Molybdenum disulfide modulated iron monatomic bionic nano-enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to molybdenum disulfide modulated iron monatomic bionic nano-enzyme as well as a preparation method and application thereof. The bionic nano-enzyme is in a hollow nano-rod shape, a carbon base anchored with iron monatomic atoms is uniformly doped with molybdenum disulfide lamellae, the iron monatomic atoms are anchored through nitrogen uniformly doped on the carbon base, the molybdenum disulfide lamellae are composed of 1T-phase molybdenum disulfide, a FeN5-MoS2 active center is formed, catalytic activity is achieved, and the carbon base and the molybdenum disulfide lamellae can be used for preparing the nano-enzyme. The heme b5-molybdenum cofactor is similar to the heme b5-molybdenum cofactor of natural sulfite oxidase, and has efficient peroxidase-like activity and good stability. Colorimetric signals are generated on the basis that the activity of iron atoms is affected by acetylcholine hydrolysate, a cascade detection platform with acetylcholin esterase as a marker is constructed, and sensitive detection of insecticide acetamiprid and distinguishing of different natural alkaloids are achieved. In order to adapt to convenient real-time detection in a complex environment, a paper-based colorimetric sensing platform is combined with a paper base, the residual content of acetamiprid is read through a mobile phone colorimetric program, and the method has the advantages of low cost, simplicity in operation, good specificity, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and specifically relates to a bionic nanozyme of molybdenum disulfide modulating a single iron atom, and a preparation method and application thereof. Background Art

[0002] In recent years, single-atom nanozymes have received great attention and development in the fields of analytical chemistry and battery energy catalysis. Compared with traditional nanozymes, single-atom nanozymes have the advantages of high atomic utilization, dispersed active sites and easy structural regulation. Among them, iron-based single-atom nanozymes have high activity and are expected to replace precious metal nanozymes. At present, most single-atom nanozymes have a Fe-Nx central structure similar to natural cytochrome P450. Although they have achieved good catalytic activity, their selectivity for substrates and the repeatability of enzyme-like activity are low. In nature, sulfite oxidase is a class of proteases widely present in animals, plants and eukaryotic cells, which are involved in the process of converting sulfite into sulfate. In animals and higher plants, researchers have found that sulfite oxidase also has peroxidase activity. This is because this type of sulfite oxidase has dual active factors: heme b5 containing Fe-N4 structure and molybdenum cofactor Moco containing Mo-O-S2 structure.

[0003] Acetylcholinesterase is a powerful hydrolase that specifically catalyzes acetylcholine. It is expressed in various organs and the central and peripheral nervous systems. At the same time, as a commonly used biomarker, acetylcholinesterase plays an important role in the research fields of pesticide poisoning, liver disease and Alzheimer's disease. Acetylcholinesterase is not only involved in the respiratory and motor systems of organisms, but is also crucial to the higher brain functions of mammals, such as learning and memory. Some environmental chemicals, such as organophosphorus (OP) and carbamate pesticides, will specifically bind to the catalytic triad site of acetylcholinesterase, thereby inhibiting the enzyme, causing permanent neural damage or even death of the organism. Based on this, acetylcholinesterase can be used in vitro to participate in the construction of biosensors to detect pesticide residues in the environment through inhibitory effects. Compared with the conventional methods currently used for pesticide residue detection: gas chromatography, high performance liquid chromatography, chromatography and chromatography-mass spectrometry, etc., the sensor developed based on acetylcholinesterase has the advantages of high sensitivity, high selectivity, simple operation, fast detection speed and low cost. With the in-depth study of acetylcholinesterase, many of its potential functions have been gradually discovered and applied, especially in the research and development of new clinical drugs. These include newly developed natural cholinesterase inhibitors, such as galantamine and huperzine A. These natural alkaloids can act as reversible inhibitors of cholinesterase and participate in the adjuvant treatment of diseases of the nervous system, urinary system and immune system. Therefore, biosensors based on acetylcholinesterase also play an indispensable role in the evaluation and testing of new drugs.

