A fullerene / metal-organic framework loaded with acetylcholinesterase, preparation method and application thereof

Abstract

The present application relates to the technical fields of preparation and application of functional materials, in particular to a fullerene / metal organic framework loaded with acetylcholinesterase, a preparation method and application thereof. 60 @PCN-222(Fe) nanoscale enzyme, based on its class oxidase OXD and class peroxidase POD activity, proposes a self-cascading amplification strategy without external energy input. Subsequently, the molecular interaction is used to combine C 60 @PCN-222(Fe) with acetylcholinesterase AChE, and further develop AChE / C 60 @PCN-222(Fe) enzyme-nanoscale enzyme array flow reactor. The sensor realizes the parallel real-time detection and distinction of multiple organophosphorus pesticides, and the sensor has a wide linear range, high sensitivity and low detection limit. The present application provides a promising strategy for the design of nanoscale enzyme and trace pollutant detection system, and the sensing platform has stable performance, low price and is convenient for commercial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparation and application of functional materials, and particularly relates to a fullerene / metal-organic framework loaded with acetylcholinesterase, a preparation method and application thereof. BACKGROUND

[0002] Various subtle biochemical cascades in living organisms are usually confined to cells, and they perform cascade flow reactions in a spatially limited and substrate channel environment, and cooperatively complete complex biological processes. Inspired by life systems, scientists use nanoscale enzymes with multiple enzyme activities and enzyme cascade catalytic properties as cascade flow reactors, which have the characteristics of low cost and batch production capability. Metal-organic framework (MOF) nanoscale enzymes are connected by organic ligands and metal ion nodes, and have the advantages of customizable pore channels, high specific surface area, and balance between rigidity and flexibility similar to the cell environment. At the same time, the meso / macroporous holes formed inside the nanoporous MOF can form a limited environment in space to simulate the flow process in the living body. However, due to the lack of enzyme-like molecular recognition units, the inherent reaction selectivity of nanoscale enzymes is poor, which limits their wide application. Therefore, it can be assumed that if the well-defined and efficient natural enzymes are integrated into the powerful nanoscale enzyme structure, 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, and due to the lack of specific binding pockets and finely tuned catalytic microenvironments, the enzymes are unevenly distributed or the enzyme activity is denatured. Fortunately, biological recognition elements can be anchored by spatial effects, π-π interactions and strong supramolecular van der Waals effects, thereby establishing structural complementarity between nanoscale enzymes and natural enzymes and providing an adaptive environment for the natural enzyme payload. Therefore, the enzyme-nanoscale enzyme endogenous system based on the integration of natural enzymes and functional nanoscale enzymes 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 substances flow directionally in the enzyme-nanoscale enzyme cascade flow reactor, and perform cascade reactions in a limited space, avoiding the disordered diffusion of molecules in traditional closed systems and eliminating the disadvantages of open systems.

[0004] Organophosphorus pesticides (OPs) are organic complex pesticides containing phosphorus elements, which are mainly used to prevent and control plant pests and weed damage, ensure crop yield and improve the quality of agricultural products. However, once the organophosphorus compounds enter the organism through the food chain, excessive use beyond the maximum permissible limit (MPLs) will cause acetylcholine accumulation, and in severe cases, can lead to pulmonary edema, respiratory paralysis and death. Therefore, it is an urgent need for disease prevention, environmental monitoring and agricultural production to establish a sensitive OPs residue analysis method. By using the inhibitory effect of acetylcholinesterase activity, various colorimetric methods for detecting OPs have been developed in environmental monitoring, agricultural production and disease prevention, but they are still limited by the "lock-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 non-specific recognition elements overcomes the limitation of the traditional "lock-key" specific recognition mechanism through mathematical analysis, which can convert the different responses of receptors to each analyte into different regional patterns with clear boundaries, just like a biological nose, which can simultaneously identify OPs with different structures and properties. SUMMARY

[0005] To solve the problems in the background art, the present application uses fullerene / metal-organic framework C 60 @PCN-222(Fe) nanoscale enzyme as a scaffold and acetylcholinesterase AChE as a biological recognition element to construct a high-efficiency AChE / C 60 @PCN-222(Fe) enzyme-nanoscale enzyme colorimetric flow sensor, which uses the inhibitory effect of OPs on AChE activity to construct a colorimetric array, and uses the response difference of the three channels to detect and distinguish multiple OPs.

