Preparation method and application of metal-sulfur coordinated single-atom nanozymes

Abstract

The present invention relates to a preparation method and application of a metal-sulfur coordinated single-atom nanozyme, and belongs to the technical field of biomimetic nanocatalyst synthesis. It includes: S1, inoculating metallothionein-expressing cells into a culture medium for cultivation; S2, adding a protein expression inducer to induce metallothionein expression after entering the growth platform phase; S3, adding a salt solution containing metal ions to the product obtained from S2, so that the metal ions bind to metallothionein, and centrifugally collecting cells containing metallothionein; S4, adding a stationary liquid to the product obtained from S3; S5, adding a volatile salt solution to the product obtained from S4, drying, and calcining at high temperature in an inert gas atmosphere; S6, cleaning the excess volatile salt solution in the product obtained from S5 and drying to obtain a metal-sulfur coordinated single-atom nanozyme. The single-atom nanozyme prepared in the embodiment of the present invention has a high peroxidase-like activity and excellent photothermal performance.

Description

Technical Field

[0001] The present invention relates to the technical field of bionic nanocatalyst synthesis, and in particular to a preparation method and application of a metal-sulfur coordinated single-atom nanozyme. Background Art

[0002] Nanozymes are nanomaterials with enzyme-like activity. Compared with natural enzymes, nanozymes show higher stability under extreme conditions (such as high temperature, high salinity, and extreme pH values). In addition, the preparation of nanozymes is usually relatively simple, and a variety of nanomaterials with specific functions, such as iron oxide and titanium oxide, can be synthesized by physical or chemical methods. Among them, single-atom nanozymes, as a new generation of enzyme-like preparations, show broad application prospects with their efficient catalytic activity, excellent stability and low-cost preparation process. They not only have important application value in biomedicine, environmental monitoring, and industrial catalysis, but also provide new perspectives and ideas for catalytic science research. Although single-atom nanozymes show excellent catalytic performance and stability, there are still some challenges in their preparation process. For example, many metal single atoms are currently coordinated with nitrogen atoms. Although this coordination structure can provide a certain degree of stability, it may also hinder the development of more enzyme activities and limit the further improvement of catalytic efficiency. In order to overcome these shortcomings, researchers are exploring the optimization of the coordination environment and the improvement of loading technology to achieve more efficient and stable single-atom nanozymes, and further promote their application in biomedicine, environmental governance, and industrial catalysis.

[0003] Proteases coordinated by metal and sulfur atoms play an important role in organisms. The most representative of these are proteases with iron-sulfur clusters as active centers. Iron-sulfur clusters are complexes containing iron and sulfur, which are widely present in many enzymes and proteins. Their unique structure enables them to have important functions in electron transfer, catalysis and regulation. These include electron transfer in the cellular respiratory chain and photosynthesis, catalytic reactions in amino acid synthesis and decomposition, gene expression regulation in iron metabolic balance, and key functions in DNA repair and protection. These enzymes not only play a key role in maintaining cellular energy metabolism and metabolic balance, but also ensure the stability of the genome and the accuracy of information transmission, revealing their indispensability for basic life processes. The development and preparation of single-atom nanozymes with metal-sulfur coordination has multiple advantages. Their efficient catalytic activity and versatility enable them to mimic the important role of natural iron-sulfur clusters in electron transfer, amino acid synthesis and gene expression regulation. In addition, these nanozymes have excellent stability and tolerance and can perform well under extreme environmental conditions. The preparation process is simple and low-cost, suitable for large-scale production, and environmentally friendly, which helps promote green chemistry and sustainable development. These characteristics make metal-sulfur coordinated single-atom nanozymes have broad application prospects in biomedicine, industry and environment.

[0004] Therefore, the development of single-atom nanozymes with metal-sulfur coordination 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

[0005] In order to solve the above technical problems, the present invention provides a preparation method and application of a metal-sulfur coordinated single-atom nanozyme. The technical solution of the present invention is as follows:

[0006] The present invention provides a method for preparing a metal-sulfur coordinated single-atom nanozyme, which comprises:

[0007] S1, inoculation of metallothionein-expressing cells into culture medium;

[0008] S2, after entering the growth station phase, protein expression inducer IPTG is added to induce metallothionein expression;

[0009] S3, adding a salt solution containing metal ions to the product obtained in S2, incubating for 0.5h-5h to allow the metal ions to bind to the metallothionein, and collecting the cells containing the metallothionein by centrifugation;

[0010] S4, adding a fixing solution to the product obtained in S3 for fixing;

[0011] S5, adding a volatile salt solution to the product obtained in S4, allowing the volatile salt solution to penetrate into the fixed cells and simultaneously encapsulate the metallothionein-expressing cells, followed by drying and calcining at high temperature in an inert gas atmosphere;

[0012] S6, washing the excess volatile salt solution in the product obtained in S5 and drying it to obtain a metal-sulfur coordinated single-atom nanozyme.

