Zinc-based monatomic nano-enzyme with multi-enzyme activity as well as preparation method and application of zinc-based monatomic nano-enzyme

Abstract

The invention belongs to the technical field of nano-enzymes and anti-biofilm materials, and discloses a zinc-based monatomic nano-enzyme with multi-enzyme activity as well as a preparation method and application of the zinc-based monatomic nano-enzyme. The preparation method comprises the following steps: by taking imidazole-2-formaldehyde and zinc salt as precursors, synthesizing ZIF-90 through a solvothermal method; and performing pyrolysis in an inert atmosphere to obtain the Zn-Nx-C monatomic nano-enzyme. The method disclosed by the invention is simple and efficient in process, the obtained Zn-Nx-C monatomic nano-enzyme has peroxidase-like activity and homoserine lactonase-like activity, the efficiency bottleneck of a traditional single-enzyme system is broken through due to the dual-function synergistic effect, and the Zn-Nx-C monatomic nano-enzyme is suitable for the scenes of food preservation, medical instrument antibiosis, environmental microorganism control and the like and has a wide application prospect. And an efficient solution is provided for biofilm prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozymes and anti-biofilm materials, and particularly relates to a zinc-based single-atom nanozyme with multi-enzyme activities, a preparation method thereof, and an application thereof. Background Art

[0002] As highly efficient biocatalysts, enzymes mediate various biochemical reactions in life activities under mild conditions. However, natural enzymes have inherent defects such as complex preparation processes, high environmental sensitivity, and low recycling efficiency, which severely restrict their industrial applications. To break through this bottleneck, the artificial enzyme mimicking technology has emerged. Among them, nanozymes have become a research hotspot of a new generation of bionic catalytic materials due to their characteristics such as scalable production, high stability, and multi-functional integration. Since Fe3O4 nanoparticles were first discovered to have peroxidase-like activity, nanozyme systems such as metal oxides, carbon-based materials, and metal-organic frameworks (MOFs) have been developed one after another. However, traditional nanozymes are difficult to accurately mimic the catalytic mechanism of natural enzymes due to problems such as uneven distribution of active sites and low catalytic selectivity.

[0003] In recent years, single-atom catalysts (SACs) have achieved the maximum utilization of active sites and precise regulation of structures by anchoring metal atoms on the surface of carriers in an atomically dispersed form. Among them, metal-nitrogen-carbon (M-N-C) type single-atom nanozymes exhibit catalytic efficiency and selectivity comparable to those of biocatalysts due to their M-Nx coordination structure similar to that of natural enzymes. Although significant progress has been made in this field, existing single-atom nanozymes are still generally limited to the simulation of single enzyme activities and are difficult to meet the requirements of complex catalytic scenarios. Research shows that integrating multiple enzyme activities can trigger cascade reactions. For example, tumor microenvironment regulation can be achieved by synergistic oxidase / peroxidase activities, or oxidative stress damage can be alleviated by combining superoxide dismutase / catalase activities. Therefore, developing single-atom nanozymes with multi-enzyme activities has become a key direction to break through the application boundaries.

[0004] In the field of biofilm prevention and control, traditional antibiotic killing strategies are prone to induce drug resistance, while blocking biofilm formation by degrading the quorum sensing signal molecule N-acyl homoserine lactone (AHLs) is regarded as a more sustainable solution. Although natural homoserine lactonase can efficiently hydrolyze AHLs, its harsh storage conditions, high preparation cost, and limited reusability restrict its practical applications. Although existing nanomaterials have achieved the simulation of activities such as peroxidase, a multi-enzyme synergistic system with both AHLs degradation function has not been reported yet. How to design a bifunctional nanozyme with high catalytic activity, wide environmental adaptability, and potential for large-scale preparation has become a technical problem to be solved urgently in this field. Summary of the Invention

[0005] To overcome the disadvantages and deficiencies of the above-mentioned existing technologies, the primary objective of the present invention is to provide a preparation method for a zinc-based single-atom nanozyme with multi-enzyme activity.

