Preparation method and application of monatomic nano-enzyme taking microbial cells as template

Abstract

The invention relates to a preparation method and application of monatomic nano-enzyme taking microbial cells as a template, and belongs to the technical field of bionic nano-catalyst synthesis. Comprising the following steps: S1, inoculating microbial cells into a culture medium for culture; s2, after the microbial cells enter a growth plateau stage, adding a pre-cooled calcium chloride solution into the microbial cells; s3, adding metal ions and a volatile salt solution into a product obtained in S2; s4, adding a stationary liquid into a product obtained in S3 for immobilization; s5, adding a salt solution into a product obtained in S4, drying, and calcining at high temperature in an inert gas atmosphere; and S6, cleaning the redundant salt solution in the product obtained in the step S5, and drying to obtain the monatomic nano-enzyme. The monatomic nano-enzyme prepared by the method has higher peroxidase-like activity and excellent photo-thermal performance, can catalyze generation of a large amount of ROS (reactive oxygen species) so as to kill 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 bionic nanocatalyst synthesis, and in particular to a preparation method of a single-atom nanozyme using microbial cells as a template and an application thereof. Background Art

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

[0003] The design of a new generation of enzyme-like preparations is no longer limited to the modification of macromolecular structures, but focuses on using the progress of nanotechnology and materials science to develop nanoenzyme-like materials with high catalytic activity and stability. Among them, single-atom nanozymes are considered to be the most promising type of nanoenzyme-like materials. The active center of single-atom nanozymes is similar to that of natural enzymes, and has a series of remarkable properties, including geometric, chemical and electronic structures. As an important component of biocatalysts, single-atom nanozymes have extraordinary activity and specificity, revealing new pathways for the complex mechanisms of catalytic reactions, surpassing the limitations of natural enzymes and nanozymes. Compared with traditional enzyme engineering, the preparation process of single-atom nanozymes is simpler and less expensive, while also having excellent catalytic performance and stability.

[0004] At present, the synthesis of single-atom nanozymes utilizes a variety of different supporting materials, 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 single-atom nanozyme carriers. Under the carbon-based material carrier, the interaction between the metal 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. Metal oxides are commonly used carrier materials, and the oxygen vacancy defects on their surfaces can serve as sites for stabilizing single-atom metals. The metal atoms loaded on two-dimensional materials can form a band structure with non-metal atoms, thereby regulating the efficiency of enzyme catalytic reactions. However, these synthesis methods have some disadvantages, such as low load atomic weight, cumbersome synthesis process, and atoms are easy to aggregate into clusters. The problems of atomic aggregation and atomic loading have resulted in the inability to mass-produce single-atom nanozymes.

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

[0006] In order 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 comprises:

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

[0009] S2, after the microbial cells enter the growth plateau phase, the microbial cells are collected, and a pre-cooled calcium chloride solution is added to the microbial cells;

[0010] 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 penetrate into the cells, and then centrifuging to collect the cells;

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

[0012] S5, adding a salt solution to the product obtained in S4, so that the salt solution wraps the microbial cells, drying, and calcining at a high temperature in an inert gas atmosphere;

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

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

[0015] The single-atom nanozyme is mixed with chloroauric acid and citric acid is added. After sufficient reaction, the single-atom cascade nanozyme is obtained.

[0016] Optionally, the S1 includes:

[0017] The microbial cells were inoculated into the culture medium using an inoculation loop, and the culture medium was placed in a shaker for culture at a temperature of 37° C. and a rotation speed of 200 rpm.

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

[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 0.1mM and 50mM, preferably 0.5mM;

[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 NH4HCO3 solution to NaCl solution is 1:0.5-1:6, preferably 1:1; the concentration of the mixture of NH4HCO3 solution and NaCl solution is 0.1M-5M, preferably 1M; 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; and the high-temperature calcination time is 0.5h-4h, preferably 2h.

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

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

[0027] The present invention also provides an application of a single-atom nanozyme using a microbial cell as a template, wherein the single-atom nanozyme using a microbial cell as a template is used to prepare an enzyme-like preparation;

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

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

[0030] All the above optional technical solutions can be combined arbitrarily, and the present invention does not provide detailed descriptions of the structures after the combinations.

[0031] By means of the above scheme, 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 embodiment of the present invention has a high peroxidase-like activity and excellent photothermal performance, which can catalyze the production of a large amount of ROS, thereby killing bacteria and having a significant antibacterial effect. At the same time, since microbial cells are easy to obtain and the expansion production method is mature, it is conducive 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 more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

[0042] Fig. 9 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] Fig.10 This is the antibacterial activity diagram of the single-atom nanozyme prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

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

[0046] S1, inoculate microbial cells into culture medium for cultivation.

