Preparation method of hydrangea-shaped cobalt coordination polymer / ferrocobalt layered double-metal hydroxide nano-enzyme with five-enzyme simulation activity

Abstract

The invention discloses a preparation method of hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nano-enzyme with five-enzyme simulation activity, which mainly comprises the following steps: preparing a Co HNFs (cobalt coordination polymer graded nanoflower) precursor from cobalt nitrate hexahydrate and pyridine-2, 5-dicarboxylic acid in a mixed solution of N, N-dimethylacetamide, methanol and water, and preparing the cobalt coordination polymer / cobalt-iron layered double hydroxide nano-enzyme with five-enzyme simulation activity. CoFe-LDH grows on the surface of the precursor through an etching method, the Co HNFs / CoFe-LDH composite material is prepared, the material is cheap in raw material, the preparation method is simple, the reaction energy consumption is low, the Co HNFs / CoFe-LDH composite material has a good industrial application prospect, and the catalyst has nano-enzymes with five enzyme activities, specifically oxidase, peroxidase and catalase, and has a good application prospect. The five natural enzymes such as superoxide dismutase and laccase have the same enzyme activity.

Description

Technical Field

[0001] The invention belongs to the technical field of nanozymes, and specifically relates to a preparation method of a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double metal hydroxide nanozyme with pentaenzyme mimicking activity. Background Art

[0002] As traditional biocatalysts, natural enzymes have high affinity and specific catalytic activity for substrates under mild conditions, and show important application value in various biological detections. However, since enzymes are mainly composed of proteins and bioorganic molecules, their enzymatic activity and stability are largely dependent on mild reaction conditions and environments. These inherent defects, coupled with the high cost of enzymes, have greatly hindered their practical application. In order to overcome these challenges, researchers have been actively looking for alternatives to natural enzymes. The emergence of nanotechnology, biotechnology, and catalytic science has paved the way for the development of nanomaterials with enzyme-like catalytic activity. As early as 2007, researchers reported a nanomaterial Fe3O4NPs with peroxidase-like activity. As an imitation of natural enzymes, nanozymes have the advantages of low cost, easy preparation, good stability, and good durability, and therefore have received widespread attention.

[0003] As the research on nanozymes deepens, people have found that the synthesized bimetallic nanomaterials have multiple catalytic centers and active sites, which accelerate electron transfer and oxygen activation. Therefore, it is expected to prepare nanozymes with a variety of enzyme activities to replace nanozymes with only one enzyme activity. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme with five enzyme mimicking activities, which has high activity, good stability, simple preparation method and low cost.

[0005] 1. Preparation of Co HNFs / CoFe-LDH Nanozymes 1) Dissolve cobalt nitrate hexahydrate and pyridine 2,5-dicarboxylic acid ligand in an organic solvent by ultrasonication to form a mixed solution, then heat the mixed solution to 120-180°C for 6-30h, and after the solution is cooled to room temperature, centrifuge to obtain a precipitate, wash and dry to obtain a Co HNFs precursor; In the above step 1), the molar ratio of cobalt nitrate hexahydrate to pyridine 2,5-dicarboxylic acid is 1:1-1:4. The organic solvent is a mixture of N,N-dimethylformamide, methanol and water, wherein the volume ratio of N,N-dimethylformamide, methanol and water is (8-10):(4-6):1. The centrifugal speed is 8000-10000 rpm; the centrifugal time is 2-5 min. Drying refers to thermal drying at 60°C for 10-24 h.

[0006] 2) The Co HNFs precursor and ferrous sulfate heptahydrate were dissolved in an ethanol solution and reacted at room temperature for 0.1-1 h under N2 protection to obtain hydrangea-shaped Co HNFs / CoFe-LDH nanozymes with pentaenzyme mimetic activity; In the above step 2), the mass ratio of the Co HNFs precursor to ferrous sulfate heptahydrate is 2:1-1:4.

[0007] Synthesis mechanism of the present invention: In the preparation process of the above-mentioned Co HNFs / CoFe-LDH, cobalt nitrate hexahydrate provides a cobalt source, and a novel Co HNFs material is constructed by a solvothermal method with a pyridine 2,5-dicarboxylic acid ligand; then ferrous sulfate heptahydrate is added, and the Co HNFs / CoFe-LDH material is synthesized by ion exchange under N2 protection. The bimetallic material has more active centers and a larger specific surface area, which is more conducive to the attachment of substrates, improves catalytic activity, and has multi-enzyme activity, providing inspiration for subsequent cascade reactions and sensor detection.