[0006] To achieve the above purpose, the present application realizes the technical scheme as follows.

[0007] C 60 The preparation method of PCN-222(Fe) comprises the following steps:

[0008] (1) dispersing zirconium chloride octahydrate ZrOCl2·8H2O, [5,10,15,20-tetra(4-carboxyphenyl) iron porphyrin FeTCPP and benzoic acid in N,N-dimethylformamide DEF, then adding a toluene solution containing fullerene C 60 80 and stirring for 0.5-1.5 h, then adding the mixed solution to a Teflon-lined autoclave and reacting at 100-140 °C for 44-52 h; wherein ZrOCl2·8H2O, FeTCPP, benzoic acid and C 601:0.67:36:0.07, with a total mass of 1.41-4.23 g; the volume ratio of DEF and toluene is 1:1, and the total volume is 15-25 mL;

[0009] (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, respectively, to exchange and remove the DEF as a volatile solvent, and finally vacuum dried at 60-80 °C for 10-16 h to obtain the product C 60 @PCN-222(Fe); wherein the volume ratio of hydrochloric acid and acetone is 1:1, and the total volume is 2-6 mL.

[0010] Exploring C 60 @PCN-222(Fe) nanoscale enzyme OXD and POD activity, characterized by comprising the following steps:

[0011] (1) Exploring C 60 @PCN-222(Fe) OXD-like activity: C 60 @PCN-222(Fe) and 3,3',5,5'-tetramethylbenzidine TMB are added to acetic acid-sodium acetate HAc-NaAc buffer, and reacted at 20-30 °C for 20-30 min, and then the absorbance of the reaction system at 652 nm is recorded by ultraviolet-visible light UV-vis spectrophotometer; wherein C 60 @PCN-222(Fe), TMB and HAc-NaAc buffer have a volume ratio of 1:30:180, and a total volume of 1.5-2.5 mL; C 60 @PCN-222(Fe) has a final concentration of 0.1-0.3 mg·mL -1 , the final concentration of TMB is 4-6 mM, and the final concentration and pH of HAc-NaAc buffer are 0.1-0.3 M and 3-5, respectively;

[0012] (2) Exploring C 60 @PCN-222(Fe) POD-like activity: C 60 @PCN-222(Fe), TMB and hydrogen peroxide H2O2 are 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 is recorded by UV-vis spectrophotometer; wherein C 60 @PCN-222(Fe), TMB, H2O2 and HAc-NaAc buffer have a volume ratio of 1:20:15:180, and a total volume of 1.5-2.5 mL; C 60 @PCN-222(Fe) has a final concentration of 0.1-0.3 mg·mL-1 The final concentration of TMB is 4-6 mM, the final concentration of H2O2 is 7-13 mM, and the final concentration and pH of the HAc-NaAc buffer are 0.1-0.3 M and 3-5, respectively.

[0013] AChE / C 60 The preparation method of the PCN-222(Fe) enzyme-nanoenzyme is characterized by comprising the following steps:

[0014] 0.5-1.5 mg of C 60 The PCN-222(Fe) is ultrasonically treated in 3-7 mL of ultrapure water for 20-40 min to obtain a uniform C 60 PCN-222(Fe) suspension, then 0.5-1.5 mg of AChE is added to the suspension, stirred for 20-40 min, and then incubated at 35-39 °C for 8-12 h to obtain AChE / C 60 The PCN-222(Fe) enzyme-nanoenzyme is stored at 2-6 °C for standby use.

