CuFe biatomic sheet nanoscale enzyme with photocatalytic antibacterial activity, and preparation method and application thereof

Abstract

This invention discloses a CuFe dual-single-atom sheet-like nanozyme with photocatalytic antibacterial activity, its preparation method, and its application, belonging to the field of bionanomaterials technology. The preparation method provided by this invention is as follows: copper salt is added to a dispersion of carbon nitride nanosheets for impregnation, followed by solid-liquid separation and drying, and then calcination under an inert atmosphere to obtain carbon nitride nanosheets loaded with copper single atoms; the copper-loaded carbon nitride nanosheets are dispersed in water, impregnated with ferrous salt, and then dried after solid-liquid separation to obtain the final product. The above preparation method of this invention can achieve a high degree of dispersion of bimetallic active sites at the atomic scale. The resulting dual-single-atom sheet-like nanozyme possesses both photocatalytic self-production of hydrogen peroxide and peroxidase-like activity. It can efficiently generate reactive oxygen species through photocatalysis and enzyme-like cascade reactions without the need for external addition of hydrogen peroxide, exhibiting excellent antibacterial properties.

Description

Technical Field

[0001] This invention relates to the field of bionanomaterials technology, and in particular to a CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity, its preparation method, and its application. Background Technology

[0002] Bacterial infections are a major challenge threatening public health, and the increasing resistance to antibiotics poses a serious challenge to traditional treatments. Nanozymes, as nanomaterials that can mimic the activity of natural enzymes, exhibit unique advantages in the field of antibacterial research, possessing characteristics such as structural stability, ease of preparation, and adaptability to complex physiological environments.

[0003] However, existing nanozyme antibacterial technologies still face key bottlenecks in practical applications. On the one hand, most nanozymes rely on the supply of exogenous hydrogen peroxide to generate reactive oxygen species (ROS), which not only limits their application in environments lacking exogenous substrates but also reduces the controllability of ROS release. On the other hand, the catalytic activity of nanozymes is significantly affected by the number and distribution of active sites, and traditional nanozymes still have considerable room for improvement in terms of atom utilization efficiency and catalytic performance. Summary of the Invention

[0004] In view of this, the present invention provides a CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity, its preparation method and application, which solves the problems of existing nanozyme antibacterial technology relying on exogenous hydrogen peroxide supply and insufficient catalytic active sites.

[0005] In a first aspect, the present invention provides a method for preparing CuFe double single-atom sheet-like nanozymes with photocatalytic antibacterial activity, comprising the following steps: Copper salt was added to the dispersion of carbon nitride nanosheets for impregnation treatment, and the solid product was obtained by drying after solid-liquid separation. The first solid product was calcined under an inert atmosphere to obtain carbon nitride nanosheets loaded with copper single atoms. The copper-loaded carbon nitride nanosheets were dispersed in water, impregnated with ferrous salt, and dried after solid-liquid separation to obtain the CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity.

[0006] Preferably, the copper salt is at least one of copper nitrate, copper sulfate, and copper acetate, and the ferrous salt is at least one of ferrous chloride, ferrous sulfate, and ferrous nitrate.

[0007] Preferably, the amount of copper salt added is 15-30% of the total mass of the carbon nitride nanosheets and copper salt; the amount of ferrous salt added is 2-5% of the total mass of the carbon nitride nanosheets loaded with copper single atoms and ferrous salt.

[0008] Preferably, in the step of adding copper salt for impregnation, the impregnation time is 1 to 4 hours; in the step of adding ferrous salt for impregnation, the impregnation time is 2 to 6 hours; the impregnation is carried out under stirring conditions.

[0009] Preferably, the heating rate of the calcination treatment is 5~15℃ / min, the calcination temperature is 450~650℃, the holding time is 0.5~2 h, and the inert atmosphere is argon atmosphere or nitrogen atmosphere.

[0010] Preferably, the preparation of the carbon nitride nanosheets includes: placing dicyandiamide in an air atmosphere and annealing it at 500~600°C to obtain bulk graphitic carbon nitride, and then acidifying and exfoliating the bulk graphitic carbon nitride with concentrated sulfuric acid, dialyzing, and drying to obtain the carbon nitride nanosheets.

[0011] In a second aspect, the present invention provides a CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity, which is prepared by the preparation method described in the first aspect.

[0012] Thirdly, the present invention provides a dual single-atom nanoenzyme hydrogel microneedle, which includes a microneedle matrix and a CuFe dual single-atom sheet nanoenzyme with photocatalytic antibacterial activity as described in the second aspect dispersed in the microneedle matrix.

[0013] Preferably, the microneedle matrix is ​​methacryloyl hyaluronic acid.

