Copper-doped carbon dots with peroxidase-like activity and preparation method and application thereof

Abstract

The application discloses copper-doped carbon dots with peroxidase-like activity and a preparation method and application thereof. The carbon dots are prepared by using cherry blossom petals and copper chloride as raw materials, using a 50% ethanol solution as a solvent, and adopting a one-step hydrothermal method. The carbon dots have excellent peroxidase-like activity, can effectively catalyze the decomposition of H2O2, generate highly oxidizable hydroxyl radicals, thus exhibit excellent antibacterial performance. The carbon dots also have very low cytotoxicity, and have application prospects of being prepared into antibacterial drugs. The preparation method disclosed by the application uses cherry blossom petals and copper chloride as raw materials, uses a 50% ethanol solution as a solvent, and adopts a one-step hydrothermal method to prepare copper-doped carbon dots with peroxidase-like activity. The use of cherry blossom petals as a carbon source is not only environmentally friendly, but also cost-effective, and is beneficial to large-scale production of the carbon dots. The method disclosed by the application is simple in operation, and is beneficial to popularization and application.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a copper-doped carbon dot with peroxidase-like activity, its preparation method, and its application. Background Technology

[0002] With the rapid development of nanotechnology, carbon dots (CDs) have become a research hotspot due to their unique physicochemical properties and biocompatibility. Carbon dots are a class of nanoscale carbon-based materials, typically possessing a spherical structure with a diameter of less than 10 nm. Compared with traditional materials, carbon dots have advantages such as low toxicity, good biocompatibility, and ease of modification, thus showing great application potential in fields such as biomedicine, energy catalysis, and optoelectronics.

[0003] The biocompatibility of carbon dots primarily stems from their carbon core structure, making them unlikely to trigger immune responses or inflammation in biological systems. Furthermore, carbon dots can be surface functionalized through various methods, introducing different functional groups to impart specific biological activities or stability. By doping carbon dots with metals, their surface chemistry and electronic structure can be tuned, thereby precisely controlling the catalytic performance of nanozymes. This synthetic strategy not only expands the variety of nanozymes but also provides researchers with a flexible and controllable method for developing nanozymes with specific catalytic activities. Therefore, as a novel approach to synthesizing nanozymes, carbon dots offer innovative ideas and methods for the design and preparation of nanozymes.

[0004] Scientists frequently synthesize carbon dots from plants and explore their biological applications. In a study by Qureshi et al., fluorescent carbon dots of 5-9 nm in size were produced using pomegranate peel as a raw material via a low-temperature carbonization method, and their antibacterial effects against certain pathogenic bacteria were found. In another study by Roy et al., curcumin-derived carbon dots (CDs) were synthesized via a hydrothermal process using curcumin as a carbon source, and their antibacterial properties were further enhanced by sulfur-functionalized carbon dots (S-CDs).

[0005] Cherry blossoms, belonging to the Rosaceae family, are renowned for their deciduous growth and are a symbolic representation of spring in Japan. Existing research indicates that cherry blossom extracts possess anti-inflammatory and anti-aging effects. Furthermore, Huang et al. were the first to synthesize carbon dots from cherry blossoms and explored their potential applications, demonstrating their potential as materials for fluorescent inks, metal ion sensors, and cell imaging agents. However, their study did not explore the peroxidase-like activity of these carbon dots. Therefore, investigating the peroxidase-like activity of cherry blossom-derived carbon dots and developing cherry blossom carbon dots with excellent peroxidase-like activity will provide new directions for the further research and development of nanozymes. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a copper-doped carbon dot with peroxidase-like activity, its preparation method and application, wherein the copper-doped carbon dot with peroxidase-like activity has excellent peroxidase-like activity and can effectively catalyze the decomposition of H2O2 to generate highly oxidizing hydroxyl radicals, thereby exhibiting excellent antibacterial properties.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a copper-doped carbon dot with peroxidase-like activity, which is prepared by a one-step hydrothermal method using cherry blossom petals and copper chloride as raw materials and 50% ethanol solution as solvent.

