A sodium-doped nanozyme, its preparation method and application

Abstract

The present invention discloses a sodium-doped nanozyme and its preparation method and application, belonging to the technical field of antibacterial new nanomaterials. The nanozyme is prepared by mixing a sodium ion salt with vitamin C and L-alanyl-L-glutamate in an inorganic solvent to form a precursor solution, followed by treatment with a one-pot hydrothermal method and then cooling, and drying to obtain the sodium-doped nanozyme. The molar ratio of sodium element : nitrogen element : carbon element in the precursor raw materials is 1 : (2 - 120) : (10 - 500). The treatment temperature of the one-pot hydrothermal method is 100°C - 300°C. The nanozyme uses sodium as the doping metal and sodium salts such as sodium chloride as the sodium source raw material, and has the advantages of being green, safe, low-cost, and excellent antibacterial ability. Moreover, it is prepared by a simple one-step hydrothermal method, avoiding the use of expensive raw materials, effectively reducing the preparation cost of the nanozyme, and simplifying the preparation process. At the same time, since the nanozyme avoids the use of heavy metals and noble metals, it also avoids the potential safety risks associated with them.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel nanomaterial antibacterial technology, and particularly relates to a sodium-doped nanozyme and its preparation method and application. Background Art

[0002] Due to the abuse of antibiotics, the emergence of drug-resistant bacteria has posed a challenge of new intractable infections. The main antibacterial mechanism of antibiotics is to inhibit the synthesis of bacterial cell walls, damage cell membranes, interfere with the synthesis of bacterial proteins, and limit the transcription and replication of bacterial nucleic acids. However, the long-term use of antibiotics can lead to the formation of biofilms around bacteria, hindering the contact between antibiotic small molecules and bacteria and resulting in multi-drug resistance, which makes treatment more challenging. The emergence of drug-resistant bacteria is a new global crisis, increasing the incidence and mortality of human infections and having a negative impact on the clinical treatment outcomes of a wide range of people, including those in intensive care units or undergoing surgery, organ transplantation, or cancer treatment. Therefore, it is urgent to find new small molecule antibacterial substances. The emergence of antibacterial nanomaterials has alleviated this crisis and effectively improved the ability to combat drug-resistant bacterial contamination. Among them, nanozymes are prominent in antibacterial materials. Due to their similar enzymatic reaction kinetic characteristics and catalytic ability to natural enzymes, as well as advantages such as simple preparation, low cost, easy industrial production, and stable reaction conditions, nanozymes have attracted great interest.

[0003] Nanozymes are a new generation of artificial mimetic enzymes. Like natural enzymes, they can efficiently catalyze enzyme substrates under mild conditions, showing catalytic efficiency and enzymatic reaction kinetic characteristics similar to natural enzymes; however, they are more stable than natural enzymes and can still maintain 85% catalytic activity even in strong acid / strong base (pH 2 - 10) or a wide temperature range (4°C - 90°C). Animal experiments have shown that nanozymes have functions such as protecting the myocardium, improving Alzheimer's disease, and ischemic stroke, indicating that the application research of nanozymes has extended from in vitro to in vivo and is expected to provide new ideas and methods for the treatment of diseases. The emergence of nanozymes has changed people's traditional concepts, and inorganic nanomaterials are no longer considered inert substances. Nanozymes have revealed the biological effects inherent in nanomaterials, which not only expands the application of nanomaterials in biomedicine but also provides new ideas and new materials for the research of artificial mimetic enzymes, and even opens up a new research direction for nanobiology.

[0004] After searching for patents closely related to this application, the specific analysis is as follows:

[0005] Yang Yaling (Patent Publication No.: CN114053473B) disclosed a preparation method and application of a magnetic iron oxide composite nanozyme (Fe3O4-MWCNT-Hemin) antibacterial agent. Its raw materials include magnetic iron oxide, hemin, and adenosine triphosphate. When this nanozyme was used in antibacterial experiments, the results showed that the nanozyme of this invention has significant peroxidase activity, has a good antibacterial effect, and has good biocompatibility at the same time.

[0006] Wei Hui (Patent Publication No.: CN114306382B) disclosed a copper-based nanozyme (CuDA@PDA) and its preparation method and application. Its raw materials include dopamine and copper ion salt. When this nanozyme was used in antibacterial experiments, the results showed that the nanozyme of this invention has significant peroxidase activity, has a good antibacterial effect, realizes good antibacterial property against drug-resistant bacteria, and this nanozyme can be widely used in the treatment of bacterial infections.

[0007] Chen Wenwen (Patent Publication No.: CN111215141A) disclosed a gold nanozyme and its preparation method and application. Its raw materials include a bovine serum albumin skeleton and gold nanoparticles attached to the bovine serum albumin skeleton. When this nanozyme was used for the detection of reducing substances, enzyme-linked immunosorbent assay technology, and antibacterial, the results showed that the nanozyme of this invention has good oxidase activity and peroxidase activity.

[0008] With the continuous development of the nanozyme industry, researchers' understanding of nanozymes is gradually deepening. The related research on nanozymes has gradually matured, but from the existing research results, these nanozymes mainly have problems such as high cost and difficulty in obtaining precursor substances, high toxicity of precursors, complex synthesis methods, poor biocompatibility, and high cytotoxicity. In addition, although there are some reports on the preparation and practical characteristics of nanozymes in the existing technology, the theoretical research on the formation mechanism of nanozymes is still immature, and there is no conclusion on the formation mechanism of nanozymes. In the process of preparing nanozymes using many other raw materials, there are often situations where there is antibacterial property but no catalytic property, there is catalytic property but no antibacterial property, and there is neither catalytic property nor antibacterial property.