[0014] Fourthly, the present invention provides the application of CuFe double single-atom sheet nanozymes with photocatalytic antibacterial activity as described in the second aspect or double single-atom nanozyme hydrogel microneedles as described in the third aspect in the preparation of antibacterial products.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention employs a stepwise loading strategy, first anchoring copper in single-atom form onto a carbon nitride nanosheet support, and then introducing iron through gentle impregnation, successfully constructing a copper-iron dual-single-atom co-loaded nanozyme structure. This preparation method effectively inhibits the migration and aggregation of metal atoms during high-temperature processing, achieving a high degree of dispersion of bimetallic active sites at the atomic scale, significantly increasing the density and atomic utilization of catalytic active sites. The resulting dual-single-atom sheet-like nanozyme possesses both photocatalytic self-production of hydrogen peroxide and peroxidase-like activity. It can efficiently generate reactive oxygen species through photocatalysis and an enzyme-like cascade reaction without the need for exogenous hydrogen peroxide, providing a structural basis for overcoming the technical bottleneck of existing nanozymes that rely on exogenous hydrogen peroxide, while simultaneously improving catalytic efficiency and antibacterial properties. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 The images show the characterization of CuFe-g-C3N4 prepared in Example 1 of this invention, where a and b are scanning electron microscope (SEM) images; c and d are transmission electron microscope (TEM) images; and e is an atomic force microscope (AFM) image. Figure 2 This is a synchrotron radiation characterization of CuFe-g-C3N4 prepared in Example 1 of this invention, where a and b are Cu K-edge XANES and K-edge EXAFS spectra; d and e are Fe K-edge XANES and K-edge EXAFS spectra; c and f are HAADF-STEM aberration-corrected images; g and h are elemental surface mapping images; i is a simulation using two-dimensional (2D) and three-dimensional (3D) resolution images, showing the positions and signals of Cu-Fe atomic pairs. Figure 3 The images show the characterization of the CuFe-g-C3N4-MN patch prepared in Example 2 of this invention, where a is a digital photograph, SEM image, and elemental surface distribution (mapping) diagram; b is a force-displacement curve; and c is a Young's modulus diagram. Figure 4 The images show the characterization results of the g-C3N4 nanosheets prepared in Comparative Example 1 of this invention, where a and b are scanning electron microscope (SEM) images; and c and d are transmission electron microscope (TEM) images. Figure 5 This is a comparison of the UV-Vis absorption spectra of catalytic TMB oxidation under different reaction systems in the experimental examples of this invention; Figure 6 This is the absorbance change curve of the CuFe-g-C3N4 nanozyme (abbreviated as CuFe-CN) catalyzing the oxidation of TMB under different light exposure times and different pH values ​​in the experimental examples of this invention; Figure 7 These are the absorbance change curves of the four CuFe-g-C3N4 nanozymes prepared in Example 1 and Comparative Examples 4-6 of this invention for the oxidation of TMB catalyzed by CuFe-g-C3N4 nanozymes. Figure 8 This is a graph showing the plate antibacterial test results of different concentrations of CuFe-g-C3N4 nanozyme from Example 1 on Staphylococcus aureus and Escherichia coli. Figure 9 This is a graph showing the plate antibacterial test results of different treatment groups against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) in the experimental examples of this invention; Figure 10 This is a graph showing the cytotoxicity and biocompatibility evaluation results of CuFe-g-C3N4 nanozymes of different concentrations in Example 1 of this invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] As noted in the background section, existing nanozyme antibacterial technologies generally rely on exogenous hydrogen peroxide supply and suffer from insufficient catalytic active sites. Therefore, this invention provides a method for preparing CuFe double single-atom sheet-like nanozymes with photocatalytic antibacterial activity, comprising the following steps: Preparation of S1 and carbon nitride nanosheets In this invention, carbon nitride nanosheets serve as a carrier material for subsequent loading of copper and iron single atoms. Various methods exist for preparing carbon nitride nanosheets, including but not limited to thermal polymerization, template methods, and ultrasonic exfoliation. This invention preferably uses a combination of thermal polymerization and chemical exfoliation to prepare ultrathin carbon nitride nanosheets, thereby obtaining a larger specific surface area and abundant defect sites, which is beneficial for the anchoring of metal single atoms.

[0020] This invention outlines a typical but non-limiting process for preparing carbon nitride nanosheets, specifically including: Dicyandiamide is placed in an air atmosphere and annealed at 500-600°C to obtain bulk graphitic carbon nitride (g-C3N4). The specific operation of the annealing process is as follows: Dicyandiamide is placed in a ceramic crucible or ceramic combustion vessel and heated from room temperature to 500-600°C, preferably 520-580°C, with the heating rate controlled at 5-15°C / min. The mixture is held at the target temperature for 3-5 hours and then allowed to cool naturally to room temperature.

[0021] The obtained bulk graphitic carbon nitride was dispersed in concentrated sulfuric acid for acid exfoliation. Specifically, bulk graphitic carbon nitride was dispersed in concentrated sulfuric acid, with the preferred ratio of graphitic carbon nitride to concentrated sulfuric acid being (2~4) g : (20~50) mL. The mixture was stirred for 0.5~2 hours, preferably 1 hour, and then water was slowly added, causing the dispersion to gradually become clear. Anhydrous ethanol was then added, and stirring continued for 12~20 hours to precipitate the nanosheets from the solution. The nanosheet suspension was dialyzed with water to remove residual sulfate ions and ethanol until the pH of the system reached 7.0~7.2, preferably 7.0. After dialyzing, the precipitate was collected by centrifugation and then vacuum dried at 40~70℃ for 10~20 hours to obtain carbon nitride nanosheets.

[0022] S2. Preparation of carbon nitride nanosheets loaded with copper single atoms The carbon nitride nanosheets prepared in step S1 are dispersed in deionized water, and then impregnated with copper salt. The copper salt is at least one of copper nitrate, copper sulfate, and copper acetate, preferably copper nitrate, or a hydrated salt of the above copper salts. The amount of copper salt added is 15-30% of the total mass of the carbon nitride nanosheets and the copper salt, preferably 20-25%. The impregnation time is 1-4 hours, preferably 2-3 hours, for example, 2 hours. The impregnation is carried out under stirring conditions to ensure that copper ions are fully adsorbed onto the surface of the carbon nitride nanosheets. After solid-liquid separation, the solid product is collected and dried to obtain the first solid product. Solid-liquid separation can be performed by centrifugation; the drying temperature is 40-70°C, and the time is 5-15 hours.

