Fullerene / metal organic framework loaded with acetylcholin esterase as well as preparation method and application of fullerene / metal organic framework

Abstract

The invention relates to the technical field of preparation and application of functional materials, in particular to a fullerene / metal organic framework loaded with acetylcholin esterase as well as a preparation method and application of the fullerene / metal organic framework. The preparation method comprises the following steps: firstly, preparing a fullerene / metal organic framework C60-coated PCN-222 (Fe) nano-enzyme with a space limited environment, and providing a self-cascade amplification strategy without external energy input based on the activities of oxidase-like OXD and peroxidase-like POD of the fullerene / metal organic framework C60-coated PCN-222 (Fe) nano-enzyme; then, the C60 atched PCN-222 (Fe) is combined with acetylcholin esterase AChE by means of molecular interaction, and an AChE / C60 atched PCN-222 (Fe) enzyme-nano enzyme array flow reactor is further developed. The sensor realizes parallel real-time detection and distinguishing of a plurality of organophosphorus pesticides, and has the advantages of wide linear range, high sensitivity and low detection limit. A promising strategy is provided for design of a nano-enzyme and trace pollutant detection system, and a sensing platform is stable in performance, low in price and convenient for commercialized application.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation and application of functional materials, and in particular to a fullerene / metal organic framework loaded with acetylcholinesterase, a preparation method and an application thereof. Background Art

[0002] Various delicate biochemical cascade processes in organisms are usually confined to cells. They carry out cascade flow reactions under spatial constraints and substrate channel environments, and work synergistically to complete complex biological processes. Inspired by life systems, scientists use nanozymes with multiple enzyme activities and enzyme cascade catalytic properties as cascade flow reactors, which are characterized by low cost and mass production capabilities. Among them, metal organic framework MOF nanozymes are connected by organic ligands and metal ion nodes, and have the advantages of customizable pore channels, high specific surface area, and a balance between rigidity and flexibility similar to the cellular environment. At the same time, the meso / macropores formed inside the nanoporous MOF can form a confined environment in space, simulating the flow process in organisms. However, due to the lack of enzyme-like molecular recognition units, the inherent reaction selectivity of nanozymes is poor, which limits their wide application. Therefore, it can be imagined that if well-defined and efficient natural enzymes are integrated into powerful nanozyme structures, the concept of continuous flow catalysis will be significantly improved to the level of practical application.

[0003] Traditional natural enzyme immobilization methods are usually based on covalent binding, which leads to uneven enzyme distribution or denaturation of enzyme activity due to the lack of specific binding pockets and finely tuned catalytic microenvironment. Fortunately, biorecognition elements can be anchored through steric effects, π-π interactions and strong supramolecular van der Waals effects, thereby establishing structural complementarity between nanozymes and natural enzymes and providing an adaptive environment for natural enzyme payloads. Therefore, the endogenous enzyme-nanozyme system based on the integration of natural enzymes and functional nanozymes does not require any external energy input to trigger the catalytic reaction, thereby avoiding the influence and limitation of external triggering factors. Specifically, the measured substance flows in a direction in the enzyme-nanozyme cascade flow reactor and performs a cascade reaction in a limited space, avoiding the disordered diffusion of molecules in traditional closed systems and eliminating the drawbacks of open systems.

[0004] Organophosphorus pesticides (OPs) are organic compound pesticides containing phosphorus, which are mainly used to control plant pests and weeds, ensure crop yields, and improve the quality of agricultural products. However, once organophosphorus compounds enter organisms through the food chain, excessive use exceeding the maximum permissible limits (MPLs) will cause acetylcholine accumulation, which can lead to pulmonary edema, respiratory paralysis, and death in severe cases. Therefore, it is urgent to establish a sensitive OPs residue analysis method for disease prevention, environmental monitoring, and agricultural production. Various colorimetric methods have been developed to detect OPs in environmental monitoring, agricultural production, and disease prevention by utilizing the inhibition of acetylcholinesterase activity, but they are still limited by the "lock-and-key" specific recognition mechanism and can only be used to detect or identify a single target substance. It is worth noting that the colorimetric sensor array using nonspecific recognition elements overcomes the limitations of the traditional "lock-and-key" specific recognition mechanism through mathematical analysis. This method can convert the different responses of the receptor to each analyte into different regional patterns with clear boundaries, just like a biological nose, and can simultaneously identify OPs with different structures and properties. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the present invention uses a fullerene / metal organic framework C having excellent oxidase-like OXD and peroxidase-like POD activities. 60 @PCN-222(Fe) nanozyme is used as a scaffold and acetylcholinesterase AChE is used as a biorecognition element to construct an efficient AChE / C 60 @PCN-222(Fe) enzyme-nanozyme colorimetric flow sensor uses the inhibitory effect of OPs on AChE activity to construct a colorimetric array, and uses the response differences of three channels to detect and distinguish multiple OPs.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions.