[0009] In summary, traditional antibiotics are prone to cause bacterial drug resistance and have high R & D costs; although antibacterial materials have improved the comprehensive effect of antibacterial treatment to a certain extent compared with antibiotics, the antibacterial effect is still far from ideal. At the same time, at present, when using metal doping to manufacture nanozymes, precious metals or heavy metals are often used, such as gold, copper, iron, etc. This not only has a high preparation cost, but also the presence of heavy metals will increase potential food safety risks. Summary of the Invention

[0010] (I) Technical problems to be solved

[0011] The present invention aims to solve one of the following technical problems existing in the prior art or related technologies:

[0012] Existing nanozymes have problems such as high preparation costs due to precious metals or heavy metals as raw materials for preparation, potential food safety risks, and unsatisfactory antibacterial effects.

[0013] (II) Technical solution

[0014] To solve the above technical problems, the present invention provides a sodium-doped Na@AGV nanozyme, and the specific technical solution adopted is as follows:

[0015] A sodium-doped Na@AGV nanozyme, which is obtained by mixing a sodium ion salt with vitamin C and L-alanyl-L-glutamate through an inorganic solvent to form a precursor solution, and then cooling after one-pot hydrothermal treatment and drying to obtain the sodium-doped nanozyme; in the sodium ion salt, vitamin C and L-alanyl-L-glutamate, the molar ratio of sodium element: nitrogen element: carbon element is: 1:(2-120):(10-500); the temperature of the one-pot hydrothermal treatment is 100°C - 300°C.

[0016] Preferably, the average particle size of the Na@AGV nanozyme is 0.1 - 10 nm.

[0017] Preferably, the sodium ion salt is sodium chloride, sodium acetate or sodium sulfate.

[0018] Another object of the present invention is to provide a preparation method of the above Na@AGV nanozyme, and the steps of this preparation method are as follows:

[0019] (1) Add the sodium ion salt, vitamin C and L-alanyl-L-glutamate to water and dissolve them, and mix them evenly to form a precursor solution;

[0020] (2) Transfer the precursor solution obtained in step (1) to a reaction kettle for reaction, and cool it after the reaction ends;

[0021] (3) Dry the reaction solution cooled in step (2) to obtain the sodium-doped Na@AGV nanozyme.

[0022] Preferably, in step (1), the concentration of vitamin C is 0.01 - 1 mol / L; the concentration of L-alanyl-L-glutamate is 0.01 - 1 mol / L; the concentration of the sodium ion salt is 0.001 - 1 mol / L.

[0023] Preferably, the reaction in step (2) is carried out at 100°C - 300°C for 2 h - 72 h.

[0024] More preferably, the reaction temperature is 140°C - 280°C, and the treatment time is 2 - 8 h.

[0025] The above-mentioned Na@AGV nanozyme can be used in the antibacterial field, for example, for preparing antibacterial agents, and can be used in industries such as food, biology, agriculture, and clinical.

[0026] Preferably, the drying in step (3) is freeze-drying, air-blowing drying, or natural air-drying.

[0027] (III) Beneficial effects

[0028] Compared with the prior art, the beneficial effects obtained by the present invention are as follows:

[0029] The present invention uses sodium as the doped metal of the Na@AGV nanozyme, and uses easily available and low-cost sodium salts such as sodium chloride as the sodium source raw material for synthesizing the nanozyme, which has the advantages of green safety and low price. And it is prepared by a one-step hydrothermal method with a simple process, avoiding the use of expensive raw materials, effectively reducing the preparation cost of the nanozyme and simplifying the preparation process. At the same time, since the nanozyme avoids the use of heavy metals and precious metals, the potential safety risks thereof are also avoided. Description of the drawings

[0030] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0031] Figure 1 It is a transmission electron microscope image (TEM) of the sodium ion-doped Na@AGV nanozyme prepared in Example 2. Among them, 1 is the morphological image under the scales of 5 nm (red square in the upper right corner) and 20 nm, 2 is the morphological image under the scale of 50 nm, and 3 is the morphological image under the scale of 100 nm.

[0032] Figure 2 It is an X-ray photoelectron spectroscopy (XPS) of the sodium ion-doped Na@AGV nanozyme prepared in Example 2. Among them, 1 is the full XPS spectrum of the Na@AGV nanozyme; 2 is the high-resolution XPS spectrum of C1s; 3 is the high-resolution XPS spectrum of O1s; 4 is the high-resolution XPS spectrum of Na1s.

[0033] Figure 3 It is an X-ray diffraction pattern (XRD) of the sodium ion-doped Na@AGV nanozyme prepared in Example 2.

[0034] Figure 4 It is a Fourier transform infrared spectroscopy image (FTIR) of the sodium ion-doped Na@AGV nanozyme prepared in Example 2.

