A bimetallic single-atom nanozyme and a preparation method and application thereof

Abstract

The present invention relates to a bimetallic single-atom nanozyme, its preparation method, and application. The bimetallic single-atom nanozyme has the simplified formula of ZnFe-NC and is composed of the following components in atomic percentage: Zn 0.5% to 5%; Fe 0.05% to 2%; C 60% to 70%; N 15% to 25%; and O 10% to 20%.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of bimetallic single atom nanozyme for inflammation wound treatment and preparation method thereof, belong to nanometer material technical field. BACKGROUND

[0002] The incidence of inflammatory wounds such as wound sepsis, diabetic foot and burn is gradually increasing, and its high mortality rate poses a great threat to people's physical and mental health. When the skin is subjected to severe external stimuli, the antioxidant system in the tissue is disturbed, oxidative stress is generated, and the level of reactive oxygen species (ROS) in the tissue is abnormally elevated, resulting in massive cell death. Therefore, removing excess ROS from skin tissue and resisting oxidative stress can effectively treat inflammatory wounds and promote wound healing and regeneration. Currently, ROS removal is mainly achieved by delivering non-enzymatic antioxidants or natural antioxidant enzymes. However, non-enzymatic antioxidants have insufficient removal capacity and need to be delivered in large doses and continuously to inhibit the abnormal increase of ROS. Natural antioxidant enzymes, although having strong removal capacity, are easily inactivated and have complex preparation and low yield, limiting their clinical application. Therefore, functional materials that can effectively remove ROS and alleviate inflammatory response need to be further designed.

[0003] In recent years, single atom nanozymes with metal atoms as enzyme-like catalytic active sites not only have a designable geometric structure and electronic coordination, but also can effectively simulate the metal active center of natural antioxidant enzymes at the atomic level, and have excellent structural stability, enabling efficient and persistent ROS removal, showing great potential in inflammation treatment. However, the existing single atom nanozymes require pyrolysis and acid washing during preparation, which is a complex process and the resulting material has poor dispersibility, which is not conducive to its medical application. At the same time, the exploration of single atom nanozymes in biomedical applications has mainly focused on peroxidase-like and oxidase-like nanozymes that generate free radicals, especially the use of such enzyme activity to respond to the acidic environment of tumors to generate free radicals for tumor treatment research is particularly widespread, and there is a lack of research and application of single atom nanozymes in free radical removal. SUMMARY

[0004] To solve the above problems in the prior art, the purpose of the present application is to provide a bimetallic single atom nanozyme with multiple free radical removal capabilities and a preparation method thereof. The bimetallic single atom nanozyme of the present application has excellent ROS removal capacity and immunomodulatory capacity, and can be effectively applied to inflammation treatment.

[0005] In one aspect, the present application provides a bimetallic single atom nanozyme, the simple formula of which is ZnFe-NC, and consists of the following components in atomic percentage: Zn 0.5% to 5%; Fe 0.05% to 2%; C 60% to 70%; N 15% to 25%; O 10% to 20%.

[0006] Preferably, the nanoszyme in the bimetallic single-atom nanoszyme has a nitrogen-carbon skeleton structure, and the bimetallic single atoms are Zn atoms and Fe atoms, wherein the Zn atoms and the Fe atoms are in an oxidation state, are distributed in a monodisperse form in the nitrogen-carbon skeleton, and are connected to the nitrogen-carbon skeleton structure in the form of Zn-N and Fe-N coordination, respectively.

[0007] Preferably, the N atoms mainly exist in the form of pyridine nitrogen and pyrrole nitrogen; and the C atoms mainly exist in the form of C=N, C-C and C-N.

[0008] Preferably, when the content of Fe is unchanged, as the content of Zn increases, the crystallinity of the bimetallic single-atom nanoszyme ZnFe-NC gradually weakens, and the micro-morphology gradually changes from a sheet shape to a granular shape.

[0009] Preferably, the size of the ZnFe-NC is ≤1 μm; and when the content of Fe is unchanged, as the content of Zn increases, the size of the bimetallic single-atom nanoszyme gradually decreases.