[0015] AChE / C 60 The PCN-222(Fe) enzyme-nanoenzyme flow reactor detects various OPs, and is characterized by comprising the following steps:

[0016] The AChE / C 60 PCN-222(Fe) and different concentrations of OPs (glyphosate / omethoate / standard solution of paraoxon) are mixed in a centrifuge tube, and then reacted at 30-40 °C for 20-40 min, then TMB, ATCh and HAc-NaAc buffer are added, and then incubated at 30-40 °C for 20-30 min, and then the absorbance of the reaction system at 652 nm is recorded by a UV-vis spectrophotometer; wherein the volume ratio of AChE / C 60 PCN-222(Fe), glyphosate / omethoate / standard solution of paraoxon, TMB, ATCh and HAc-NaAc buffer is 1:0.3:1:0.5:8, and the total volume is 0.8-1.3 mL; the final concentration of AChE / C 60 PCN-222(Fe) is 1.5-2.5 mg·mL -1 The final concentration of TMB is 3-7 mM, the final concentration of ATCh is 4-8 mM, and the final concentration and pH of the HAc-NaAc buffer are 0.1-0.3 mM and 3-5, respectively.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] (1) The C 60The preparation method of PCN-222(Fe) is simple, and post-processing is relatively easy, and the unique nano-structured cavity and rich molecular diffusion path form a space-restricted environment, which provides potential for effective flow reaction under specific nanoscale;

[0019] (2) The application is based on C 60 The unique host-guest interaction of PCN-222(Fe) optimizes the OXD and POD activities, and a cascade catalytic strategy without external energy input is provided;

[0020] (2) In the application, the AChE is successfully fixed in AChE / C 60 In PCN-222(Fe), cascade reactions are carried out in a limited domain space, avoiding the disordered diffusion of molecules in a traditional closed system and eliminating the disadvantages of an open system;

[0021] (4) Compared with the prior art, the AChE / C 60 The enzyme-nanoplasma flow reactor of PCN-222(Fe) not only realizes the simultaneous detection of glyphosate, omethoate and parathion, but also effectively distinguishes the three kinds of organophosphorus pesticides by using the response difference of the three channels, has a wide linear range, a low detection limit, and obvious improvement in stability and repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is (A) C 60 The synthesis diagram of PCN-222(Fe) and (B) the principle diagram for colorimetric detection of glyphosate, omethoate and parathion;

[0023] Figure 2 is C 60 The scanning electron microscope diagram of PCN-222(Fe);

[0024] Figure 3 is C 60 The transmission electron microscope diagram of PCN-222(Fe);

[0025] Figure 4 is C 60 The element analysis diagram of PCN-222(Fe);

[0026] Figure 5 is C 60 The X-ray photoelectron spectroscopy diagram of PCN-222(Fe);

[0027] Figure 6 is C 60 The thermogravimetric analysis diagram of PCN-222(Fe);

[0028] Figure 7 is C 60 @UV-vis absorption spectra of PCN-222(Fe) class OXD and class POD activities;

[0029] Figure 8 is AChE, C 60 @PCN-222(Fe) and AChE / C 60 @Infrared spectra of PCN-222(Fe);

[0030] Figure 9 is AChE / C 60 @PCN-222(Fe) and free AChE with C 60 @Activity comparison of PCN-222(Fe) mixtures;

[0031] Figure 10 is AChE / C 60 @UV-vis absorption spectra of PCN-222(Fe) enzyme-nanoparticle reactor against different concentrations of (A) glyphosate, (B) omethoate and (C) parathion;

[0032] Figure 11 is AChE / C 60 @Linear relationship plot of absorbance at 652 nm of PCN-222(Fe) enzyme-nanoparticle reactor against concentrations of (A) glyphosate, (B) omethoate and (C) parathion;

[0033] Figure 12 is AChE / C 60 @Thermal map of PCN-222(Fe) enzyme-nanoparticle reactor against different concentrations of glyphosate, omethoate and parathion;

[0034] Figure 13 is AChE / C 60 @Stability and repeatability of PCN-222(Fe) enzyme-nanoparticle reactor for detection of (A) glyphosate, (B) omethoate and (C) parathion;

[0035] Figure 14 is AChE / C 60 @Data of PCN-222(Fe) enzyme-nanoparticle reactor for detection of glyphosate in real samples;

[0036] Figure 15 is AChE / C 60 @Data of PCN-222(Fe) enzyme-nanoparticle reactor for detection of omethoate in real samples;

[0037] Figure 16 is AChE / C60 Data for the detection of paraoxon in real samples using the PCN-222(Fe) enzyme-nanoreactor flow reactor. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be clearly and completely described with reference to the drawings in the present application. The content of the present application is not limited to the following embodiments, and each detail in the description can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application.