[0035] Figure 5 The fluorescence image (PL) of sodium ion-doped Na@AGV nanozyme prepared in Example 2. In the figure, 1 is the measurement spectrum of the optimal excitation wavelength and emission wavelength, and 2 is the fluorescence emission spectrum of Na@AGV nanozyme when the excitation wavelength varies from 310 nm to 370 nm.

[0036] Figure 6 The in vitro bactericidal effect diagram of sodium ion-doped nanozyme prepared in Example 2 on Escherichia coli (E. coli). In the figure, A is the control group, B is the Na@AGV nanozyme experimental group, and C is the Na@AGV nanozyme + H2O2 experimental group.

[0037] Figure 7 The in vitro bactericidal effect diagram of sodium ion-doped Na@AGV nanozyme prepared in Example 2 on Staphylococcus aureus (S. aureus). In the figure, A is the control group, B is the Na@AGV nanozyme experimental group, and C is the Na@AGV nanozyme + H2O2 experimental group.

[0038] Figure 8 The in vitro bactericidal effect diagram of iron ion-doped nanozyme prepared in Example 12 on E. coli. In the figure, A is the control group, B is the Fe@AGV nanozyme experimental group, and C is the Fe@AGV nanozyme + H2O2 experimental group.

[0039] Figure 9 The in vitro bactericidal effect diagram of iron ion-doped nanozyme prepared in Example 12 on S. aureus. In the figure, A is the control group, B is the Fe@AGV nanozyme experimental group, and C is the Fe@AGV nanozyme + H2O2 experimental group. Detailed implementation manners

[0040] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which will be obvious to those skilled in the art.

[0044] The materials, reagents, instruments, etc. used in the following examples are all conventional materials, reagents, and instruments in this field, unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0045] The calculated molecular weights of the substances in the following examples are as follows:

[0046] Sodium chloride: 58.44, sodium sulfate: 142.04, sodium acetate (CH3COONa): 82.03,

[0047] VC (C6H8O6): 176.12, Ala-Gln (C8H 13 N3O3): 199.21.

[0048] Example 1 Preparation of Sodium-Doped Nanozymes from Different Raw Materials

[0049] When the inventors were studying the preparation of sodium metal-doped nanozymes, they considered a variety of different carbon-based and nitrogen-based raw materials. The raw materials selected were: L-alanyl-L-glutamate, ascorbic acid, citric acid, L-histidine, L-lysine, L-methionine, carboxymethyl chitosan, β-cyclodextrin, dopamine hydrochloride, usnic acid, kojic acid, glycine, glucose, and urea. Using the above raw materials, 8 pairs of combinations were formed, and the sodium source used in each combination was sodium chloride. The specific preparation method was as follows: Weighed precursor substances (including 0.01 g of sodium chloride) were dissolved together in 50 ml of deionized water. After ultrasonicating the mixture for 60 min, a uniformly dispersed solution was obtained. The solution was transferred into a high-pressure reaction kettle, and the reaction kettle was heated to 140 °C and reacted for 6 hours. The obtained solution was vacuum freeze-dried for 72 h, and the resulting powder was the "nanozyme". At the same time, the antibacterial and catalytic properties of the obtained 8 pairs of "nanozymes" were measured. The antibacterial and catalytic properties of the nanozymes were verified by the in vitro antibacterial effects on S. aureus and E. coli. The specific method was as follows:

[0050] 1) Minimum inhibitory concentration experiment of E. coli: Transfer the E. coli monoclonal on the solid Luria - Bertani (LB) agar plate to 10 mL of LB liquid medium, and then place the medium in a constant temperature oscillator at 37 °C and culture for 6 h at a rotation speed of 260 rpm. After the culture, transfer the above bacterial liquid to 4 mL of LB liquid medium again with a 100 - fold dilution factor and continue to culture under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After the activation is completed according to the above steps, centrifuge the obtained bacterial liquid at 1000 rpm for 15 min, remove the supernatant, and wash it with normal saline three times or more. Dilute the washed bacterial liquid ten - fold and place it in an enzyme - linked immunosorbent assay (ELISA) reader to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm). The measured value should be about 0.1 (the experimental measured value is OD 595 = 0.13). After obtaining the OD value, dilute the original bacterial dispersion stock solution by 500 - fold to obtain the E. coli bacterial dispersion for the experiment. Subsequently, in different 1.5 - mL centrifuge tubes, add 0.25 mL of the nanozyme prepared from different raw materials and the bacterial liquid respectively. At the same time, set up the H2O2 experimental group, and add 0.01 mL of H2O2 (C = 100 mM) additionally in the H2O2 experimental group to test the catalytic activity of the nanozyme. All samples are placed in an incubator at 37 °C for incubation. After the incubation, take out 100 μL of each group of dispersion and spread it on the solid LB agar plate medium. Place the plates of each group after spreading in an incubator at 37 °C for 6 h. After the culture, place the plates of each group in a gel imager for photographing and recording to obtain the in vitro antibacterial results of E. coli against the Na@AGV nanozyme.

