Cu / Cu2O / CuO-Ti3C2Tx nano material as well as preparation method and application thereof

Abstract

The invention relates to a Cu / Cu2O / CuO-Ti3C2Tx nano-material and a preparation method and application thereof, and belongs to the technical field of biological medicines.The Cu / Cu2O / CuO-Ti3C2Tx nano-material is prepared by preparing a Cu / Cu2O / CuO mixture with hydrazine hydrate as a reducing agent and self-assembling the Cu / Cu2O / CuO mixture and Ti3C2Tx to prepare a Cu2O / CuO-Ti3C2Tx compound with peroxidase (POD-like) activity, and the Cu2O / CuO-Ti3C2Tx compound is used for preparing the Cu2O / CuO-Ti3C2Tx nano-material. According to the compound, the plasma resonance effect of Ti3C2Tx and semiconductors and redox characteristics of Cu with different valence states are utilized, efficient space separation of photo-induced electron-hole pairs is achieved under near-infrared (NIR) excitation, and therefore the peroxidase-like activity and the glutathione-like dual activity of the compound are synchronously activated. The double-enzyme activity synergistic system can synergistically catalyze H2O2 and endogenous glutathione (GSH) to be converted into hydroxyl free radicals (OH) with high cytotoxicity and oxidized glutathione (GSSG), and through an oxidative stress'double-channel 'mechanism, the excellent effects of multi-dimensional broad-spectrum killing of gram-negative bacteria, gram-positive bacteria and fungi and removal of biological membranes are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial and a preparation method and application thereof. Background Art

[0002] With the increasing prevalence of drug-resistant bacteria caused by overuse of antibiotics, the highly adaptable nature of bacteria, fungi, and the biofilms they form poses significant challenges for conventional antimicrobial treatments, including insufficient penetration, the development of drug resistance, and environmental residues. Against this backdrop, the development of novel, highly effective, safe, and sustainable antimicrobial materials is becoming increasingly urgent. In this quest, near-infrared (NIR)-responsive materials, owing to their unique photothermal / photodynamic synergistic effects, have emerged as promising candidates. These materials effectively leverage the deep tissue penetration of NIR light to achieve precise and controlled bactericidal effects while significantly minimizing damage to normal tissue. Furthermore, NIR-responsive materials demonstrate significant advantages in anti-infective therapy, such as high bactericidal efficiency and reduced resistance development, bringing revolutionary new approaches to this field. With continued research and technological advancement, NIR-responsive materials are poised to play an increasingly important role in future anti-infective treatments, contributing significantly to global public health.

[0003] Metal oxide semiconductors Cu2O and CuO nanomaterials have attracted much attention in the field of catalysis due to their unique redox activity and narrow energy gap. However, Cu2O and CuO nanomaterials also have problems such as insufficient photostability and limited catalytic efficiency, which undoubtedly greatly hinder their promotion and development in practical applications. Summary of the Invention

[0004] To overcome the problems existing in the prior art, the present invention provides a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial, its preparation method, and application. A Cu / Cu2O / CuO mixture is prepared using hydrazine hydrate as a reducing agent. This mixture self-assembles with Ti3C2Tx to form a Cu / Cu2O / CuO-Ti3C2Tx complex with peroxidase-like (POD-like) activity. This complex utilizes the plasmon resonance effect of Ti3C2Tx and the semiconductor and redox properties of Cu in different valence states to achieve efficient spatial separation of photogenerated electron-hole pairs under near-infrared (NIR) excitation, thereby simultaneously activating its POD-like activity and glutathione oxidase-like (GSH) dual activity. This dual-enzyme activity synergistic system can synergistically catalyze the conversion of H2O2 and endogenous glutathione (GSH) into highly cytotoxic hydroxyl radicals (·OH) and oxidized glutathione (GSSG). Through a "dual pathway" mechanism of oxidative stress, it achieves anti-Gram-negative effects (e.g.,E. coli ), Gram-positive bacteria (such as S. aureus ) and fungi (such as C. albicans ) has excellent effects in multi-dimensional broad-spectrum killing and removal of biofilm.

