Cu / cu2o / cuo-ti3c2tx nanomaterial and preparation method and application thereof

Abstract

The present application relates to a kind of Cu / Cu2O / CuO-Ti3C2Tx Nanomaterial and its preparation method and application, belong to biological medicine technical field, the Cu / Cu2O / CuO mixture prepared with hydrazine hydrate as reducing agent, with Ti3C2Tx self-assembly, prepare Cu2O / CuO-Ti3C2Tx complex with peroxidase-like (POD-like) activity, the complex utilizes the plasmonic resonance effect of Ti3C2Tx and the semiconductor and redox characteristics of different valence Cu, under near infrared (NIR) excitation Realize the efficient spatial separation of photoinduced electron-hole pairs, to simultaneously activate its peroxidase-like activity and glutathione oxidase-like (GSH oxidase-like) double activity. The double enzyme activity synergistic system can synergistically catalyze H2O2 And endogenous glutathione (GSH) be converted into high cytotoxicity hydroxyl radical (OH) and oxidized glutathione (GSSG), by oxidative stress "double channel" mechanism, realize the excellent effect of multi-dimensional broad-spectrum killing and removing biofilm to gram-negative bacteria, gram-positive bacteria and fungi.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial and a preparation method and application thereof. BACKGROUND

[0002] With the increasingly serious problem of drug-resistant bacteria caused by antibiotic abuse, bacteria, fungi and biofilms formed by them have high adaptability. Traditional antibacterial methods often face challenges such as insufficient penetration, drug resistance and environmental residues. In this context, it is particularly urgent to develop new antibacterial materials that are efficient, safe and sustainable. In this exploration process, near-infrared (NIR) responsive materials have gradually stood out due to their unique photothermal / photodynamic synergistic effect. This kind of material can effectively utilize the deep tissue penetration ability of NIR light to achieve precise and controllable sterilization effect, while significantly reducing damage to normal tissues. In addition, NIR-responsive materials have shown significant advantages in anti-infection treatment, such as efficient sterilization and reduced drug resistance, which has brought a revolutionary new idea to the field of anti-infection treatment. It can be predicted that with the continuous deepening of research and the continuous progress of technology, NIR-responsive materials will play an increasingly important role in future anti-infection treatment and contribute new strength to the field of global public health.

[0003] Metal oxide semiconductors Cu2O and CuO nanomaterials have been the focus of researchers in the field of catalysis due to their unique redox activity and narrow energy gap. However, Cu2O and CuO nanomaterials also have the problems of insufficient light stability and limited catalytic efficiency, which undoubtedly greatly hinder their promotion and development in practical applications. SUMMARY

[0004] In order to overcome the problems in the background art, the application provides a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial and a preparation method and application thereof. Hydrazine hydrate is used as a reducing agent to prepare a Cu / Cu2O / CuO mixture, which is self-assembled with Ti3C2Tx to prepare a Cu / Cu2O / CuO-Ti3C2Tx complex with POD-like activity. The complex utilizes the plasmonic resonance effect of Ti3C2Tx and the semiconductor and redox properties of different valence states of Cu to achieve efficient spatial separation of photo-generated electron-hole pairs under near-infrared (NIR) excitation, thereby simultaneously activating its POD-like activity and GSH oxidase-like dual activity. The 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), realizing the "double-pathway" mechanism of oxidative stress to kill Gram-negative bacteria (such asE. coli gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.), gram-positive bacteria (such as Staphylococcus aureus, Enterococcus faecalis, etc.) and fungi (such as Candida albicans, Aspergillus niger, etc. S. aureus ) have excellent effects of multi-dimensional broad-spectrum killing and removing biofilm. C. albicans

[0005] To achieve the above-mentioned purposes, the first purpose of the present application is to provide a preparation method of Cu / Cu2O / CuO-Ti3C2Tx nanomaterial, comprising the following steps:

[0006] (1) 1.00-1.50 g of LiF and 20-30 mL of 9 M HCl solution are mixed, stirred for 1-2 h, then 1.00-1.50 g of Ti3AlC2 is slowly added, continuously stirred at 35℃ for 48-50 h, centrifuged, washed with deionized water until pH 4.5-5.5, and freeze-dried to obtain Ti3C2Tx;

[0007] (2) 4.00-5.00 g of polyvinylpyrrolidone PVP powder is weighed and added into 100-120 mL of 0.01 M CuCl2·2H2O blue solution, stirred until PVP is dissolved, 10-15 mL of 2 M NaOH is added dropwise to generate a blue precipitate, continuously stirred at room temperature for 30-40 min, then 5-10 mL of hydrazine hydrate solution is added to form a brown-black precipitate, and continuously stirred in a 50-55℃ water bath for 3-4 h, then cooled to room temperature, centrifuged, and the solid phase obtained by centrifugation is washed with water and ethanol alternately for 3-4 times, and vacuum dried to obtain a Cu / Cu2O / CuO mixture;

[0008] (3) Cu / Cu2O / CuO and Ti3C2Tx are dispersed in 10-15 mL of deionized water at a mass ratio of 3-4:1, stirred for 12-15 h, centrifuged, washed with water and ethanol alternately for 3-4 times, and vacuum dried to obtain Cu / Cu2O / CuO-Ti3C2Tx nanoscale enzyme.

[0009] The present application also provides the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method.

[0010] The present application also provides the application of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method as an antibacterial nanoscale enzyme.

[0011] The present application also provides the application of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial obtained by the above preparation method as a near-infrared light responsive antibacterial nanoscale enzyme.

[0012] Further, the wavelength of the near-infrared light is 808 nm, and the power is 0.50-1.0 W / cm 2 . ​

[0013] Beneficial effects of the present invention:

[0014] 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.

[0015] 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. 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

[0016] Figure 1 This is a SEM image of Cu / Cu2O / CuO-Ti3C2Tx prepared in Example 1 of the present invention;

[0017] Figure 2 This is the Cu 2p high-resolution XPS spectrum of Cu / Cu2O / CuO-Ti3C2Tx prepared in Example 1 of the present invention;

[0018] Figure 3 UV-Vis absorption spectra of Cu / Cu2O / CuO-Ti3C2Tx and Cu / Cu2O / CuO-Ti3C2Tx+NIR in Example 1 of the present application;

[0019] Figure 4 Photothermal performance curve of Cu / Cu2O / CuO-Ti3C2Tx in Example 1 of the present application, Figure a is the temperature curve of Cu / Cu2O / CuO-Ti3C2Tx with different concentrations, Figure b is the temperature curve of Cu / Cu2O / CuO-Ti3C2Tx with different NIR light powers;

[0020] Figure 5 Photothermal conversion efficiency curve of 150 μg / mL Cu / Cu2O / CuO-Ti3C2Tx;

[0021] Figure 6 UV absorption spectra of Cu / Cu2O / CuO-Ti3C2Tx+H2O2+TMB under 1.0 W / cm 2 , 808 nm irradiation for 7 min and no irradiation;

[0022] Figure 7 Steady-state kinetic analysis of Cu / Cu2O / CuO-Ti3C2Tx;

[0023] Figure 8 Enzymatic activity of Cu / Cu2O / CuO-Ti3C2Tx, Figure a is the GSH oxidase-like enzymatic activity of Cu / Cu2O / CuO-Ti3C2Tx, Figure b is the influence of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx on GSH oxidase-like enzymatic activity;

[0024] Figure 9 Influence of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx on the survival rate of Hep G2 cells and HUVECs cells at 24 h and 48 h;

[0025] Figure 10 Antibacterial performance of different groups of materials under 808 nm NIR irradiation and no irradiation on different pathogenic bacteria and fungi compared with positive drugs;

[0026] Figure 11 Antibacterial performance of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx under 808 nm NIR irradiation and no irradiation on different pathogenic bacteria and fungi;

[0027] Figure 12Antibacterial rates of different concentrations of Cu / Cu2O / CuO-Ti3C2Tx under 808nm NIR irradiation and non-irradiation on different pathogenic bacteria and fungi;