[0051] 2) In vitro antibacterial experiment of S. aureus: Transfer the S. aureus monoclonal on the solid LB agar plate to 10 mL of LB liquid medium, place the medium in a constant temperature oscillator at 37 °C and culture for 6 h at a rotation speed of 260 rpm. After the culture, transfer the above bacterial liquid to 4 mL of LB liquid medium again with a 100 - fold dilution factor and continue to culture under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After the activation is completed according to the above steps, centrifuge the obtained bacterial liquid at 1000 rpm for 15 min, remove the supernatant, and wash it with normal saline three times or more. Dilute the washed bacterial liquid ten - fold and place it in an ELISA reader to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm) to be about 0.1 (the experimental reference OD 595

[0052] (when OD = 0.12), after obtaining the OD value, dilute the original bacterial dispersion solution by 500 times to obtain the S.aureus bacterial dispersion solution for the experiment. Set the in vitro antibacterial experiment of S.aureus into two types of experimental groups: (1) Na@AGV with different raw materials + S.aureus; (2) different raw materials Na@AGV + S.aureus + H2O2. Subsequently, in a 1.5 mL centrifuge tube, according to the above experimental settings, add Na@AGV nanozyme (the nanozyme is gradient diluted) and 0.25 ml of the bacterial solution to each 1.5 mL centrifuge tube. In the second group, additionally add 0.01 mL of H2O2 (C = 100 mM) to test the catalytic activity of the nanozyme. Both groups are placed in an environment of 37 °C for incubation. After incubation, take out 100 μL of each group of dispersion solution and coat it on a solid LB agar plate medium. Place the coated culture media of each group in an incubator at 37 °C for 6 h. After the culture is completed, place the culture media of each group in a gel imager for photographing and recording to obtain the in vitro antibacterial results of S.aureus of the Na@AGV nanozyme.

[0053] The antibacterial experimental results of the nanozymes prepared from the above 8 different raw materials against two bacteria are shown in Table 1.

[0054] Table 1 Comparison results of antibacterial and catalytic properties of nanozymes prepared from different raw materials

[0055] As can be seen from Table 1, most of the nanozymes prepared from different raw materials cannot simultaneously have antibacterial and catalytic properties. Only the nanozyme with L-alanyl-L-glutamate and ascorbic acid as raw materials has both antibacterial and catalytic properties.

[0056] Example 2 Synthesis of sodium ion-doped nanozyme

[0057] This example provides a sodium ion-doped Na@AGV nanozyme and a preparation method of the Na@AGV nanozyme. This method uses sodium chloride (NaCl), L-alanyl-L-glutamate (Ala-Gln) and ascorbic acid (Vitamin C VC) as precursors to prepare the Na@AGV nanozyme by self-assembly. Among them, the molar ratio of sodium, nitrogen and carbon elements is: 1:111.7:476.6. The specific method is: dissolve 0.7 g of Ala-Gln, 0.5 g of VC and 0.01 g of NaCl in 50 mL of deionized water, ultrasonically treat the mixed solution for 60 min to obtain a uniformly dispersed solution. Then, transfer the solution into a high-pressure reaction kettle, heat the reaction kettle to 280 °C, react for 2 h, and perform vacuum freeze-drying on the obtained solution for 72 h. The obtained powder is the Na@AGV nanozyme.

[0058] After the preparation is completed, the single-preparation raw material cost of the Na@AGV nanozyme prepared in this example is calculated with that of several nanozymes introduced in the background technology, namely Fe3O4-MWCNT-Hemin, CuDA@PDA, and gold nanozyme. The specific results are shown in Table 2.

[0059] Table 2 Comparison of single-preparation raw material costs of different nanozymes

[0060] The above reagent price data comes from the official website of Aladdin Reagents (https: / / www.aladdin-e.com / zh_cn / ). The unit price in Table 2 is the unit price per gram of the precursor, and the total price is the cost required for synthesizing a nanozyme once according to the disclosed specific method. According to the cost accounting comparison of the precursor substances, the raw materials of the Na@AGV nanozyme of this application are not only common and easily available, but also the cost is significantly lower than that of other existing nanozymes.

[0061] Example 3 Synthesis of sodium-ion doped nanozyme

[0062] This example provides a sodium-ion doped Na@AGV nanozyme and a preparation method thereof. The method uses sodium sulfate (Na2SO4), Ala-Gln, and VC as precursors to prepare the Na@AGV nanozyme by self-assembly. Among them, the molar ratio of sodium, nitrogen, and carbon elements is: 1:7.5:32. The specific method is as follows: Dissolve 0.7 g of Ala-Gln, 0.5 g of VC, and 0.1 g of Na2SO4 in 50 mL of deionized water, and ultrasonicate the mixture for 60 min to obtain a uniformly dispersed solution. Then, transfer the solution into a high-pressure reaction kettle, heat the reaction kettle to 140 °C, react for 5 h, and subject the obtained solution to vacuum freeze-drying for 72 h. The obtained powder is the Na@AGV nanozyme.

[0063] Example 4 Synthesis of sodium-ion doped nanozyme

[0064] This example provides a sodium-ion doped Na@AGV nanozyme and a preparation method thereof. The method uses sodium acetate (NaAc), Ala-Gln, and VC as precursors to prepare the Na@AGV nanozyme by self-assembly. Among them, the molar ratio of sodium, nitrogen, and carbon elements is: 1:2.48:64.64. The specific method is as follows: Dissolve 0.2 g of Ala-Gln, 2 g of VC, and 0.1 g of NaAc in 50 mL of deionized water, and ultrasonicate the mixture for 60 min to obtain a uniformly dispersed solution. Then, transfer the solution into a high-pressure reaction kettle, heat the reaction kettle to 200 °C, react for 5 h, and subject the obtained solution to vacuum freeze-drying for 72 h. The obtained powder is the Na@AGV nanozyme.