[0004] In addition, the signal output methods of biosensors are generally divided into colorimetry, fluorescence, electrochemistry and chromatography, among which colorimetry has low dependence on instruments, simple operation and fast analysis speed. However, in the face of the actual complex detection environment, the requirements for colorimetric sensors are further improved, and UV-visible spectrophotometers cannot meet the daily real-time detection needs. Therefore, paper-based colorimetric sensors have gained broad application prospects due to the following advantages:

[0005] 1. Low cost. Paper as the base material of the sensor is low-cost and easily available. It can be prepared on a large scale, thus reducing the cost of the sensor.

[0006] 2. Flexibility: Paper has good flexibility and plasticity, can be easily processed and prepared, and is suitable for sensor designs of various shapes and sizes.

[0007] 3. Biocompatibility. Paper as a sensor substrate has good biocompatibility and can be used in the preparation of biosensors to detect biological molecules.

[0008] 4. Environmentally friendly and sustainable. Paper is a renewable resource, and the preparation of paper-based sensors has a lower environmental impact and meets the requirements of sustainable development. Summary of the invention

[0009] The present invention aims to provide a bionic nanozyme of iron single atom modulated by molybdenum disulfide and its application, using MoS2 to simulate Moco in natural enzymes, providing more electrons for FeN5 active center, accelerating the catalytic reaction of H2O2, and combining acetylcholinesterase to construct a rapid detection and evaluation platform for acetamiprid and natural alkaloids. Using colorimetric paper-based sensors, portable detection of pesticide residues can be achieved.

[0010] The present invention provides a bionic nanozyme of molybdenum disulfide modulating single iron atoms, in which a carbon base anchored with single iron atoms is uniformly doped with molybdenum disulfide sheets to present a hollow nanorod shape, and the single iron atoms are anchored by nitrogen uniformly doped on the carbon base, and the molybdenum disulfide sheets are composed of molybdenum disulfide in 1T phase to form a FeN5-MoS2 active center.

[0011] The outer diameter width of the bionic nanozyme is 80-220 nm, preferably 100-200 nm, and more preferably 150-180 nm.

[0012] The valence state of the iron is between +2 and +3, and the valence state of the molybdenum is mainly +4.

[0013] The method for preparing the bionic nanozyme of molybdenum disulfide modulating iron single atom provided by the present invention comprises the following steps:

[0014] (1) mixing nitric acid and ammonium molybdate in a solution and performing a hydrothermal reaction to generate MoO3;

[0015] (2) adding ammonium ferric sulfate to a solution containing MoO3 to react and generate MoO3@FeOOH;

[0016] (3) adding ammonia water to the solution containing MoO3@FeOOH and suspending it at room temperature to generate HoMoO3@FeOOH;

[0017] (4) Mixing a methanol solution containing hexachlorotripolyphosphinate (HCCP), bisphenol sulfide (BPS) and HoMoO3@FeOOH, and then adding triethylamine (TEA) to react and generate HoMoO3@FeOOH@PZS;

[0018] (5) After calcination, HoMoO3@FeOOH@PZS was acid washed to obtain FeN5-MoS2.

[0019] In step (1), the molar ratio of molybdenum element to nitric acid is 1:11-14, preferably 1:12.5-13; the molar concentration of nitric acid is 2.5-3 mol / L, preferably 2.8-2.9 mol / L.

[0020] In step (1), the hydrothermal reaction is carried out in an autoclave at 180° C. for 20 hours.

[0021] In step (1), the ammonium molybdate includes ammonium dimolybdate, ammonium tetramolybdate, ammonium paramolybdate, and ammonium octamolybdate, preferably ammonium paramolybdate.

[0022] In step (2), the molar ratio of molybdenum to iron is 1:1.8-3, preferably 1:2-2.5, and more preferably 1:2; the molar concentration of iron is 20-40 mmol / L, preferably 22-30 mmol / L, and more preferably 22-23 mmol / L.

[0023] In step (2), the solution is a mixture of water and ethanol, and the volume ratio of water to ethanol is 7-9:1, preferably 9:1.

[0024] In step (2), the reaction was carried out at 70°C for 3 hours.

[0025] In step (3), the mass ratio of ammonia to MoO3@FeOOH is 25-30:1, preferably 27:1; the mass concentration of MoO3@FeOOH is 4-5 mg / mL, preferably 4.3-4.5 mg / mL.