[0006] Another objective of the present invention is to provide a zinc-based single-atom nanozyme with multi-enzyme activity prepared by the above method.

[0007] A further objective of the present invention is to provide the application of the above zinc-based single-atom nanozyme with multi-enzyme activity in the field of biofilm prevention and control.

[0008] The objectives of the present invention are achieved through the following solutions:

[0009] A preparation method for a zinc-based single-atom nanozyme with multi-enzyme activity, comprising the following steps: using imidazole-2-carbaldehyde and a zinc salt as precursors, synthesizing ZIF-90 through a solvothermal method; then pyrolyzing in an inert atmosphere to obtain a Zn-Nx-C single-atom nanozyme.

[0010] The preparation method for the zinc-based single-atom nanozyme with multi-enzyme activity specifically comprises the following steps: mixing an imidazole-2-carbaldehyde solution with a zinc salt solution, reacting, and separating to obtain ZIF-90 crystals; pyrolyzing the ZIF-90 crystals under an inert atmosphere to obtain a Zn-Nx-C single-atom nanozyme.

[0011] The imidazole-2-carbaldehyde solution is prepared by adding imidazole-2-carbaldehyde to a solvent and heating to 65 - 75 °C until dissolved.

[0012] The solvent of the imidazole-2-carbaldehyde solution includes at least one of dimethylformamide (DMF) and methanol.

[0013] The concentration of the imidazole-2-carbaldehyde solution is 0.4 - 0.8 mol / L.

[0014] The zinc salt includes at least one of zinc nitrate, zinc acetate, and zinc chloride.

[0015] The solvent of the zinc salt solution is methanol.

[0016] The concentration of the zinc salt solution is 0.1 - 0.2 mol / L.

[0017] The temperature of the reaction is 20 - 30 °C; the time is 30 - 60 min.

[0018] The inert atmosphere includes at least one of nitrogen and helium.

[0019] The temperature of the pyrolysis is 700 - 800 °C; the time is 2 - 3 h; the heating rate is 5 °C / min.

[0020] A zinc-based single-atom nanozyme with multi-enzyme activity prepared by the above method.

[0021] The multi-enzyme activity is the dual enzyme activities of homoserine lactonase-like and peroxidase-like.

[0022] Application of the above zinc-based single-atom nanozyme with multi-enzyme activity in the field of biofilm prevention and control.

[0023] The mechanism of the present invention is as follows:

[0024] The present invention has the following synergistic mechanism: When the material contacts with bacteria, the local microenvironment is transiently acidified (pH 4.5 - 6.5), activating the peroxidase-like activity, catalyzing H2O2 to generate strongly oxidizing ·OH free radicals, and rapidly killing free bacteria (survival rate < 10%). In this stage, the metabolic balance of bacteria is disrupted, which may trigger a bacterial stress response, prompting the remaining bacteria to accelerate the transformation into the biofilm form. As the acidic substances are consumed, the local pH gradually increases, activating the homoserine lactonase-like. At this time, the remaining bacteria attempt to form a biofilm by secreting AHLs signaling molecules, but the enzyme activity degrades 80 - 95% of N-acyl homoserine lactones (AHLs), blocking quorum sensing, resulting in the biofilm being unable to mature or disintegrating.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The preparation method of the present invention is simple, and Zn-Nx-C is simply and efficiently prepared by a one-pot method of high-temperature calcination.

[0027] (2) The Zn-Nx-C prepared by the present invention has the dual enzyme activities of homoserine lactonase-like and peroxidase-like.

[0028] (3) When the Zn-Nx-C prepared by the present invention is used as a catalyst to catalyze the reaction of homoserine lactonase substrate and peroxidase substrate, it has a high affinity for the substrate.

[0029] (4) The Zn-Nx-C prepared by the present invention has strong tolerance to high temperature, alkaline pH, organic solvents and ionic strength, and has a wide applicability.

[0030] (5) The Zn-Nx-C prepared by the present invention uses ZIF-90 as a precursor, breaking through the limitation of using common metal-organic framework materials in the traditional preparation of Zn-Nx-C. The unique structure and chemical properties of ZIF-90 endow Zn-Nx-C with new performance.

