Silver monatomic doped carbon quantum dot nano-enzyme as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: (1) synthesis of nitrogen-doped carbon dots: dissolving anhydrous citric acid and urea in formic acid, uniformly mixing, transferring the solution into a polytetrafluoroethylene reaction kettle for reaction, cooling to room temperature after the reaction, and filtering to obtain nitrogen-doped carbon dots; adding the same amount of ethanol, standing overnight, centrifuging, and freeze-drying precipitates obtained after centrifuging to obtain nitrogen-doped carbon dots; and (2) synthesis of the silver monatomic nano-enzyme: dissolving the synthesized nitrogen-doped carbon dots in deionized water, uniformly mixing, adjusting the pH value, dropwise adding a solution containing silver ions while stirring, uniformly stirring, sequentially adding trisodium citrate and sodium borohydride, mixing, and carrying out oil bath reaction, dialysis and freeze drying to obtain the silver monatomic nano-enzyme. The nano-enzyme prepared by the invention can organically combine active targeting, red light imaging and collaborative treatment, and can greatly improve the treatment effect on renal injury.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme materials technology, and relates to a silver single-atom doped carbon quantum dot nanoenzyme, its preparation method and application. Background Technology

[0002] Reactive oxygen species (ROS) are chemical substances formed during incomplete oxidation-reduction processes, mainly including hydrogen peroxide (H2O2) and superoxide anion (O3). 2- Oxidative oxygen species (ROS) include singlet oxygen (O2) and hydroxyl radicals (OH). In the human system, ROS influence various physiological activities and play a crucial role in diverse life processes. ROS are important secondary messengers in cell signaling pathways, mitosis, cell proliferation / migration / differentiation, and the body's resistance to pathogen invasion. However, high levels of ROS can easily cause lipid peroxidation, protein denaturation, DNA damage, and oxidative damage to other biomolecules, thus having harmful effects on cells and tissues. ROS-induced oxidative stress refers to the imbalance between oxidation and antioxidation in the body and is an important indicator of aging and disease. Excessive ROS production can trigger cell damage, inflammatory responses, and tissue fibrosis.

[0003] Acute kidney injury (AKI) is a critical illness characterized by a rapid deterioration of kidney function. It is characterized by the sudden loss of renal excretory function in previously healthy individuals and can lead to long-term, severe kidney damage if not treated promptly. Oxidative stress is one of the core pathological mechanisms of AKI. Therefore, efficient clearance of ROS from AKI cells is a primary means of treating acute kidney injury caused by oxidative stress.

[0004] Nanozymes, as novel functional nanomaterials possessing natural enzyme activity, offer advantages such as high catalytic efficiency, stable activity, tunable activity (adjustable through size and surface modification), and large-scale preparation, leading to their widespread application in biosensing, biomedicine, food safety, and environmental engineering. Recent studies have discovered that silver atoms or nanoclusters also exhibit enzyme-like activities, such as POD and SOD-like activities, offering potential for their translational applications in medicine. However, research on silver atom nanozymes in the treatment of oxidative stress-related diseases, particularly AKI, has not been systematically reported and remains in a gap. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a silver single-atom-doped carbon quantum dot nanozyme for efficient ROS removal in AKI cells and its preparation method.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] The first aspect of this invention is to provide a method for preparing silver single-atom-doped carbon quantum dot nanozymes, comprising the following steps:

[0008] (1) Synthesis of nitrogen-doped carbon dots

[0009] Anhydrous citric acid and urea were dissolved in formic acid and mixed evenly. The solution was then transferred to a polytetrafluoroethylene reactor for reaction. After the reaction, the mixture was cooled to room temperature, and an equal amount of ethanol was added and allowed to stand overnight. The mixture was then centrifuged, and the precipitate obtained after centrifugation was freeze-dried to obtain nitrogen-doped carbon dots.

[0010] (2) Synthesis of silver single-atom nanozymes

[0011] The synthesized nitrogen-doped carbon dots were dissolved in deionized water, mixed well, and the pH was adjusted. Then, a solution containing silver ions was added dropwise while stirring. After stirring evenly, trisodium citrate and sodium borohydride were added in sequence and mixed. After oil bath reaction, dialysis, and freeze-drying, silver single-atom nanozymes were obtained.

[0012] In an optional embodiment, the method further includes step (3), in which carboxylated triphenylphosphine, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) are added to DMSO for reaction, and then the silver single-atom nanozyme prepared in step (2) is added to the system for reaction. After dialysis and freeze-drying, silver single-atom nanozyme powder with triphenylphosphine linkage is obtained.

[0013] In one alternative embodiment, in step (1), the mass ratio of anhydrous citric acid to urea is 1:2.

[0014] In one optional embodiment, in step (1), the reaction conditions in the reactor are: temperature 150-180°C, time 2-6h.

[0015] In an optional embodiment, in step (2), the nitrogen-doped carbon dot solution: silver ion solution = (0.15~0.35) mg / mL: 1 mM.

[0016] In an alternative embodiment, in step (2), the ratio of trisodium citrate to sodium borohydride is 7 mM to 5 mM.

[0017] In an optional embodiment, in step (2), the conditions for the oil bath reaction are: temperature 60-80°C, time 4-6h; and the conditions for dialysis are: molecular weight cutoff 450-550 Da, time 48-96h.

[0018] In an optional embodiment, the carboxylated triphenylphosphine: N-hydroxysuccinimide (NHS): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): silver single-atom nanozyme = 2.7 mg: 1.35 mg: 1.35 mg: 1 mg.

[0019] A second aspect of the present invention is to provide a silver single-atom-doped carbon quantum dot nanozyme, which is prepared according to the preparation method described above.

