Three-dimensional metal-doped molybdenum disulfide-based nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a three-dimensional metal-doped molybdenum disulfide-based nano-enzyme and a preparation method and application thereof, and belongs to the field of nano-enzyme catalysis, the nano-enzyme is of a three-dimensional flower-shaped structure, has extremely strong affinity to hydrogen peroxide, presents superstrong peroxidase-like performance, and also shows glutathione (GSH) consumption activity, so that the nano-enzyme can be used for preparing the nano-enzyme. The preparation method is simple, effective, low in cost and easy for large-scale industrial application, a molybdenum source, a sulfur source and a metal source are dissolved in a deionized water solvent, a precipitate is generated through a heating reaction, the precipitate is separated, and the three-dimensional metal-doped molybdenum disulfide-based nano-enzyme is obtained. The obtained nano-material has a good treatment effect and a potential application prospect in the aspect of nano-enzyme catalysis.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme catalysis technology, and in particular to a three-dimensional metal-doped molybdenum disulfide-based nanozyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are a type of enzyme mimics that have both the unique properties of nanomaterials and catalytic functions. They work by inducing the production of a large amount of reactive oxygen species, including metal oxide nanozymes, metal nanozymes, and carbon-based nanozymes. Compared with natural enzymes, nanozymes are inexpensive to prepare and have stable chemical properties. Even in strong acid / base (pH 2-10) or a large temperature range (4-90°C), they can still maintain 85% catalytic activity. They have been widely used in various fields. The peroxidase-like activity of metal-based nanomaterials such as iron-based nanomaterials was discovered in 2007 and attracted widespread attention from researchers. It can catalyze the conversion of H2O2 into highly toxic ·OH, showing excellent antibacterial properties. In addition, compared with other types of metal-based nanozymes, the final product of iron-based nanozymes in the body is iron ions with higher biosafety, avoiding the potential toxicity of traditional nano drug carriers that remain in the body for a long time. However, the decomposition efficiency of iron-based nanozymes for H2O2 is still at a very low level (<30%). In order to increase the reaction rate, it is often necessary to add a large amount of H2O2 (30-6000mmol / L) and Fe 2+ (18~410mmol / L) to generate sufficiently high concentrations of active species such as ·OH, which not only increases the reaction cost, but high concentrations of iron ions are also prone to generate "iron mud" and cause catalytic reaction poisoning. In addition, H2O2, as an oxidizing agent, also has safety risks when used in large quantities. Coincidentally, the above problems also exist in other types of metal nanozyme catalysis. Therefore, improving the catalytic activity of metal nanozymes is crucial to improving their safety.

[0003] Metal sulfides have been reported to be excellent co-catalysts to improve the decomposition efficiency of metal-based nanozymes for H2O2, reducing the input of H2O2 and the content of metal ions. Molybdenum disulfide nanosheets (MoS2) have been widely explored as nanocarriers in various catalytic and biomedical applications due to their large specific surface area, unique planar structure and abundant surface active sites. However, they also have the problem of low catalytic activity. Therefore, how to improve the catalytic activity of nanozymes and expand their applications has become a problem to be solved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a three-dimensional metal-doped molybdenum disulfide-based nanozyme and a preparation method and application thereof, so as to improve the catalytic performance of the nanozyme and expand its application.

[0005] The first purpose of the present invention is to provide a method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme, which comprises the following steps: adding a molybdenum source and a sulfur source to deionized water, stirring to form a uniform solution one, adding a metal source to deionized water to prepare a solution two, adding the solution two dropwise to the solution one, mixing thoroughly and placing into a hydrothermal synthesis reactor, heating at a temperature of 150-250°C for 8-24h, cooling naturally and then centrifuging and washing with water to obtain the three-dimensional metal-doped molybdenum disulfide-based nanozyme.

[0006] Preferably, the molybdenum source comprises ammonium molybdate tetrahydrate.

[0007] Preferably, the sulfur source comprises thiourea.

[0008] Preferably, the metal source includes ferric chloride hexahydrate, cupric chloride dihydrate, and zinc chloride.

