absorbance of the reaction solution at 405 nm. This experiment used a GSH kit to assess the GSH consumption of FC-Z. Figure 4c As shown, FC-Z exhibits a certain capacity to consume glutathione (GSH), indicating that FC-Z possesses glutathionein-like activity. In summary, FC-Z possesses both hydrogen peroxide-supplied peroxidase-like and glutathionein-like activities.

[0050] like Figure 4a , Figure 4b As shown, compared to FC-Z nanozymes, under the conditions of high-frequency ultrasound-assisted FC-Z (after adjusting the volume of the appropriate buffer, high-frequency ultrasound treatment and subsequent processes were performed simultaneously, with ultrasound power density and time of 1.5 W / cm²), -2 (After 15 min), the reaction products showed higher absorbance values ​​at 585 nm and 652 nm, indicating that ultrasound can promote the production of more H2O2 by FC-Z and enhance the peroxidase-like activity of FC-Z. Furthermore, under ultrasound-assisted conditions (after adjusting the volume with the appropriate buffer, high-frequency ultrasound was started simultaneously with subsequent processes, with an ultrasound power density and time of 1.5 W / cm²), the reaction product exhibited higher absorbance values ​​at 585 nm and 652 nm, indicating that ultrasound can promote the production of more H2O2 by FC-Z and enhance the peroxidase-like activity of FC-Z. -2 The absorbance of FC-Z at 405 nm decreased significantly after 15 min, indicating a reduction in the GSH content in the reaction system, suggesting a significantly enhanced ability to consume GSH. Figure 4c In summary, ultrasound-assisted synthesis can enhance the dual enzyme activity of FC-Z.

[0051] To understand the effects of ultrasound-assisted hydrogen peroxide generation and peroxidase-like activity enhancement in FC-Z, this example investigated the reaction of FC-Z with TMB and the generation of H2O2 in the presence of ultrasound at different powers and durations. As the ultrasound power increased ( Figure 5a , Figure 5c ) and the extension of time ( Figure 5b , Figure 5d The fluorescence intensity at 585 nm and absorbance at 652 nm of the product gradually increased, indicating that with increasing H2O2 concentration in the reaction system, ultrasound can assist in improving the activity of FC-Z peroxidase, and this effect is power- and time-dependent. Meanwhile, with increasing power ( Figure 5e ) and the extension of time ( Figure 5f The absorbance values ​​at 405 nm decreased, indicating a reduction in GSH content in the reaction solution. This suggests a positive correlation between GSH consumption and the frequency and duration of ultrasound, indicating that ultrasound can enhance the glutathionease activity of FC-Z. In conclusion, ultrasound can enhance the dual-enzyme activity of FC-Z in a power- and time-dependent manner.

[0052] Example 2 The antibacterial application of ultrasound-assisted Fe / Cu-ZnO2 nanozymes, see Escherichia coli. Figure 6a Staphylococcus aureus (see also: Figure 6b .

[0053] The specific operation is as follows: Use a pipette to pick up single colonies of Escherichia coli and Staphylococcus aureus, inoculate them into liquid LB medium for activation, and place them in a shaker (37°C, 200 rpm) for overnight incubation. Then take 2 mL of each of Escherichia coli and Staphylococcus aureus into a 10 mL sterile centrifuge tube, centrifuge and wash three times with sterile water to remove residual culture medium. Then add 1 mL of NaAc-HAc buffer to provide a slightly acidic environment. Then add the bacterial suspension to a 12-well sterile plate and perform the following different treatments on the bacterial suspension: (1) blank control (C), (2) US, (3) FC-Z (i.e., add FC-Z), (4) US / FC-Z, (5) FC-Z+US, (6) FC-Z+US. Among them, (1) blank control (C) bacterial suspension was not treated in any way; (2) US was used to sonicate the bacterial suspension for 15 min; (3) FC-Z was added to the bacterial suspension and incubated for 15 min; (4) US / FC-Z was used to sonicate the bacterial suspension for 15 min and then FC-Z was added to the treatment solution; (5) FC-Z / US was used to add FC-Z to the bacterial suspension and incubated for 15 min, and then the incubated sample was sonicated for 15 min; (6) FC-Z+US was used to add FC-Z to the bacterial suspension, and after mixing, it was sonicated for 15 min while incubating. The ultrasonic power density and time involved were 1.5 W / cm², respectively. -2 After 15 min, the treated samples were placed in a 37°C constant temperature shaking incubator and incubated for 4 h. After incubation, plate counts were performed.

