Fe / mn-snc bi-atom nanomaterial with multiple enzyme activities, and preparation method and use thereof

Abstract

The application belongs to the technical field of medical nanomaterials, and particularly relates to a Fe / Mn-SNC double-atom nanomaterial with multiple enzyme activities and a preparation method and application thereof. The Fe / Mn-SNC double-atom nanomaterial is designed and developed by using a local fine regulation strategy, fine regulation of a host surface and a spatial structure is carried out to bear the relationship between metal double active sites, a Yolk-Shell structure double-atom nanoenzyme is synthesized by using a one-step pyrolysis method and a ligand etching pyrolysis strategy, the nanoenzyme has stable structural properties, rich active sites and maximum enzyme-like activity. The nanomaterial has multiple enzyme activities, can induce a cascade reaction of itself, generate rich ROS, has an antibacterial effect and does not have drug resistance, has strong universality, and therefore has important scientific significance and great commercial value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical nanomaterials, and particularly relates to a Fe / Mn-SNC double monatomic nanomaterial with multiple enzyme activities and a preparation method and application thereof. BACKGROUND

[0002] After billions of years of evolution, natural enzymes exhibit multifunctional catalytic ability and can perform efficient cascade reactions in different biological systems while maintaining excellent product selectivity. However, some inherent fragilities of natural enzymes, such as difficulty in preparation, variability, high cost, and difficulty in recovery and preservation, hinder their practical application in the biomedical field.

[0003] In order to solve these problems, since the 1970s, people have been trying to artificially mimic enzymes by integrating metal cofactors and catalytic active sites into scaffolds (mainly proteins), which are called artificial nanoscale enzymes. Under this background, many nanocatalysts called single-atom catalysts (SAC) with uniform coordination catalytic sites have been widely synthesized, characterized and realized. This constantly developing toolbox provides new ways for biological scientists to re-examine complex enzyme catalysis and design advanced artificial enzymes.

[0004] Based on this, the inventors designed and developed a new Fe / Mn-SNC double monatomic nanomaterial with multiple enzyme activities. Research has found that the Fe / Mn-SNC can undergo cascade reactions and generate ROS without external ROS. It can quickly kill bacteria and has no drug resistance, and has strong universality. SUMMARY

[0005] In order to overcome the defects and deficiencies existing in the prior art, the application provides a new Fe / Mn-SNC double monatomic nanomaterial with multiple enzyme activities and a preparation method and application thereof. The material can quickly kill bacteria (especially methicillin-resistant Staphylococcus aureus, MRSA), and has no drug resistance, so it has important scientific significance and great commercial value.

[0006] In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

[0007] In a first aspect, the application provides a preparation method of a Fe / Mn-SNC double monatomic nanomaterial with multiple enzyme activities, which comprises the following steps:

[0008] Step 1: Preparation of powder FeMn-MOF: 1-5 g Zn(N03)2-6H20 and 20-80 mg Mn(OAc)2and 20-80 mg Fe(N03)2-9H20 were simultaneously added into 50-100 mL organic solvent, stirring, to prepare a metal precursor solution; 1-5 g 2-methylimidazole was dissolved in 50-100 mL organic solvent to prepare a ligand solution; then, the above metal precursor solution was immediately added into the above ligand solution, continuous stirring, washing with organic solvent for several times, and drying in a vacuum oven overnight to obtain powder FeMn-MOF;

[0009] Step 2: Preparation of FeMn-SNC DASC by one-step pyrolysis strategy: 10-50 mg of sulfur powder was uniformly mixed with 0.5-5 g of dicyandiamide, loaded into a porcelain boat and placed in the upstream of a tube furnace, 50-150 mg of FeMn MOF powder was placed in a porcelain boat and placed in the downstream of the tube furnace, then pyrolysis at 800-1200 °C under Ar atmosphere at a rate of 5 °C min -1 for a period of time to obtain FeMn-SNC DASC.