[0009] Preferably, the molar ratio of the molybdenum source, the sulfur source and the metal source is 1:2-5:0.01-0.1. Preferably, the molar ratio of the molybdenum source, the sulfur source and the metal source is 1:5:0.03.

[0010] The second object of the present invention is to provide a three-dimensional metal-doped molybdenum disulfide-based nanozyme, wherein the structure of the three-dimensional metal-doped molybdenum disulfide-based nanozyme is a three-dimensional nanoflower composed of several sheets.

[0011] The three-dimensional metal-doped MoS2-based nanozyme has a strong peroxidase-like activity. In an acidic buffer, the three-dimensional metal-doped MoS2-based nanozyme can rapidly catalyze the decomposition of H2O2 to produce a large amount of ·OH, and has a strong affinity for H2O2.

[0012] The three-dimensional metal-doped molybdenum disulfide-based nanozyme has excellent GSH consumption performance. After co-incubation with GSH, the metal-doped molybdenum disulfide-based nanozyme can consume GSH in the system and significantly reduce the content of GSH in the system.

[0013] The present invention also provides an application of three-dimensional metal-doped molybdenum disulfide-based nanozymes in antibacterial aspects. When co-incubated with different types of bacteria, the nanozymes have excellent antibacterial effects on S. aureus, showing the characteristics of specificity for S. aureus, and are assisted by GSH consumption and ·OH to achieve efficient removal of S. aureus in vivo and in vitro.

[0014] Studies have shown that the metal-doped molybdenum disulfide-based nanozyme has excellent peroxidase-like activity and GSH consumption performance, and can be made hydrophilic and biocompatible through various chemical modifications known to those skilled in the art, thereby being applied in the field of nanozyme catalysis.

[0015] The present invention has the following beneficial effects: 1) Compared with metal-based nanozymes, the three-dimensional metal-doped molybdenum disulfide-based nanozyme prepared by the present invention has more active sites exposed due to the metal doping and the presence of the flower-like structure, resulting in the nanozyme having a strong affinity for H2O2, exhibiting super strong peroxidase-like activity, and being able to catalyze the production of a large amount of ·OH.

[0016] 2) The three-dimensional metal-doped molybdenum disulfide-based nanozyme prepared by the present invention has GSH consumption performance.

[0017] 3) The preparation method provided by the present invention is convenient and effective, has simple steps, low cost, mild and controllable synthesis conditions, and is easy for large-scale industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Transmission electron microscopy (a) and XRD (b) patterns of three-dimensional iron-doped MoS2-based nanozymes.

[0019] Figure 2 : Determination of peroxidase-like activity of three-dimensional iron-doped MoS2-based nanozymes.

[0020] Figure 3 : Enzyme kinetics determination of three-dimensional iron-doped MoS2-based nanozymes ((a) kinetic curve; (b) double reciprocal curve graph).

[0021] Figure 4 : Determination of GSH consumption performance of three-dimensional iron-doped MoS2-based nanozymes.

[0022] Figure 5 : In vitro antibacterial results of three-dimensional iron-doped MoS2-based nanozymes ((a) standard plate colony counting image; (b) colony number statistical results).

[0023] Figure 6 : XPS image of three-dimensional copper-doped MoS2-based nanozyme.

[0024] Figure 7 : XPS image of three-dimensional zinc-doped molybdenum disulfide-based nanozyme. DETAILED DESCRIPTION

[0025] For a better understanding of the present invention, the following examples are further descriptions of the present invention. The following descriptions are illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0026] Ammonium molybdate tetrahydrate and thiourea were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ferric chloride hexahydrate was purchased from Aladdin; 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Bede Pharmaceuticals; H2O2 was purchased from Nanjing Chemical Reagent Co., Ltd.; and the GSH kit was purchased from Jiangsu KeyGen Biotech Co., Ltd.

[0027] S. aureus (ATCC 25923) was obtained from the School of Life Science and Technology, China Pharmaceutical University.