[0054] After ultrasonic treatment, the levels of *E. coli* and *Staphylococcus aureus* decreased by 0.14 log and 0.32 log, respectively, indicating negligible antibacterial activity. After FC-Z treatment (50 μg / mL FC-Z for 15 min followed by incubation for 4 h), the levels of *E. coli* and *Staphylococcus aureus* decreased by 1.60 log and 1.85 log, respectively. On one hand, FC-Z possesses peroxidase-like activity that catalyzes the production of cytotoxic ROS. On the other hand, FC-Z's glutathione reductase activity reduces intracellular oxidized glutathione, disrupting bacterial homeostasis and leading to bacterial death due to metabolic abnormalities. Furthermore, the metal ions released by FC-Z also have some toxicity to bacteria, accelerating bacterial death. Data from different ultrasonic-assisted nanozyme group treatments showed that the order of ultrasonic treatment significantly affected the bacterial killing effect, with simultaneous treatment of the material and ultrasonication yielding the best bactericidal effect. Figure 6c , Figure 6dCompared to the control group, ultrasound-assisted FC-Z treatment resulted in 3.89 log and 3.69 log in the inactivation of Escherichia coli and Staphylococcus aureus, respectively. Compared to the bactericidal effects of ultrasound alone and the materials alone, the combined treatment group showed a greater bactericidal effect than the single treatment group, indicating a synergistic bactericidal effect between ultrasound and FC-Z. This synergistic effect is related to the precise control of ultrasound and the increased catalytic activity of the two enzymes.

[0055] Example 3 Application of ultrasound-assisted Fe / Cu-ZnO2 nanozymes in antibacterial biofilms This embodiment uses the crystal violet staining method (which also involves a buffer volume adjustment process) to evaluate ultrasound-assisted ultrasound (after adjusting the volume with the appropriate buffer, high-frequency ultrasound is started simultaneously with the subsequent process; the ultrasound power density and time are 1.5 W / cm²). -2 The biofilm damage performance of FC-Z (and 15 min) was compared. Figure 7a and Figure 7b As shown, in the control group, *Escherichia coli* and *Staphylococcus aureus* appeared deep purple, indicating that a complete biofilm had formed. In contrast, the biofilm in the ultrasound-assisted FC-Z group was a lighter purple, and its absorbance was significantly lower than that of the other groups (p<0.05). This result indicates that ultrasound-assisted FC-Z can disrupt biofilm formation and limit bacterial proliferation.

[0056] Example 4 A method for preparing Fe / Cu-ZnO2 nanozymes with self-supplying hydrogen peroxide and dual enzyme activity specifically includes the following steps: Step 1. Preparation of ZnO2: Under stirring conditions, 0.809 g of Zn(NO3)2·6H2O and 1.4 g of polyvinylpyrrolidone (PVP) were dissolved in 60 mL of anhydrous ethanol. After stirring evenly on a magnetic stirrer, 500 μL of 15% ammonia solution was added to the above solution, followed immediately by 800 μL of H2O2 aqueous solution (1 M) to the above mixture. After stirring for 30 min, the reaction solution was centrifuged (10000 rpm, 20 min), and the precipitate was washed three times with ethanol and dried in an oven at 50 °C for later use.

[0057] Step 2. Preparation of Fe / Cu-ZnO2: First, 0.08 g of CuCl2·2H2O was dissolved in 30 mL of anhydrous ethanol to prepare a mother liquor for later use. Then, 120 mg of ZnO2 was dispersed in 80 mL of deionized water, and 0.03 g of FeCl2·4H2O was added under magnetic stirring until the mixture became clear. The prepared mother liquor was then added to the mixture, and stirring was continued for 2.5 h. The mixture was collected by centrifugation, washed five times with anhydrous ethanol, and dried in a 55°C oven for 10 h for later use.