[0020] A third aspect of the present invention is to provide a silver single-atom-doped carbon quantum dot nanozyme as an antioxidant enzyme mimic in the preparation of drugs for treating cellular oxidative stress damage.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The silver single-atom nanozyme provided by this invention uses nitrogen-doped carbon dots as a carrier, on which silver atoms are loaded and linked to triphenylphosphine. This nanoformulation exhibits good dispersibility, small particle size, and fluorescence properties, enabling highly efficient targeted delivery to mitochondria in renal tubular epithelial cells. This invention improves the precision of kidney injury treatment by combining active targeting of mitochondria with scale-driven passive targeting of the kidney by triphenylphosphine. Red light imaging using carbon dots avoids biological autofluorescence, improving imaging contrast and sensitivity. Silver-triggered chemodynamic therapy specifically reduces intracellular superoxide radicals and hydrogen peroxide, synergistically targeting mitochondria to clear ROS, enhancing therapeutic efficacy. The organic combination of active targeting, red light imaging, and synergistic therapy can significantly improve the therapeutic effect on kidney injury. Attached Figure Description

[0023] Figure 1 The N-CDs and Ag synthesized in Example 1 of this invention SA - Transmission electron micrograph of CDs;

[0024] Figure 2 The N-CDs and Ag synthesized in Example 1 of this invention SA XPS full spectrum of CDs;

[0025] Figure 3 The N-CDs and Ag synthesized in Example 1 of this invention SA FTIR spectra of -CDs;

[0026] Figure 4 The N-CDs and Ag synthesized in Example 1 of this invention SA X-ray diffraction (XRD) patterns of CDs;

[0027] Figure 5 Ag synthesized in Example 1 of this invention SA -Fluorescence spectra of CDs;

[0028] Figure 6 The T-Ag synthesized in Example 1 of this invention SA -NMR spectra of CDs;

[0029] Figure 7 The N-CDs and Ag synthesized in Example 1 of this invention SA -The antioxidant activity effect of CDs;

[0030] Figure 8 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA -Diagram showing the cellular uptake effect of CDs;

[0031] Figure 9 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA -Cytotoxicity test results of CDs;

[0032] Figure 10 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA -Cellular activity protection test results of CDs;

[0033] Figure 11 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA - Targeted mitochondrial test results for CDs;

[0034] Figure 12 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA -CDs cellular ROS assay results;

[0035] Figure 13 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA -CDs cellular mitochondrial MitoSox assay results;

[0036] Figure 14 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA Apoptosis assay results of -CDs;

[0037] Figure 15 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-AgSA - Changes in body weight, serum urea nitrogen, and creatinine in model mice after treatment with CDs;

[0038] Figure 16 The N-CDs and Ag synthesized in Example 1 of this invention SA -CDs and T-Ag SA - Distribution of CDs in the heart, brain, liver, spleen, lung and kidney tissues of mice;

[0039] Figure 17 The N-CDs and Ag synthesized in Example 1 SA -CDs and T-Ag SA -Cellular tissue sections of mouse kidneys after CDs treatment. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0041] In the following experiments, the materials used were as follows: citric acid and urea were purchased from Aladdin; formic acid, silver nitrate, sodium borohydride, and citrate trihydrate were purchased from Cilon Scientific; the SOD assay kit was purchased from Digiwin Biotechnology Co., Ltd.; NHS (4-carboxybutyl)triphenylphosphine bromide, ABTS, glutathione peroxidase assay kit, Annexin V-FITC apoptosis assay kit, JC-1 mitochondrial membrane potential assay kit, and LDH cytotoxicity assay kit were purchased from Beyotime Biotech Co., Ltd.; DAPI, Mito-tracker, Lyso-tracker, and Mitosox-green were purchased from Thermo Fisher Scientific. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was purchased from McKinnon Biotech Co., Ltd.; DDPH and DCFH-DA were purchased from Macquarie Biotech Co., Ltd.; the cell counting kit-8 and markers (10 kDa to 180 kDa) were purchased from Bosch Registrars Co., Ltd.; and the antibodies used in the experiments were provided by Wuhan Sanying Biotech Co., Ltd.

[0042] Example 1

[0043] This embodiment provides a method for preparing silver single-atom-doped carbon quantum dot nanozymes, including the following steps:

[0044] (1) Synthesis of nitrogen-doped carbon dots

[0045] The synthesis was performed using a bottom-up hydrothermal molecular fusion method, with the following specific steps:

[0046] Weigh 6g of anhydrous citric acid (TCI, purity 98%) and 12g of urea, add 60mL of formic acid, mix for 0.5h with magnetic stirring at 600 rpm, then transfer the solution to a polytetrafluoroethylene reactor and react at 160℃ for 4h. After cooling to room temperature, add an equal amount of ethanol and let stand overnight. Then, centrifuge three times at 13000rpm for 20min each time, and freeze-dry the precipitate to obtain nitrogen-doped carbon dots (N-CDs).

[0047] (2) Synthesis of silver single-atom nanozymes

[0048] The specific steps for preparing atomic silver nanozymes using a wet chemical method are as follows:

[0049] Weigh 60 mg of nitrogen-doped carbon dots (N-CDs), add 24 ml of deionized water, mix well, and adjust the pH to 12 with sodium hydroxide. Add 4.8 ml of AgNO3 (10 mM) dropwise while stirring at 1000 rpm. After stirring for 30 min, add 3.6 ml of trisodium citrate (14 mM) and 4.8 ml of sodium borohydride (10 mM) sequentially at room temperature. Mix and incubate in an oil bath at 60 °C for 3 h. After the reaction is complete, dialyze with deionized water in the dark (MW: 500 Da) for 72 h, and freeze-dry to obtain silver single-atom nanozyme (Ag). SA -CDs).

[0050] (3) Triphenylphosphine linkage

[0051] TPP-carboxyl groups were coupled to Ag using NHS chemical methods. SA -CDs surface, the specific steps are as follows:

[0052] 54 mg of carboxylated triphenylphosphine (COOH-TPP), 27 mg of N-hydroxysuccinimide (NHS), and 27 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added to 20 ml of DMSO and reacted for 1 h. Then, 20 mg of Ag was added. SA -CDs were added to the system, and the pH was adjusted to 7.2. The reaction was carried out under a nitrogen atmosphere for 24 h, followed by dialysis (MW: 500 Da) for 72 h, and then freeze-drying to obtain a silver single-atom nanozyme (T-Ag) linked to triphenylphosphine. SA -CDs).

