MnOx-CeOx nano-enzyme rich in oxygen vacancy structure as well as preparation method and application of MnOx-CeOx nano-enzyme

Abstract

The invention discloses MnOx-CeOx nano-enzyme rich in oxygen vacancy structures as well as a preparation method and application of the MnOx-CeOx nano-enzyme. The preparation method comprises the following steps: synthesizing CeO2 nano-rods by adopting a hydrothermal method; mnOx nano-particles are deposited on the surface of the CeO2 nano-rod by adopting an atomic layer deposition technology, and MnOx-CeO2 nano-enzyme is obtained; and reducing the MnOx-CeO2 nano-enzyme with high-temperature H2 to obtain the MnOx-CeOx nano-enzyme rich in the oxygen vacancy structure. The MnOx-CeOx nano-enzyme has high peroxidase activity, can kill bacteria under the condition of low dosage of hydrogen peroxide, not only can effectively kill staphylococcus aureus, but also has an excellent sterilization effect on escherichia coli, and has broad-spectrum antibacterial characteristics.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial nanozymes, and in particular to a MnOx-CeOx nanozyme rich in oxygen vacancy structure, a preparation method thereof, and applications thereof. Background Art

[0002] Bacterial infections are a worldwide public health problem, posing a significant threat to healthcare systems and human health. Bacterial infections cause millions of morbidity and mortality each year. Individuals infected with pathogenic bacteria can develop serious illnesses such as meningitis, sepsis, and bacteremia. Antibiotics are by far the most common and effective treatment for bacterial infections. They can kill pathogens or inhibit bacterial growth by disrupting bacterial cell membrane / wall formation, increasing bacterial cell membrane permeability, interfering with bacterial protein synthesis, and inhibiting bacterial deoxyribonucleic acid (DNA) replication / transcription. However, the overuse and misuse of antibiotics inevitably lead to the development of robust antibiotic resistance in many bacteria. The rapid spread of antibiotic resistance (AMR) has become one of the greatest threats to global health in less than a century. Given the considerable time required to discover antibiotics, which lags far behind the emergence of resistant bacteria, there is an urgent need to develop effective alternative antibiotic strategies to address this problem.

[0003] In recent years, researchers have developed many broad-spectrum antibacterial and antibacterial platforms to replace antibiotics. Among them, nanozymes have shown great potential in antibiotic alternative therapies due to their advantages such as low cost, easy regulation, ideal bactericidal effect, and reduced risk of drug resistance. Nanozymes have the unique structure and physical properties of nanomaterials and can be specifically synthesized and modified. Compared with natural enzymes, they show obvious advantages, including high cost-effectiveness, easy production, good stability, and customizable catalytic activity. Nanozymes combine nanomaterials and enzyme-like activity. They are enzyme mimics with super-strong reactive oxygen species (ROS) generation capabilities. The ROS generated can destroy the bacterial cell membrane and further lead to the irreversible degradation of essential substances such as proteins and nucleic acids. In recent years, a variety of nanozymes have been developed for ROS antibacterial therapy, including carbon nanomaterials, metal-organic frameworks (MOFs), metal nanoparticles, layered nanomaterials, metal oxide nanozymes, etc.

[0004] However, most nanozymes in related technologies need to catalyze H2O2 to produce ·OH under high concentrations of H2O2. High concentrations of H2O2 are highly toxic to cells and affect the catalytic activity of nanozymes. In addition, these nanozymes also lack broad-spectrum antibacterial effects. Summary of the Invention

[0005] In order to solve the above problems, the present invention proposes a MnOx-CeOx nanozyme rich in oxygen vacancy structure and its preparation method and application. The MnOx-CeOx nanozyme has high peroxidase activity and can kill bacteria under low doses of H2O2. It can not only effectively kill Staphylococcus aureus, but also has excellent bactericidal effect against Escherichia coli, and has broad-spectrum antibacterial properties.

[0006] To achieve the above objectives, the embodiments of the present invention provide, in a first aspect, a method for preparing a MnOx-CeOx nanozyme rich in oxygen vacancy structure, which comprises the following steps:

[0007] CeO2 nanorods were synthesized by hydrothermal method;

[0008] MnOx nanoparticles are deposited on the surface of the CeO2 nanorods using atomic layer deposition technology to obtain MnOx-CeO2 nanozymes;

[0009] High-temperature H2 reduction of the MnOx-CeO2 nanozyme yields a MnOx-CeOx nanozyme rich in oxygen vacancy structure.

