Preparation method of graded nano-flower-shaped CeO2 / CoCPNFs nano-enzyme with five-enzyme activity

Abstract

The invention discloses a preparation method of a graded nano-flower-shaped CeO2 / CoCPNFs (CeO2 / CoCPNFs) nano-enzyme with five-enzyme activity. The preparation process adopts a two-step hydrothermal method, CoCPNFs with a nanoflower structure are prepared from a mixed solution of cobalt nitrate hexahydrate, isonicotinic acid, N, N-dimethylacetamide, ethanol and water, then the CeO2 / CoCPNFs nano composite material is prepared from a mixed solution of a cobalt precursor, cerium nitrate hexahydrate, hexamethylenetetramine and ethanol, and the CeO2 / CoCPNFs nano composite material belongs to the technical field of bionic nano enzymes. The nano material is simple in preparation process, cheap in raw materials, low in cost and high in yield, has mimic activity of five enzymes including peroxidase, oxidase, superoxide dismutase, laccase and catalase, and is high in catalytic activity and stable in performance. In conclusion, the CeO2 / CoCPNFs prepared by the method disclosed by the invention is a multienzyme active nano material with a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a hierarchical nanoflower-shaped CeO with five enzyme activities. 2 / CoCPNFs nanozyme preparation method. Background Art

[0002] Compared with the emerging nanozymes, natural enzymes have poor stability, are extremely sensitive to environmental factors, have harsh action conditions, are expensive, and are difficult to store. In contrast, nanozymes have better stability, can maintain activity over a wider temperature and pH range, are more environmentally adaptable, are low-cost and easy to store, and show great potential in industry, medicine, biosensors, etc. However, the functions of single-activity nanozymes are relatively limited, and they only catalyze a specific chemical reaction, and cannot be used in complex environments; while nanozymes with multiple enzyme activities, with their rich functions, not only show better environmental applicability and stability, but also provide new directions and hopes for solving many complex problems through cascade reactions and synergistic effects between enzymes. Therefore, the preparation of nanozymes with multiple enzyme activities has been favored by more and more researchers.

[0003] Although nanozymes have made great achievements, there are still some bottlenecks that limit the development and further application of nanozymes. Nanozymes mainly have the following defects: First, the catalytic activity of nanozymes is usually low, and it is difficult to achieve the efficient catalysis of natural enzymes. Second, the existing nanozyme preparation methods usually require expensive equipment and complex processes, with high cost, low yield, and it is difficult to control the size and morphology of nanozymes. Third, the enzyme-like activity of nanozymes is single, and there is mutual interference between multi-enzyme mimics. Therefore, the development of multi-enzyme active nanozymes with simple operation, high yield, low cost and stable performance still has important practical significance. Summary of the invention

[0004] The purpose of the present invention is to provide a hierarchical nano-flower-shaped CeO with five enzyme activities, which has simple preparation process, low cost, high yield and stable catalytic performance. 2 / CoCPNFs nanozyme preparation method.

[0005] 1. CeO 2 Preparation of / CoCPNFs nanozymes 1) Cobalt nitrate hexahydrate is used as the cobalt salt and isonicotinic acid is used as the ligand. They are dissolved in a mixed solution of N,N-dimethylformamide, anhydrous ethanol and water, and ultrasonicated to form a uniform mixed solution. The mixed solution is then hydroheated at 120°C~180°C for 6~20h, naturally cooled to room temperature, and the precipitate is centrifuged, washed, and dried to obtain CoCPNFs.

[0006] The molar ratio of cobalt nitrate hexahydrate to isonicotinic acid is 1:1-1:4. The volume ratio of N,N-dimethylformamide, anhydrous ethanol and water is 8-10:4-6:1. The centrifugal speed is 8000r / min, and the centrifugal time is 1-3min. Drying refers to 40℃-60℃, and the time is 6-12h.

[0007] 2) Dissolve CoCPNFs in anhydrous ethanol, ultrasonically treat to form a uniform solution, add hexamethylenetetramine and cerium nitrate hexahydrate in sequence under magnetic stirring to form a uniform mixed solution, then hydrothermally react the mixed solution at 140℃~200℃ for 4~12h, cool naturally to room temperature, centrifuge to obtain the precipitate, wash, and dry to obtain a hierarchical nanoflower-like CeO with five enzyme activities. 2 / CoCPNFs nanozymes.

[0008] The mass ratio of CoCPNFs to cerium nitrate hexahydrate is 3:2, and the molar mass ratio of cerium nitrate hexahydrate to hexamethylenetetramine is 1:1-1:5. The magnetic stirring time is 30-60 minutes. The centrifugal speed is 8000r / min, and the time is 1-3 minutes. Drying refers to 40°C-60°C, and the time is 6-12 hours.

