Preparation Method and Application of Single-Atom Nanozyme Using Microbial Cells as Templates

Abstract

The present invention relates to a preparation method and application of a single-atom nanozyme using microbial cells as a template, belonging to the technical field of bionic nanocatalyst synthesis. It includes: S1, inoculating microbial cells into a culture medium for cultivation; S2, after the microbial cells enter the growth plateau phase, adding a pre-cooled calcium chloride solution to the microbial cells; S3, adding a metal ion and a volatile salt solution to the product obtained in S2; S4, adding a fixing solution to the product obtained in S3 for fixation; S5, adding a salt solution to the product obtained in S4, drying, and performing high-temperature calcination in an inert gas atmosphere; S6, washing the excess salt solution in the product obtained in S5 and drying to obtain a single-atom nanozyme. The single-atom nanozyme prepared by the present invention has high peroxidase-like activity and excellent photothermal properties, can catalyze the generation of a large amount of ROS, thereby killing bacteria, has an obvious antibacterial effect, and is beneficial to large-scale batch production.

Description

Technical Field

[0001] The present invention relates to the technical field of synthesis of bionic nano-catalysts, and particularly to a preparation method and application of single-atom nanozymes using microbial cells as templates. Background Art

[0002] Traditional enzyme engineering mainly focuses on improving the performance and adaptability of enzymes by modifying the macromolecular structure, such as changing the amino acid sequence of enzymes through recombinant DNA technology. Although this method has improved the activity, stability, and specificity of enzymes to a certain extent, its preparation process is very cumbersome and challenging, requiring in-depth understanding of the structure and function of enzymes, and the cost is relatively high. Therefore, it is urgent to develop enzyme-like preparations with simple preparation methods, low costs, and excellent performance.

[0003] The design of the new generation of enzyme-like preparations is no longer limited to the modification of macromolecular structures, but focuses on utilizing the progress of nanotechnology and materials science to develop nanozyme-mimicking materials with high catalytic activity and stability. Among them, single-atom nanozymes are considered to be one of the most promising nanozyme-mimicking materials. The active center of single-atom nanozymes is similar to that of natural enzymes and has a series of remarkable characteristics, including geometric, chemical, and electronic structures. As an important part of biocatalysts, single-atom nanozymes possess extraordinary activity and specificity, revealing new ways to understand the complex mechanisms of catalytic reactions and transcending the limitations of natural enzymes and nanozymes. Compared with traditional enzyme engineering, the preparation process of single-atom nanozymes is simpler, the cost is lower, and they also have excellent catalytic performance and stability.

[0004] Currently, various different support materials are used in the synthesis of single-atom nanozymes, such as carbon-based materials, metal oxides, MOF materials, and two-dimensional materials. In biomedical applications, carbon-based materials are one of the most widely studied carriers of single-atom nanozymes. Under the carbon-based material carrier, the interaction between metals and the carrier promotes the separation and transfer efficiency, and the coexistence of metal single atoms and nitrogen vacancies improves the catalytic efficiency, showing a synergistic catalytic effect. MOF materials have large cavities and can effectively capture metal single atoms. As a commonly used carrier material, the oxygen vacancy defects on the surface of metal oxides can serve as sites for stabilizing single-atom metals. Metal atoms loaded on two-dimensional materials can form banded structures with non-metal atoms, thereby regulating the efficiency of enzyme-catalyzed reactions. However, these synthesis methods have some disadvantages. For example, the loading amount of atoms is low, the synthesis process is cumbersome, and atoms are prone to aggregation into clusters. The problems of atomic aggregation and atomic loading still prevent the large-scale batch production of single-atom nanozymes.