[0065] Synthesis of Sodium-Ion-Doped Nanozyme in Example 5

[0066] This example provides a sodium-ion-doped Na@AGV nanozyme and a preparation method thereof. The method prepares the Na@AGV nanozyme by self-assembly using NaCl, Ala-Gln, and VC as precursors. Among them, the molar ratio of sodium, nitrogen, and carbon elements is: 1:8.82:103.29. The specific method is as follows: Dissolve 0.5 g of Ala-Gln, 2 g of VC, and 0.05 g of NaCl in 50 mL of deionized water, and ultrasonicate the mixture for 60 min to obtain a uniformly dispersed solution. Then, transfer the solution into a high-pressure reaction kettle, heat the reaction kettle to 140 °C, and react for 5 h. Add 0.1 g of Ala-Gln to the reacted solution, ultrasonicate it under vacuum for 2 h, and then place the solution in an oven at 50 °C for drying for 72 h. The obtained powder is the Na@AGV nanozyme.

[0067] TEM Experiment of Na@AGV Nanozyme in Example 6

[0068] Dilute the Na@AGV nanozyme prepared in Example 2 with water to colorless and transparent according to the method disclosed in the national standard GB / T 42208-2022 "Nanotechnology - Transmission Electron Microscopy Image Method for Measuring the Particle Size of Nanoparticles in Multiphase Systems". Then, drop the solution onto a clean copper grid, dry it thoroughly, and prepare a sample to be measured. Use a transmission electron microscope (acceleration voltage of 200 kV) to measure the sample to be measured, and obtain the transmission electron microscope image of the Na@AGV nanozyme. The results are as Figure 1 shown. Among them Figure 1-1 are the morphological diagrams of the Na@AGV nanozyme with 5 nm (upper right corner) and 20 nm, Figure 1-2 and 1-3 are the morphological diagrams of the Na@AGV nanozyme with 50 nm and 100 nm respectively. It can be seen from Figure 1 that the Na@AGV nanozyme is a spherical shape with uniform morphology, which conforms to the morphological characteristics of the nanozyme.

[0069] XPS Experiment of Na@AGV Nanozyme in Example 7

[0070] Place the dried Na@AGV nanozyme in Example 2 on an X-ray photoelectron spectrometer (the measurement method refers to GB / T 19500-2004 "General Rules for X-Ray Photoelectron Spectroscopy Analysis Method") to obtain the XPS energy spectrum of the Na@AGV nanozyme and obtain the atomic valence state and molecular structure of the Na@AGV nanozyme. The results are as Figure 2 shown, Figure 2 is the XPS diagram of the Na@AGV nanozyme. It can be seen from Figure 2-4It can be seen that obvious peaks appear in the XPS spectrum, which confirms the existence of Na in the Na@AGV nanozyme, and the Na content in the Na@AGV nanozyme is measured to be 0.83 wt% by ICP-MS. From the high-resolution XPS spectra of C1s and O 1s of the Na@AGV nanozyme ( Figure 2-3 , Figure 2-2 ), a new peak can be observed at about 288.5 eV and 532.5 eV respectively, which belongs to Na-C and Na-O, and this confirms the coordination form of Na in the Na-CDs ( Figure 2-2 , Figure 2-3 ). As shown in Figure 2-2 , Figures 2-3, the peak separation results of C and O in Na@AGV show that the two have similar coordination, that is, the coordination forms of C and O are confirmed.

[0071] Example 8 XRD Experiment of Na@AGV Nanozyme

[0072] The Na@AGV nanozyme prepared in Example 2 was fully freeze-dried and pressed into a sample tablet. The Na@AGV nanozyme sample was measured using a Rigaku Ultima diffractometer (with Cu Kα X-rays as the excitation source and a scanning rate of 5° / min) to obtain an X-ray diffraction image. The results are shown in Figure 3 . As can be seen from Figure 3 , the curve of the X-ray diffraction pattern of the Na@AGV nanozyme nanoparticles bulges at the characteristic position of 25.7°, proving that the Na@AGV nanoparticles were successfully prepared.

[0073] Example 9 FTIR Experiment of Na@AGV Nanozyme

[0074] The Na@AGV nanozyme prepared in Example 2 was fully freeze-dried and pressed into a sample tablet, and a Fourier transform infrared spectrometer was used to measure the Na@AGV nanozyme (for the specific measurement method, see GB / T21186-2007 "Fourier Transform Infrared Spectrometer") to obtain the FTIR image of the sample. The results are shown in Figure 4 . Through FTIR spectral analysis, the bond energy vibrations of the Na@AGV nanozyme in the range of 500-4000 cm -1 were compared. The FTIR spectrum of the Na@AGV nanozyme shows peaks at 3229.7 cm -1 and 2927.89 cm -1 , which are attributed to N-H / O-H and C-H stretching and bending vibrations respectively; the peaks at 2375.87 cm -1 and 1666.19 cm -1 correspond to the stretching and bending vibrations of C-Na and C=O / C=C respectively; the peaks at 1593.87 cm -1 and 1357.15 cm-1 correspond to N-H and -CH3 respectively; the peaks are at 1228.43 cm -1 and 1107.9 cm -1 which are attributed to the stretching vibrations of C-N and C-O respectively. The XPS spectrum of Na@AGV nanozyme ( Figure 2-1 ) shows four main peaks: Na 2p (1071.25 eV), C 1s (284.8 eV) and O 1s (532.15 eV). The O 1s spectrum of Na@AGV nanozyme shows three peaks at 533.25, 532.55 and 531.65 eV, corresponding to C=O / C-OH, O-Na and -OH. The C 1s spectrum shows three peaks at 288.6, 286.15 and 284.75 eV, corresponding to C-Na, C=O bond and C-C bond respectively. The results of the FT-IR spectrum are consistent with those of the XPS spectrum, confirming the groups and chemical composition of Na@AGV nanozyme.

