Preparation method and application of G / Cu / GDY three-dimensional structure nano-enzyme

Abstract

The invention provides a simple, convenient and efficient three-dimensional structure nano-enzyme preparation method. The nano enzyme has remarkable laccase activity, can be used for efficiently detecting various phenol pollutants, and shows excellent performance in the aspect of degradation of micro-plastic pollutants. Ultra-small copper nanoparticles (CuNPs) are embedded between graphdiyne (GDY) and graphene (Graphene) to form a three-dimensional nano enzyme (G / Cu / GDY) with a sandwich structure, and the structure simulates a local electric field and three-dimensional geometric characteristics of natural laccase. Compared with a natural enzyme, the nano enzyme shows higher catalytic efficiency and stability, can adapt to pollutant treatment in a complex environment, and particularly has a wide application prospect in the fields of water quality monitoring and micro-plastic degradation.

Description

Technical Field

[0001] The invention relates to a preparation method of a novel G / Cu / GDY three-dimensional structure nanozyme and application thereof in pollutant detection and degradation, belonging to the technical field of nano material catalysts. Background Art

[0002] Natural enzymes play an irreplaceable role in biological, environmental and industrial applications due to their efficient catalytic ability and high specificity. However, the application of natural enzymes is limited by their structural fragility, high cost and easy inactivation in complex environments. As an alternative to natural enzymes, nanozymes have attracted widespread attention due to their excellent stability, controllability and low cost. However, traditional nanozymes still have difficulty reaching the level of natural enzymes in catalytic activity and selectivity, which is mainly attributed to their lack of the complex three-dimensional structure and local electric field distribution of natural enzymes.

[0003] Constructing nanozymes with three-dimensional structures is an important strategy to mimic the complex structure and function of natural enzymes. By constructing nanozymes with three-dimensional hierarchical structures, the exposed area of ​​active sites can be significantly increased, and the contact efficiency between reactants and catalytic centers can be optimized. This three-dimensional design not only provides a higher specific surface area, but also destroys the axial symmetry of electrons, enhances the pathway of electron transfer during catalysis, and thus effectively improves enzyme activity and stability. In addition, the axial asymmetry of electrons can produce a local electric field effect (LEF), regulate electron distribution through mechanisms such as orbital hybridization, and simulate the electric field environment in natural enzymes. This local electric field helps to reduce the reaction energy barrier, promote electron transfer, and thus accelerate the catalytic reaction. Overall, the three-dimensional construction of nanozymes and the electric field effect inside the enzyme synergistically enhance the catalytic efficiency, so that it still has excellent catalytic performance under complex environmental conditions, and expands its application potential in environmental monitoring, biosensing, and industrial catalysis.

[0004] Patent application CN115920935A discloses a method for synthesizing a carbon-based sandwich structure nanozyme, which forms a stable MnO2@C@Fe3C three-dimensional structure nanozyme by embedding metal nanoparticles between graphene or other carbon materials. The prepared nanozyme has excellent catalytic performance and can be used for colorimetric detection of biological small molecules such as glucose. It can quickly and accurately detect glucose content with a low detection limit and has high application value. However, the disadvantage of this process is that it is difficult to control the size of nanoparticles, and the preparation process involves multi-step synthesis, which is costly and limited in industrial application.

[0005] Patent application CN108130321A introduces a three-dimensional nanoflower structure of a protease and an inorganic material composite. The nanoenzyme is embedded in a hydrogel through immobilization technology. The unique three-dimensional network structure allows the enzyme to be stably present in the catalytic system, thereby achieving the purpose of secondary immobilization to improve its stability and reusability. The disadvantage of this process is that after being embedded in the hydrogel, the exposure of the active site of the enzyme may be limited, reducing the catalytic efficiency. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a method for preparing a graphene / copper nanoparticle / graphene (G / Cu / GDY) three-dimensional structure nanozyme, which uses graphene to solve the problem that the above three-dimensional structure nanozyme is difficult to prepare, and at the same time, the electron distribution of the antibonding orbit in the oxygen molecule is adjusted through orbital hybridization during the catalytic process, thereby reducing the free energy required for the reaction, reducing the reaction energy barrier, and accelerating the catalytic process of the hydroquinone substance, showing high sensitivity and a wide detection range. At the same time, the stability of the graphene surface ensures recyclability in complex environments, and the stability at high temperatures shows great potential in the degradation of microplastic pollutants.