[0010] As an optional way, in the above preparation method, the preparation method comprises the following steps:

[0011] Step 1: Preparation of powder FeMn-MOF: 2-4 g Zn(N03)2-6H20 and 50-70 mg Mn(OAc)2and 50-70 mg Fe(N03)2-9H20 were simultaneously added into 70-90 mL organic solvent, stirring for 20-40 min, to prepare a metal precursor solution; 2-4 g 2-methylimidazole was dissolved in 70-90 mL organic solvent to prepare a ligand solution; then, the above metal precursor solution was immediately added into the above ligand solution, continuous stirring for 4-6 h, washing with organic solvent for several times, and drying in a vacuum oven overnight to obtain powder FeMn-MOF;

[0012] Step 2: Preparation of FeMn-SNC DASC by one-step pyrolysis strategy: 20-40 mg of sulfur powder was uniformly mixed with 0.5-2 g of dicyandiamide, loaded into a porcelain boat and placed in the upstream of a tube furnace, 80-120 mg of FeMn MOF powder was placed in a porcelain boat and placed in the downstream of the tube furnace, then pyrolysis at 900-1100 °C under Ar atmosphere at a rate of 5 °C min -1 for 1-5 h to obtain FeMn-SNC DASC.

[0013] As an optional way, in the above preparation method, the preparation method comprises the following steps:

[0014] Step 1: Preparation of powder FeMn-MOF: 2.94 g Zn(N03)2-6H20 and 60 mg Mn(OAc)2+ 60 mg Fe(N03)2-9H20 were simultaneously added into 80 mL anhydrous methanol, stirred for 30 min, and a metal precursor solution was prepared. 3.24 g 2-methylimidazole was dissolved in 80 mL methanol solution, and a ligand solution was prepared. Then, the above metal precursor solution was immediately added into the above ligand solution, and stirring was continued for 5 h. Washing with methanol was performed three times, and drying in a vacuum oven at 60 °C overnight gave powder FeMn-MOF.

[0015] Step 2: Preparation of FeMn-SNC DASC using one-step pyrolysis strategy: 30 mg of sulfur powder was uniformly mixed with 1 g of dicyanediamine, loaded into a ceramic boat, and placed in the upstream of a tube furnace. 100 mg of FeMn MOF powder was placed in a ceramic boat and placed in the downstream of the tube furnace, and then pyrolysis was performed at 1000 °C under Ar atmosphere at a rate of 5 °C min -1 for 2 h to obtain FeMn-SNC DASC.

[0016] As an optional mode, in the above preparation method, the organic solvent is methanol.

[0017] In a second aspect, the present application provides a Fe / Mn-SNC biatomic nanomaterial prepared by the preparation method of the first aspect.

[0018] As an optional mode, in the above nanomaterial, the nanomaterial has a Yolk-Shell structure.

[0019] As an optional mode, in the above nanomaterial, the multiple enzyme activities include activities of oxidase-like (OXD), glutathione peroxidase-like (GPx), superoxide dismutase-like (SOD), and peroxidase-like (POD).

[0020] As an optional mode, in the above nanomaterial, the multiple enzyme activities are activities of oxidase-like (OXD), glutathione peroxidase-like (GPx), superoxide dismutase-like (SOD), and peroxidase-like (POD).

[0021] In a third aspect, the present application provides a use of the nanomaterial of the second aspect in preparing an antibacterial agent for removing bacteria.

[0022] As an optional mode, in the above use, the antibacterial agent has a broad-spectrum antibacterial effect.

[0023] As an optional mode, in the above use, the bacteria include Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

[0024] Compared with the prior art, the present application has the following advantages and positive effects:

[0025] (1) The Fe / Mn-SNC double monatomic atom designed and developed by the present application adopts a local fine regulation strategy, finely adjusts the relationship between the host surface and the space structure to bear the metal double active sites, and through a one-step pyrolysis method, a Yolk-Shell structure double monatomic nanometer enzyme is synthesized by the strategy of ligand etching pyrolysis, which has stable structure and property and rich active sites, and can maximize the enzyme-like activity.