[0010] According to an embodiment of the present invention, a method for preparing a MnOx-CeOx nanozyme rich in oxygen vacancy structure uses atomic layer deposition (ALD) technology to uniformly modify MnOx onto CeO2 nanorods, and realizes the construction of oxygen vacancies in one step through hydrogen reduction, which simplifies the preparation process of the nanozyme, improves the preparation efficiency and repeatability, and is conducive to large-scale production and application; the prepared MnOx-CeOx nanozyme has high peroxidase activity and can kill bacteria under low doses of H2O2. It can not only effectively kill Staphylococcus aureus, but also has excellent bactericidal effects on Escherichia coli, and has broad-spectrum antibacterial properties.

[0011] Optionally, the steps of synthesizing CeO2 nanorods by hydrothermal method include:

[0012] NaOH and Ce(NO3)3·6H2O were dissolved in deionized water and kept at 100℃ for 24h. The solid product was collected by centrifugation, washed and dried to obtain CeO2 nanorod precursor.

[0013] The CeO2 nanorod precursor is dispersed in deionized water and subjected to hydrothermal treatment at 170°C for 12 hours. The yellow product is collected by centrifugation, washed, and dried to obtain CeO2 nanorods.

[0014] Optionally, the operating conditions of the atomic layer deposition reactor are:

[0015] The deposition temperature of MnOx was set at 250 °C, nitrogen was used as the carrier gas and purge gas, the pulse time and exposure time of di(ethylcyclopentadienyl)manganese(II) and O3 were 1.2 s, 9 s and 25 s, respectively, and the number of ALD cycles of MnOx was 30.

[0016] Optionally, the conditions for high-temperature H2 reduction of the MnOx-CeO2 nanozyme are: activating the MnOx-CeO2 nanozyme at 300°C for 1.5h under a 10% H2 / Ar atmosphere to obtain the MnOx-CeOx nanozyme rich in oxygen vacancy structure.

[0017] In a second aspect, an embodiment of the present invention provides a MnOx-CeOx nanozyme rich in oxygen vacancy structure, which is prepared using the above-mentioned method for preparing the MnOx-CeOx nanozyme rich in oxygen vacancy structure.

[0018] According to the antibacterial results of the MnOx-CeOx nanozyme according to the embodiments of the present invention, it can completely inactivate Escherichia coli and Staphylococcus aureus under weakly acidic conditions, has broad-spectrum antibacterial properties, and has great potential in antibacterial treatment.

[0019] In a third aspect, an embodiment of the present invention proposes the use of the above-mentioned MnOx-CeOx nanozyme rich in oxygen vacancy structure in the preparation of antibacterial materials.

[0020] Optionally, the antibacterial material is used to disinfect Staphylococcus aureus and Escherichia coli.

[0021] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a SEM image of CeO2 nanorods according to an embodiment of the present invention;

[0023] Figure 2 2 are SEM images (a) of the MnOx-CeO2 nanozyme according to an embodiment of the present invention and EDS mapping images (b)-(d) of different elements in the MnOx-CeO2 nanozyme;

[0024] Figure 3 Figures 1 and 2 are morphological and structural characterizations of the MnOx-CeOx nanozyme according to an embodiment of the present invention. (a) and (b) are TEM images of the MnOx-CeOx nanozyme; (c) and (d) are HAADF-STEM images of the MnOx-CeOx nanozyme.

[0025] Figure 4Figure 2 is a structural characterization of the nanozymes according to the embodiments of the present invention: (a) is the XRD spectra of CeO2, MnOx-CeO2 and MnOx-CeOx nanozymes; (b) is the XPS full spectrum and XPS fine spectrum of MnOx-CeOx nanozymes; (c) is Ce 3d, (d) is O1s and (e) is Mn 2p, and (f) is the EPR spectra of CeO2, MnOx-CeO2 and MnOx-CeOx nanozymes;

[0026] Figure 5 (a) is the UV-visible absorption spectrum of oxTMB generated by the MnOx-CeOx nanozyme catalytic system according to an embodiment of the present invention over time (0-5 minutes); (b) is the UV-visible absorption spectrum of oxTMB generated by CeO2, MnOx-CeO2 and MnOx-CeOx nanozyme catalytic systems at the fifth minute; (c) is the change in the UV absorption of oxTMB at 652nm in different nanozyme systems from 0 to 5 minutes; the initial reaction rate as a function of (d) [H2O2] and ( e) Relationship between 1 / V and 1 / [H2O2] in the Lineweaver-Burk plot; (f) UV-visible absorption spectra of oxOPD generated by the MnOx-CeOx nanozyme catalytic system over time (0-5 minutes); (g) Changes in the UV absorption of oxOPD at 450 nm in different nanozyme systems from 0 to 5 minutes; (h) Comparison of the efficiency of MB degradation by different nanozyme systems from 0 to 30 minutes; (i) EPR spectra of DMPO capturing ·OH under different conditions;

