Molybdenum nitride nano-enzyme material with multi-enzyme activity as well as preparation method and application of molybdenum nitride nano-enzyme material

Abstract

The invention discloses a molybdenum nitride nano-enzyme material with multi-enzyme activity as well as a preparation method and application thereof, and particularly relates to the field of nano-materials. The molybdenum nitride nano-enzyme material disclosed by the invention is MoN nano-enzyme with a nano-structure, and the size range is 5nm to 2mu m. The method has great application in the fields of biosensing detection and antibacterial treatment. The molybdenum nitride nano-enzyme material with multienzyme activity prepared by the invention has a unique electronic structure and excellent nano-enzyme catalytic performance, so that the molybdenum nitride nano-enzyme material has huge application potential in the aspects of biosensing, antibacterial treatment and the like.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and particularly to a molybdenum nitride nanozyme material with multi-enzyme activity, a preparation method thereof, and an application thereof. Background Art

[0002] With the rapid development of nanotechnology and the biomedical field, nanozymes have gradually become ideal substitutes for natural enzymes due to their high stability, low cost, and easy batch preparation. Natural enzymes (such as horseradish peroxidase HRP) are prone to inactivation, have high purification costs, and have strict requirements for storage conditions in practical applications, which limit their wide application in complex environments. At the same time, common nanozymes (such as Fe3O4, CeO2) usually only mimic a single enzyme activity, making it difficult to meet the needs of multiple catalytic reactions, and the overall catalytic efficiency still needs to be improved. In addition, traditional antibacterial materials (such as silver nanoparticles) have the risks of inducing bacterial drug resistance and high cytotoxicity, and the antibacterial mechanism based on reactive oxygen species (ROS) is limited by the low ROS generation efficiency of existing materials, affecting the antibacterial performance and stability. Summary of the Invention

[0003] In view of the above disadvantages of the prior art, the purpose of the present invention is to provide a molybdenum nitride nanozyme material with multi-enzyme activity, a preparation method thereof, and an application thereof, which can effectively solve the problems of single nanozyme activity and low nanozyme activity existing in the prior art.

[0004] To achieve the above purpose or other purposes, the present invention is realized through the following technical solutions.

[0005] A molybdenum nitride nanozyme material with multi-enzyme activity, wherein the molybdenum nitride nanozyme material is a MoN nanozyme with a nanostructure, and the size range is 5 nm to 2 μm.

[0006] The molybdenum nitride nanozyme material of the present invention is rich in active sites on the surface and has peroxidase, catalase, and oxidase catalytic activities at the same time.

[0007] The molybdenum nitride nanozyme material of the present invention has a nanostructure, including but not limited to nanoparticles, nanoflowers, nanosheets, and quantum dots.

[0008] The present invention also provides a use of the above molybdenum nitride nanozyme material with multi-enzyme activity in the fields of biosensing detection and antibacterial therapy.

[0009] The preparation method of the molybdenum nitride nanozyme material with multi-enzyme activity protected by the present invention includes the following steps:

[0010] Dissolve a molybdenum source and a nitrogen source in a solvent, and generate a precursor containing molybdenum and nitrogen elements through a hydrothermal / solvothermal reaction; calcine the precursor under an inert atmosphere to obtain the molybdenum nitride nanozyme material;

[0011] Alternatively, the molybdenum source is directly calcined in an ammonia atmosphere to directly obtain the molybdenum nitride nanozyme material.

[0012] In an example of the present invention, the molybdenum source is selected from one or more of ammonium molybdate, sodium molybdate, molybdenum powder, molybdenum sulfide, ammonium tetrathiomolybdate, phosphomolybdic acid, and molybdenum chloride.

[0013] In an example of the present invention, the nitrogen source is selected from one or more of urea, melamine, ammonia water, and ammonia gas.

[0014] In an example of the present invention, the dosage ratio of the molybdenum source to the nitrogen source satisfies that the ratio of molybdenum atoms to nitrogen atoms is 1:(1 - 10).