[0053] Example 2

[0054] This embodiment provides a method for preparing silver single-atom-doped carbon quantum dot nanozymes, including the following steps:

[0055] (1) Synthesis of nitrogen-doped carbon dots

[0056] The synthesis was performed using a bottom-up hydrothermal molecular fusion method, with the following specific steps:

[0057] Weigh 6g of anhydrous citric acid (TCI, purity 98%) and 18g of urea, add 60mL of formic acid, mix for 0.5h with magnetic stirring at 600 rpm, then transfer the solution to a polytetrafluoroethylene reactor and react at 150℃ for 6h. After cooling to room temperature, add an equal amount of ethanol and let stand overnight. Then, centrifuge three times at 13000rpm for 20min each time, and freeze-dry the precipitate to obtain nitrogen-doped carbon dots (N-CDs).

[0058] (2) Synthesis of silver single-atom nanozymes

[0059] The specific steps for preparing atomic silver nanozymes using a wet chemical method are as follows:

[0060] Weigh 36 mg of nitrogen-doped carbon dots (N-CDs), add 24 ml of deionized water, mix well, and adjust the pH to 12 with sodium hydroxide. Add 4.8 ml of AgNO3 (10 mM) dropwise while stirring at 1000 rpm. After stirring for 30 min, add 3.6 ml of trisodium citrate (14 mM) and 4.8 ml of sodium borohydride (10 mM) sequentially. Mix and incubate in an oil bath at 60 °C for 3 h. After the reaction is complete, dialyze against light using deionized water (MW: 500 Da) for 72 h, and freeze-dry to obtain silver single-atom nanozyme (Ag). SA -CDs).

[0061] (3) Triphenylphosphine linkage

[0062] 54 mg of carboxylated triphenylphosphine (COOH-TPP), 27 mg of N-hydroxysuccinimide (NHS), and 27 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added to 20 ml of DMSO and reacted for 1 h. Then, 20 mg of AgSA-CDs were added to the system and reacted for 48 h. The mixture was dialyzed (MW: 500 Da) for 72 h, and then freeze-dried to obtain a silver single-atom nanozyme (T-Ag) linked to triphenylphosphine. SA -CDs).

[0063] Example 3

[0064] (1) Synthesis of nitrogen-doped carbon dots

[0065] The synthesis was performed using a bottom-up hydrothermal molecular fusion method, with the following specific steps:

[0066] Weigh 6g of anhydrous citric acid (TCI, purity 98%) and 8g of urea, add 60mL of formic acid, mix for 0.5h with magnetic stirring at 600 rpm, then transfer the solution to a polytetrafluoroethylene reactor and react at 180℃ for 3h. After cooling to room temperature, add an equal amount of ethanol and let stand overnight. Then, centrifuge three times at 13000rpm for 20min each time, and freeze-dry the precipitate to obtain nitrogen-doped carbon dots (N-CDs).

[0067] (2) Synthesis of silver single-atom nanozymes

[0068] The specific steps for preparing atomic silver nanozymes using a wet chemical method are as follows:

[0069] Weigh 84 mg of nitrogen-doped carbon dots (N-CDs), add 24 ml of deionized water, mix well, and adjust the pH to 12 with sodium hydroxide. Add 4.8 ml of AgNO3 (10 mM) dropwise while stirring at 1000 rpm. After stirring for 30 min, add 3.6 ml of trisodium citrate (14 mM) and 4.8 ml of sodium borohydride (10 mM) sequentially. Mix and incubate in an oil bath at 60 °C for 3 h. After the reaction is complete, dialyze against light using deionized water (MW: 500 Da) for 72 h, and freeze-dry to obtain silver single-atom nanozyme (Ag). SA -CDs).

[0070] (3) Triphenylphosphine linkage

[0071] 54 mg of carboxylated triphenylphosphine (COOH-TPP), 27 mg of N-hydroxysuccinimide (NHS), and 27 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added to 20 ml of DMSO and reacted for 1 h. Then, 20 mg of Ag was added. SA -CDs were added to the system and reacted for 48 h, followed by dialysis (MW: 500 Da) for 72 h, and freeze-drying to obtain a silver single-atom nanozyme (T-Ag) linked to triphenylphosphine. SA -CDs).

[0072] Test case

[0073] I. Microscopic Morphological Characterization

[0074] The nitrogen-doped carbon dots (N-CDs) and silver single-atom nanozymes (Ag) synthesized in Example 1 were used in the experiment. SA After diluting the CDs to an appropriate concentration, 10 μL of each sample was dropped onto a copper grid. After evaporating the moisture, the grid containing the sample was placed inside the TEM machine, and its microstructure was observed. The results are as follows: Figure 1 As shown.

[0075] Depend on Figure 1It can be seen that nitrogen-doped carbon dots (N-CDs) have good dispersibility, exhibiting monodispersity with a particle size of approximately 5 nm; silver single-atom nanozymes (Ag) also show good dispersibility. SA The -CDs exhibit good dispersion properties, with spherical particles and a particle size of approximately 5 nm, indicating that silver is doped into the carbon dots in the form of atomic-level metal clusters.

[0076] II. Characterization by X-ray photoelectron spectroscopy (XPS)

[0077] The nitrogen-doped carbon dots (N-CDs) and silver single-atom nanozymes (Ag) synthesized in Example 1 were used in the experiment. SA -CDs) were used as samples for XPS analysis.

[0078] The samples were subjected to full-spectrum and fine-spectrum analysis using an X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha, USA) source of Al Kα (1486.6 eV), calibrated using 284.8 eV of adsorbed carbon C1s as a standard. The results are as follows: Figure 2 As shown. In Figure 2 In the figure, a represents N-CDs and Ag. SA Figure 1 shows the XPS full spectrum of CDs; Figure 2 shows the peak division of the C1s peak; Figure 3 shows the peak division of the N1s peak; Figure 4 shows the peak division of the O1s peak; Figure 5 shows the detailed division of the Ag3d peak.

