CN114345349A - Preparation method and application of bimetallic nanoenzyme - Google Patents
Preparation method and application of bimetallic nanoenzyme Download PDFInfo
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- CN114345349A CN114345349A CN202210017747.2A CN202210017747A CN114345349A CN 114345349 A CN114345349 A CN 114345349A CN 202210017747 A CN202210017747 A CN 202210017747A CN 114345349 A CN114345349 A CN 114345349A
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Abstract
The invention relates to a preparation method and application of bimetallic nanoenzyme, which comprises the following steps of (a) preparing a hexagonal metal organic framework material ZIF-67 by adopting a solvent method; (b) synthesis of nickel cobaltate (NiCo) by ion exchange method2O4) (ii) a (c) By one stepOne-step self-assembly template method for preparing NiCo2O4The @ PVP bimetal nanoenzyme can well simulate the activity of superoxide dismutase (SOD enzyme) and catalase (CAT enzyme), and can effectively remove OH. The invention has the beneficial effects that: the prepared bimetallic nanoenzyme NiCo with oxygen vacancy2O4After macromolecular polyvinylpyrrolidone (PVP) is wrapped, the nano enzyme NiCo is obviously improved2O4Stability in simulated gastric fluid and simulated intestinal fluid. Nano enzyme NiCo2O4The @ PVP can well simulate the activity of SOD enzyme and the activity of CAT enzyme, and can effectively remove OH. Nano enzyme NiCo2O4@ PVP exhibits electronegativity under neutral and alkaline conditions and particle size<300 nm. Nano enzyme NiCo2O4The @ PVP has no toxic or side effect, and can effectively eliminate the generation of active oxygen in cells.
Description
Technical Field
The invention belongs to the research field of nano enzyme, and relates to a preparation method and catalytic application of bimetallic nano enzyme.
Background
Reactive Oxygen Species (ROS) mainly include types of superoxide anions, nitric oxide, hydrogen peroxide, hydroxyl radicals, singlet oxygen molecules, and the like. Low level of ROS is necessary for the body, physiological processes such as signal transduction, cell differentiation and apoptosis need to be involved, and excessive ROS can generate damage to the body through oxidation. The accumulation of ROS is known as a "timed bomb" for the body, and can cause more serious disease at any time. For example, excess ROS can cause oxidative damage to macromolecules such as DNA, proteins, lipids, etc., and can also induce caspase activation, leading to apoptosis. At least 60 diseases have been reported to be associated with ROS. Dopamine activity is reduced, as in patients with parkinson's disease. Dopamine on Cu as a good metal ion chelating agent2+And Fe3+The strong affinity is provided, and after the dopamine is combined with metal ions, the ROS generated by the metal ions through Fenton reaction can be reduced. However, a decrease in dopamine activity increases ROS production, thus exacerbating parkinson's disease. In addition, ROS are involved in diabetes, cancer, atherosclerosis, rheumatoid arthritis, and other diseases.
The nano enzyme is a nano material with natural enzyme activity, is used as a novel mimic enzyme, has the unique property of the nano material, and can mimic the high-efficiency catalytic function of the natural enzyme. Compared with natural enzymes, the nano-enzyme has the advantages of low cost, strong tolerance, high stability, easy long-term storage, large-scale production and the like. So far, it has been reported that various nanomaterials have catalytic activity of natural enzymes, and these nanomaterials can simulate various enzymes such as GPXs, CAT, SOD, POD, protease, nuclease, etc. The nano enzyme is successfully applied to the aspects of disease diagnosis, antioxidant treatment, bacterial elimination, cancer treatment and the like.