[0026] (2) The nanomaterial designed and developed by the present application has multi-enzyme activity, can trigger its own cascade reaction, thereby generating abundant ROS, playing an antibacterial (especially methicillin-resistant Staphylococcus aureus, MRSA) role, and has no drug resistance, strong universality, and therefore has important scientific significance and great commercial value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Preparation and characterization of Fe / Mn-SNC double monatomic nanomaterials. Figure 1 A: flow chart. Process of synthesizing Fe / Mn-SNC. Figure 1 B and Figure 1 C: Transmission electron microscopy (TEM) images clearly show that Fe / Mn-SNC has a Yolk-Shell structure, including a rough, concave shell and an internal dense carbon layer. By scanning electron microscopy, similar phenomena can be observed on a larger scale. Figure 1 D: Energy dispersive x-ray spectroscopy mapping shows that Fe and Mn are uniformly dispersed, and so are S, C and N elements. Figure E: In order to further determine the dispersion of metal species on the yolk-shell carbon framework, AC-STEM (aberration-corrected STEM) was used. Figure F: The yellow oval-shaped marked atomic dispersed bright spots can be assigned to S, N, C and other heavier Fe-Mn double sites, confirming that the Fe-Mn pairs on the yolk-shell carbon framework are indeed atomically dispersed. Figures G and H: In order to visualize the Fe-Mn atomic pairs, two-dimensional (2D) and three-dimensional (3D) resolution maps were used to simulate them, and the results showed that the positions of the Fe-Mn pairs showed strong color signals.

[0028] Figure 2 Multi-enzyme simulation activity of Fe / Mn-SNC double monatomic nanomaterials. Figure 2 A: Oxidase-like principle diagram. It is illustrated that Fe / Mn-SNC can oxidize the most common substrates TMB\OPD\ABTS under the action of O2 without adding H2O2. Figure 2 B: Ultraviolet wavelength diagram of oxidizing three substrates. Figure 3C: The main sample is compared with the control sample to compare the properties of oxidase using TMB oxidation as an example. The absorbance from high to low: Fe / Mn-SNC > Fe-N4S > Mn-N4S > Fe-N4 > Mn-N4 > control. Figure 2 D: Glutathione-like oxidation schematic diagram. Figure 2 E: The ability of the main sample and the control sample to oxidize GSH is detected. The absorbance from high to low: control > Mn-N4 > Fe-N4 >> Mn-N4S > Fe-N4S > Fe / Mn-SNC. The ability to consume GSH: Fe / Mn-SNC > Fe-N4S > Mn-N4S > Fe-N4 > Mn-N4 > control. Figure 2 F: The ability of the main sample to consume GSH. Change the enzyme amount of Fe / Mn-SNC to 0, 10, 15, 20 μg / mL. The kinetic curve of the main sample's ability to consume GSH, detect the decrease of absorbance between 0-400S. Figure 2 G: Superoxide dismutase-like schematic diagram. Superoxide dismutase can react with ·O2 - to generate H2O2. Figure 2 H: SOD kit principle curve for Fe / Mn-SNC. Figure 2 I: The higher the inhibition rate of SOD kit to ·O2 - , the better the SOD-like activity. Figure 2 J: H2O2 and TMB react under the action of enzyme schematic diagram. Figure 2 K: Comparison of absorbance of Fe / Mn-SNC + H2O2 + pH + TMB, Fe / Mn-SNC + pH + TMB, pH + TMB. Figure 2 L: The main sample is compared with the control sample to compare the properties of peroxidase using TMB oxidation as an example. The absorbance from high to low: Fe / Mn-SNC > Fe-N4S > Mn-N4S > Fe-N4 > Mn-N4 > control. Figure 2 M: Summary of peroxidase enzyme activity and Michaelis constant of main sample and control sample. Figure 2 N: ESR measurement spectrum of peroxidase system. Figure 3 O: ESR measurement spectrum of oxidase system.

