Preparation and application of N-doped porous carbon nanotube loaded Fe monatomic nano-enzyme

Abstract

The invention relates to the technical field of nano-drugs, in particular to preparation and application of N-doped porous carbon nanotube loaded Fe monatomic nano-enzyme, the preparation method comprises the following steps: step S1, Fe (acac) 3 and ZIF-8 are used for preparing and synthesizing Fe-doped ZIF-8 nano-particles; s2, obtaining a composite porous nanotube material through an electrostatic spinning technology; and S3, preparing the N-doped porous carbon nanotube loaded Fe monatomic nano-enzyme in a thermal annealing manner. The Fe monatomic nano-enzyme FeNC with Fe monatomic loaded on N-doped porous carbon nanotubes prepared by the invention has excellent activity of CAT, POD and GPx enzymes and good photo-thermal performance, has a photodynamic effect of generating singlet oxygen 1O2 under near-infrared illumination (808nm), can generate a large amount of ROS, alleviates an anoxic environment, improves the sterilization temperature of an infected lesion, and has a good application prospect. Therefore, a better sterilization effect is achieved by cooperating with PTT, PDT and CDT therapies, and a new strategy is provided for promoting the antibacterial aspect of infected wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to the preparation and application of nanozymes containing N-doped porous carbon nanotubes loaded with Fe single atoms. Background Art

[0002] Bacterial infection-related diseases remain one of the most serious health problems worldwide. Currently, antibiotic therapy remains the mainstay of treatment for bacterial infections. However, the widespread use and misuse of antibiotics has led to the emergence of multidrug-resistant bacteria, severely reducing the therapeutic effectiveness of antibiotics. Therefore, the development of alternative antimicrobial agents is considered a promising approach to prevent bacterial resistance.

[0003] In recent years, nanozymes have been considered as promising antimicrobial agents. They can act as catalysts to produce reactive oxygen species (ROS) and achieve broad-spectrum antimicrobial therapy. Single-atom nanozymes (SAzymes) are nanozymes with theoretically high catalytic performance. They have a structure similar to that of natural enzymes and exhibit a clear coordination structure. By maximizing the use of dispersed metal atoms with catalytic activity, they exhibit catalytic activity 10 to 100 times higher than traditional nanozymes and have better stability. However, compared with the excellent performance of natural enzymes, single-atom nanozymes still face challenges and opportunities in future research. For example, the catalytic activity still needs to be further improved, and they need to have better biocompatibility and low toxicity, as well as better selectivity.

[0004] Chinese invention patent CN119770681A (publication date: April 8, 2025) discloses a biomimetic nanomedicine for treating subcutaneous drug-resistant bacterial infections, as well as its preparation method and application. The invention adds dopamine hydrochloride during the synthesis of a metal-organic framework to prepare a dandelion-shaped carbon nanozyme precursor, which is then carbonized to obtain a carbon nanozyme with targeting, photoresponsiveness, and peroxidase activity. Finally, berberine hydrochloride is loaded on the carbon nanozyme to obtain the biomimetic nanomedicine. The biomimetic nanomedicine proposed in this invention first has good biocompatibility, and secondly has a unique dandelion-like morphology, which is conducive to targeting. Finally, it has a strong light response ability in the near-infrared region, and can synergistically kill bacteria through the action mechanism of antibacterial drugs, photothermal therapy, and chemokinetics. It has important application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections and provides a new strategy for nanozyme treatment of bacterial infections.