[0027] Figure 6 2 is a diagram showing the TMB oxidation color according to an embodiment of the present invention, and the UV-visible absorption spectra of TMB oxidized by free radicals produced by CeO2 (a) and MnOx-CeO2 (b) catalyzed by hydrogen peroxide over time;

[0028] Figure 7 2 is a graph showing the color development of OPD oxidation according to an embodiment of the present invention, showing the UV-visible absorption spectra of the free radical oxidation of OPD produced by CeO2 (a) and MnOx-CeO2 (b) catalyzing hydrogen peroxide over time; and comparing the UV-visible absorption curves of hydrogen peroxide catalyzed by CeO2, MnOx-CeO2, and MnOx-CeOx at the 5th minute (c);

[0029] Figure 8 (a) shows the growth of bacteria on agar plates after different treatments according to an embodiment of the present invention; (b) is a histogram of bacterial survival based on the plate count method; (c) is a live / dead staining image of bacteria after different treatments; (d) is a SEM image of bacteria killed by MnOx-CeOx nanozymes under different pH conditions;

[0030] Figure 9 The antibacterial effect of the nanozyme according to the embodiment of the present invention is shown in the following photos of Escherichia coli on agar plates treated with CeO2, MnOx-CeO2, and MnOx-CeOx under different pH environments;

[0031] Figure 10 The survival rate of Escherichia coli is calculated using the plate count method according to an embodiment of the present invention: (a) blank control; (b) H2O2; (c) CeO2; (d) MnOx-CeO2; (e) MnOx-CeOx;

[0032] Figure 11 Schematic diagram of the synthesis path of MnOx-CeOx nanozyme rich in oxygen vacancy structure and its application in catalytic hydrogen peroxide sterilization according to an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.

[0034] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0035] The materials of the present invention are all commercially available and used without further purification. All solvents used were of analytical grade.

[0036] Ce(NO₃)₃·6H₂O, NaOH, and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) were purchased from Shanghai MacLean Biochemical Co., Ltd. Mn raw materials were purchased from Strem Chemicals (J&K Scientific, Beijing, China). 3,3′,5,5′-Tetramethylbenzidine (TMB), 1,2-phenylenediamine (OPD), and hydrogen peroxide (H₂O₂, 30%) were also purchased from MacLean Biochemical Co., Ltd. Acetic acid (CH₃COOH) was purchased from Sinopharm Chemical Reagent Co., Ltd. Glutaraldehyde was purchased from Sigma-Aldrich. Calcein-AM / propidium iodide (PI) and SYTO9 were provided by Beyotime.

[0037] In the following examples, scanning electron microscopy (SEM) images of the characterization samples were obtained using a field emission scanning electron microscope (Ve rios G4, FEI, USA). High-angle annular dark field scanning TEM (HAADF-STEM) images were collected on an operating FEI Talos F200X TEM instrument. X-ray diffraction patterns were collected on a Bruker diffractometer (D8 DISCOVERA25) with Co Kα radiation 2θ = 5-90°. X-ray photoelectron spectroscopy (BSD-PS(M)) was used to record XPS spectra. NanoDrop (Thermo Scientific) was used to measure UV-Vis-NIR absorption spectra. The generation of ·OH was measured using an electron paramagnetic resonance (EPR) spectrometer (Bruker EPR A300) with DMPO as a scavenger.

[0038] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0039] Example 1

[0040] like Figure 11 As shown, CeO2 nanorods were synthesized by a hydrothermal method; MnOx nanoparticles were deposited on the surface of the CeO2 nanorods by atomic layer deposition technology to obtain MnOx-CeO2 nanozymes; further, the MnOx-CeO2 nanozymes were reduced by high-temperature H2 to obtain MnOx-CeOx nanozymes rich in oxygen vacancy structures.