[0029] Figure 3 : Biocompatibility and in vitro antibacterial activity of Fe / Mn-SNC double-atom nanomaterial. Figure 3 A: Survival rate of E. coil and MRSA treated with Fe / Mn-SNC. Figure 3 B: Cell viability of L929 cells treated with Fe / Mn-SNC. Figure 3 C: Live / dead cell staining fluorescence image of L929 cells treated with Fe / Mn-SNC for 3 days. Figure 3D: Hemolysis results of NaCl, Triton X and Fe / Mn-SNC. Figure 3 E: Photographs of MRSA and E. coil colonies after different treatments. Figure 3 F: SEM images of MRSA and E. coil after different treatments. Yellow arrows indicate the destruction of bacterial cell membranes. Figure 3 G: Live / dead staining fluorescence images of MRSA and E. coil after Fe / Mn-SNC treatment. Figure 3 H: Quantitative analysis of ROS in MRSA after Fe / Mn-SNC treatment. ****p<0.0001. Figure 4 I: UV-Vis absorption spectra and photographs of DNTB in MRSA treated with Fe / Mn-SNC for different times. Figure 4 J: Survival rate of MRSA bacteria after adding different free radical scavengers.

[0030] Figure 4 : Therapeutic effect of Fe / Mn-SNC bi-monatomic nanomaterials on MRSA infected skin wounds. Figure 4 A: Schematic diagram of MRSA infected skin wound mouse model and treatment process. Figure 4 B: Photographs of mouse wounds at different treatment times. Figure 4 C: Changes in wound area of mice after different treatments at different time periods (1st, 5th, 8th, 12th day). **p<0.01 and ****p<0.0001. Figure 4 D: Rheograms of wounds treated by different methods at different time periods. Figure 4 E: Photographs of agar plates of MRSA colonies after different treatments on the 1st and 12th days. Figure 1 F and Figure 2 G: H&E staining and Masson staining images of wound tissues of mice after different treatments on the 12th day. DETAILED DESCRIPTION

[0031] The present application will be further described below with reference to specific examples. It should be understood that the specific examples described herein are intended to explain the present application and are not intended to limit the scope of the present application.

[0032] Unless otherwise specified, the techniques or conditions in the examples are in accordance with those described in the literature or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available products unless otherwise specified.

[0034] Example 1: Preparation and characterization of Fe / Mn-SNC bi-atom nanomaterials

[0035] Preparation method:

[0036] Step 1: Preparation of powder FeMn-MOF: 2.94 g Zn(NO3)2·6H2O and 60 mg Mn(OAc)2+ 60 mg Fe(NO3)2·9H2O were simultaneously added to 80 mL of anhydrous methanol, stirred for 30 min, and a metal precursor solution was prepared. 3.24 g of 2-methylimidazole was dissolved in 80 mL of methanol solution to prepare a ligand solution. Then, the above metal precursor solution was immediately added to the above ligand solution, and stirring was continued for 5 h. Washing with methanol three times and drying in a vacuum oven at 60°C overnight to obtain powder FeMn-MOF.

[0037] Step 2: Preparation of FeMn-SNC DASC using one-step pyrolysis strategy: 30 mg of sulfur powder was uniformly mixed with 1 g of dicyandiamide, loaded into a porcelain boat and placed upstream of the tube furnace, 100 mg of FeMn MOF powder was placed in a porcelain boat and placed downstream of the tube furnace, then pyrolysis was carried out at 1000°C under Ar atmosphere at a rate of 5°C / min for 2 h to obtain FeMn-SNC DASC. -1

[0038] In the above preparation method, the specific preparation process of Fe / Mn-SNC is as shown in Figure 2 A. Fe and Mn precursors were used as metal nodes, and 2-methylimidazole was used as a coordination linker to synthesize a typical Fe-Mn bimetallic organic framework (FeMn-MOF) polyhedron in a methanol solvent through a self-assembly process. A one-step pyrolysis method was used, in which sulfur powder and dicyandiamide were placed upstream of the tube furnace, and FeMn-MOF was placed downstream. Pyrolysis was carried out at 1000°C under argon for two hours. In the process of pyrolysis, the original ordered structure of MOF was destroyed, the etching of gas S ligand to the surface structure of MOF was accelerated, the initial shrinkage of the internal cavity of MOF was promoted, N2 promoted the shrinkage of the internal carbon layer, and a yolk-shell structure was formed, in which the surface was surrounded by a graphene-like framework and the inside was a carbon core. Due to the precise control of the feedstock, atomic-level Fe and Mn can be accurately formed. Quantitatively, inductively coupled plasma optical emission spectrometry (ICP OES) confirmed that the mass ratio of Fe (1.35 wt%) and Mn (1.24 wt%) was close to 1:1.