[0075] Example 10 PL experiment of Na@AGV nanozyme

[0076] The Na@AGV nanozyme solution was fully diluted and dropped into a microcuvette. The excitation and emission wavelengths of Na@AGV nanozyme were measured using a fluorescence spectrometer. After the measurement, the PL energy spectrum of Na@AGV nanozyme was obtained (for the specific measurement method, see GB / T 40359-2021 "Test Methods and Requirements for Photoluminescent Coatings of Timekeeping Instruments"). The results are as Figure 5 shown, Figure 5 which is the PL energy spectrum of Na@AGV nanozyme. It can be seen from Figure 5-1 that the optimal excitation wavelength and emission wavelength for the extensive absorption of Na@AGV nanozyme in the spectrum are located at 368 nm and 441 nm respectively, Figure 5-2 which is the fluorescence emission spectrum of Na@AGV nanozyme when the excitation wavelength varies from 310 - 370 nm. It can be seen from Figure 5 that when the excitation is at 370 nm, Na@AGV nanozyme has the best fluorescence emission intensity. And when the excitation wavelength varies from 310 - 370 nm, the shift of the fluorescence emission peak of Na@AGV nanozyme with the change of the excitation wavelength is very small, which may be because Na@AGV nanozyme has a relatively uniform surface state.

[0077] Example 11 Determination of in vitro antibacterial effects of Na@AGV nanozyme against E. coli and S. aureus

[0078] In this example, S. aureus and E. coli were selected to verify the in vitro antibacterial effects of the Na@AGV nanozyme prepared in Example 1. The specific method is as follows:

[0079] 1) Minimum inhibitory concentration experiment of E. coli: Transfer the E. coli monoclonal on the solid LB agar plate to 10 mL of LB liquid medium, and then place the medium in a constant temperature oscillator at 37 °C and culture it at a rotation speed of 260 rpm for 6 h. After the culture is completed, dilute the above bacterial liquid by 100 times and transfer it to 4 mL of LB liquid medium again, and continue to culture it under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After activation according to the above steps, centrifuge the obtained bacterial liquid at 1000 rpm for 15 min, remove the supernatant, and wash it with normal saline three times or more. Dilute the washed bacterial liquid by ten times and place it in an enzyme-labeling instrument to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm), and the measured value can be about 0.1 (the actual measured value of the experiment is OD 595 = 0.13). After obtaining the OD value, dilute the original bacterial dispersion stock solution by 500 times to obtain the E. coli bacterial dispersion for the experiment. Set the in vitro antibacterial experiment of E. coli into two groups: (1) Na@AGV + E. coli; (2) Na@AGV + E. coli + H2O2. Subsequently, in 1.5 mL centrifuge tubes, according to the above experimental settings, add 0.25 mL of Na@AGV nanozyme (the nanozyme is serially diluted) and bacterial liquid to each 1.5 mL centrifuge tube. In the second group, additionally add 0.01 mL of H2O2 (C = 100 mM) to test the catalytic activity of the nanozyme. Both groups are placed in an environment at 37 °C for incubation. After the incubation is completed, take out 100 μL of the dispersion of each group and spread it on the solid LB agar plate medium. Place the plates of each group of culture media in an incubator at 37 °C and culture for 6 h. After the culture is completed, place the culture media of each group in a gel imaging system for photographing and recording to obtain the in vitro antibacterial results of E. coli of Na@AGV nanozyme, and the results are as Figure 6 shown.

[0080] Figure 6 is the in vitro antibacterial effect diagram of Na@AGV nanozyme on E. coli, where (A) is the control group before and after the catalysis of E. coli, (B) is the plate coating diagram of the in vitro antibacterial of Na@AGV nanozyme on E. coli, and (C) is the antibacterial efficiency diagram of the in vitro antibacterial of Na@AGV nanozyme on E. coli after adding the substrate H2O2. It can be Figure 6 seen that compared with the control group, Na@AGV nanozyme itself has good antibacterial properties. Under the action of H2O2, Na@AGV nanozyme generates ROS, showing a better peroxidase effect. The in vitro antibacterial effect of Na@AGV nanozyme on E. coli is further improved, and it can effectively kill most bacteria.