[0007] The preparation method of the G / Cu / GDY three-dimensional structure nanozyme is:

[0008] 1. Preparation of graphene / copper:

[0009] First, copper nanoparticles were prepared using the polyol method. Briefly, CuCl2 (0.106 g) was dissolved in 20 mL of ethylene glycol solution, and then 30 mL of NaOH (0.1 M) in ethylene glycol solution was added. The mixed solution was kept in a 160 ° C oil bath for 3 h, and N2 was continuously passed to maintain an inert atmosphere. Then, graphene (50 mg) was mixed with the prepared Cu NPs in a mass ratio of 10:1 and physically stirred at room temperature for 24 h. After completion, the reaction mixture was centrifuged and washed several times with ethanol and ultrapure water. The precipitate was dried in a freeze dryer to obtain a G / Cu sample. The preferred concentration of ethanol is 95%.

[0010] 2. Preparation of graphene / copper / graphene:

[0011] GDY was synthesized mainly by coupling reaction with hexaethynylbenzene (HEB) as monomer. First, a solution of HEB-TMS and tetrabutylammonium fluoride (TBAF, 1 M in tetrahydrofuran (THF)) was prepared under argon atmosphere and stirred at 0°C for 15 min. Subsequently, the mixture was diluted with ethyl acetate, washed twice with saturated sodium chloride (NaCl) solution, dried with anhydrous magnesium sulfate (MgSO4), and filtered. The solvent was then evaporated under vacuum while keeping the temperature below 20°C. The resulting residue was diluted with 50 mL of acetone and added dropwise to a three-necked flask containing acetone (100 mL), pyridine (5 mL) and G / Cu (20 mg) over 10 hours, and the reaction mixture was kept under argon atmosphere at 60°C for 15 hours. After the reaction was completed, it was washed with N,N-dimethylformamide (DMF), acetone and ethanol in sequence. Finally, the graphene / copper / graphene acetylene composite was freeze-dried.

[0012] Preferably, the concentration of ethyl acetate is 99.5%, the concentration of acetone is 99.5%, the concentration of pyridine is 99.5%, and the concentration of DMF is 99.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the synthesis route of G / Cu / GDY;

[0014] Figure 2 This is the TEM image of G / Cu / GDY;

[0015] Figure 3 This is the electrochemical sensitivity performance test diagram of G / Cu / GDY;

[0016] Figure 4 is a diagram of a G / Cu / GDY microfluidic electrical sensor device;

[0017] Figure 5 This is the TEM image of LDPE microplastic degradation of G / Cu / GDY. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0019] Embodiment 1:

[0020] G / Cu / GDY was characterized and hydroquinone was detected and microplastic degradation experiments were performed. Transmission electron microscopy was used for characterization. G / Cu / GDY nanozymes synthesized using different proportions of copper nanoparticles were used for hydroquinone detection and microplastic degradation experiments.

[0021] The catalytic performance of G / Cu / GDY nanozyme was evaluated by the electrochemical signal response of hydroquinone (HQ). -1 ) solution was added dropwise to the glassy carbon electrode surface to form the working electrode, and the Ag / AgCl (3MKCl) electrode and the platinum wire electrode were used as the reference electrode and the counter electrode, respectively. -1 ) was added dropwise to the electrolyte (PBS buffer solution, pH = 7), and the electrochemical signal changes were detected by differential pulse voltammetry (DPV). -1 ) were used to conduct sensitivity test. In addition, 10 mg L -1 of (catechol (CC) and resorcinol (RC)) and 20 mg L -1 Other interfering agents (epinephrine, glucose, ascorbic acid, p-nitrophenol and bisphenol A).

[0022] These compounds were subjected to broad spectrum testing and stability testing according to the methods described above.

[0023]

[0024] Example 2: Based on the sensitive detection capability of G / Cu / GDY for hydroquinone substances, a microfluidic electrosensor device for rapid detection of hydroquinone pollutants in wastewater was established. The device comprises a microfluidic device, an electrochemical workstation, a peristaltic pump and a filter membrane. First, the injection hose of the peristaltic pump is opened, and under the action of the peristaltic pump, the wastewater containing hydroquinone pollutants enters the microfluidic sensor device from the wastewater bottle. Subsequently, the circulation hose of the peristaltic pump and the valve at the bottom of the glass tube are opened to allow the wastewater to circulate between the sensor and the glass tube. An electrochemical workstation is connected to the microfluidic sensor to monitor the changes in electrochemical signals in real time. After the reaction, the valve at the bottom of the glass tube is closed and the valve at the top of the glass tube is opened. The detected wastewater flows into the collection bottle from the top of the glass tube under the action of the peristaltic pump, and the large contaminants that are insoluble in water are retained in the filtration system by the filter membrane. After completion, the injection hose of the peristaltic pump is reopened to start the next cycle. (The initial concentration of HQ is 10ppm)

[0025] According to the above method, different electrolyte media were tested to verify the effect of G / Cu / GDY in practical applications. According to the method of Example 1, the detection performance of G / Cu / GDY nanozyme was evaluated using DPV electrochemical signal response.