[0039] ​Transmission electron microscopy (TEM) and high-angle annular dark-field scanning TEM (HAADF-STEM) images clearly show that Fe / Mn-SNC has a yolk-shell structure (Figs. B and C), consisting of a rough, concave outer shell and a dense inner carbon layer. Energy-dispersive X-ray spectroscopy (EDS, Fig. D) mapping shows that Fe and Mn are uniformly dispersed, as are S, C, and N elements, providing evidence for the successful incorporation of sulfur into the carbon material. To further determine the dispersion of metal species within the yolk-shell carbon framework, anomaly-corrected STEM (AC-STEM) was used to analyze Fe / Mn-SNC (Fig. E). As shown in Fig. F, many bright Fe / Mn biatomic pairs are uniformly dispersed, indicated by yellow circles. Analysis of the biatomic distances between Fe and Mn atoms indicates a direct interaction between them. To visualize the Fe-Mn atomic pairs, three-dimensional (3D) and two-dimensional (2D) resolution maps were fabricated, revealing a strong color signal representing the location of the Fe-Mn pairs (Figs. G and H).

[0040] Example 2: Multienzyme mimicry activity of Fe / Mn-SNC dual single-atom nanomaterials

[0041] In Example 2, the multi-enzyme mimicry activities of the Fe / Mn-SNC prepared in Example 1 were investigated, including the activities of oxidase-like (OXD), glutathione oxidase-like (GPx), superoxide dismutase-like (SOD), and peroxidase-like (POD).

[0042] First, the OXD-simulated activity of Fe / Mn-SNC was evaluated, demonstrating its ability to catalyze the oxidation of the most common chromogenic substrates (TMB, OPD, and ABTS) in the presence of O2. Figure 2 As shown in Figure A, Fe / Mn-SNC can rapidly oxidize O2 in the absence of any oxidant, producing ·O2 - This causes the colors of OPD, TMB, and ABTS to range from colorless to yellow, blue, and green, and the characteristic peaks to appear in the UV-Vis absorption spectra at 417, 652, and 420 nm. Figure 2 B). In Figure 2C. In C, the absorbance of five catalysts was compared using oxidized substrate TMB as an example. The order of the experimental sequence of the oxidase-like activity was Fe / Mn-SNC > Fe-N4S > Mn-N4S > Fe-N4 > Mn-N4 > control. Obviously, Fe / Mn-SNC has significantly higher oxidase-like activity compared with other reference catalysts. It is well known that GSH (0.1-10 mM) exists in bacterial cells, which can neutralize ROS and reduce the antibacterial efficiency. Therefore, the consumption of GSH can enhance the ROS-based antibacterial effect. With the increase of Fe / Mn-SNC concentration, the absorption intensity at 412 nm decreased significantly, which confirmed its GPx mimetic activity Figure 2 F. In addition, Fe / Mn-SNC showed intrinsic SOD mimetic activity, which can be demonstrated from the measurement of hydrogen peroxide produced by ·O2 - consumption Figure 2 G. The experiment showed that ·O2 - Nitro blue tetrazolium (NBT) can be reduced to blue formate salt, which has a characteristic absorption peak at about 600 nm. Fe / Mn-SNC can eliminate ·O2 - , inhibit the generation of formate salt, resulting in a decrease in absorbance compared with the control (without Fe / Mn-SNC) Figure 2 H. The higher the inhibition rate, the better the SOD-like activity. The order of SOD-like activity of the samples was Fe / Mn-SNC > Fe-N4S > Fe-N4 > Mn-N4S > Mn-N4 Figure 2 I. The POD mimetic activity of Fe / Mn-SNC was detected by TMB oxidation colorimetry. Fe / Mn-SNC effectively catalyzed the generation of ·OH from hydrogen peroxide Figure 2 J. As shown in Figure 2 k, Fe / Mn-SNC-TMB group has a significant absorption peak at 652 nm, while the absorption peak of Fe / Mn-SNC-TMB-H2O2 at 652 nm can be further enhanced. The specific activity (SA) was determined to verify the synergistic catalysis between Fe and Mn single atoms. As shown in Figure 2 M, the SA of Fe / Mn-SNC (54.63 U / mg) is higher than that of Fe-N4S (35.70 U / mg), Mn-N4S (11.34 U / mg), Fe-N4 (2.16 U / mg) and Mn-N4 (0.62 U / mg), respectively, indicating that Fe / Mn-SNC has excellent enzymatic performance. The effect of Fe, Mn double atom sites on enzyme activity and the steady-state kinetics of the main sample (Fe / Mn-SNC) and reference groups (Fe-N4S, Mn-N4S, Fe-N4 and Mn-N4) were studied Figure 2M). As expected, Fe / Mn-SNC nanoszyme has better Michaelis constant (K m , 0.17 mM, V max , 47.46 x 10 -8 Ms -1 ) for TMB substrate and Michaelis constant (K m , 18.79 mM, V max , 95.37 x 10 -8 Ms -1 ) for H2O2 Figure 3 M) than other reported nanoszymes. In order to evaluate the synergistic enzyme catalytic mechanism of electron spin resonance (ESR), the generated ROS in the reaction process was characterized by electron spin resonance (ESR) and fluorescence method. According to the previous reports, the trapping agent 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO) was used to verify the existence of active substances (·O2 - and ·OH). In Figure 3 N, in addition to detecting ·O2 - with a specific six-peak pattern, ·OH with a specific four-peak pattern was also detected by ESR determination of OXD-like reaction without hydrogen peroxide. Therefore, it is proved that the inventors' hypothesis that Fe / Mn-SNC has multiple enzyme properties, and due to the SOD mimetic activity of Fe / Mn-SNC, part of the generated part ·O2 - is decomposed into hydrogen peroxide. Similarly, ·OH with a specific four-peak pattern was detected by ESR in the POD-like reaction Figure 3 O).