[0005] Based on the existing technology, in order to better carry out sterilization treatment, it is urgent to design a single-atom nanozyme with efficient multi-type enzyme activities. The nanozyme should include peroxidase (POD), oxidase (OXD), catalase (CAT) and glutathione peroxidase (GPx), etc., so as to generate a large amount of ROS (O2, ·OH, 1 O2) for chemodynamic therapy (CDT) to fight bacteria, and produce O2 to relieve hypoxia in infected tissues, consume glutathione to reduce the antioxidant capacity of bacteria, and further enhance the bactericidal effect of ROS; at the same time, the nanozyme should also have excellent photothermal performance (absorb light energy and convert it into heat energy) and have photothermal therapy (PTT) effect. PTT can increase the temperature of the reaction system, on the one hand, for photothermal sterilization, on the other hand, the temperature increase can significantly enhance the catalytic performance of the single-atom nanozyme; in addition, under light irradiation, the nanozyme can also have photodynamic therapy effect, producing singlet oxygen ( 1 O2), enhancing the antibacterial effect of ROS. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides the preparation and application of N-doped porous carbon nanotube-loaded Fe single-atom nanozymes. Fe(acac)3 and ZIF-8 are prepared to synthesize Fe-doped ZIF-8 nanoparticles, and then a composite porous nanotube material is obtained through electrospinning technology. Finally, the Fe single-atom nanozyme FeNC is successfully prepared by thermal annealing, which proposes a new strategy for the field of wound antibacterial treatment.

[0007] The preparation method of the nanozyme containing N-doped porous carbon nanotubes and Fe single atoms provided by the present invention comprises the following steps:

[0008] Step S1: preparing and synthesizing Fe-doped ZIF-8 nanoparticles using Fe(acac)3 and ZIF-8;

[0009] Step S2: obtaining a composite porous nanotube material through electrospinning technology;

[0010] Step S3: Prepare the nanozyme containing N-doped porous carbon nanotubes loaded with Fe single atoms by thermal annealing.

[0011] Furthermore, the step S1 includes the following steps:

[0012] Step S11: dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol, respectively, stirring at room temperature, centrifuging and drying to obtain ZIF-8;

[0013] Step S12: Dispersing ZIF-8 nanoparticles and Fe(acac)3 in ethanol with continuous stirring;

[0014] Step S13: washing by centrifugation, and vacuum drying at 40° C. to 60° C. to obtain Fe-doped ZIF-8 nanoparticles.

[0015] Furthermore, step S2 includes the following steps:

[0016] Step S21: adding Fe-doped ZIF-8 nanoparticles and polyacrylonitrile into N-N-dimethylformamide solvent and stirring;

[0017] Step S22: the mixed solution is transferred into a plastic syringe, and a composite porous nanotube material is obtained by electrospinning, and the composite porous nanotube material is vacuum dried at 40° C. to 60° C. for 12 h to 15 h.

[0018] Furthermore, step S3 includes the following steps:

[0019] After the composite nanotubes were heated in air, they were immediately thermally annealed under argon at a heating rate of 5°C / min, ultimately obtaining N-doped porous carbon nanotubes loaded with Fe single atoms as nanozymes.

[0020] Furthermore, in step S11, the stirring time is 2 hours to 4 hours.

[0021] Furthermore, in step S12, the mass ratio of the ZIF-8 nanoparticles to Fe(acac)3 is in the range of 5:3 to 15:1, the concentration of the ZIF-8 nanoparticles in ethanol is in the range of 5 mg / ml to 3.75 mg / ml, and the continuous stirring time is 10 h to 14 h.

[0022] Furthermore, in step S13, the vacuum drying time is 12 hours to 24 hours.

[0023] Furthermore, in step S21, the mass ratio of the Fe-doped ZIF-8 nanoparticles to polyacrylonitrile is in the range of 1:2 to 2:1, the concentration of the Fe-doped ZIF-8 nanoparticles in the NN dimethylformamide solvent is in the range of 0.2 mg / ml to 0.1 mg / ml, and the stirring time is 20 h to 24 h.

[0024] Furthermore, in step S3, the composite nanotubes are heated in air at a temperature of 200° C. to 250° C. for 2 to 3 hours, the argon gas temperature is 700° C. to 900° C., and the thermal annealing treatment time is 2 to 4 hours.

[0025] The present invention also provides a nanozyme, which is a nanozyme prepared by the above-mentioned preparation method and in which N-doped porous carbon nanotubes are loaded with Fe single atoms, namely FeNC.

[0026] The present invention also provides a method for preparing a nanozyme containing a single Fe atom and a N-doped porous carbon nanotube, and uses the nanozyme prepared in the preparation of antibacterial drugs.