[0041] Specifically, the synthesis of CeO2 nanorods:

[0042] CeO2 nanorods were synthesized by an improved two-step hydrothermal method. First, NaOH (19.2 g) and Ce(NO3)3·6H2O (1.367 g) were added sequentially to a beaker containing 70 mL of deionized water, stirred vigorously until completely dissolved, and then transferred to an oven and kept warm at 100°C for 24 h to obtain a CeO2 nanorod precursor. After naturally cooling to room temperature, the solid product was collected by centrifugation, washed three times with deionized water and ethanol, and then dried at 60°C overnight. The CeO2 nanorod precursor (2 g) was ultrasonically dispersed in 300 mL of deionized water, then transferred to a stainless steel high-pressure reactor and hydrothermally treated at 170°C for 12 h. The yellow product was collected by centrifugation, washed three times with deionized water and ethanol, and vacuum dried overnight to obtain CeO2 nanorods. The scanning electron microscope image of CeO2 nanorods is shown in FIG. Figure 1 shown.

[0043] Synthesis of MnOx-CeO2:

[0044] MnOx-CeO2 was prepared using an atomic layer deposition (ALD) reactor. MnOx atomic layer deposition was performed by continuously exposing a ceria substrate to di(ethylcyclopentadienyl)manganese(II) at 60°C and ozone (O3) produced by an O3 generator. The deposition temperature of MnOx was set to 250°C, with nitrogen as the carrier gas and purge gas. The pulse times of di(ethylcyclopentadienyl)manganese(II) and O3 were 1.2s, 9s, and 25s, respectively. The number of ALD cycles for MnOx was 30; MnOx-CeO2 was obtained. MnOx nanoparticles were deposited on the surface of CeO2 nanorods by ALD deposition technology. The content of MnOx can be gradually increased by increasing the number of ALD cycles, and the MnOx deposited by ALD technology is highly dispersed. As Figure 2 As shown in Figure a, after ALD deposition of MnOx, the CeO2 nanorods still maintain a good structure and the morphology does not change, indicating that ALD technology is a mild deposition technology that will not destroy the structure of CeO2. The EDS spectrum shows that MnOx is successfully deposited on the CeO2 nanorods and is highly dispersed, as shown in Figure 2. Figure 2 As shown in b-2d.

[0045] Preparation of MnOx-CeOx nanozymes by H2 reduction method:

[0046] The preparation method of MnOx-CeOx is to reduce MnOx-CeO2 under H2 atmosphere. First, the prepared MnOx-CeO2 is inverted in a rectangular ceramic boat, then transferred to a tube furnace and activated at 300℃ for 1.5 hours under 10% H2 / Ar atmosphere. Finally, the MnOx-CeOx nanozyme with rich oxygen vacancy structure is obtained. The MnOx-CeOx nanozyme still maintains the rod-like morphology and porous structure, such as Figure 3As shown in Figures 3a and 3b, high temperature H2 reduction conditions did not destroy the crystal structure of CeO2. Figure 3 As shown in Figures c and 3d, the lattice spacing of CeO2 is about 0.27nm, corresponding to the (200) crystal plane of CeO2 crystal. Figure 4 As shown in Figure 2, the crystal structures of CeO2, MnOx-CeO2 and MnOx-CeOx were further studied by X-ray diffraction (XRD). The diffraction peaks corresponding to the (111), (200) and (311) crystal planes can be clearly seen in the XRD spectrum of CeO2. Figure 4 a), indicating that CeO2 was successfully synthesized. In addition, the diffraction peaks of MnOx-CeO2 and MnOx-CeOx samples obtained by ALD deposition and H2 reduction are basically consistent with those of CeO2, indicating that they have similar crystal structures. There are no characteristic peaks of MnOx in the XRD pattern. This is because the MnOx particles are relatively small, which also shows that MnOx is highly dispersed. The surface structure of all samples was further analyzed by X-ray photoelectron spectroscopy (XPS). The full XPS spectrum of MnOx-CeOx ( Figure 4 b) There is little change compared with MnOx-CeO2. The six Ce 3d peaks at 916.07eV, 897.87eV, 907.02eV, 888.42eV, 900.47eV and 881.92eV indicate the presence of Ce 4+ The other two peaks at 903.02eV and 884.8eV are related to Ce 3+ Related. 3+ The formation of CeOx and MnOx-CeOx indicates that there are surface bonding defects on the surface ( Figure 4 c). Figure 4 d is the O1s XPS spectrum of MnOx-CeOx. The O 1s characteristic peak is divided into two peaks, corresponding to OH (531.6eV) and Ce-O-Mn (529.8eV) bonds, which indicates that there is a strong chemical bond between CeOx and MnOx. A significant spin-orbit splitting energy difference is observed between the Mn 2p1 / 2 and Mn 2p3 / 2 orbitals, confirming the presence of Mn 3+ and Mn 4+ Valence ( Figure 4 e). In order to study whether MnOx-CeO2 generates abundant oxygen vacancies during H2 reduction, electron paramagnetic resonance (EPR) was used for characterization. Figure 4 As shown in Figure f, an obvious peak (g = 2.004) was observed in the MnOx-CeOx spectrum, which was attributed to oxygen vacancies, indicating that there were a large number of oxygen vacancies in MnOx-CeOx. At the same time, a large number of Ce 3+ peak, further verifying the role of H2 reduction. Figure 4f It can be seen that the oxygen vacancy peak of CeO2 sample is lower, and Ce 3 + -O-Ce 4+ Defect peak. After depositing MnOx on CeO2 sample, Ce 3+ -O-Ce 4+ The defect peak gradually disappears. The above results indicate that H2 can generate oxygen-rich vacancies on CeO2, and MnOx can act as a catalyst to promote the generation of oxygen vacancies.