[0079] Depend on Figure 2 As can be seen from a, the XPS full spectrum of N-CDs mainly contains three elements: C, NO, and Ag. SA The Ag3d peaks of the N-CDs indicate the presence of Ag in the carbon dots (N-CDs), demonstrating successful Ag loading onto the carbon dots. The Ag3d peaks are further subdivided as follows: Figure 2 As shown in e. From Figure 2 From b, we can see that the C1s peak mainly has three sub-peaks: C=O / COOH, CO / CN, and CC / C=C; from Figure 2 As can be seen from c, the =NH and N-(C)3 peaks of N1s are gradually translated into Ag-N peaks after silver doping; from Figure 2 As can be seen from d, the O1s peak is mainly composed of two peaks: CO / OH and C=O.

[0080] III. Infrared Spectroscopy (FTIR) Characterization

[0081] The nitrogen-doped carbon dots (N-CDs) and silver single-atom nanozymes (Ag) synthesized in Example 1 were used in the experiment. SA -CDs) were used as samples for FTIR analysis. The results are as follows: Figure 3 As shown.

[0082] from Figure 3As can be seen from this, compared with undoped Ag N-CDs, Ag SA -CDs appeared / enhanced in the low wavenumber region by approximately 800 cm⁻¹ -1 The absorption peak is approximately 800 cm⁻¹. -1 The absorption peaks provide evidence of coordination between silver and nitrogen-containing sites (such as pyridine / amide N) on the surface of carbon dots, further indicating that silver acts as an atom / site anchored on the surface of carbon dots.

[0083] IV. X-ray diffraction (XRD) characterization

[0084] X-ray powder diffraction was used to determine the nitrogen-doped carbon dots (N-CDs) and silver single-atom nanozymes (Ag) synthesized in Example 1. SA XRD patterns of CDs. Results are as follows. Figure 4 As shown.

[0085] Figure 4 XRD results confirmed that Ag SA -CDs simultaneously exhibit characteristic diffraction patterns of both the carbon matrix and elemental silver. The broadened peak of the carbon phase is located at 2θ≈23.8°, consistent with PDF card 04-007-8496 (graphitized carbon); while sharp diffraction peaks appear at 38.2°, 44.4°, and 64.6°, corresponding to the (111), (200), and (220) crystal planes of face-centered cubic silver, respectively, perfectly consistent with PDF 04-006-6516. The multi-peak structure with both broad and sharp peaks not only indicates the amorphous / graphitized coexistence of the carbon dot framework, but also confirms that silver is uniformly embedded in the carbon matrix in the form of highly crystalline single atoms / nanoclusters, providing a structural basis for subsequent catalysis and fluorescence enhancement.

[0086] V. Fluorescence Spectroscopy Characterization

[0087] The silver single-atom nanozyme (Ag) synthesized in Example 1 was used. SA -CDs) were used as samples for fluorescence spectroscopy analysis. The results are as follows: Figure 5 As shown.

[0088] from Figure 5 From this, we can see that Ag SA -CDs have the optimal excitation light at around 560 nm and emit light at around 640 nm, exhibiting typical red fluorescence characteristics.

[0089] VI. Nuclear Magnetic Resonance (NMR) Characterization

[0090] The T-Ag synthesized in Example 1 SA -CDs were used as samples for NMR analysis. Results are as follows: Figure 6 As shown, Ag modified with TPP SA -CDs(T-Ag SAThe presence of typical TPP NMR peaks on the -CDs indicates that TPP has been successfully ligated to Ag. SA -CDs on.

[0091] Effect verification example

[0092] The following tests were all conducted using N-CDs and Ag prepared in Example 1. SA -CDs, T-Ag SA -CDs were used as test samples.

[0093] I. Antioxidant Activity Test

[0094] (I) As a SOD mimicry for superoxide anion (O2) ·- ) clearing ability

[0095] Superoxide dismutase (SOD) can catalyze O2 ·- SOD mimics are considered potential therapeutic agents against oxidative stress-related diseases because they can be disproportionated into O2 and H2O2 and finely regulate intracellular ROS levels.

[0096] Ag was assessed using the classic xanthine-xanthine oxidase (XOD)-WST-1 system. SA -CDs and N-CDs, as SOD mimics, demonstrate the scavenging ability of superoxide anions. The principle of the WST-1 method is that WST-1 can react with the superoxide anion (O2) produced by xanthine oxidase catalyzing xanthine. ·- The reaction produces water-soluble formazan dye absorbed at 440 nm. Since SOD can catalyze the disproportionation of superoxide anions, this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye produced. Thus, the enzyme activity of SOD can be calculated by colorimetric analysis of the WST-1 product.

[0097] The experiment was conducted using a superoxide dismutase (SOD) assay kit. Test samples were diluted with phosphate-buffered saline (PBS) to concentration gradients of 0.125, 0.25, 0.5, 1, 2, 4, and 8 g / mL, with three replicates for each concentration. Following the kit instructions, samples were incubated at 37°C for 20 minutes, and absorbance was immediately measured at 450 nm. The SOD inhibition rate was calculated using a formula and converted to activity units (U / mL). Results are shown below. Figure 7 As shown in Figure a.

[0098] Depend on Figure 7 As can be seen from this, with the increase of Ag-CDs mimicry concentration, its inhibition rate also increases, approaching 100%, meaning that the SOD mimicry has a significant effect on O2 inhibition. ·-The inhibition effect of WST-1 reduction became increasingly significant, and the amount of formazan produced decreased significantly; while the inhibition rate of N-CDs mimics was half that of Ag-CDs mimics, indicating that Ag-CDs have superior antioxidant activity compared to N-CDs.

[0099] (ii) Catalytic ability of GPx simulants for H2O2

[0100] Glutathione peroxidase (GPx) belongs to the peroxidase family. It can convert H2O2 into H2O with the help of the reducing equivalent provided by the intracellular tripeptide glutathione (GSH), while oxidizing GSH to GSSG. Subsequently, glutathione reductase (GR) uses NADPH to reduce GSSG back to GSH, completing the cycle.