[0005] Therefore, the development of single-atom nanozymes that can efficiently load metal atoms and can be mass-produced is of great significance for the further large-scale development and application of single-atom nanozymes in various fields of biomedicine, as well as for the further development of enzyme-mimicking preparations with higher catalytic activity. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for preparing a single-atom nanozyme using microbial cells as a template and its application. The technical solution of the present invention is as follows:

[0007] The present invention provides a method for preparing a single-atom nanozyme using microbial cells as a template, which includes:

[0008] S1, inoculating microbial cells into a culture medium for cultivation;

[0009] S2, after the microbial cells enter the growth plateau phase, collecting the microbial cells and adding a precooled calcium chloride solution to the microbial cells;

[0010] S3, adding metal ions and a volatile salt solution to the product obtained in S2, dispersing the metal ions inside and on the surface of the microbial cells, and at the same time allowing the volatile salt to penetrate into the cell interior, and then centrifuging to collect the bacterial cells;

[0011] S4, adding a fixing solution to the product obtained in S3 for fixation;

[0012] S5, adding a salt solution to the product obtained in S4, wrapping the microbial cells with the salt solution, then drying, and performing high-temperature calcination in an inert gas atmosphere;

[0013] S6, washing the excess salt solution in the product obtained in S5 and drying to obtain a single-atom nanozyme.

[0014] Optionally, the method for preparing a single-atom nanozyme using microbial cells as a template further includes S7:

[0015] Mixing the single-atom nanozyme with chloroauric acid and adding citric acid, and fully reacting to obtain a single-atom cascade nanozyme.

[0016] Optionally, the S1 includes:

[0017] Inoculating microbial cells into the culture medium with an inoculation loop, placing the culture medium in a shaker for cultivation, the temperature of the shaker being 37 °C and the rotation speed being 200 rpm.

[0018] Optionally, the precooled calcium chloride solution refers to a calcium chloride solution formed after cooling a calcium chloride solution on ice for more than 30 minutes; the concentration of the calcium chloride solution is 0.05 M - 1 M, preferably 0.1 M.

[0019] Optionally, the metal ions are metal ions directly soluble in water or metal ions contained in metal compounds, including V 3+ 、V 4+ 、V 5+ Cr 3+ , Mn 2+ , Mn 4+ , Mn 7+ , Fe 2+ , Fe 3+ 、Co 2+ 、Co 3+ 、Ni 2+ 、Ni 3+ , Cu 2+ 、Zr 2+ 、Zr 3+ 、Zr 4 + 、Mo 2+ 、Mo 4+ 、Mo 6+ 、Ru 2+ 、Ru 3+ , Rh 2+ , Rh 3+ , Rh 4+ , Pd 2+ , Pd 4+ , W 4+ , W 6+ 、Re 5+ 、Re 6+ 、Re 7+ , Ir 3+ , Ir 4+ , Pt 2 + , Pt 4+ 、Au 3+ 、Au 5+ 、Ce 3+ 、Ce 4+ , Gd 3+ , Tb 3+ and Tb 4+ At least one of, preferably Mn 2+ ;

[0020] The concentration of the metal ion is between 0.1 mM and 50 mM, preferably 0.5 mM;

[0021] The volatile salt solution is NH4HCO3 solution with a concentration of 0.1M-5M, preferably 0.5M.

[0022] Optionally, the salt solution is a mixture of volatile inorganic salts and non-volatile inorganic salts, preferably a mixture of NH4HCO3 solution and NaCl solution, and the volume ratio of the NH4HCO3 solution to the NaCl solution is 1:0.5 - 1:6, preferably 1:1; the concentration of the mixture of the NH4HCO3 solution and the NaCl solution is 0.1 M - 5 M, preferably 1 M; the fixing solution is 4% paraformaldehyde.

[0023] Optionally, the microbial cells are cells of bacteria, fungi or algae; the bacteria are Gram-positive bacteria, Gram-negative bacteria or Escherichia coli, preferably Escherichia coli.

[0024] Optionally, the drying in S5 is vacuum freeze-drying; the high-temperature calcination temperature is 500 °C - 1000 °C, preferably 800 °C; the high-temperature calcination time is 0.5 h - 4 h, preferably 2 h.

[0025] Optionally, the concentration of chloroauric acid is 0.2 mM - 10 mM, preferably 1 mM, and the volume is 5 ml - 50 ml;

[0026] The concentration of citric acid is 10 mM - 100 mM, preferably 45 mM, and the volume is 1 ml - 10 ml.