[0005] To achieve the above objectives, the first object of the present invention is to provide a method for preparing a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial, comprising the following steps: (1) Mix 1.00-1.50 g of LiF and 20-30 mL of 9 M HCl solution, stir for 1-2 h, then slowly add 1.00-1.50 g of Ti3AlC2, continue stirring at 35°C for 48-50 h, centrifuge, wash with deionized water to pH 4.5-5.5, and freeze-dry to obtain Ti3C2Tx; (2) Weigh 4.00-5.00 g of polyvinylpyrrolidone (PVP) powder and add it to 100-120 mL of 0.01 M CuCl2·2H2O blue solution. Stir until the PVP dissolves. Add 10-15 mL of 2 M NaOH dropwise to form a blue precipitate. Stir continuously at room temperature for 30-40 min. Then add 5-10 mL of hydrazine hydrate solution to form a brown-black precipitate. Stir continuously in a 50-55 °C water bath for 3-4 h. Then cool to room temperature and centrifuge. Wash the solid phase obtained by centrifugation alternately with water and ethanol 3-4 times and vacuum dry to obtain a Cu / Cu2O / CuO mixture. (3) Cu / Cu2O / CuO and Ti3C2Tx were dispersed in 10-15 mL of deionized water at a mass ratio of 3-4:1, stirred for 12-15 h, and then centrifuged. After washing alternately with water and ethanol for 3-4 times, the mixture was vacuum-dried to obtain Cu / Cu2O / CuO-Ti3C2Tx nanozyme.

[0006] The present invention also provides a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method.

[0007] The present invention also provides the use of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method as an antibacterial nanozyme.

[0008] The present invention also provides the use of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method as a near-infrared light-responsive antibacterial nanozyme.

[0009] Furthermore, the near-infrared light has a wavelength of 808 nm and a power of 0.50-1.0 W / cm 2 .

[0010] Beneficial effects of the present invention: The present invention uses hydrazine hydrate as a reducing agent to prepare a Cu / Cu2O / CuO mixture, which is then self-assembled with Ti3C2Tx to prepare a Cu / Cu2O / CuO-Ti3C2Tx complex with peroxidase-like (POD-like) activity. By utilizing the plasmon resonance effect of Ti3C2Tx and the semiconductor and redox properties of Cu in different valence states, efficient spatial separation of photogenerated electron-hole pairs is achieved under near-infrared (NIR) excitation, thereby simultaneously activating its POD-like activity and glutathione oxidase-like (GSH oxidase-like) dual activity. This dual-enzyme activity synergistic system can synergistically catalyze the conversion of H2O2 and endogenous glutathione (GSH) into highly cytotoxic hydroxyl radicals (·OH) and oxidized glutathione (GSSG), achieving a dual-pathway mechanism of oxidative stress against Gram-negative bacteria (such as E. coli ), Gram-positive bacteria (such as S. aureus ) and fungi (such as C. albicans ) has excellent effects in multi-dimensional broad-spectrum killing and removal of biofilm.