[0039] It should be understood that the terms used in the examples of the present application are used to describe specific specific embodiments, and are not intended to limit the scope of protection of the present application. The methods in the following examples are not specified unless the specific conditions are specified. The methods are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] When the numerical range is given in the examples, it should be understood that, unless otherwise specified in the present application, 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 application are based on the mastery of the prior art by those skilled in the art and the description of the present application. Any method, equipment and material of the prior art similar or equivalent to the method, equipment and material described in the examples of the present application can be used to realize the present application.

[0041] Example 1C 60 Synthesis and characterization of PCN-222(Fe)

[0042] 1.1.C 60 Synthesis of PCN-222(Fe)

[0043] (1) Figure 1 (A) is C 60 Synthesis of PCN-222(Fe) scheme: ZrOCl2·8H2O, FeTCPP and benzoic acid were dispersed in DEF containing, and then C 60 containing toluene solution was added and stirred for 1.0 h, then the mixed solution was added to a Teflon-lined autoclave and reacted at 120 °C for 48 h;

[0044] (2) After the reaction was completed, the precipitate was centrifuged and washed three times with ethanol and DEF, respectively, and then soaked in hydrochloric acid and acetone for 24 h, respectively, to exchange and remove the DEF as a volatile solvent, and finally dried at 70 °C under vacuum for 12 h to obtain the product C 60 PCN-222(Fe);

[0045] 1.2. C 60 Characterization of PCN-222(Fe)

[0046] (1) Figure 2 and Figure 3 They are C 60 Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of @PCN-222(Fe): From Figure 2 and Figure 3 It can be known that C 60 @PCN-222(Fe) exhibits a typical rod-like structure with dimensions of 1.9 × 0.5 μm and a relatively smooth surface. This smooth surface and well-defined morphology indicate that C 60 Instead of being loaded on the PCN-222(Fe) surface, it is encapsulated inside PCN-222(Fe) as a guest to form a host-guest interaction, thus preventing functional molecules from attaching to the outer surface of the MOF.

[0047] (2) Figure 4 and Figure 5 They are C 60 Elemental analysis chart and X-ray photoelectron spectrum of @PCN-222(Fe): From Figure 4 It can be seen that C, N, O and Fe are evenly distributed in C 60 From @PCN-222(Fe), Figure 5 It can be seen from C 60 @PCN-222(Fe) corresponds to the binding energies of Zr 4d, C 1s, N 1s, O 1s and Fe 2p at 183.1, 283.9, 399.2, 530.8 and 722.3 eV;

[0048] (3) Figure 6 It is C 60 Thermogravimetric analysis plot of @PCN-222(Fe): The plot shows that when the temperature is raised from room temperature to 800°C, C 60 The mass of @PCN-222(Fe) gradually decreases, with a total weight loss of only 59.6%, indicating high stability;

[0049] (4) The above results confirm the successful synthesis of C. 60 @PCN-222(Fe).

[0050] Example 2 Investigating C 60 OXD-like and POD activities of @PCN-222(Fe)

[0051] (1) Investigating C 60 @OXD-like activity of PCN-222(Fe): 10 μL of C 60 @PCN-222(Fe) (0.2 mg·mL) -1) and 300 μL of TMB (5 mM) were added into 1800 μL of HAc-NaAc buffer (0.2 M, pH 4.0), and the reaction was carried out at 25 °C for 25 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer;

[0052] (2) To explore the C 60 POD-like activity of PCN-222(Fe): 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 into 1800 μL of HAc-NaAc buffer (0.2 M, pH 4.0), and the reaction was carried out at 25 °C for 20 min, and then the absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer;

[0053] (3) Figure 7 is C 60 UV-vis absorption spectra of PCN-222(Fe) OXD-like and POD-like activities: It can be seen that the absorbance of TMB system and TMB + H2O2 system at 652 nm is negligible, and there is no color change, while C 60 @PCN-222(Fe) + TMB system has a significant blue color change, and the absorbance at 652 nm is high, which proves that C 60 @PCN-222(Fe) has excellent OXD-like activity, and due to the presence of H2O2, C 60 @PCN-222(Fe) + TMB + H2O2 has much higher absorbance at 652 nm than C 60 @PCN-222(Fe) + TMB, and C 60 @PCN-222(Fe) + TMB + H2O2 system has a significant deepening of blue color, which indicates that C 60 @PCN-222(Fe) not only has OXD-like activity, but also maintains significant POD-like activity.