[0023] The first solid product is calcined under an inert atmosphere. The heating rate of the calcination treatment is 5~15℃ / min, preferably 8~12℃ / min, for example, 10℃ / min; the calcination temperature is 450~650℃, preferably 500~600℃, for example, 550℃; the holding time is 0.5~2 hours, preferably 1~1.5 hours, for example, 1 hour. The inert atmosphere is an argon atmosphere or a nitrogen atmosphere. After calcination, it is naturally cooled to room temperature to obtain carbon nitride nanosheets loaded with copper single atoms.

[0024] This step involves high-temperature calcination, which anchors copper atoms onto the carbon nitride support in single-atom form. During calcination, copper ions are reduced and coordinate with nitrogen atoms in the carbon nitride, forming stable Cu-N coordination bonds. Simultaneously, the introduction of copper atoms creates vacancy defects in the carbon nitride lattice. These vacancies provide crucial active sites for the subsequent anchoring of iron atoms, forming the structural basis for achieving dual single-atom co-loading.

[0025] S3. Preparation of CuFe double single-atom sheet-like nanozymes with photocatalytic antibacterial activity The copper-loaded carbon nitride nanosheets prepared in step S2 are dispersed in deionized water, and then impregnated with a ferrous salt. The ferrous salt is at least one of ferrous chloride, ferrous sulfate, and ferrous nitrate, preferably ferrous chloride (FeCl2), or a hydrated salt of the aforementioned ferrous salts. The amount of ferrous salt added is 2-5% of the total mass of the copper-loaded carbon nitride nanosheets and the ferrous salt, preferably 2.5-3.5%, for example, 3.0%. The impregnation time is 2-6 hours, preferably 3-5 hours, for example, 4 hours, and the impregnation is carried out under stirring conditions.

[0026] After solid-liquid separation, the solid product is collected, dried, and ground into a fine powder to obtain the CuFe double-monatomic sheet-like nanozyme with photocatalytic antibacterial activity. Solid-liquid separation can be performed by centrifugation, with a drying temperature of 40–70°C and a preferred drying time of 8–20 hours.

[0027] This invention employs a method of solid-liquid separation followed by drying, rather than direct drying, to prevent the migration and aggregation of metal ions during the drying process. This ensures that iron is anchored to the carrier in a coordinated form rather than existing as free ions. Since the number of vacancies formed after calcination in step S2 is limited, adsorption stops once the iron load reaches saturation. Excess iron ions can be removed through solid-liquid separation, thereby ensuring that the iron element is dispersed in single-atom form.

[0028] In the final product, copper atoms form Cu-N coordination bonds with nitrogen atoms in carbon nitride, while iron atoms form Cu-Fe and Fe-N coordination bonds with adjacent copper and nitrogen atoms respectively through vacancy anchoring. This unique double single-atom coordination structure enables the material to possess both the ability to photocatalyze the self-production of hydrogen peroxide and peroxidase-like activity, with the two working synergistically to form a highly efficient cascade catalytic system.

[0029] This invention also provides a CuFe dual single-atom sheet nanozyme with photocatalytic antibacterial activity, prepared by the above-described method. In this nanozyme, both copper and iron are dispersed in single-atom form on a carbon nitride nanosheet support, and the nanozyme possesses both visible light photocatalytic self-production of hydrogen peroxide activity and peroxidase-like activity.

[0030] According to inductively coupled plasma (ICP) analysis, the CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity prepared by the above method has an iron mass fraction of 0.2~0.5wt% and a copper mass fraction of 1~2wt%.

[0031] This invention also provides a dual-single-atom nanozyme hydrogel microneedle, comprising a microneedle matrix and the aforementioned CuFe dual-single-atom sheet-like nanozyme with photocatalytic antibacterial activity dispersed in the microneedle matrix. The microneedle matrix is ​​methacrylamide hyaluronic acid (MeHA). Methacrylamide hyaluronic acid has good biocompatibility and biodegradability, and can serve as a framework material for the microneedle, supporting the formation of a mechanically strong microneedle structure that facilitates transdermal drug delivery.

[0032] In this invention, the preferred method for preparing dual single-atom nanoenzyme hydrogel microneedles includes the following steps: First, methacrylamide hyaluronic acid is prepared. Hyaluronic acid is dissolved in water, and methacrylic anhydride is added dropwise. The preferred ratio of hyaluronic acid, water, and methacrylic anhydride is (1.8~2.5) g : (80~120) mL : (1.5~1.8) mL. The pH is adjusted to 8~9 with sodium hydroxide solution, and the reaction is stirred for 20~28 hours. After the reaction is complete, the methacrylamide hyaluronic acid is precipitated with 0.4~0.6 M sodium chloride solution, washed 2~5 times with ethanol to remove unreacted substances. The product is redissolved in water, dialyzed for 1~3 days using a dialysis bag with a molecular weight cutoff of 8000~12000 Da, and then lyophilized to obtain methacrylamide hyaluronic acid.

[0033] Next, a pre-hydrogel solution is prepared. The CuFe double-single-atom sheet-like nanozyme with photocatalytic antibacterial activity is uniformly dispersed in deionized water to obtain a nanozyme suspension. The nanozyme suspension is mixed with a methacrylamide hyaluronic acid solution, and a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), is added. The mixture is then ultrasonically treated to ensure uniform dispersion of the nanozyme, forming a pre-hydrogel solution. The mass fraction of methacrylamide hyaluronic acid is 4-6 wt%, preferably 5 wt%; the mass fraction of LAP is 0.2-0.3 wt%, preferably 0.25 wt%.

