Preparation method and application of Cu / Pt bimetallic monatomic nano-enzyme

Abstract

The invention discloses a preparation method and application of Cu / Pt bimetal monatomic nano-enzyme, and relates to the technical field of nano-enzyme, the preparation method comprises the following steps: S1, placing urea in a tubular furnace for reaction, and uniformly grinding to obtain thermally stripped g-C3N4; s2, acidizing the g-C3N4 with concentrated nitric acid, performing ultrasonic stripping after repeated water washing, centrifugation and ethanol washing, and finally performing centrifugal drying to obtain acidized and stripped g-C3N4; and S3, preparing the Cu / Pt bimetallic monatomic nano-enzyme through a solvothermal reaction and a high-temperature calcination method. Compared with g-C3N4, the nano-enzyme has the advantages that the SOD enzyme activity and the CAT enzyme activity are obviously improved, and ROS can be effectively removed. The Cu / Pt bimetallic monatomic nano-enzyme prepared by the invention has good SOD enzyme and CAT enzyme activity, and can efficiently remove ROS on a wound surface, reduce inflammation, remodel a tissue regeneration microenvironment and promote wound surface healing.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of nanoenzymes, in particular to a preparation method and application of a Cu / Pt bimetallic single-atom nanoenzyme. BACKGROUND

[0002] Diabetic wounds are one of the common and serious complications of diabetes, and are mainly characterized by high blood sugar, persistent inflammatory response, impaired angiogenesis, reduced blood flow and impaired tissue epithelialization. These pathological factors lead to a decrease in the wound healing ability of patients, which easily causes infection, results in lower limb amputation or death. One of the core mechanisms of the difficulty in healing of diabetic wounds is that high blood sugar induces excessive accumulation of ROS. Reactive oxygen species (ROS) are a class of chemically active oxygen-containing molecules or free radicals, such as superoxide anions, peroxide radicals and hydrogen peroxide, which are involved in signal transduction, immune defense and maintenance of redox homeostasis. Excessive ROS can cause cell damage, while antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) can keep ROS in balance. At present, the common clinical treatment methods are debridement, negative pressure wound therapy and surgical skin grafting treatment. Although these treatment methods can alleviate the symptoms to a certain extent, they have the disadvantages of limited efficacy, large side effects and causing secondary trauma. In the prior art, there are methods for realizing wound repair through biological dressings, negative pressure suction or growth factor regulation. There are limitations in the treatment of diabetic wounds, and natural antioxidant enzymes are effective but easy to inactivate and high in cost. In view of these, nanoenzymes provide a new solution.

[0003] For example, Chinese Patent CN114345349A (published on April 15, 2022) discloses a preparation method and application of a bimetallic nanoenzyme, which comprises the following steps: (a) preparing a hexagonal metal organic framework material ZIF-67 by using a solvent method; (b) synthesizing nickel cobalt oxide (NiCo2O4) by using an ion replacement method; and (c) preparing a NiCo2O4@PVP bimetallic nanoenzyme by using a step-by-step self-assembly template method. The nanoenzyme can well simulate the activity of superoxide dismutase (SOD enzyme) and catalase (CAT enzyme), and can effectively remove ·OH. The nanoenzyme NiCo2O4@PVP can well simulate the activity of SOD enzyme and CAT enzyme, and can effectively remove ·OH. The nanoenzyme NiCo2O4@PVP has negative electric properties under neutral and alkaline conditions, and the particle size is less than 300 nm. The nanoenzyme NiCo2O4@PVP has no toxic side effects and can effectively remove the generation of active oxygen in cells.

[0004] For example, Chinese patent CN119386044A (published on February 7, 2025) discloses a preparation method of zinc-dihydromyricetin nanozyme and its application in preparing a drug for promoting diabetic wound healing. The invention uses PVP as a surfactant to coordinate metal element zinc (Zn) with natural product dihydromyricetin (DHM) through a one-step method to obtain zinc-dihydromyricetin metal polyphenol nanozyme (Zn-DHM). The Zn-DHM nanozyme prepared by the invention has antioxidant properties and can promote cell proliferation and migration, and can maintain the metabolic and immune homeostasis of diabetic wounds, thus accelerating the early healing of diabetic wounds.