[0027] The present invention also provides an application of a single-atom nanozyme using microbial cells as a template, and the single-atom nanozyme using microbial cells as a template is used for preparing an enzyme-like preparation;

[0028] The enzyme-like preparation is an oxidase-like preparation, a peroxidase-like preparation, a superoxide dismutase-like preparation or a haloperoxidase-like preparation;

[0029] The enzyme-like preparation is an antibacterial agent, an anti-tumor drug, a wastewater treatment agent, a tissue repair drug or a detection reagent.

[0030] All of the above optional technical solutions can be arbitrarily combined, and the present invention does not elaborate on the structures after the combination one by one.

[0031] By means of the above solutions, the beneficial effects of the present invention are as follows:

[0032] Through performance testing, it can be obtained that the single-atom nanozyme prepared by the preparation method provided by the embodiments of the present invention has high peroxidase-like activity, and at the same time has excellent photothermal performance, can catalyze the generation of a large amount of ROS, thereby killing bacteria, and has an obvious antibacterial effect. At the same time, since microbial cells are easy to obtain and the large-scale production method is mature, it is beneficial to the large-scale batch production of single-atom nanozymes.

[0033] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and be implemented in accordance with the content of the specification, the following describes in detail with reference to the preferred embodiments of the present invention and the accompanying drawings. Brief Description of the Drawings

[0034] Figure 1 It is a flow chart of a method for preparing a single-atom nanozyme using a microbial cell as a template provided by an embodiment of the present invention;

[0035] Figure 2 It is a sample diagram of the single-atom nanozyme prepared in Example 1 of the present invention;

[0036] Figure 3 It is a Zeta potential diagram of the single-atom nanozyme prepared in Example 1 of the present invention;

[0037] Figure 4 It is a particle size diagram of the single-atom nanozyme prepared in Example 1 of the present invention;

[0038] Figure 5 It is a schematic diagram of the peroxidase activity measurement result of the single-atom nanozyme prepared in Example 1 of the present invention;

[0039] Figure 6 It is a schematic diagram of the oxidase activity measurement result of the single-atom nanozyme prepared in Example 1 of the present invention;

[0040] Figure 7 It is a schematic diagram of the catalase activity measurement result of the single-atom nanozyme prepared in Example 1 of the present invention;

[0041] Figure 8 It is a schematic diagram of the cascade activity measurement result of the single-atom cascade nanozyme prepared in Example 1 of the present invention;

[0042] Figure 9 It is a schematic diagram of the photothermal performance measurement results of the single-atom nanozyme and the single-atom nano-cascade enzyme prepared in Example 1 of the present invention;

[0043] Figure 10 It is the antibacterial activity diagram of the single-atom nanozyme prepared in Example 1 of the present invention. Detailed Description of the Embodiments

[0044] The following further describes in detail the specific embodiments of the present invention with reference to the drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0045] As Figure 1 shown, an embodiment of the present invention provides a method for preparing a single-atom nanozyme using a microbial cell as a template, which includes:

[0046] S1, inoculate microbial cells into a culture medium and culture.

[0047] In a specific embodiment, the S1 includes: inoculating microbial cells into a culture medium with an inoculation loop, and placing the culture medium in a shaker for culturing. The temperature of the shaker is 25 °C - 37 °C, preferably 37 °C, and the rotation speed is 150 rpm - 2500 rpm, preferably 200 rpm.

[0048] In a specific embodiment, the microbial cells are cells such as bacteria, fungi or algae; the bacteria are Gram-positive bacteria, Gram-negative bacteria or Escherichia coli, preferably Escherichia coli.

[0049] In the embodiments of the present invention, the microbial cells serve as an atomically dispersed template.

[0050] S2, after the microbial cells enter the growth plateau phase, collect the microbial cells, and add a pre-cooled calcium chloride solution to the microbial cells to make the microbial cells form competent cells, which is convenient for absorbing external substances.

[0051] Among them, when determining whether to enter the growth plateau phase, it is achieved by the OD value of the bacterial solution. When the OD value of the bacterial solution is 0.5 - 1.0, it is determined that the microbial cells enter the growth plateau phase.