[0034] Finally, the microneedle patch was prepared. The obtained pre-hydrogel solution was cast into a polydimethylsiloxane (PDMS) microneedle mold, and after vacuum degassing to remove air bubbles, continuous drying cycles were performed to shape the microneedles. Finally, using a gelatin solution as a substrate, after drying and stabilization, the microneedle was demolded from the PDMS mold to obtain the dual single-atom nanoenzyme hydrogel microneedle patch. In the prepared microneedles, CuFe dual single-atom sheet-like nanoenzymes with photocatalytic antibacterial activity were uniformly distributed in the microneedle matrix, realizing local transdermal delivery of antibacterial functional materials.

[0035] The present invention also provides the application of the above-mentioned CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity or the above-mentioned double single-atom nanozyme hydrogel microneedles in the preparation of antibacterial products.

[0036] In this invention, the antibacterial product is used to generate reactive oxygen species through a cascade catalytic reaction under light irradiation. Specifically, the cascade catalytic reaction includes the nanozyme generating hydrogen peroxide in situ under light irradiation, and utilizing its peroxidase-like activity to convert the hydrogen peroxide into hydroxyl radicals. This application is particularly suitable for treating bacterial skin infections caused by Staphylococcus aureus, Escherichia coli, or methicillin-resistant Staphylococcus aureus.

[0037] In use, the dual single-atom nanoenzyme hydrogel microneedle patch is applied to the infected site. The microneedles penetrate the stratum corneum of the skin, delivering the nanoenzyme directly to the infected area. Then, visible light irradiation is applied to initiate a cascade catalytic reaction, achieving highly efficient antibacterial treatment.

[0038] This invention provides a CuFe dual-single-atom sheet-like nanozyme with photocatalytic antibacterial activity. Through a stepwise loading strategy, it achieves high atomic-scale dispersion of the copper and iron bimetallic compounds. The calcination treatment after copper loading creates vacancy defects on the support, providing active sites for iron anchoring, ultimately forming a unique structure with coexisting Cu-N, Fe-N, and Cu-Fe coordination bonds. This structure endows the material with excellent photocatalytic self-generation of hydrogen peroxide and peroxidase-like activity. These two elements synergistically form a cascade catalytic system, efficiently generating reactive oxygen species without the need for exogenous hydrogen peroxide. Encapsulating this nanozyme in methacrylamide hyaluronic acid microneedles creates a wearable transdermal drug delivery platform that enables painless delivery and locally controlled antibacterial activity, showing broad application prospects in the field of antibacterial therapy.

[0039] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0040] Example 1 This embodiment provides a method for preparing CuFe dual single-atom sheet nanozymes (CuFe-g-C3N4) with photocatalytic antibacterial activity.

[0041] (1) Preparation of carbon nitride nanosheets: Six g of dicyandiamide was placed in a ceramic crucible and heated from room temperature to 550 °C in air at a rate of 5 °C / min. The mixture was held at 550 °C for 4 hours and then allowed to cool naturally to room temperature to obtain bulk graphitic carbon nitride (g-C3N4). Three g of the bulk graphitic carbon nitride was dispersed in 30 mL of concentrated sulfuric acid and stirred for 1 hour. Then, 30 mL of deionized water was slowly added, and the dispersion gradually became clear. 90 mL of anhydrous ethanol was added, and stirring continued for 18 hours to precipitate nanosheets. The nanosheet suspension was dialyzed with deionized water to remove residual sulfate ions and ethanol until the pH reached 7.0. After dialyzing, the precipitate was collected by centrifugation at 9000 r / min for 5 minutes and then vacuum dried at 60 °C for 12 hours to obtain carbon nitride (g-C3N4) nanosheets.

[0042] (2) Preparation of carbon nitride nanosheets loaded with copper single atoms: 2 g of the carbon nitride nanosheets prepared in step (1) were dispersed in deionized water, and 0.65 g of Cu(NO3)2·6H2O was added (the amount of copper salt added was 24.5% of the total mass of carbon nitride nanosheets and copper salt). The mixture was stirred for 2 hours for impregnation treatment. The solid product was collected by centrifugation at 9000 r / min for 10 minutes and dried at 45℃ for 8 hours to obtain the first solid product. The first solid product was placed in a porcelain boat and heated to 550℃ at a heating rate of 10℃ / min under an argon atmosphere. The temperature was maintained for 1 hour for calcination treatment, and the mixture was naturally cooled to room temperature to obtain carbon nitride nanosheets loaded with copper single atoms (Cu-g-C3N4).

[0043] (3) Preparation of CuFe double single-atom sheet nanozymes with photocatalytic antibacterial activity: 500 mg of the copper-loaded carbon nitride nanosheets prepared in step (2) were dispersed in 50 mL of deionized water, and ferrous chloride tetrahydrate (FeCl2·4H2O) was added. The amount of ferrous salt added was 3.0% of the total mass of the copper-loaded carbon nitride nanosheets and ferrous salt. The mixture was stirred for 4 hours for impregnation treatment. The product was collected by centrifugation at 9000 r / min for 10 minutes, dried at 45℃ for 12 hours, and ground into a fine powder to obtain CuFe double single-atom sheet nanozyme (CuFe-g-C3N4) with photocatalytic antibacterial activity, denoted as CuFe-g-C3N4-1. Inductively coupled plasma (ICP) analysis showed that the mass fraction of iron in the product obtained in this example was 0.31 wt%, and the mass fraction of copper was 1.12 wt%.

