Nickel-doped histidine carbon nano enzyme and preparation method thereof

Abstract

The invention relates to the technical field of carbon nano-enzymes, in particular to a nickel-doped histidine carbon nano-enzyme and a preparation method thereof. Conventional metal-doped carbon nano-enzyme has POD activity and is not beneficial to later-stage healing of bacterial infected wounds. In order to solve the problems, the invention provides the nickel-doped histidine carbon nano-enzyme which is a product generated by high-temperature hydrothermal reaction of histidine and soluble nickel salt, and nickel ions and histidine form coordination chemical bonds under the high-temperature hydrothermal condition. The obtained carbon nano-enzyme has a good photo-thermal sterilization effect, does not have POD activity, and is more beneficial to later-stage healing of a wound surface.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon nanozymes, and in particular to a nickel-doped histidine carbon nanozyme and a preparation method thereof. Background Art

[0002] The application of carbon materials in the biomedical field is showing its extraordinary clinical therapeutic potential, especially as an effective alternative to antibiotics, opening up a new horizon for anti-infection treatment. These tiny nanoparticles not only have excellent biocompatibility and low toxicity, but also show efficient targeting and killing capabilities for pathogens, providing an innovative solution to the increasingly serious problem of drug resistance.

[0003] Although traditional antibiotics play a key role in treating bacterial infections, the increased drug resistance caused by long-term and widespread use has become a major challenge to global public health. In contrast, carbon materials, with their unique optical, electrical and chemical properties, can be activated under light or under specific conditions to release precise attack power against bacteria, thereby reducing the risk of drug resistance while achieving more accurate and efficient treatment.

[0004] However, although metal-doped carbon nanozymes have been explored for use in the biomedical field and exhibit peroxidase (POD) activity, this property has application value in some occasions, but the continuous generation of reactive oxygen species (ROS) brings side effects that cannot be ignored. The excessive generation of ROS not only aggravates the inflammatory response at the wound, but also seriously interferes with the normal regeneration process of skin tissue, which poses a major obstacle to the late healing of bacterial-infected wounds. Therefore, how to avoid or alleviate these adverse reactions while maintaining antibacterial efficacy has become a key issue that needs to be solved urgently.

[0005] It is worth noting that despite the increasing application of metal-doped carbon nanozymes in the biomedical field, there are few reports on the research of metal-doped carbon nanozyme sterilization materials without POD activity. The development of such materials will hopefully achieve safer and more efficient bacterial infection control without triggering excessive inflammatory responses, creating a more favorable microenvironment for wound healing and tissue regeneration. In the future, with the deepening of relevant research, metal-doped carbon nanozyme sterilization materials without POD activity are expected to become an important supplement to antibiotic treatment. Summary of the invention

[0006] The problem in the prior art is that conventional metal-doped carbon nanozymes have POD activity. In view of the above technical problems, the present invention provides a nickel-doped histidine carbon nanozyme, which is a product generated by a high-temperature hydrothermal reaction of histidine and a soluble nickel salt. Under the high-temperature hydrothermal conditions, nickel ions and histidine form a coordination chemical bond. The particle size range is 100-300nm.

[0007] Preferably, the soluble nickel salt includes one or a combination of two or more of nickel chloride, nickel acetate, nickel nitrate or nickel sulfate.

[0008] Preferably, the temperature of the hydrothermal reaction is 150-220°C.

[0009] Preferably, the preparation method of the nickel-doped histidine carbon nanozyme comprises the following steps:

[0010] The histidine was completely dissolved in water, a soluble nickel salt was added, and the mixture was stirred and dissolved evenly. The obtained mixture was transferred to a hydrothermal reactor for high-pressure hydrothermal reaction. After the reaction was completed, the obtained product was taken out from the hydrothermal reactor, ultrasonically mixed evenly, and freeze-dried to obtain the doped histidine carbon nanozyme.

[0011] Preferably, the ratio of histidine to soluble nickel salt is 1-10:1.