[0031] (6) The Zn-Nx-C prepared by the present invention can target and degrade the quorum sensing signal molecules that mediate biofilm formation, inhibit biofilm formation while killing bacteria, and is not easily induced to develop antibiotic resistance. It breaks through the limitations of traditional nanozymes in preventing and controlling biofilms by killing bacteria, and has a mild, efficient, green and environmentally friendly action mode, making it suitable for the prevention and control of biofilms in fields such as food, medicine, and the environment. Description of the Drawings

[0032] Figure 1 SEM and TEM images of the Zn-Nx-C obtained in Example 1 (a is the SEM image, b is the TEM image).

[0033] Figure 2 Graph of the catalytic performance of the Zn-Nx-C obtained in Example 1 for homoserine lactonase-like activity (a is the reaction time graph, B is the reaction pH graph, c is the reaction temperature graph).

[0034] Figure 3 Graph of the catalytic kinetics of the Zn-Nx-C obtained in Example 1 for homoserine lactonase-like activity (a is the Michaelis-Menten equation graph, b is the double-reciprocal graph).

[0035] Figure 4 Graph of the catalytic stability of the Zn-Nx-C obtained in Example 1 for homoserine lactonase-like activity (a is the pH graph, b is the temperature graph, c is the NaCl concentration graph, d is the ethanol content graph).

[0036] Figure 5 Graph of the catalytic performance of the Zn-Nx-C obtained in Example 1 for peroxidase-like activity (a is the graph of different chromogenic substrates, b is the graph of reactions in different systems).

[0037] Figure 6 Graph of the catalytic kinetics of the Zn-Nx-C obtained in Example 1 for peroxidase-like activity (a, b are the kinetics graphs of H2O2, c, d are the kinetics graphs of TMB).

[0038] Figure 7 Graph of the optimal conditions for the catalytic activity of the Zn-Nx-C obtained in Example 1 for peroxidase-like activity (a is the pH graph, b is the temperature graph, c is the time graph, d is the material concentration graph, e is the H2O2 concentration graph, f is the TMB concentration graph).

[0039] Figure 8 Graph of the anti-biofilm performance of the Zn-Nx-C obtained in Example 1 (a is the survival rate graph, b is the degradation rate graph, c is the biomass graph). Detailed Description of the Invention

[0040] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0041] The reagents used in the examples can be conventionally purchased from the market without special instructions.

[0042] Example 1 Synthesis of Zn-Nx-C single-atom nanozyme

[0043] Add 20 mmol of imidazole-2-carbaldehyde to 50 mL of dimethylformamide (DMF), and heat to 70 °C until dissolved. After cooling to room temperature, quickly pour the mixture of zinc nitrate hexahydrate (5 mmol) dissolved in 50 mL of methanol into the imidazole-2-carbaldehyde solution, and react at 25 °C for 30 min. The ZIF-90 crystals are separated from the turbid suspension by centrifugation three times and washing with methanol. Then the ZIF-90 sample is pyrolyzed in a nitrogen atmosphere at 700 °C for 2 hours with a heating program of 5 °C / min to obtain the Zn-Nx-C sample.

[0044] Use SEM and TEM to observe the morphology of the synthesized nanozyme. Figure 1 It can be seen that SEM ( Figure 1 a in it) shows that it has a uniform polyhedron structure with an average particle size of 100 ± 5 nm; TEM ( Figure 1 b in it) confirms that Zn is dispersed as single atoms.