[0009] The above CeO 2 In the preparation process of CoCPNFs, cobalt nitrate hexahydrate provides a cobalt source, which is chelated with isonicotinic acid containing a carboxylic acid ligand to construct a novel nanomaterial; then hexamethylenetetramine (ligand) and cerium nitrate hexahydrate (cerium source) are added in sequence to synthesize CeO 2 / CoCPNFs nanozymes. CeO 2 ) is a typical rare earth metal oxide. 3+ and Ce 4 + The flexible conversion between the two, the reversible exchange of surface oxygen ions, the high oxygen storage capacity and oxygen vacancies, etc., can greatly improve the catalytic activity of the composite material by loading cerium dioxide as an active site on the nanomaterial, providing more possibilities for simulating multi-active nanozymes. At the same time, this preparation process also meets the requirements of low material cost.

[0010] 2. CeO 2 Study on the five enzyme activities of CoCPNFs Investigation of peroxidase-like activity: Establishment of CeO under acidic conditions 2 / CoCPNFs-H 2 O 2 -3,3',5,5'-tetramethylbenzidine (TMB) system, CeO 2 / CoCPNFs can effectively catalyze H 2 O 2The decomposition of hydroxyl radicals (·OH) produces hydroxyl radicals (·OH), which oxidize the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (oxidized TMB). A 652 nm The peroxidase activity of the nanozyme was studied by measuring the absorbance changes at

[0011] Investigation of oxidase activity: In the absence of H 2 O 2 In the presence of CeO, the reaction time is prolonged and the oxygen in the air is absorbed by CeO 2 / CoCPNFs are reduced to generate reactive oxygen species, which further react with TMB molecules to oxidize the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (ox-TMB). 652nm The absorbance change at 4° was used to verify the oxidase activity.

[0012] Investigation of superoxide dismutase activity: The activity was evaluated by measuring the inhibition of nitro blue tetrazolium (NBT) photoreduction. In the NBT-riboflavin-methionine-EDTA reaction system, light causes riboflavin to generate superoxide anion free radicals (O 2 ·- ), which can reduce NBT to a blue-purple formazan product, A 560 nm The absorbance at the reaction point increases significantly. If CeO is added to this reaction system 2 / CoCPNFs, eliminated the generated superoxide anion free radicals, A 560nm The decrease in absorbance at 4° indicated that the nanozyme had superoxide dismutase activity.

[0013] Investigation of laccase activity: Dopamine hydrochloride (DA) was used as the chromogenic substrate. Since dopamine hydrochloride would remove the hydrochloric acid group of dopamine hydrochloride to form dopamine under weak alkaline conditions, a chromogenic reaction of dopamine hydrochloride (HCl-DA) and CeO was constructed under weak alkaline conditions. 2 / CoCPNFs-HCl-DA reaction system, CeO 2 / CoCPNFs can catalyze dopamine oxidation in A 475nm Characteristic peaks appear.

[0014] Investigation of catalase activity: Due to hydrogen peroxide (H 2 O 2 ) is unstable and can be decomposed into water (H 2 O) and oxygen (O 2 ), thus constructing CeO 2 / CoCPNFs-H 2 O 2The absorbance value of the test system at 240nm was used as the evaluation 2 O 2 Decomposed indicators.

[0015] The present invention discloses a method for preparing hierarchical nano-flower-shaped CeO with five enzyme activities by a simple two-step hydrothermal method. 2 / CoCPNFs nanozyme method, which has potential biological application value. 2 / CoCPNFs nanozymes have antioxidant activity (catalase-like, superoxide dismutase-like), which can effectively remove reactive oxygen species through cascade reactions and treat various diseases caused by oxidative stress. 2 / CoCPNFs nanozymes have pro-oxidative activity (peroxidase-like, oxidase-like), which can catalyze the production of a large number of reactive oxygen species through cascade reactions and synergistic effects, and are widely used in biosensing, anti-cancer and antibacterial treatments. However, antioxidant or pro-oxidative nanozymes alone are also limited in biomedical applications. For example, in anti-tumor applications, the role of pro-oxidative nanozymes is limited due to the hypoxia of the tumor microenvironment. Antioxidant enzymes and pro-oxidative enzymes work synergistically to provide oxygen and promote the production of reactive oxygen species to kill tumor cells. In the treatment of acute kidney injury, the synergistic effect of antioxidant enzymes and pro-oxidative enzymes can effectively control the increase in the production of reactive oxygen species caused by the disease. Therefore, CeO 2 The synergistic effect of the antioxidant and pro-oxidant nanozymes of / CoCPNFs can greatly expand the application range of nanozymes. 2 / CoCPNFs are a kind of multi-enzyme active nanomaterials with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The CeO prepared in the present invention 2 Schematic diagram of the five enzyme activities of / CoCPNFs.