[0012] The present invention has the following beneficial effects:

[0013] (1) The carbon nanozyme obtained by the present invention does not have POD activity, and does not generate ROS during use, but can remove ROS. The nanozyme uses photothermal effect, CAT activity and SOD activity for sterilization;

[0014] (2) The carbon nanozyme obtained by the present invention does not generate ROS while sterilizing, can significantly reduce the inflammatory response at the wound surface, promote the formation of blood vessels at the wound surface, and is more conducive to the late healing of bacterially infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Particle size distribution diagram of NH Nanozyme obtained in Example 1

[0016] Figure 2 : Graph showing the test results of the ability of NH Nanozyme obtained in Example 1 to scavenge ABTS free radicals.

[0017] Figure 3 : Result of the test on the ability of NH Nanozyme obtained in Example 1 to scavenge DPPH free radicals.

[0018] Figure 4 : Result of the test on the ability of NH Nanozyme to remove OH obtained in Example 1.

[0019] Figure 5 : SOD activity diagram of NH Nanozyme obtained in Example 1.

[0020] Figure 6 : POD activity diagram of Example 1 and Comparative Examples 1 and 2.

[0021] Figure 7 : Antibacterial effect diagram of NH Nanozyme obtained in Example 1. DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to the examples. However, it should be understood that the following examples are only illustrative of the embodiments of the present invention, and are not intended to limit the scope of the present invention.

[0023] Example 1

[0024] 0.030 g of histidine was dissolved in 20 mL of distilled water, and then 0.009 g of nickel chloride hexahydrate was added. After stirring to completely dissolve, the mixture was fully mixed and stirred for 10 minutes to obtain a mixed solution. After that, the mixed solution was placed in a hydrothermal reactor and subjected to hydrothermal reaction at 180° C. for 12 hours. After the reaction was completed, the obtained product was taken out from the hydrothermal reactor, ultrasonically mixed evenly, and freeze-dried to obtain nickel-doped histidine carbon nanozyme, which was recorded as NH Nanozyme.

[0025] The particle size distribution diagram of NH Nanozyme obtained in Example 1 is shown in the attached specification. Figure 1 As shown, the particle size of the NH Nanozyme is about 139.6 nm.

[0026] The effect diagram of the test of the ability of NH Nanozyme to scavenge ABTS free radicals obtained in Example 1 is as shown in the attached manual. Figure 2 shown.

[0027] The effect diagram of the test of the ability of NH Nanozyme to scavenge DPPH free radicals obtained in Example 1 is as shown in the attached manual. Figure 3 shown.

[0028] The effect diagram of the test of the ability of NH Nanozyme to scavenge hydroxyl radicals (·OH) obtained in Example 1 is as shown in the attached manual. Figure 4 shown.

[0029] The SOD activity diagram of NH Nanozyme obtained in Example 1 is shown in the attached manual. Figure 5 shown.

[0030] The POD activity diagram of NH Nanozyme obtained in Example 1 is as shown in the attached manual. Figure 6 shown.

[0031] The antibacterial effect diagram of NH Nanozyme obtained in Example 1 is as shown in the attached manual. Figure 7 shown.

[0032] Example 2 is the same as Example 1, except that the hydrothermal reaction temperature in Example 2 is 200°C.

[0033] Example 3 is the same as Example 1, except that the hydrothermal reaction time in Example 3 is 8 hours.

[0034] Example 4

[0035] Dissolve 0.030 g of histidine in 20 mL of distilled water, then add 0.018 g of nickel nitrate hexahydrate, stir until completely dissolved, and then fully mix and stir for 10 minutes to obtain a mixed solution. Then, place the mixed solution in a hydrothermal reactor and perform a hydrothermal reaction at 180°C for 12 hours. After the reaction, take out the product from the hydrothermal reactor, mix it evenly by ultrasonication, and freeze-dry to obtain nickel-doped histidine carbon nanozyme.