[0047] Example 2

[0048] 1. The product obtained in Example 1 was tested for hydroxyl radicals (·OH): 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were used to evaluate the peroxidase-like activity of different samples in the presence of hydrogen peroxide. Specifically, TMB dissolved in DMSO was added to 1 mL of a solution of different samples CeO2, MnOx-CeO2, and MnOx-CeOx (200 ppm), and then 1 mM H2O2 solution was added. The change in ultraviolet absorbance at 652 nm was detected at different time points (0, 1, 2, 3, 4, 5 min). OPD was then added in the same way, and the absorbance of the solution at 450 nm was detected.

[0049] MB degradation experiments can also qualitatively detect the presence of OH. CeO₂, MnOx-CeO₂, and MnOx-CeOx at 200 ppm concentrations were added to solutions containing MB and H₂O₂. UV-visible spectra of three test samples were analyzed at specific time intervals (0, 5, 10, 20, and 30 minutes), and the changes in absorbance at 664 nm were recorded.

[0050] In an electron paramagnetic resonance (EPR) spectrometer, dimethylpyridine oxide (DMPO) was used as a scavenger to detect hydroxyl radicals (·OH) generated in the system. CeO2, MnOx-CeO2, and MnOx-CeOx (500 ppm) were each mixed with 0.1 mM H2O2. DMPO (5 mM) was then added to the reaction system. After reacting for 5 minutes, equal amounts of the solution were collected using a capillary tube and placed into the EPR instrument for detection.

[0051] 2. Michaelis-Menten kinetics The TMB method was used to study the kinetic characteristics of MnOx-CeOx nanozymes. MnOx-CeOx (20 ppm), TMB (1 mM) and different concentrations of hydrogen peroxide (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mM) were reacted at 37°C, and the absorbance of TMB at 652 nm was detected by a microplate reader.

[0052] Since both CeOx and MnOx are rich in oxygen vacancies and have high nanozyme activity, MnOx-CeOx is expected to become an ideal catalyst for catalyzing H2O2 to produce reactive oxygen species (ROS). In order to explore the nanozyme activity of MnOx-CeOx, the ROS generation efficiency of CeO2, MnOx-CeO2 and MnOx-CeOx under the same conditions was compared through the colorimetric reaction of TMB and OPD. Under acidic conditions, the catalyst can catalyze H2O2 to produce ·OH, and ·OH can oxidize TMB to oxTMB. The ultraviolet absorption peak of oxTMB can be detected at 652nm. Figure 5 As shown in Figure a, in the presence of MnOx-CeOx and H2O2, the UV absorption peak of oxTMB gradually increases over time, indicating that MnOx-CeOx can continuously catalyze H2O2 to generate ·OH. In addition, it can be seen that under the same conditions, CeO2 and MnOx-CeO2 both exhibit certain enzyme activity: as time goes by, the two reaction systems produce more and more ·OH, as shown in Figure 2. Figure 6 a and Figure 6 As shown in b. Figure 5 As shown in b, at 5 min, the UV absorption peak of the MnOx-CeO2 system is higher than that of CeO2 under the same conditions, indicating that CeO2 can produce more ROS after MnOx deposition, and MnOx-CeO2 has higher nanozyme activity. The MnOx-CeOx obtained under high temperature H2 reduction conditions has the highest nanozyme activity compared with CeO2 and MnOx-CeO2, which is attributed to the presence of abundant oxygen vacancies in MnOx-CeOx ( Figure 5 c). Given its good POD activity, Michaelis-Menten steady-state kinetic studies were conducted to further determine the catalytic efficiency of MnOx-CeOx: Under the conditions of fixed MnOx-CeOx and TMB concentrations, steady-state kinetic experiments were conducted by varying the hydrogen peroxide concentration. The Vmax and Km of MnOx-CeOx were 30.4×10 -6 Ms -1 and 0.89 mM ( Figure 5 d and Figure 5 e). Compared with other reported nanomaterials, MnOx-CeOx exhibits a higher Vmax and lower Km, demonstrating its excellent catalytic efficiency.