[0045] Study on the catalytic performance of the nanozyme in Example 1 for homoserine lactonase-like activity

[0046] Use a high-performance liquid chromatograph to determine the homoserine lactonase-like activity of Zn-Nx-C obtained in Example 1. The typical experimental process is as follows: Weigh 300 μg of N-octanoyl-L-homoserine lactone (C8-HSL) and dissolve it in 5 mL of methanol to obtain a C8-HSL solution (60 μg / mL); Weigh an appropriate amount of Zn-Nx-C and ultrasonically disperse it in deionized water to obtain a Zn-Nx-C solution (0.4 mg / mL). Add 500 μL of the Zn-Nx-C solution (0.4 mg / mL) to 500 μL of the C8-HSL solution (60 μg / mL), mix well, and react at 37 °C and 220 rpm for 4 h. Then centrifuge and take the supernatant, filter it through a 0.22 μm filter membrane, and detect the change in the peak area of C8-HSL before and after the reaction on a high-performance liquid chromatograph. The catalytic performance of the Zn-Nx-C catalytic reaction under changes in time, pH, and temperature was studied.

[0047] From Figure 2It can be seen that the degradation rate of the Zn-Nx-C nanomaterial generally shows an upward trend with the increase of the reaction time. Zn-Nx-C exhibits the highest catalytic activity at pH 9.0. Zn-Nx-C has a low sensitivity to the pH value and maintains a catalytic activity of over 60% in the pH range of 4 to 11. Zn-Nx-C shows the highest catalytic activity at 50 °C and retains over 70% of the initial activity when the reaction temperature exceeds 60 °C.

[0048] Catalytic Kinetics Study on the Activity of Nanozyme with Homoserine Lactonase-like Activity in Test Example 2

[0049] Using the Zn-Nx-C + C8-HSL obtained in Example 1 as the reaction system, the kinetic parameters of the enzymatic reaction of Zn-Nx-C with homoserine lactonase-like activity were determined under the same reaction conditions as in Test Example 1. First, a standard curve of the C8-HSL concentration versus the peak area was established. The concentration of Zn-Nx-C was fixed at 0.4 mg / mL and the dosage was 500 μL. 500 μL of C8-HSL with concentrations of 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 300 μM, and 400 μM was taken and reacted at 37 °C and 220 rpm for 4 h. Subsequently, the supernatant was taken by centrifugation, passed through a 0.22 μm filter membrane, and the change in the peak area of C8-HSL before and after the reaction was detected by a high-performance liquid chromatograph. The kinetic parameters were determined by the following formula: 1 / ν = Km / Vmax(1 / [S] + 1 / Km); where ν represents the initial velocity, Km represents the Michaelis constant, [S] is the substrate concentration, and Vmax is the maximum reaction velocity.

[0050] From Figure 3 it can be calculated that the maximum initial velocity Vmax is 4.6×10 -8 M -1 s -1 and the Michaelis constant Km is 155 μM.

[0051] Catalytic Stability Study on the Activity of Nanozyme with Homoserine Lactonase-like Activity in Test Example 3

[0052] Studying the catalytic stability of the research-type homoserine lactonase and maintaining high activity are the keys to the application of single-atom nanozymes. The catalytic activities of Zn-Nx-C obtained in Example 1 were studied under different pH values, temperatures, ionic strengths, and organic solvent conditions. In terms of temperature, 500 μL of 0.4 mg / mL Zn-Nx-C and native homoserine lactonase were incubated in a water bath at 20, 30, 40, 50, 60, 70, 80, and 90 °C for 4 h, respectively, and then reactions were carried out and analyzed by high-performance liquid chromatography. In terms of pH, 500 μL of 0.4 mg / mL Zn-Nx-C and native homoserine lactonase were incubated in buffer solutions (0.1 M) with pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 for 4 h, respectively, and then reactions were carried out and analyzed by high-performance liquid chromatography. In terms of ionic strength, 500 μL of 0.4 mg / mL Zn-Nx-C and native homoserine lactonase were incubated in NaCl solutions with concentrations of 50, 100, 150, 200, 250, and 300 mM for 4 h, respectively, and then reactions were carried out and analyzed by high-performance liquid chromatography. In terms of organic solvents, Zn-Nx-C and native homoserine lactonase were incubated in ethanol solutions containing 0%, 10%, 30%, 50%, 70%, and 90% for 4 h, respectively, and then reactions were carried out and analyzed by high-performance liquid chromatography.