[0008] 2. Study on the enzyme activity of Co HNFs / CoFe-LDH Investigation of oxidase-like activity: In the Co HNFs / CoFe-LDH / TMB reaction system constructed under acidic conditions, Co CPNSs / CoFe-LDH showed the same catalytic properties as natural oxidase activity. .- The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized to generate blue oxidized TMB (ox-TMB). The oxidase activity can be determined by analyzing the absorbance at 652 nm.

[0009] Investigation of peroxidase-like activity: In the Co HNFs / CoFe-LDH / H2O2 / TMB reaction system constructed under acidic conditions, Co HNFs / CoFe / LDH exhibited the same catalytic properties as natural peroxidase activity. In the presence of hydrogen peroxide (H2O2), Co CPNSs / CoFe-LDH effectively catalyzed the decomposition of H2O2 to produce hydroxyl radicals (•OH), oxidizing TMB to ox-TMB. The peroxidase activity of the nanomaterial can be obtained by analyzing the absorbance change at 652 nm.

[0010] Investigation of catalase-like activity: H2O2 is an unstable compound that can be decomposed to produce O2 and H2O under the action of catalase. Therefore, in a closed neutral environment, H2O2 and Co HNFs / CoFe-LDH-H2O2 reaction systems were constructed, and the pressure values ​​of the two systems were measured as an indicator to evaluate the decomposition rate of H2O2. Compared with the H2O2 system, Co HNFs / CoFe-LDH in the Co HNFs / CoFe-LDH / H2O2 reaction system effectively decomposed H2O2, resulting in the production of a large amount of gas and increasing the air pressure in the space.

[0011] Investigation of laccase-like activity: 2,4-dichlorophenol (2,4-DP) is a chromogenic substrate. In the presence of 4-aminoantipyrine (4-AP), laccase can catalyze the oxidation of 2,4-DP / 4-AP to produce a rose-red substance. Therefore, the 2,4-DP / 4-AP and Co HNFs / CoFe-LDH / 2,4-DP / 4-AP reaction systems were constructed under weak alkaline conditions. As a result, the catalytic oxidation of 2,4-DP / 4-AP by Co HNFs / CoFe-LDH showed a characteristic peak at 510 nm.

[0012] Research on superoxide dismutase-like enzymes: The research on superoxide dismutase uses the classic nitrotetrazole blue photoreduction method. First, a nitrotetrazole blue chloride (NBT)-riboflavin-methionine-ethylenediaminetetraacetic acid (EDTA) reaction system is constructed under weak alkaline conditions. When this system is exposed to light, riboflavin will produce superoxide anion free radicals (O2 .- ) and then reduce nitro blue tetrazolium chloride to a blue-purple formazan product. Once superoxide dismutase is introduced into the above-constructed system, the amount of blue-purple formazan product formed is greatly reduced, because superoxide dismutase eliminates the generated superoxide anion free radicals. The constructed Co HNFs / CoFe-LDH-NBT-riboflavin-methionine-EDTA reaction system shows weak absorption at 560nm, indicating that Co HNFs / CoFe-LDH has superoxide dismutase-like properties.

[0013] Nanozymes have potential biological applications due to the multi-enzyme activity of hydrangea-like Co HNFs / CoFe-LDH nanoflowers. Nanozymes (catalase-like, superoxide dismutase-like) based on the antioxidant activity of Co HNFs / CoFe-LDH nanoflowers can effectively remove reactive oxygen species through cascade reactions and treat various diseases caused by oxidative stress. On the other hand, nanozymes (peroxidase-like) based on the pro-oxidant activity of CoHNFs / CoFe-LDH nanoflowers can catalyze the production of a large number of reactive oxygen species in vivo through cascade reactions and synergistic effects, and are widely used in biosensing, anticancer and antibacterial treatment. However, antioxidant or pro-oxidant nanozymes alone are also limited in biomedical applications. For example, in anti-tumor applications, the role of pro-oxidant nanozymes is limited due to the hypoxia of the tumor microenvironment. Antioxidant and pro-oxidant 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 and pro-oxidant enzymes can effectively control the increase in the production of reactive oxygen species caused by the disease. Therefore, the synergistic effects of the antioxidant-like and pro-oxidant-like nanozymes of Co HNFs / CoFe-LDH nanoflowers can greatly expand the application scope of nanozymes.