[0053] In addition, the peroxidase activity of CeO2, MnOx-CeO2 and MnOx-CeOx was determined using a typical o-phenylenediamine (OPD) catalytic system. They can all be oxidized by ·OH, with a characteristic absorption peak at 452nm. The ·OH generation efficiency mediated by the Fenton reaction determines the quality of the catalytic performance. Therefore, the ·OH generation efficiency of representative CeO2, MnOx-CeO2 and MnOx-CeOx was monitored using OPD, as shown in Figure 2. Figure 6 a. Figure 6 b and Figure 5 f. Figure 6 As can be seen from a and 6b, under the same conditions, the absorption peak of CeO2 can be ignored, the absorption peak of MnOx-CeO2 gradually increases, and the absorption peak of MnOx-CeOx is the highest. MnOx-CeOx has a peroxidase-like activity, which is about 3.7 times that of CeO2 ( Figure 5 g), which showed a similar trend to the TMB catalytic system. Subsequently, the catalytic activity of CeO2, MnOx-CeO2, and MnOx-CeOx was evaluated using methylene blue (MB) as a ROS indicator. Under acidic conditions in the presence of CeO2 and H2O2, the absorption of MB did not change much. In the MnOx-CeO2 reaction system, the absorption peak of MB decreased ( Figure 5 h). In the presence of MnOx-CeOx and H2O2, the intensity of the characteristic absorption peak of MB decreased significantly over time, indicating that a large amount of ROS was generated and participated in the degradation of MB. Therefore, it can be seen that: MnOx-CeO2 retains the ability to generate ROS, and under the action of a large number of oxygen vacancies generated by H2 reduction, the catalytic activity of MnOx-CeOx is greatly enhanced. Finally, electron paramagnetic resonance (EPR) spectroscopy was used to further determine the type and ability of CeO2, MnOx-CeO2 and MnOx-CeOx nanozymes to generate ROS. 5,5-Dimethyl-1-pyrrolidine-N-oxide (DMPO) was used as a capture agent to detect the generation of ·OH, such as Figure 5 As shown in Figure 1, the characteristic peak of 1:2:2:1 corresponds to the signal of OH. The EPR signal of MnOx-CeOx is the highest, which is consistent with the results of TMB, OPD, and MB, indicating that MnOx-CeOx has a strong ability to generate ROS. These results show that the MnOx-CeOx nanozyme with oxygen vacancies has the highest enzymatic activity.

[0054] Example 3

[0055] 1. Evaluation of the antibacterial effect of the product of Example 1:

[0056] The plate count method was used to evaluate the antibacterial properties of nanozymes. Specifically, Staphylococcus aureus was cultured in liquid culture medium for 16 hours to reach the logarithmic growth phase, and then the cultured bacteria were diluted to an OD value of 1 at 600nm for subsequent experiments. The bacteria were then treated as follows: a: Control, b: H2O2, c: CeO2, d: MnOx-CeO2, e: MnOx-CeOx. The concentrations of CeO2, MnOx-CeO2, and MnOx-CeOx were all 200ppm, the concentration of H2O2 was 0.1mM, and the concentration of bacteria was 1×10 7 CFU / mL. Each bacterial suspension was incubated in a shaker at 37°C for 30 min. 100 μL of the treated bacterial suspension was evenly spread on an agar plate. The agar plate was then placed in a 37°C incubator and incubated overnight. The colony count was observed, and the antibacterial activity of the nanozyme against E. coli was evaluated using the same method.

[0057] 2. Characterization of bacterial morphology The morphology of bacterial cells was observed using a scanning electron microscope (SEM) to study the antibacterial mechanism of the nanozyme. First, the cultured bacteria were treated with MnOx-CeOx nanozymes and H2O2, and untreated bacteria were used as a control. The bacterial suspension was centrifuged at a certain speed, and the resulting precipitate was fixed with 2.5% glutaraldehyde at 4°C for 2h, then washed three times with phosphate-buffered saline (PBS), and then dehydrated with 25%, 50%, 75%, 90% and 100% ethanol. Finally, the sample was completely dried on a silicon wafer under natural conditions and sprayed with gold for morphological observation.