[0081] 2) In vitro antibacterial experiment of S. aureus: Transfer the S. aureus monoclonal on the solid LB agar plate to 10 mL of LB liquid medium. Place the medium in a constant temperature oscillator at 37 °C and culture it at a rotation speed of 260 rpm for 6 h. After the culture, transfer the above bacterial solution to 4 mL of LB liquid medium again with a 100-fold dilution factor and continue to culture it under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After activation according to the above steps, centrifuge the obtained bacterial solution at a rotation speed of 1000 rpm for 15 min, remove the supernatant, and wash it with normal saline three times or more. Dilute the washed bacterial solution tenfold and place it in an enzyme-labeled instrument to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm) to be about 0.1 (the experimental reference OD 595 = 0.12). After obtaining the OD value, dilute the original bacterial dispersion stock solution by 500 times to obtain the S. aureus bacterial dispersion for the experiment. Set the in vitro antibacterial experiment of S. aureus into two groups: (1) Na@AGV + S. aureus; (2) Na@AGV + S. aureus + H2O2. Subsequently, in 1.5 mL centrifuge tubes, according to the above experimental settings, add 0.25 ml of Na@AGV nanozyme (the nanozyme is serially diluted) and bacterial solution to each 1.5 mL centrifuge tube. In the second group, additionally add 0.01 mL of H2O2 (C = 100 mM) to test the catalytic activity of the nanozyme. Both groups are placed in an environment at 37 °C for incubation. After incubation, take out 100 μL of the dispersion from each group and coat it on a solid LB agar plate medium. Place the coated culture media of each group in an incubator at 37 °C for 6 h. After the culture, place the culture media of each group in a gel imaging system for photographing and recording to obtain the in vitro antibacterial results of S. aureus of the Na@AGV nanozyme. The results are as Figure 7 shown.

[0082] Figure 7 is the in vitro antibacterial effect diagram of the Na@AGV nanozyme on S. aureus. Among them, (A) is the control group before and after the catalysis of S. aureus, (B) is the plate coating diagram of the Na@AGV nanozyme for in vitro antibacterial of S. aureus, and (C) is the antibacterial efficiency diagram of the Na@AGV nanozyme for in vitro antibacterial of S. aureus when catalyzed by the substrate H2O2. It can be Figure 7 seen that the Na@AGV nanozyme itself has good antibacterial properties. Under the action of H2O2, ROS is generated, showing good peroxidase effects. The in vitro antibacterial effect of the Na@AGV nanozyme on S. aureus is further improved, and it can effectively kill most bacteria.

[0083] Example 12 In vitro bactericidal effect of Fe@AGV nanozyme on E. coli and S. aureus

[0084] In addition to using sodium ions as raw materials for preparing nanozymes, the inventors also selected iron, which is used more frequently, as a raw material for preparing nanozymes to prepare Fe@AGV nanozymes. The preparation process of Fe@AGV nanozymes is as follows: Dissolve 0.7 g of Ala-Gln, 0.5 g of VC, and 0.01 g of FeSO4 in 50 mL of deionized water, and ultrasonically treat the mixture for 60 min to obtain a uniformly dispersed solution. Then, transfer the solution into a high-pressure reaction kettle, heat the reaction kettle to 280 °C, and react for 2 h. Vacuum freeze-dry the obtained solution for 72 h, and the obtained powder is the Fe@AGV nanozyme. After obtaining the Fe@AGV nanozyme, S. aureus and E. coli were selected to verify the in vitro antibacterial effect of the Fe@AGV nanozyme. The specific method is as follows:

[0085] 1) E. coli minimum inhibitory concentration experiment: Transfer the E. coli monoclonal on the solid LB agar plate to 10 mL of LB liquid medium, place the medium in a constant temperature oscillator at 37 °C, and culture it at a rotation speed of 260 rpm for 6 h. After the culture is completed, transfer the above bacterial solution to 4 mL of LB liquid medium again with a 100-fold dilution factor, and continue to culture it under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After activation according to the above steps, centrifuge the obtained bacterial solution at a rotation speed of 1000 rpm for 15 min, remove the supernatant, and wash it with physiological saline three times or more. Dilute the washed bacterial solution ten times and place it in an enzyme-labeling instrument to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm) to be about 0.1 (the experimental reference OD 595 = 0.11). After obtaining the OD value, dilute the original bacterial dispersion stock solution by 500 times to obtain the experimental E. coli bacterial dispersion for use. Set the E. coli in vitro antibacterial experiment into two groups: (1) Fe@AGV nanozyme + E. coli; (2) Fe@AGV nanozyme + E. coli + H2O2. Subsequently, in 1.5 mL centrifuge tubes, according to the above experimental settings, add Fe@AGV nanozyme (the nanozyme is gradient-diluted) and 0.25 mL of the bacterial solution to each 1.5 mL centrifuge tube. In the second group, additionally add 0.01 mL of H2O2 (C = 100 mM) to test the catalytic activity of the nanozyme. Both groups are placed in an incubator at 37 °C for incubation. After the incubation is completed, take out 100 μL of each group of dispersion liquid and spread it on a solid LB agar plate medium. Place the plated culture media of each group in an incubator at 37 °C for 6 h. After the culture is completed, place the culture media of each group in a gel imaging instrument for photographing and recording to obtain the in vitro antibacterial results of Fe@AGV nanozyme against E. coli, as Figure 8 shown.

[0086] Figure 8Figure for the in vitro antibacterial effect of Fe@AGV nanozyme against E. coli. Among them, (A) is the control group before and after the catalysis of E. coli, (B) is the plate coating diagram of the in vitro antibacterial effect of Fe@AGV nanozyme against E. coli, and (C) is the antibacterial efficiency diagram of the in vitro antibacterial effect of Fe@AGV nanozyme against E. coli after adding the substrate H2O2. It can be seen from Figure 8 that the Fe@AGV nanozyme itself also has a certain degree of antibacterial property. However, under the action of H2O2, the antibacterial effect shown is significantly lower than that of the Na@AGV nanozyme.