[0013] Optionally, the method for preparing the metal-sulfur coordinated single-atom nanozyme further comprises S7:

[0014] The metal-sulfur coordinated single-atom nanozyme is mixed with chloroauric acid, and citric acid is added. After sufficient reaction, the metal-sulfur coordinated single-atom cascade nanozyme is obtained.

[0015] Optionally, the S1 includes:

[0016] The metallothionein-expressing 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.

[0017] Optionally, the concentration of IPTG is between 0.1-1 mM, preferably 0.1 mM;

[0018] 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 Fe 2+ ;

[0019] The concentration of the metal ion is between 0.1mM and 50mM, preferably 0.5mM;

[0020] Optionally, the fixing liquid is 4% paraformaldehyde; the volatile salt solution is a volatile inorganic salt, preferably NH4HCO3 solution, with a concentration of 0.1M-5M, preferably 1M.

[0021] Optionally, the metallothionein expression cell is a bacterium, a fungus, an insect cell or an animal cell; preferably, it is Escherichia coli among bacteria.

[0022] Optionally, the drying in S5 is vacuum freeze drying; the temperature of the high-temperature calcination is 500°C-1000°C, preferably 800°C; and the high-temperature calcination time is 0.5h-4h, preferably 2h.

[0023] Optionally, the concentration of chloroauric acid is 0.2mM-10mM, preferably 1mM, and the volume is 5ml-50ml;

[0024] The concentration of the citric acid is 10mM-100mM, preferably 45mM, and the volume is 1ml-10ml.

[0025] The present invention also provides an application of a single-atom nanozyme using a microbial cell as a template, wherein the metal-sulfur coordinated single-atom nanozyme is used to prepare an enzyme-like preparation;

[0026] The enzyme-like preparation is an oxidase-like preparation, a peroxidase-like preparation, a superoxide dismutase-like preparation or a haloperoxidase-like preparation;

[0027] The enzyme preparation is an antibacterial agent, an antitumor drug, a wastewater treatment agent, a tissue repair drug or a detection reagent.

[0028] 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.

[0029] By means of the above scheme, the beneficial effects of the present invention are as follows:

[0030] Through performance testing, it can be obtained that the metal-sulfur coordinated 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 metallothionein expression 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.

[0031] 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

[0032] Figure 1 This is a flow chart of a method for preparing a metal-sulfur coordinated single-atom nanozyme provided in an embodiment of the present invention;

[0033] Figure 2 This is a sample image of the single-atom nanozyme prepared in Example 1 of the present invention;

[0034] Figure 3This is the Zeta potential diagram of the single-atom nanozyme prepared in Example 1 of the present invention;

[0035] Figure 4 This is a particle size diagram of the single-atom nanozyme prepared in Example 1 of the present invention;

[0036] 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;

[0037] Figure 6 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;

[0038] Figure 7 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;

[0039] Figure 8 This is a schematic diagram of the photothermal performance measurement results of the single-atom nanozyme prepared in Example 1 of the present invention;

[0040] Fig. 9 This is the antibacterial activity diagram of the single-atom nanozyme prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] The embodiment of the present invention provides a method for preparing a metal-sulfur coordinated single-atom nanozyme, which comprises:

[0043] S1, inoculate metallothionein-expressing cells into the culture medium and culture them.

[0044] In a specific embodiment, S1 comprises: inoculating the metallothionein-expressing cells into the culture medium using an inoculation loop, and culturing the culture medium in a shaker, wherein the shaker temperature is 25°C-37°C, preferably 37°C, and the rotation speed is 150rpm-500rpm, preferably 200rpm.

[0045] In a specific embodiment, the metallothionein expression cell is a common protein expression system, including protein expression systems such as bacteria, fungi, insect cells, and animal cells; preferably, it is an Escherichia coli protein expression system in bacteria.