[0027] Compared with the existing technology, the advantages and effects of this application are as follows:

[0028] 1. The preparation and application of the nanozyme of N-doped porous carbon nanotubes loaded with Fe single atoms provided by the present invention have excellent OXD, CAT, POD, and GPx enzyme activities, and can generate a large amount of ROS (O2, ·OH, 1 O2) for chemodynamic therapy (CDT) to fight bacteria, and produce O2 to relieve hypoxia in infected tissues, consume glutathione to reduce the antioxidant capacity of bacteria, and further enhance the bactericidal effect of ROS.

[0029] 2. The preparation and application of the nanozyme of N-doped porous carbon nanotubes loaded with Fe single atoms provided by the present invention have good photothermal performance and PPT effect, which increases the temperature of the reaction system, on the one hand, for photothermal sterilization, and on the other hand, the temperature increase can significantly enhance the catalytic performance of the single-atom nanozyme; at the same time, it has PDT effect and can generate singlet oxygen under near-infrared light (808nm). 1 The photodynamic effect of O2 can enhance the antibacterial effect of ROS.

[0030] 3. The preparation and application of the nanozyme of N-doped porous carbon nanotubes loaded with Fe single atoms provided by the present invention achieve the synergistic and efficient antibacterial effects of chemodynamic therapy (CDT), photodynamic therapy (PDT) and photothermal therapy (PTT) by combining the above enzyme-like catalytic activity with near-infrared light (808nm) irradiation, while having good biocompatibility, providing a new strategy for promoting antibacterial effects on infected wounds.

[0031] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiment of the present application in conjunction with the accompanying drawings.

[0032] Based on the detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without inventive work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0034] in:

[0035] Figure 1 Schematic diagram of the preparation of FeNC prepared in this application;

[0036] Figure 2 is a scanning electron microscope (SEM) image of the N-doped porous carbon nanotube support NC of FeNC prepared in the present application;

[0037] Figure 3 is a scanning electron microscope (SEM) image of FeNC prepared in this application;

[0038] Figure 4 is a transmission electron microscopy (TEM) image of FeNC prepared in this application;

[0039] Figure 5 A is the specific surface area and pore structure of FeNC prepared in this application, and B is the specific surface area and pore structure of N-doped porous carbon nanotube support NC;

[0040] Figure 6 is the electron energy X-ray dispersion spectrum (EDS-mapping) elemental map of FeNC prepared in this application;

[0041] Figure 7 is a high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of FeNC prepared in this application;

[0042] Figure 8 is a high-resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of FeNC prepared in this application;

[0043] Figure 9 is the X-ray diffraction (XRD) pattern of FeNC and NC prepared in this application;

[0044] Figure 10 is a photothermal heating effect diagram of FeNC prepared in this application, where (A) is the photothermal heating effect of FeNC with different concentrations under 808nm near-infrared laser irradiation for 10min (1.0W / cm 2) is a temperature-increasing thermal imaging diagram of FeNC; (B) is a time-dependent temperature-increasing curve diagram corresponding to Figure A.

[0045] Figure 11 This is a graph analyzing the peroxidase-like (POD) activity of FeNC prepared in the present application;

[0046] Figure 12 This is a graph analyzing the catalase-like (CAT) activity of FeNC prepared in the present application;

[0047] Figure 13 The FeNC prepared in this application produces singlet oxygen 1 Activity analysis diagram of O2;

[0048] Figure 14 Analytical graphs showing the glutathione (GSH) consumption capacity of FeNC prepared in the present application, wherein (A) is a graph showing the GSH (10 mM) consumption curve of FeNC at 10 μg / ml, (B) is a graph showing the GSH (10 mM) consumption curve of FeNC at 50 μg / ml, (D) is a graph showing the GSH (10 mM) consumption curve of NC at 10 μg / ml, (E) is a graph showing the GSH (10 mM) consumption curve of NC at 50 μg / ml, (F) is a graph showing the GSH (10 mM) consumption curve of NC at 10 μg / ml, (E) is a graph showing the GSH (10 mM) consumption curve of NC at 50 μg / ml;

[0049] Figure 15 This is a detection diagram of the reactive oxygen species (ROS) produced in bacteria during the sterilization process of the FeNC prepared in this application;

[0050] Figure 16 This is a graph showing the in vitro antibacterial activity test of FeNC prepared in this application. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. In the following description, specific details such as specific configurations and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear 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, for clarity and brevity, the description of known functions and structures has been omitted in the embodiments.