[0005] Bimetallic single-atom nanozyme is a kind of artificial enzyme that anchors two metal atoms on a carrier, has diversified catalytic active sites, and is expected to realize the complementary function and synergistic effect of different metal atoms, can simulate multiple enzyme functions to improve catalytic activity, and regulate the microenvironment of the wound. It has important significance for the treatment of diabetic wounds. SUMMARY

[0006] To solve the above problems, the present application provides a preparation method and application of Cu / Pt bimetallic single-atom nanozyme, which provides a new strategy for the treatment of diabetic wounds.

[0007] The present application provides a preparation method of Cu / Pt bimetallic single-atom nanozyme, comprising the following steps:

[0008] Step S1: placing urea in a tube furnace reaction and grinding uniformly to obtain thermally exfoliated g-C3N4;

[0009] Step S2: acidifying the g-C3N4 with concentrated nitric acid, repeatedly washing with water, centrifuging, ethanol washing, and then ultrasonic exfoliation, and finally centrifuging and drying to obtain acid-exfoliated g-C3N4;

[0010] Step S3: preparing Cu / Pt bimetallic single-atom nanozyme by solvothermal reaction and high-temperature calcination.

[0011] Further, the step S1 comprises the following steps:

[0012] Step S11: weighing urea in a quartz boat using an analytical balance, and performing a tube furnace reaction;

[0013] Step S12: taking out the sample, grinding it uniformly in an agate mortar, and reinserting it into the quartz boat for tube furnace reaction;

[0014] Step S13: grinding uniformly in the agate mortar to obtain thermally exfoliated g-C3N4, and storing it in the dark after drying.

[0015] Further, the step S2 comprises the following steps:

[0016] Step S21: Analytical balance takes the heat-stripped g-C3N4 in a glass beaker, and adds concentrated nitric acid under magnetic stirring, continuously stirs at room temperature;

[0017] Step S22: Stand overnight, collect the precipitate and transfer to a 1L glass beaker, add appropriate amount of ultrapure water to resuspend, after stratification, discard the supernatant, resuspend with ultrapure water, repeat the operation until the pH of the suspension is ≥6;

[0018] Step S23: Centrifuge the suspension in the centrifuge, collect the precipitate;

[0019] Step S24: Wash the precipitate with anhydrous ethanol and ultrapure water respectively, transfer the precipitate to a centrifuge tube, add appropriate amount of ultrapure water to resuspend, seal and ultrasonic stripping;

[0020] Step S25: Centrifuge the suspension in the centrifuge, collect the precipitate, and dry in a vacuum drying machine to obtain acid-stripped g-C3N4.

[0021] Further, the step S3 comprises the following steps:

[0022] Step S31: Analytical balance takes 1,10-phenanthroline in a beaker, adds copper acetate solution, chloroplatinic acid solution and dimethyl sulfoxide (DMSO), stirs at room temperature for 0.7-1.5h, until the crystals are completely dissolved;

[0023] Step S32: Add acid-stripped g-C3N4, seal and transfer to an oil bath pot, continuously stir at 50-70℃ for 3-5h;

[0024] Step S33: Increase the temperature to 190℃ to evaporate DMSO, and dry in a vacuum drying oven;

[0025] Step S34: After complete drying, transfer the material to an agate mortar, grind uniformly and put into a ceramic crucible;

[0026] Step S35: Under the protection of argon, react at 500-700℃ for 1.5-2.5h in a tube furnace to obtain Cu / Pt-DAzymes.

[0027] Further, in the step S11, the tube furnace has a heating rate of 5℃ / min, and after the tube furnace is heated to 550℃, react for 4h.

[0028] Further, in the step S12, the tube furnace has a heating rate of 5℃ / min, and after the tube furnace is heated to 550℃, react for 5h.

[0029] Further, in the step S21, the ratio of the thermal exfoliated g-C3N4 and concentrated nitric acid is 1:100, the rotating speed is 350-650 rpm, and the stirring time is 17-31 h.

[0030] Further, in the step S23, the rotating speed of the centrifuge is 3000-5000 rpm, and the centrifugation time is 15-25 min.

[0031] Further, in the step S24, the ultrasonic exfoliation time is 34-60 h.