[0010] In another aspect, the present application provides a preparation method of a bimetallic single-atom nanoszyme, comprising:

[0011] (1) adding iron salt and zinc salt into formamide or a mixture of formamide and other organic solvents, ultrasonically dissolving to obtain solution A;

[0012] (2) carrying out a solvothermal reaction of the solution at 200-240°C, and then cooling, washing and drying to obtain the bimetallic single-atom nanoszyme ZnFe-NC. The preparation principle is explained as follows: formamide contains both amino and aldehyde groups. Under specific conditions, N atoms attack C atoms to occur intermolecular nucleophilic addition reaction, i.e. Schiff base reaction, to form unsaturated N-C=N bonds, and meanwhile, the lone pair electrons of the outer layer of the N atoms are chelated with metal atoms (M) to form M-N coordination bonds. The preparation method is a one-step solvothermal method, and does not need to be subjected to calcination and other deoxidation treatments, so that the metal nitrogen-carbon (M-NC) single-atom nanoszyme is prepared while retaining a large amount of oxygen-containing functional groups. The large amount of oxygen-containing functional groups ensures the stability of the single-atom nanoszyme in a physiological environment, and also can act as an electron donor to directly scavenge active oxygen.

[0013] Preferably, in step (1), the iron salt is at least one selected from ferric chloride, ferrous chloride, ferric sulfate and ferric acetate; and the zinc salt is at least one selected from zinc chloride, zinc sulfate, zinc nitrate and zinc acetate.

[0014] Preferably, in step (1), the iron salt is at least one selected from ferric chloride, ferrous chloride, ferric sulfate and ferric acetate; and the zinc salt is at least one selected from zinc chloride, zinc sulfate, zinc nitrate and zinc acetate.

[0015] Preferably, in step (1), the molar ratio of the iron salt to the zinc salt is (1-20 mM):(5-200 mM).

[0016] Preferably, in step (1), the molar ratio of the iron salt to the zinc salt is (1-20 mM):(5-200 mM).

[0017] The ratio of the formamide or mixture of formamide and other organic solvents to the iron salt is (30-50) mL:(1-20) mM;

[0018] The power of the ultrasonic dissolution is 40-320 W, and the time is 5-60 minutes

[0019] Preferably, in step (2):

[0020] The time of the solvothermal reaction is 12-24 hours.

[0021] The cooling method is air cooling, water cooling, or furnace cooling.

[0022] The washing method is differential centrifugation, with a low centrifugal speed rlow=3000-10000 rpm, a high centrifugal speed rhigh=10000-12000 rpm, rlow

[0023] The drying method is drying at 50-100 DEG C or freeze-drying at -50 DEG C to -5 DEG C.

[0024] In another aspect, the application also provides a use of the bimetallic single-atom nanoenzyme, which has oxidase-like (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities; wherein Fe-N is an enzyme-like catalytic active site, and Zn-N is a coenzyme site. The enzyme-like catalytic reaction equation is as follows:

[0025]

[0026] Preferably, the enzyme-like activity of the bimetallic single-atom nanoenzyme has pH and temperature dependence, and when the pH changes from acidic to alkaline, the enzyme-like activity changes from OXD and POD to CAT and SOD, and under physiological conditions, the bimetallic single-atom nanoenzyme exhibits CAT and SOD activities; the bimetallic single-atom nanoenzyme realizes the removal of H2O2 and ·O2 - by a CAT and SOD enzyme catalytic reaction.

[0027] Preferably, the bimetallic single-atom nanoenzyme directly removes ·OH as an electron donor.

[0028] Preferably, the bimetallic single-atom nanoenzyme is used to prepare an inflammation treatment material, so that the ZnFe-NC shows an inflammation treatment effect in various cell antioxidant models; the inflammation treatment effect includes removing ROS, down-regulating pro-inflammatory gene expression, and up-regulating anti-inflammatory gene expression; the cell antioxidant models include H2O2 antioxidant models, ROSUP antioxidant models, and LPS antioxidant models.

[0029] Further, preferably, the inflammation treatment material prepared by the bimetallic single-atom nanozyme shows an inflammation treatment effect in an animal inflammation model; the effect includes improving the survival rate of the animal, relieving multi-organ failure of the animal, and slowing down the inflammatory response; the inflammation model includes inflammatory wounds (wound sepsis, burns, diabetic wounds), kidney injury, colitis, pneumonia, central nervous system diseases, and periodontitis.

[0030] The present application has the following beneficial effects:

[0031] The present application uses a simple one-step solvothermal method to synthesize bimetallic single-atom nanozymes from inexpensive and non-toxic formamide. The synthesis of the single-atom nanozyme does not require the pyrolysis and acid washing process in the traditional single-atom nanozyme synthesis process. The synthesis method is simple, low in cost, mild and stable in conditions, and easy to mass-produce.