[0044] Figure 1 In this example, 'a' and 'b' are scanning electron microscope (SEM) images of CuFe-g-C3N4 prepared in this embodiment. Figure 1c and d in the figure are transmission electron microscope (TEM) images of CuFe-g-C3N4 prepared in this embodiment. It can be seen that the material has a porous sheet structure.

[0045] Figure 1 In this example, 'e' represents the atomic force microscope (AFM) image of CuFe-g-C3N4 prepared in this embodiment and the corresponding nanosheet thickness analysis. It can be seen that the material has a nanosheet structure with a thickness of approximately 5-6 nm.

[0046] X-ray absorption near-edge structure (XANES) and Fourier transform extended X-ray absorption fine structure (FT-EXAFS) analyses further clarified the coordination environment and chemical state of Cu and Fe atoms in the CuFe-g-C3N4 prepared in this embodiment, such as... Figure 2 The values ​​a, b, d, and e are shown in the figure. The Cu K-edge XANES spectrum lies between that of Cu foil and CuPc, indicating that the oxidation state of Cu in CuFe-CN is positive, with an average oxidation state above 0 but below +2. Similarly, the Fe K-edge XANES spectrum lies between that of FePc and Fe2O3, indicating that the average oxidation state of Fe in CuFe-CN is above +2 but below +3. In the FT-EXAFS spectrum of CuFe-CN, a major peak appears at approximately 1.48 Å. Figure 2 (b) can be attributed to Cu-N coordination. In contrast, no Cu-Cu scattering signal was detected at approximately 2.24 Å, indicating that the Cu species are atomically dispersed. Notably, a weak peak appeared at approximately 2.04 Å, which can be attributed to the formation of Cu-Fe pairs. Similarly, the main peak in the Fe K-edge FT-EXAFS spectrum appeared at approximately 1.48 Å, corresponding to Fe-N coordination. No Fe-Fe contribution was detected at approximately 2.12 Å, confirming the atomically dispersed nature of Fe. A small peak was observed at approximately 2.31 Å, attributed to Fe-Cu interaction. EXAFS fitting analysis showed that the average coordination numbers of Fe-N, Cu-N, and Fe-Cu in CuFe-CN were 4.4, 4.1, and 1.1, respectively, while the average coordination number of Cu-Fe was approximately 0.3. These results indicate that Cu is more abundant than Fe, and that Fe atoms mainly exist in the form of Fe-Cu pairs. Therefore, the atomic structure of CuFe-CN was determined to be an N4-Cu-Fe-N4 structure.

[0047] Figure 2The c and f atoms in the image were shown as uniformly dispersed on the support by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). Isolated bright spots, marked by yellow circles and green rectangles, were identified as Cu-Fe atom pairs. Energy-dispersive X-ray spectroscopy (EDS) elemental distribution maps confirmed the uniform distribution of C, N, Fe, and Cu in the CuFe-CN structure (e.g., ...). Figure 2 (As shown by g and h). Figure 2 As shown in Figure i, the magnified 3D rendering of the region and the Gaussian fitting of overlapping atomic features further demonstrate the presence of neighboring Fe and Cu sites in CuFe-CN. Intensity profiles show that the interatomic distance between the bimetallic pairs is approximately 2.37–2.48 Å, consistent with the relatively short intervals that favor interatomic interactions. However, it is worth noting that HAADF-STEM is a projection-based imaging technique; therefore, the measured 2D distance may be significantly smaller than the actual 3D separation. In some cases, extremely short projection distances can lead to atomic contrast overlap, hindering the clear identification of individual atoms. Conversely, some bright spot pairs with projection distances exceeding 3 Å may not originate from actual bimetallic sites, but rather appear to be adjacent due to projection overlap, making two atoms that are far apart in 3D space appear close together.

[0048] Example 2 This embodiment provides the preparation of dual single-atom nanozyme hydrogel microneedles (CuFe-g-C3N4-MN).

[0049] (1) Preparation of methacrylamide hyaluronic acid (MeHA): 2.0 g of hyaluronic acid was dissolved in 100 mL of deionized water, and 1.6 mL of methacrylic anhydride was added dropwise. The pH was adjusted to 8-9 with 5 M sodium hydroxide solution, and the reaction was stirred at 4 °C for 24 hours. After the reaction was completed, the methacrylated hyaluronic acid was precipitated with 0.5 M sodium chloride aqueous solution, washed three times with ethanol to remove unreacted substances. The product was redissolved in deionized water, dialyzed for 2 days using a dialysis bag with a molecular weight cutoff of 10000 Da, and then lyophilized to obtain methacrylated hyaluronic acid.

[0050] (2) Preparation of pre-hydrogel solution: The CuFe-g-C3N4 nanozyme prepared in Example 1 was uniformly dispersed in deionized water to obtain a nanozyme suspension. This suspension was mixed with a solution of methacrylamide hyaluronic acid, and a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), was added. The mixture was then sonicated to ensure uniform dispersion of the nanozyme, forming a pre-hydrogel solution. In the pre-hydrogel solution, the mass fraction of CuFe-g-C3N4 nanozyme was 10 wt%, the mass fraction of methacrylamide hyaluronic acid was 5 wt%, and the mass fraction of LAP was 0.25 wt%.