[0011] In vitro experiments showed that Cu / Cu2O / CuO-Ti3C2Tx has excellent biocompatibility and low toxicity. Combined with its POD-like activity and photothermal properties, the nanomaterial exhibits broad-spectrum antibacterial properties under NIR activation and can effectively inactivate Gram-negative bacteria ( E. coli , 1 μg / mL, 99.0%), Gram-positive bacteria ( S. aureus , 0.8 μg / mL, 99.57%) and fungi ( C. albicans , 10 μg / mL, 98.32%); 80 μg / mL of Cu / Cu2O / CuO-Ti3C2Tx can make E. coli 、 S. aureus and C. albicans The biofilm percentage dropped to about 13%. This "catalysis-depletion-photothermal" three-modal synergistic system provides a new model for the development of smart nanomaterials for antimicrobial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a SEM image of Cu / Cu2O / CuO-Ti3C2Tx prepared in Example 1 of the present invention; Figure 2 This is the Cu 2p high-resolution XPS spectrum of Cu / Cu2O / CuO-Ti3C2Tx prepared in Example 1 of the present invention; Figure 3The UV-visible absorption spectra of Cu / Cu2O / CuO-Ti3C2Tx and Cu / Cu2O / CuO-Ti3C2Tx+NIR in Example 1 of the present invention are as follows; Figure 4 Figure 1 is the photothermal performance curve of Cu / Cu2O / CuO-Ti3C2Tx in Example 1 of the present invention. Figure a is the temperature curve of Cu / Cu2O / CuO-Ti3C2Tx at different concentrations, and Figure b is the temperature curve of Cu / Cu2O / CuO-Ti3C2Tx at different near-infrared light powers. Figure 5 The photothermal conversion curve of 150 μg / mL Cu / Cu2O / CuO-Ti3C2Tx; Figure 6 Cu / Cu2O / CuO-Ti3C2Tx+H2O2+TMB at 1.0 W / cm 2 , UV absorption spectra with 808 nm irradiation for 7 min and without irradiation; Figure 7 Steady-state kinetic analysis of Cu / Cu2O / CuO-Ti3C2Tx; Figure 8 Figure 2 is the enzyme activity of Cu / Cu2O / CuO-Ti3C2Tx. Figure a is the GSHoxidase-like enzyme activity of Cu / Cu2O / CuO-Ti3C2Tx. Figure b is the effect of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx on the GSHoxidase-like enzyme activity. Figure 9 Effects of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx on the viability of Hep G2 cells and HUVECs cells at 24h and 48h; Figure 10 The antibacterial properties of different groups of materials against different pathogens and fungi under 808nm NIR irradiation and without irradiation were compared with positive drugs; Figure 11 The antibacterial performance of Cu / Cu2O / CuO-Ti3C2Tx with different concentrations against different pathogens and fungi under 808nm NIR irradiation and without irradiation; Figure 12 The antibacterial rates of Cu / Cu2O / CuO-Ti3C2Tx with different concentrations against different pathogens and fungi under 808nm NIR irradiation and without irradiation; Figure 13 Cu / Cu2O / CuO-Ti3C2Tx inhibits C. albicans 、 S. aureus and C. albicans Biofilm properties; Figure 14Cu / Cu2O / CuO-Ti3C2Tx inhibits C. albicans 、 S. aureus and C. albicans Biofilm performance CV staining results; Figure 15 Cu / Cu2O / CuO-Ti3C2Tx inhibits E. coli , S. aureus and C. albicans 3D biofilm3D biofilm imaging; Figure 16 The damage of bacterial cell membranes was determined by NPN in Example 1 of the present invention; Figure 17 This is the bacterial protein leakage situation in Example 1 of the present invention; Figure 18 The ROS generation in different bacterial cells treated with Cu / Cu2O / CuO-Ti3C2Tx; Figure 19 This is the SYTO-9 / PI double staining fluorescence imaging analysis in Example 1 of the present invention; Figure 20 These are SEM images of different bacteria treated with Cu / Cu2O / CuO-Ti3C2Tx in Example 1 of the present invention. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention will be described in detail below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0014] The preparation method of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial of the present invention comprises the following steps: (1) Place 1.00-1.50 g of LiF and 20-30 mL of 9 M HCl solution in a polytetrafluoroethylene beaker, stir for 1-2 h, then slowly add 1.00-1.50 g of Ti3AlC2, continue stirring at 35°C for 48-50 h, centrifuge, wash with deionized water to pH 4.5-5.5, and freeze-dry to obtain Ti3C2Tx; (2) Weigh 4.00-5.00 g of polyvinylpyrrolidone (PVP) powder and add it to 100-120 mL of 0.01 M CuCl2·2H2O blue solution. Stir until the PVP dissolves. Add 10-15 mL of 2 M NaOH dropwise to form a blue precipitate. Continue stirring at room temperature for 30-40 min. Then add 5-10 mL of hydrazine hydrate solution to form a brown-black precipitate. Continue stirring in a 50-55 °C water bath for 3-4 h. Then cool to room temperature, centrifuge, wash the precipitate with water and ethanol alternately 3-4 times, and vacuum dry to obtain a Cu / Cu2O / CuO mixture. (3) Disperse Cu / Cu2O / CuO and Ti3C2Tx in 10-15 mL of deionized water at a mass ratio of 3-4:1, stir for 12-15 h, centrifuge, wash alternately with water and ethanol 3-4 times, and vacuum dry to obtain Cu / Cu2O / CuO-Ti3C2Tx nanomaterials; Application of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials: The Cu / Cu2O / CuO-Ti3C2Tx nanozyme was mixed with a peroxide and ultrasonically treated for 5-10 minutes, wherein the weight ratio of Cu / Cu2O / CuO-Ti3C2Tx to peroxide was 1-1:5. When used, the nanozyme was irradiated with 808 nm near-infrared light for 10-15 minutes. The peroxide was hydrogen peroxide, sodium persulfate, or ammonium persulfate; and the power of the near-infrared light was 0.50-1.0 W / cm 2 .