[0052] The pre-cooled calcium chloride solution refers to the calcium chloride solution formed after cooling the calcium chloride solution on ice for more than 30 minutes; the concentration of the calcium chloride solution is 0.05 M - 1 M, preferably 0.1 M.

[0053] S3, add metal ions and a volatile salt solution to the product obtained in S2, so that the metal ions are dispersed inside and on the surface of the microbial cells, and at the same time, the volatile salt penetrates into the cells, and then centrifuge to collect the thalli.

[0054] In a specific embodiment, the metal ions are metal ions directly dissolved in water or metal ions contained in metal compounds, including V 3+ 、V 4+ 、V 5+ 、Cr 3+ 、Mn 2+ 、Mn 4+ 、Mn 7+ 、Fe 2+ 、Fe 3+ 、Co 2+ 、Co 3+ 、Ni 2+ 、Ni 3+ 、Cu 2+ 、Zr 2+ 、Zr 3+ 、Zr 4+ 、Mo2+ 、Mo 4+ 、Mo 6+ 、Ru 2+ 、Ru 3+ 、Rh 2+ 、Rh 3+ 、Rh 4+ 、Pd 2+ 、Pd 4+ 、W 4+ 、W 6+ 、Re 5+ 、Re 6+ 、Re 7+ 、Ir 3+ 、Ir 4+ 、Pt 2+ 、Pt 4+ 、Au 3+ 、Au 5+ 、Ce 3+ 、Ce 4+ 、Gd 3+ 、Tb 3+ and Tb 4+ and at least one of them, preferably Mn 2+ ; the concentration of the metal ions is between 0.1 mM and 50 mM, preferably 0.5 mM.

[0055] The volatile salt solution is a salt compound that is commonly volatile at high temperatures, preferably an NH4HCO3 solution, with a concentration of 0.1 M - 5 M, preferably 0.5 M.

[0056] In order to ensure that the metal ions can be uniformly dispersed inside and on the surface of the microbial cells, in the embodiments of the present invention, after adding the metal ions and the volatile salt solution to the microbial cells, they are incubated for 0.5 h - 5 h.

[0057] Specifically, the process of centrifuging and collecting the bacterial cells is as follows: centrifuging the bacterial solution, discarding the supernatant to obtain the bacterial cells; preferably, the centrifugation speed is 5000 rmp - 10000 rmp, preferably 6000 rmp, and the centrifugation time is 5 min - 30 min, preferably 10 min.

[0058] S4. Adding a fixing solution to the product obtained in S3 for fixation.

[0059] In a specific embodiment, the fixing solution is 4% paraformaldehyde.

[0060] Among them, the volume ratio of the fixing solution to the bacterial cells is 1:1 - 1:10, and the fixing time is 10 min - 30 min.

[0061] Further, after fixation, it also includes centrifuging to remove the excess fixing solution.

[0062] S5. Add a salt solution to the product obtained in S4. After the salt solution wraps the microbial cells, perform drying and then high-temperature calcination in an inert gas atmosphere.

[0063] Among them, the purpose of drying is to ensure that the salt solution can wrap the microbial cells.

[0064] The inert gas atmosphere is nitrogen, argon, etc., and preferably argon.

[0065] In another specific embodiment, the salt solution is a mixture of a volatile inorganic salt and a non-volatile inorganic salt, preferably a mixture of an NH4HCO3 solution and an NaCl solution, and the volume ratio of the NH4HCO3 solution to the NaCl solution is 1:0.5 - 1:6, preferably 1:1; the concentration of the mixture of the NH4HCO3 solution and the NaCl solution is 0.1 M - 5 M, preferably 0.5 M. During the subsequent carbonization process of high-temperature calcination, the volatilization of the volatile inorganic salt is beneficial to forming a porous and fluffy structure of the material, which is more conducive to the exertion of the enzyme-like activity.

[0066] In a specific embodiment, the drying in S5 is vacuum freeze-drying, and the temperature of vacuum freeze-drying is -70 °C - 80 °C; the high-temperature calcination temperature is 500 °C - 1000 °C, preferably 800 °C; the high-temperature calcination time is 0.5 h - 4 h, preferably 2 h.