[0051] (3) Preparation of microneedle patches: The pre-hydrogel solution obtained in step (2) was cast into a polydimethylsiloxane (PDMS) microneedle mold, and the air bubbles were removed by vacuum degassing. The microneedles were then shaped by continuous drying cycles. Using a gelatin solution as a base, the microneedles were demolded from the PDMS mold after drying and stabilization to obtain a double single-atom nanoenzyme hydrogel microneedle (CuFe-g-C3N4-MN) patch.

[0052] Figure 3 These are digital photographs, SEM images, elemental mapping diagrams, force-displacement curves, and Young's modulus diagrams of the CuFe-g-C3N4-MN patch prepared in this embodiment. It can be seen that the CuFe-g-C3N4 nanozyme is uniformly distributed within the microneedle patch. Testing showed that the minimum force required for the CuFe-g-C3N4-MN patch to penetrate the stratum corneum in this embodiment is 0.045 N, and its compressive force when the needle tip is fully deformed is 0.084 N / needle, ensuring effective penetration of the skin and subcutaneous tissue. From... Figure 3 As can be seen from Figure c, compared with pure HAMA hydrogel, CuFe-CN / HAMA hydrogel exhibits higher modulus and viscosity, indicating that the incorporation of CuFe-CN nanoparticles significantly enhances the mechanical strength of the hydrogel matrix.

[0053] Comparative Example 1 The difference between this comparative example and Example 1 is that this comparative example does not perform steps (2) and (3), and only obtains carbon nitride (g-C3N4) nanosheets.

[0054] Figure 4 In the figures, a and b are scanning electron microscope (SEM) images of the g-C3N4 nanosheets prepared in this comparative example. Figure 4 c and d in the image are transmission electron microscope (TEM) images of the g-C3N4 nanosheets prepared in this comparative example. It can be observed that the g-C3N4 nanosheets exist in the form of thin sheets and have a certain amount of pores, but the pores are not as dense as those of the CuFe-g-C3N4 nanosheets in Example 1.

[0055] Comparative Example 2 The difference between this comparative example and Example 1 is that step (3) is not performed in this comparative example, and only carbon nitride nanosheets loaded with copper single atoms (Cu-g-C3N4) are obtained.

[0056] Comparative Example 3 This comparative example provides a method for preparing carbon nitride nanosheets (Fe-g-C3N4) loaded with iron single atoms. The specific steps are as follows: (1) Following the same steps as in Example 1, carbon nitride (g-C3N4) nanosheets were obtained.

[0057] (2) Preparation of carbon nitride nanosheets loaded with iron single atoms (Fe-g-C3N4): A certain amount of calcined carbon nitride was dissolved in water, and 3% ferrous chloride tetrahydrate was added with stirring. After centrifugation and vacuum drying, Fe-g-C3N4 was obtained, denoted as Fe-CN.

[0058] Comparative Example 4 This comparative example provides a method for preparing CuFe-g-C3N4 nanozymes by first loading iron and then loading copper.

[0059] (1) Preparation of carbon nitride nanosheets loaded with iron single atoms: Two g of carbon nitride nanosheets prepared in step (1) of Example 1 were dispersed in deionized water, and ferrous chloride tetrahydrate (FeCl2·4H2O) was added (the amount of ferrous salt added was 24.5% of the total mass of carbon nitride nanosheets and ferrous salt). The mixture was stirred for 2 hours for impregnation treatment. The solid product was collected by centrifugation at 9000 r / min for 10 minutes and dried at 45°C for 8 hours to obtain the first solid product. The first solid product was placed in a porcelain boat and heated to 550°C at a heating rate of 10°C / min under an argon atmosphere. The temperature was maintained for 1 hour for calcination treatment, and the mixture was naturally cooled to room temperature to obtain carbon nitride nanosheets loaded with iron single atoms (Fe-g-C3N4).

[0060] (2) Preparation of CuFe double single-atom sheet nanozymes with photocatalytic antibacterial activity: 500 mg of the iron-loaded carbon nitride nanosheets prepared in step (1) were dispersed in 50 mL of deionized water, and Cu(NO3)2·6H2O was added. The amount of copper salt added was 3.0% of the total mass of the copper-loaded carbon nitride nanosheets and copper salt. The mixture was stirred for 4 hours for impregnation treatment. The product was collected by centrifugation at 9000 r / min for 10 minutes, dried at 45℃ for 12 hours, and ground into a fine powder to obtain CuFe double single-atom sheet nanozyme (CuFe-g-C3N4) with photocatalytic antibacterial activity, denoted as CuFe-g-C3N4-2.

[0061] Comparative Example 5 This comparative example prepared co-blended CuFe-g-C3N4 nanosheets via a one-step blending-calcination method. The specific preparation method is as follows: Two g of carbon nitride nanosheets prepared in step (1) of Example 1 were dispersed in deionized water, and 0.65 g of Cu(NO3)2·6H2O and 0.06 g of FeCl2·4H2O were added simultaneously. The mixture was stirred for 2 hours. The solid product was collected by centrifugation at 9000 r / min for 10 minutes and dried at 45°C for 8 hours. The dried powder was placed in a porcelain boat and calcined at 550°C at a heating rate of 10°C / min under an argon atmosphere for 1 hour. After natural cooling to room temperature, the powder was ground to obtain co-blended CuFe-g-C3N4 nanosheets prepared by a one-step blending and calcination method, denoted as CuFe-g-C3N4-3.

[0062] Comparative Example 6 This comparative example uses a blending and impregnation treatment method, without calcination.