[0007] C 60 The preparation method of @PCN-222(Fe) is characterized by comprising the following steps: (1) Disperse zirconium chloride octahydrate ZrOCl2·8H2O, [5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin iron FeTCPP and benzoic acid in N,N-diethylformamide DEF, and then add fullerene C 60 The toluene solution was stirred for 0.5-1.5 h, and then the mixed solution was added to a Teflon-lined autoclave and reacted at 100-140 °C for 44-52 h; ZrOCl2·8H2O, FeTCPP, benzoic acid and C 60 The mass ratio of DEF and toluene is 1:0.67:36:0.07, and the total mass is 1.41-4.23 g; the volume ratio of DEF and toluene is 1:1, and the total volume is 15-25 mL; (2) After the reaction is completed, the precipitate is centrifuged and washed three times with ethanol and DEF respectively, and then soaked in hydrochloric acid and acetone for 20-30 h to exchange and remove DEF as a volatile solvent, and finally dried in vacuum at 60-80 °C for 10-16 h to obtain product C 60 @PCN-222(Fe); the volume ratio of hydrochloric acid to acetone is 1:1, and the total volume is 2-6 mL.

[0008] Explore C 60 @PCN-222(Fe) nanozyme has OXD-like and POD-like activity, characterized in that it comprises the following steps: (1) Explore C 60 @ OXD-like activity of PCN-222(Fe): C 60 @PCN-222(Fe) and 3,3',5,5'-tetramethylbenzidine TMB were added to acetic acid-sodium acetate HAc-NaAc buffer and reacted at 20-30 °C for 20-30 min. The absorbance of the reaction system at 652 nm was then recorded by UV-vis spectrophotometer; C 60 @The volume ratio of PCN-222(Fe), TMB and HAc-NaAc buffer is 1:30:180, and the total volume is 1.5-2.5 mL; C 60 The final concentration of @PCN-222(Fe) is 0.1-0.3 mg·mL -1 , the final concentration of TMB was 4-6 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 M and 3-5, respectively; (2) Explore C 60 @PCN-222(Fe) POD-like activity: C 60 @PCN-222(Fe), TMB and hydrogen peroxide H2O2 were added to HAc-NaAc buffer and reacted at 20-30 °C for 15-25 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; C 60 The volume ratio of PCN-222(Fe), TMB, H2O2 and HAc-NaAc buffer was 1:20:15:180, and the total volume was 1.5-2.5 mL; C 60 The final concentration of @PCN-222(Fe) is 0.1-0.3 mg·mL -1 , the final concentration of TMB was 4-6 mM, the final concentration of H2O2 was 7-13 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 M and 3-5, respectively.

[0009] AChE / C 60 @PCN-222 (Fe) enzyme-nanozyme preparation method, characterized in that it comprises the following steps: Add 0.5-1.5 mg of C 60 @PCN-222(Fe) was ultrasonicated in 3-7 mL ultrapure water for 20-40 min to obtain uniform C 60 @PCN-222(Fe) suspension, then add 0.5-1.5 mg of AChE to the suspension, stir for 20-40 min, and incubate at 35-39 °C for 8-12 h to obtain AChE / C 60 @PCN-222(Fe) enzyme-nanozyme, store at 2-6 °C for future use.