[0054] Example 3 AChE / C 60 Preparation, characterization and activity exploration of PCN-222(Fe) enzyme-nanoplasma

[0055] It is an effective strategy to develop a powerful catalyst to reasonably immobilize fragile enzymes on a scaffold, and on this basis, C 60@PCN-222(Fe) nanozyme serves as a scaffold, forming strong supramolecular interactions through hydrogen bond bridges between abundant carboxyl and amino groups, especially protein surface groups, and MOF organic linkers, thereby immobilizing AChE enzyme molecules and maintaining their biological activity.

[0056] 3.1.AChE / C 60 Preparation of PCN-222(Fe) enzyme-nanozymes

[0057] 1.0 mg of C 60 @PCN-222(Fe) was sonicated in 5 mL of ultrapure water for 30 min to obtain homogeneous C. 60 @PCN-222(Fe) suspension, 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, store at 4 °C for later use;

[0058] 3.2.AChE / C 60 Characterization of @PCN-222(Fe) enzyme-nanozyme

[0059] Figure 8 It is AChE, C 60 @PCN-222(Fe) and AChE / C 60 The second derivative FT-IR spectrum of @PCN-222(Fe): The figure shows AChE and AChE / C... 60 The amide I band of @PCN-222(Fe) (700-1600 cm⁻¹) -1 ) and amide II band (1600-1500 cm) -1 This proves the successful fixation of AChE;

[0060] 3.3.AChE / C 60 Activity Study of @PCN-222(Fe) Enzyme-Nanozyme

[0061] Figure 9 It is AChE / C 60 @PCN-222(Fe) and free AChE with C 60 Activity comparison of @PCN-222(Fe) mixture: As shown in the figure, compared with mixtures containing free AChE and C 60 Compared to the mixture of @PCN-222(Fe), AChE / C 60 The cascade activity of @PCN-222(Fe) was increased by approximately 1.3 times.

[0062] Example 4 AChE / C 60Detection of various OPs by PCN-222(Fe) enzyme-nanocatalyst flow reactor and sensing mechanism

[0063] 4.1. AChE / C 60 Sensing mechanism of PCN-222(Fe) flow reactor for detection of OPs

[0064] Figure 1 (B) Schematic diagram of colorimetric detection of glyphosate, omethoate and parathion, based on C 60 OXD and POD activities of PCN-222(Fe) and the inhibition of AChE activity by OPs, using AChE / C 60 Determination of OPs by PCN-222(Fe) enzyme-nanocatalyst continuous flow reactor

[0065] 4.2. AChE / C 60 Detection of various OPs by PCN-222(Fe) flow reactor

[0066] (1) 100 μL of AChE / C 60 PCN-222(Fe) (2.0 mg·mL -1 ) and 30 μL of different concentrations of glyphosate / omethoate / parathion standard solution were mixed well in a centrifuge tube, and 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, and the mixture was incubated at 35 ℃ for 25 min. The absorbance of the reaction system at 652 nm was recorded by UV-vis spectrophotometer.