[0053] From Figure 4 It can be seen that the Zn-Nx-C nanomaterial maintains a catalytic activity higher than 60% at 20 - 90 °C, pH 4 - 11, an ionic strength of 50 - 300 mM, and 0 - 90% organic solvents. These results indicate that the Zn-Nx-C nanomaterial can effectively catalyze the hydrolysis reaction of AHL molecules under a wide range of reaction conditions, while native homoserine lactonase only functions under physiological conditions.

[0054] Study on the homoserine lactonase-like catalytic selectivity performance of the nanozyme in Test Example 4

[0055] To evaluate the catalytic selectivity of the single-atom nanozyme, the single-atom nanozyme was reacted with C6-HSL, C7-HSL, C10-HSL, C12-HSL, 3-ox-C6-HSL which are similar in structure to C8-HSL, and with cethivoir, rosiglitazone, oxiracetam, and levetiracetam which have different structures from C8-HSL. The degradation effects of Zn-Nx-C obtained in Example 1 on these substances were evaluated by high-performance liquid chromatography.

[0056] Table 1

[0057]

[0058] As can be seen from Table 1, Zn-Nx-C effectively catalyzes substrates such as C6-HSL, C7-HSL, C10-HSL, C12-HSL, and 3-oxo-C6-HSL, and the degradation rate is above 65%. However, it shows limited catalytic effects on cetirizone, rosiglitazone, and oxiracetam, levetiracetam. These results indicate that Zn-Nx-C has similar catalytic activities for AHLs with different acyl chain lengths, but cannot catalyze C-N-C and C-S-C bonds.

[0059] Study on the Catalytic Performance of the Peroxidase-like Activity of the Nanozyme in Test Example 5

[0060] The Zn-Nx-C with peroxidase-like activity obtained in Example 1 can oxidize substrates in the presence of H2O2. Add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of H2O2 (1.5 M), 50 μL of 3,3,5,5-tetramethylbenzidine (TMB) (10 mM), and 50 μL of 10 mM substrate (tetramethylbenzidine (TMB) or o-phenylenediamine (OPD) or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)) to 350 μL of HAc-NaAc buffer (0.1 M, pH 4.5). TMB is dissolved in dimethyl sulfoxide (DMSO), OPD is dissolved in ethanol, and ABTS is dissolved in water. React the reaction system at room temperature for 10 min, and finally measure the ultraviolet absorption at 652 nm.

[0061] To verify the feasibility of this experiment, the ultraviolet-visible absorption spectra under different conditions were measured in this experiment. It includes the system containing only TMB, the system containing TMB and H2O2, the system containing TMB + material, and the system containing TMB + H2O2 + material, and the ultraviolet absorption of different systems at 652 nm was measured.

[0062] From Figure 5 It can be seen that in the case of adding H2O2, Zn-Nx-C can catalyze different chromogenic substrates TMB, OPD, ABTS to oxidize them into blue, yellow, and green, which indicates that Zn-Nx-C has the catalytic characteristics of peroxidase-like. The system containing only TMB, the system containing TMB and H2O2, and the system containing TMB + material have no characteristic absorption peaks at 652 nm. The system containing TMB + H2O2 + material has an obvious characteristic absorption peak of TMBox at 652 nm, because Zn-Nx-C catalyzes H2O2 to generate highly oxidizing hydroxyl radicals, thereby oxidizing TMB to present blue. This further proves that the Zn-Nx-C material has peroxidase-like activity.

[0063] Study on the Catalytic Kinetics of the Peroxidase-like Activity of the Nanozyme in Test Example 6

[0064] Using TMB-H2O2 as the reaction system, the enzymatic reaction kinetic parameters of the Zn-Nx-C peroxidase-like enzyme obtained in Example 1 were measured under the same reaction conditions as in Test Example 5. First, fix the concentration of TMB at 10 mM and the dosage at 50 μL. Take 50 μL of H2O2 with concentrations of 10 mM, 20 mM, 30 mM, 50 mM, 70 mM, 100 mM, and 150 mM respectively. Take 50 μL of Zn-Nx-C (0.5 mg / mL) and 350 μL of HAc-NaAc buffer (0.1 M, pH 4.5). The total reaction system is 500 μL. React at room temperature for 10 min. Measure the absorbance of the blue reaction product oxTMB at 652 nm and calculate Km and Vmax. Fix the concentration of H2O2 at 1.5 M and the dosage at 50 μL. Take 50 μL of TMB with concentrations of 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM, and 1 mM respectively. Take 50 μL of Zn-Nx-C and 350 μL of HAc-NaAc buffer (0.1 M, pH 4.5). The total reaction system is 500 μL. React at room temperature for 10 min. Measure the absorbance of the blue reaction product oxTMB at 652 nm and calculate Km and Vmax.