[0010] AChE / C 60 @PCN-222 (Fe) enzyme-nanozyme flow reactor detects multiple OPs, characterized by comprising the following steps: AChE / C 60 @PCN-222(Fe) and OPs (glyphosate / omethoate / paraoxon standard solution) of different concentrations were fully mixed in a centrifuge tube, reacted at 30-40°C for 20-40 min, then TMB, ATCh, and HAc-NaAc buffer were added, incubated at 30-40°C for 20-30 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; AChE / C 60 The volume ratio of PCN-222(Fe), glyphosate / omethoate / paraoxon standard solution, TMB, ATCh and HAc-NaAc buffer was 1:0.3:1:0.5:8, and the total volume was 0.8-1.3 mL; AChE / C 60 The final concentration of @PCN-222(Fe) is 1.5-2.5 mg·mL -1 , the final concentration of TMB was 3-7 mM, the final concentration of ATCh was 4-8 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 mM and 3-5, respectively.

[0011] Compared with the existing technology, the present invention has the following beneficial effects: (1) C provided by the present invention 60 @PCN-222(Fe) is simple to prepare and relatively easy to post-process. Its unique nanostructured cavities and abundant molecular diffusion pathways form a spatially confined environment, providing potential for effective flow reactions at a specific nanoscale. (2) The present invention is based on C 60The unique host-guest interaction of @PCN-222(Fe) optimizes OXD-like and POD-like activities, suggesting a cascade catalytic strategy without external energy input; (2) In the present invention, AChE was successfully immobilized on AChE / C by the enzyme in situ assembly strategy. 60 In @PCN-222(Fe), cascade reactions are carried out in a limited domain space, avoiding the disordered diffusion of molecules in traditional closed systems and eliminating the drawbacks of open systems; (4) Compared with the prior art, the AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor not only realizes the simultaneous detection of glyphosate, omethoate and paraoxon, but also effectively distinguishes these three organophosphorus pesticides by utilizing the response differences of the three channels. It has a wide linear range, low detection limit, and significant improvements in stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Yes (A) C 60 @PCN-222(Fe) synthesis diagram and (B) schematic diagram of the colorimetric detection of glyphosate, omethoate and paraoxon; Figure 2 It is C 60 @Scanning electron micrograph of PCN-222(Fe); Figure 3 It is C 60 @ Transmission electron micrograph of PCN-222(Fe); Figure 4 It is C 60 @Elemental analysis chart of PCN-222(Fe); Figure 5 It is C 60 @X-ray photoelectron spectrum of PCN-222(Fe); Figure 6 It is C 60 @Thermogravimetric analysis of PCN-222(Fe); Figure 7 It is C 60 @UV-vis absorption spectra of OXD-like and POD-like activities of PCN-222(Fe); Figure 8 AChE, C 60 @PCN-222(Fe) and AChE / C 60 @Infrared spectrum of PCN-222(Fe); Fig. 9 AChE / C 60 @PCN-222(Fe) and free AChE and C60 @Comparison of the activity of PCN-222(Fe) mixtures; Fig.10 AChE / C 60 UV-vis absorption spectra of the PCN-222(Fe) enzyme-nanozyme flow reactor for different concentrations of (A) glyphosate, (B) omethoate and (C) paraoxon; Fig.11 AChE / C 60 @Linear relationship between the absorbance of the PCN-222(Fe) enzyme-nanozyme flow reactor at 652 nm and the concentrations of (A) glyphosate, (B) omethoate, and (C) paraoxon; Fig.12 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor heat map of different concentrations of glyphosate, methoxyphosphine and paraoxon; Fig.13 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor for the stability and repeatability of (A) glyphosate, (B) omethoate, and (C) paraoxon detection; Fig.14 AChE / C 60 @Data of PCN-222(Fe) enzyme-nanozyme flow reactor detecting glyphosate in real samples; Fig.15 AChE / C 60 @Data of PCN-222(Fe) enzyme-nanozyme flow reactor for detecting omethoate in real samples; Fig.16 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor data for detecting paraoxon in real samples. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. However, the content of the present invention is not limited to the following embodiments. The details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0014] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. The methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or under conditions recommended by various manufacturers.