[0009] Furthermore, the carbon dots are uniformly spherical with a size within the range of 10 nm and a lattice spacing of 0.23 nm; the Fourier transform infrared absorption peak is at 3440 cm⁻¹. -1 2910cm -1 2850cm -1 1790cm -1 1620cm -1 1380cm -1 527cm -1 The X-ray photoelectron spectral peaks are 285.08 eV, 398.08 eV, 532.08 eV, and 933.91 eV; the ultraviolet absorption peaks are 280 nm and 323 nm; the optimal excitation and emission wavelengths are 418 nm and 330 nm, respectively; and the peak value of the Zeta potential is 3.11 mV.

[0010] Further, the preparation method of the carbon dots includes the following: 0.5g of dried cherry blossom petal powder and 0.0914g of copper chloride dihydrate are added to 20ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20min, and then transferred to a polytetrafluoroethylene-lined hydrothermal reactor. The mixture is heated to 180℃ and maintained for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22-micron microfiltration membrane. The filtrate is dialyzed against a 1000Da dialysis membrane for three days to obtain copper-doped carbon dots with peroxidase-like activity.

[0011] The present invention also provides the application of the copper-doped carbon dots with peroxidase-like activity in the preparation of antibacterial drugs.

[0012] The present invention also provides a method for preparing the above-mentioned copper-doped carbon dots with peroxidase-like activity. The method uses cherry blossom petals and copper chloride as raw materials and 50% ethanol solution as solvent to prepare copper-doped carbon dots with peroxidase-like activity in a one-step hydrothermal method.

[0013] Further, the method includes the following steps: 0.5g of dried cherry blossom petal powder and 0.0914g of copper chloride dihydrate are added to 20ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20min, then transferred to a polytetrafluoroethylene-lined hydrothermal reactor and heated to 180℃ for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22-micron microfiltration membrane. The filtrate is dialyzed for three days using a 1000Da dialysis membrane to obtain copper-doped carbon dots with peroxidase-like activity.

[0014] The beneficial effects of this invention are as follows: This invention discloses a copper-doped carbon dot with peroxidase-like activity. The carbon dot is prepared using cherry blossom petals and copper chloride as raw materials, with 50% ethanol solution as solvent, via a one-step hydrothermal method. The carbon dot exhibits excellent peroxidase-like activity, effectively catalyzing the decomposition of H₂O₂ to generate highly oxidizing hydroxyl radicals, thus demonstrating excellent antibacterial properties. Furthermore, the carbon dot of this invention also exhibits very low cytotoxicity, showing promise for application as an antibacterial drug. This invention also discloses a method for preparing copper-doped carbon dots with peroxidase-like activity. This method uses cherry blossom petals and copper chloride as raw materials, with 50% ethanol solution as solvent, via a one-step hydrothermal method to obtain copper-doped carbon dots with peroxidase-like activity. This method uses cherry blossom petals as a carbon source, which is not only environmentally friendly but also cost-effective, facilitating large-scale production of carbon dots. Moreover, the method of this invention is simple to operate and conducive to widespread application. Attached Figure Description

[0015] Figure 1 The following are the characterization results of carbon dots, where a is the transmission electron microscope (TEM) and high-resolution transmission electron microscope (HRTEM) images and the average diameter; b is the Fourier transform infrared (FT-IR) absorption spectrum; c is the full spectrum of X-ray photoelectron spectroscopy (XPS); d is the ultraviolet-visible (UV-Vis) absorption spectrum, emission spectrum and excitation spectrum; e is the fluorescence spectrum at different excitation wavelengths; and f is the zeta potential distribution.

[0016] Figure 2 To illustrate the peroxidase-like activity characteristics of carbon dots, a) shows the observation and absorption spectra of TMB solutions oxidized by different carbon dots; b) shows the ESR spectra of different carbon dots; c) shows the absorption spectra of Cu-CDs oxidized TMB solutions in PBS (phosphate buffer) buffer at different pH values; d) shows the absorption spectra of Cu-CDs oxidized TMB solutions at different temperatures.

[0017] Figure 3The results are kinetic analysis results for Cu-CDs, where a represents the reaction rate of Cu-CD oxidation at different TMB concentrations, b represents the reaction rate of Cu-CD oxidation at different H2O2 concentrations, c represents the reciprocal plot of Cu-CD activity at different TMB concentrations, and d represents the reciprocal plot of Cu-CD activity at different H2O2 concentrations.