[0067] (2) Figure 10 and Figure 11 are the UV-vis absorption spectra of AChE / C 60 PCN-222(Fe) enzyme-nanocatalyst flow reactor for different concentrations of glyphosate, omethoate and parathion, and the linear relationship between the absorbance at 652 nm and the concentration of glyphosate, omethoate and parathion: from Figure 10 it can be seen that with the increase of OPs concentration, the corresponding absorbance at 652 nm increases, Figure 11 showing that there is a good linear relationship between the absorbance and the concentration of glyphosate, omethoate and parathion, and the linear range is 2.0-800 ng·ml -1 , 0.5-800 ng·ml -1 and 1.0-800 ng·ml -1 , respectively, and the corresponding detection limits are 0.65 ng·ml -1, 0.16 ng·ml -1 and 0.32 ng·ml -1 (3σ / S), most importantly, the different OPs can be directly observed to exhibit distinct responses at 371 nm (peak I), 568 nm (peak II), and 652 nm (peak III), three channels, which can be used for qualitative identification;

[0068] (3) Figure 12 is AChE / C 60 @PCN-222(Fe) Enzyme-nanosome flow reactor for different concentrations of glyphosate, methamidophos and parathion: The heat map is a visualization tool for optical data, which converts the absorbance of three OPs at different concentrations at different peaks into the corresponding color, thereby obtaining a training data matrix, as shown in the figure, AChE / C 60 @PCN-222(Fe) different responses to three OPs are converted into different area patterns, with clear boundaries, indicating that AChE / C 60 @PCN-222(Fe) array sensor is an ideal tool for simultaneous distinguishing and detecting multiple OPs through pattern recognition;

[0069] 4.3. AChE / C 60 @PCN-222(Fe) flow reactor for multiple OPs stability and repeatability

[0070] Figure 13 is AChE / C 60 @PCN-222(Fe) Enzyme-nanosome flow reactor for glyphosate, omethoate and parathion detection stability and repeatability: From Figure 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 parathion after being placed at room temperature for 15 days, indicating that it has good stability and repeatability.

[0071] Example 5 Detection of glyphosate, omethoate and parathion in real samples

[0072] Figures 14-16 are AChE / C 60 @PCN-222(Fe) Enzyme-nanosome flow reactor for glyphosate, omethoate and parathion detection data in real samples; In order to evaluate the actual availability of AChE / C 60 @PCN-222(Fe) sensor in real samples, as a proof of concept, three parallel measurements of glyphosate, omethoate and parathion in food and environmental samples were made by standard addition method; as shown in the attached Figures 14-16As shown, the recovery rates of the three OPs were between 97.46%-103.79%, and the relative standard deviation RSD was not more than 4.10%, indicating that the established sensing platform had reliable performance in actual samples.

[0073] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A C 60 @PCN-222(Fe) nano-enzyme-based construction of acetylcholinesterase AChE / C 60 @PCN-222(Fe) enzyme-nano-enzyme preparation method, characterized in that: (1) the C 60 A process for the preparation of PCN-222(Fe) comprising the steps of: ①Zirconium chloride octahydrate, 5,10,15,20-tetra(4-carboxyphenyl)porphyrin iron FeTCPP and benzoic acid were dispersed in N,N-dimethylformamide, then a toluene solution containing C 60 was added and stirred for 0.5-1.5h, then the mixed solution was added to a Teflon-lined autoclave and reacted at 100-140℃ for 44-52h; wherein the mass ratio of zirconium chloride octahydrate, FeTCPP, benzoic acid and C 60 was 1:0.67:36:0.07, and the total mass was 1.41-4.23g; the volume ratio of N,N-dimethylformamide and toluene was 1:1, and the total volume was 15-25mL; (ii) after completion of the reaction, the obtained precipitate is centrifuged and washed with ethanol and N,N-dimethylformamide each three times, then is soaked in hydrochloric acid and acetone successively for 20-30 h to exchange and remove N,N-dimethylformamide as a volatile solvent, and finally is vacuum dried at 60-80 °C for 10-16 h to obtain product C 60 @PCN-222 (Fe); wherein the volume ratio of hydrochloric acid and acetone is 1:1, and the total volume is 2-6 mL; (2) the AChE / C 60 A method of preparing PCN-222(Fe) comprising the steps of: C 60 @PCN-222(Fe) was ultrasonicated in 3-7 mL ultrapure water for 20-40 min to obtain a homogeneous C 60 @PCN-222(Fe) suspension, 0.5-1.5 mg of AChE was added into the suspension, and after stirring for 20-40 min, the mixture was incubated at 35-39 °C for 8-12 h to obtain AChE / C 60 @PCN-222(Fe), and the obtained product was stored at 2-6 °C for later use.

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