[0067] S6. Wash the excess salt solution in the product obtained in S5 and dry it to obtain a single-atom nanozyme.

[0068] During specific washing, perform ultrasonic fragmentation for 15 min - 120 min. The temperature during drying in this step S6 is 20 °C - 80 °C.

[0069] Furthermore, after obtaining the single-atom nanozyme, the embodiment of the present invention may further include S7: Mix the single-atom nanozyme with chloroauric acid (HAuCl4), and add citric acid. After fully reacting, obtain a single-atom cascade nanozyme. In a specific embodiment, the concentration of the chloroauric acid is 0.2 mM - 10 mM, preferably 1 mM, and the volume is 5 ml - 50 ml; the concentration of the citric acid is 10 mM - 100 mM, preferably 45 mM, and the volume is 1 ml - 10 ml; the reaction time is 30 min - 12 h, and the reaction temperature is 37 °C - 160 °C.

[0070] Through step S7, the cascade of single-atom nanozyme and gold nanoparticles Au NPs can be realized to grow Au NPs on the single-atom nanozyme. Au NPs have glucose oxidase activity and can form a cascade reaction with the peroxidase of the single-atom nanozyme. In the process of the cascade reaction, glucose is oxidized by Au NPs to produce H2O2. The single-atom nanozyme uses the produced H2O2 for peroxidase activity and generates hydroxyl radicals, which can kill bacteria or tumor cells, or can be used for the detection of bacteria or tumor cells, etc.

[0071] To facilitate the description of the performance of the single-atom nanozyme prepared by the embodiments of the present invention, a specific embodiment is provided below. Example

[0072] The preparation method of the single-atom nanozyme using microbial cells as a template provided by the embodiments of the present invention includes the following steps:

[0073] Inoculate Escherichia coli strains into LB medium, place the medium in a shaker, and culture in the shaker for 8 - 10 hours at a culture temperature of 37 °C and a rotation speed of 200 rmp; after the OD value of the bacterial liquid reaches 0.6 - 1.0 and enters the growth plateau phase, collect the microbial cells, add pre-cooled 0.1 M CaCl2 solution to resuspend the cells, add 0.5 mM MnCl2 solution and 0.5 M NH4HCO3 solution with a final concentration, incubate for 5 min - 30 min, and then centrifuge to collect the bacterial cells; add 4% paraformaldehyde to the bacterial cells according to the volume ratio of the fixative to the bacterial cells of 1:1, mix well and fix at 4 °C, centrifuge to discard the excess paraformaldehyde; add 0.5 M NaCl solution and 0.5 M NH4HCO3 solution, mix well and place in a -80 °C refrigerator overnight, freeze-dry the frozen bacterial liquid to obtain a white powder, place it in an argon atmosphere, calcine at 800 °C for 2 h, and perform natural annealing; wash away the excess NaCl solution, place the washed product in a vacuum drying oven, and dry at 40 °C to obtain a black powder, which is the single-atom nanozyme using microbial cells as a template; mix the single-atom nanozyme with 20 mL of 1 mM chloroauric acid (HAuCl4), add 1 mL of 40 mM citric acid, react under light protection at 37 °C - 160 °C for 30 min - 12 h, after sufficient reaction, the solution color changes from dark yellow to dark red, and the single-atom cascade nanozyme is obtained.

[0074] As Figure 2 shown in Figure a and Figure b in Figure 2 In Example 1, the product after freeze-drying is in the form of a white powder; as

[0075] As shown Figure 3 in the figure, it is the Zeta potential diagram of the single-atom nanozyme prepared in Example 1. Figure 3 The Zeta potential results in Figure 3 show that the potential of the bacteria themselves is about -20 mV. When metal ions are adsorbed and carbonized, there are obvious changes in the potential, indicating that the metal ions are dispersed inside and on the surface of the bacteria.