[0028] Figure 13 Performance of Cu / Cu2O / CuO-Ti3C2Tx in inhibiting C. albicans 、 S. aureus and C. albicans biofilms;

[0029] Figure 14 Performance of Cu / Cu2O / CuO-Ti3C2Tx in inhibiting C. albicans 、 S. aureus and C. albicans biofilms CV staining results;

[0030] Figure 15 3D biofilm 3D biofilm imaging of Cu / Cu2O / CuO-Ti3C2Tx; E. coli , S. aureus and C. albicans 3D biofilm 3D biofilm imaging of Cu / Cu2O / CuO-Ti3C2Tx;

[0031] Figure 16 NPN determination of bacterial cell membrane damage in Example 1 of the application;

[0032] Figure 17 Bacterial protein leakage in Example 1 of the application;

[0033] Figure 18 ROS production in different bacterial cells treated with Cu / Cu2O / CuO-Ti3C2Tx;

[0034] Figure 19 SYTO-9 / PI double staining fluorescence imaging analysis in Example 1 of the application;

[0035] Figure 20 SEM images of different bacteria treated with Cu / Cu2O / CuO-Ti3C2Tx in Example 1 of the application. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the technical scheme of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0037] The preparation method of the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial of the present application comprises the following steps:

[0038] (1) Put 1.00-1.50 g LiF and 20-30 mL 9 M HCl solution into a polytetrafluoroethane beaker in turn, stir for 1-2 h, then slowly add 1.00-1.50 g Ti3AlC2, continuously stir at 35℃ for 48-50 h, centrifuge, wash with deionized water until pH 4.5-5.5, freeze-dry, and Ti3C2Tx is obtained;

[0039] (2) Take 4.00-5.00 g polyvinylpyrrolidone (PVP) powder and add it to 100-120 mL 0.01 M CuCl2·2H2O blue solution, stir until PVP dissolves, add 10-15 mL 2 M NaOH to form a blue precipitate, continuously stir at room temperature for 30-40 min, then add 5-10 mL hydrazine hydrate solution to form a brown-black precipitate, and continue to stir in a 50-55℃ water bath for 3-4 h, then cool to room temperature, centrifuge, and wash the precipitate with water and ethanol alternately for 3-4 times, and vacuum dry to obtain a Cu / Cu2O / CuO mixture;

[0040] (3) Disperse the Cu / Cu2O / CuO and Ti3C2Tx in a mass ratio of 3-4:1 in 10-15 mL deionized water, stir for 12-15 h, centrifuge, wash with water and ethanol alternately for 3-4 times, and vacuum dry to obtain a Cu / Cu2O / CuO-Ti3C2Tx nanomaterial;

[0041] Application of Cu / Cu2O / CuO-Ti3C2Tx nanomaterial:

[0042] Mix the Cu / Cu2O / CuO-Ti3C2Tx nanomaterial with a peroxide, ultrasonic treat for 5-10 min, wherein the weight ratio of Cu / Cu2O / CuO-Ti3C2Tx to peroxide is 1-1:5, and when applied, irradiate with 808 nm near-infrared light for 10-15 min. The peroxide is hydrogen peroxide, persulfate, or ammonium persulfate; the power of the near-infrared light is 0.50-1.0 W / cm 2 .

[0043] In order to make the present application clearer, the following examples are used for detailed description.

[0044] Example 1

[0045] Preparation of Cu / Cu2O / CuO-Ti3C2Tx nanomaterial, including the following steps:

[0046] (1) Preparation of Ti3C2Tx

[0047] Put 1.00 g LiF and 20 mL 9 M HCl solution into a polytetrafluoroethane beaker in turn, stir for 1 h, then slowly add 1.00 g Ti3AlC2, continue stirring at 35℃ for 48 h, then centrifuge at 8000 r / min for 10 min, wash with deionized water until pH is about 5, and freeze-dry to obtain Ti3C2Tx.