[0063] 2 g of carbon nitride nanosheets prepared in step (1) of Example 1 were dispersed in deionized water, and 0.65 g of Cu(NO3)2·6H2O and 0.06 g of FeCl2·4H2O were added simultaneously. The mixture was stirred for 2 hours. The solid product was collected by centrifugation at 9000 r / min for 10 minutes. The product was dried at 45°C for 8 hours without calcination and was directly ground to obtain CuFe-g-C3N4 nanosheets prepared by omitting the calcination step, denoted as CuFe-g-C3N4-4.

[0064] Test case 1. Verification of peroxidase-like activity of CuFe-g-C3N4 nanozymes (1) Test method: The CuFe-g-C3N4 nanozyme (abbreviated as CuFe-CN) prepared in Example 1 was dissolved in deionized water to form a 1 mg / mL dispersion. Different reaction systems were prepared according to Table 1, with a total volume of 1 mL. After thorough mixing, the mixtures were reacted at room temperature. The UV-Vis absorption spectra of each mixed solution were measured using a UV-Vis spectrophotometer, and the absorbance at a wavelength of 652 nm was recorded.

[0065] Table 1. Components and conditions of different reaction systems

[0066] (2) Test results: like Figure 5As shown, systems 1 and 2 showed no obvious TMB oxidation characteristic peaks; system 3 (material only, no H2O2, no light) also did not produce obvious characteristic peaks; systems 4 (material only + light), 5 (material + H2O2), and 6 (material + H2O2 + light) all showed obvious characteristic absorption peaks at 652 nm. Particularly noteworthy is that system 4 (material only + light) achieved a catalytic effect comparable to system 5 (material + H2O2), demonstrating that CuFe-g-C3N4 can self-produce H2O2 under light irradiation and drive a peroxidase-like reaction without the need for external H2O2 addition.

[0067] Figure 5 In the figure, b represents the H2O2 content detected at different times using potassium iodide and potassium hydrogen phthalate solutions after adding different materials, thus proving that the system can generate H2O2 under light irradiation. The results show that all materials can generate H2O2 under light irradiation, and the amount of H2O2 increases with time. Comparative Example 3's Fe-CN can generate 244.81 μM of H2O2 in 30 min. Combined with TMB colorimetric testing, this confirms that there is no consumption of H2O2 during this process. Compared with g-C3N4, the introduction of Fe promotes H2O2 generation. H2O2 can be detected in CuFe-CN of Example 1. We believe that the H2O2 generated in this process is immediately consumed to generate ROS, which is an H2O2-mediated ROS generation process.

[0068] Figure 6 The absorbance curves of the CuFe-g-C3N4 nanozyme (CuFe-CN) catalyzing the oxidation of TMB under different light exposure times and pH values ​​in Example 1 are shown. It can be seen that as the light exposure time increases, the TMB color becomes darker and the absorbance becomes stronger; the lower the pH, the darker the TMB color becomes and the stronger the absorbance becomes.

[0069] 2. Comparison of enzyme-like activities of materials prepared by different methods (1) Test method: Four CuFe-g-C3N4 nanozymes prepared in Example 1 and Comparative Examples 4-6 were dissolved in deionized water to prepare dispersions of 100 μg / mL. 100 μL of 10 mmol / L TMB solution and 100 μL of nanozyme dispersion were added to 800 μL of MES buffer (0.1 mol / L, pH 6.2), mixed thoroughly, and irradiated with light for 30 minutes. The absorbance of each mixed solution at 652 nm was measured using a UV-Vis spectrophotometer.

[0070] (2) Test results: like Figure 7As shown, the CuFe-g-C3N4 nanozyme prepared in Example 1 exhibited the highest absorbance at 652 nm, significantly superior to Comparative Examples 4-6. The results indicate that the stepwise loading strategy of this invention (first loading Cu and calcining, then loading Fe) is key to obtaining high enzyme-like activity; changing the loading order or omitting the calcination step significantly reduces the catalytic performance of the material.

[0071] 3. Evaluation of the antibacterial properties of CuFe-g-C3N4 nanozymes Staphylococcus aureus and Escherichia coli were selected as representative strains, with an initial concentration of 1×10⁻⁶. 7 CFU / mL. The CuFe-g-C3N4 nanozyme from Example 1 was prepared into solutions of different concentrations (0, 20, 40, 60, 80, 100 μg / mL) using MES buffer. The final bacterial concentration was 1×10⁻⁶. 6 In a CFU / mL buffer system, the mixture was incubated under light for 30 minutes. 20 μL of the bacterial culture was evenly spread onto an LB agar plate and incubated at 37°C for 16 hours. Results were as follows... Figure 8 As shown, it can be seen that as the concentration of nanozyme material added during the antibacterial process increases, the number of colonies decreases during the plate antibacterial process.

[0072] Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) were selected as representative strains, with an initial concentration of 1×10⁻⁶. 7 CFU / mL. The experiment was divided into 5 groups: (I) control group (MES buffer), (II) g-C3N4 group of Comparative Example 1, (III) Fe-g-C3N4 group of Comparative Example 3, (IV) Cu-g-C3N4 group of Comparative Example 2, and (V) CuFe-g-C3N4 group of Example 1. The material concentration in each group was 100 μg / mL, and the final bacterial concentration was 1×10⁻⁶. 6 In a CFU / mL buffer system, irradiate with light for 30 minutes. Spread 20 μL of bacterial suspension evenly onto LB solid agar plates and incubate at 37°C for 16 hours. Count the colony-forming units on each agar plate and calculate the antibacterial rate.