[0028] Example 1 Preparation of three-dimensional iron-doped molybdenum disulfide-based nanozymes

[0029] Dissolve 353 mg of ammonium molybdate tetrahydrate and 651 mg of thiourea in 5 mL of deionized water and stir to form a uniform solution 1; dissolve 16 mg of ferric chloride hexahydrate in 5 mL of deionized water to prepare solution 2; add solution 2 dropwise into solution 1, stir evenly and transfer into a 25 mL reactor, seal it, and heat it at 200 ° C for 10 hours. Cool naturally to room temperature (25 ° C), wash the product with deionized water, and collect the iron-doped molybdenum disulfide-based nanozyme sample. Combined characterization shows that the iron-doped molybdenum disulfide-based nanozyme presents a three-dimensional nanoflower shape ( Figure 1 a).

[0030] Example 2 Preparation of three-dimensional copper-doped molybdenum disulfide-based nanozymes

[0031] 353 mg of ammonium molybdate tetrahydrate and 651 mg of thiourea were dissolved in 5 mL of deionized water and stirred to form a uniform solution 1; 10 mg of cupric chloride dihydrate was dissolved in 5 mL of deionized water to prepare solution 2; solution 2 was added dropwise to solution 1, stirred evenly, transferred to a 25 mL reactor, sealed, and hydrothermaled at 200 ° C for 10 h. After cooling naturally to room temperature, the product was washed with deionized water and collected to obtain a copper-doped molybdenum disulfide-based nanozyme sample.

[0032] Example 3 Preparation of three-dimensional zinc-doped molybdenum disulfide-based nanozymes

[0033] Dissolve 353 mg of ammonium molybdate tetrahydrate and 651 mg of thiourea in 5 mL of deionized water and stir to form a uniform solution 1; dissolve 8 mg of zinc chloride in 5 mL of deionized water to prepare solution 2; add solution 2 dropwise to solution 1, stir evenly, transfer to a 25 mL reactor, seal, and hydrothermal at 200 ° C for 10 h. Cool naturally to room temperature, wash the product with deionized water, and collect the zinc-doped molybdenum disulfide-based nanozyme sample.

[0034] Experimental Example 1 Related properties of three-dimensional iron-doped molybdenum disulfide-based nanozymes

[0035] 1) Characterization of three-dimensional iron-doped MoS2-based nanozymes

[0036] Figure 1 a shows the TEM image of the three-dimensional iron-doped MoS2-based nanozyme, proving that the three-dimensional iron-doped MoS2-based nanozyme is a three-dimensional nanoflower. Figure 1b is the XRD pattern of the three-dimensional iron-doped molybdenum disulfide-based nanozyme, which proves that the crystal phase of the nanozyme is 1T phase and has stronger catalytic activity.

[0037] 2) Peroxidase-like activity of three-dimensional iron-doped MoS2-based nanozymes

[0038] TMB can be oxidized by highly active ·OH to produce a colorless to blue color reaction. The blue absorbance is detected at 652nm, and the enzyme activity is evaluated based on the absorbance value. Figure 2 The graph shows the change of the ultraviolet absorption curve after TMB was incubated with three-dimensional iron-doped MoS2 nanozyme and H2O2. The results show that after the three-dimensional iron-doped MoS2-based nanozyme was co-incubated with H2O2, there was obvious ultraviolet absorption at 652nm, indicating that the three-dimensional iron-doped MoS2-based nanozyme can catalyze the decomposition of H2O2 to produce ·OH, and has peroxidase-like activity.

[0039] The concentration of H2O2 in the reaction system was changed, the reaction rate at different H2O2 concentrations was measured, and the affinity of iron-doped molybdenum disulfide-based nanozymes for H2O2 was calculated. Figure 3 The results showed that the three-dimensional iron-doped MoS2-based nanozyme had a strong affinity for H2O2, with a Km value of 32 μM, which was significantly lower than that of traditional iron-based nanozymes, such as single-atom iron-based nanozymes (K m value is 18.8mM) and MoS2 nanozyme (the Km value of MoS2 nanozyme is 327.35μm).