[0101] The experiment was performed using a total glutathione peroxidase assay kit. The test samples were diluted with PBS (pH 7.4) to a concentration of 10 μg / mL. -1 Three replicate wells were then set up. The reaction system was prepared at 37°C according to the kit instructions. An enzyme-linked immunosorbent assay (ELISA) reader was used in kinetic mode to monitor the absorbance at 340 nm every 40 seconds. The real-time decrease in NADPH absorbance at 340 nm was used to monitor Ag. SA The GPx-like activity of -CDs and N-CDs was continuously monitored for 14 minutes. Results are as follows: Figure 7 As shown in b, the absorbance of the N-CDs group remained almost unchanged, indicating that it lacked significant GPx simulation function; while the absorbance of the Ag-CDs group NADPH decreased rapidly, indicating that it had efficient GPx-like catalytic ability.

[0102] (III) Clear ABTS + The ability of free radicals

[0103] The ABTS method is a general indirect method for determining free radical scavenging ability, and it can simultaneously evaluate the antioxidant capacity of both hydrophilic and lipophilic substances. ABTS reacts with an oxidizing agent to generate a stable green free radical cation, ABTS· + It exhibits characteristic absorption at 734 nm or 405 nm; in the presence of antioxidants, ABTS· + The generation of [something] is suppressed, and the system fades.

[0104] Mix 7 mM ABTS with 2.45 mM potassium persulfate (K₂S₂O₈) at a ratio of 1:1 (v / v). Let the mixture stand at room temperature in the dark for 12 hours to generate ABTS· +Stock solution. Before use, dilute the stock solution with PBS (pH 7.4) to achieve an absorbance of 0.70 ± 0.02 at 734 nm. In a 96-well plate, add 20 μL of sample solution at different concentrations (2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, 15 μg / mL, 17.5 μg / mL, 20 μg / mL) and 180 μL of PBS to each well sequentially. + After thoroughly mixing the working solution, incubate in the dark for 30 minutes, and immediately measure the absorbance at 734 nm. Each sample was tested three times, with PBS used as a blank control. The clearance rate was calculated using the following formula:

[0105] Clearance rate (%) = [(A(blank) - A(sample)) / A(blank)] × 100

[0106] The results are expressed as mean ± SD (n = 3).

[0107] The results are as follows Figure 7 As shown in c, Ag SA -CDs in 15 μg mL -1 At concentrations of ABTS and above, + The clearance rate is over 90%.

[0108] Ag was determined by the DTNB method. SA - The GSH elimination ability of CDs at different time periods. For example... Figure 7 As shown in d, the GSH content gradually decreases over time; GR can reduce GSSG to GSH, and GSH reacts with the chromogenic substrate DTNB to generate yellow TNB and GSSG. The degree of decrease in the absorbance of TNB at 412nm is proportional to the amount of free radical scavenging.

[0109] (iv) Ability to scavenge DPPH free radicals

[0110] DPPH radicals are deep purple with a maximum absorption wavelength of 515 nm. When they encounter antioxidants, their lone pairs of electrons are removed, the solution color lightens, and the absorbance decreases. By monitoring the change in absorbance at 515 nm, the ability of a sample to scavenge DPPH radicals can be quantitatively evaluated.

[0111] Weigh out DPPH reagent and dissolve it in anhydrous ethanol to prepare a 0.1 mM working solution. Add different concentrations of sample solution (2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, 15 μg / mL, 17.5 μg / mL, 20 μg / mL) and 180 μL of DPPH working solution sequentially to a 96-well plate. Mix immediately and incubate at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm (sample group). Set up an equal volume of PBS as a blank control (blank group) and an equal volume of anhydrous ethanol instead of DPPH as the sample background (background group). Set up three replicate wells for each concentration. Calculate the clearance rate using the following formula:

[0112] Scan rate (%) = [1 - (sample - background) / blank] × 100

[0113] The results are expressed as mean ± SD (n = 3).

[0114] The results are as follows Figure 7 As shown in e, Ag SA -CDs in 10 μg / mL -1 At concentrations of 100% and above, the DPPH scavenging rate can reach over 90%.

[0115] (V) Superoxide anion (O2) ·- ESR determination

[0116] To further confirm Ag SA To detect the SOD-like activity of -CDs, we used electron spin resonance (ESR) technology, which has excellent sensitivity and specificity, to directly detect O2. ·- .

[0117] Background signal: FeSO4 (1 mg / mL) -1 The sample (1 mg / mL) was instantaneously mixed with H2O2 (5 mM) at a 1:1 ratio. -1 After mixing the background solution at a 1:1 ratio, it is then mixed with DMPO scavenger at a 1:1 ratio and immediately loaded onto the instrument. Using DMPO as a spin trapping agent, a characteristic DMPO / O2 structure can be formed. ·- Adduct.

[0118] The results are as follows Figure 7 As shown in f, Ag is added. SA -CDs after DMPO / O2 ·- The ESR signal was significantly reduced, confirming its efficient SOD-like scavenging effect.

[0119] II. Cell Experiments

[0120] 293T cells were cultured. 293T cells were added to high-glucose DMEM medium containing 10% FBS, 1% streptomycin-penicillin, and 1% non-essential amino acids, and cultured at 37°C, 95% humidity, and 5% CO2. When cell confluence reached 90%, cells were passaged using 0.25% trypsin-EDTA.

[0121] (I) Cell uptake experiment

[0122] To investigate the uptake of test samples by 293T cells, the cells were seeded in 12-well plates and cultured overnight. Subsequently, they were treated with N-CDs and Ag. SA -CDs, T-Ag SA Cells were treated with CDs (50 μg / mL) for 6 hours. After incubation, cells were washed three times with PBS to remove excess material, collected, and resuspended in PBS for analysis. Intracellular fluorescence intensity of the carbon dot channels was detected using the PerCP channel, and FlowJo software was used to analyze the fluorescence intensity to assess cellular uptake. Results are as follows: Figure 8 As shown.