[0052] It should be understood that references throughout this specification to "one embodiment" or "this embodiment" mean that a particular feature, structure, or characteristic associated with the embodiment is included in at least one embodiment of the present application. Therefore, the appearance of "one embodiment" or "this embodiment" throughout this specification does not necessarily refer to the same embodiment. Furthermore, these particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0053] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0054] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the related objects before and after are in an "or" relationship.

[0055] The term "at least one" in this article is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0056] It should also be noted that, in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include," "comprises," or any other variations thereof are intended to cover non-exclusive inclusion.

[0057] Example 1

[0058] This example introduces a method for preparing a nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes under a specific situation. The preparation diagram is shown in Figure 1 , including the following steps:

[0059] Step S1: preparing and synthesizing Fe-doped ZIF-8 nanoparticles using Fe(acac)3 and ZIF-8;

[0060] Step S2: obtaining a composite porous nanotube material through electrospinning technology;

[0061] Step S3: Prepare the nanozyme containing N-doped porous carbon nanotubes loaded with Fe single atoms by thermal annealing.

[0062] Preferably, step S1 includes the following steps:

[0063] Step S11: dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol, respectively, stirring at room temperature, centrifuging and drying to obtain ZIF-8;

[0064] Step S12: Dispersing ZIF-8 nanoparticles and Fe(acac)3 in ethanol with continuous stirring;

[0065] Step S13: washing by centrifugation, and vacuum drying at 40° C. to 60° C. to obtain Fe-doped ZIF-8 nanoparticles.

[0066] Preferably, step S2 includes the following steps:

[0067] Step S21: adding Fe-doped ZIF-8 nanoparticles and polyacrylonitrile into N-N-dimethylformamide solvent and stirring;

[0068] Step S22: the mixed solution is transferred into a plastic syringe, and a composite porous nanotube material is obtained by electrospinning, and the composite porous nanotube material is vacuum dried at 40° C. to 60° C. for 12 h to 15 h.

[0069] Preferably, step S3 includes the following steps:

[0070] After the composite nanotubes were heated in air, they were immediately thermally annealed under argon at a heating rate of 5°C / min, ultimately obtaining the N-doped porous carbon nanotube-loaded Fe single-atom nanozyme FeNC.

[0071] Preferably, in step S11, the stirring time is 2 hours to 4 hours.

[0072] Preferably, in step S12, the ZIF-8 nanoparticles are 50 mg to 150 mg, Fe(acac)3 is 10 mg to 30 mg, ethanol is 10 ml to 40 ml, and the continuous stirring time is 10 h to 14 h.

[0073] Preferably, in step S13, the vacuum drying time is 12 hours to 24 hours.

[0074] Preferably, in step S21, the Fe-doped ZIF-8 nanoparticles are 0.2 g to 0.4 g, the polyacrylonitrile is 0.2 g to 0.4 g, the N-N-dimethylformamide solvent is 5 ml to 10 ml, and the stirring time is 20 h to 24 h.

[0075] Preferably, in step S3, the composite nanotubes are heated in air at a temperature of 200° C. to 250° C. for 2 to 3 hours, the argon gas temperature is 700° C. to 900° C., and the thermal annealing treatment time is 2 to 4 hours.

[0076] The technical effects achieved by this embodiment are as follows: This embodiment provides a method for preparing a nanozyme containing N-doped porous carbon nanotubes and Fe single atoms, which uses N-doped carbon materials as a carrier for constructing single-atom nanozymes and simultaneously loads Fe single atoms, and uses a spatial confinement strategy to prepare N-doped porous carbon nanotubes with high loading capacity of monodisperse Fe single-atom nanozymes.

[0077] Example 2

[0078] Based on Example 1, this example also provides a nanozyme, which is a nanozyme of N-doped porous carbon nanotubes loaded with Fe single atoms, i.e., FeNC, prepared by the preparation method described in Example 1. For a general diagram of FeNC, please refer to Figure 1 .