[0046] S2, after entering the growth station phase, IPTG was added to induce metallothionein expression.

[0047] The OD value is used to determine whether the plant has entered the growth plateau phase. When the OD value is between 0.5 and 1.0, the plant has entered the growth plateau phase.

[0048] The concentration of IPTG is between 0.1-1 mM, preferably 0.1 mM.

[0049] The IPTG induction conditions are: temperature of 30° C., rotation speed of 150 rpm-500 rpm, preferably 200 rpm.

[0050] S3, adding a salt solution containing metal ions to the product obtained in S2, incubating for 0.5h-5h to allow the metal ions to bind to the metallothionein, and collecting the cells containing the metallothionein by centrifugation.

[0051] 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+ , Tb3+ and Tb 4+ At least one of Fe 2+ ; The concentration of the metal ion is between 0.1mM-50mM, preferably 0.5mM.

[0052] In the embodiment of the present invention, the incubation time is 0.5 h to 5 h, which can ensure that the metal ions can bind to the metallothionein.

[0053] Specifically, the process of collecting the cells containing metallothionein by centrifugation is: centrifuging the incubated solution, discarding the supernatant, and obtaining the cells containing metallothionein; preferably, the centrifugal speed is 5000rmp-10000rmp, preferably 6000rmp, and the centrifugal time is 5min-30min, preferably 10min.

[0054] S4, adding a fixing solution to the product obtained in S3 for fixing.

[0055] In a specific embodiment, the fixing solution is 4% paraformaldehyde.

[0056] The volume ratio of the fixing solution to the product obtained from S3 is 1:1-1:10, and the fixing time is 10 min-30 min.

[0057] Furthermore, after fixation, centrifugation is also performed to remove excess fixative.

[0058] S5, adding a volatile salt solution to the product obtained in S4, allowing the volatile salt solution to penetrate into the fixed cells and simultaneously encapsulate the metallothionein-expressing cells, followed by drying and calcining at high temperature in an inert gas atmosphere.

[0059] The purpose of drying is to remove water molecules in the cells containing metallothionein while ensuring that the volatile salt solution can wrap the metallothionein-expressing cells.

[0060] The inert gas atmosphere is nitrogen or argon, etc., preferably argon.

[0061] The volatile salt solution is a volatile inorganic salt, preferably a NH4HCO3 solution, with a concentration of 0.1M-5M, preferably 1 M. In the subsequent high-temperature calcination carbonization process, the volatilization of the volatile inorganic salt is conducive to forming a porous fluffy structure of the material, which is more conducive to the enzyme-like activity.

[0062] In a specific embodiment, the drying in S5 is vacuum freeze drying, and the temperature of the cold trap of the vacuum freeze dryer 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.

[0063] S6, washing the excess volatile salt solution in the product obtained in S5 and drying it to obtain a metal-sulfur coordinated single-atom nanozyme.

[0064] Specifically, the washing is carried out by ultrasonication for 15-120 minutes. The drying temperature in step S6 is 20-80°C.

[0065] Further, after the metal-sulfur coordinated single-atom nanozyme is obtained, the embodiment of the present invention may also include S7: mixing the metal-sulfur coordinated single-atom nanozyme with chloroauric acid (HAuCl4), and adding citric acid, and obtaining the metal-sulfur coordinated single-atom cascade nanozyme after sufficient reaction. 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℃.

[0066] Through step S7, the metal-sulfur coordinated single-atom nanozyme and gold nanoparticles Au NPs can be cascaded to grow Au NPs on the metal-sulfur coordinated single-atom nanozyme. Au NPs have glucose oxidase activity and can form a cascade reaction with the peroxidase of the metal-sulfur coordinated single-atom nanozyme. In the process of the cascade reaction, glucose is oxidized by AuNPs to produce H2O2, and the metal-sulfur coordinated single-atom nanozyme uses the generated 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, etc.

[0067] To facilitate the description of the performance of the metal-sulfur coordinated single-atom nanozyme prepared by the embodiment of the present invention, a specific example is provided below.