[0032] The synthesized bimetallic single-atom nanozyme ZnFe-NC has OXD, POD, CAT, and SOD four kinds of natural enzyme simulation activities. Compared with single-metal single-atom nanozymes Fe-NC and Zn-NC, the four kinds of enzyme-like activities of ZnFe-NC are significantly improved, solving the problems of single catalytic substrate, low efficiency, and poor stability. At the same time, the problems of instability and easy deactivation of natural enzymes, and the need for strict catalytic conditions are also solved. Among them, Fe-NC has enzyme-like activity, and Zn-NC basically has no enzyme-like activity, indicating that the enzyme-like activity of ZnFe-NC is derived from the Fe-N active site. The introduction of Zn on the one hand greatly reduces the size of Fe-NC, making the Fe-N active site fully exposed and improving its active site utilization rate; on the other hand, it forms a Zn-N structure as a coenzyme site of Fe-N, which synergistically improves its enzyme-like activity. The enzyme-like activity of the prepared ZnFe-NC has pH and temperature dependence, and under physiological conditions (pH = 7.4, T = 37℃), it exhibits CAT and SOD activities, and can realize the removal of H2O2, ·OH, and ·O2 - .

[0033] The synthesized bimetallic single-atom nanozyme ZnFe-NC has multiple free radical scavenging capacity, and the introduction of Zn can regulate its size, has good biological safety and dispersion, and has excellent inflammation treatment effect, solving the problem that the traditional single-atom nanozyme needs to be further modified with a surfactant after preparation for biomedical use. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The bimetallic single-atom nanozyme Zn 30 Fe-NC, Zn 60 Fe-NC, Zn 120TEM images of Fe-NC, NC, Zn-NC and Fe-NC obtained in Comparative Example 1, Comparative Example 2 and Comparative Example 3 (scale bar: 500 nm);

[0035] Figure 2 Bimetallic single-atom nanoszyme Zn 60 OXD-like activity of Fe-NC at different pH (abscissa: wavelength (nm), ordinate: absorbance (a.u.));

[0036] Figure 3 Bimetallic single-atom nanoszyme Zn 60 POD-like activity of Fe-NC at different pH (abscissa: wavelength (nm), ordinate: absorbance (a.u.));

[0037] Figure 4 Bimetallic single-atom nanoszyme Zn 30 Fe-NC, Zn 60 Fe-NC, Zn 120 Comparison of OXD-like activity of Fe-NC and NC, Zn-NC and Fe-NC obtained in Comparative Example 1, Comparative Example 2 and Comparative Example 3 60 CAT-like activity of Fe-NC at different concentrations (abscissa: wavelength (nm), ordinate: absorbance (a.u.));

[0038] Figure 5 Bimetallic single-atom nanoszyme Zn 30 Fe-NC, Zn 60 Fe-NC, Zn 120 Comparison of POD-like activity of Fe-NC and NC, Zn-NC and Fe-NC obtained in Comparative Example 1, Comparative Example 2 and Comparative Example 3 (abscissa: wavelength (nm), ordinate: absorbance (a.u.));

[0039] Figure 6 Bimetallic single-atom nanoszyme Zn 30 Fe-NC, Zn 60 Fe-NC, Zn 120Comparison of CAT-like activity of Fe-NC and NC, Zn-NC, Fe-NC obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3 (abscissa: Wavelength (nm), ordinate: Absorbance (a.u.));

[0040] Figure 7 Bimetallic single-atom nanoszyme Zn 60 CAT-like activity of Fe-NC at different concentrations (abscissa: Time (s), ordinate: Dissolved Oxygen (mg / L));

[0041] Figure 8 Bimetallic single-atom nanoszyme Zn 60 SOD-like activity of Fe-NC at different concentrations (abscissa: Wavelength (nm), ordinate: Absorbance);

[0042] Figure 9 Bimetallic single-atom nanoszyme Zn 60 EPR spectrum of Fe-NC scavenging ·OH (abscissa: Magnetic Field (mT), ordinate: Intensity (a.u.));

[0043] Figure 10 Bimetallic single-atom nanoszyme Zn 60 Schematic diagram of Fe-NC scavenging free radicals;

[0044] Figure 11 Bimetallic single-atom nanoszyme Zn 60 Survival curve of Fe-NC treating wound sepsis (abscissa: Time (Day), ordinate: Survival Rate (percent));

[0045] Figure 12 Bimetallic single-atom nanoszyme Zn 60 Healing of Fe-NC treating wound sepsis. DETAILED DESCRIPTION

[0046] The present application is further illustrated by the following examples, which should be understood as merely illustrative of the present application and not limiting the present application.