[0079] The FeNC provided in this embodiment is shown by scanning electron microscopy (SEM) to be porous nanotubes interwoven with NC and FeNC. The SEM image of the FeNC N-doped porous carbon nanotube support NC is shown in FIG. Figure 2 , the scanning electron microscope (SEM) image of FeNC can be found in Figure 3 TEM images of FeNCs show porous nanotubes. Figure 4 .

[0080] The porous structure of FeNC tubes can fully contact with the reaction substrate, which is conducive to the adsorption of small molecules and improves the catalytic efficiency.

[0081] The technical effects achieved by this embodiment are as follows: This embodiment provides an N-doped porous carbon nanotube-loaded Fe single atom nanozyme, which is in the shape of a porous nanotube and can fully contact the reaction substrate, which is conducive to the adsorption of small molecules and improves the catalytic efficiency.

[0082] Example 3

[0083] Based on the above Examples 1-2, this example uses nitrogen (N2) adsorption / desorption method and Barrett-Joyner-Halenda (BJH) method to analyze the specific surface area and pore size of NC and FeNC; its N2 adsorption-desorption isotherm and pore size distribution diagram can be found in Figure 5 , A is the specific surface area and pore structure diagram of FeNC prepared in this application, and B is the specific surface area and pore structure diagram of N-doped porous carbon nanotube support NC.

[0084] When the P / Po ratio is small (<0.5), the isotherms of NC and FeNC show similar behavior. When the P / Po ratio is large (>0.5), the desorption curves of the two show slight differences, but the isotherms both rise. When the relative pressure is close to 1.0, adsorption occurs on the macropores, and the curve rises rapidly.

[0085] The specific surface areas of NC and FeNC are 418.78 m 2 / g and 559.22m 2 / g, and the average pore diameter is 3.31nm.

[0086] The specific surface area of FeNC is larger than that of NC because more pores are generated during the high-temperature preparation process. The larger the specific surface area, the stronger its adsorption capacity, which is more conducive to the catalytic reaction.

[0087] The technical effects achieved by this embodiment are as follows: It proves that FeNC is beneficial for catalytic reactions.

[0088] Example 4

[0089] Based on the above examples 1-3, this example verifies the presence of Fe, N, and C elements by scanning transmission electron microscopy (HAADF-STEM) images and electron energy X-ray dispersion spectroscopy (EDS-mapping) elemental maps; the electron energy X-ray dispersion spectroscopy (EDS-mapping) elemental map of FeNC can be found in Figure 6 , high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images of FeNC can be found in Figure 7 , high-resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images of FeNC can be found in Figure 8 .

[0090] Elemental mapping images confirmed that Fe, N and C were uniformly distributed in the sample.

[0091] Figure 8 The bright white dots in the middle are monodispersed Fe atoms (red circles), which indicates that the single-atom nanozyme FeNC with Fe single atoms dispersed on the carrier NC has been successfully prepared.

[0092] In this example, the chemical structures of the carrier NC and FeNC nanozymes were analyzed and verified using powder X-ray diffraction (XRD) technology. The X-ray diffraction (XRD) patterns of FeNC and NC are shown in Figure 9 .

[0093] The XRD patterns show that the supports NC and FeNC are similar, and no crystal characteristic peaks of metals, metal oxides and metal carbides (such as Fe, Zn, etc.) are detected.

[0094] The characteristic peak at about 25° in the XRD pattern is attributed to the 002 crystal plane of C.

[0095] The XRD pattern of FeNC showed no peaks of Fe or its oxides, indicating that there were no metal nanoparticles in FeNC, further proving the successful synthesis of Fe single atoms.

[0096] The technical effects achieved in this embodiment are as follows: It proves that the single-atom nanozyme FeNC in which single Fe atoms are dispersed on the carrier NC has been successfully prepared.

[0097] Example 5

[0098] Based on the above Examples 1-4, this Example verifies the temperature-raising effect of FeNC. Please refer to FIG10 for a diagram of the photothermal temperature-raising effect of FeNC.

[0099] Different concentrations of FeNC (0, 5, 10, 20, 30, 40, 50 μg / ml) were irradiated with an 808 nm near-infrared laser. It can be seen that the temperature of the FeNC nanozyme increased with the increase of concentration under the same irradiation conditions, providing a good photothermal effect for sterilization in skin soft tissue.