[0015] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention are familiar to those skilled in the art and the description of the present invention. The present invention can also be implemented using any methods, equipment and materials in the prior art that are similar or equivalent to the methods, equipment and materials described in the embodiments of the present invention.

[0016] Example 1C 60 Synthesis and characterization of @PCN-222(Fe) 1.1.C 60 Synthesis of @PCN-222(Fe) (1) Figure 1 (A) is C 60 @PCN-222(Fe) Synthesis diagram: ZrOCl2·8H2O, FeTCPP and benzoic acid are dispersed in DEF, and then C 60 The toluene solution was stirred for 1.0 h, and then the mixed solution was added to a Teflon-lined autoclave and reacted at 120 °C for 48 h; (2) After the reaction was completed, the precipitate was centrifuged and washed three times with ethanol and DEF, and then soaked in hydrochloric acid and acetone for 24 h to exchange and remove DEF as a volatile solvent. Finally, it was dried in vacuum at 70 °C for 12 h to obtain product C. 60 @PCN-222(Fe); 1.2. C 60 @PCN-222(Fe) Characterization (1) Figure 2 and Figure 3 C 60 @Scanning electron microscope and transmission electron microscope images of PCN-222(Fe): Figure 2 and Figure 3 It can be seen that C 60 @PCN-222(Fe) is a typical rod-like structure with a size of 1.9×0.5 μm and a relatively smooth surface. This smooth surface and clear morphology indicate that C 60 Instead of being loaded on the surface of PCN-222(Fe), they are encapsulated inside PCN-222(Fe) as guests to form host-guest interactions, hindering the functional molecules from attaching to the outer surface of MOF; (2) Figure 4 and Figure 5 C 60 @PCN-222(Fe) elemental analysis and X-ray photoelectron spectrum: Figure 4 It can be seen that C, N, O and Fe are evenly distributed in C60 @PCN-222(Fe), from Figure 5 It can be seen that C 60 @PCN-222(Fe) at 183.1, 283.9, 399.2, 530.8, and 722.3 eV corresponding to the binding energies of Zr 4d, C 1s, N 1s, O 1s, and Fe 2p; (3) Figure 6 It is C 60 @Thermogravimetric analysis of PCN-222(Fe): From the figure, it can be seen that when the temperature is raised from room temperature to 800°C, 60 The mass of @PCN-222(Fe) gradually decreased, with a total weight loss of only 59.6%, which showed high stability; (4) The above results confirm that C 60 @PCN-222(Fe).

[0017] Example 2 Investigation of C 60 OXD-like and POD-like activities of @PCN-222(Fe) (1) Explore C 60 @PCN-222(Fe)-like OXD activity: 10 μL of C 60 @PCN-222(Fe)(0.2 mg·mL -1 ) and 300 μL of TMB (5 mM) were added to 1800 μL of HAc-NaAc buffer (0.2 M, pH 4.0), reacted at 25 °C for 25 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; (2) Explore C 60 @PCN-222(Fe) POD-like activity: 10 μL of C 60 @PCN-222(Fe)(0.2 mg·mL -1 ), 200 μL of TMB (5 mM) and 150 μL of H2O2 (10 mM) were added to 1800 μL of HAc-NaAc buffer (0.2 M, pH 4.0), reacted at 25 °C for 20 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; (3) Figure 7 It is C 60 @PCN-222(Fe) UV-vis absorption spectra of OXD-like and POD-like activities: It can be seen that the absorbance of the TMB system and TMB + H2O2 system at 652 nm is negligible without any color change, while C 60The color of the @PCN-222(Fe)+ TMB system turned blue, and the absorbance at 652 nm was high, which proved that C 60 @PCN-222(Fe) has excellent OXD-like activity. Due to the presence of H2O2, C 60 @PCN-222(Fe) + TMB + H2O2 has a much higher absorbance at 652 nm than C 60 @PCN-222(Fe) + TMB absorbance, and C 60 @PCN-222(Fe) + TMB + H2O2 system has a significantly darker blue color, indicating that C 60 @PCN-222(Fe) not only has OXD-like activity, but also maintains significant POD-like activity.