[0018] Figure 4 The antibacterial activity of carbon dots is shown in Figure 1. Image 'a' shows E. coli and Staphylococcus aureus treated with different concentrations of CDs. Images 'b' and 'c' show the bacterial survival rates of E. coli and Staphylococcus aureus treated with different concentrations of CDs. Images 'd' and 'e' show E. coli and Staphylococcus aureus treated with different carbon dots and with and without H2O2. Images 'f' and 'g' show the survival rates of E. coli and Staphylococcus aureus treated with different carbon dots and H2O2. Data are expressed as mean ± standard deviation (n = 3), ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0019] Figure 5 The cell survival rate results are shown for carbon dots at different final concentrations. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the content of this invention are covered within the scope of protection intended by this invention. Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0021] This invention discloses a copper-doped carbon dot with peroxidase-like activity, which is prepared by a one-step hydrothermal method using cherry blossom petals and copper chloride as raw materials and 50% ethanol solution as solvent.

[0022] In some embodiments, the carbon dots are uniformly spherical with a size in the range of 10 nm and a lattice spacing of 0.23 nm; the Fourier transform infrared absorption peak is at 3440 cm⁻¹. -1 2910cm -1 2850cm -1 1790cm -1 1620cm -1 1380cm -1 527cm -1The X-ray photoelectron spectral peaks are 285.08 eV, 398.08 eV, 532.08 eV, and 933.91 eV; the ultraviolet absorption peaks are 280 nm and 323 nm; the optimal excitation and emission wavelengths are 418 nm and 330 nm, respectively; and the peak value of the Zeta potential is 3.11 mV.

[0023] In some embodiments, the preparation method of the carbon dots includes the following: 0.5 g of dried cherry blossom petal powder and 0.0914 g of copper chloride dihydrate are added to 20 ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20 min, and then transferred to a polytetrafluoroethylene-lined hydrothermal reactor. The mixture is heated to 180°C and maintained for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22-micron microfiltration membrane. The filtrate is dialyzed against a 1000 Da dialysis membrane for three days to obtain copper-doped carbon dots with peroxidase-like activity.

[0024] This invention also discloses the application of copper-doped carbon dots with peroxidase-like activity in the preparation of antibacterial drugs.

[0025] The present invention also discloses a method for preparing copper-doped carbon dots with peroxidase-like activity. The method uses cherry blossom petals and copper chloride as raw materials and 50% ethanol solution as solvent to prepare copper-doped carbon dots with peroxidase-like activity in a one-step hydrothermal method.

[0026] In some embodiments, the method includes the following steps: 0.5 g of dried cherry blossom petal powder and 0.0914 g of copper chloride dihydrate are added to 20 ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20 min, then transferred to a polytetrafluoroethylene-lined hydrothermal reactor and heated to 180°C for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22-micron microfiltration membrane, and the filtrate is dialyzed for three days using a 1000 Da dialysis membrane to obtain copper-doped carbon dots with peroxidase-like activity.

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0028] 1. Materials and Methods

[0029] (1) Experimental materials: Cherry blossom petals were hand-picked on the campus of Shanghai University; ferric chloride hexahydrate (FeCl3·6H2O) was purchased from SIGMA-ALDRICH Ltd.; other reagents, including 3,3',5,5'-tetramethylbenzidine (TMB), cobalt chloride hexahydrate (CoCl2·6H2O) and copper chloride dihydrate (CuCl2·2H2O), were purchased from Aladdin Chemical Reagent Co., Ltd.; culture media, including Luria broth (LB), agar powder and tryptone soybean broth (TSB), were all from Solarbio Science & Technology Co., Ltd.; fetal bovine serum (FBS), Dulbecco modified Eagle medium (DMEM) and antibiotic mixture penicillin / streptomycin (PS) were all purchased from Shanghai SangGene Biotechnology Co., Ltd.; phosphate buffered saline (PBS) was purchased from Legen Biotechnology Co., Ltd.; to ensure the absolute purity and accuracy of the experimental procedures, all solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ·cm from the Milli-Q system.