[0047] In the present invention, the bimetallic single-atom nanozyme ZnFe-NC for treating inflammation is composed of the following components by element mass percentage: Zn 0.5% to 5%, Fe 0.05% to 2%, C 60% to 70%, N 15% to 25%, and O 10% to 20%.

[0048] Among them, when the Fe content of ZnFe-NC is constant, its morphology changes accordingly with the change of Zn addition amount.

[0049] In the present invention, the bimetallic single-atom nanozyme ZnFe-NC has oxidase (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities at the same time. It exhibits CAT and SOD activities under physiological conditions and can realize the oxidation of H2O2, ·OH, ·O2 - of removal.

[0050] In an optional embodiment, the bimetallic single-atom nanozyme ZnFe-NC showed excellent inflammatory treatment effects in the ROSUP antioxidant model. After ZnFe-NC treatment, cell activity recovered from 30% to more than 90%.

[0051] In an optional embodiment, the bimetallic single-atom nanozyme ZnFe-NC shows excellent therapeutic effects in a mouse wound sepsis model, including improving mouse survival rate and promoting inflammatory wound healing.

[0052] The following is an illustrative example of the preparation method of the bimetallic single-atom nanozyme ZnFe-NC.

[0053] Add iron salt and zinc salt to formamide and dissolve them by ultrasonication to obtain solution A. Preferably, the iron salt and zinc salt do not contain water of crystallization, the concentration of the iron salt is 1-10mM, and the concentration of the zinc salt is not less than 60mM. Preferably, the power of the ultrasonic dissolution is 40-320W, the time is 5-30min, and the ultrasonic temperature is not higher than 40°C. As an example, 1-20mM iron salt and 5-200mM zinc salt are added to 30-50mL formamide and dissolved by ultrasonication for 5-60min to obtain solution A. The iron salt includes but is not limited to ferric chloride, ferrous chloride, ferric sulfate, ferric acetate, or a mixture of two thereof, and the zinc salt includes but is not limited to zinc chloride, zinc sulfate, zinc nitrate, or zinc acetate, or a mixture of two thereof.

[0054] Transfer Solution A to a stainless steel hydrothermal reactor, place it in an oven, and set the temperature to begin the solvothermal reaction. Preferably, the solvothermal reaction time is 12-18 hours at a temperature of 200-240°C. The reaction temperature and time are regulated based on the amount of iron salt. Higher iron content increases the reaction temperature and time.

[0055] After the reaction is completed, cool to room temperature, open the reactor, transfer the reaction liquid to a centrifuge tube, and wash and dry the obtained reaction product with a mixed solution of ultrapure water and ethanol by differential centrifugation. Preferably, the cooling method is air cooling, and the cooling speed is less than 20℃ / min. Preferably, the cut-off interval of the differential centrifugation method is 10000-12000rpm, and the number of washing times is not less than 4. Preferably, the volume ratio of the mixed solution of ultrapure water and ethanol is 1:1. Preferably, the drying method is freeze-drying at-50~5℃.

[0056] The present inventors have not found a one-step solvothermal method for directly synthesizing single-atom nanoszyme materials with multiple free radical scavenging capabilities, and there is no report on single-atom nanoszyme for treating inflammatory wounds such as wound sepsis.

[0057] The following examples are further illustrated to explain the present application in detail. It should also be understood that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present application are within the scope of protection of the present application. The specific process parameters in the following examples are only one example in the appropriate range, i.e. those skilled in the art can select within the appropriate range according to the description herein, and are not limited to the specific values in the following examples.

[0058] Example 1

[0059] Dissolve 48.66mg of anhydrous ferric chloride and 122.6mg of zinc chloride hexahydrate in 30mL of formamide, and ultrasonicate for 30min to completely dissolve, and prepare a solution with Fe 3+ , Zn 2+ concentrations of 10mM and 30mM respectively. Then transfer to a stainless steel hydrothermal reactor, and react at 220℃ in a blast drying oven for 12h. After the reaction is completed, air cool to room temperature, open the reactor, transfer the reaction liquid to a centrifuge tube, set the speed to 10000rpm, and centrifuge for 5min; wash the precipitate obtained by centrifugation with a 1:1 mixed solution of ultrapure water and ethanol for 3 times, and freeze-dry at-20℃ for 48h. The prepared bimetallic single-atom nanoszyme is denoted as Zn 30 Fe-NC. According to the mass percentage of elements, the material is composed of Zn 0.41%, Fe 0.21%, C 60.17%, N 27.58%, and O 11.63%.