[0018] Example 3 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme preparation, characterization, and activity study Rationally immobilizing fragile enzymes on scaffolds is an effective strategy for developing powerful catalysts. 60 @PCN-222(Fe) nanozyme as a scaffold forms a strong supramolecular interaction through abundant carboxyl and amino groups, especially the hydrogen bond bridge formed between the protein surface groups and the MOF organic connector, fixes the AChE enzyme molecules and maintains biological activity; 3.1.AChE / C 60 Preparation of @PCN-222(Fe) enzyme-nanozyme 1.0 mg of C 60 @PCN-222(Fe) was ultrasonicated in 5 mL ultrapure water for 30 min to obtain uniform C 60 @PCN-222(Fe) suspension, and then 1 mg of AChE was added to the suspension, stirred for 30 min, and incubated at 37 °C for 10 h to obtain AChE / C 60 @PCN-222(Fe) enzyme-nanozyme, stored at 4 °C for future use; 3.2.AChE / C 60 Characterization of @PCN-222(Fe) enzyme-nanozyme Figure 8 AChE, C 60 @PCN-222(Fe) and AChE / C 60 @The second derivative FT-IR spectrum of PCN-222(Fe): From the figure, we can see that AChE and AChE / C 60 @PCN-222(Fe) Amide I band (700-1600 cm -1) and amide II band (1600–1500 cm -1 ) demonstrated the successful immobilization of AChE; 3.3.AChE / C 60 @PCN-222(Fe) enzyme-nanozyme activity study Fig. 9 AChE / C 60 @PCN-222(Fe) and free AChE and C 60 @PCN-222(Fe) mixture activity comparison: As shown in the figure, compared with the mixture containing free AChE and C 60 @PCN-222(Fe) mixture, AChE / C 60 The cascade activity of @PCN-222(Fe) was increased by about 1.3 times.

[0019] Example 4 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor for detection of various OPs and sensing mechanism 4.1.AChE / C 60 @PCN-222(Fe) Sensing mechanism of OPs detection using flow reactor Figure 1 (B) Schematic diagram of the principle of colorimetric detection of glyphosate, omethoate and paraoxon, based on C 60 @PCN-222(Fe) has OXD-like and POD-like activities, and combined with the inhibitory effect of OPs on AChE activity, it utilizes AChE / C 60 @PCN-222(Fe) enzyme-nanozyme continuous flow reactor was used to measure OPs; 4.2. AChE / C 60 @PCN-222(Fe) flow reactor for detection of various OPs (1) Add 100 μL of AChE / C 60 @PCN-222(Fe)(2.0 mg·mL -1 ) and 30 μL of glyphosate / omethoate / paraoxon standard solutions of different concentrations were fully mixed in a centrifuge tube, reacted at 35 °C for 30 min, then 100 μL of TMB (5 mM), 50 μL of ATCh (6 mM), and 800 μL of HAc-NaAc (0.2 mM, pH 4.0) buffer were added, incubated at 35 °C for 25 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; (2) Fig.10 and Fig.11 They are AChE / C 60UV-vis absorption spectra of @PCN-222(Fe) enzyme-nanozyme flow reactor for different concentrations of glyphosate, omethoate and paraoxon and the linear relationship between the absorbance at 652 nm and the concentrations of glyphosate, omethoate and paraoxon: Fig.10 It can be seen that as the concentration of OPs increases, the corresponding absorbance at 652nm increases. Fig.11 The results showed that there was a good linear relationship between the absorbance and concentration of glyphosate, omethoate and paraoxon, and the linear range was 2.0-800 ng·ml -1 , 0.5-800 ng·ml -1 and 1.0-800 ng·ml -1 , and the corresponding detection limits were 0.65 ng·ml -1 , 0.16 ng·ml -1 and 0.32 ng·ml -1 (3σ / S), the most important thing is that it can be directly observed that different OPs show obvious different responses in the three channels of 371 nm (peak I), 568 nm (peak II) and 652 nm (peak III), which can be qualitatively identified; (3) Fig.12 AChE / C 60 Heat map of the enzyme-nanozyme flow reactor for different concentrations of glyphosate, methoxyphosphine and paraoxon: Heat map is a visualization tool for optical data. It converts the absorbance of different peaks of the three OPs at different concentrations into corresponding colors to obtain the training data matrix. As shown in the figure, AChE / C 60 The different responses of @PCN-222(Fe) to the three OPs were transformed into different regional patterns with clear boundaries, indicating that AChE / C 60 @PCN-222(Fe) array sensor is an ideal tool for distinguishing and detecting multiple OPs simultaneously through pattern recognition; 4.3. AChE / C 60 @PCN-222(Fe) flow reactor stability and repeatability for various OPs Fig.13 AChE / C 60 Stability and repeatability of PCN-222(Fe) enzyme-nanozyme flow reactor for glyphosate, omethoate and paraoxon detection: from Fig.13 It can be seen that the three different batches of AChE / C 60 @PCN-222(Fe) flow reactor can maintain more than 82% relative activity for glyphosate, omethoate and paraoxon after being placed at room temperature for 15 days, indicating that it has good stability and repeatability.