[0030] (2) Experimental Apparatus: The physical properties of the samples were characterized using a transmission electron microscope (TEM) (FEI, USA, Tecnai G2 F20 S-Twin TMP). Absorbance was measured using a UV-Vis spectrophotometer (Perkin-Elmer, USA, Lambda 750). Fluorescence properties were evaluated using an FS5 spectrofluorometer (Edinburgh Instruments, UK). X-ray photoelectron spectroscopy (XPS) analysis was performed on a Kratos Axis Ultra DLD (KRATOS, UK). Furthermore, Fourier transform infrared (FTIR) spectroscopy was performed using a Nicolet iS50 FTIR spectrometer (Thermo Fisher Scientific, USA), while Zeta potential measurements were performed using a Litesizer 500 (AntonPaar, Austria). Electron spin resonance (ESR) measurements were performed using a Bruker EMX ESR spectrometer (Bruker, Germany).

[0031] Example 1: Preparation of copper-doped carbon dots Cu-CDs with peroxidase-like activity

[0032] The collected cherry blossom petals were placed in an oven and dried at 60℃ for 48 hours. After thorough drying, the cherry blossom petals were ground into a fine powder using a grinding device. 0.5g of the cherry blossom petal powder and 0.0914g of copper chloride dihydrate were added to 20ml of 50% ethanol solution and mixed. The mixture was ultrasonically treated for 20min, then transferred to a 50ml hydrothermal reactor lined with polytetrafluoroethylene (PTFE). The mixture was heated to 180℃ and maintained for 8 hours. After the reaction was completed, the ethanol was removed by rotary evaporation. The resulting brown solution was filtered through a 0.22-micron microfiltration membrane. The filtrate was dialyzed against a 1000Da dialysis membrane for three days to obtain copper-doped carbon dots with peroxidase-like activity.

[0033] Comparative Example 1: Preparation of Cherry Blossom Carbon Dot CDs

[0034] The preparation method is similar to that in Example 1, except that copper chloride dihydrate is not added during the preparation process.

[0035] Comparative Example 2: Preparation of Iron-Doped Carbon Dots (Fe-CDs)

[0036] The preparation method is similar to that in Example 1, except that copper chloride dihydrate is not added during the preparation process, but 0.08g FeCl3·6H2O is added instead.

[0037] Comparative Example 3: Preparation of Cobalt-Doped Carbon Dots (Co-CDs)

[0038] The preparation method is similar to that in Example 1, except that copper chloride dihydrate is not added during the preparation process, but 0.0843g of CoCl2·6H2O is added instead.

[0039] Test Example 1 Characteristic Characterization Analysis

[0040] Structural characterization was performed using transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM), and the results are as follows: Figure 1 As shown in Figure a. Transmission electron microscopy (TEM) analysis showed that the prepared CDs, Fe-CDs, Co-CDs, and Cu-CDs all had uniform spherical shapes, good dispersion, and sizes within the range of 10 nm. High-resolution TEM (HR-TEM) analysis showed that CDs, Fe-CDs, Co-CDs, and Cu-CDs all had clear crystal structures, with a lattice spacing corresponding to the (100) crystal plane of graphite carbon, which was 0.23 nm.

[0041] The composition and chemical bonding state of the prepared carbon dots were characterized by Fourier transform infrared spectroscopy (FTIR), and the results are as follows: Figure 1 As shown in b. At 3440cm -1 2910cm -1 2850cm -1 1790cm -11620cm -1 and 1380cm -1 The absorption peaks at 592 cm⁻¹ are attributed to OH / NH, CH, CH₂, C=O, C=C, and CN groups, respectively. For Fe-CDs, Co-CDs, and Cu-CDs, the absorption peaks at 592 cm⁻¹ are respectively attributed to OH / NH, CH, CH₂, C=O, C=C, and CN groups. For Fe-CDs, Co-CDs, and Cu-CDs, the absorption peaks at 592 cm⁻¹ are respectively... -1 576cm -1 and 527cm -1 The peaks at the carbon dots are attributed to Fe-O, Co-N, and Cu-O groups. These results indicate the presence of nitrogen-containing groups (pyridine-type N, pyrrole-type N, and alkylamines) and oxygen-containing groups (hydroxyl and carboxyl groups) in the carbon dots, providing preliminary evidence for successful metal doping into the carbon dots.