[0015] In order to illustrate the present invention more clearly, the following examples are provided for detailed description.

[0016] Example 1

[0017] The preparation of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials includes the following steps: (1) Preparation of Ti3C2Tx 1.00 g of LiF and 20 mL of 9 M HCl solution were placed in a polytetrafluoroethylene beaker in sequence. After stirring for 1 h, 1.00 g of Ti3AlC2 was slowly added. After continuous stirring at 35°C for 48 h, the mixture was centrifuged at 8000 r / min for 10 min, washed with deionized water to about pH 5, and freeze-dried to obtain Ti3C2Tx.

[0018] (2) Preparation of Cu / Cu2O / CuO 4.00 g of polyvinylpyrrolidone (PVP) powder was weighed and added to 100 mL of a 0.01 M blue CuCl2·2H2O solution. The mixture was stirred until the PVP dissolved. 10 mL of 2 M NaOH was added dropwise to form a blue precipitate. Stirring was continued at room temperature for 30 minutes. Subsequently, 8 mL of hydrazine hydrate solution was added to form a brown-black precipitate. Stirring was continued in a 50°C water bath for 4 hours. The mixture was then cooled to room temperature and centrifuged at 8000 rpm for 10 minutes. The precipitate was washed alternately with water and ethanol three to four times and then dried in vacuo at 60°C for 12 hours to obtain a Cu / Cu2O / CuO mixture.

[0019] (3) Preparation of Cu / Cu2O / CuO-Ti3C2Tx 30 mg of Cu / Cu2O / CuO and 10 mg of Ti3C2Tx were dispersed in 10 mL of deionized water, stirred for 12 h, centrifuged at 10000 r / min for 5 min, washed alternately with water and ethanol 3-4 times, and then vacuum dried at 60 °C for 12 h to obtain Cu / Cu2O / CuO-Ti3C2Tx nanomaterials.

[0020] Performance analysis of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials: (1) Through SEM ( Figure 1 ) verified the cluster microsphere morphology of Cu / Cu2O / CuO-Ti3C2Tx, and SEM showed that there were many small microspheres aggregated on the surface of Cu / Cu2O / CuO-Ti3C2Tx.

[0021] (2) High-resolution spectra of Cu 2p of Cu / Cu2O / CuO-Ti3C2Tx (attached Figure 2 ) It can be concluded that Cu 0 The bond energy peaks at 932.1 eV, 951.7 eV and 933.9 eV are attributed to Cu + , while the peak at 955.1 eV is attributed to Cu 2+ , which confirms the simultaneous existence of three valence states of copper in Cu / Cu2O / CuO-Ti3C2Tx.

[0022] (3) Photothermal performance evaluation of Cu / Cu2O / CuO-Ti3C2Tx In order to evaluate the NIR-triggered photothermal performance of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials, the temperature change of the nanomaterial solution under 808 nm NIR laser irradiation was measured. The ultraviolet-visible-NIR (UV-Vis-NIR) absorption spectroscopy was used to characterize the Figure 3) found that Cu / Cu2O / CuO-Ti3C2Tx exhibits a significant absorption peak at 808 nm, which is directly related to its photothermal energy conversion performance. Photothermal heating curve ( Figure 4 ab) confirmed that the temperature increase is related to the concentration and infrared intensity of Cu / Cu2O / CuO-Ti3C2Tx. Under 808 nm wavelength light irradiation, the temperature of the Cu / Cu2O / CuO-Ti3C2Tx solution with a concentration of 200 µg / mL rose to 57.2°C. This temperature range can effectively induce damage to the bacterial membrane structure. In contrast, the temperature increase of pure water under 808 nm infrared irradiation is negligible. These results indicate that Cu / Cu2O / CuO-Ti3C2Tx has the ability to convert NIR light energy into thermal energy, providing a basis for further research on the photothermal properties of Cu / Cu2O / CuO-Ti3C2Tx. In addition, the photothermal conversion efficiency (η) of Cu / Cu2O / CuO-Ti3C2Tx was calculated according to the following formula:

[0023] in T max is the equilibrium temperature of the sample solution, T surr Corresponding to the ambient temperature of the experiment, Q dis is the heat loss caused by the container absorbing light, where Q dis = (5.4×10 -4 ), I represents the power density of 808 nm laser (1.0 W / cm 2 ), the absorbance of the sample solution at 808 nm was recorded as A λ ;for hs The determined value is obtained according to the following formula:

[0024] in m represents the mass of the sample solution, C water Corresponding to the heat capacity of water (4.2 J kg -1 K -1 ), τ s is the time constant of the system. Therefore, the photothermal conversion efficiency of the prepared Cu / Cu2O / CuO-Ti3C2Tx is 49.8% ( Figure 5 ab), showing high photothermal conversion efficiency.