Disclosure of Invention
Aiming at the requirements of the research methods and the blank of the field, the bimetallic nanoenzyme is introduced. The nano enzyme is a nano material with natural enzyme activity, is used as a novel mimic enzyme, has the unique property of the nano material, and can mimic the high-efficiency catalytic function of the natural enzyme. Compared with natural enzymes, the nano-enzyme has the advantages of low cost, strong tolerance, high stability, easy long-term storage, large-scale production and the like. Under different environmental conditions, some nanoenzymes can show distinct catalytic activities, and under acidic conditions, some nanoenzymes can be used as a pro-oxidant to generate ROS (reactive oxygen species) and promote oxidative apoptosis of cells. Under neutral conditions, the nanoenzymes can also be used as antioxidants to scavenge ROS and relieve oxidative stress of cells. The nano enzyme has various properties, so that the nano enzyme can be applied to different fields, and in tumor cells, the nano enzyme can inhibit the growth of the tumor cells and promote the apoptosis of the cells by generating high-activity ROS.
In the research, ZIF-67 is used as a template, and a simple ion replacement method is adopted to synthesize NiCo Layered Double Hydroxide (LDH). Subsequently, NiCo-LDH is calcined to produce NiCo2O4Nano particles, a bimetal nano enzyme containing oxygen vacancy is constructed, and finally, polyvinylpyrrolidone is used for coating NiCo2O4Nanoparticles to improve biocompatibility and acid resistance thereof. The ability of the newly synthesized nanobody to scavenge ROS in vitro is determined.
The invention aims to research the catalytic activity of the bimetallic nanoenzyme, the activity of the nanoenzyme is not influenced by external factors, such as pH, temperature, substrate concentration and the like, but the catalytic activity, such as size, shape and the like, can be influenced by the unique properties of the nanomaterial. Under different environmental conditions, some nanoenzymes may exhibit distinct catalytic activities. In tumor cells, the nanoenzyme can inhibit the growth of tumor cells and promote apoptosis by generating high-activity ROS. In some enteritis cells, there is a close relationship between ROS and IBD, excess ROS may cause IBD, and ROS produced in IBD may also cause exacerbation of the disease. The nano enzyme can relieve and treat IBD by scavenging ROS.
The specific technical scheme provided by the invention is as follows:
the preparation and catalytic application of the bimetallic nanoenzyme comprise the following steps:
(a) preparing a hexagonal metal organic framework material ZIF-67 by a solvent method;
(b) synthesis of nickel cobaltate NiCo by ion replacement method2O4；
(c) Preparing NiCo by a one-step self-assembly template method2O4The @ PVP bimetal nano enzyme can well simulate the activity of SOD enzyme and the activity of CAT enzyme, and can effectively remove OH.
Further, the step (a) adopts a solvent method, and the process for preparing the hexagonal metal organic framework material ZIF-67 is as follows:
cobalt nitrate hexahydrate and 2-methylimidazole were weighed into a 250mL triangular flask, and then 100mL of methanol was added to dissolve them sufficiently. The two solutions were then mixed and stirred at room temperature for 13min, followed by 24h at room temperature. And after the reaction is finished, recovering the precipitate by centrifugation, then cleaning the precipitate by using ethanol, and drying the precipitate in a vacuum drying oven to obtain purple ZIF-67.
Furthermore, the dosage of the cobalt nitrate hexahydrate in the step is 1.436g, and the dosage of the 2-methylimidazole is 3.244 g.
Further, the step (b) is used for synthesizing nickel cobaltate NiCo by an ion replacement method2O4The process of (2) is as follows:
accurately weighing 100mg of ZIF-67 obtained in the step (a) and nickel chloride hexahydrate, placing the ZIF-67 and the nickel chloride hexahydrate in a 50mL reaction kettle, adding ethanol, and performing ultra-high pressure distillationTreating under sonic condition for 30 min. Subsequently, the reaction vessel was transferred to an electrically heated forced air oven and heated at 90 ℃ for 1 h. After the reaction is finished, carrying out centrifugal drying and recycling to obtain the green NiCo layered double hydroxide. Then, a certain amount of NiCo layered double hydroxide is weighed and placed in a crucible, then the crucible is transferred into a muffle furnace, and the mixture is calcined for 3 hours at the temperature of 300 ℃, and the heating rate is set to be 2.5 ℃/min. Finally obtaining NiCo2O4(ii) particulate matter.