[0048] (2) Preparation of Cu / Cu2O / CuO

[0049] Weigh 4.00 g of polyvinylpyrrolidone PVP powder into 100 mL of 0.01 M CuCl2·2H2O blue solution, stir until PVP is dissolved. Add 10 mL of 2 M NaOH to form a blue precipitate, continue stirring at room temperature for 30 min, then add 8 mL of hydrazine hydrate solution to form a brown-black precipitate, continue stirring in a 50℃ water bath for 4 h, then cool to room temperature, centrifuge at 8000 r / min for 10 min, wash the precipitate with water and ethanol alternately for 3-4 times, and vacuum dry at 60℃ for 12 h to obtain a Cu / Cu2O / CuO mixture.

[0050] (3) Preparation of Cu / Cu2O / CuO-Ti3C2Tx

[0051] Disperse 30 mg of Cu / Cu2O / CuO and 10 mg of Ti3C2Tx in 10 mL of deionized water, stir for 12 h, then centrifuge at 10000 r / min for 5 min, wash with water and ethanol alternately for 3-4 times, and vacuum dry at 60℃ for 12 h to obtain Cu / Cu2O / CuO-Ti3C2Tx nanomaterials.

[0052] Performance analysis of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials:

[0053] (1) The cluster microspherical morphology of Cu / Cu2O / CuO-Ti3C2Tx is verified by SEM Figure 1 ).

[0054] (2) The high-resolution spectrum of Cu 2p of Cu / Cu2O / CuO-Ti3C2Tx (attached Figure 2 ) can be obtained. 0 The binding energy peak of Cu + is at 932.1 eV, 951.7 eV and 933.9 eV, and the peak at 955.1 eV is attributed to Cu 2+This confirms the presence of three valence states of copper in Cu / Cu2O / CuO-Ti3C2Tx.

[0055] (3) Evaluation of the photothermal performance of Cu / Cu2O / CuO-Ti3C2Tx

[0056] 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. It was found by ultraviolet-visible-near infrared (UV-Vis-NIR) absorption spectroscopy characterization ( Figure 3 ) that Cu / Cu2O / CuO-Ti3C2Tx exhibited a significant absorption peak at 808 nm wavelength, which was directly related to its photothermal energy conversion performance. The photothermal heating curve ( Figure 4 a-b) confirmed that the temperature rise was 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℃, which was in the temperature range that could effectively induce bacterial membrane structure damage. In contrast, the temperature rise of pure water under 808 nm infrared irradiation was negligible. These results showed that Cu / Cu2O / CuO-Ti3C2Tx had the ability to convert NIR light energy into heat energy, providing a basis for further study of the photothermal performance of Cu / Cu2O / CuO-Ti3C2Tx. In addition, the photothermal conversion efficiency (η) of Cu / Cu2O / CuO-Ti3C2Tx was calculated according to the following formula:

[0057]

[0058] wherein 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 of the container absorbing light, wherein Q dis = (5.4×10 -4 ), I represents the power density of the 808 nm laser (1.0 W / cm 2 ), and the absorbance of the sample solution at 808 nm is denoted as A λ ; in order to hs determine the value, the following formula is used:

[0059]

[0060] whereinm 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.

[0061] (4) Evaluation of Cu / Cu2O / CuO-Ti3C2Tx peroxidase activity

[0062] The peroxidase-like activity (POD-like) of Cu / Cu2O / CuO-Ti3C2Tx was evaluated using TMB as the catalytic reaction substrate as follows:

[0063] 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 as follows 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 we applied NIR irradiation to the system for 10 minutes, we observed that the POD-like activity of Cu / Cu2O / CuO-Ti3C2Tx was significantly enhanced, with an increase of nearly 4 times. Cu / Cu2O / CuO-Ti3C2Tx exhibits the characteristics of near-infrared light-enhanced peroxidase-like activity.