[0015] In an example of the present invention, the hydrothermal / solvothermal reaction temperature is 100°C to 250°C, and the reaction time is 5 h to 25 h.

[0016] In an example of the present invention, the solvent is selected from N,N-dimethylformamide. The dosage of the solvent satisfies that the concentration of the formed molybdenum source solution is 0.01 mM to 10 mM.

[0017] In an example of the present invention, the calcination temperature is 200°C to 1200°C, the calcination time is 3 h to 12 h, and the heating rate is 1 - 50°C / min.

[0018] The molybdenum nitride nanozyme material with multi-enzyme activity prepared by the present invention exhibits great application potential in biosensing and antibacterial therapy due to its unique electronic structure and excellent nanozyme catalytic performance. By optimizing the synthesis conditions of molybdenum nitride, such as precursor selection, reaction temperature, and nitridation process, its electronic structure and surface chemical state are precisely regulated, thereby endowing the material with multiple enzyme activities, including peroxidase, oxidase, and superoxide dismutase activities. This multiple enzyme activity can not only effectively replace natural enzymes but also significantly expand the application potential of the material in the fields of multifunctional detection and antibacterial.

[0019] In the present invention, the molybdenum nitride nanozyme material with multi-enzyme activity is obtained by hydrothermal / solvothermal and direct calcination methods. The obtained molybdenum nitride nanozyme material has a uniform nanostructure and has the characteristics of high catalytic activity, stability, and low cost. Using the molybdenum nitride nanozyme material prepared by the present invention, in the detection field, through the catalytic substrate color reaction, it is possible to achieve the detection of hydrogen peroxide (H2O2), glucose, nitrate (NO3 - ) and nitrite (NO2 -Sensitive detection of biomarkers such as ; in the field of antibacterial and antibacterial therapy, it shows broad-spectrum antibacterial effects against Gram-positive / negative bacteria by generating reactive oxygen species, supplying oxygen, and decomposing bacterial biofilms, and can relieve the hypoxic condition in the infected area. Therefore, the molybdenum nitride nanozyme material of the present invention solves the problems of poor stability, high cost of traditional natural enzymes, and single catalytic function of existing nanozymes, and has significant application potential. Brief Description of the Drawings

[0020] Figure 1 It is a scanning electron microscope (SEM) image of the molybdenum nitride nanozyme material prepared in Example 1 of the present invention.

[0021] Figure 2 It is the test of peroxidase-like activity of the materials prepared in Examples 1-4 and Comparative Examples 1-4 of the present invention.

[0022] Figure 3 It is the glucose detection result of the molybdenum nitride nanozyme material prepared in Example 1 of the present invention.

[0023] Figure 4 It is the test result of the antibacterial performance of the materials prepared in Examples 1-4 and Comparative Examples 1-4 of the present invention.

[0024] Figure 5 It is the test of catalase-like activity of the materials prepared in Examples 1-4 and Comparative Examples 1-4 of the present invention. Detailed Description of the Invention

[0025] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, without conflict, the following examples and the features in the examples can be combined with each other. It should also be understood that the terms used in the embodiments of the present invention are for describing specific specific implementation manners, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0026] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", and "one" cited in this specification are only for the convenience of clear description and are not used to limit the scope of implementation of the present invention. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope of implementation of the present invention.

[0027] The present invention provides a molybdenum nitride nanozyme material with multi-enzyme activity. The molybdenum nitride nanozyme material is a MoN nanozyme with a nanostructure, and the size range is 5 nm to 2 μm. The nanostructure of the molybdenum nitride nanozyme material includes but is not limited to nanoparticles, nanoflowers, nanosheets, and quantum dots. The surface of the molybdenum nitride nanozyme material of the present invention is rich in active sites and simultaneously has peroxidase, catalase, and oxidase catalytic activities.

[0028] The molybdenum nitride nanozyme material with multi-enzyme activity provided by the present invention has good application uses in the fields of biosensing detection and antibacterial therapy.