[0076] As shown Figure 4 in the figure, it is the particle size diagram of the single-atom nanozyme prepared in Example 1. It can be seen Figure 4 from Figure 4 that the particle size of the single-atom nanozyme is about 1 μm, with a uniform distribution, and subsequent research can be carried out.

[0077] Furthermore, the embodiments of the present invention also measured the activity of the single-atom nanozyme prepared in Example 1, specifically:

[0078] 1. Determination of oxidase activity, including:

[0079] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to a cuvette. Then add the single-atom nanozyme prepared in Example 1 with a final concentration of 10 μg / mL. After mixing evenly, place it in a UV-Vis-NIR spectrophotometer to scan the baseline (330 - 1000 nm). Then add 20 μL of TMB (50 mM), quickly mix well, and measure the absorption curve every five minutes for 30 min.

[0080] 2. Determination of peroxidase activity, including:

[0081] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to a cuvette. Then add the single-atom nanozyme prepared in Example 1 with a final concentration of 10 μg / mL. After mixing evenly, place it in a UV-Vis-NIR spectrophotometer to scan the baseline (330 - 1000 nm). Then add 1 μL of H2O2 (10 M) and 20 μL of TMB (50 mM), quickly mix well, and measure the absorption curve every five minutes for 30 min.

[0082] 3. Determination of catalase activity, including:

[0083] Take 2 mL of phosphate buffer solution (0.1 M, pH = 8.0) and add it to a cuvette. Then add the single-atom nanozyme prepared in Example 1 with a final concentration of 10 μg / mL. After mixing evenly, measure the oxygen concentration in the solution with an oxygen meter. Then add 1 μL of H2O2 (10 M), quickly mix well, and measure the oxygen concentration every five minutes for 30 min.

[0084] As shown Figures 5-7As shown, they are respectively the schematic diagrams of the peroxidase activity assay, oxidase activity assay, and catalase activity assay of the single-atom nanozyme prepared in Example 1. Figure 6 Figures a and b in Figures 5 to 7 show the absorbance values measured after the single-atom nanozyme oxidizes TMB, and the schematic diagram of the comparison between the single-atom nanozyme after oxidizing TMB and the control group. It can be seen that

[0085] the single-atom nanozyme prepared in Example 1 has excellent peroxidase-like activity, oxidase-like activity, and catalase-like activity.

[0086] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to a cuvette. Then add a certain amount of the single-atom cascade nanozyme prepared in Example 1. Next, add 20 μL of glucose (1 M) and 20 μL of TMB (50 mM). After rapid mixing, incubate at 37 °C for 30 min. After the incubation ends, place it in a UV-Vis-NIR spectrophotometer to measure the absorbance value of the solution at a wavelength of 652 nm. As Figure 8 shown, it is the schematic diagram of the cascade activity assay of the single-atom cascade nanozyme prepared in Example 1 of the present invention. It can be seen from Figure 8 this that the single-atom cascade nanozyme has good cascade performance, can catalyze the decomposition of glucose, and further oxidize TMB to achieve multi-enzyme activity cascade.

[0087] Furthermore, the present invention's examples also tested the photothermal properties of the single-atom nanozyme and the single-atom cascade nanozyme prepared in Example 1, including: using a thermometer to measure the temperature changes of the control group, the single-atom nanozyme group, and the single-atom cascade nanozyme group respectively after being irradiated with an 808 laser (2 W / cm 2 , 6 min). The measurement results are as Figure 9 shown. It can be seen from Figure 9 this that the temperature of the single-atom nanozyme and the single-atom cascade nanozyme can reach about 45 °C within one minute after laser irradiation, showing good photothermal properties.

[0088] In addition, the present invention's examples also conducted an antibacterial activity test on the single-atom nanozyme prepared in Example 1, specifically using Escherichia coli for the antibacterial test. The steps are as follows: First, inoculate the bacterial strain into a standard LB medium and culture it until OD = about 1.0. Incubate the bacterial solution with sodium acetate buffer, single-atom nanozyme, 808 laser, and single-atom nanozyme + 808 laser respectively, and plate to calculate the antibacterial efficiency. The results are as Figure 10 shown. It can be seen from Figure 10It can be seen that the single-atom nanozyme can effectively kill Escherichia coli under 808 laser irradiation and is an efficient antibacterial material.