[0064] (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 m0.603 mM and 5.582 mM, with a reaction rate constant of 4.027 x 10 -6 M / s and 0.309 x 10 -6 M / s, respectively, and the absorbance value at 420 nm decreased significantly after NIR irradiation, indicating that NIR irradiation increased the affinity and reaction rate of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials with the substrate. K m 0.531 mM and 5.115 mM, with a reaction rate constant of 7.789 x 10 -6 M / s and 0.356 x 10 -6 M / s, indicating that NIR irradiation increased the affinity and reaction rate of Cu / Cu2O / CuO-Ti3C2Tx nanomaterials with the substrate.

[0065] Table 1 Michaelis-Menten kinetic parameters

[0066]

[0067] (6) Evaluation of glutathione oxidase-like activity

[0068] To 50 μL of a mixture of 100 mM 5,5'-dithiobisnitrobenzoic acid (DTNB) and 1 mL of 2 mg / mL GSH, 50 μL of 2 mg / mL Cu / Cu2O / CuO-Ti3C2Tx was added, and then the change in absorbance at 420 nm was observed. As shown in Figure 8 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 had significant GSH scavenging activity. In addition, as shown in Figure 8 b, under NIR irradiation, by testing different concentrations of Cu / Cu2O / CuO-Ti3C2Tx, it was found that the GSH oxidase-like enzyme activity increased with increasing concentration, indicating that the enzyme activity was positively correlated with the concentration, and the above results all indicated that Cu / Cu2O / CuO-Ti3C2Tx could effectively oxidize GSH to oxidized glutathione (GSSG).

[0069] (7) Cytotoxicity test

[0070] The cytotoxicity of the nanomaterials was detected using a CCK-8 cell viability kit. 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.

[0071] (8) Antibacterial test of Cu / Cu2O / CuO-Ti3C2Tx

[0072] The following strains were obtained from Beina Chuanglian Biotechnology Co., Ltd.

[0073] 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.

[0074] 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 gentamicin, C. albicans fluconazole) and blank control group, where the concentration of Cu / Cu2O / CuO-Ti3C2Tx was 5 μg / mL and the concentration of H2O2 was 0.5 mmol / L. The cultured bacteria were added to phosphate buffer solution as a blank control group, and the other groups were mixed with the nanomaterials, which were then treated with 1.0 W / cm2, 808 nm irradiation for 10 min or without infrared light irradiation treatment, and incubated at 37 °C for 30 min. After dilution (100 μL), the bacterial suspension was uniformly coated on LB solid medium and nutrient broth solid medium, and incubated at 37 °C for 24 h. The number of colonies was counted to determine the antibacterial performance. The results are shown in 2 , 808 nm irradiation for 10 min or without infrared light irradiation treatment, and incubated at 37 °C for 30 min. After dilution (100 μL), the bacterial suspension was uniformly coated on LB solid medium and nutrient broth solid medium, and incubated at 37 °C for 24 h. The number of colonies was counted to determine the antibacterial performance. The results are shown in Figure 10 , under the same concentration, the Cu / Cu2O / CuO-Ti3C2Tx + H2O2 + NIR group showed the best antibacterial effect, and the antibacterial effect on the three kinds of bacteria was better than that of the positive drug E. coli , S. aureus and C. albicans The positive drugs were gentamicin, cephalexin and fluconazole, respectively). This phenomenon confirmed that NIR light irradiation can significantly enhance the antibacterial performance of Cu / Cu2O / CuO-Ti3C2Tx composite materials, and the mechanism may be related to the local thermal effect and ROS increase induced by NIR light. Through the antibacterial performance test of Cu / Cu2O / CuO-Ti3C2Tx at different concentrations on different pathogenic bacteria and fungi under 808 nm irradiation and without irradiation, the results are shown in Figure 11 and Figure 12 Cu / Cu2O / CuO-Ti3C2Tx showed significant concentration-dependent antibacterial effect on different microorganisms. The experimental results showed that: (1) for gram-negative bacteria E. coli , when the concentration of Cu / Cu2O / CuO-Ti3C2Tx was 1 μg / mL, the sterilization rate was 99.0%; (2) for gram-positive bacteria S. aureus , at a concentration of 0.8 μg / mL, the inhibition rate reached 99.57%; (3) for fungi C. albicans , at a concentration of 10 μg / mL, the sterilization rate was 98.32%.