[0058] 3. Staining of live and dead bacteria SYTO 9 is a green fluorescent nucleic acid dye that can completely penetrate the cell membrane. PI is a nuclear dye that can only penetrate cells with damaged cell membranes and cell walls. 4 CFU / mL) were treated with PBS, H₂O₂, CeO₂, MnOx-CeO₂, and MnOx-CeOx (200 ppm) for 4 hours. Each mixture was then incubated with the dye at room temperature in the dark for 15 minutes. After centrifugation to remove excess dye, the stained bacterial suspension (10 μL) was placed on a glass slide. Finally, the stained bacteria were observed using a fluorescence microscope.

[0059] In order to evaluate the antibacterial effect of MnOx-CeOx nanozymes, Escherichia coli and Staphylococcus aureus were used as representatives of Gram-negative and Gram-positive bacteria, respectively, and the survival rates of bacteria under different conditions were studied. Figure 8 As shown, the number of colony forming units of Staphylococcus aureus on Luria-Bertani (LB) agar plates ( Figure 8a). Regardless of whether it is under neutral or weakly acidic conditions, the bacteria treated with hydrogen peroxide (H2O2) remain active, indicating that the use of H2O2 alone has no significant effect on bacterial activity. The bacterial activity decreased after treatment with H2O2 and CeO2, but it was still strong under neutral and weakly acidic conditions. This may be because the activity of CeO2 nanozymes is low and cannot cause damage to the bacteria. After the deposition of MnOx particles on the CeO2 surface, the activity of MnOx-CeO2 nanozymes was significantly improved. Under neutral conditions, the activity of bacteria treated with H2O2 and MnOx-CeO2 was significantly reduced, and the bacterial survival rate was 78.2% (compared with the control group). Figure 8 b) Under weakly acidic conditions of pH 5.0, the activity of the MnOx-CeO2 nanozyme was further enhanced, while the bacterial survival rate decreased to 62.8%, demonstrating that weakly acidic conditions can enhance nanozyme activity. This is due to the higher efficiency of MnOx-CeO2 in catalyzing H2O2 to produce ·OH under acidic conditions. Further hydrogen reduction was used to generate MnOx-CeOx nanozymes with oxygen vacancies, which exhibited significantly enhanced enzymatic activity compared to MnOx-CeO2. Under neutral conditions, the bacterial survival rate after treatment with H2O2 and MnOx-CeOx nanozymes was only 34.5%. However, under weakly acidic conditions, the MnOx-CeOx nanozyme killed 99.9% of the bacteria, attributed to its high enzymatic activity. This demonstrates that oxygen vacancies can significantly enhance the enzymatic activity of the MnOx-CeOx nanozyme.

[0060] The antibacterial properties of MnOx-CeOx nanozymes against Gram-negative bacteria were also evaluated. Figure 9 As shown in the figure, the antibacterial properties of MnOx-CeOx nanozymes also show pH dependence. Under weakly acidic conditions, the antibacterial properties of MnOx-CeOx nanozymes are much higher than those under neutral conditions. Under weakly acidic conditions, the bacterial survival rate after treatment with MnOx-CeO2 nanozymes is 34.6%, while the bacterial survival rate after treatment with MnOx-CeOx nanozymes is only 4.7% ( Figure 10 Compared with MnOx-CeO2, the POD activity of the MnOx-CeOx nanozyme was significantly enhanced, which was attributed to the presence of oxygen vacancies. The MnOx-CeOx nanozyme not only killed Staphylococcus aureus but also had a good antibacterial effect on Escherichia coli, indicating that the MnOx-CeOx nanozyme has broad-spectrum antibacterial properties.