[0065] From Figure 6 the maximum initial velocity Vmax and the Michaelis constant Km can be calculated. When using H2O2 as the substrate, the Km value of Zn-Nx-C is 1.3 mM, which is much lower than the Km value of HRP (Km = 3.7 mM), indicating that Zn-Nx-C has a strong affinity for H2O2. The Vmax value of Zn-Nx-C is 2.65×10 -8 M -1 s -1 . In addition, when using TMB as the substrate, the Km value of Zn-Nx-C is 0.1 mM, which is much lower than the Km value of HRP (Km = 0.4 mM), indicating that Zn-Nx-C has a strong affinity for TMB. The Vmax value of Zn-Nx-C is 3.05×10 -8 M -1 s -1 .

[0066] Table 2

[0067]

[0068] Study on the Optimal Conditions for the Peroxidase-like Activity Catalysis of the Nanozyme in Test Example 7

[0069] Determine the optimal reaction temperature: Use the HAc-NaAc buffer solution with pH = 4.5 as the reaction system, add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of TMB (10 mM), and 50 μL of H2O2 (1.5 M), and supplement the buffer solution to make the total system volume 500 μL. Control the temperature at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C using a water bath, and the reaction time is 10 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of oxTMB at 652 nm to evaluate the optimal reaction temperature of the Zn-Nx-C peroxidase-like enzyme.

[0070] Determine the optimal reaction pH: Prepare HAc-NaAc buffer solutions with pH = 3, 4, 4.5, 5, 5.5, and 6, and prepare Tris-HCl buffer solutions with pH = 7, 8, and 9. Respectively use the above buffer solutions as the reaction system, add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of TMB (10 mM), and 50 μL of H2O2 (1.5 M), and supplement the buffer solution to make the total system volume 500 μL. React at room temperature for 10 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of the reaction product oxTMB at 652 nm to evaluate the optimal reaction pH of the Zn-Nx-C peroxidase-like enzyme.

[0071] Determine the optimal reaction time: Use the HAc-NaAc buffer solution with pH = 4.5 as the reaction system, add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of TMB (10 mM), and 50 μL of H2O2 (1.5 M), and supplement the buffer solution to make the total system volume 500 μL. React at room temperature for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, and 60 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of the reaction product TMBox at 652 nm to evaluate the optimal reaction time of the Zn-Nx-C peroxidase-like enzyme.

[0072] Determine the optimal dosage of the material: Use the HAc-NaAc buffer solution with pH = 4.5 as the reaction system, add 50 μL of Zn-Nx-C with different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1 mg / mL), 50 μL of TMB (10 mM), and 50 μL of H2O2 (1.5 M), and supplement the buffer solution to make the total system volume 500 μL. React at room temperature for 10 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of the reaction product oxTMB at 652 nm to evaluate the optimal dosage of the Zn-Nx-C peroxidase-like enzyme material.

[0073] Determination of the optimal dosage of H2O2: Using the HAc-NaAc buffer solution with pH = 4.5 as the reaction system, add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of TMB (10 mM), and 50 μL of H2O2 with different concentrations (0.1, 0.3, 0.5, 0.7, 1, 1.5, 2 M), and supplement the buffer solution to make the total volume of the system 500 μL. React at room temperature for 10 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of the reaction product oxTMB at 652 nm to evaluate the optimal dosage of H2O2 for the Zn-Nx-C peroxidase-like enzyme.