[0036] Example 5

[0037] Dissolve 0.030 g of histidine in 20 mL of distilled water, then add 0.003 g of nickel acetate tetrahydrate, stir until completely dissolved, and then fully mix and stir for 10 minutes to obtain a mixed solution. Then, place the mixed solution in a hydrothermal reactor and perform a hydrothermal reaction at 180°C for 12 hours. After the reaction, take out the product from the hydrothermal reactor, mix it evenly by ultrasonication, and freeze-dry to obtain nickel-doped histidine carbon nanozyme.

[0038] Comparative Example 1 is the same as Example 1, except that histidine is replaced by cysteine ​​in Comparative Example 1.

[0039] Comparative Example 2 is the same as Example 1, except that in Comparative Example 2, nickel chloride hexahydrate is replaced by ferrous chloride tetrahydrate, and the molar amount of ferrous ions in ferrous chloride tetrahydrate is equal to the molar amount of nickel ions in nickel chloride hexahydrate in Example 1.

[0040] Comparative Examples 1 and 2 have POD activity, can generate hydroxyl radicals, and can be made to appear blue after adding TMB. However, Example 1 does not have POD activity, cannot generate hydroxyl radicals, and cannot be made to appear blue after adding TMB.

[0041] The POD activity diagrams of NH Nanozyme obtained in Comparative Examples 1-2 are shown in the attached manual. Figure 6 shown.

[0042] Performance Testing

[0043] The carbon nanozymes obtained in the examples of the present invention and the comparative examples were respectively subjected to relevant performance tests, and the specific test results are shown in Table 1.

[0044] The test method for photothermal sterilization performance is as follows:

[0045] Take 200 μL of 10 8 CFU·mL -1Staphylococcus aureus was mixed evenly with 200 μL of a 50-150 μg / mL carbon nanozyme aqueous solution, then incubated at 37°C for 1 hour, irradiated with a laser of 1.5 W and 808 nm for 10 minutes, and then 100 μL of the mixed solution was diluted 20,000 times and spread on the plate. The plate was then cultured in an incubator at 37°C overnight, and the number of colonies on the plate was counted.

[0046] The test method for peroxidase (POD) activity is as follows:

[0047] The nanozymes obtained in the examples of the present invention and the comparative examples were added to the same volume of H2O2 aqueous solution with a concentration of 100 mM. The same mass of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each test solution, and after standing at room temperature for 20 minutes, the ultraviolet absorption of the solution at 650 nm was tested.

[0048] The test principle is: if the material has POD activity, it will catalyze H2O2 to generate a large amount of ·OH in a short time. At this time, 3,3',5,5'-tetramethylbenzidine (TMB) in the test solution will combine with hydroxyl radicals and be oxidized to ox-TMB, which will show a characteristic ultraviolet absorption peak at 650nm. On the contrary, if the material does not have POD activity, it will not be able to catalyze H2O2 to generate a large amount of hydroxyl radicals, and ox-TMB will not be generated in the test solution, so there will be no ultraviolet characteristic absorption peak at 650nm.

[0049] The test method for the ability to scavenge ABTS free radicals is as follows:

[0050] The same mass of the carbon nanozymes obtained in the examples of the present invention and the comparative example were added to the ABTS working solution of the same volume and concentration, respectively. After standing at room temperature for 20 minutes, the ultraviolet absorption of the solution at 734 nm was tested.

[0051] The test principle is: when 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt reacts with potassium persulfate, a blue-green free radical ABTS is generated. + The free radical has obvious ultraviolet characteristic absorption at a wavelength of 734nm. When the antioxidant acts on the free radical, the antioxidant will provide electrons to react with ABTS· + The free radicals combine, causing the color of the solution to lighten, and the absorbance at 734nm will decrease or disappear.

[0052] The ABTS working solution is prepared by dissolving ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) in deionized water, then adding an equal volume of potassium persulfate aqueous solution (completely dissolved by ultrasonication), mixing well, and placing at room temperature away from light for 12 hours to obtain the ABTS working solution.

[0053] The test method for the ability to scavenge DPPH free radicals is as follows:

[0054] The same mass of carbon nanozymes obtained in the examples of the present invention and the comparative examples were added to the same volume and concentration of DPPH alcohol solution, and the solution was left to stand at room temperature for 20 minutes to test whether the solution had characteristic ultraviolet absorption at 519 nm. The DPPH alcohol solution is a uniform solution formed by dissolving DPPH in anhydrous ethanol.