[0068] Embodiment 1:

[0069] like Figure 1 As shown, the method for preparing the metal-sulfur coordinated single-atom nanozyme provided in an embodiment of the present invention comprises the following steps:

[0070] Inoculate Escherichia coli into LB medium, place the medium in a shaker, and culture it in the shaker for 8-10 hours at a temperature of 37°C and a speed of 200 rpm; when the OD value of the bacterial solution reaches 0.6-1.0 and enters the growth plateau, lower the culture temperature to 30°C, add IPTG with a final concentration of 0.1mM to induce metallothionein expression, add 0.5mM FeSO4 solution after 1 hour, continue to culture at 30°C, and collect the cells expressing metallothionein by centrifugation after 6 hours; add 4% paraformaldehyde to the bacterial solution at a volume ratio of 1:1 between the fixative and the bacterial solution, mix well, and fix at 4°C, and discard the excess paraformaldehyde by centrifugation; add 0.5M NH4HCO3 solution, incubate for 30 minutes and then place in a -80℃ refrigerator overnight. The frozen bacterial solution is vacuum freeze-dried to obtain a white powder, which is placed in an argon atmosphere and calcined at 800℃ for 2h, and naturally annealed; the excess NH4HCO3 solution is washed away, and the washed product is placed in a vacuum drying oven and dried at 40℃ to obtain a black powder, which is the metal-sulfur coordinated single-atom nanozyme; the single-atom nanozyme is mixed with 20mL 1mM chloroauric acid, and 1mL 40mM citric acid is added, protected from light and reacted at 37℃-160℃ for 30min-12h. After sufficient reaction, the color of the solution changes from dark yellow to dark red, and the metal-sulfur coordinated single-atom cascade nanozyme is obtained.

[0071] like Figure 2 As shown, after freeze-drying and high-temperature calcination and carbonization, the metallothionein prokaryotic expression cells in Example 1 become black powder.

[0072] 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 -22mV. When metal ions are added for adsorption and carbonization, the potential changes significantly, indicating that the metal elements are dispersed inside the single-atom nanomaterial.

[0073] 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 the single-atom nanozyme is about 0.8 μm and is evenly distributed, which can be used for subsequent research.

[0074] Furthermore, the present invention also carried out an activity test on the single-atom nanozyme prepared in Example 1, specifically:

[0075] 1. Oxidase activity assay, including:

[0076] 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.

[0077] 2. Peroxidase activity assay, including:

[0078] 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.

[0079] 3. Catalase activity assay, including:

[0080] 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.

[0081] like Figure 5-6 As shown, they are respectively a schematic diagram of the peroxidase activity measurement result and a schematic diagram of the catalase activity measurement result of the single-atom nanozyme prepared in Example 1. Figure 5 and Figure 6 It can be obtained that the single-atom nanozyme prepared in Example 1 has excellent peroxidase-like activity and catalase-like activity.

[0082] In addition, the present invention also conducted a performance test on the single-atom cascade nanozyme prepared in Example 1, specifically including:

[0083] 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 7 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 7 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.

[0084] Furthermore, the present invention also tests the photothermal performance of the single-atom nanozyme prepared in Example 1, including: using a thermometer to measure the photothermal performance of the single-atom nanozyme after 808 laser irradiation (2W / cm 2 , 6min), the temperature changes of the control group and the single-atom nanozyme group are shown in the following measurement results: Figure 8 As shown. Figure 8 It can be found that the temperature of the single-atom nanozyme can reach about 35°C within one minute after laser irradiation, and it has good photothermal properties.

[0085] In addition, the present invention also tests the antibacterial activity of the single-atom nanozyme prepared in Example 1, and the steps are as follows: first, the bacterial species (Escherichia coli ATCC25922, Staphylococcus aureus CMCC26003) are inoculated into a standard LB medium and cultured to about OD = 1.0. The bacterial solution is incubated with sodium acetate buffer, hydrogen peroxide, 808 laser, 808 laser + hydrogen peroxide, single-atom nanozyme, single-atom nanozyme + hydrogen peroxide, single-atom nanozyme + 808 laser, single-atom nanozyme + 808 laser + hydrogen peroxide, and the antibacterial efficiency is calculated by coating the plate. The results are as follows: Fig. 9 shown.

[0086] Depend on Fig. 9 It can be found that single-atom nanozymes can effectively kill Escherichia coli and Staphylococcus aureus under 808 laser irradiation, and are a highly effective bactericidal material.

[0087] 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)

[0088] Combined with the above performance test results, it can be seen that the metal-sulfur coordinated single-atom nanozymes and single-atom nanocascade enzymes 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 its 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.