[0123] from Figure 8 As can be seen from this, compared with the unmodified TPP Ag SA Compared to CDs, TPP functionalized Ag SA -CDs(T-Ag SA -CDs) showed higher cellular uptake efficiency in 239T cells.

[0124] (II) Cytotoxicity Test

[0125] This study used the CCK-8 assay kit to evaluate N-CDs and Ag. SA -CDs, T-Ag SA -CDs showed in vitro toxicity to 293T cells. The specific procedure was as follows: 293T cells were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to promote complete cell adhesion. The old culture medium was then discarded, and different concentrations (3.2 μg / mL, 6.4 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of N-CDs and Ag were added. SA -CDs, T-Ag SA Fresh, complete culture medium for CDs was added. After incubation for 24 hours, the cells were gently washed twice with sterile PBS. 100 μL of fresh culture medium and 10 μL of CCK-8 reagent were added to each well, and the cells were incubated at 37°C in the dark for 2 hours. Finally, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 The formula for calculating cell viability is:

[0126] Cell viability (%) = (experimental group (OD)) 450 - Blank group (OD) 450 )) / (Control group (OD) 450 - Blank group (OD) 450 ))×100%

[0127] The blank group consisted of cell-free culture medium, while the control group consisted of cells without the test sample.

[0128] The results are as follows Figure 9 As shown, from Figure 9 As can be seen from this, with Ag SA -CDs and T-Ag SA Increased concentrations of -CDs resulted in cells exhibiting a certain degree of viability. The results indicated that Ag... SA -CDs and T-Ag SA -CDs have no significant toxic effects on cells.

[0129] (III) Assay of cellular hydrogen peroxide scavenging capacity

[0130] Cells were seeded into 96-well plates according to the method described in the "Cytotoxicity Test" above. Each group was then treated with DMEM medium containing 400 μM hydrogen peroxide for 3 hours to induce oxidative stress. Fresh complete culture medium was then added, along with different concentrations (3.2 μg / mL, 6.4 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of N-CDs and Ag. SA -CDs, T-Ag SA -CDs were cultured in fresh, complete medium for 12 hours. Cell viability (cell survival rate) was then calculated using the method described above. Results are as follows: Figure 10 As shown.

[0131] from Figure 10 It can be seen that cell viability decreased to about 50% after H2O2 treatment alone; no significant recovery of viability was observed after N-CDs intervention; and 12.5 μg mL -1 Ag SA -CDs can restore cell viability to about 70%, the same dose of T-Ag SA -CDs further increased the activity to approximately 90%. The results fully demonstrate that Ag SA -CDs and T-Ag SA -CDs all have good oxidative stress damage repair capabilities, but T-Ag SA -CDs have a superior ability to repair oxidative stress damage.

[0132] (iv) Organelle Colocalization Test

[0133] Add 50 μg mL to the 293T cells on the culture plate -1 N-CDs, Ag SA -CDs, T-Ag SA -CDs were incubated at 37°C in the dark for 6 hours. After gentle washing twice with PBS, the medium was replaced with serum-free DMEM containing either mitochondrial tracking agent green or lysosomal tracking agent green, and incubated at 37°C in the dark for another 30 minutes. After washing once with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cells were then washed twice more with PBS, and the nuclei were stained with DAPI for 10 minutes, followed by two more washes. Imaging was immediately performed using a laser scanning confocal microscope (LSCM). Results are as follows: Figure 11 As shown, Figure 11 In the image, a is a fluorescence micrograph of mitochondria; b is a fluorescence micrograph of lysosome escape.

[0134] from Figure 11 a and Figure 11 As shown in b, Ag after the connection of triphenylphosphine (TPP) SA -CDs nanozyme (T-Ag) SA -CDs can target mitochondria and escape from lysosomes.

[0135] (V) Determination of cellular ROS scavenging capacity

[0136] The intracellular ROS level in H2O2-induced 293T cells was monitored using the DCFH-DA probe.

[0137] To evaluate N-CDs, Ag SA -CDs, T-Ag SA The ability of CDs to scavenge reactive oxygen species (ROS) induced by hydrogen peroxide was detected using 293T cells. Cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 1 cells / well in 12-well plates and cultured at 37°C and 5% CO2 for 24 hours. After removing the old culture medium, serum-free DMEM medium containing 250 μM H2O2 was added and incubated for another 4 hours to establish an oxidative stress model. The H2O2 culture medium was then aspirated, and the cells were gently washed twice with PBS buffer. Finally, fresh complete culture medium was added. The fresh complete culture medium contained 50 μg / mL N-CDs and Ag. SA -CDs, T-Ag SA-CDs. The incubation process lasted 6 hours. After drug treatment, the sample was washed twice with PBS, followed by incubation in serum-free DMEM medium containing 10 μM DCFH-DA at 37°C in the dark for 30 minutes. After sample addition, the sample was thoroughly rinsed three times with cold PBS to remove unbound probes, and then observed under a confocal fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The results are as follows: Figure 12 As shown.

[0138] from Figure 12 Fluorescence imaging showed that H2O2 stimulation alone significantly enhanced green fluorescence; Ag... SA After treatment with -CDs, the fluorescence darkened significantly, while after treatment with T-Ag... SA After treatment with CDs, ROS-related fluorescence almost completely disappeared, which directly confirms its excellent reactive oxygen species scavenging ability.

[0139] (vi) Assay of mitochondrial MitoSOX clearance capacity

[0140] The level of mitochondrial superoxide in H2O2-induced 293T cells was assessed using the MitoSOX-Green probe.

[0141] The construction and material processing steps of the oxidative stress model were completely consistent with the DCFH-DA detection method described previously in the section on "Determination of Cellular ROS Scavenging Capacity". N-CDs and Ag were added. SA -CDs, T-Ag SA After co-incubating CDs with cells for 6 hours, gently wash twice with PBS. Add serum-free DMEM medium containing 5 μM MitoSOX Green and incubate at 37°C in the dark for 20 minutes. Then wash thoroughly three times with cold PBS and immediately observe using a confocal fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). Results are as follows: Figure 13 As shown.