[0060] Example 2

[0061] Dissolve 48.66mg of anhydrous ferric chloride and 122.6mg of zinc chloride hexahydrate in 30mL of formamide, and ultrasonicate for 30min to completely dissolve, and prepare a solution with Fe 3+, Zn 2+ concentrations of 10 mM and 60 mM, respectively. Then transferred into a stainless steel hydrothermal reactor, reacted at 220 °C in a blast drying oven for 12 h. After the reaction was completed, air-cooled to room temperature, opened the reactor, transferred the reaction liquid to a centrifuge tube, set the speed to 10000 rpm, centrifuged for 5 min; the precipitate obtained by centrifugation was washed with a 1:1 mixture of ultrapure water and ethanol solution for 3 times, and freeze-dried at -20 °C for 48 h. The prepared bimetallic single-atom nanoenzyme is denoted as Zn 60 Fe-NC. According to the mass percentage of elements, the material is composed of the following components: Zn 0.68%, Fe 0.17%, C 62.19%, N 24.22%, O 12.74%.

[0062] Example 3

[0063] Dissolve 48.66 mg of anhydrous ferric chloride and 490.4 mg of zinc chloride hexahydrate in 30 mL of formamide, ultrasonic for 30 min to completely dissolve, and prepare Fe 3+ , Zn 2+ concentrations of 10 mM and 120 mM, respectively. Then transferred into a stainless steel hydrothermal reactor, reacted at 220 °C in a blast drying oven for 12 h. After the reaction was completed, air-cooled to room temperature, opened the reactor, transferred the reaction liquid to a centrifuge tube, set the speed to 10000 rpm, centrifuged for 5 min; the precipitate obtained by centrifugation was washed with a 1:1 mixture of ultrapure water and ethanol solution for 3 times, and freeze-dried at -20 °C for 48 h. The prepared bimetallic single-atom nanoenzyme is denoted as Zn 120 Fe-NC. According to the mass percentage of elements, the material is composed of the following components: Zn 0.96%, Fe 0.12%, C 63.70%, N 20.87%, O 14.35%.

[0064] Comparative Example 1

[0065] Transfer 30 mL of formamide directly into a stainless steel hydrothermal reactor, react at 220 °C in a blast drying oven for 12 h. After the reaction was completed, air-cooled to room temperature, opened the reactor, transferred the reaction liquid to a centrifuge tube, set the speed to 10000 rpm, centrifuged for 5 min; the precipitate obtained by centrifugation was washed with a 1:4 mixture of ultrapure water and ethanol solution for 3 times, and oven-dried at 60 °C for 6 h. The prepared material is denoted as NC.

[0066] Comparative Example 2

[0067] Dissolve 245.2 mg of zinc chloride hexahydrate in 30 mL of formamide, ultrasonic for 30 min to completely dissolve, and prepare Zn 2+The solution was then transferred to a stainless steel hydrothermal reactor and reacted at 220°C in a forced air drying oven for 12 hours. After the reaction was completed, the reaction mixture was air-cooled to room temperature, opened, and the reaction liquid transferred to a centrifuge tube. The tube was centrifuged at 10,000 rpm for 5 minutes. The resulting precipitate was washed three times with a 1:1 mixture of ultrapure water and ethanol and freeze-dried at -20°C for 48 hours. The resulting single-metal single-atom nanozyme was designated Zn-NC. The material was composed of the following elements by mass: 1.13% Zn, 61.35% C, 21.22% N, and 16.30% O.

[0068] Comparative Example 3

[0069] Dissolve 48.66 mg of anhydrous ferric chloride in 30 mL of formamide and ultrasonicate for 30 min to dissolve it completely. 3+ The solution was then transferred to a stainless steel hydrothermal reactor and reacted at 220°C in a forced air drying oven for 12 hours. After the reaction was completed, the reaction mixture was air-cooled to room temperature, opened, and the reaction liquid transferred to a centrifuge tube. The centrifugation was set at 10,000 rpm and centrifuged for 5 minutes. The resulting precipitate was washed three times with a 1:1 mixture of ultrapure water and ethanol and freeze-dried at -20°C for 48 hours. The prepared single-metal single-atom nanozyme was designated Fe-NC. The material is composed of the following elements by mass: Fe 0.29%, C 68.24%, N 15.24%, and O 16.23%.