[0089] It should be noted that in the above content of the embodiments of the present invention, statistical analysis was performed using Graphpad 10.1.1 software for data analysis. The independent sample T-test was used for comparison between two groups, and one-way analysis of variance was used for comparison among multiple groups. P < 0.05 was considered statistically significant. The control group in the above embodiments used phosphate buffer solution (0.1 M, pH = 8.0).

[0090] Combined with the above performance test results, it can be known that the single-atom nanozyme and single-atom nano-cascade enzyme prepared with microbial cells as templates by the embodiments of the present invention can be used in the preparation of enzyme-like preparations; the enzyme-like preparations can be oxidase-like preparations, peroxidase-like preparations, superoxide dismutase-like preparations or haloperoxidase-like preparations, and can be used to prepare enzyme-mimicking preparations with high catalytic activity; combined with their photothermal properties, the enzyme-like preparations can be antibacterial agents, anti-tumor drugs, wastewater treatment agents, tissue repair drugs or detection reagents, and have broad application prospects in the fields of antibacterial, anti-tumor, wastewater treatment, tissue repair or detection.

[0091] In summary, the preparation method of the single-atom nanozyme with microbial cells as templates provided by the embodiments of the present invention creatively uses microbial cells as carbonization templates to prepare single-atom nanozymes. The entire preparation process is simple and easy to perform, and has high repeatability. Microbial cells such as Escherichia coli can be used after cultivation. After large-scale application, the size and shape of the templates can be maintained in a uniform state, which is very conducive to large-scale development and preparation, and solves the problem that most of the current preparation methods require the prior preparation of carriers suitable for loading, and these carriers are only suitable for small-scale preparation, the preparation method is cumbersome, and it is extremely easy for atoms to agglomerate. In addition, in terms of metal loading, the embodiments of the present invention can prepare different types of single-atom nanozymes by changing the types of metal ions added. Metals are the active centers of single-atom nanozymes, and the activities of single-atom nanozymes prepared with different types of metal ions will also change. Therefore, the activity of the target single-atom nanozyme can be easily regulated by controlling the types of metal ions added, or adding one or more types, or controlling the loading amount of metal ions. Different functions and uses can be realized by regulating the activities of different single-atom nanozymes.

[0092] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a single-atom nanozyme using a microbial cell as a template, characterized in that, Comprising: S1, inoculating microbial cells into a culture medium for cultivation; the microbial cells are cells of bacteria, fungi or algae; S2, after the microbial cells enter the growth plateau phase, collecting the microbial cells and adding a pre-cooled calcium chloride solution to the microbial cells; S3, adding metal ions and volatile salt solution to the product obtained in S2, so that the metal ions are dispersed in and on the surface of the microbial cells, and the volatile salts are infiltrated into the cells, and then the cells are collected by centrifugation, wherein the volatile salt solution is NH4HCO3 solution; the metal ions are metal ions directly dissolved in water or metal ions contained in metal compounds, including V 3+ 、V 4 + 、V 5+ Cr 3+ , Mn 2+ , Mn 4+ , Mn 7+ , Fe 2+ , Fe 3+ 、Co 2+ 、Co 3+ 、Ni 2+ 、Ni 3+ , Cu 2+ 、Zr 2+ 、Zr 3+ 、Zr 4+ 、Mo 2+ 、Mo 4+ 、Mo 6 + 、Ru 2+ 、Ru 3+ , Rh 2+ , Rh 3+ , Rh 4+ , Pd 2+ , Pd 4+ , W 4+ , W 6+ 、Re 5+ 、Re 6+ 、Re 7+ , Ir 3+ , Ir 4+ , Pt 2+ , Pt 4+ 、Au 3+ 、Au 5 + 、Ce 3+ 、Ce 4+ , Gd 3+ , Tb 3+ and Tb 4+ At least one of; S4, adding a fixing solution to the product obtained in S3 for fixation; S5, adding a salt solution to the product obtained in S4, allowing the salt solution to wrap the microbial cells, then drying and performing high-temperature calcination in an inert gas atmosphere; the salt solution is a mixture of a volatile inorganic salt and a non-volatile inorganic salt, the volatile inorganic salt is an NH4HCO3 solution, and the non-volatile inorganic salt is an NaCl solution; the high-temperature calcination temperature is 500 °C - 1000 °C; S6, washing and drying the excess salt solution in the product obtained in S5 to obtain a single-atom nanozyme.