Furthermore, the dosage of the nickel chloride hexahydrate is 150mg, and the volume of the ethanol is 30 mL.
Further, the step (c) is to prepare NiCo by a one-step self-assembly template method2O4The @ PVP bimetal nano enzyme can well simulate the activity of SOD enzyme and the activity of CAT enzyme, and the process of effectively removing OH is as follows:
accurately weighing 200mg of NiCo prepared in the step (b)2O4And 200mg of polyvinylpyrrolidone were placed in a 150mL Erlenmeyer flask. Then, 60mL of ethanol was added and sonication was performed for 30min to dissolve it sufficiently. The mixture was then stirred at room temperature for 1 h. After the reaction is finished, centrifuging, recovering and drying the precipitate to finally obtain NiCo2O4@ PVP particulate material.
Further, the obtained bimetallic nano enzyme NiCo2O4@ PVP simulates SOD enzyme activity, i.e. nanoenzyme NiCo2O4@ PVP scavenging. O2 -Further by measuring nanoenzyme and H2O2The amount of oxygen generated by the reaction is evaluated by the nano enzyme NiCo2O4@ PVP simulates the activity of CAT, followed by evaluation of the nanoenzyme NiCo2O4The ability of @ PVP to scavenge. OH, the effect of scavenging active oxygen under neutral conditions is achieved;
the application of the invention is that the average particle size of the prepared bimetallic nano-enzyme is about 268nm, and oxygen vacancies exist in the nano-material, which is beneficial to the capture of the material to Reactive Oxygen Species (ROS), and meanwhile, the bimetallic nano-enzyme has good stability and extremely low toxic and side effects in digestive juice, and has stronger effect of eliminating the reactive oxygen under the neutral condition.
The reported nanoenzyme, such as carbon-based nanoenzyme, has the defects of poor stability, poor catalytic performance and the like, and has very limited application in practice. The bimetallic nano-enzyme NiCo with multiple enzyme activities prepared by the invention2O4The nano enzyme has oxygen vacancy which is beneficial to the capture of ROS, the stability of the nano enzyme in simulated gastrointestinal digestive juice is obviously improved after the nano enzyme is coated with macromolecule PVP, and the nano enzyme can simulate various enzyme activities under the neutral condition to achieve the aim of removing O2 -，H2O2And OH, which is a promising intervention substance.
The invention has the beneficial effects that:
(1) the bimetallic nano-enzyme NiCo with multiple enzyme activities prepared by the invention2O4And the determination through XPS characterization shows that the nano material has oxygen vacancy, which is helpful for the capture of the material to Reactive Oxygen Species (ROS).
(2) After the nano enzyme developed by the invention is coated with the macromolecular substance PVP, the nano enzyme NiCo is obviously improved2O4Stability of @ PVP in simulated gastric fluid and simulated intestinal fluid; the simulated enzyme activity determination shows that the synthetic material is a pH dependent nano enzyme, can simulate SOD enzyme activity and CAT enzyme activity under a neutral condition, can effectively remove OH, and can simulate horse radish peroxidase activity and oxidase activity under an acidic condition; NiCo2O4@ PVP exhibits electronegativity under neutral and alkaline conditions and particle size<300nm, which shows that the nano enzyme NiCo2O4@ PVP may target effects through charge and particle size mediated mechanisms of action.
(3) The quantity of each substance is reasonably selected, the optimum reaction proportion and reaction conditions are obtained through detailed comparison, analysis and optimization in the experimental process, and the catalytic performance of the bimetallic nano-enzyme obtained through the reaction is optimum.