[0020] Example 5 Detection of glyphosate, omethoate and paraoxon in actual samples Figure 14-16 AChE / C 60 @PCN-222(Fe) enzyme-nanozyme flow reactor for the detection of glyphosate, omethoate and paraoxon in real samples; in order to evaluate the AChE / C 60 The practical usability of the @PCN-222(Fe) sensor in real samples was demonstrated. As a proof of concept, three replicate measurements of glyphosate, omethoate, and paraoxon in food and environmental samples were performed by the standard addition method; see Appendix Figure 14-16 As shown, the recoveries of the three OPs were between 97.46% and 103.79%, and the relative standard deviation (RSD) did not exceed 4.10%, indicating that the established sensing platform had reliable performance in actual samples.

[0021] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A fullerene / metal organic framework C 60 @The preparation method of PCN-222(Fe), characterized in that: The following steps are involved: (1) Disperse zirconium chloride octahydrate ZrOCl2·8H2O, [5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin iron FeTCPP and benzoic acid in N,N-diethylformamide DEF, and then add fullerene C 60 The toluene solution was stirred for 0.5-1.5 h, and then the mixed solution was added to a Teflon-lined autoclave and reacted at 100-140 °C for 44-52 h; ZrOCl2·8H2O, FeTCPP, benzoic acid and C 60 The mass ratio of DEF and toluene is 1:0.67:36:0.07, and the total mass is 1.41-4.23 g; the volume ratio of DEF and toluene is 1:1, and the total volume is 15-25 mL; (2) After the reaction is completed, the precipitate is centrifuged and washed three times with ethanol and DEF respectively, and then soaked in hydrochloric acid and acetone for 20-30 hours to exchange and remove DEF as a volatile solvent, and finally dried in vacuum at 60-80 °C for 10-16 hours to obtain product C 60 @PCN-222(Fe); the volume ratio of hydrochloric acid to acetone is 1:1, and the total volume is 2-6 mL.

2. A C according to claim 1 60 @The preparation method of PCN-222(Fe), characterized in that: Preparation of C 60 @PCN-222(Fe) has unique nanostructure cavities and abundant molecular diffusion paths, and the spatially confined environment formed provides conditions for flow reactions at a specific nanoscale.

3. C prepared according to claim 1 60 @PCN-222(Fe) explores its oxidase OXD activity and peroxidase POD activity, characterized by: Here’s how: (1) Explore C 60 @ OXD-like activity of PCN-222(Fe): C 60 @PCN-222(Fe) and 3,3',5,5'-tetramethylbenzidine TMB were added to acetic acid-sodium acetate HAc-NaAc buffer and reacted at 20-30 °C for 20-30 min. The absorbance of the reaction system at 652 nm was then recorded by UV-vis spectrophotometer; C 60 @The volume ratio of PCN-222(Fe), TMB and HAc-NaAc buffer is 1:30:180, and the total volume is 1.5-2.5 mL; C 60 The final concentration of @PCN-222(Fe) is 0.1-0.3 mg·mL -1 , the final concentration of TMB was 4.0-6.0 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 M and 3.0-5.0, respectively; (2) Explore C 60 @PCN-222(Fe) POD-like activity: C 60 @PCN-222(Fe), TMB and hydrogen peroxide H2O2 were added to HAc-NaAc buffer and reacted at 20-30°C for 15-25 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer; C 60 The volume ratio of PCN-222(Fe), TMB, H2O2 and HAc-NaAc buffer was 1:20:15:180, and the total volume was 1.5-2.5 mL; C 60 The final concentration of @PCN-222(Fe) is 0.1-0.3 mg·mL -1 , the final concentration of TMB was 4.0-6.0 mM, the final concentration of H2O2 was 7.0-13.0 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 M and 3.0-5.0, respectively.