[0100] The near-infrared laser irradiation power is 1.0 W / cm 2 , a total of 600s.

[0101] The technical effects achieved in this example are as follows: FeNC demonstrates its excellent photothermal effect. The FeNC nanozyme achieved a photothermal conversion rate of 41.95%. Compared to the control (ultrapure water without FeNC), at room temperature (25°C), after irradiation with an 808nm near-infrared laser for 10 minutes at different concentrations of FeNC (5, 10, 20, 30, 40, and 50 μg / ml), the temperature in the FeNC group increased by 1.4-2.3 times, ranging from 35°C to 58°C. This indicates that the temperature increased with irradiation time and was positively correlated with the material concentration.

[0102] Example 6

[0103] Based on the above examples 1-5, this example studies the peroxidase (POD)-like activity, catalase (CAT)-like activity and singlet oxygen production of FeNC. 1 The activity of O2 was analyzed; the peroxidase (POD) activity analysis diagram of FeNC is shown in Figure 11 , please refer to the analysis diagram of catalase (CAT) activity of FeNC Figure 12 , FeNC produces singlet oxygen 1 Please refer to the O2 activity analysis chart Figure 13 .

[0104] FeNC catalyzes H2O2 to produce hydroxyl radicals ·OH through peroxidase-like (POD) activity. 3,3',5,5'-tetramethylbenzidine (TMB) is then used as a ·OH probe to monitor the absorption peak of its blue oxidation product TMBOX at 652 nm and visualize the color change.

[0105] Figure 11 Figure (A) in the middle shows that FeNC has stronger peroxidase-like catalytic activity in the presence of H2O2; the absorption peak of TMBOX at 652nm produced by the FeNC+TMB+H2O2 group in the presence of H2O2 is significantly increased by 1.6 times compared with the FeNC+TMB group in the absence of H2O2, and the color is darker blue, indicating that the catalytic activity of FeNC's POD is stronger in the presence of H2O2.

[0106] Figure 11 Figure (B) shows that the peroxidase (POD) catalytic activity of FeNC is concentration-dependent with H₂O₂. When 30 μg / ml FeNC is treated with different H₂O₂ concentrations (0.05, 0.1, 0.2, 0.5, and 1.0 mM) via POD activity, the absorption peak at 652 nm for TMBOX production gradually increases, increasing by 1.2- to 1.8-fold compared to the control (0 mM).

[0107] To analyze the catalase-like (CAT) activity of FeNC nanozymes, a dissolved oxygen meter was used to measure the amount of O2 produced by FeNC-catalyzed H2O2;

[0108] Figure 12 Figure (A) shows the curve of FeNC nanozyme (30 μg / ml) and carrier NC (30 μg / ml) catalyzing H2O2 to produce O2, indicating that FeNC can effectively catalyze H2O2 to produce a large amount of O2, indicating that FeNC has catalase (CAT)-like activity;

[0109] Figure 12 Figure (B) shows the O2 production curve of H2O2 catalyzed by FeNC nanozymes at different concentrations (0, 10, 20, 30, 40 and 50 μg / ml), indicating that the amount of O2 produced increases rapidly with the increase of FeNC concentration and reaction time, indicating that FeNC has excellent catalase (CAT)-like activity; after 10 minutes of action, compared with the control group (FeNC is 0 μg / ml), the amount of O2 produced by H2O2 (5mM) catalyzed by FeNC nanozymes at different concentrations (10, 20, 30, 40 and 50 μg / ml) gradually increased by 2.7 to 4.2 times.

[0110] The control group was pure water; H2O2 was 5mM.

[0111] In order to analyze the generation of singlet oxygen by FeNC under 808 nm near-infrared laser irradiation 1 O2 capacity, using 1,3-diphenylbenzisofuran (DPBF) 1 O2 is detected once DPBF and 1 When combined with O2, DPBF will be irreversibly oxidized, and the absorption intensity of UV-visible light at 420nm will decrease rapidly;

[0112] Figure 13 Middle (A) shows that with the extension of the action time, FeNC nanozyme can rapidly reduce the UV absorption peak of DPBF at 420nm, and the UV absorption peak almost drops to 0 at 12min, while the carrier NC cannot reduce the UV absorption peak of DPBF;

[0113] Figure 13 (B) shows that with the increase of FeNC nanozyme concentration, the UV absorption peak of DPBF gradually decreases, which shows that under 808nm near-infrared laser irradiation, FeNC can catalyze H2O2 to produce singlet oxygen 1 O2.