[0017] Figure 2 AB is CeO prepared in the embodiment of the present invention. 2 / CoCPNF scanning electron microscope image; CF is CeO prepared in the embodiment of the present invention 2 / CoCPNF transmission electron microscopy images at different magnifications, and selected area electron diffraction images; G is CeO prepared in the embodiment of the present invention 2 Energy dispersive X-ray spectrum of / CoCPNF, (G1-G5) represent C, N, O, Co and Ce elements, respectively.

[0018] Figure 3 A is CeO prepared in the embodiment of the present invention. 2 / CoCPNFs X-ray diffraction pattern, BC is CeO prepared in the embodiment of the present invention 2 / CoCPNFsN 2 Adsorption-desorption isotherms and pore size distribution, DI is CeO prepared in the embodiment of the present invention 2 / CoCPNFsX-ray photoelectron spectrum.

[0019] Figure 4 CeO prepared in the embodiment of the present invention 2 / Peroxidase activity test diagram of CoCPNFs.

[0020] Figure 5 CeO prepared in the embodiment of the present invention 2 / Oxidase activity test diagram of CoCPNFs.

[0021] Figure 6 CeO prepared in the embodiment of the present invention 2 / Superoxide dismutase activity test diagram of CoCPNFs.

[0022] Figure 7 CeO prepared in the embodiment of the present invention 2 / CoCPNFs laccase activity test diagram.

[0023] Figure 8 CeO prepared in the embodiment of the present invention 2 / CoCPNFs catalase activity test diagram. DETAILED DESCRIPTION

[0024] The present invention is further explained below with reference to specific embodiments.

[0025] Preparation of CoCPNFs First, 0.1161 g of cobalt nitrate hexahydrate and 0.024 g of isonicotinic acid were weighed and dissolved in a mixed solution of 18 mL of N,N-dimethylformamide, 10 mL of anhydrous ethanol and 2 mL of ultrapure water in that order. The mixture was ultrasonically treated for 10 min to completely dissolve it. The mixed solution was then heated to 160 ° C in an oven, reacted for 12 h, naturally cooled to room temperature, and centrifuged at 8000 r / min to collect the precipitate. The solid in the previous step was then washed three times with ultrapure water and anhydrous ethanol solution respectively and the precipitate was collected to precipitate CoCPNFs. Finally, it was dried at 60 ° C for 10 h to obtain a CoCPNFs powder sample.

[0026] CeO 2 Preparation of / CoCPNFs Take 0.0390g of CoCPNFs and dissolve it in 15mL of anhydrous ethanol. Ultrasonicate to form a uniformly dispersed solution. Then add 0.0252g of hexamethylenetetramine and 0.0261g of cerium nitrate hexahydrate in sequence, stir magnetically for 40min, and then heat the mixed solution to 180℃ in an oven, react for 6h, cool naturally to room temperature, and collect the precipitate by centrifugation at 8000r / min to obtain CeO 2 / CoCPNFs, then the solid was washed three times with ultrapure water and anhydrous ethanol respectively and the precipitate was collected to precipitate CeO 2 / CoCPNFs, dried at 60℃ for 10h, and obtained CeO 2 / CoCPNFs powder samples.

[0027] CeO 2 Structural characterization of / CoCPNFs Figure 2 AF is the prepared CeO 2 / CoCPNFs scanning electron microscopy, transmission electron microscopy and selected area electron diffraction patterns. It can be seen that CeO 2 / CoCPNFs have a nanoflower structure and confirmed the successful loading of ceria. Figure 2 G (G1-G5) is CeO 2 / CoCPNFs energy dispersion X-ray spectrum, showing that C, N, O, Co, and Ce elements are evenly dispersed on the surface of the nanoflowers, further confirming that CeO 2 / CoCPNFs were successfully prepared. 3A is CeO 2 X-ray diffraction pattern of CeO / CoCPNFs. 2 / CoCPNFs and CeO 2 (JCPDS no. 43-1002), further proving that CeO 2 of successful load. Figure 3 BC is CeO 2 / N of CoCPNFs 2 The adsorption-desorption isotherms and pore size distribution show that the sample exhibits a typical IV-type isotherm, indicating that the sample has a multi-level pore structure. The pore size is mainly concentrated in 2-40nm, further proving the rich mesoporous structure of the catalyst. Figure 3 DI is CeO prepared in Example 1 of the present invention 2 X-ray photoelectron spectroscopy of CeO / CoCPNFs further proves that 2 / CoCPNFs were successfully prepared.

[0028] CeO 2Study on the five enzyme activities of CoCPNFs Peroxidase activity: TMB was used as the chromogenic substrate to 2 / CoCPNFs (100uL, 150ug / mL) and TMB (100uL, 0.5mM) were added to a citric acid-sodium citrate buffer solution with a pH of 4.2 in sequence, and the final volume was maintained at 2mL. The mixture was reacted at room temperature for 3min, and the absorbance of the mixed solution at 652nm was recorded by a UV spectrophotometer; Figure 4 CeO 2 / CoCPNFs-H 2 O 2 -TMB reaction system in A 652nm The absorbance of H 2 O 2 In the presence of CeO 2 / CoCPNFs can quickly oxidize TMB to ox-TMB. 2 / CoCPNFs have peroxidase activity.