[0075] To further evaluate the antibiofilm performance of Cu / Cu2O / CuO-Ti3C2Tx, we explored its antibiofilm ability by crystal violet (CV) staining, and measured the absorbance at 590 nm to quantify the biofilm. The CV staining results Figure 13 and Figure 14 showed that in the presence of H2O2, Cu / Cu2O / CuO-Ti3C2Tx had a significant antibiofilm effect on E. coli , S. aureus and C. albicansCu / Cu2O / CuO-Ti3C2Tx+NIR had better effect than Cu / Cu2O / CuO-Ti3C2Tx. Different concentrations of Cu / Cu2O / CuO-Ti3C2Tx were mixed with E. coli 、 S. aureus and C. albicans , and after 808 nm irradiation for 30 min, they were put into the incubator for 24 h. Compared with the group without Cu / Cu2O / CuO-Ti3C2Tx and without infrared, the production rate of bacterial biofilm was reduced. The results showed that the increase of Cu / Cu2O / CuO-Ti3C2Tx content led to the decrease of biofilm production. Cu / Cu2O / CuO-Ti3C2Tx at 100 μg / mL could reduce the biofilm to about 11.35%, E. coli and 12.36% and 13.49% respectively, indicating that Cu / Cu2O / CuO-Ti3C2Tx had excellent inhibitory ability to biofilm production. S. aureus C. albicans Visual 3D biofilm test: the destruction of biofilm is an important parameter to determine the membrane effect of antibiotics. To further evaluate the destruction performance of Cu / Cu2O / CuO-Ti3C2Tx to ,

[0076] and E. coli , Alexa Fluor 647 dextran dye with a final concentration of 1 mM was added during the biofilm culture process to label the polysaccharides in the extracellular matrix as red, and SYTO-9 fluorescent nucleic acid stain with a final concentration of 2.5 mM to label the bacterial cells in the biofilm as green. Confocal laser scanning microscope was used for 3D biofilm imaging. S. aureus From the C. albicans , it can be clearly seen that in the blank group, Figure 15 , Figure 15 and E. coli were very dense and spherical protrusions, and the bacteria and EPS were very rich. The most intuitive manifestation was that the thickness of the biofilm of the three groups showed a downward trend, which proved the destruction of Cu / Cu2O / CuO-Ti3C2Tx to biofilm. S. aureus C. albicans Then, the permeability of the outer membrane of various pathogenic bacteria was measured by hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN), which showed weak fluorescence in aqueous solution and strong fluorescence intensity in hydrophobic environment. As

[0077] Then, the permeability of the outer membrane of various pathogenic bacteria was measured by hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN), which showed weak fluorescence in aqueous solution and strong fluorescence intensity in hydrophobic environment. As Figure 16 ​As shown, Cu / Cu2O / CuO-Ti3C2Tx+NIR group exhibited significantly higher fluorescence intensity than the control group, indicating that NIR excitation can significantly enhance the membrane damage performance of Cu / Cu2O / CuO-Ti3C2Tx materials, and the photothermal-photodynamic synergistic effect leads to the disintegration of the membrane lipid bilayer structure, promoting more NPN probe molecules to penetrate into the hydrophobic inner core.

[0078] Another way to verify the damage to the pathogenic bacteria membrane is the leakage of intracellular substances, including some key proteins, and the protein leakage concentration of pathogenic bacteria and fungi treated by Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR was detected by bicinchoninic acid assay (BCA) method. Under alkaline conditions, proteins will form a purple complex with BCA molecules. As shown in 2+ reduced to Cu + , Cu + forms a purple complex with BCA molecules. As shown in Figure 17 , the extracellular protein concentration of three kinds of bacteria after interaction with different concentrations of Cu / Cu2O / CuO-Ti3C2Tx was determined by protein quantification method. The extracellular protein concentration of the three kinds of bacteria gradually increased with the increase of Cu / Cu2O / CuO-Ti3C2Tx, showing a certain concentration dependence. In addition, the rupture of bacterial cell membrane may be related to the sharp edge of Ti3C2Tx, and Ti3C2Tx can physically cut the bacterial cell membrane. These results show that Cu / Cu2O / CuO-Ti3C2Tx can cause the deperforation of bacterial cell membrane, leading to the leakage of cytoplasmic components, damage the integrity of bacterial cell wall and cell membrane, and cause bacterial death.