[0025] (4) Evaluation of Cu / Cu2O / CuO-Ti3C2Tx peroxidase activity The peroxidase-like activity (POD-like) of Cu / Cu2O / CuO-Ti3C2Tx was evaluated using TMB as the catalytic reaction substrate as follows: In a 5 mL stoppered colorimetric tube, 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H2O2, and 50 μL of 1 mg / mL Cu / Cu2O / CuO-Ti3C2Tx were added to 2 mL of 0.1 mol / L HAc-NaAc buffer solution (pH 4.0). The mixture was thoroughly mixed and the volume was adjusted to 3 mL. After reacting for 10 min, the absorbance was measured. At the same time, the test was carried out at 1.0 W / cm 2 The enzyme activity after irradiation at 808 nm for 10 min was compared, and the absorbance was measured at 654 nm. The results are shown in Figure 2. Figure 6 When Cu / Cu2O / CuO-Ti3C2Tx and H2O2 were present in the system, TMB underwent a significant oxidation reaction. This experimental phenomenon fully demonstrated that Cu / Cu2O / CuO-Ti3C2Tx possesses POD-like catalytic activity, mimicking the function of natural peroxidases and catalyzing the oxidation of TMB by H2O2 to produce a blue product. Furthermore, when the system was exposed to NIR irradiation for 10 minutes, the POD-like activity of Cu / Cu2O / CuO-Ti3C2Tx was observed to be significantly enhanced, by nearly 4 times, indicating that Cu / Cu2O / CuO-Ti3C2Tx exhibits near-infrared light-enhanced peroxidase-like activity.

[0026] (5) The experiment also carried out the determination of Michaelis-Menten catalytic kinetic parameters ( Figure 7 (ad) and Table 1), the Michaelis constants of Cu / Cu2O / CuO-Ti3C2Tx for substrates TMB and H2O2 K m The reaction rates were 0.603 mM and 5.582 mM, respectively, and the reaction rate constant was 4.027×10 -6 M / s and 0.309×10 -6 M / s, after infrared light irradiation K m are 0.531 mM and 5.115 mM, and the reaction rate constant is 7.789×10 -6 M / s and 0.356×10 -6 M / s, indicating that infrared light irradiation increases the affinity and reaction rate of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials with the substrate.

[0027] Table 1 Michaelis-Menten catalytic kinetic parameters

[0028] (6) Evaluation of glutathione oxidase (GSH oxidase-like) activity To a mixed solution of 50 μL of 100 mM 5,5′-dithiodinitrophenol (DTNB) and 1 mL of 2 mg / mL GSH, 50 μL of 2 mg / mL Cu / Cu2O / CuO-Ti3C2Tx was added, and the change in absorbance at 420 nm was observed. Figure 8 As shown in a, compared with the blank group, the absorbance value of the Cu / Cu2O / CuO-Ti3C2Tx reaction system at 420 nm decreased significantly, indicating that Cu / Cu2O / CuO-Ti3C2Tx has significant GSH scavenging activity. Figure 8 As shown in Figure b, under NIR irradiation, by testing different concentrations of Cu / Cu2O / CuO-Ti3C2Tx, it was found that its GSH oxidase-like enzyme activity increased with increasing concentration, indicating that the enzyme activity was positively correlated with concentration. The above results all indicate that Cu / Cu2O / CuO-Ti3C2Tx can effectively oxidize GSH to oxidized glutathione (GSSG).

[0029] (7) Cytotoxicity test The CCK-8 cell viability kit was used to detect the cytotoxicity of the nanomaterials. In the specific experiment, human umbilical vein endothelial cells and hepatocellular carcinoma cells (HUVECs and Hep G2, Beina Chuanglian Biotechnology Co., Ltd.) were seeded in 96-well plates and cultured for 24 hours. They were then incubated with different concentrations of Cu / Cu2O / CuO-Ti3C2Tx for 24 and 48 hours, respectively. The cells were rinsed with PBS in batches, and CCK-8 solution was added to each well to a concentration of 10%. The cells were incubated at 37°C, and the absorbance was measured at 450 nm. The CCK-8 assay ( Figure 9 ab) showed that Cu / Cu2O / CuO-Ti3C2Tx had no toxicity to cells.