[0070] The bimetallic single-atom nanozyme Zn obtained in Example 1, Example 2, and Example 3 30 Fe-NC、Zn 60 Fe-NC、Zn 120 TEM images of Fe-NC and NC, Zn-NC and Fe-NC obtained in Comparative Examples 1, 2 and 3 are shown in FIG. Figure 1 As shown, the morphology of each material obtained is flaky or granular. Compared with Fe-NC, the doping of Zn reduces the size of the material and weakens its crystallinity. When the Fe content is constant, as the addition of Zn increases from 0mM to 60mM, its morphology gradually changes from a highly crystalline flake to a fibrous network, and its size gradually decreases, which is conducive to the full exposure of the enzyme-like catalytic active sites and improves its enzyme-like catalytic activity; however, when the addition of Zn increases from 60mM to 120mM, its size increases instead. This is because excessive Zn incorporation makes the formed nanozymes too small in size and the surface energy too high, thus agglomerating to form large particles. Zn acts as a coordination site, and its large introduction increases the density of coordination sites, which in turn affects the polymerization process of formamide, resulting in a decrease in the degree of formamide polymerization and a reduction in the size of the material.

[0071] Application Example 1: Enzyme-like activity of bimetallic single-atom nanozyme

[0072] The OXD-like and POD-like activities of the bimetallic single-atom nanozyme were evaluated by 3,3',5,5'-tetramethylbenzidine (TMB) color development method.

[0073] OXD-like activity evaluation: 100 μL of 1 mg / mL dispersion of different materials, 100 μL of 20 mM TMB-DMSO solution, and 100 μL of 20 mM H2O2 solution were added to 700 μL of acetic acid-sodium acetate buffer solution (HAc-NaAc) at different pH (pH = 4, pH = 6.5, pH = 7.4), and completely mixed. After 30 min of reaction at 37°C, 100 μL was taken and the spectral absorption at 500-800 nm was measured using an enzyme marker, as shown in Figure 2 and Figure 4 .

[0074] POD-like activity evaluation: 100 μL of 1 mg / mL dispersion of different materials, 100 μL of 20 mM TMB-DMSO solution, and 100 μL of 20 mM H2O2 solution were added to 700 μL of acetic acid-sodium acetate buffer solution (HAc-NaAc) at different pH (pH = 4, pH = 6.5, pH = 7.4), and completely mixed. After 30 min of reaction at 37°C, 100 μL was taken and the spectral absorption at 500-800 nm was measured using an enzyme marker, as shown in Figure 3 and Figure 5 .

[0075] The Zn 60 The ability of Fe-NC to catalyze H2O2 to produce O2 was measured, and the ability of the material to consume H2O2 was measured by the color reaction of titanium sulfate with H2O2 to evaluate its CAT-like activity: O2 production ability evaluation: 200 μL of 0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL Zn 60 Fe-NC dispersion was added to 1600 μL of PBS buffer solution (pH = 7.4, 20 mM), and then 200 μL of 100 mM H2O2 solution was added. After complete mixing, the change in O2 content in the reaction system was immediately measured using a dissolved oxygen meter, recording every 5 s for a total of 300 s, as shown in Figure 7 .

[0076] H2O2 consumption ability evaluation: 100 μL of 1 mg / mL dispersion of different materials was added to 900 μL of PBS buffer solution (pH = 7.4, 20 mM), and completely mixed. After 8 h of reaction at 37°C, 200 μL was taken and added to a 96-well white plate, and then 20 μL of 0.6% Ti(SO4)2 solution was added. After uniform blowing, the spectral absorption at 300-550 nm was measured using an enzyme marker, as shown inFigure 6 .

[0077] The total SOD activity was evaluated by using SOD activity assay kit (WST-8 method) to evaluate the Zn 60 SOD-like activity of Fe-NC: SOD-like activity evaluation: The SOD-like activity of different concentrations of Zn 60 Fe-NC was evaluated according to the kit instructions. After incubation at 37°C for 30 min, the full spectrum absorption of 410-550 nm was measured using a microplate reader. The lower the absorption peak, the higher the SOD-like activity. For example, the SOD-like activity of Zn Figure 8 .