[0043] Example 3: Biocompatibility and in vitro antibacterial activity of Fe / Mn-SNC biogenic single-atom nanomaterials

[0044] The minimum inhibitory concentration of Fe / Mn-SNC was explored by taking gram-negative bacteria-E. coli and gram-positive bacteria-methicillin-resistant Staphylococcus aureus (MRSA) as representative strains, and the results showed that when the concentration of Fe / Mn-SNC reached 100 μg mL -1 , the survival rate of the two strains was less than 5%, which proved that it had good antibacterial effect. Therefore, 100 μg mL -1 of Fe / Mn-SNC was selected for subsequent experiments Figure 3 A).

[0045] Good biocompatibility is an important factor for the development of biomaterials. In the first part of the experiment, the cytotoxicity of the minimum inhibitory concentration of Fe / Mn-SNC was evaluated by cell proliferation-cytotoxicity detection kit (CCK-8). The L929 cells from ATCC were mixed with 100 μg mL -1After 1, 2, 3 days of co-incubation with Fe / Mn-SNC, it was found that the cell survival rate of L929 cells co-cultured with Fe / Mn-SNC had no significant difference compared with the blank control L929 cells, and the cell survival rate was more than 95% during the three-day culture period Figure 3 B). In order to further observe the effect of Fe / Mn-SNC on the proliferation of L929 cells, the cells co-cultured with Fe / Mn-SNC for 1, 2, 3 days were subjected to fluorescence staining, and the staining results showed that L929 cells maintained healthy cell morphology and the number of cells increased during the three-day culture period, with good proliferation activity Figure 3 C). This indicates that Fe / Mn-SNC has good biocompatibility at the cellular level.

[0046] In addition, in vitro hemolysis test was used to evaluate the blood compatibility of Fe / Mn-SNC. Figure 3 The pictures in D show the obvious color difference between the negative control (NaCl), Fe / Mn-SNC and the positive control (Triton X). The color of the Fe / Mn-SNC group is transparent, similar to the negative control group, while the Triton X group is bright red. The quantitative results show that the hemolysis rate of the Fe / Mn-SNC group is very low (2.68%), which has no significant difference with the negative control group, indicating that Fe / Mn-SNC has good blood compatibility.