[0055] The test principle is: DPPH alcohol solution is purple, and DPPH has characteristic ultraviolet absorption at 519nm. It is known that in the presence of antioxidants, DPPH free radicals will be eliminated, causing the solution color to become lighter and the ultraviolet absorption at 519nm to decrease or disappear.

[0056] The test method for the ability to scavenge hydroxyl radicals (·OH) is as follows:

[0057] FeCl2 was added to the H2O2 aqueous solution and allowed to react for 5-10 minutes. The carbon nanozymes obtained in the examples of the present invention and the comparative examples were added to the reaction solution and allowed to stand for 5-10 minutes. 3,3',5,5'-tetramethylbenzidine (TMB) was then added and allowed to stand for 20 minutes at room temperature, and the ultraviolet absorption of the solution at 650 nm was tested.

[0058] The test principle is: using Fenton reaction to produce OH: H2O2+Fe 2+ =OH+H2O+Fe 3+ If ·OH is present in the test solution, 3,3',5,5'-tetramethylbenzidine (TMB) will combine with ·OH and be oxidized to ox-TMB, which will show a characteristic UV absorption peak at 650nm. On the contrary, if ·OH is not present in the test solution, H2O2 cannot be catalyzed to generate a large number of hydroxyl radicals, and ox-TMB will not be generated in the test solution, so there will be no UV characteristic absorption peak at 650nm.

[0059] The test method for superoxide dismutase (SOD) activity is as follows:

[0060] Methioninase and riboflavin were dissolved in PBS, and the carbon nanozymes obtained in the examples of the present invention and the comparative examples were added. NBT was added after 5 minutes, and the mixture was allowed to stand under ultraviolet light for 30 minutes. The ultraviolet absorption of the solution at 560 nm was tested.

[0061] The test principle is: in the presence of oxidizing substances, riboflavin can be reduced by light, and the reduced riboflavin is easily reoxidized under aerobic conditions to produce superoxide anion free radicals (O2 - ), superoxide anion free radical (·O2 - ) can reduce nitroblue tetrazolium (NBT) to blue formazan, which has a maximum absorption at 560nm. SOD can remove superoxide anion free radicals, thereby inhibiting the formation of formazan, resulting in a decrease in ultraviolet absorption at 560nm. Therefore, after the photoreduction reaction, the darker the blue color of the reaction solution, the lower the enzyme activity, and vice versa.

[0062] Table 1

[0063]

[0064] As shown in Table 1, the carbon nanozymes obtained in the examples of the present invention do not have POD activity, but have good bactericidal effects, while the carbon nanozymes obtained in the comparative examples all have POD activity.

[0065] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A nickel-doped histidine carbon nanozyme, characterized in that: It is a product generated by high-temperature hydrothermal reaction of histidine and soluble nickel salt. Under high-temperature hydrothermal conditions, nickel ions and histidine form coordination chemical bonds.

2. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that: The particle size of histidine carbon nanozyme ranges from 100 to 300 nm.

3. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that: The soluble nickel salt includes one or a combination of two or more of nickel chloride, nickel acetate, nickel nitrate or nickel sulfate.

4. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that: The temperature of the hydrothermal reaction is 150-220°C.

5. The nickel-doped histidine carbon nanozyme according to claim 4, characterized in that: The temperature of the hydrothermal reaction was 200°C.

6. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that: The preparation method comprises the following steps: The histidine was completely dissolved in water, a soluble nickel salt was added, and the mixture was stirred and dissolved evenly. The obtained mixture was transferred to a hydrothermal reactor for high-pressure hydrothermal reaction. After the reaction was completed, the obtained product was taken out from the hydrothermal reactor, ultrasonically mixed evenly, and freeze-dried to obtain the doped histidine carbon nanozyme.

7. The nickel-doped histidine carbon nanozyme according to claim 6, characterized in that: The mass ratio of histidine to soluble nickel salt is 1-10:1.

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