[0078] As shown in Figure 1, Zn Figures 2-8 Fe-NC has four natural enzyme mimetic activities of OXD, POD, CAT and SOD, and has pH and material concentration dependence. The SOD-like activity of Zn 60 Fe-NC is the strongest at pH = 4, and almost negligible at pH = 7.4, indicating that the material will not produce toxic free radicals in physiological environment, indicating that ZnFe-NC has excellent biological safety and shows great application potential in the field of biological medicine.

[0079] Figure 4 , Figure 5 , Figure 6 ZnFe-NC, ZnFe-NC, ZnFe-NC obtained in Example 1, Example 2, Example 3, respectively 30 Fe-NC, Zn 60 Fe-NC, Zn 120 Fe-NC and NC, Zn-NC, Fe-NC obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3. From Figure 4 , Figure 5 , Figure 6It can be seen that Zn-NC lacks enzyme-like activity, while Fe-NC does, indicating that Zn-N is not the source of enzyme-like activity, but Fe-N. Furthermore, compared with NC, Zn-NC, and Fe-NC, the OXD-, POD-, and CAT-like activities of ZnFe-NC are significantly enhanced, with the highest enzyme-like activity achieved at a Zn doping concentration of 60 mM. Zn incorporation reduces the size of the material, exposing the catalytic active sites more fully and enhancing interaction with the substrate, thereby enhancing the enzyme-like catalytic ability of Fe-NC. Furthermore, Zn and Fe atoms compete for coordination during formamide self-polymerization. Increased Zn doping increases the number of N atoms that coordinate with Zn rather than Fe, reducing the number of Fe-N coordination sites. In other words, Zn doping reduces the number of Fe-N active sites that serve as enzyme-like sites, thereby reducing the enzyme-like catalytic ability. The combined effects of increased exposure and decreased number of catalytic active sites demonstrate the impact of Zn doping on the enzyme-like catalytic ability of Fe-NC. When the Zn doping amount is less than or equal to 60 mM, the effectiveness of fully exposed active sites in improving catalytic activity is greater than the effectiveness of reduced active sites in reducing catalytic activity, thereby improving the enzyme-like activity of ZnFe-NC. When the Zn doping amount is greater than 60 mM, the number of Fe-N active sites is greatly reduced, and the aggregation of small particles prevents the active sites from being fully exposed, thereby weakening the enzyme-like activity of ZnFe-NC.

[0080] Application Example 2: Free radical scavenging ability of bimetallic single-atom nanozymes

[0081] · OH scavenging ability evaluation: Electron paramagnetic resonance spectroscopy was used to analyze the Zn 60 The ·OH scavenging ability of Fe-NC was analyzed by EPR. ·OH was generated by Fenton reaction. A mixture of 1mM FeSO4 and 100μM H2O2 was reacted for 5 minutes, and DMPO was added. The ESR signal was immediately collected. The final concentration was 5mg / mL Zn 60 A mixture of Fe-NC, 1 mM FeSO₄, and 100 μM H₂O₂ was reacted for 5 minutes before DMPO was added and ESR signals were immediately collected. The ·OH group in the aqueous solution was captured by the DMPO probe, forming a spin adduct DMPO / OH. This compound exhibits a characteristic EPR spectrum. The spectral intensity comparison before and after the addition of the material can be used to evaluate the ·OH scavenging ability of the material.

[0082] The H2O2 scavenging ability corresponds to the CAT-like activity of ZnFe-NC. - The scavenging ability of ZnFe-NC corresponds to the SOD-like activity of ZnFe-NC.

[0083] like Figure 9 As shown, Zn 60Fe-NC can effectively scavenge more toxic ·OH. Figure 10 The radical scavenging process of ZnFe-NC was plotted, which can not only catalyze the scavenging of H2O2 and ·O2 - by using its CAT-like and SOD-like enzyme activities, but also directly scavenge more toxic ·OH, which can be used for the treatment of inflammatory wounds.

[0084] Application Example 3: Inflammatory treatment effect of bimetallic single-atom nanozyme

[0085] A wound sepsis model of C57BL / 6 mice was constructed by inducing inflammation with 20 μL of 2 mg / mL LPS, and the survival of 20 μL of 2 mg / mL Zn 60 Fe-NC treated mice within 11 days and the wound healing were recorded. The experimental groups were divided into 4 groups, namely normal wound group (Control group), ZnFe-NC group, LPS group and LPS+ZnFe-NC group.