[0030] (8) Antibacterial test of Cu / Cu2O / CuO-Ti3C2Tx The following strains were obtained from Beina Chuanglian Biotechnology Co., Ltd.

[0031] Experimental method: Staphylococcus aureus ( S. aureus , ATCC 43300), Escherichia coli ( E. coli , ATCC-8099), Candida albicans ( C. albicans , ATCC 90028) were used to represent Gram-positive and Gram-negative strains and fungi. The antibacterial activity of Cu / Cu2O / CuO-Ti3C2Tx was determined by counting the number of CFU using the plate count method.

[0032] First, the above strains E. coli In Luria-Bertani (LB) solid medium and S. aureus Incubate in solid nutrient broth for 24 h. C. albicans After incubation in Sabouraud's solid medium for 48 h, the formed colonies were picked with an inoculation loop and transferred into the corresponding liquid medium (5 mL). Then, the bacterial suspension (1 × 10 8 CFU / mL), diluted to 1 × 10 5 CFU / mL. The materials were divided into the following groups: Ti3C2Tx+ H2O2, Ti3C2Tx+ H2O2+NIR, Cu / Cu2O / CuO +H2O2, Cu / Cu2O / CuO+H2O2+NIR, Cu / Cu2O / CuO-Ti3C2Tx +H2O2、Cu / Cu2O / CuO-Ti3C2Tx +H2O2+NIR and positive control group ( E. coli Cephalexin, S. aureus For gentamicin, C. albicans The cultured bacteria were added to phosphate buffer as the blank control group, and the other groups were mixed with nanomaterials and subjected to 1.0 W / cm 2 The cells were irradiated with 808 nm for 10 min or without infrared light treatment and incubated at 37°C for 30 min. The bacterial suspension was diluted (100 μL) and evenly spread on LB solid medium and nutrient broth solid medium. The cells were cultured at 37°C for 24 h, and the number of colonies was counted to determine the antibacterial properties. The results were as follows: Figure 10 As shown in Figure 2, at the same concentration, the Cu / Cu2O / CuO-Ti3C2Tx+ H2O2+NIR group showed the best antibacterial effect, and its antibacterial effect on the three bacteria was better than that of the positive drug ( E. coli , S. aureus and C. albicans The positive drugs for the Cu / Cu2O / CuO-Ti3C2Tx composites were gentamicin, cephalexin, and fluconazole. This phenomenon confirms that NIR light irradiation can significantly enhance the antibacterial properties of Cu / Cu2O / CuO-Ti3C2Tx composites. The mechanism may be related to the local thermal effect and ROS increase induced by NIR light. The antibacterial properties of Cu / Cu2O / CuO-Ti3C2Tx with different concentrations were tested against different pathogens and fungi under 808 nm irradiation and without irradiation. Figure 11 and Figure 12 As shown in the figure, Cu / Cu2O / CuO-Ti3C2Tx exhibited significant concentration-dependent antibacterial effects on different microorganisms. The experimental results showed that: (1) against Gram-negative bacteria E. coli When the concentration of Cu / Cu2O / CuO-Ti3C2Tx was 1 μg / mL, the bactericidal rate was 99.0%; (2) for Gram-positive bacteria S. aureus , the inhibition rate can reach 99.57% at a concentration of 0.8 μg / mL; (3) C. albicans At a concentration of 10 μg / mL, the bactericidal rate was 98.32%.