[0014] In summary, the present invention discloses a one-step solvothermal method for preparing multi-enzyme active nanomaterials, and the prepared hydrangea-shaped graded nanoflowers with five enzyme mimicking activities have the same enzyme activities as five natural enzymes, namely oxidase, peroxidase, catalase, superoxide dismutase and laccase. Among them, the oxidase, peroxidase, superoxide dismutase and laccase activities of Co HNFs / CoFe-LDH have been fully demonstrated by constructing a classic colorimetric system, while the catalase activity of Co HNFs / CoFe-LDH was evaluated by the degree of pressure change of the system. In summary, the Co HNFs / CoFe-LDH prepared by the present invention is a multi-enzyme active nanomaterial with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of five enzyme activities of Co HNFs / CoFe-LDH prepared by the present invention; Figure 2 AF are SEM images of Co HNFs / CoFe-LDH; F1-F5 are elemental atlas images of Co HNFs / CoFe-LDH; Figure 3 is the X-ray powder diffraction spectrum of Co HNFs / CoFe-LDH; Figure 4 is the UV-visible absorption spectrum of Co HNFs / CoFe-LDH / TMB, TMB reaction system; Figure 5 It is the UV-visible absorption spectra of Co HNFs / CoFe-LDH / H2O2 / TMB, H2O2 / TMB, H2O2, and TMB reaction systems; Figure 6 It is a comparison chart of the gas pressure of the two reaction systems of Co HNFs / CoFe-LDH / H2O2 / TMB and H2O2; Figure 7 It is the UV-visible absorption spectra of the reaction systems of Co HNFs / CoFe-LDH / 2,4-DP / 4-AP, 2,4-DP / 4-AP, 4-AP, 2,4-DP; Figure 8 It is the UV-visible absorption spectrum of NBT-riboflavin-methionine-EDTA and Co HNFs / CoFe-LDH / NBT-riboflavin-methionine-EDTA reaction system. DETAILED DESCRIPTION

[0016] The present invention is further explained below with reference to specific embodiments. Example

[0017] Preparation of Co HNFs / CoFe-LDH First, 116.4 mg Co(NO3)2·6H2O and 33.4 mg pyridine 2,5-dicarboxylic acid were ultrasonically mixed in a mixed solution of 30 mL of N,N-dimethylformamide (18 mL), methanol (10 mL) and water (2 mL); then the mixed solution was heated to 160°C in an oven, and the precipitate was collected by centrifugation after 18 h, washed three times with water and ethanol solution respectively, and dried in an oven at 60°C to obtain the Co HNFs precursor.

[0018] 10 mg of Co CPNSs was dissolved in ethanol to form solution A, 10 mg of FeSO4·7H2O was dissolved in a mixed solution of water and ethanol to form solution B, solution B was injected into solution A, and the mixture was reacted at room temperature for 15 min under N2 protection. The mixture was washed twice with ethanol solution and dried in an oven at 60 °C to obtain hydrangea-shaped hierarchical nanoflowers Co HNFs / CoFe-LDH.

[0019] Structural characterization of Co HNFs / CoFe-LDH Figure 2 A, B, C, D, E, and F are scanning electron micrographs of Co HNFs / CoFe-LDH. Transmission electron microscopy imaging characterized the morphology of Co HNFs / CoFe-LDH. It can be seen that Co HNFs / CoFe-LDH has the morphology of hydrangea-like hierarchical nanoflowers. Figure 2From F1-F5, we can see that Co HNFs / CoFe-LDH contains C, N, O, Fe and Co elements, and element mapping imaging shows that the elements are evenly distributed in Co HNFs / CoFe-LDH.

[0020] Figure 3 : is the X-ray powder diffraction spectrum of Co HNFs / CoFe-LDH. The characteristic peaks of Co HNFs / CoFe-LDH in the spectrum are consistent with the characteristic diffraction peaks of CoFe-LDH (JCPDS# 50-0235), indicating that the formation of Co HNFs / CoFe-LDH is composed of Co HNFs and CoFe-LDH.

[0021] Study on the five enzyme activities of Co HNFs / CoFe-LDH Oxidase activity of Co HNFs / CoFe-LDH: Co HNFs / CoFe-LDH solution (80 μL, 2 mg / mL) was added to a mixed solution containing 150 μL TMB solution (0.75 μM) and 2 mL acetic acid-sodium acetate buffer solution (0.2 M, pH = 4.2). The mixture was incubated at room temperature for 5 minutes, and the absorbance at 655 nm was recorded by a UV-Vis spectrophotometer. Figure 4 The absorbance of the Co HNFs / CoFe-LDH / TMB reaction system at 652 nm was significantly higher than that of TMB and Co HNFs / CoFe-LDH, proving that Co HNFs / CoFe-LDH catalyzed the oxidation of TMB to generate blue ox-TMB.

[0022] The peroxidase activity of Co HNFs / CoFe-LDH catalyzes the oxidation of the chromogenic substrate TMB to ox-TMB in the presence of H2O2. H2O2 (0.4 M), TMB (0.4 mM) and Co HNFs / CoFe-LDH (80 μL, 2 mg / mL) were added to an acetate buffer solution at pH = 4.4 and incubated at 25°C for 3 minutes. The absorbance at 652 nm was recorded by a UV-Vis spectrophotometer. Figure 5 The absorbance of the Co HNFs / CoFe-LDH / TMB / H2O2 system was significantly higher than that of Co HNFs / CoFe-LDH / TMB, TMB / H2O2, H2O2 and TMB, indicating that TMB was oxidized to form blue oxTMB in the presence of H2O2.