[0086] As shown in Figure 11 , the survival rates of mice in the Control group and the ZnFe-NC group were both around 80%, while all the mice treated with LPS died on the 4th day after the operation, with a survival rate of 0%, indicating the successful establishment of the wound sepsis mouse model. After ZnFe-NC treatment, the survival rate of sepsis mice was significantly improved, indicating the excellent inflammatory treatment effect of ZnFe-NC.

[0087] As shown in Figure 12 , for normal wound mice, the wound healing of the ZnFe-NC group was significantly better than that of the Control group, and for wound sepsis mice, the addition of ZnFe-NC not only improved the postoperative survival rate of mice, but also significantly promoted the healing of inflammatory wounds.

[0088] The specific embodiments described above further illustrate the purposes, technical solutions and beneficial effects of the present application. It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or modifications according to the above description, all of which should belong to the protection scope of the appended claims of the present application.

Claims

1. A bimetallic single-atom nanozyme, characterized in that: The simple formula of the bimetallic single-atom nanozyme is ZnFe-NC, which is composed of the following components in atomic percentage: Zn 0.5% to 5%; Fe 0.05% to 2%; C 60% to 70%; N 15% to 25%; O 10% to 20%; The preparation method of the bimetallic single-atom nanozyme comprises: (1) Adding an iron salt and a zinc salt to formamide or a mixture of formamide and other organic solvents, and dissolving them by ultrasonication to obtain a solution A; wherein the molar ratio of the iron salt to the zinc salt is (1-20 mM): (30-60 mM); and the ratio of the formamide or the mixture of formamide and other organic solvents to the iron salt is (30-50) mL: (1-20) mM; (2) Solution A is subjected to a solvothermal reaction at 200-240° C., and then cooled, washed, and dried to obtain the bimetallic single-atom nanozyme ZnFe-NC.

2. The bimetallic single-atom nanozyme according to claim 1, characterized in that The nanozyme in the bimetallic single-atom nanozyme has a nitrogen-carbon skeleton structure, and the bimetallic single atoms are Zn atoms and Fe atoms, wherein the Zn atoms and Fe atoms are in an oxidized state and are distributed in the nitrogen-carbon skeleton in a monodispersed form, and are connected to the nitrogen-carbon skeleton structure in the coordination form of Zn-N and Fe-N, respectively.

3. The bimetallic single-atom nanozyme according to claim 1, characterized in that N atoms mainly exist in the form of pyridinic nitrogen and pyrrolic nitrogen; C atoms mainly exist in the form of C=N, CC, and CN.

4. The bimetallic single-atom nanozyme according to claim 1, characterized in that The bimetallic single-atom nanozyme ZnFe-NC, when the Fe content remains unchanged, as the Zn content increases, the crystallinity of the bimetallic single-atom nanozyme gradually weakens, and the microscopic morphology gradually changes from lamellar to granular.

5. The bimetallic single-atom nanozyme according to claim 1, characterized in that The size of the ZnFe-NC is ≤1 μm; when the Fe content remains unchanged, the size of the bimetallic single-atom nanozyme gradually decreases with the increase of Zn content.

6. The bimetallic single-atom nanozyme according to claim 1, characterized in that In step (1): The iron salt is selected from at least one of ferric chloride, ferrous chloride, ferric sulfate, and ferric acetate; The zinc salt is selected from at least one of zinc chloride, zinc sulfate, zinc nitrate and zinc acetate; The other organic solvent is selected from at least one of ethanol, ethylene glycol, glycerol, ethanolamine, N, N-dimethylformamide, and N, N-dimethylacetamide, wherein the volume ratio of formamide to the other organic solvent is not 0; The power of the ultrasonic dissolution is 40 to 320 W, and the time is 5 to 60 minutes.

7. The bimetallic single-atom nanozyme according to claim 1, characterized in that In step (2): The solvent thermal reaction time is 12 to 24 hours; The cooling method is air cooling, water cooling, furnace cooling; The washing method is differential centrifugation, with a low centrifugal speed r low = 3000-10000 rpm, a high centrifugal speed r high = 10000-12000 rpm, r low < r high, and the number of washing times is not less than 4 times; The drying method is drying at 50-100°C or freeze drying at -50--5°C.

8. Use of the bimetallic single-atom nanozyme according to any one of claims 1 to 5 in the preparation of a drug for improving wound inflammation.

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