[0087] 2) In vitro antibacterial experiment of S. aureus: Transfer the S. aureus monoclonal on the solid LB agar plate to 10 mL of LB liquid medium. Place the medium in a constant temperature oscillator at 37 °C and culture it at a rotation speed of 260 rpm for 6 h. After the culture is completed, transfer the above bacterial liquid to 4 mL of LB liquid medium again with a 100-fold dilution factor and continue to culture it under the same conditions in a constant temperature oscillator at 37 °C for 15 h. After the activation is completed according to the above steps, centrifuge the obtained bacterial liquid at a rotation speed of 1000 rpm for 15 min, remove the supernatant, and wash it with physiological saline three times or more. Dilute the washed bacterial liquid ten times and place it in a microplate reader to measure the absorbance of the bacterial dispersion at 595 nm (OD at 595 nm) to be about 0.1 (the experimental reference OD 595 = 0.12). After obtaining the OD value, dilute the original bacterial dispersion stock solution by 500 times to obtain the experimental S. aureus bacterial dispersion for use. Set the in vitro antibacterial experiment of S. aureus into two groups: (1) Fe@AGV nanozyme + S. aureus; (2) Fe@AGV nanozyme + S. aureus + H2O2. Subsequently, in 1.5 mL centrifuge tubes, respectively according to the above experimental settings, add Fe@AGV nanozyme (the nanozyme is gradient diluted) and 0.25 ml of bacterial liquid to each 1.5 mL centrifuge tube. Add an additional 0.01 mL of H2O2 (C = 100 mM) to the second group to test the catalytic activity of the nanozyme. Both groups are placed in an environment at 37 °C for incubation. After the incubation is completed, take out 100 μL of each group of dispersion and coat it on a solid LB agar plate medium. Place the coated culture media of each group in an incubator at 37 °C for 6 h. After the culture is completed, place the culture media of each group in a gel imaging system for photographing and recording to obtain the in vitro antibacterial results of S. aureus of Fe@AGV nanozyme. The results are as Figure 9 shown.

[0088] Figure 9The figure shows the in vitro antibacterial effect of Fe@AGV nanozyme on S. aureus. Among them, (A) is the control group before and after the catalysis of S. aureus, (B) is the plate coating diagram of the in vitro antibacterial effect of Fe@AGV nanozyme on S. aureus, and (C) is the antibacterial efficiency diagram of Fe@AGV nanozyme on the in vitro antibacterial effect of S. aureus when catalyzed by the substrate H2O2. As can be seen from Figure 9 it that the Fe@AGV nanozyme itself has a certain resistance to S. aureus. Under the action of H2O2, it does not show good peroxidase effect, and the in vitro antibacterial effect of Fe@AGV on S. aureus has not been significantly improved, and it cannot effectively kill most bacteria.

[0089] According to Figure 6 , Figure 7 , Figure 8 and Figure 9 the results show that the same raw materials synthesis will form corresponding catalytic groups, and the catalytic groups will be adapted to different metals, and only the appropriate metals can show the catalytic activity of the nanozyme.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sodium-doped Na@AGV nanozyme, characterized in that, A sodium-doped nanozyme is obtained by mixing a sodium ion salt with vitamin C and L-alanyl-L-glutamate in an inorganic solvent to form a precursor solution, followed by cooling after one-pot hydrothermal treatment and drying. The molar ratio of sodium element: nitrogen element: carbon element in the sodium ion salt, vitamin C and L-alanyl-L-glutamate is 1: (2 to 120): (10 to 500). The temperature of the one-pot hydrothermal treatment is 100 °C to 300 °C.

2. The sodium-doped Na@AGV nanozyme according to claim 1, wherein, The average particle size of the Na@AGV nanozyme is 0.1 to 10 nm.

3. The sodium-doped Na@AGV nanozyme according to claim 1, wherein The sodium ion salt is sodium chloride, sodium acetate or sodium sulfate.

4. The preparation method of the Na@AGV nanozyme according to any one of claims 1-3, characterized in that, The steps are as follows: (1) Add the sodium ion salt, vitamin C and L-alanyl-L-glutamate together into water and dissolve them, and mix well to form a precursor solution; (2) Transfer the precursor solution obtained in step (1) to a reaction kettle for reaction, and cool it after the reaction ends; (3) Dry the reaction solution cooled in step (2) to obtain the sodium-doped nanozyme.

5. The preparation method according to claim 4, characterized in that, In step (1), the concentration of vitamin C is 0.01 - 1 mol / L; the concentration of L-alanyl-L-glutamate is 0.01 - 1 mol / L; the concentration of the sodium ion salt is 0.001 - 1 mol / L.

6. The preparation method according to claim 4, wherein, The reaction in step (2) is carried out at 100 °C - 300 °C for 2 h - 72 h.

7. The preparation method according to claim 6, characterized in that, The reaction temperature is 140 °C - 280 °C, and the treatment time is 2 - 8 h.

8. Use of the Na@AGV nanozyme according to any one of claims 1 - 3 in the field of antibacterial.

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