Description of the drawings:
FIG. 1: (A) bimetallic nanoenzyme NiCo2O4Synthetic scheme for @ PVP (B) ZIF-67(C) NiCo LDH (D) NiCo2O4And (E, F) NiCo2O4TEM image of @ PVP(G) Nano enzyme NiCo2O4@ PVP particle size distribution (H) Nanomase NiCo2O4High resolution transmission electron microscopy picture (I) nano enzyme NiCo of @ PVP2O4EDX elemental analysis chart of @ PVP
FIG. 2: bimetallic nanoenzyme NiCo2O4@ PVP multiplex enzyme activity assay: (A) SOD mimic Activity (B) CAT mimic Activity (C) Activity for mimicking the scavenging activity of oxidase and horseradish peroxidase (pH 4) (D). OH under acidic conditions
FIG. 3: (A) nano enzyme NiCo under different pH values2O4And the nano enzyme NiCo2O4The grain size (B) of @ PVP simulates gastric juice and simulates nano enzyme NiCo after intestinal juice treatment2O4XRD pattern (C) of @ PVP simulates nanoenzyme NiCo after gastric juice treatment2O4The residual enzyme activity (D) of @ PVP simulates the nano enzyme NiCo after intestinal juice treatment2O4The residual enzyme activity (E) of @ PVP and the nano enzyme NiCo under different pH values2O4@ potential of PVP
FIG. 4: (A) nano enzyme NiCo with different concentrations2O4Influence of @ PVP on macrophage RAW264.7 Activity (B) Co content determination (C) Ni content determination
Detailed Description
In order that the above features and advantages of the present invention will be readily understood and appreciated, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
Example 1
Preparation and catalytic application of bimetallic nanoenzyme
(1) And preparing the hexagonal metal organic framework ZIF-67 by adopting a solvent method.
1.436g of cobalt nitrate hexahydrate and 3.244g of 2-methylimidazole were weighed into a 250mL triangular flask, and then 100mL of methanol was added to dissolve them sufficiently. The two solutions were then mixed and stirred at room temperature for 13min, followed by 24h at room temperature. And after the reaction is finished, recovering the precipitate by centrifugation, then cleaning the precipitate by using ethanol, and drying the precipitate in a vacuum drying oven to obtain purple ZIF-67.
(2) By means of ionsSynthesis of nickel cobaltate NiCo by displacement method2O4。
Accurately weighing 100mg of ZIF-67 obtained in step (a) and 150mg of NiCl2·6H2And O, placing the mixture into a 50mL reaction kettle, adding 30mL ethanol, and treating for 30min under ultrasonic conditions. Subsequently, the reaction vessel was transferred to an electrically heated forced air oven and heated at 90 ℃ for 1 h. After the reaction is finished, carrying out centrifugal drying and recycling to obtain the green NiCo layered double hydroxide. Then, a certain amount of NiCo layered double hydroxide is weighed and placed in a crucible, then the crucible is transferred into a muffle furnace, and the mixture is calcined for 3 hours at the temperature of 300 ℃, and the heating rate is set to be 2.5 ℃/min. Finally obtaining NiCo2O4(ii) particulate matter.
(2) Preparing NiCo by a one-step self-assembly template method2O4The @ PVP bimetal nano enzyme can well simulate the activity of SOD enzyme and the activity of CAT enzyme, and can effectively remove OH.
Accurately weighing 200mg of NiCo prepared in the step (b)2O4And 200mg of polyvinylpyrrolidone were placed in a 150mL Erlenmeyer flask. Then, 60mL of ethanol was added and sonication was performed for 30min to dissolve it sufficiently. The mixture was then stirred at room temperature for 1 h. After the reaction is finished, centrifuging, recovering and drying the precipitate to finally obtain NiCo2O4@ PVP particulate material.
Example 2
The material obtained in example 1 was used to simulate SOD enzyme activity, and riboflavin 20. mu.M, methionine 0.013M, NBT 75. mu.M and NiCo at various concentrations were mixed2O4@ 0-100. mu.g/mL PBS (pH 7.4, 100mM) buffer was irradiated under an ultraviolet lamp of constant light intensity for 30min, and the absorbance at 560nm of the mixed solution was measured immediately after the ultraviolet irradiation.
Example 3
The material obtained in example 1 was used to mimic oxidase and horseradish peroxidase activities, and 3mL of sodium acetate buffer (1M, pH 4) and 200. mu.L of NiCo were added2O4@ PVP (0.25mg/L) was mixed in the tube. Then, 100 μ L H was added to the tube2O2And 200. mu.L of TMB (5mM, DMSO), incubationAfter 2 minutes, the absorbance of the mixture was measured with an ultraviolet-visible spectrophotometer.