4. C according to claim 3 60 @PCN-222(Fe) nanozyme has excellent OXD-like and POD-like activities, characterized by: C 60 @PCN-222(Fe) not only has OXD-like activity, but also maintains significant POD-like activity. 60 @PCN-222(Fe)'s dual enzyme activity constitutes a cascade reaction.

5. C according to claim 3 60 @PCN-222(Fe) Nanozyme with OXD-like and POD-like Activities to Construct Fullerene / Metal-Organic Framework Loaded with Acetylcholinesterase AChE / C 60 @PCN-222 (Fe) enzyme-nanozyme preparation method, characterized in that, The following steps are involved: Add 0.5-1.5 mg of C 60 @PCN-222(Fe) was ultrasonicated in 3-7 mL ultrapure water for 20-40 min to obtain uniform C 60 @PCN-222(Fe) suspension, then add 0.5-1.5 mg of AChE to the suspension, stir for 20-40 min, and incubate at 35-39°C for 8-12 h to obtain AChE / C 60 @PCN-222(Fe) enzyme-nanozyme, store at 2-6 °C for future use.

6. AChE / C constructed according to claim 5 60 @PCN-222(Fe) constructs enzyme-nanozyme flow reactor to detect various OPs, characterized in that: The following steps are involved: AChE / C 60 @PCN-222(Fe) and organophosphorus pesticides OPs (glyphosate / omethoate / paraoxon standard solution) of different concentrations were fully mixed in a centrifuge tube and reacted at 30-40°C for 20-40 min. Then TMB, ATCh, and HAc-NaAc buffer were added and incubated at 30-40°C for 20-30 min. The absorbance of the reaction system at 652 nm was then recorded by a UV-vis spectrophotometer. 60 The volume ratio of PCN-222(Fe), glyphosate / omethoate / paraoxon standard solution, TMB, ATCh and HAc-NaAc buffer was 1:0.3:1:0.5:8, and the total volume was 0.8-1.3 mL; AChE / C 60 The final concentration of @PCN-222(Fe) is 1.5-2.5 mg·mL -1 , the final concentration of TMB was 3-7 mM, the final concentration of ATCh was 4-8 mM, and the final concentration and pH of HAc-NaAc buffer were 0.1-0.3 mM and 3.0-5.0, respectively.

7. AChE / C according to claim 6 60 @PCN-222(Fe) constructs enzyme-nanozyme flow reactor to detect various OPs, characterized by: As the concentration of OPs increased, the corresponding absorbance at 652 nm increased. There was a good linear relationship between the absorbance and concentration of glyphosate, omethoate and paraoxon, and the linear range was 2.0-800 ng·ml -1 , 0.5-800 ng·ml -1 and 1.0-800 ng·ml -1 , and the corresponding detection limits were 0.65 ng·ml -1 , 0.16 ng·ml -1 and 0.32 ng·ml -1 (3σ / S), the most important thing is that it can be directly observed that different OPs show obvious different responses in the three channels of 371 nm (peak I), 568 nm (peak II) and 652 nm (peak III), which can be used for qualitative identification.

8. AChE / C according to claim 6 60 @PCN-222(Fe) constructs an enzyme-nanozyme flow reactor to detect OPs in actual samples, characterized by: The recoveries of the three OPs were between 97.46% and 103.79%, and the relative standard deviation (RSD) did not exceed 4.10%, indicating that the established sensing platform had reliable performance in actual samples.

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