[0061] The good antibacterial effect of MnOx-CeOx nanozymes has greatly aroused the interest of the inventors in studying its mechanism of action. First, the bacteria treated with different methods were observed by live / dead staining ( Figure 8c). SYTO9 can label all cells with green fluorescence, while PI can only label bacterial cells with damaged cell membranes with red fluorescence. In the control group, Staphylococcus aureus emitted completely green fluorescence, which was consistent with expectations. Bacteria treated with H2O2 and CeO2 showed almost no red fluorescence, indicating that they did not damage the bacterial cell membrane. The MnOx-CeO2 group showed a weak red fluorescence signal in the PI channel, indicating that the bacteria treated with the MnOx-CeO2 nanozyme had some damage, but not enough to kill all bacteria. In the MnOx-CeOx nanozyme-treated group, almost all bacteria showed red fluorescence, indicating that the cell membranes of most bacteria were severely damaged, further proving that the MnOx-CeOx nanozyme has excellent antibacterial effects. In addition, the morphological changes of Staphylococcus aureus were directly observed by scanning electron microscopy, and the bactericidal mechanism of MnOx-CeOx nanozyme was further studied, such as Figure 8 As shown in Figure d, SEM images reveal that the bacteria in the control group maintained intact morphology and smooth surfaces under both weakly acidic and neutral conditions, indicating that pH alone does not affect bacterial growth. However, the MnOx-CeOx nanozyme exhibited moderate antibacterial activity under neutral conditions. After weakly acidic treatment, numerous depressions appeared on the bacterial surface, indicating the most severe bacterial death, confirming the MnOx-CeOx nanozyme's best antibacterial properties. This is consistent with the results of plate colony counts and live / dead staining, demonstrating that the MnOx-CeOx nanozyme possesses broad-spectrum antibacterial properties and exhibits great potential for further antibacterial applications.

[0062] In summary, according to an embodiment of the present invention, MnOx was successfully modified on CeO2 nanorods using an ALD deposition strategy, and a highly active MnOx-CeOx nanozyme with an oxygen vacancy structure was obtained by H2 reduction. The generation of oxygen vacancies was promoted during the H2 reduction process. At the same time, the oxygen vacancies provided binding sites for MnOx, established a strong metal-support interaction, thereby preventing their agglomeration and enhancing their catalytic activity. The metal in MnOx-CeO2 can catalyze hydrogen peroxide to produce cytotoxic ·OH. More importantly, compared with MnOx-CeO2, the construction of oxygen vacancies during the H2 reduction process greatly enhanced the POD activity of the MnOx-CeOx nanozyme. The antibacterial results showed that the MnOx-CeOx nanozyme can completely inactivate Escherichia coli and Staphylococcus aureus under weakly acidic conditions, has broad-spectrum antibacterial properties, and has great potential in antibacterial therapy.

[0063] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0064] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a MnOx-CeOx nanozyme rich in oxygen vacancy structure, characterized in that: The following steps are involved: CeO2 nanorods were synthesized by hydrothermal method; MnOx nanoparticles are deposited on the surface of the CeO2 nanorods using atomic layer deposition technology to obtain MnOx-CeO2 nanozymes; High-temperature H2 reduction of the MnOx-CeO2 nanozyme yields a MnOx-CeOx nanozyme rich in oxygen vacancy structure.

2. The method for preparing the MnOx-CeOx nanozyme rich in oxygen vacancy structure according to claim 1, wherein: The steps of synthesizing CeO2 nanorods by hydrothermal method include: NaOH and Ce(NO3)3·6H2O were dissolved in deionized water and kept at 100℃ for 24h. The solid product was collected by centrifugation, washed and dried to obtain CeO2 nanorod precursor. The CeO2 nanorod precursor is dispersed in deionized water and subjected to hydrothermal treatment at 170°C for 12 hours. The yellow product is collected by centrifugation, washed, and dried to obtain CeO2 nanorods.

3. The method for preparing the MnOx-CeOx nanozyme rich in oxygen vacancy structure according to claim 1, wherein: The operating conditions of the atomic layer deposition technology are: The deposition temperature of MnOx was set at 250 °C, nitrogen was used as the carrier gas and purge gas, the pulse time and exposure time of di(ethylcyclopentadienyl)manganese(II) and O3 were 1.2 s, 9 s and 25 s, respectively, and the number of ALD cycles of MnOx was 30.

4. The method for preparing the MnOx-CeOx nanozyme rich in oxygen vacancy structure according to claim 1, wherein: The conditions for high-temperature H2 reduction of the MnOx-CeO2 nanozyme are: activating the MnOx-CeO2 nanozyme at 300°C for 1.5h in a 10% H2 / Ar atmosphere to obtain the MnOx-CeOx nanozyme rich in oxygen vacancy structure.

5. A MnOx-CeOx nanozyme rich in oxygen vacancy structure, characterized in that: The nanozyme is prepared by the method for preparing the MnOx-CeOx nanozyme rich in oxygen vacancy structure according to any one of claims 1 to 4.

6. Use of the MnOx-CeOx nanozyme rich in oxygen vacancy structure as claimed in claim 5 in the preparation of antibacterial materials.

7. The use according to claim 6, characterized in that The antibacterial material is used for disinfecting Staphylococcus aureus and Escherichia coli.

Images