[0047] In the second part of the experiment, the antibacterial activity of Fe / Mn-SNC against MRSA and E. coli was first studied by plate culture method. Figure 3 E shows that no colonies were formed in the Fe / Mn-SNC and Ag groups, while the untreated bacteria still had a large number of colonies growing. This indicates that Fe / Mn-SNC has good antibacterial effect on MRSA and E. coli. In order to further explore its antibacterial performance, scanning electron microscopy was used to observe the morphology of the bacteria, as shown in Figure 3 F, MRSA and E. coli were smooth before treatment, and were spherical and rod-shaped, respectively. In contrast, the surfaces of the two bacteria treated with Fe / Mn-SNC became rough and wrinkled or severely ruptured, lost the complete skeleton, and even caused some mutual adhesion between adjacent bacteria, with yellow arrows indicating different degrees of damage. At the same time, confocal laser scanning microscopy (CLSM) was used to directly evaluate the antibacterial activity of Fe / Mn-SNC, and the bacteria were stained using a bacterial live / dead staining kit. The live bacteria showed green color, and the dead bacteria showed red color. As shown in Figure 4As shown in FIG. 6, before Fe / Mn-SNC treatment, almost all bacteria were alive, while after 2 h Fe / Mn-SNC treatment, clear red fluorescence was observed for both MRSA and E. coli, indicating that most of the bacteria were killed. These results confirmed that Fe / Mn-SNC had good antibacterial activity against MRSA and E. coli.

[0048] In addition, the antibacterial mechanism of Fe / Mn-SNC was further explored. To evaluate the ROS production, dichlorofluorescein (DCFH-DA) was used as an indicator of intracellular ROS. Compared with the untreated group, the fluorescence intensity of MRSA after Fe / Mn-SNC treatment increased by 2.2 times, indicating that Fe / Mn-SNC induced the production of ROS in MRSA strains Figure 4 H) It is known that high glutathione (GSH) levels at the site of bacterial infection can consume a large amount of ROS produced by nanoszymes. Therefore, consumption of GSH at the site of infection can enhance the antibacterial treatment effect. The results of the GSH detection kit showed that the concentration of GSH decreased with the increase of incubation time of MRSA with Fe / Mn-SNC, indicating that Fe / Mn-SNC had effective GSH consumption ability Figure 4 I) The results of free radical scavenging experiments showed that after H2O2, ·OH, ·O2 - and 1 O2 were scavenged, the survival rate of MRSA bacteria increased, which indicated that H2O2, ·OH, ·O2 - and 1 O2 played a crucial role in the antibacterial process of Fe / Mn-SNC Figure 4 J).

[0049] These results are consistent with the above-mentioned enzyme catalytic activity and antibacterial performance, and fully demonstrate that Fe / Mn-SNC has good multi-enzyme bactericidal performance and can produce abundant toxic ROS and consume GSH. It is worth noting that Fe / Mn-SNC has a wide range of antibacterial effects.

[0050] Example 4: Therapeutic effect of Fe / Mn-SNC double-atom nanomaterials on MRSA-infected skin wounds

[0051] Inspired by the excellent antibacterial activity of Fe / Mn-SNC in vitro, the inventors constructed a mouse model of MRSA-infected skin wounds to evaluate the in vivo therapeutic effect of Fe / Mn-SNC Figure 4A). 6-8 weeks old female Balb / C mice were chosen, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and placed in the animal barrier laboratory of China Institute of Rehabilitation Science under controlled conditions. Specifically, a MASR infection wound with a diameter of about 0.8 cm was established on the back of the mouse, and wound photos under different treatments were provided to evaluate the healing progress. The experimental animals were randomly divided into PBS group, Ag treatment group and Fe / Mn-SNC treatment group, 5 in each group.

[0052] Notably, compared with the PBS group and the Ag group, the wound area of the Fe / Mn-SNC group decreased significantly faster, showing faster wound healing and less scar formation on the 12th day ( Figure 4 B、 Figure 4 C、 ​ D). The wound tissue of the mouse was collected at the 1st and 12th day of treatment for plate counting experiment. ​ The photos in E show that at the 1st day of treatment, a large number of colonies were produced in the three groups, which indicates the successful construction of the MRSA infection model. However, at the 12th day of treatment, the number of colonies in the Fe / Mn-SNC group was significantly lower than that in the other groups, which explains why the wound healing in the Fe / Mn-SNC group was the fastest.