Example 4
The material obtained in example 1 was used to simulate the CAT enzyme activity, with different concentrations of NiCo2O4@ PVP and H2O2The mixture was mixed in PBS buffer (pH 7.4, 50mM), and the oxygen content (mg/L) in the solution was measured within 5 min.
Example 5
The material obtained in example 1 was assayed for OH scavenging activity and contained 0.5mM terephthalic acid, H2O2Nano enzyme NiCo with 20mM and different concentration2O4@ PVP in PBS buffer (pH 7.4, 20mM) was irradiated under an ultraviolet lamp of constant intensity for 30min, and then the fluorescence intensity was measured with a fluorescence spectrophotometer, the excitation wavelength of fluorescence was 315nm, and the emission wavelength was 425 nm.
FIG. 1 shows a bimetallic nanoenzyme NiCo2O4The preparation and characterization of @ PVP show that the nano material has oxygen vacancy, and the trapping of the material to Reactive Oxygen Species (ROS) is facilitated.
FIG. 2 shows a bimetallic nanoenzyme NiCo2O4The multiple simulated enzyme activity determination of @ PVP shows that the prepared bimetallic nanoenzyme is a pH-dependent nanoenzyme, can simulate SOD enzyme activity and CAT enzyme activity under a neutral condition, can effectively remove OH, and can simulate horse radish peroxidase activity and oxidase activity under an acidic condition.
FIG. 3 shows a bimetallic nanoenzyme NiCo2O4The stability of @ PVP in simulated digestive intestinal juice shows that the prepared nano enzyme NiCo is obviously improved after the nano enzyme is coated with the macromolecular substance PVP2O4@ PVP stability in simulated gastric fluid and simulated intestinal fluid.
FIG. 4 shows a bimetallic nanoenzyme NiCo2O4The cytotoxicity and endocytosis of @ PVP show that the prepared nano enzyme NiCo2O4The @ PVP has no toxic or side effect, and can inhibit the generation of inflammatory factors of LPS (LPS) stimulated cells by endocytosis for H2O2The stimulated cells play a role in protection and effectively eliminate the generation of active oxygen in the cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.
Claims (8)
1. The preparation method of the bimetallic nano-enzyme is characterized by comprising the following steps of:
(a) preparing a hexagonal metal organic framework material ZIF-67 by a solvent method;
(b) synthesis of nickel cobaltate NiCo by ion replacement method2O4；
(c) Preparing NiCo by a one-step self-assembly template method2O4The @ PVP bimetal nano enzyme can well simulate the activity of SOD enzyme and the activity of CAT enzyme, and can effectively remove OH.
2. The method for preparing the bimetallic nanoenzyme as in claim 1, wherein the step (a) adopts a solvent method, and the process for preparing the metal-organic framework material is as follows:
respectively weighing cobalt nitrate hexahydrate and 2-methylimidazole, placing the cobalt nitrate hexahydrate and the 2-methylimidazole in a 250mL triangular flask, and then respectively adding 100mL of methanol to fully dissolve the cobalt nitrate hexahydrate and the 2-methylimidazole; then mixing the two solutions, stirring for 13min at room temperature, and standing for 24h at room temperature; and after the reaction is finished, recovering the precipitate by centrifugation, then cleaning the precipitate by using ethanol, and drying the precipitate in a vacuum drying oven to obtain purple ZIF-67.
3. The method for preparing bimetallic nanoenzyme as in claim 2, wherein the amount of the cobalt nitrate hexahydrate is 1.436g, and the amount of the 2-methylimidazole is 3.244 g.