[0029] The method for preparing the molybdenum nitride nanozyme material with multi-enzyme activity of the present invention can select hydrothermal / solvothermal reaction or direct calcination method. The steps include: Hydrothermal / solvothermal reaction: Dissolve the molybdenum source and the nitrogen source in a solvent, and generate a precursor containing molybdenum element and nitrogen element through hydrothermal / solvothermal reaction; calcine the precursor under an inert atmosphere to obtain the molybdenum nitride nanozyme material. Direct calcination method: Directly calcine the molybdenum source under an ammonia atmosphere to directly obtain the molybdenum nitride nanozyme material.

[0030] In one embodiment, the molybdenum source is selected from one or more of ammonium molybdate, sodium molybdate, molybdenum powder, molybdenum sulfide, ammonium tetrathiomolybdate, phosphomolybdic acid, molybdenum chloride, that is, the molybdenum source can be selected from any one of the above-listed molybdenum source types. For example, the molybdenum source is ammonium molybdate or sodium molybdate or molybdenum powder or molybdenum sulfide or ammonium tetrathiomolybdate or phosphomolybdic acid or molybdenum chloride, etc. The molybdenum source can also be a combination of any two or more of the above-listed molybdenum source types. For example, the molybdenum source is a composition of ammonium molybdate and sodium molybdate, or a composition of ammonium molybdate and molybdenum powder, or a composition of molybdenum powder, ammonium molybdate, and sodium molybdate, or a composition of ammonium molybdate, sodium molybdate, molybdenum powder, and molybdenum sulfide, etc., and will not be listed one by one here. It should be noted that the molybdenum source includes but is not limited to the above-listed types, and it can also select molybdenum sources not listed above. When the molybdenum source is a composition of two or more, the ratio between the components in the composition is not limited and can be mixed in any ratio, as long as the ratio requirement of the total amount of the molybdenum source and the amount of the nitrogen source is met.

[0031] In one embodiment, the nitrogen source is selected from one or more of urea, melamine, ammonia water, and ammonia gas, that is, the nitrogen source can be selected from any one of the above-listed nitrogen source types. For example, the nitrogen source is urea or melamine or ammonia water or ammonia gas. The nitrogen source can also be a combination of any two or more of the above-listed nitrogen source types. For example, the nitrogen source is a composition of urea and melamine, or a composition of urea and ammonia water, or a composition of urea, melamine, and ammonia water, etc., which will not be listed one by one here. It should be noted that the nitrogen source includes but is not limited to the above-listed types, and it can also select nitrogen sources not listed above. When the nitrogen source is a composition of two or more components, the ratio between the components in the composition is not limited and can be mixed in any ratio, as long as the ratio requirement of the total amount of molybdenum source to the total amount of nitrogen source is met.

[0032] In one embodiment, the dosage ratio of the molybdenum source to the nitrogen source satisfies that the ratio of molybdenum atoms to nitrogen atoms is 1:(1 - 10).

[0033] In one embodiment, the solvent is selected from N,N-dimethylformamide. The dosage of the solvent satisfies that the concentration of the formed molybdenum source solution is 0.01 mM - 10 mM.

[0034] In one embodiment, the hydrothermal / solvothermal reaction temperature is 100°C - 250°C, and the reaction time is 5 h - 25 h.

[0035] In one embodiment, the calcination temperature is 200°C - 1200°C, the calcination time is 3 h - 12 h, and the heating rate is 1 - 50°C / min. If the calcination temperature is too low, the molybdenum nitride structure cannot be formed, and it remains a nitride precursor or molybdenum oxide, and its enzyme activity performance is low. If the calcination temperature is too high, the molybdenum nitride will decompose at high temperature to form molybdenum metal nanospheres, etc., which do not have enzyme activity.

[0036] In one embodiment, in the direct calcination method, the ammonia gas atmosphere is flowing to ensure an excess of ammonia gas.

[0037] The technical solutions of the present invention will be described in detail below through several specific examples and comparative examples. Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by conventional methods in the art. Unless otherwise specified in the examples of the present invention, the detection methods used are conventional detection methods in the industry.