[0053] Subsequently, hematoxylin-eosin (H&E) and Masson staining were used to observe the histological changes of the mouse wound after 12 days of different treatment, to further evaluate the wound healing. The results showed that the wound in the Fe / Mn-SNC group had no signs of inflammation, with more new hair follicles and a more complete epidermis formed ( ​ F), and the collagen fibers were more dense ( ​ G). These results show that Fe / Mn-SNC treatment has better bactericidal effect, thus promoting wound healing.

[0054] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for preparing Fe / Mn-SNC dual single-atom nanomaterials with multi-enzyme activity, characterized in that: The preparation method comprises the following steps: Step 1: preparing the powder FeMn-MOF: 1-5 g of Zn(NO3)2·6H2O and 20-80 mg of Mn(OAc)2 and 20-80 mg of Fe(NO3)2·9H2O are simultaneously added into 50-100 mL of an organic solvent, stirring to prepare a metal precursor solution; 1-5 g of 2-methylimidazole is dissolved in 50-100 mL of an organic solvent to prepare a ligand solution; then, the above metal precursor solution is immediately added into the above ligand solution, continuous stirring, washing with an organic solvent for several times, and drying in a vacuum oven overnight to obtain the powder FeMn-MOF; Step 2: Preparation of FeMn-SNC double-atom (DASC) using one-step pyrolysis strategy: 10-50 mg of sulfur powder was uniformly mixed with 0.5-5 g of dicyandiamide in a porcelain boat and placed in the upstream of the tube furnace, 50-150 mg of FeMn MOF powder was placed in a porcelain boat and placed in the downstream of the tube furnace, then pyrolysis at 800-1200 °C for a period of time at a rate of 5 °C min -1 under Ar atmosphere to obtain FeMn-SNC DASC.

2. The method of claim 1, wherein: The preparation method comprises the following steps: Step 1: preparing the powder FeMn-MOF: 2-4 g of Zn(NO3)2·6H2O and 50-70 mg of Mn(OAc)2 and 50-70 mg of Fe(NO3)2·9H2O are simultaneously added into 70-90 mL of an organic solvent, stirring for 20-40 min to prepare a metal precursor solution; 2-4 g of 2-methylimidazole is dissolved in 70-90 mL of an organic solvent to prepare a ligand solution; then, the above metal precursor solution is immediately added into the above ligand solution, continuous stirring for 4-6 h, washing with an organic solvent for several times, and drying in a vacuum oven overnight to obtain the powder FeMn-MOF; Step 2: Preparation of FeMn-SNC DASC using one-step pyrolysis strategy: 20-40 mg of sulfur powder was homogeneously mixed with 0.5-2 g of dicyandiamide in a porcelain boat and placed in the upstream of the tube furnace, 80-120 mg of FeMn MOF powder was placed in a porcelain boat and put in the downstream of the tube furnace, then pyrolysis was carried out at 900-1100 °C under Ar atmosphere at a rate of 5 °C min -1 for 1-5 h to obtain FeMn-SNC DASC.

3. The method of claim 1, wherein: The organic solvent is methanol.

4. The Fe / Mn-SNC biatomic nanomaterial prepared by the preparation method according to any one of claims 1 to 3.

5. The nanomaterial of claim 4, wherein: The nanomaterial has a Yolk-Shell structure.

6. The nanomaterial of claim 4, wherein: The multi-enzyme activity comprises the activities of oxydase (OXD), glutathione peroxidase (GPx), superoxide dismutase (SOD) and peroxidase (POD).

7. Use of the nanomaterial according to any one of claims 4 to 6 in the preparation of an antibacterial agent for scavenging bacteria.

8. Use according to claim 7, characterized in that: The antibacterial agent has a broad-spectrum antibacterial effect.

9. Use according to claim 7, characterized in that: The bacteria include Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

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