[0142] from Figure 13 Fluorescence imaging showed that H2O2 stimulation alone significantly enhanced green fluorescence; after Ag... SA -CDs treatment significantly weakened the intensity of mitochondrial superoxide-related fluorescence, while T-Ag treatment significantly reduced it. SA After treatment with CDs, mitochondrial superoxide-related fluorescence almost completely disappeared, which directly confirms its excellent mitochondrial ROS scavenging ability.

[0143] (vii) Apoptosis detection

[0144] The oxidative stress model and material processing procedures were as described in the previous section on "Determination of Cellular ROS Scavenging Capacity". After co-incubating N-CDs, AgSA-CDs, and T-AgSA-CDs with cells for 12 hours, the supernatant was discarded and the cells were washed twice with cold PBS. EDTA-free trypsin was added, and the cells were gently detached by shaking and collected in flow cytometry tubes. The cells were centrifuged at 400 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 195 μL of buffer, and 5 μL of Annexin V-FITC fluorescent dye was added, followed by 10 μL of PI dye. The mixture was gently shaken and incubated on ice in the dark for 20 minutes. Immediate flow cytometry analysis was performed: FL1 (530 / 30 nm) was used for FITC detection, and FL2 (585 / 42 nm) was used for PI detection. Results are as follows: Figure 14 As shown.

[0145] Figure 14 This image demonstrates a dual staining assay for the cytotoxicity of U251-TR cells induced primarily by apoptosis / necrosis, using fluorescein-annexin V and propidium iodide (PI). From left to right, the image represents the H2O2 control group, the H2O2+N-CDs group, and the H2O2+Ag group. SA -CDs group, H2O2+T-Ag SA -CDs group, PBS group.

[0146] from Figure 14 It can be seen that, compared with the untreated H2O2 control group, N-CDs and Ag... SA -CDs and T-Ag SA -CDs group showed a significant reduction in late-stage apoptosis. T-Ag SA Late apoptosis was less in the -CDs group than in the H2O2 group (14.7%), N-CDs group (10.6%), and Ag group (14.7%). SA -CDs group (6.5%), 2.22%.

[0147] III. Mouse Treatment Effect Experiment

[0148] (I) Modeling and Treatment of Acute Kidney Injury (AKI) in Mice

[0149] Male C57BL / 6 mice (8-10 weeks old) were selected and given a single tail vein injection of cisplatin (CDDP, 20 mg / kg). -1 Dissolved in 0.9% physiological saline, a mouse model of acute kidney injury (AKI) was established. Figure 15 As shown in Figure a.

[0150] Injection of cisplatin (20 mg / kg) -1Afterwards, the mice exhibited the following typical characteristics: (1) a rapid decrease in body weight of approximately 25% within 72 hours; (2) serum urea nitrogen (BUN) and creatinine (Scr) levels increased to approximately 5 times that of the control group within 72 hours, indicating severe impairment of glomerular filtration function. Subsequently, four interventions were administered: N-acetylcysteine ​​(NAC), nitrogen-doped carbon dots (N-CDs), silver single-atom nanozymes (Ag... SA -CDs) and T-Ag targeting renal tubular epithelial cells SA -CDs. The results are as follows: Figure 15 The figures are shown in diagrams b, c, and d.

[0151] The results showed that the NAC and N-CDs groups only partially reversed the above biochemical indicators; at 72 hours, BUN and Scr were still about 2-4 times higher than baseline. Meanwhile, T-Ag... SA - The CDs group performed significantly better than the previous two, with their weight recovering to 90% of their initial value; Ag SA The CDs group recovered its body weight, BUN, and Scr to control levels within 72 hours, indicating that it had the best overall renal protective effect.

[0152] (II) Study on biological distribution

[0153] AKI mice were randomly divided into three groups and intravenously injected with N-CDs, Ag, etc., respectively. SA -CDs or T-Ag SA -CDs (all doses are 10 mg / kg) -1 Animals were euthanized at 1, 3, 6, 12, and 24 hours post-injection, and tissue samples from the heart, brain, liver, spleen, lungs, and kidneys were collected. Fluorescence images of each organ were acquired using a Kino small animal imaging system (Spectroscopic Instruments Imaging, Inc., USA), with the excitation / emission filters matched to the emission peak wavelength of the carbon dot material. N-CDs and Ag were injected. SA -CDs, T-Ag SA Fluorescence images of kidney tissue samples containing -CDs, as shown below. Figure 16 As shown in Figure a, T-Ag was injected. SA Fluorescence images of heart, brain, liver, spleen, lung, and kidney tissue samples containing CDs, as shown below. Figure 16 As shown in Figure b.

[0154] Figure 16 a shows that N-CDs, Ag SA -CDs and T-Ag SA N-CDs uptake in mouse kidneys was time-dependent. N-CDs were rapidly internalized 1 hour after injection, distributed as fluorescent dots in the renal parenchyma, and remained there for up to 24 hours; Ag SAThe kinetic curves of -CDs are similar, but thanks to the silver-mediated fluorescence enhancement effect, both their brightness and fluorescence intensity are significantly improved; T-Ag SA -CDs have the highest uptake efficiency, with the signal peak shifting further forward and reaching its maximum intensity.

[0155] Figure 16 b indicates that T-Ag SA CDs are mainly enriched in the renal cortex and medulla, reaching their fluorescence peak in 6-12 hours. The uptake in other organs (brain, heart, lungs, liver, spleen) is extremely low, highlighting their excellent renal targeting properties.

[0156] Journey to the West T-Ag SA Heart, brain, liver, spleen, lung, and kidney tissues from mice injected with CDs were weighed and homogenized with PBS buffer at a weight-to-volume ratio of 1:3. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C, and the fluorescence intensity of the supernatant was then measured (excitation wavelength 560 nm / emission wavelength 640 nm). The final concentration was calculated based on the standard curve prepared from the control tissue homogenate.