[0038] Example 1

[0039] Place 1 g of ammonium tetrathiomolybdate in a tube furnace, calcine it at 700°C for 6 h in an NH3 atmosphere with a heating rate of 10°C / min to obtain a molybdenum nitride nanozyme material.

[0040] Example 2

[0041] Place 1 g of ammonium tetrathiomolybdate in a tube furnace, calcine it at 800 °C for 4 h in an NH3 atmosphere with a heating rate of 5 °C / min to obtain molybdenum nitride nanoparticles.

[0042] Example 3

[0043] (1) Dissolve 0.5 g of ammonium molybdate and 2 g of urea in 30 mL of DMF, stir evenly at room temperature, transfer the above precursor solution to a high-pressure reactor, and react at 180 °C for 12 hours.

[0044] (2) Centrifuge, collect, wash, dry, and then place it in a tube furnace, calcine it at 650 °C for 3 h in an Ar atmosphere to obtain molybdenum nitride nanosheets.

[0045] Example 4

[0046] (1) Dissolve 0.5 g of ammonium tetrathiomolybdate in 30 mL of DMF, stir evenly, transfer the above solution to a high-pressure reactor, and react at 200 °C for 15 hours.

[0047] (2) Centrifuge, collect, wash, dry, and then place it in a tube furnace, calcine it at 650 °C for 2 h in an NH3 atmosphere to obtain molybdenum nitride nanosheets.

[0048] Example 5

[0049] The difference between this example and Example 1 is that it is calcined at 200 °C for 12 h with a heating rate of 50 °C / min.

[0050] Example 6

[0051] The difference between this example and Example 1 is that it is calcined at 1200 °C for 3 h with a heating rate of 40 °C / min.

[0052] Example 7

[0053] The difference between this example and Example 1 is that it is calcined at 500 °C for 12 h with a heating rate of 5 °C / min.

[0054] Example 8

[0055] The difference between this example and Example 3 is that sodium molybdate and melamine with a molybdenum atom to nitrogen atom ratio of 1:5 are selected, the reaction temperature is 250 °C, the reaction time is 5 h, and it is calcined at 1200 °C for 3 h in an Ar atmosphere.

[0056] Example 9

[0057] The difference between this example and Example 3 is that molybdenum sulfide and urea with a molybdenum atom to nitrogen atom ratio of 1:2 are selected, the reaction temperature is 100 °C, the reaction time is 25 h, and it is calcined at 200 °C for 12 h in an Ar atmosphere.

[0058] Example 10

[0059] The difference between this example and Example 4 is as follows: Phosphomolybdic acid with a molybdenum atom to nitrogen atom ratio of 1:1 and ammonia water are selected. The reaction temperature is 200 °C, the reaction time is 10 h, and it is calcined at 1000 °C for 6 h in an Ar atmosphere.

[0060] Comparative Example 1

[0061] Place 1 g of ammonium molybdate in a tube furnace, calcine it at 150 °C for 3 h in an NH3 atmosphere with a heating rate of 10 °C / min to obtain molybdenum nitride nanoparticles.

[0062] Comparative Example 2

[0063] Place 1 g of ammonium tetrathiomolybdate in a tube furnace, calcine it at 150 °C for 1 h in an NH3 atmosphere with a heating rate of 5 °C / min to obtain molybdenum nitride nanoparticles.

[0064] Comparative Example 3

[0065] (1) Dissolve 0.5 g of ammonium molybdate and 2 g of urea in 30 mL of DMF, stir evenly at room temperature, transfer the above precursor solution to a high-pressure reaction kettle, and react at 180 °C for 12 hours.

[0066] (2) Centrifuge, collect, wash, dry, and then place it in a tube furnace, calcine it at 150 °C for 3 h in an Ar atmosphere to obtain molybdenum nitride nanosheets.

[0067] Comparative Example 4

[0068] (1) Dissolve 0.5 g of ammonium tetrathiomolybdate in 30 mL of DMF, stir evenly, transfer the above solution to a high-pressure reaction kettle, and react at 200 °C for 15 hours.