[0032] Further, in the step S25, the rotating speed of the centrifuge is 9000-11000 rpm, the centrifugation time is 25-40 min, the temperature of the vacuum drying machine is 50-70 DEG C, and the drying time is 24-48 h.

[0033] Further, in the step S31, the mass ratio of the 1,10-phenanthroline and copper acetate is 1:0.024-0.036, the mass ratio of the 1,10-phenanthroline and chloroplatinic acid is 1:0.07-0.1, and the mass ratio of the 1,10-phenanthroline and DMSO is 1:1350-2000.

[0034] Further, in the step S32, the mass ratio of the 1,10-phenanthroline and acidified exfoliated g-C3N4 is 1:3.1-4.6.

[0035] Further, in the step S33, the drying time is 24-48 h.

[0036] The application further provides application of the Cu / Pt bimetallic single-atom nanoenzyme prepared by the preparation method of the Cu / Pt bimetallic single-atom nanoenzyme in treatment of a wound of diabetes.

[0037] Compared with the prior art, the application has the following advantages and effects:

[0038] 1. The application provides a preparation method and application of a Cu / Pt bimetallic single-atom nanoenzyme, Cu and Pt are dispersed on a carrier in a single-atom form, the theoretical utilization rate of metal atoms is close to 100%, the amount of noble metal Pt is reduced, costs are saved, the CAT enzyme activity and the SOD enzyme activity of the nanoenzyme of the application are more than 90% at a concentration of 50 mu g / mL, which is about 30% higher than that of the prior art.

[0039] 2, The preparation method and application of the Cu / Pt bimetallic single-atom nanoenzyme provided by the application have excellent CAT enzyme activity. The H2O2 removal capacity of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR is analyzed by using a hydrogen peroxidase detection kit, and the results show that the CAT enzyme activity is significantly higher than that of g-C3N4, and ROS (such as H2O2, O2 - ) can be effectively removed by decomposing H2O2 into H2O and O, remodeling the tissue regeneration microenvironment, and promoting wound healing.

[0040] 3, The preparation method and application of the Cu / Pt bimetallic single-atom nanoenzyme provided by the application have excellent SOD enzyme activity. The H2O2 removal capacity of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR is analyzed by using a SOD enzyme detection kit, and the results show that the SOD enzyme activity is significantly higher than that of g-C3N4, and O2 - can be removed, and the ROS can be removed in cooperation with the CAT enzyme, inflammation can be inhibited, and wound healing can be promoted.

[0041] The above description is only a summary of the technical solutions of the application, in order to more clearly understand the technical means of the application, so as to implement according to the content of the description, and in order to make the above and other purposes, characteristics and advantages of the application more obvious and easy to understand, the following will be described in detail with the preferred embodiments of the application and the accompanying drawings.

[0042] According to the detailed description of the specific embodiments of the application in the following text combined with the drawings, those skilled in the art will more clearly understand the above and other purposes, advantages and characteristics of the application. DETAILED DESCRIPTION

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion.

[0044] Among them:

[0045] Figure 1 The transmission electron microscope graph of each group of nanometer materials in the application;

[0046] Figure 1 A is the transmission electron microscope graph of g-C3N4;

[0047] Figure 1 B is the transmission electron micrograph of Cu / Pt-DAzymes;

[0048] Figure 2 It is the metal atom type and element distribution map of Cu / Pt bimetallic single atom nanozyme in the application;

[0049] Figure 2 A is the high-resolution transmission electron micrograph of Cu / Pt-Dazymes;

[0050] Figure 2 B is the energy dispersive X-ray spectrum element map of Cu / Pt-Dazymes;

[0051] Figure 2 C is the EDS spectrum analysis map of Cu / Pt-Dazymes;

[0052] Figure 2 D and Figure 2 E is the spherical aberration correction high-angle annular dark field scanning transmission electron micrograph of Cu / Pt-Dazymes;

[0053] Figure 3 It is the near-infrared spectrum of g-C3N4 and Cu / Pt bimetallic single atom nanozyme in the application;

[0054] Figure 4 It is the X-ray diffraction spectrum of g-C3N4 and Cu / P bimetallic single atom nanozyme in the application;

[0055] Figure 5 It is the CAT enzyme activity experimental result of Cu / Pt bimetallic single atom nanozyme in the application;