[0058] Example 5 A method for preparing Fe / Cu-ZnO2 nanozymes with self-supplying hydrogen peroxide and dual enzyme activity specifically includes the following steps: Step 1. Preparation of ZnO2: Under stirring conditions, 0.809 g of Zn(NO3)2·6H2O and 1.4 g of polyvinylpyrrolidone (PVP) were dissolved in 60 mL of anhydrous ethanol. After stirring evenly on a magnetic stirrer, 500 μL of 15% ammonia solution was added to the above solution, followed immediately by 800 μL of H2O2 aqueous solution (1 M) to the above mixture. After stirring for 30 min, the reaction solution was centrifuged (10000 rpm, 20 min), and the precipitate was washed three times with ethanol and dried in an oven at 50 °C for later use.

[0059] Step 2. Preparation of Fe / Cu-ZnO2: First, 0.06 g of CuCl2·2H2O was dissolved in 20 mL of anhydrous ethanol to prepare a mother liquor for later use. Then, 80 mg of ZnO2 was dispersed in 60 mL of deionized water, and 0.02 g of FeCl2·4H2O was added under magnetic stirring until the mixture became clear. The prepared mother liquor was then added to the mixture, and stirring was continued for 1.5 h. The mixture was collected by centrifugation, washed four times with anhydrous ethanol, and dried in a 45°C oven for 14 h for later use.

Claims

1. A method for preparing an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity, characterized in that, Includes the following steps: S1, dissolve the divalent copper salt to obtain the mother liquor; ZnO2 and ferrous salts were dispersed to obtain a mixed solution; S2, the mother liquor and the mixed liquor are dispersed evenly, and the mass ratio of divalent copper salt, ZnO2 and divalent iron salt is (6~8):(8~12):(2~3). The resulting precipitate is then washed and dried to obtain an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity.

2. The method for preparing iron / copper-zinc peroxide nanozymes with self-supplying hydrogen peroxide and dual-enzyme activity according to claim 1, characterized in that, The divalent copper salt mentioned in S1 is CuCl2·2H2O, and the divalent iron salt is FeCl2·4H2O.

3. The method for preparing iron / copper-zinc peroxide nanozymes with self-supplying hydrogen peroxide and dual enzyme activity according to claim 2, characterized in that, In S1, CuCl2·2H2O is dissolved in anhydrous ethanol to obtain the mother liquor; ZnO2 and FeCl2·4H2O were dispersed in deionized water to obtain a mixed solution.

4. The method for preparing iron / copper-zinc peroxide nanozymes with self-supplying hydrogen peroxide and dual enzyme activity according to claim 3, characterized in that, S1 dissolves 0.06~0.08 g of CuCl2·2H2O in 20~30 mL of anhydrous ethanol to obtain the mother liquor.

5. The method for preparing iron / copper-zinc peroxide nanozymes with self-supplying hydrogen peroxide and dual enzyme activity according to claim 3, characterized in that, S1 first disperses ZnO2 in deionized water, then adds FeCl2·4H2O while stirring, and stirs until clear to obtain a mixed solution.

6. The method for preparing iron / copper-zinc peroxide nanozymes with self-supplying hydrogen peroxide and dual enzyme activity according to claim 5, characterized in that, The ratio of ZnO2 to deionized water in S1 is 80~120 mg: 60~80 mL.

7. The method for preparing iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity according to claim 1, characterized in that, S2: The mother liquor and the mixture are stirred continuously for 1.5 to 2.5 hours, and then the resulting precipitate is washed and dried.

8. The method for preparing iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity according to claim 1, characterized in that, S2 The obtained precipitate was washed 3-5 times with anhydrous ethanol and then dried at 45-55℃ for 10-14h to obtain an iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity.

9. An iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual-enzyme activity obtained by the preparation method of iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual-enzyme activity according to any one of claims 1 to 8, characterized in that, The dual enzyme activities described are peroxidase-like activities and glutathionease-like activities.

10. The application of the iron / copper-zinc peroxide nanozyme with self-supplying hydrogen peroxide and dual enzyme activity as described in claim 9 in antibacterial and antimicrobial applications, characterized in that, After adjusting the volume with the appropriate buffer solution, high-frequency sonication was initiated, with an ultrasonic power density and duration of 0.5–1.5 W / cm². -2 5~15 min.

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