[0157] Biodistribution is expressed as percentage of injected dose per gram of tissue (%ID g) -1 = [(CD content per gram of tissue based on fluorescence) / (total injection dose)] × 100%.

[0158] The results are as follows Figure 16 As shown in c, T-Ag SA -CDs accumulate in the kidneys, peaking at 45% ID / g within 3 hours, followed by rapid clearance, decreasing to 20% ID / g within 24 hours, with target organ residues <5% ID / g. Liver uptake shows only transient increases, while lung, heart, and brain uptake remain consistently below 5% ID / g. This pharmacokinetic profile ensures efficient accumulation of nanozymes in AKI lesions while maintaining extremely low systemic exposure, providing strong assurance for safe treatment.

[0159] (III) Evaluation of in vivo therapeutic effects

[0160] Male C57BL / 6 mice were randomly divided into six groups (n=6 per group): (1) healthy PBS group; (2) AKI+PBS group; (3) AKI+N-acetylcysteine ​​(NAC) group; (4) AKI+(N-CD) group; (5) AKI+(Ag) group. SA -CD) group; (6)AKI+(T-Ag SA -CD) group.

[0161] Cisplatin (20 mg / kg) was administered via a single tail vein injection. -1AKI induction. The dosing regimen is as follows: intravenous injection 1 hour before cisplatin administration, followed by repeated administration at 24 and 48 hours (total dose 10 mg / kg). -1 NAC dosage and T-Ag SA -CD equivalent). Mice were sacrificed after 72 hours, and blood was collected and centrifuged (4℃, 3000rpm, 20min). Serum samples were sent to Wuhan Cyber ​​Biotechnology Co., Ltd. for automated detection of blood urea nitrogen and creatinine. Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4μm). Histopathological evaluation was performed under light microscopy using hematoxylin-eosin (H&E) staining. Results are as follows. Figure 17 As shown in Figure a.

[0162] from Figure 17 As shown in a, diffuse swelling of renal tubular epithelial cells and significant dilation of the lumen were observed in the renal cortex of the AKI group; Bowman's capsule in the glomeruli was widened, and T-Ag was detected. SA - After CDs treatment, renal tubular epithelial edema completely subsided, and the brush border structure returned to clear; the glomerular outline was regular, with no significant difference from the control group.

[0163] Paraffin-embedded kidney sections (4 μm) were dewaxed and hydrated before being processed with 20 μg mL of [agent / method / treatment]. -1 Proteinase K was permeabilized at 37°C for 15 minutes. After rinsing with PBS, apoptotic cells were labeled by incubating with TUNEL reaction mixture at 37°C in the dark for 60 minutes. Sections were washed three times with PBS, counterstained with DAPI, and imaged under a fluorescence microscope. Results are as follows: Figure 17 As shown in Figure b.

[0164] from Figure 17 b indicates that Ag SA -CDs and T-Ag SA -CDs showed lower fluorescence intensity than other treatment groups, T-Ag SA -CDs exhibited the lowest fluorescence intensity, indicating that they can accelerate the repair of renal tubular structure and function by inhibiting apoptosis.

[0165] (iv) Statistical methods

[0166] All statistical tests (including one-way and two-way ANOVA, supplemented by Tukey's multiple comparison test and Student's t-test) were performed in OriginPro 2022 (evaluation version). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.001 represent significance levels. Unless otherwise stated, error bars in the figures are mean ± standard error (SEM). Biodistribution and weight analysis were performed in Microsoft Excel 2016.

[0167] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A method for preparing silver single-atom-doped carbon quantum dot nanozymes, characterized in that, Includes the following steps: (1) Synthesis of nitrogen-doped carbon dots Anhydrous citric acid and urea were dissolved in formic acid and mixed thoroughly. The solution was then transferred to a reaction vessel for reaction. After the reaction, the mixture was cooled to room temperature, and an equal amount of ethanol was added and allowed to stand overnight. The mixture was then centrifuged, and the precipitate obtained after centrifugation was freeze-dried to obtain nitrogen-doped carbon dots. (2) Synthesis of silver single-atom nanozymes The synthesized nitrogen-doped carbon dots were dissolved in deionized water, mixed well, and the pH was adjusted. Then, a solution containing silver ions was added dropwise while stirring. After stirring evenly, trisodium citrate and sodium borohydride were added in sequence and mixed. After oil bath reaction, dialysis, and freeze-drying, silver single-atom nanozymes were obtained.

2. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, The process also includes step (3), in which carboxylated triphenylphosphine, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) are added to DMSO for reaction. Then, the silver single-atom nanozyme prepared in step (2) is added to the system for reaction. After dialysis and freeze-drying, silver single-atom nanozyme powder with triphenylphosphine linkage is obtained.

3. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, In step (1), the mass ratio of anhydrous citric acid to urea is 1:(1-3).

4. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, In step (1), the reaction conditions in the reactor are: temperature 150-180℃, time 2-6h.

5. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, In step (2), the nitrogen-doped carbon dot solution: silver ion solution = (0.15~0.35) mg / mL: 1 mM.

6. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, In step (2), the ratio of trisodium citrate to sodium borohydride is 7 mM to 5 mM.

7. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 1, characterized in that, In step (2), the conditions for the oil bath reaction are: temperature 60-80℃, time 4-6h; the conditions for dialysis are: molecular weight cutoff 450-550 Da, time 48-96h.

8. The method for preparing silver single-atom-doped carbon quantum dot nanozymes according to claim 2, characterized in that, The carboxylated triphenylphosphine: N-hydroxysuccinimide (NHS): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): silver single-atom nanozyme = 2.7 mg: 1.35 mg: 1.35 mg: 1 mg.

9. A silver single-atom-doped carbon quantum dot nanozyme, characterized in that, Prepared by the preparation method according to any one of claims 1-8.

10. The application of the silver single-atom doped carbon quantum dot nanozyme according to claim 9 as an antioxidant enzyme mimic in the preparation of drugs for treating cellular oxidative stress damage.

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