[0056] Figure 5 A is the CAT enzyme activity detection map of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR;

[0057] Figure 5 B is the influence of Cu / Pt-DAzymes concentration on CAT enzyme activity;

[0058] Figure 6 It is the SOD enzyme activity experimental result of Cu / Pt bimetallic single atom nanozyme in the application;

[0059] Figure 6 A is the SOD enzyme activity detection map of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR;

[0060] Figure 6 B is the influence of Cu / Pt-DAzymes concentration on SOD enzyme activity;

[0061] Figure 7 Fluorescence detection of ROS level in cells of different treatment groups;

[0062] Figure 8 Quantitative analysis of ROS fluorescence intensity in cells of different treatment groups by ImageJ;

[0063] Figure 9 Flow cytometry detection of ROS level in cells of different groups. DETAILED DESCRIPTION

[0064] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some but not all of the embodiments of the present application. In the following description, specific details such as specific configurations and components are provided only to help a comprehensive understanding of the embodiments of the present application. Therefore, it should be apparent to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, descriptions of known functions and configurations are omitted in the embodiments for clarity and conciseness.

[0065] It should be understood that the "one embodiment" or "the embodiment" mentioned throughout the specification means that the specific features, structures or characteristics related to the embodiment are included in at least one embodiment of the present application. Therefore, "one embodiment" or "the embodiment" appearing throughout the specification does not necessarily mean the same embodiment. In addition, these specific features, structures or characteristics can be combined in one or more embodiments in any suitable manner.

[0066] In addition, reference numerals and / or letters can be repeated in different examples in the present application. Such repetition is for the purpose of simplification and clarity, and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0067] The term "and / or" herein is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that A exists alone, B exists alone, and A and B exist together. The term "and" herein is a description of another association relationship of the associated objects, which means that there can be two relationships, for example, A and B can mean that A exists alone and A and B exist together. In addition, the character " / " herein generally indicates that the associated objects before and after the " / " are in an "or" relationship.

[0068] The term "at least one" herein is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, at least one of A and B can mean that A exists alone, A and B exist together, and B exists alone.

[0069] It is also important to note that the terms "first" and "second" and similar relational terms are used solely to distinguish one entity or action from another without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion.

[0070] Embodiment 1

[0071] The application provides a preparation method of a Cu / Pt bimetallic single-atom nanozyme, comprising the following steps:

[0072] Step S1: placing urea in a tube furnace reaction and grinding uniformly to obtain heat-stripped g-C3N4;

[0073] Step S2: acidizing the g-C3N4 by using concentrated nitric acid, repeatedly washing, centrifuging, ethanol washing, ultrasonic stripping, and finally centrifugally drying to obtain acid-stripped g-C3N4;

[0074] Step S3: preparing the Cu / Pt bimetallic single-atom nanozyme by a solvothermal reaction and a high-temperature calcination method.

[0075] Further, the step S1 comprises the following steps:

[0076] Step S11: weighing urea in a quartz boat by using an analytical balance, and performing a tube furnace reaction;

[0077] Step S12: taking out the sample, grinding uniformly in an agate mortar, re-putting into the quartz boat, and performing a tube furnace reaction;

[0078] Step S13: grinding uniformly in the agate mortar to obtain heat-stripped g-C3N4, and avoiding light to dry and store.

[0079] Further, the step S2 comprises the following steps:

[0080] Step S21: weighing heat-stripped g-C3N4 in a glass beaker by using an analytical balance, and adding concentrated nitric acid under magnetic stirring, and continuously stirring at room temperature;

[0081] Step S22: standing overnight, collecting the precipitate and transferring to a 1L glass beaker, adding appropriate amount of ultrapure water to resuspend, discarding the supernatant after layering, re-adding ultrapure water to resuspend, and repeatedly operating until the pH of the suspension is greater than or equal to 6;

[0082] Step S23: centrifuging the suspension in a centrifuge, and collecting the precipitate;

[0083] Step S24: precipitate, transferring the precipitate to a centrifuge tube, adding appropriate amount of ultrapure water to resuspend, sealing after ultrasonic stripping;

[0084] Step S25: centrifuging the suspension in a centrifuge, collecting the precipitate, and drying in a vacuum drying machine to obtain the acidized exfoliated g-C3N4.