[0089] In summary, the preparation method of the metal-sulfur coordinated single-atom nanozyme provided in the embodiment of the present invention is innovative in that biological cells are used to express metallothionein, and metallothionein is used as a metal-sulfur coordinated template for preparing metal-sulfur coordinated single-atom nanozyme. The entire preparation process is simple and easy, with high repeatability. Microbial cells such as Escherichia coli can be used after culture and adding protein expression inducers. 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 require the preparation of carriers suitable for loading in advance, and these carriers are only suitable for small-scale preparation, the preparation method is cumbersome, and it is very easy to cause atoms to agglomerate, and the coordination of metal-sulfur cannot be achieved well. In addition, in terms of metal loading, the embodiments of the present invention can prepare different types of single-atom nanozymes by changing the type of metal ions added. Metal is the active center of single-atom nanozymes. The activity 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 controlled by regulating the type of metal ions added, or adding one or more, or regulating the loading amount of metal ions. Different functions and uses can be achieved by regulating the activity of different single-atom nanozymes.

[0090] In summary, the metal-sulfur coordinated single-atom nanozyme prepared in the present invention has the characteristics of simulating the activity of natural peroxidase, and can be used to prepare enzyme-like preparations with high catalytic activity. Combined with its photothermal properties, it has broad application prospects in the fields of antibacterial, anti-tumor, wastewater treatment, tissue repair or detection.

[0091] 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 metal-sulfur coordinated single-atom nanozyme, characterized in that: include: S1, inoculation of metallothionein-expressing cells into culture medium; S2, after entering the growth station phase, protein expression inducer IPTG is added to induce metallothionein expression; S3, adding a salt solution containing metal ions to the product obtained in S2, incubating for 0.5h-5h to allow the metal ions to bind to the metallothionein, and collecting the cells containing the metallothionein by centrifugation; S4, adding a fixing solution to the product obtained in S3 for fixing, wherein the fixing solution is 4% paraformaldehyde; S5, adding a volatile salt solution to the product obtained in S4, allowing the volatile salt solution to penetrate into the fixed cells and wrap the metallothionein-expressing cells, drying, and calcining at high temperature in an inert gas atmosphere; the volatile salt solution is a volatile inorganic salt; the high-temperature calcination temperature is 500° C.-1000° C., and the high-temperature calcination time is 0.5 h-4 h; S6, washing the excess volatile salt solution in the product obtained in S5 and drying it to obtain a metal-sulfur coordinated single-atom nanozyme.

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

3. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The S1 includes: The metallothionein-expressing 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 the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The concentration of IPTG is between 0.1-1 mM.

5. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The concentration of IPTG was 0.1 mM.

6. The method for preparing the metal-sulfur coordinated single-atom nanozyme 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; The concentration of the metal ions is between 0.1 mM and 50 mM.

7. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The metal ion is Fe 2+ ; The concentration of the metal ion is 0.5mM.

8. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The volatile salt solution is NH4HCO3 solution with a concentration of 0.1M-5M.

9. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 8, characterized in that: The concentration of the NH4HCO3 solution is 1M.

10. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The metallothionein expressing cells are bacteria, fungi, insect cells or animal cells.

11. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The metallothionein expression cell is Escherichia coli among bacteria.

12. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 1 or 2, characterized in that: The drying in S5 is vacuum freeze drying; the temperature of the high temperature calcination is 800° C.; and the high temperature calcination time is 2 hours.

13. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 2, characterized in that: The concentration of the chloroauric acid is 0.2mM-10mM, and the volume is 5mL-50mL; The concentration of the citric acid is 10mM-100mM, and the volume is 1mL-10mL.

14. The method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 2, characterized in that: The concentration of the chloroauric acid is 1 mM; the concentration of the citric acid is 45 mM.

15. An application of a metal-sulfur coordinated single-atom nanozyme prepared by the method for preparing a metal-sulfur coordinated single-atom nanozyme according to any one of claims 1 to 14, characterized in that: The metal-sulfur coordinated single-atom nanozyme is used to prepare enzyme-like preparations.

16. The use of the metal-sulfur coordinated single-atom nanozyme prepared by the method for preparing the metal-sulfur coordinated single-atom nanozyme according to claim 15, characterized in that: 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 preparation is an antibacterial agent, an antitumor drug, a wastewater treatment agent, a tissue repair drug or a detection reagent.

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