[0069] (2) Centrifuge, collect, wash, dry, and then place it in a tube furnace, calcine it at 150 °C for 2 h in an NH3 atmosphere to obtain molybdenum nitride nanosheets.

[0070] Performance Test

[0071] 1. Take the molybdenum nitride nanozyme material prepared in Example 1 for scanning electron microscopy (SEM) testing. The results are as Figure 1 shown. It can be seen from the figure that the size range of the prepared nanoparticles is 20 nm - 2 μm.

[0072] 2. 20 μg of the materials prepared in Examples 1-4 and Comparative Examples 1-4 were respectively taken, and 20 μL of 40 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to 1 mL of acetic acid-sodium acetate buffer solution with a pH of 3.6. Then 10 μL of 1 M H2O2 solution was added, and after shaking for 1 min, the absorbance was measured at a wavelength of 652 nm using a UV-visible spectrophotometer. The peroxidase-like activity of the nanozyme material was reflected by the intensity of the absorbance. The reason is that peroxidase-like activity can decompose H2O2 to generate hydroxyl radicals, and the reaction between hydroxyl radicals and TMB will produce a blue product with an absorption peak at 652 nm. Therefore, the size of the enzyme activity can be illustrated by observing the intensity of the absorption peak at 652 nm. The absorbances measured for the systems with the molybdenum nitride nanozyme materials of Examples 1-4 were 1.28, 1.26, 1.19, and 1.04 respectively, and the absorbances measured for the materials of Comparative Examples 1-4 were 0.16, 0.08, 0.10, and 0.11 respectively. The specific results are as Figure 2 shown. It can be seen from Figure 2 that, compared with the comparative examples, the examples of the present invention have better peroxidase-like activity.

[0073] 3. The molybdenum nitride nanozyme material prepared in Example 1 was taken for glucose detection test. The specific test method was as follows: 1 mL of glucose solutions with concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM were respectively placed in different reaction vessels. Subsequently, 20 μL of 10 mg / mL glucose oxidase was added to each reaction vessel, and incubated at 37 °C for 20 minutes to allow glucose oxidase to decompose glucose and produce hydrogen peroxide. Then 20 μL of 40 mM TMB solution and 20 μg of the molybdenum nitride nanozyme material prepared in Example 1 were respectively added to the above glucose systems. The nanozyme activity would catalyze hydrogen peroxide to produce reactive oxygen species, triggering the oxidation of TMB. After TMB was oxidized, the solution would turn blue. After 3 min, 10 μL of H2SO4 (20%, v / v) concentrated sulfuric acid was added to the solution for reaction, and the solution turned yellow with an obvious absorption peak at 450 nm. The absorbances of the systems with different glucose concentrations were measured at a wavelength of 450 nm using a UV-visible spectrophotometer. The obtained absorbances were combined with the glucose concentration gradient to obtain a standard curve, as Figure 3 shown. Through linear fitting, it can be seen that its detection range is from 0.1 - 6 mM, with a good linear relationship. Through Figure 3 the glucose content in the sample can be quantitatively analyzed. At the same time, it can be seen from Figure 3 that the detection limit of the molybdenum nitride nanozyme material prepared in the examples of the present invention for glucose detection can reach 0.2 μM.

[0074] 4. The antibacterial effect of the nanozyme material was determined by the plate counting method. The specific method was as follows: 1.0 mL of pH buffer solution containing Gram-negative bacterium Escherichia coli was prepared, with a pH value of 5.4 and a bacterial concentration of 10 6 / ml. The above bacterial solution was taken and divided into 8 portions. 100 μg of molybdenum nitride nanomaterials of Examples 1 to 4 and Comparative Examples 1 to 4 and 50 μL of H2O2 (concentration 1 mM) were added to the above 8 portions of bacterial solution respectively to prepare different bacterial solution samples. After shaking well and standing for 30 min, then the above different bacterial solution samples were successively subjected to plate counting to obtain the bacterial concentration C after sterilization. The material sterilization efficiency = (1 - C / 10 6 ) × 100%. After calculation, the sterilization efficiencies of Examples 1 to 4 were 98%, 99%, 82%, and 89% respectively, and the sterilization efficiencies of Comparative Examples 1 to 4 were 6%, 9%, 5%, and 4% respectively, as specifically shown in Figure 4 shown, Figure 4 which was the statistical result of the sterilization efficiency of different materials. It can be seen from the figure that the sterilization effects of Examples 1 - 4 were all above 80%, and the sterilization capabilities of the comparative examples were all lower than 10%.