[0040] 3) GSH consumption performance of three-dimensional iron-doped MoS2-based nanozymes

[0041] 2.5 mL of 1.88 μg / mL carbonate buffer solution of three-dimensional iron-doped molybdenum disulfide-based nanozyme and 2.5 mL of 1 mM GSH solution were mixed, stirred at room temperature for a certain period of time (150 rpm), and the GSH content in the reaction system was determined using a GSH kit. Figure 4 The results showed that the GSH content in the reaction system containing the nanozyme was reduced, proving that the nanozyme had the property of GSH consumption.

[0042] Experimental Example 2 Enzyme catalytic application of three-dimensional iron-doped molybdenum disulfide-based nanozymes

[0043] Dilute the suspension of S. aureus (ATCC 25923) in the logarithmic growth phase with fresh LB liquid medium to a concentration of 1×10 8 CFU / mL. They were divided into 2 groups: (I) control group; (II) MFH; the final concentration of iron-doped molybdenum disulfide-based nanozymes was 8 μg / mL. After incubation at 37°C for 12 h, the bacterial solution was diluted with sterile saline (bacterial solution: saline = 1:10 6), take 5 μL of bacterial solution and spot it on the plate, incubate at 37℃ for 12 h, and record the number of colonies on the plate.

[0044] like Figure 5 As shown, compared with the control group, the number of bacteria in the MFH group was reduced by 2 orders of magnitude, proving that the nanozyme produced a large amount of ·OH with the help of its own peroxidase-like activity, while consuming bacterial GSH to prevent it from clearing ·OH, ultimately leading to the death of bacteria, showing excellent antibacterial properties.

[0045] Experimental Example 3 Related properties of three-dimensional copper-doped molybdenum disulfide-based nanozymes

[0046] Figure 6 This is the XPS image of the three-dimensional copper-doped MoS2-based nanozyme, proving the successful preparation of the three-dimensional copper-doped MoS2-based nanozyme.

[0047] Experimental Example 4 Related properties of three-dimensional zinc-doped molybdenum disulfide-based nanozymes

[0048] Figure 7 This is the XPS image of three-dimensional zinc-doped molybdenum disulfide-based nanozyme, proving the successful preparation of three-dimensional zinc-doped molybdenum disulfide-based nanozyme.

[0049] The above is only a preferred embodiment of the present invention, and certainly cannot be used to limit the scope of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, some improvements can be made using the technical content suggested above, and these improvements and changes are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme, characterized in that: The method comprises the following steps: adding a molybdenum source and a sulfur source into deionized water, stirring to form a uniform solution one, adding a metal source into the deionized water to prepare a solution two, adding the solution two dropwise into the solution one, mixing thoroughly and placing in a hydrothermal synthesis reactor, heating at a temperature of 150-250° C. for 8-24 hours, cooling naturally and then centrifuging and washing with water to obtain a three-dimensional metal-doped molybdenum disulfide-based nanozyme.

2. The method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme according to claim 1, characterized in that: The molybdenum source includes ammonium molybdate tetrahydrate.

3. The method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme according to claim 1, characterized in that: The sulfur source includes thiourea.

4. The method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme according to claim 1, characterized in that: The metal source includes ferric chloride hexahydrate, cupric chloride dihydrate and zinc chloride.

5. The method for preparing a three-dimensional metal-doped molybdenum disulfide-based nanozyme according to claim 1, characterized in that: The molar ratio of the molybdenum source, the sulfur source and the metal source is 1:2-5:0.01-0.

1.

6. A three-dimensional metal-doped molybdenum disulfide-based nanozyme, characterized in that: The three-dimensional metal-doped molybdenum disulfide-based nanozyme is a three-dimensional nanoflower-like structure composed of a plurality of sheets, and is prepared by the method described in any one of claims 1-5.

7. The use of the three-dimensional metal-doped molybdenum disulfide-based nanozyme according to claim 6, characterized in that: Three-dimensional metal-doped MoS2-based nanozymes have GSH-consuming properties and peroxidase-like activity and can be used for antibacterial applications.

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