[0085] Further, the step S3 comprises the following steps:

[0086] Step S31: 18 mg of 1,10-phenanthroline was weighed on an analytical balance and placed in a beaker, 60.4 μl of copper acetate solution, 156 μl of chloroplatinic acid solution and 30 mL of dimethyl sulfoxide (DMSO) were added, and stirred at room temperature for 1 h until the crystals were completely dissolved;

[0087] Step S32: 69.6 mg of acidized exfoliated g-C3N4 was added, sealed and transferred to an oil bath, and stirred at 60°C for 4 h;

[0088] Step S33: the temperature was raised to 190°C to evaporate the DMSO, and dried in a vacuum drying oven;

[0089] Step S34: after complete drying, the material was transferred to an agate mortar, ground uniformly and placed in a ceramic crucible;

[0090] Step S35: under argon protection, the tube furnace was reacted at 600°C for 2 h to obtain Cu / Pt-DAzymes.

[0091] Further, in the step S11, the tube furnace was heated at a rate of 5°C / min to 550°C and reacted for 4 h.

[0092] Further, in the step S12, the tube furnace was heated at a rate of 5°C / min to 550°C and reacted for 5 h.

[0093] Further, in the step S21, the concentrated nitric acid was 50 mL, the rotation speed was 500 rpm, and the stirring time was 24 h.

[0094] Further, in the step S23, the centrifuge speed was 4000 rpm and the centrifugation time was 20 min.

[0095] Further, in the step S24, the ultrasonic exfoliation time was 48 h.

[0096] Further, in the step S25, the centrifuge speed was 10000 rpm, the centrifugation time was 30 min, the vacuum drying machine temperature was 60°C, and the drying time was 24-48 h.

[0097] Further, in the step S31, the concentrations of the copper acetate solution and the chloroplatinic acid solution were both 10 mg / mL.

[0098] Further, the drying time in step S33 is 24-48h.

[0099] Effects achieved by the present embodiment are as follows: The present embodiment provides a preparation method of Cu / Pt bimetallic single-atom nanozyme. g-C3N4 is used as a carrier, and Cu / Pt bimetallic single-atom nanozyme is prepared by a solvothermal reaction and a high-temperature calcination method. In the nanozyme, Cu and Pt are dispersed in the form of single atoms on the carrier, and the theoretical utilization rate of metal atoms is close to 100%, thereby reducing the amount of noble metal Pt and saving costs.

[0100] Embodiment 2

[0101] Based on embodiment 1, the present embodiment further provides a nanozyme, which is a Cu / Pt bimetallic single-atom nanozyme prepared by the preparation method described in embodiment 1, i.e., Cu / Pt-DAzymes.

[0102] The Cu / Pt bimetallic single-atom nanozyme provided by the present embodiment is observed by transmission electron microscopy, and no obvious metal particles are observed on the surface of Cu / Pt-DAzymes. The transmission electron microscopy observation diagram is shown in Figure 1 ; energy dispersive X-ray spectroscopy (EDS) element mapping results and spherical aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) results show that Cu, Pt, C, N and O exist in the Cu / Pt-DAzymes system, and Cu and Pt exist in the form of atoms. The HAADF-STEM and EDS mapping diagrams are shown in Figure 2 ; near-infrared spectroscopy results show that the original structure of g-C3N4 is not damaged after loading copper and platinum metal atoms. The near-infrared spectrogram is shown in Figure 3 ; X-ray diffraction (XRD) results show that no metal particles of nanoscale are formed in Cu / Pt-DAzymes. The X-ray diffraction spectrogram is shown in Figure 4 .

[0103] Effects achieved by the present embodiment are as follows: The Cu / Pt bimetallic single-atom nanozyme provided by the present embodiment has no nano-particles, thereby reducing cell stress. The nanozyme has a thin-layer structure, which does not block active sites, thereby improving catalytic efficiency.

[0104] Embodiment 3

[0105] The present embodiment is CAT enzyme activity detection of Cu / Pt bimetallic single-atom nanozyme.

[0106] Based on the above embodiment 1-2, this embodiment is to analyze the H2O2 removal ability of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR by using catalase detection kit. The CAT enzyme activity analysis results are shown in the following table A. Figure 5 , Figure 5 A is the analysis of the H2O2 removal ability of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR, Figure 5 B is the removal effect of H2O2 under different Cu / Pt-DAzymes concentrations.