[0029] Oxidase activity: CeO 2 / CoCPNFs (100uL, 150ug / mL) and TMB (100uL, 0.5mM) were added to a citric acid-sodium citrate buffer solution at pH = 4.2 in sequence, and the final volume was kept at 2mL. The reaction was carried out at room temperature for 1h, and the temperature of the mixed solution at A was recorded. 652nm The absorbance at Figure 5 CeO 2 / CoCPNFs-TMB system has the highest absorbance, which proves that 2 / CoCPNFs have oxidase activity.

[0030] Superoxide dismutase activity: CeO 2 / CoCPNFs (120uL, 180ug / mL), riboflavin (40uL, 0.02mM), methionine (60uL, 3mM), EDTA (60uL, 0.3mM), NBT (60uL, 0.3mM) were added to pH=8 Tris-HCl buffer in sequence, and the final volume was kept at 2mL. The mixture was reacted in the dark for 5min at room temperature and under LED light for 5min. The mixed solution A was recorded. 560nm The absorbance at Figure 6 See the addition of CeO 2 / CoCPNFs, the reduction of NBT was well inhibited. 560nm The absorbance at the 2 / CoCPNFs have superoxide dismutase activity.

[0031] Laccase activity: CeO 2 / CoCPNFs (100uL, 150ug / mL) and HCl-DA (80uL, 0.4mM) were added to Tris-HCl buffer at pH=7.5 in sequence, and the final volume was maintained at 2mL. The reaction was carried out at room temperature for 20min. Test A 475nm The absorbance at Figure 7 CeO 2 / CoCPNFs can effectively oxidize HCl-DA, proving that CeO 2 / CoCPNFs have laccase activity.

[0032] Catalase activity: For CeO 2 Steady-state kinetic analysis of catalase-like activity of CeO / CoCPNFs 2 / CoCPNFs (150ug / mL), H 2 O 2 (20uL, 0.1M) was added to Tris-HCl buffer solution at pH = 8, incubated at room temperature for 5 minutes, and tested A 240nm The absorbance at Figure 8 CeO 2 / CoCPNFs-H 2 O 2 The absorbance of the system is significantly lower than that of pure H 2 O 2 System, CeO 2 The addition of CoCPNFs can achieve 2 O 2 The effective decomposition of CeO 2 / CoCPNFs have catalase activity.

Claims

1. A method for preparing a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities, comprising the following steps: (1) Cobalt nitrate hexahydrate is used as the cobalt salt and isonicotinic acid is used as the ligand. They are dissolved in a mixed solution of N,N-dimethylformamide, anhydrous ethanol and water, and ultrasonically mixed to obtain CoCPNFs. The mixed solution is then hydrothermaled at 120°C to 180°C for 6 to 20 hours, cooled naturally to room temperature, and precipitated by centrifugation. The precipitate is washed and dried to obtain CoCPNFs.

2. (2) CoCPNFs were dissolved in anhydrous ethanol and subjected to ultrasonic treatment to form a uniform solution. Hexamethylenetetramine and cerium nitrate hexahydrate were added in sequence under magnetic stirring to form a uniform mixed solution. The mixed solution was then subjected to hydrothermal reaction at 140-200°C for 4-12 hours, naturally cooled to room temperature, centrifuged to obtain the precipitate, washed, and dried to obtain nanoflower-shaped CeO2 / CoCPNFs nanozyme with pentaenzyme activity.

3. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (1), the molar mass ratio of cobalt nitrate hexahydrate to isonicotinic acid is 1:1~1:

4.

4. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (1), the volume ratio of N,N-dimethylformamide, anhydrous ethanol and water is 8~10:4~6:

1.

5. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (1), the centrifugal speed is 8000 r / min and the centrifugal time is 1-3 min.

6. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (1), drying refers to 40°C~60°C and time 6~12h.

7. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (2), the mass ratio of CoCPNFs to cerium nitrate hexahydrate is 3:1~3:3; the molar mass ratio of cerium nitrate hexahydrate to hexamethylenetetramine is 1:1~1:

5.

8. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (2), the magnetic stirring time is 30 to 60 minutes.

9. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, characterized in that: In step (2), the centrifugal speed is 8000 r / min and the time is 1 to 3 min.

10. The preparation of a hierarchical nanoflower-shaped CeO2 / CoCPNFs nanozyme with five enzyme activities as claimed in claim 1, wherein in step (2), drying is performed at 40°C to 60°C for 6 to 12 hours.

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