[0074] Determination of the optimal dosage of TMB: Using the HAc-NaAc buffer solution with pH = 4.5 as the reaction system, add 50 μL of Zn-Nx-C (0.5 mg / mL), 50 μL of TMB with different concentrations (1, 5, 10, 15, 20, 15 mM), and 50 μL of H2O2 (1.5 M), and supplement the buffer solution to make the total volume of the system 500 μL. React at room temperature for 10 min. After the reaction, use a UV-visible spectrophotometer to measure the absorbance of the reaction product oxTMB at 652 nm to evaluate the optimal dosage of TMB for the Zn-Nx-C peroxidase-like enzyme.

[0075] From Figure 7 It can be seen that the optimal catalytic conditions are as follows: the pH of the HAc-NaAc buffer solution is 4.5, the reaction temperature is 40 °C, the reaction time is 10 min, the material concentration is 0.5 mg / mL, the H2O2 concentration is 1.5 M, and the TMB concentration is 10 mM.

[0076] Study on the anti-biofilm performance of the nanozyme in Test Example 8

[0077] Add the Zn-Nx-C obtained in Example 1 to deionized water to prepare Zn-Nx-C solutions with different concentrations (0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL).

[0078] Take the Zn-Nx-C solution and mix it with the Cronobacter sakazakii bacterial solution (10 6 CFU / mL) in a volume ratio of 1:9, incubate together, and use the plate counting method to determine the survival rate of planktonic bacteria. The results are shown in Figure 8 a as shown.

[0079] Mix the Zn-Nx-C solution and the C8-HSL solution in equal volume, react at 37 °C for 24 h, and after centrifugation, detect the residual concentration of AHLs by HPLC and calculate the degradation rate. The results are shown in Figure 8 b as shown.

[0080] Add 100 μL of Zn-Nx-C solution to the Cronobacter sakazakii biofilm model (incubated in Luria-Bertani medium (LB medium) at 37 °C for 24 h), and measure the biofilm biomass (OD570) using the crystal violet staining method. The results are as Figure 8 shown in c below. After incubation at 37 °C for 24 hours, the biofilm biomass decreased by ≥ 90%.

[0081] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of a zinc-based single-atom nanozyme with multi-enzyme activity, characterized in that, It includes the following steps: Using imidazole-2-carboxaldehyde and zinc salt as precursors, ZIF-90 is synthesized by solvothermal method; then pyrolyzed in an inert atmosphere to obtain Zn-Nx-C single-atom nanozyme.

2. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 1, characterized in that, Specifically, it includes the following steps: Mix the imidazole-2-carboxaldehyde solution with the zinc salt solution, react, and separate to obtain ZIF-90 crystals; pyrolyze the ZIF-90 crystals under an inert atmosphere to obtain Zn-Nx-C single-atom nanozyme.

3. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 2, characterized in that: The solvent of the imidazole-2-carboxaldehyde solution includes at least one of dimethylformamide and methanol; the concentration of the imidazole-2-carboxaldehyde solution is 0.4 - 0.8 mol / L.

4. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 1 or 2, characterized in that: The zinc salt includes at least one of zinc nitrate, zinc acetate, and zinc chloride.

5. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 2, characterized in that: The solvent of the zinc salt solution is methanol; the concentration of the zinc salt solution is 0.1 - 0.2 mol / L.

6. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 2, characterized in that: The temperature of the reaction is 20 - 30 °C; the time is 30 - 60 min.

7. The preparation method of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 1 or 2, characterized in that: The inert atmosphere includes at least one of nitrogen and helium; The temperature of the pyrolysis is 700 - 800 °C; the time is 2 - 3 h; the heating rate is 5 °C / min.

8. The zinc-based single-atom nanozyme with multi-enzyme activity prepared by the method according to any one of claims 1 - 7.

9. The zinc-based single-atom nanozyme with multi-enzyme activity according to claim 8, wherein: The multi-enzyme activity is the dual enzyme activities of homoserine lactonase-like and peroxidase-like.

10. The application of the zinc-based single-atom nanozyme with multi-enzyme activity according to claim 8 in the field of biofilm prevention and control.

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