[0114] The technical effects achieved in this embodiment are as follows: It is demonstrated that FeNC has peroxidase (POD)-like catalytic activity, catalase (CAT)-like activity and the ability to generate singlet oxygen under 808nm near-infrared laser irradiation. 1 O2's capabilities.

[0115] Example 7

[0116] Based on the above Examples 1-6, this Example analyzes the ability of FeNC to consume glutathione (GSH); the analysis chart of the ability of FeNC to consume glutathione (GSH) can be found in Figure 14 , where (A) is the curve change diagram of FeNC consumption of GSH (10 mM) at 10 μg / ml, (B) is 30 μg / ml, and (C) is 50 μg / ml, and (D) is the curve change diagram of NC consumption of GSH (10 mM) at 10 μg / ml, (E) is 30 μg / ml, and (F) is 50 μg / ml.

[0117] 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a probe to detect GSH. When the absorption peak at 412 nm gradually decreased, it indicated that GSH was gradually consumed by FeNC nanozymes.

[0118] like Figure 14As shown in (A), (B), and (C), the consumption of GSH by FeNC nanozymes is time-dependent. 10 μg / ml FeNC nanozymes requires 25 min to basically consume all GSH, while 30 μg / ml and 50 μg / ml FeNC nanozymes can consume all GSH in a shorter time, 15 min and 7 min, respectively. This indicates that the higher the concentration of FeNC nanozymes, the faster the consumption of GSH.

[0119] like Figure 14 As shown in (D), (E), and (F), in comparison, the carrier NC with the same concentration is far from consuming all GSH within the same period of time, which indicates that the ability to rapidly consume GSH mainly comes from the catalytic Fe single atom FeNC nanozyme.

[0120] The technical effects achieved by this embodiment are as follows: it is proved that FeNC has the ability to quickly consume GSH, and the higher the concentration, the faster the consumption of GSH.

[0121] Example 8

[0122] Based on the above Examples 1-7, this Example detects the generation of reactive oxygen species (ROS) in bacteria during the sterilization process by FeNC. For a diagram showing the generation of reactive oxygen species (ROS) in bacteria during the sterilization process by FeNC, see Figure 15 .

[0123] In order to detect the ability of FeNC nanozymes to produce ROS in cells during the bactericidal process, 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as a chemical probe to evaluate the changes in ROS production in bacteria;

[0124] like Figure 15 As shown in (A) and (B), in MRSA and E. coli, the FeNC+NIR, FeNC+H2O2, and FeNC+H2O2+NIR groups showed strong green fluorescence, indicating that ROS were generated in the cells during the bactericidal process of FeNC nanozymes;

[0125] Among them, the FeNC+H2O2+NIR group produced a large amount of ROS through photothermal effect and enzyme-like catalysis. The green fluorescence intensity of ROS produced in the FeNC+H2O2+NIR group of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was 3.1 times and 3.4 times that of the control group, respectively.

[0126] This example also tested the in vitro antibacterial activity of FeNC.

[0127] To investigate the in vitro antibacterial properties of FeNC, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) were used as models;

[0128] Figure 16 Middle (A) shows the antibacterial effect of FeNC nanozyme alone. It can be found that as the concentration of FeNC nanozyme gradually increases, the bactericidal effect on MRSA and E. coli becomes stronger, indicating that FeNC nanozyme itself has a bactericidal effect, and its strength is related to its concentration;

[0129] Figure 16 Figures (B) and (C) show that the FeNC+H2O2 group further enhanced its bactericidal effect against MRSA and E. coli, suggesting that FeNC catalyzes H2O2 to produce toxic reactive oxygen species (ROS) (hydroxyl radicals ·OH) that kill bacteria. In the absence of NIR irradiation, the FeNC+H2O2 group achieved bactericidal rates of 39.5% and 37.5% against MRSA and E. coli, respectively, compared to the control group.