[0079] (9) Monitoring of ROS in bacterial cells

[0080] DCFH-DA was used to capture the generation of reactive oxygen species (ROS) in bacteria, and the imaging results are shown in Figure 18 As shown, the experimental group showed significantly enhanced green fluorescence signal after Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR treatment, confirming that the composite system can effectively improve the intracellular ROS level. It is worth noting that under the condition of NIR light excitation, the catalytic activity of the composite material is significantly improved, which reveals the synergistic catalysis mechanism of Cu / Cu2O / CuO-Ti3C2Tx in photothermal synergistic catalysis - by enhancing the conversion efficiency of H2O2 to high-toxicity ROS, thereby achieving efficient killing of pathogenic bacteria.

[0081] (10) Dead / live bacterial staining

[0082] SYTO-9 / PI double staining method was used to evaluate the dead / alive state of bacteria. As shown in Figure 19 , Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR can effectively kill bacteria, and the killing rate is positively correlated with the concentration of Cu / Cu2O / CuO-Ti3C2Tx.E. coli , S. aureus and C. albicans showed significant antibacterial effect, and no significant red fluorescence emission was detected in the experimental group and the blank control group, indicating that the single component system failed to effectively activate the antibacterial effect. After the introduction of H2O2, the characteristic red fluorescence was observed in the nano material and H2O2 co-treatment group, and the fluorescence was enhanced, which confirmed that it realized the strengthening of antibacterial function by catalyzing the conversion of H2O2. It is worth noting that under the condition of NIR light activation, the fluorescence intensity of the composite system reaches the peak, which is consistent with the mechanism of NIR catalytic synergistic effect enhancing ROS generation. The antibacterial efficiency of the material is verified from the molecular level.

[0083] (11) SEM observation of bacterial morphology

[0084] Scanning electron microscopy SEM characterization revealed ( Figure 20 ), the control group bacteria showed typical rod / spherical ultrastructure, and the cell membrane integrity was confirmed to be unaffected by the nano material. After Cu / Cu2O / CuO-Ti3C2Tx+H2O2+NIR treatment, the bacteria showed characteristic structural damage: ①membrane surface wrinkles; ②cell collapse; ③intracellular material leakage. This multi-level structure damage mode is closely related to the cascade catalytic characteristics of the nano material - the enhanced electron transfer efficiency of NIR increases the quantum yield of H2O2 converted to ·OH, which in turn triggers the lipid peroxidation cascade reaction of bacterial membrane. The results revealed by this SEM are mutually confirmed with the previous ROS detection and protein leakage experiment, and the antibacterial action path of the material is fully revealed.

[0085] The above results show that the nano material Cu / Cu2O / CuO-Ti3C2Tx prepared by the present application exhibits high peroxidase activity and photothermal performance under the irradiation of NIR, and catalyzes H2O2 to generate ROS with bactericidal effect, thereby producing good antibacterial effect on E. E. coli , S. aureus and C. albicans The light response characteristic characterization shows that the nano material system presents a bimodal antibacterial mechanism under the excitation of 808 nm NIR: ①POD-like activity is improved; ②photothermal conversion efficiency η=55.4%, which can realize local temperature rise. The research results show that: (1) for 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 of 99.57% can be achieved at a concentration of 0.8 μg / mL; (3) for fungi ( C. albicans ), the bactericidal rate is 98.32% at a concentration of 10 μg / mL.

[0086] Finally, it should be noted that the above preferred embodiments are merely intended to illustrate the technical solutions of the present application, not to limit the present application. Even though the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details thereof without departing from the scope of the present application as defined by the appended claims.

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 in the preparation of antibacterial nanozyme drugs.

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

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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