[0033] To further evaluate the anti-biofilm performance of Cu / Cu2O / CuO-Ti3C2Tx, we investigated its anti-biofilm ability by crystal violet (CV) staining and measured the absorbance at 590 nm to quantify the biofilm. Figure 13 and Figure 14 As shown in the presence of H2O2, Cu / Cu2O / CuO-Ti3C2Tx E. coli 、 S. aureus and C. albicans The biofilms of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx were inhibited by NIR, and the effect of Cu / Cu2O / CuO-Ti3C2Tx+NIR was better than that of Cu / Cu2O / CuO-Ti3C2Tx. E. coli 、 S. aureus and C. albicans The mixture was irradiated at 808 nm for 30 min and then placed in a constant temperature incubator for 24 h. The production rate of bacterial biofilm was compared with that of the group without Cu / Cu2O / CuO-Ti3C2Tx and the group without infrared. The results showed that the increase of Cu / Cu2O / CuO-Ti3C2Tx content led to a decrease in biofilm production. 100 μg / mL of Cu / Cu2O / CuO-Ti3C2Tx could E. coli The biofilm decreased to about 11.35%, S. aureus Hehe C. albicans were 12.36% and 13.49%, respectively, indicating that Cu / Cu2O / CuO-Ti3C2Tx had excellent inhibitory ability on the formation of biofilm.

[0034] Visualized 3D biofilm test: The destruction of biofilm is an important parameter to determine the effectiveness of antibiotic film. E. coli , S. aureus and C. albicansTo improve the destructive performance of the biofilm, Alexa Fluor 647 dextran dye was added at a final concentration of 1 mM during the biofilm culture process to label the polysaccharides in the extracellular matrix red, and SYTO-9 fluorescent nucleic acid dye at a final concentration of 2.5 mM was added to label the bacterial cells in the biofilm green. 3D biofilm imaging was performed using a confocal laser scanning microscope ( Figure 15 ).from Figure 15 It can be clearly seen that in the blank group, E. coli , S. aureus and C. albicans It is very dense, with spherical protrusions, and is rich in bacterial species and EPS. The most intuitive manifestation is that the thickness of the biofilm of the three is on a downward trend, proving that Cu / Cu2O / CuO-Ti3C2Tx destroys the biofilm.

[0035] Next, the penetration of the outer membrane of various pathogenic bacteria was measured using the hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN), which exhibits weak fluorescence in aqueous solution but strong fluorescence intensity in hydrophobic environments. Figure 16 As shown in the figure, the Cu / Cu2O / CuO-Ti3C2Tx+NIR group showed a fluorescence intensity significantly higher than that of the control group, indicating that NIR excitation can significantly enhance the membrane damage performance of the Cu / Cu2O / CuO-Ti3C2Tx material, and the synergistic photothermal-photodynamic effect causes the disintegration of the membrane lipid bilayer structure, promoting more NPN probe molecules to penetrate into the hydrophobic core.

[0036] Another way to verify the destruction of pathogenic bacteria membranes is the leakage of intracellular substances, including some key proteins. The diquinoline formic acid assay (BCA) method was used to detect the leakage concentration of pathogenic bacteria and fungal proteins treated with Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR. Under alkaline conditions, the protein will transfer Cu 2+ Reduction to Cu + , Cu + It forms a purple complex with BCA molecules. Figure 17 As shown in the figure, the protein quantification method was used to determine the extracellular protein concentrations of the three bacteria after interaction with different concentrations of Cu / Cu2O / CuO-Ti3C2Tx. The extracellular protein concentrations of the three bacteria gradually increased with the increase of Cu / Cu2O / CuO-Ti3C2Tx, showing a certain concentration dependence. In addition, the rupture of the bacterial cell membrane may be related to the sharp edges of Ti3C2Tx, which can physically cut the bacterial cell membrane. These results indicate that Cu / Cu2O / CuO-Ti3C2Tx can cause decellularization and perforation of the bacterial cell membrane, leading to the leakage of cytoplasmic components, destroying the integrity of the bacterial cell wall and cell membrane, and causing bacterial death.

[0037] (9) Monitoring of ROS in bacterial cells DCFH-DA was used to capture the generation of reactive oxygen species (ROS) in bacteria. The imaging results are shown in Figure 2. Figure 18 As shown, after treatment with Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR, the experimental group exhibited a significantly enhanced green fluorescence signal, confirming that the composite system can effectively increase intracellular ROS levels. Notably, under NIR light excitation conditions, the catalytic activity of the composite material was significantly enhanced, revealing the synergistic mechanism of Cu / Cu2O / CuO-Ti3C2Tx in photothermal synergistic catalysis - by enhancing the conversion efficiency of H2O2 to highly toxic ROS, thereby achieving a highly effective killing effect on pathogens.