[0130] The photothermal effect of FeNC has a relatively strong bactericidal effect. The FeNC+H2O2+NIR group produces reactive oxygen species (ROS) through photothermal effect and enzyme-like catalysis, which has a very strong synergistic antibacterial effect.

[0131] The technical effects achieved in this example are as follows: FeNC nanozymes generate ROS intracellularly during the bactericidal process, demonstrating that the photothermal effect of FeNC has a relatively strong bactericidal effect. The FeNC + H2O2 + NIR treatment, through photothermal and enzyme-like catalysis, generates a large amount of ROS, resulting in a powerful synergistic antibacterial effect. Under NIR irradiation, the synergistic antibacterial effect of the FeNC + H2O2 + NIR treatment achieved a 100% bactericidal rate against both MRSA and E. coli, compared to the control group.

[0132] The foregoing description is merely a preferred embodiment of the present invention and does not limit the scope of protection of the present invention. It will be apparent to those skilled in the art that various variations, modifications, substitutions, integrations, and parameter changes are possible with the present invention. Any variation, modification, substitution, integration, and parameter changes that fall within the spirit and principles of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a nanozyme containing a single Fe atom loaded on a N-doped porous carbon nanotube, characterized in that: The following steps are involved: Step S1: preparing and synthesizing Fe-doped ZIF-8 nanoparticles using Fe(acac)3 and ZIF-8; Step S2: obtaining a composite porous nanotube material through electrospinning technology; Step S3: Prepare the nanozyme containing N-doped porous carbon nanotubes loaded with Fe single atoms by thermal annealing.

2. The method for preparing the nanozyme containing N-doped porous carbon nanotubes and Fe single atoms according to claim 1, wherein: The step S1 comprises the following steps: Step S11: dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol, respectively, stirring at room temperature, centrifuging and drying to obtain ZIF-8; Step S12: Dispersing ZIF-8 nanoparticles and Fe(acac)3 in ethanol with continuous stirring; Step S13: washing by centrifugation, and vacuum drying at 40° C. to 60° C. to obtain Fe-doped ZIF-8 nanoparticles.

3. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 1, characterized in that: The step S2 comprises the following steps: Step S21: adding Fe-doped ZIF-8 nanoparticles and polyacrylonitrile into N-N-dimethylformamide solvent and stirring; Step S22: the mixed solution is transferred into a plastic syringe, and a composite porous nanotube material is obtained by electrospinning, and the composite porous nanotube material is vacuum dried at 40° C. to 60° C. for 12 h to 15 h.

4. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 1, characterized in that: The step S3 comprises the following steps: After the composite nanotubes were heated in air, they were immediately thermally annealed under argon at a heating rate of 5°C / min, ultimately obtaining N-doped porous carbon nanotubes loaded with Fe single atoms as nanozymes.

5. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 2, characterized in that: In the step S11, the stirring time is 2 hours to 4 hours.

6. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 2, characterized in that: In step S12, the mass ratio of the ZIF-8 nanoparticles to Fe(acac)3 is in the range of 5:3 to 15:1, the concentration of the ZIF-8 nanoparticles in ethanol is in the range of 5 mg / ml to 3.75 mg / ml, and the continuous stirring time is 10 h to 14 h.

7. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 2, characterized in that: In the step S13, the vacuum drying time is 12 hours to 24 hours.

8. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 3, characterized in that: In step S21, the mass ratio of the Fe-doped ZIF-8 nanoparticles to polyacrylonitrile is in the range of 1:2 to 2:1, the concentration of the Fe-doped ZIF-8 nanoparticles in the NN dimethylformamide solvent is in the range of 0.2 mg / ml to 0.1 mg / ml, and the stirring time is 20 h to 24 h.

9. The method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to claim 4, characterized in that: In step S3, the composite nanotubes are heated in air at a temperature of 200° C. to 250° C. for 2 to 3 hours, the argon gas temperature is 700° C. to 900° C., and the thermal annealing treatment time is 2 to 4 hours.

10. Use of the nanozyme prepared by the method for preparing the nanozyme containing Fe single atoms loaded on N-doped porous carbon nanotubes according to any one of claims 1 to 9 in the preparation of antibacterial drugs.

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