[0023] The catalase activity of Co HNFs / CoFe-LDH was demonstrated based on the gas production by the catalase-catalyzed decomposition of H2O2. 100 μL H2O2 (0.1 M) and 80 μL Co HNFs / CoFe-LDH (2 mg / mL) were added to 900 μL ultrapure water, and the gas pressure was measured with a barometer. Figure 6 The gas pressure of the Co HNFs / CoFe-LDH / H2O2 system is significantly higher than that of the single H2O2 system, indicating that CoHNFs / CoFe-LDH can catalyze H2O2 to accelerate its decomposition.

[0024] The laccase activity of Co HNFs / CoFe-LDH was determined by the classic 2,4-DP and 4-AP colorimetric method. 2,4-DP (100 μl 1 mg / mL), 4-AP (100 μl 1 mg / mL) and Co HNFs / CoFe-LDH (80 μl 2 mg / mL) solutions were added to tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution (pH=7.5). The reaction was carried out in a shaker at 25°C for 1.5 h. The absorbance of the reaction system at 510 nm was recorded using a UV-visible spectrophotometer. Figure 7 The absorbance of the Co HNFs / CoFe-LDH / 2,4-DP / 4-AP system was significantly higher than that of 2,4-DP / 4-AP, 2,4-DP and 4-AP, indicating that Co HNFs / CoFe-LDH catalyzed the generation of rose-red quinone compounds in the presence of 2,4-DP and 4-AP.

[0025] The superoxide dismutase-like performance of Co HNFs / CoFe-LDH was investigated by colorimetry to investigate its inhibitory effect on the photoreduction of nitro blue tetrazolium (NBT). First, 0.3 mM NBT, 15 mM methionine, and 0.15 mM Na2EDTA were mixed in tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution (pH=8) and 15 μM riboflavin, with or without the addition of Co HNFs / CoFe-LDH (2 mg / mL). The mixture was then reacted in the dark for 5 min and then irradiated under LED light for 5 min. The absorbance of the supernatant at 560 nm was recorded by a UV-visible spectrophotometer. Figure 8 The absorbance of the Co HNFs / CoFe-LDH-NBT-riboflavin-methionine-EDTA system was significantly lower than that of NBT-riboflavin-methionine-EDTA, indicating that Co HNFs / CoFe-LDH eliminated the superoxide anion radicals produced in the presence of riboflavin under light, resulting in a significant decrease in the absorbance at 560 nm.

Claims

1. A method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme with five enzyme mimicking activities, characterized in that: The following steps are involved: 1) Dissolve cobalt nitrate hexahydrate and pyridine 2,5-dicarboxylic acid ligand in an organic solvent by ultrasonication to form a mixed solution, then heat the mixed solution to 120-180°C for 6-30h, and after the solution is cooled to room temperature, centrifuge to obtain a precipitate, wash and dry to obtain a Co HNFs precursor; 2) The Co HNFs precursor and ferrous sulfate heptahydrate were co-dissolved in an ethanol solution and reacted at room temperature for 0.1-1 h under N2 protection to obtain hydrangea-shaped Co HNFs / CoFe-LDH nanozymes with pentaenzyme mimetic activity.

2. The method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme having five enzyme mimicking activities as claimed in claim 1, characterized in that: In step 1), the molar ratio of cobalt nitrate hexahydrate to pyridine 2,5-dicarboxylic acid is 1:1 to 1:

4.

3. The method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme having five enzyme mimicking activities as claimed in claim 1, characterized in that: In step 1), the organic solvent is a mixture of N,N-dimethylformamide, methanol and water, wherein the volume ratio of N,N-dimethylformamide, methanol and water is (8-10):(4-6):

1.

4. The method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme having five enzyme mimicking activities as claimed in claim 1, characterized in that: In step 1), the centrifugal speed is 8000-10000 rpm and the centrifugal time is 2-5 min.

5. The method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme having pentaenzyme mimicking activity as claimed in claim 1, characterized in that: In step 1), drying refers to thermal drying at 60°C for 10 to 24 hours.

6. The method for preparing a hydrangea-shaped cobalt coordination polymer / cobalt-iron layered double hydroxide nanozyme having five enzyme mimicking activities as claimed in claim 1, characterized in that: In step 2), the mass ratio of the Co HNFs precursor to ferrous sulfate heptahydrate is 2:1 to 1:4.

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