[0073] Table 2 Comparison of antibacterial rates of different materials

[0074] As shown in Table 2 and Figure 9As shown, the CuFe-g-C3N4 group exhibited antibacterial rates of 99.79%, 99.35%, and 93.92% against E. coli, S. aureus, and MRSA, respectively, all significantly higher than the g-C3N4 group, Fe-g-C3N4 group, and Cu-g-C3N4 group. These results indicate that the CuFe dual-single-atom sheet-like nanozyme with photocatalytic antibacterial activity prepared in Example 1 of this invention possesses excellent broad-spectrum antibacterial properties, especially demonstrating highly efficient killing ability against the drug-resistant bacterium MRSA.

[0075] 4. Biocompatibility evaluation of CuFe-g-C3N4 nanozymes The biocompatibility and cytotoxicity of CuFe-CN were assessed using human umbilical vein endothelial cells (HUVECs). First, HUVECs were cultured overnight in 96-well plates at 37°C under a 5% (v / v) CO2 atmosphere. Then, different concentrations (0, 20, 40, 60, 80, and 100 μg / mL) of CuFe-CN suspension were added to the culture plates, and incubation was continued for 24, 48, and 96 hours. After incubation, cells were washed with PBS, and Cell Counting Kit-8 (CCK-8, Dojindo, Japan) reagent was added to each well, followed by incubation for 2 hours to assess cell viability. Finally, the absorbance of the supernatant at λ = 450 nm was measured using a microplate reader. HUVEC cell viability was calculated using the following formula: Cell viability = (AT / AC) × 100%, where AT is the absorbance of the test cells treated with the antimicrobial agent, and AC is the absorbance of the untreated control cells. Each treatment group underwent three replicate experiments.

[0076] Hemolysis assay: Fresh mouse whole blood was used in the hemolysis assay. Fresh blood was centrifuged at 2000 rpm for 2 minutes to collect red blood cells (RBCs), which were then washed three times with 0.9% biological NaCl solution. Subsequently, 200 µL of a 4% RBC suspension was incubated with different concentrations of CuFe-CN nanozymes for 2 hours. Negative and positive controls were treated with PBS and Triton-X100 solution, respectively. After incubation, the samples were centrifuged, and the absorbance of the supernatant was measured at 545 nm. All materials used in this study were dissolved in 0.9% NaCl solution. The hemolysis rate (%) was calculated using the following formula: Hemolysis rate (%) = (AM) AN) / (AW AN) × 100%; AM, AN, and AW represent the absorbance of red blood cells incubated with the material, PBS, and Triton-X100, respectively.

[0077] Figure 10 The results showed that the survival rate of fibroblasts remained above 90% within the concentration range of 0~100 µg / mL, indicating that CuFe-g-C3N4 nanozyme has good biocompatibility and low cytotoxicity, providing a safety guarantee for its application in the biomedical field. Figure 10 The hemolysis test results showed that the supernatant of CuFe-CN (500 µg / mL) was colorless, and the calculated hemolysis rate was only 0.06%. The supernatant of the positive control (Triton-X100) was bright red, showing obvious hemolysis.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing CuFe double single-atom sheet-like nanozymes with photocatalytic antibacterial activity, characterized in that, Includes the following steps: Copper salt was added to the dispersion of carbon nitride nanosheets for impregnation treatment, and the solid product was obtained by drying after solid-liquid separation. The first solid product was calcined under an inert atmosphere to obtain carbon nitride nanosheets loaded with copper single atoms. The copper-loaded carbon nitride nanosheets were dispersed in water, impregnated with ferrous salt, and dried after solid-liquid separation to obtain the CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity.

2. The preparation method according to claim 1, characterized in that, The copper salt is at least one of copper nitrate, copper sulfate, and copper acetate, and the ferrous salt is at least one of ferrous chloride, ferrous sulfate, and ferrous nitrate.

3. The preparation method according to claim 1, characterized in that, The amount of copper salt added is 15-30% of the total mass of the carbon nitride nanosheets and copper salt; the amount of ferrous salt added is 2-5% of the total mass of the carbon nitride nanosheets loaded with copper single atoms and ferrous salt.

4. The preparation method according to claim 1, characterized in that, In the step of adding copper salt for impregnation, the impregnation time is 1~4 h; in the step of adding ferrous salt for impregnation, the impregnation time is 2~6 h; the impregnation is carried out under stirring conditions.

5. The preparation method according to claim 1, characterized in that, The heating rate of the calcination treatment is 5~15℃ / min, the calcination temperature is 450~650℃, the holding time is 0.5~2 h, and the inert atmosphere is argon atmosphere or nitrogen atmosphere.

6. The preparation method according to claim 1, characterized in that, The preparation of the carbon nitride nanosheets includes: placing dicyandiamide in an air atmosphere and annealing it at 500~600℃ to obtain bulk graphitic carbon nitride; and then acidifying and exfoliating the bulk graphitic carbon nitride with concentrated sulfuric acid, dialyzing, and drying it to obtain the carbon nitride nanosheets.

7. A CuFe double single-atom sheet-like nanozyme with photocatalytic antibacterial activity, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. A dual single-atom nanoenzyme hydrogel microneedle, characterized in that, It includes a microneedle matrix and a CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity as described in claim 7, dispersed in the microneedle matrix.

9. The dual single-atom nanoenzyme hydrogel microneedles as described in claim 8, characterized in that, The microneedle matrix is ​​methacrylamide hyaluronic acid.

10. The application of the CuFe double single-atom sheet nanozyme with photocatalytic antibacterial activity as described in claim 7, or the double single-atom nanozyme hydrogel microneedles as described in claim 8 or 9, in the preparation of antibacterial products.

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