2. The preparation method of the single-atom nanozyme using the microbial cell according to claim 1, wherein Also including S7: Mixing the single-atom nanozyme with chloroauric acid and adding citric acid, and fully reacting to obtain a single-atom cascade nanozyme.

3. The method for preparing a single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that, The S1 includes: Inoculating microbial cells into the culture medium with an inoculation loop, and placing the culture medium in a shaker for cultivation, the shaker temperature is 37 °C and the rotation speed is 200 rpm.

4. The method for preparing a single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that The pre-cooled calcium chloride solution refers to a calcium chloride solution formed after cooling a calcium chloride solution on ice for more than 30 minutes; the concentration of the calcium chloride solution is 0.05 M - 1 M.

5. The preparation method of the single-atom nanozyme using the microbial cell according to claim 4, wherein, The concentration of the calcium chloride solution is 0.1 M.

6. The preparation method of the single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that The metal ion is Mn 2+ ; The concentration of the metal ions is between 0.1 mM - 50 mM; The concentration of the NH4HCO3 solution is 0.1 M - 5 M.

7. The preparation method of the single-atom nanozyme using the microbial cell according to claim 6, characterized in that, The concentration of the metal ions is 0.5 mM; the concentration of the NH4HCO3 solution is 0.5 M.

8. The preparation method of the single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that, The volume ratio of the NH4HCO3 solution to the NaCl solution is 1:0.5 - 1:6; the concentration of the mixture of the NH4HCO3 solution and the NaCl solution is 0.1 M - 5 M; the fixing solution is 4% paraformaldehyde.

9. The method for preparing a single-atom nanozyme using the microbial cell according to claim 8, characterized in that, The volume ratio of the NH4HCO3 solution to the NaCl solution is 1:1; the concentration of the mixture of the NH4HCO3 solution and the NaCl solution is 1 M.

10. The method for preparing a single-atom nanozyme using the microbial cell according to claim 1 or 2 as a template, characterized in that, The bacteria are Gram-positive bacteria, Gram-negative bacteria or Escherichia coli.

11. The preparation method of the single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that, The bacteria are Escherichia coli.

12. The preparation method of the single-atom nanozyme using the microbial cell according to claim 1 or 2, characterized in that The drying in S5 is vacuum freeze-drying; the high-temperature calcination temperature is 800 °C; the high-temperature calcination time is 0.5 h - 4 h.

13. The preparation method of the single-atom nanozyme using the microbial cell according to claim 12, characterized in that, The high-temperature calcination time is 2 h.

14. The preparation method of the single-atom nanozyme using the microbial cell according to claim 2, characterized in that, The concentration of the chloroauric acid is 0.2 mM - 10 mM, and the volume is 5 ml - 50 ml; The concentration of the citric acid is 10 mM - 100 mM, and the volume is 1 ml - 10 ml.

15. The preparation method of the single-atom nanozyme using the microbial cell according to claim 14, characterized in that, The concentration of the chloroauric acid is 1 mM; the concentration of the citric acid is 45 mM.

16. Use of the single-atom nanozyme prepared by the method for preparing a single-atom nanozyme using the microbial cell according to any one of claims 1 to 15 as a template, characterized in that, The single-atom nanozyme using the microbial cells as a template is used in the preparation of an enzyme-like preparation; The enzyme-like preparation is an oxidase-like preparation, a peroxidase-like preparation, a superoxide dismutase-like preparation or a haloperoxidase-like preparation; The enzyme-like preparation is an antibacterial agent, an antitumor drug, a wastewater treatment agent, a tissue repair drug or a detection reagent.

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