[0038] (10) Dead / live bacteria staining The SYTO-9 / PI double staining method was used to evaluate the viability of bacteria. Figure 19 As shown, Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR E. coli , S. aureus and C. albicans The material exhibited a significant antibacterial effect, with no significant red fluorescence emission detected in either the experimental or blank control groups, indicating that the single-component system failed to effectively activate the antibacterial effect. After the introduction of H2O2, characteristic red fluorescence was observed in the nanomaterial and H2O2 co-treatment group, with enhanced fluorescence confirming that the antibacterial function was enhanced by catalyzing the conversion of H2O2. Notably, under NIR light activation conditions, the fluorescence intensity of the composite system reached a peak, consistent with the mechanism by which its NIR catalytic synergistic effect enhances ROS generation. The antibacterial efficacy of the material was verified at the molecular level.

[0039] (11) SEM observation of bacterial morphology Scanning electron microscopy (SEM) characterization revealed that ( Figure 20 ). Control bacteria exhibited a typical rod-shaped / spherical ultrastructure and maintained cell membrane integrity, confirming that the nanomaterial was unaffected. After treatment with Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR, the bacteria exhibited characteristic structural damage: ① membrane surface wrinkling; ② cell collapse; and ③ intracellular leakage. This multi-level structural damage pattern is closely related to the nanomaterial's cascade catalytic properties. The NIR-enhanced electron transfer efficiency increases the quantum yield of H2O2 conversion to ·OH, triggering a cascade of lipid peroxidation in the bacterial membrane. These SEM findings corroborate previous ROS detection and protein leakage experiments, fully revealing the material's antimicrobial action pathway.

[0040] The above results show that the nanomaterial Cu / Cu2O / CuO-Ti3C2Tx prepared by the present invention exhibits high peroxidase activity and photothermal performance under NIR irradiation, catalyzes H2O2 to produce ROS with bactericidal effect, thereby E. coli 、 S. aureus and C. albicans It has a good antibacterial effect. The characterization of the light response characteristics shows that the nanomaterial system exhibits a dual-mode antibacterial mechanism under 808 nm NIR excitation: ① POD-like activity is improved; ② Photothermal conversion efficiency η = 55.4%, which can achieve a local temperature rise. The research results show that: (1) against Gram-negative bacteria ( E. coli ), when the concentration of Cu / Cu2O / CuO-Ti3C2Tx is 1 μg / mL, the bactericidal rate is 99.0%; (2) for Gram-positive bacteria ( S. aureus ), the inhibition rate can reach 99.57% at a concentration of 0.8 μg / mL; (3) for fungi ( C. albicans ), at a concentration of 10 μg / mL, the bactericidal rate was 98.32%.

[0041] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing Cu / Cu2O / CuO-Ti3C2Tx nanomaterial, characterized in that: The following steps are involved: (1) Mix 1.00-1.50 g of LiF and 20-30 mL of 9 M HCl solution, stir for 1-2 h, then slowly add 1.00-1.50 g of Ti3AlC2, continue stirring at 35°C for 48-50 h, centrifuge, wash with deionized water to pH 4.5-5.5, and freeze-dry to obtain Ti3C2Tx; (2) Weigh 4.00-5.00 g of polyvinylpyrrolidone (PVP) powder and add it to 100-120 mL of 0.01 M CuCl2·2H2O blue solution. Stir until PVP dissolves. Add 10-15 mL of 2 M NaOH dropwise to form a blue precipitate. Stir continuously at room temperature for 30-40 min. Then add 5-10 mL of hydrazine hydrate solution to form a brown-black precipitate. Stir continuously in a 50-55°C water bath for 3-4 h. Then cool to room temperature and centrifuge. Wash the solid phase obtained by centrifugation alternately with water and ethanol 3-4 times and vacuum dry to obtain a Cu / Cu2O / CuO mixture. (3) Cu / Cu2O / CuO and Ti3C2Tx were dispersed in 10-15 mL of deionized water at a mass ratio of 3-4:1, stirred for 12-15 h, and then centrifuged. After washing with water and ethanol alternately for 3-4 times, the mixture was vacuum-dried to obtain Cu / Cu2O / CuO-Ti3C2Tx nanomaterials.

2. The Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the preparation method according to claim 1.

3. Use of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial as claimed in claim 2 as an antibacterial nanozyme.

4. Use of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial as claimed in claim 2 as a near-infrared light-responsive antibacterial nanozyme.

5. The use according to claim 4, characterized in that The near-infrared light has a wavelength of 808 nm and a power of 0.50-1.0 W / cm 2 .

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