[0042] X-ray photoelectron spectroscopy (XPS) was used to characterize the four carbon points, and the results are as follows: Figure 1 As shown in Figure c, among the four types of carbon dots—CDs, Fe-CDs, Co-CDs, and Cu-CDs—the peaks for C 1s, N 1s, and O 1s elements appear at 285.08 eV, 398.08 eV, and 532.08 eV, respectively, which is consistent with the FTIR results, confirming the enrichment of N and O elements in the synthesized carbon dots. Furthermore, the absorption peaks for Fe-CDs, Co-CDs, and Cu-CDs are at 707.19 eV, 782.30 eV, and 933.91 eV, respectively, consistent with the FTIR analysis, confirming the successful doping of metal elements and thus verifying the preparation of Fe-CDs, Co-CDs, and Cu-CDs.

[0043] The optical properties of four carbon dots in deionized water were studied using UV-Vis absorption spectroscopy and fluorescence spectroscopy. The results are as follows: Figure 1 As shown in d-1e, the UV-Vis absorption spectra reveal a broad absorption band: the peaks near 280 nm and 323 nm for the four carbon dots are likely attributed to π-π* transitions in the C=C bond and n-π* transitions in the C=O bond, which are related to the carbon nucleus and carbonyl functional group. All carbon dots exhibit excellent fluorescence properties, with optimal excitation and emission wavelengths of 345 nm and 445 nm for CDs, 340 nm and 435 nm for Fe-CDs (iron-doped carbon dots), 342 nm and 432 nm for Co-CDs (cobalt-doped carbon dots), and 418 nm and 330 nm for Cu-CDs (copper-doped carbon dots).

[0044] Zeta potential characterization was performed on the four carbon points, and the results are as follows: Figure 1As shown in f, the peak Zeta potentials of the four types of carbon points—CDs, Fe-CDs, Co-CDs, and Cu-CDs—are 1.96 mV, -0.856 mV, 2.78 mV, and 3.11 mV, respectively. Notably, only Fe-CDs show a slightly negative value, while Cu-CDs exhibit the highest positive value.

[0045] Test Example 2: Peroxidase Activity Analysis

[0046] The peroxidase-like (POD) activity of carbon dots was evaluated using a UV-260 spectrophotometer, and the absorbance of the reaction system was measured at a wavelength of 652 nm.

[0047] First, 3,3',5,5'-tetramethylbenzidine (TMB) was used as the colorimetric substrate and oxidized in phosphate-buffered saline (PBS) at pH 3.5 containing different carbon points, with a final working concentration of H₂O₂ 5 mmol / L. -1 TMB 0.5 mmol / L -1 50 μg mL of carbon dots -1 The reaction temperature was room temperature (20℃). The carbon dots with the strongest peroxidase activity were selected, and the results are as follows: Figure 2 As shown in Figure a, the solution with added CDs was almost colorless; the reaction rate was slightly increased with the addition of Co-CDs and Fe-CDs, but not significantly, and the solution with added Fe-CDs eventually turned a lighter blue; the decomposition rate of the TMB substrate increased sharply with the addition of Cu-CDs, and the solution turned a bright dark blue, indicating that Cu-CDs catalyzed the decomposition of H2O2 most effectively and had the strongest peroxidase activity.

[0048] Secondly, copper-doped carbon dots (Cu-CDs) exhibiting the highest peroxidase (POD) activity were dissolved in PBS buffer solutions with different pH values ​​(4.5, 5.5, 6.5, 7.4). While maintaining constant final working concentrations of H2O2, TMB, and CDs, as well as constant temperature, the pH value at which Cu-CDs exhibited the fastest catalytic rate was determined to be pH = 4.5. Figure 2 c).

[0049] Next, copper-doped carbon dots (Cu-CDs) were dissolved in PBS buffer at the optimal pH (pH=4.5) for POD activity, while keeping the concentrations of H2O2, TMB, and carbon dots constant. The reaction rate was tested at different temperatures (20, 30, 40, 50, 60, 70, 80℃), and the optimal reaction temperature was determined to be 50℃. Figure 2 d).