[0075] 5. 50 μg of the materials prepared in Examples 1 to 4 and Comparative Examples 1 to 4 were taken respectively and added to 5 mL of acetic acid - sodium acetate buffer solution with a pH of 3.6. Subsequently, 50 μL of 1M H2O2 solution was added successively. After shaking well for 1 min, the dissolved oxygen content in the above 8 portions of solution was detected with a dissolved oxygen detector. The mimetic catalase activity can catalyze hydrogen peroxide to produce oxygen, thereby increasing the dissolved oxygen content in the solution. Therefore, the level of the detected dissolved oxygen content can indicate the strength of the mimetic catalase activity. Through detection, the dissolved oxygen contents corresponding to the molybdenum nitride nanozyme materials of Examples 1 to 4 were 26 mg / L, 24 mg / L, 25 mg / L, and 23 mg / L respectively, and the dissolved oxygen contents corresponding to the materials of Comparative Examples 1 to 4 were 6 mg / L, 7 mg / L, 5 mg / L, and 8 mg / L respectively, as specifically shown in Figure 5 shown. It can be seen that, compared with the comparative examples, the examples of the present invention had better mimetic catalase activity.

[0076] In summary, it can be seen that the molybdenum nitride nanozyme material with multi - enzyme activity of the present invention has multiple enzyme activities, including peroxidase, oxidase, and superoxide dismutase activities, and shows great application potential in aspects such as biosensing and antibacterial therapy. Therefore, the present invention effectively overcomes some practical problems in the prior art and thus has high utilization value and practical significance.

[0077] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A molybdenum nitride nanozyme material with multi-enzyme activity, characterized in that, The molybdenum nitride nanozyme material is a MoN nanozyme with a nanostructure, and the size range is 5 nm to 2 μm.

2. Use of the molybdenum nitride nanozyme material with multi-enzyme activity according to claim 1 in the fields of biosensing detection and antibacterial therapy.

3. A method for preparing the molybdenum nitride nanozyme material according to claim 1, characterized in that, It includes the following steps: Dissolve the molybdenum source and the nitrogen source in a solvent, and generate a precursor containing molybdenum element and nitrogen element through hydrothermal / solvothermal reaction; calcine the precursor under an inert atmosphere to obtain the molybdenum nitride nanozyme material; Or, directly calcine the molybdenum source under an ammonia atmosphere to directly obtain the molybdenum nitride nanozyme material.

4. The method according to claim 3, wherein The molybdenum source is selected from one or more of ammonium molybdate, sodium molybdate, molybdenum powder, molybdenum sulfide, ammonium tetrathiomolybdate, phosphomolybdic acid, and molybdenum chloride.

5. The method according to claim 3, wherein The nitrogen source is selected from one or more of urea, melamine, ammonia water, and ammonia gas.

6. The method according to claim 3, wherein The dosage ratio of the molybdenum source to the nitrogen source satisfies that the ratio of molybdenum atoms to nitrogen atoms is 1:(1-10).

7. The method according to claim 3, characterized in that, The hydrothermal / solvothermal reaction temperature is 100 °C to 250 °C, and the reaction time is 5 h to 25 h.

8. The method according to claim 3, characterized in that, The solvent is selected from N,N-dimethylformamide.

9. The method according to claim 3, characterized in that, The calcination temperature is 200 °C to 1200 °C, the calcination time is 3 h to 12 h, and the heating rate is 1-50 °C / min.

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