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

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

[0049] In an embodiment of the present invention, microbial cells are used as atomic dispersion templates.

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

[0051] When determining whether the microorganisms have entered the growth platform phase, the OD value of the bacterial solution is used. When the OD value of the bacterial solution is 0.5-1.0, it is determined that the microbial cells have entered the growth platform phase.

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

[0053] 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 penetrate into the cells, and then the bacteria are collected by centrifugation.

[0054] In a specific embodiment, the metal ion is a metal ion directly soluble in water or a metal ion contained in a metal compound, 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+ ; The concentration of the metal ion is between 0.1mM-50mM, preferably 0.5mM.

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

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

[0057] Specifically, the process of collecting bacteria by centrifugation is: centrifuging the bacterial solution, discarding the supernatant, and obtaining the bacteria; preferably, the centrifugal speed is 5000rmp-10000rmp, preferably 6000rmp, and the centrifugal time is 5min-30min, preferably 10min.

[0058] S4, adding a fixing solution to the product obtained in S3 for fixing.

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

[0060] The volume ratio of the fixative to the bacteria is 1:1-1:10, and the fixation time is 10 min-30 min.

[0061] Furthermore, after fixation, centrifugation is also performed to remove excess fixative.

[0062] S5, adding a salt solution to the product obtained in S4, so that the salt solution wraps the microbial cells, drying, and calcining at a high temperature 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 or argon, etc., 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 a 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 0.1M-5M, preferably 0.5M. In the subsequent high-temperature calcination carbonization process, the volatilization of the volatile inorganic salt is conducive to the formation of a porous fluffy structure of the material, which is more conducive to the performance of 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 0.5h-4h, preferably 2h.

[0067] S6, washing the excess salt solution in the product obtained in S5 and drying it to obtain the single-atom nanozyme.

[0068] Specifically, the washing is carried out by ultrasonication for 15-120 minutes. The drying temperature in step S6 is 20-80°C.

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

[0070] Through step S7, the single-atom nanozyme and the gold nanoparticles Au NPs can be cascaded 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 AuNPs to produce H2O2. The single-atom nanozyme uses the produced H2O2 to perform peroxidase activity, which will generate hydroxyl radicals, thereby killing bacteria or tumor cells, or can be used for the detection of bacteria or tumor cells.

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

[0072] Embodiment 1:

[0073] The method for preparing a single-atom nanozyme using microbial cells as a template provided in an embodiment of the present invention comprises the following steps:

[0074] Inoculate Escherichia coli into LB medium, place the medium in a shaker, and culture it in the shaker for 8-10 hours at a culture temperature of 37°C and a rotation speed of 200 rpm; after the OD value of the bacterial solution reaches 0.6-1.0 and enters the growth platform phase, collect the microbial cells, add pre-cooled 0.1M CaCl2 solution to the microbial cells to resuspend the cells, add a final concentration of 0.5mM MnCl2 solution and 0.5M NH4HCO3 solution, incubate for 5min-30min, and collect the cells by centrifugation; add 4% paraformaldehyde to the cells at a volume ratio of 1:1 between the fixative and the cells, mix well, and fix at 4°C, and discard the excess paraformaldehyde by centrifugation; add 0.5M NaCl solution and 0.5M NH4HCO3 solution, mix well and place in a -80℃ refrigerator overnight, vacuum freeze-dry the frozen bacterial solution to obtain a white powder, place it in an argon atmosphere, calcine at 800℃ for 2h, and anneal naturally; wash to remove excess NaCl solution, place the washed product in a vacuum drying oven, and dry it at 40℃ to obtain a black powder, which is the single-atom nanozyme with microbial cells as a template; mix the single-atom nanozyme with 20mL 1mM chloroauric acid (HAuCl4), and add 1mL 40mM citric acid, protect from light and react at 37℃-160℃ for 30min-12h. After sufficient reaction, the color of the solution changes from dark yellow to dark red, and the single-atom cascade nanozyme is obtained.

[0075] like Figure 2 As shown in Figures a and b in Example 1, the product after freeze-drying is a white powder; Figure 2 As shown in Figures a and c, after high-temperature calcination and carbonization, the product becomes a black powder.

[0076] like Figure 3 As shown, it is the Zeta potential diagram of the single-atom nanozyme prepared in Example 1. Figure 3 The Zeta potential results show that the potential of the bacteria itself is about -20mV. When metal ions are added for adsorption and carbonization, the potential changes significantly, indicating that the metal ions are dispersed in the bacteria and on the surface.

[0077] like Figure 4 As shown, it is a particle size diagram of the single-atom nanozyme prepared in Example 1. Figure 4 It can be obtained that the particle size of single-atom nanozymes is about 1 μm and is evenly distributed, which can be used for subsequent research.