4. The method for preparing the bimetallic nanoenzyme according to claim 1, wherein the step (b) is carried out by synthesizing by ion exchange methodNickel cobaltite NiCo2O4The process of (2) is as follows:
accurately weighing 100mg of ZIF-67 obtained in the step (a) and nickel chloride hexahydrate, placing the ZIF-67 and the nickel chloride hexahydrate in a 50mL reaction kettle, adding ethanol, and treating for 30min under an ultrasonic condition; then, transferring the reaction kettle into an electric heating air blast oven, and heating for 1h at the temperature of 90 ℃; after the reaction is finished, centrifugally drying and recovering to obtain green NiCo layered double hydroxide; then weighing a certain amount of NiCo layered double hydroxide, placing the NiCo layered double hydroxide in a crucible, transferring the crucible into a muffle furnace, calcining the NiCo layered double hydroxide for 3 hours at the temperature of 300 ℃, setting the temperature rise rate to be 2.5 ℃/min, and finally obtaining the NiCo2O4(ii) particulate matter.
5. The method for preparing bimetallic nanoenzyme as in claim 4, wherein the amount of nickel chloride hexahydrate is 150mg, and the volume of ethanol is 30 mL.
6. The method for preparing the bimetallic nanoenzyme as in claim 1, wherein the step (c) is a one-step self-assembly template method for preparing NiCo2O4@ PVP bimetal nanoenzyme, the process is as follows:
accurately weighing 200mg of NiCo prepared in the step (b)2O4And polyvinylpyrrolidone were placed in a 150mL Erlenmeyer flask; then adding ethanol, and carrying out ultrasonic treatment for 30min to fully dissolve the ethanol; then stirring the mixed solution for 1h at room temperature; after the reaction is finished, centrifuging, recovering and drying the precipitate to finally obtain NiCo2O4@ PVP particulate material.
7. The method for preparing the bimetallic nanoenzyme as in claim 4, wherein the polyvinylpyrrolidone is used in an amount of 200mg, and the volume of the ethanol is 60 mL.
8. The bimetallic nanoenzyme obtained by the preparation method of any one of claims 1 to 7 is used for scavenging active oxygen.
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| CN115121277A (en) * | 2022-07-05 | 2022-09-30 | 合肥工业大学 | Iodine-doped monoatomic nanoenzyme CoCNI and preparation method and application thereof |
| CN115463151A (en) * | 2022-08-18 | 2022-12-13 | 湖北文理学院 | Nano enzyme, preparation method and application thereof, and bacteriostatic agent |
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| CN115121277A (en) * | 2022-07-05 | 2022-09-30 | 合肥工业大学 | Iodine-doped monoatomic nanoenzyme CoCNI and preparation method and application thereof |
| CN115121277B (en) * | 2022-07-05 | 2024-01-09 | 合肥工业大学 | Iodine doped monoatomic nano enzyme CoCNI and preparation method and application thereof |
| CN115463151A (en) * | 2022-08-18 | 2022-12-13 | 湖北文理学院 | Nano enzyme, preparation method and application thereof, and bacteriostatic agent |
| CN115463151B (en) * | 2022-08-18 | 2023-09-19 | 湖北文理学院 | Nano-enzyme, preparation method and application thereof, and bacteriostat |
| CN115927175A (en) * | 2023-01-13 | 2023-04-07 | 四川大学 | Oxidation-resistant catalytic material and preparation method and application thereof |
| CN115927175B (en) * | 2023-01-13 | 2023-10-03 | 四川大学 | Antioxidant catalytic material and preparation method and application thereof |
| CN116173071A (en) * | 2023-02-17 | 2023-05-30 | 四川农业大学 | Preparation method and application of MnNiPCN@PVP nano enzyme |
| CN116173071B (en) * | 2023-02-17 | 2024-02-06 | 四川农业大学 | Preparation method and application of MnNiPCN@PVP nano enzyme |
| CN117159791A (en) * | 2023-11-01 | 2023-12-05 | 云南伦扬科技有限公司 | Hedgehog flower-shaped nano enzyme hydrogel for healing diabetic wounds |
| CN117159791B (en) * | 2023-11-01 | 2023-12-26 | 云南伦扬科技有限公司 | Hedgehog flower-shaped nano enzyme hydrogel for healing diabetic wounds |
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