[0050] Further electron spin resonance (ESR) tests were performed on four types of carbon points—CDs, Fe-CDs, Co-CDs, and Cu-CDs—to determine the concentration of ·OH produced by their catalytic decomposition in a solution with the same H₂O₂ concentration. The results are as follows: Figure 2 As shown in b. The results show that CDs did not show obvious ·OH detection peaks, Co-CDs also had almost no obvious ·OH detection peaks, while Fe-CDs showed relatively obvious ·OH detection peaks, and Cu-CDs showed the most obvious ·OH detection peaks.

[0051] Kinetic Study of Cu-CDs in Test Example 3

[0052] Based on steady-state kinetics, the catalytic performance of Cu-CDs (copper-doped carbon dots) was further investigated. Kinetic data were obtained by keeping one substrate concentration constant while varying the concentration of another substrate, TMB or H2O2. Michaelis-Menten curves were obtained by fitting time and absorbance data. Kinetic parameters, including the Michaelis constant (Km) and the maximum initial reaction rate (Vmax), were calculated using a double reciprocal plot of the Michaelis-Menten equation, namely the Lineweaver-Burk plot.

[0053] Specifically, at the optimal operating temperature of 50℃, Cu-CDs were dissolved in a PBS buffer solution with pH=4.5 to prepare H2O2 of different concentrations (final working concentrations: 1, 2, 3, 4, 5 mmol L). -1 ) and TMB (final working concentrations: 0.1, 0.2, 0.3, 0.4, 0.5 mmol L) -1 The final working concentration of Cu-CDs was set at 50 μg / mL for the solution. -1 The rate of enzyme-catalyzed reaction was measured. The final working concentration of TMB used to determine the H2O2 kinetics was 0.5 mmol / L. -1 The final working concentration of H2O2 used to determine TMB kinetics was 5 mmol / L. -1 The dynamics of this process are calculated using the Michaelis-Menten equations, which are as follows:

[0054]

[0055] Where Km represents the Michaelis constant, V represents the initial reaction rate, Vmax represents the maximum reaction rate, and C represents the substrate concentration.

[0056] When TMB was used as the substrate, the determined Km value was 0.65139 mM and Vmax was 11.952 × 10⁻⁶ mM. - 8M / s Figure 3a,c).

[0057] When H2O2 is used as the substrate, the Km value is 1.9138 mM and the Vmax is 7.0582 × 10⁻⁶. - 8M / s Figure 3 b,d).

[0058] Test Example 3: In vitro antibacterial performance test

[0059] Given the excellent peroxidase-like activity exhibited by Cu-CDs (copper-doped carbon dots), their potential in antibacterial applications was further explored. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were selected as representative bacterial strains, and their in vitro antibacterial effects were evaluated using the plate count method.

[0060] Specifically, *Escherichia coli* (E. coli) was cultured in Luria-Bertani (LB) broth, and *Staphylococcus aureus* (S. aureus) was cultured in soybean broth. The bacterial cultures were diluted to specific concentrations (OD). 600 =0.1, equivalent to 10 8 CFU mL -1 First, the bacterial solutions were divided into different groups, each with a different final concentration (500, 250, 125, 62.5, 31.25, 15.625 μg / mL). -1 The bacteria were co-cultured with CDs, with an incubation period of 12 hours and a temperature of 37℃. Bacterial growth was observed, and the results are as follows: Figure 4 As shown in Figure a, absorbance was measured at a wavelength of 600 nm using a microplate reader, and the results are as follows. Figure 4 As shown in b-4c, the results indicate that CDs have poor antibacterial effects against Escherichia coli and Staphylococcus aureus. Then, the bacterial solution was mixed with water to a final concentration of 250 μg / mL. -1 The bacteria were co-cultured in solutions of Fe-CDs, Co-CDs, and Cu-CDs at various concentrations for 12 hours at 37°C. Bacterial growth was observed, and the results are as follows: Figure 4 As shown in d, the results indicate that CDs, Fe-CDs, Co-CDs, and Cu-CDs have poor antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0061] The CDs, Fe-CDs, Co-CDs, and Cu-CDs solutions with a final concentration of 250 μg / mL were mixed with a 10 μg / mL solution. -3 molL -1The H2O2 solution was mixed with bacteria and co-cultured. The incubation period was set at 12 hours and the temperature was 37℃. The growth of the bacteria was observed, and the results were as follows: Figure 4 As shown in e, absorbance was measured at a wavelength of 600 nm using a microplate reader, and the results are shown in 4f-4g. The results show that, compared with H2O2 solution, the addition of Cu-CDs reduced the number of Gram-negative bacteria Escherichia coli by nearly 50% and the number of Gram-positive bacteria Staphylococcus aureus by about 45%. The bactericidal rates of Escherichia coli and Staphylococcus aureus reached 67.24% and 64.42%, respectively, demonstrating significant antibacterial effects.