[0078] Furthermore, the present invention also carried out an activity test on the single-atom nanozyme prepared in Example 1, specifically:

[0079] 1. Oxidase activity assay, including:

[0080] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to the cuvette, then add the single-atom nanozyme prepared in Example 1 at a final concentration of 10 μg / mL, mix well and put it into the UV-visible near-infrared spectrophotometer to scan the baseline (330-1000 nm), then add 20 μL of TMB (50 mM), mix quickly and measure the absorption curve every five minutes for 30 minutes.

[0081] 2. Peroxidase activity assay, including:

[0082] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to the cuvette, then add the single-atom nanozyme prepared in Example 1 at a final concentration of 10 μg / mL, mix well and put it into the UV-visible near-infrared spectrophotometer to scan the baseline (330-1000 nm), then add 1 μL of H2O2 (10 M) and 20 μL of TMB (50 mM), mix quickly and measure the absorption curve every five minutes for 30 minutes.

[0083] 3. Catalase activity assay, including:

[0084] Take 2 mL of phosphate buffer solution (0.1 M, pH = 8.0) and add it to the cuvette, then add the single-atom nanozyme prepared in Example 1 at a final concentration of 10 μg / mL, mix well and measure the oxygen concentration in the solution with a dissolved oxygen meter, then add 1 μL of H2O2 (10 M), mix quickly and measure the oxygen concentration every five minutes for 30 minutes.

[0085] like Figure 5-7As shown, they are respectively a schematic diagram of the peroxidase activity measurement result, a schematic diagram of the oxidase activity measurement result and a schematic diagram of the catalase activity measurement result of the single-atom nanozyme prepared in Example 1, Figure 6 Figures a and b in the figure are the absorbance values ​​measured after single-atom nanozymes oxidized TMB, and the schematic diagram of the comparison between single-atom nanozymes oxidized TMB and the control group. Figures 5 to 7 It can be obtained that the single-atom nanozyme prepared in Example 1 has excellent peroxidase-like activity, oxidase-like activity and catalase-like activity.

[0086] In addition, the present invention also conducted a performance test on the single-atom cascade nanozyme prepared in Example 1, specifically including:

[0087] Take 2 mL of acetate buffer solution (0.1 M, pH = 4.0) and add it to the cuvette, then add a certain amount of the single-atom cascade nanozyme prepared in Example 1, then add 20 μL of glucose (1 M) and 20 μL of TMB (50 mM), quickly mix well and incubate at 37°C for 30 min. After the incubation, put it into the UV-visible near-infrared spectrophotometer to measure the solution absorption value at a wavelength of 652 nm. Figure 8 As shown, it is a schematic diagram of the cascade activity measurement results of the single-atom cascade nanozyme prepared in Example 1 of the present invention. Figure 8 It can be seen that the single-atom cascade nanozyme has good cascade performance, can catalyze the decomposition of glucose, and further oxidize TMB to achieve a multi-enzyme activity cascade.

[0088] Furthermore, the present invention also tests the photothermal performance of the single-atom nanozyme and the single-atom cascade nanozyme prepared in Example 1, including: using a thermometer to measure the photothermal performance of the single-atom nanozyme and the single-atom cascade nanozyme after 808 laser irradiation (2W / cm 2 , 6min), the temperature changes of the control group, the single-atom nanozyme group and the single-atom cascade nanozyme group are shown in the following measurement results: Fig. 9 As shown. Fig. 9 It can be found that the temperature of single-atom nanozymes and single-atom cascade nanozymes can reach about 45°C within one minute after laser irradiation, and they have good photothermal properties.

[0089] In addition, the present invention also tested the antibacterial activity of the single-atom nanozyme prepared in Example 1, specifically using Escherichia coli for antibacterial testing. The steps are as follows: First, inoculate the bacteria into standard LB medium and culture until OD=1.0. The bacterial solution is incubated with sodium acetate buffer, single-atom nanozyme, 808 laser, and single-atom nanozyme + 808 laser, and the antibacterial efficiency is calculated by coating the plate. The results are as follows: Fig.10 As shown. Fig.10It can be seen that single-atom nanozyme can effectively kill Escherichia coli under 808 laser irradiation and is a highly effective bactericidal material.