[0026]

[0027] Example 3: The obtained G / Cu / GDY was subjected to microplastic degradation test. G / Cu / GDY (1 mg mL-1 ) and low-density polyethylene (LDPE, 100 mg) were added to 100 mL of ultrapure water and then ultrasonically treated for 5 minutes. The above solution was then transferred to a hydrothermal reactor and reacted at 90°C for 20 hours. The solution was removed, washed three times with ultrapure water and ethanol, and dried in a vacuum drying oven. The dried sample was weighed to obtain the degradation rate.

[0028] The G / Cu / GDY nanozyme was synthesized according to the preparation method, and the degradation ability of the G / Cu / GDY nanozyme on low-density polyethylene was verified by changing the catalyst concentration.

[0029]

[0030] Embodiment 4:

[0031] According to the method of Example 3, LDPE microplastics of different masses were changed to verify the degradation ability of G / Cu / GDY nanozyme on low-density polyethylene.

[0032]

[0033] Embodiment 5:

[0034] According to the method of Example 3, the reaction temperature was changed to verify the degradation ability of G / Cu / GDY nanozyme on low-density polyethylene.

[0035]

[0036]

Claims

1. A preparation method and application of a G / Cu / GDY three-dimensional structure nanozyme, characterized in that: The method comprises the following steps: (1) Preparation of graphene / copper: First, copper nanoparticles were prepared using the polyol method. Briefly, CuCl2 (0.106 g) was dissolved in 20 mL of ethylene glycol solution, and then 30 mL of NaOH (0.1 M) in ethylene glycol solution was added. The mixed solution was kept in a 160 ° C oil bath for 3 h, and N2 was continuously passed to maintain an inert atmosphere. Then, graphene (50 mg) was mixed with the prepared Cu NPs in a mass ratio of 10:1 and physically stirred at room temperature for 24 h. After completion, the reaction mixture was centrifuged and washed several times with ethanol and ultrapure water. The precipitate was dried in a freeze dryer to obtain a G / Cu sample. The preferred concentration of ethanol is 95%. (2) Preparation of graphene / copper / graphene: GDY was mainly synthesized by coupling reaction with hexaethynylbenzene (HEB) as monomer. First, a solution of HEB-TMS and tetrabutylammonium fluoride (TBAF, 1 M in tetrahydrofuran (THF)) was prepared under argon atmosphere and stirred at 0 °C for 15 min. Subsequently, the mixture was diluted with ethyl acetate, washed twice with saturated sodium chloride (NaCl) solution, dried with anhydrous magnesium sulfate (MgSO4), and filtered. The solvent was then evaporated under vacuum while keeping the temperature below 20 °C. The resulting residue was diluted with 50 mL of acetone and added dropwise to a three-necked flask containing acetone (100 mL), pyridine (5 mL) and G / Cu (20 mg) over 10 h, and the reaction mixture was kept at 60 °C for 15 h under argon atmosphere. It is worth noting that all processes from deprotection to addition should be carried out under dark and low temperature conditions to prevent HEB from decomposing. After the reaction was completed, it was washed with N,N-dimethylformamide (DMF), acetone and ethanol in sequence. Finally, the graphene / copper / graphene yne composite material was freeze-dried.

2. The preparation method according to claim 1, wherein The graphyne layer is formed by in-situ polymerization of hexynylbenzene monomer (HEB), and the three-dimensional structure destroys the electronic axial symmetry by d-π orbital hybridization with copper nanoparticles, and enriches the electrons on the surface of copper nanoparticles to the graphyne surface, thereby promoting the adsorption process of the catalytic reaction and reducing the free energy barrier of the reaction.

3. The preparation method as claimed in claim 1 is used in a biosensor to electrochemically detect phenolic pollutants in environmental samples. The concentration of phenolic compounds in environmental samples is detected by differential pulse voltammetry (DPV), and the minimum detection limit reaches 0.16 μM.

4. The nanozyme according to claim 1 can be used for microplastic degradation, and the decomposition of low-density polyethylene (LDPE) microplastics is achieved through the reactive oxygen species (ROS) generation mechanism. During the degradation process, the nanozyme activates O2 under high temperature conditions and produces O2· - and ·OH, free radicals can effectively destroy the polyethylene chain structure.

5. The nanozyme according to claim 1 still maintains a high catalytic activity after multiple cycles of use, and the enzyme activity remains above 89% of the initial level after 30 cycles.

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