[0062] Test Example 4: Cytotoxicity Test

[0063] Since antimicrobial agents need to be low in toxicity, low cytotoxicity is particularly important for the biological applications of Cu-CDs.

[0064] The cytotoxicity of four carbon dots (CDs, Fe-CDs, Co-CDs, and Cu-CDs) on L929 mouse fibroblasts was evaluated using the MTT assay. Specifically, L929 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The four carbon dots were introduced into the cell culture at 37°C and 5% CO2 to achieve final concentrations of 500, 250, and 125 μg / mL, respectively. -1 After a 24-hour incubation period, add 20 μl of 5 mg mL solution to each well. -1 After incubation in MTT solution for 4 hours, the resulting product was dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader. Cells cultured without added carbon dots served as a parallel negative control. Wells containing cell-free culture medium were used as blank controls. Cell viability was calculated using the following formula:

[0065] Cell viability (%) = (Abs) (sample) -Abs (blank) ) / (Abs (control) -Abs (blank) )*100%

[0066] The results are as follows Figure 5 As shown, the results indicate that the cell viability of L929 cells decreased slightly with increasing carbon dot concentration. When the final concentration reached 500 μg / mL, the cell viability of the four carbon dot treatments (CDs, Fe-CDs, Co-CDs, and Cu-CDs) remained above 85% after 24 hours. Among them, Cu-CDs showed the lowest cytotoxicity, with a cell viability exceeding 90%.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A copper-doped carbon dot with peroxidase-like activity, characterized in that, The carbon dots were prepared using cherry blossom petals and copper chloride as raw materials, and 50% ethanol solution as solvent, via a one-step hydrothermal method. The carbon dots are uniformly spherical with a size within 10 nm and a lattice spacing of 0.23 nm. The Fourier transform infrared absorption peak is at 3440 cm⁻¹. −1 2910 cm −1 2850 cm −1 1790 cm −1 1620 cm −1 1380 cm −1 527 cm −1 The X-ray photoelectron spectroscopy peaks are 285.08 eV, 398.08 eV, 532.08 eV, and 933.91 eV; the ultraviolet absorption peaks are 280 nm and 323 nm; the Zeta potential peak value is 3.11 mV. The preparation method of the carbon dots includes the following: 0.5 g of dried cherry blossom petal powder and 0.0914 g of copper chloride dihydrate are added to 20 ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20 min, and then transferred to a hydrothermal reactor lined with polytetrafluoroethylene. The mixture is heated to 180 °C and maintained for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22 micrometer microfiltration membrane. The filtrate is dialyzed with a 1000 Da dialysis membrane for three days to obtain copper-doped carbon dots with peroxidase-like activity.

2. The application of the copper-doped carbon dots with peroxidase-like activity as described in claim 1 in the preparation of antibacterial drugs.

3. The method for preparing copper-doped carbon dots with peroxidase-like activity as described in claim 1, characterized in that, The method uses cherry blossom petals and copper chloride as raw materials, and 50% ethanol solution as solvent to prepare copper-doped carbon dots with peroxidase-like activity using a one-step hydrothermal method.

4. The method according to claim 3, characterized in that, The method includes the following steps: 0.5g of dried cherry blossom petal powder and 0.0914g of copper chloride dihydrate are added to 20ml of 50% ethanol solution and mixed. The mixture is ultrasonically treated for 20min and then transferred to a polytetrafluoroethylene-lined hydrothermal reactor. The mixture is heated to 180℃ and maintained for 8 hours. After the reaction is completed, the ethanol is concentrated and removed. The resulting brown solution is filtered through a 0.22-micron microfiltration membrane. The filtrate is dialyzed against a 1000 Da dialysis membrane for three days to obtain copper-doped carbon dots with peroxidase-like activity.

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