[0090] It should be noted that in the above content of the present invention, the statistical analysis was performed using Graphpad10.1.1 software for data analysis, the comparison between two groups was performed using the independent sample T test, and the comparison between multiple groups was performed using one-way analysis of variance. P<0.05 was considered statistically significant. The control group in the above embodiment used phosphate buffer solution (0.1M, pH=8.0)

[0091] Combined with the above performance test results, it can be seen that the single-atom nanozymes and single-atom nanocascade enzymes using microbial cells as templates prepared by the embodiments of the present invention can be used to prepare enzyme-like preparations; the enzyme-like preparations can be oxidase-like preparations, peroxidase-like preparations, peroxidase-like preparations, superoxide dismutase-like preparations or haloperoxidase-like preparations, which can be used to prepare enzyme-like preparations with high catalytic activity; combined with their photothermal properties, the enzyme-like preparations can be antibacterial agents, antitumor drugs, wastewater treatment agents, tissue repair drugs or detection reagents, and have broad application prospects in the fields of antibacterial, antitumor, wastewater treatment, tissue repair or detection.

[0092] In summary, the preparation method of the single-atom nanozyme with microbial cells as templates provided in the embodiment of the present invention is a pioneering use of microbial cells as carbonization templates to prepare single-atom nanozymes. The whole preparation process is simple and easy, with high repeatability. Microbial cells such as Escherichia coli can be used after cultivation. After large-scale application, the size and shape of the template can be maintained in a uniform state, which is very conducive to large-scale development and preparation. It solves the problem that most of the current preparation methods need to prepare suitable carriers for loading in advance, and these carriers are only suitable for small-scale preparation, and the preparation method is cumbersome, which easily causes the problem of atomic agglomeration. In addition, in terms of metal loading, the embodiment of the present invention can prepare different types of single-atom nanozymes by changing the type of metal ions added. Metals are the active centers of single-atom nanozymes, and the activity of single-atom nanozymes prepared by different types of metal ions will also change, so the activity of the target single-atom nanozyme can be easily regulated by regulating the type of metal ions added, or adding one or more, or regulating the load of metal ions, and different functions and uses can be achieved by regulating the activity of different single-atom nanozymes.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a single-atom nanozyme using microbial cells as templates, characterized in that: include: S1, inoculating microbial cells into culture medium for cultivation; S2, after the microbial cells enter the growth plateau phase, the microbial cells are collected, and a pre-cooled calcium chloride solution is added 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 penetrate into the cells, and then centrifuging to collect the cells; S4, adding a fixing solution to the product obtained in S3 for fixing; S5, adding a salt solution to the product obtained in S4, so that the salt solution wraps the microbial cells, drying, and calcining at a high temperature in an inert gas atmosphere; S6, washing the excess salt solution in the product obtained in S5 and drying it to obtain the single-atom nanozyme.

2. The method for preparing a single-atom nanozyme using microbial cells as templates according to claim 1, characterized in that: Also includes S7: The single-atom nanozyme is mixed with chloroauric acid and citric acid is added. After sufficient reaction, the single-atom cascade nanozyme is obtained.

3. The method for preparing a single-atom nanozyme using a microbial cell as a template according to claim 1 or 2, characterized in that: The S1 includes: The microbial cells were inoculated into the culture medium using an inoculation loop, and the culture medium was placed in a shaker for culture at a temperature of 37° C. and a rotation speed of 200 rpm.

4. The method for preparing a single-atom nanozyme using a microbial cell as a template according to claim 1 or 2, characterized in that: The pre-cooled calcium chloride solution refers to a calcium chloride solution formed by cooling a calcium chloride solution on ice for more than 30 minutes; the concentration of the calcium chloride solution is 0.05M-1M, preferably 0.1M.

5. The method for preparing a single-atom nanozyme using a microbial cell as a template according to claim 1 or 2, characterized in that: 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+ ; The concentration of the metal ion is between 0.1mM and 50mM, preferably 0.5mM; The volatile salt solution is NH4HCO3 solution with a concentration of 0.1M-5M, preferably 0.5M.

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

7. The method for preparing a single-atom nanozyme using a microbial cell as a template according to claim 1 or 2, characterized in that: 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.

8. The method for preparing a single-atom nanozyme using a microbial cell as a template according to claim 1 or 2, characterized in that: 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 0.5h-4h, preferably 2h.

9. The method for preparing a single-atom nanozyme using microbial cells as templates according to claim 2, characterized in that: The concentration of the chloroauric acid is 0.2mM-10mM, preferably 1mM, and the volume is 5ml-50ml; The concentration of the citric acid is 10mM-100mM, preferably 45mM, and the volume is 1ml-10ml.

10. An application of a single-atom nanozyme using a microbial cell as a template as claimed in any one of claims 1 to 9, characterized in that: The single-atom nanozyme using the microbial cell as a template is used to prepare enzyme-like preparations; The enzyme-like preparation is an oxidase-like preparation, a peroxidase-like preparation, a peroxidase-like preparation, a superoxide dismutase-like preparation or a haloperoxidase-like preparation; The enzyme preparation is an antibacterial agent, an antitumor drug, a wastewater treatment agent, a tissue repair drug or a detection reagent.

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