[0107] The removal ability of g-C3N4 is the lowest, only 5.7%; the removal ability of Cu / Pt-DAzymes+NIR is the highest, reaching 95.9%; and the removal ability of Cu / Pt-DAzymes is 80.1%.

[0108] The higher the concentration of Cu / Pt-DAzymes, the stronger the ability to remove H2O2.

[0109] The technical effects achieved by this embodiment are as follows: it is proved that Cu / Pt-DAzymes has good CAT enzyme activity, and the ability to remove H2O2 is significantly higher than that of g-C3N4.

[0110] Embodiment 4

[0111] This embodiment is the CAT enzyme activity detection of Cu / Pt bimetallic single-atom nanoscale enzyme.

[0112] Based on the above embodiment 1-3, this embodiment is to analyze the H2O2 removal ability of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR by using SOD enzyme detection kit. The SOD enzyme activity analysis results are shown in the following table A. Figure 6 , Figure 6 A is the analysis of the H2O2 removal ability of g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR, Figure 6 B is the removal effect of H2O2 under different Cu / Pt-DAzymes concentrations.

[0113] The removal ability of g-C3N4 is the lowest, only 9.3%; the removal ability of Cu / Pt-DAzymes+NIR is the highest, reaching 65.75%; and the removal ability of Cu / Pt-DAzymes is 58.48%.

[0114] The higher the concentration of Cu / Pt-DAzymes, the stronger the ability to remove H2O2.

[0115] The technical effects achieved by the embodiment are as follows: it is proved that the Cu / Pt-DAzymes have good SOD enzyme activity, and the ability to remove H2O2 is significantly higher than that of g-C3N4.

[0116] Embodiment 5

[0117] This embodiment verifies the ROS removal effect of the Cu / Pt bimetallic single-atom nanoscale enzyme.

[0118] Based on the above embodiments 1-4, this embodiment verifies the ROS removal effect of the Cu / Pt bimetallic single-atom nanoscale enzyme, please refer to Figure 7 ; ROS level fluorescence quantitative diagram, please refer to Figure 8 ; flow cytometry detection ROS level result diagram please refer to Figure 9 .

[0119] H2O2 and LPS / IFN-γ are used to induce cells to produce a large amount of ROS, and the green fluorescence intensity of the normal group, the g-C3N4 group, the Cu / Pt-DAzymes group and the Cu / Pt-DAzymes+NIR group is observed and fluorescence quantification is performed, and it is found that the normal group has almost no green fluorescence; the green fluorescence intensity of the g-C3N4 group is basically the same as that of the H2O2 group and the LPS / IFN-γ group, indicating that g-C3N4 cannot remove intracellular ROS; the fluorescence intensity of the Cu / Pt-DAzyme group is significantly reduced, and the fluorescence intensity of the Cu / Pt-DAzymes+NIR group is basically the same as that of the normal group. The flow cytometry detection results show that the ROS of the cells in the NC, H2O2, g-C3N4, Cu / Pt-DAzymes and Cu / Pt-DAzymes+NIR groups accounts for 9.40%, 99.2%, 98.8%, 48.1% and 17.9% respectively, indicating that the catalytic performance of Cu / Pt-DAzymes is enhanced under near-infrared irradiation.

[0120] In summary, the Cu / Pt bimetallic single-atom nanoscale enzyme prepared by the present application has good CAT enzyme activity and SOD enzyme activity, and the ROS content of the Cu / Pt bimetallic single-atom nanoscale enzyme group is decreased by 51.32% compared with g-C3N4, indicating that the Cu / Pt bimetallic single-atom nanoscale enzyme can effectively remove ROS, relieve oxidative stress, inhibit inflammation, restore normal immune microenvironment and promote diabetic wound healing.

[0121] The above specific embodiments further illustrate the purpose, technical solutions and advantages of the present application, and it should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing Cu / Pt bimetallic single-atom nanozymes, characterized in that, Includes the following steps: Step S1: Urea is placed in a tube furnace for reaction and then ground evenly to obtain thermally exfoliated g-C3N4; Step S2: The g-C3N4 is acidified with concentrated nitric acid, and after repeated washing with water, centrifugation, and washing with ethanol, it is ultrasonically peeled off, and finally centrifuged and dried to obtain acid-peeled g-C3N4. Step S3: Prepare Cu / Pt bimetallic single-atom nanozymes by solvothermal reaction and high-temperature calcination.

2. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 1, characterized in that, Step S1 includes the following steps: Step S11: Weigh urea into a quartz boat using an analytical balance, set the heating rate to 5℃ / min, and wait for the tube furnace to heat up to 550℃ and react for 4 hours. Step S12: Take out the sample, grind it evenly in an agate mortar, put it back into the quartz boat, set the heating rate to 5℃ / min, and wait for the tube furnace to heat up to 550℃ and react for 5h. Step S13: Grind evenly in an agate mortar to obtain heat-exfoliated g-C3N4, and store it in a dark, dry place.

3. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 1, characterized in that, Step S2 includes the following steps: Step S21: Weigh the thermally exfoliated g-C3N4 into a glass beaker using an analytical balance, add concentrated nitric acid while stirring magnetically, and continue stirring at room temperature; Step S22: Let stand overnight, collect the precipitate and transfer it to a glass beaker, add an appropriate amount of ultrapure water to resuspend it, discard the supernatant after separation, add ultrapure water again to resuspend it, repeat the operation until the pH of the suspension is ≥6. Step S23: Centrifuge the suspension in a centrifuge and collect the precipitate; Step S24: Wash the precipitate with anhydrous ethanol and ultrapure water respectively, transfer the precipitate to a centrifuge tube, add an appropriate amount of ultrapure water to resuspend it, seal the tube and then ultrasonically peel it off. Step S25: Centrifuge the suspension in a centrifuge, collect the precipitate, and dry it in a vacuum dryer to obtain acid-exfoliated g-C3N4.

4. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 1, characterized in that, Step S3 includes the following steps: Step S31: Weigh 1,10-phenanthroline into a beaker using an analytical balance, add copper acetate solution, chloroplatinic acid solution and dimethyl sulfoxide (DMSO), and stir at room temperature for 0.7-1.5 h until all crystals are dissolved; Step S32: Add the acid-exfoliated g-C3N4, seal it, and transfer it to an oil bath. Stir continuously at 50-70℃ for 3-5 hours. Step S33: Heat to 190℃ to evaporate DMSO, and then dry in a vacuum drying oven; Step S34: After thorough drying, transfer the material to an agate mortar, grind it evenly, and then place it in a ceramic crucible; Step S35: Under argon protection, react in a tube furnace at 500-700℃ for 1.5-2.5h to obtain Cu / Pt-DAzymes.

5. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 3, characterized in that, In step S21, the ratio of the thermally stripped g-C3N4 to concentrated nitric acid is 1:100, the rotation speed is 350-650 rpm, and the stirring time is 17-31 h.

6. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 3, characterized in that, In step S23, the centrifuge speed is 3000-5000 rpm and the centrifugation time is 15-25 min; in step S24, the ultrasonic peeling time is 34-60 h.

7. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 3, characterized in that, In step S25, the centrifuge speed is 9000-11000 rpm, the centrifugation time is 25-40 min, the vacuum dryer temperature is 50-70℃, and the drying time is 24-48 h.

8. The method for preparing a Cu / Pt bimetallic single-atom nanozyme according to claim 4, characterized in that, In step S31, the mass ratio of 1,10-phenanthroline to copper acetate is 1:0.024-0.036; the mass ratio of 1,10-phenanthroline to chloroplatinic acid is 1:0.07-0.1; and the mass ratio of 1,10-phenanthroline to DMSO is 1:1350-2000. In step S32, the mass ratio of 1,10-phenanthroline to acid-exfoliated g-C3N4 is 1:3.1-4.6; the concentrations of both the copper acetate solution and the chloroplatinic acid solution are 10 mg / mL. In step S33, the drying time is 24-48 h.

9. A Cu / Pt bimetallic single-atom nanozyme prepared by the preparation method according to any one of claims 1-8.

10. The application of the Cu / Pt bimetallic single-atom nanozyme prepared by the preparation method of claim 9 in the treatment of diabetic wounds.

Images