[0026] In step (4), the mass ratio of HoMoO3@FeOOH, hexachlorotripolyphosphine (HCCP) and bisphenol sulfide (BPS) is 1:1-1.6:2.5-4, preferably 1:1.3-1.5:2.9-3.5, and more preferably 1:1.4:3.2; the mass concentration of HoMoO3@FeOOH is 0.8-2 mg / mL, preferably 1-1.5 mg / mL, and more preferably 1 mg / mL.

[0027] In step (4), triethylamine is slowly added, and the final concentration of triethylamine is 1.5-2 mM, preferably 1.7 mM.

[0028] In step (4), the reaction is carried out at room temperature for 12 hours.

[0029] In step (5), calcination was performed at 750°C for 2 hours with a heating rate of 2°C min -1 .

[0030] In step (5), pickling is performed in 0.5 M hydrochloric acid.

[0031] The bionic nanozyme of iron single atom modulated by molybdenum disulfide provided by the present invention can not only accelerate the decomposition of H2O2, but also catalyze the oxidation of the color indicator (TMB) in the presence of H2O2, and can also directly oxidize the color indicator (TMB). Therefore, the bionic nanozyme has the same or similar catalytic activity as catalase, peroxidase, and oxidase, and can be used to prepare catalase-like, peroxidase-like, oxidase-like, catalase mimetic enzyme, peroxidase mimetic enzyme, and oxidase mimetic enzyme.

[0032] The bionic nanozyme of the iron unit modulated by molybdenum disulfide provided by the present invention can also be used to prepare a detection product, which can be used for qualitative and / or quantitative detection of oxidative active substances, reductive active substances, substances associated with oxidative active substances, substances associated with reductive active substances, and substances associated with the bionic nanozyme. For example, the reductive active substance can reduce the oxidized color indicator (TMB), reverse the oxidation of the color indicator (TMB) in the presence of H2O2, and convert the detection of the reductive active substance into a qualitative and / or detection based on the redox reaction of the color indicator (TMB); the substance associated with the reductive active substance can be a precursor for generating the reductive active substance, or a substance that can react chemically with the reductive active substance to weaken its reductive activity, that is, a substance that is directly or indirectly associated with the reductive active substance, and its detection can be converted into a qualitative and / or detection based on the redox reaction of the color indicator (TMB) through the reductive active substance. For example, oxidative active substances can dominate the generation of H2O2, and then the detection of oxidative active substances can be converted into TMB redox detection; substances associated with oxidative active substances can be substances that regulate the activity of H2O2 generating enzymes, or substances that regulate the raw materials used for H2O2 generation, that is, substances that are directly or indirectly associated with oxidative active substances, and their detection can be converted through oxidative active substances into qualitative and / or detection based on the redox reaction of the color indicator (TMB). Substances associated with bionic nanozymes can be substances that weaken or enhance the activity of bionic nanozymes, or substances that compete with bionic nanozymes, that is, substances that are directly or indirectly associated with bionic nanozymes, and their detection can be converted through bionic nanozymes into qualitative and / or detection based on the redox reaction of the color indicator (TMB). As shown in the present invention, the hydrolysis product of acetylcholine catalyzed by acetylcholinesterase, thiocholine, can undergo a complex reaction with a single iron atom, shield the active site of the nanozyme, and reduce the catalytic activity. Therefore, acetylcholinesterase and iron single-atom nanozyme can jointly construct a biosensor system for pesticide residue detection and anticholinesterase drug effect evaluation.

[0033] The bionic nanozyme with iron units modulated by molybdenum disulfide provided by the present invention can be used to detect oxidatively active substances, reductively active substances, substances associated with oxidatively active substances, substances associated with reductively active substances, substances associated with the bionic nanozyme, pesticides, and anticholinesterase drugs.

[0034] The detection product prepared by the bionic nanozyme with iron units modulated by molybdenum disulfide provided by the present invention can be a reagent, a test kit, a material, a consumable material, a sensing platform, a sensing system, a sensor, etc.

[0035] The detection product prepared by the bionic nanozyme of iron unit modulated by molybdenum disulfide provided by the present invention comprises at least one of the following:

[0036] (1) Catalase-like enzymes or catalase-mimicking enzymes;

[0037] (2) Peroxidase-like or peroxidase-mimicking enzymes;

[0038] (3) Oxidase-like or oxidase-mimicking enzymes;

[0039] (4) Products for detecting oxidatively active substances or substances related to oxidatively active substances;

[0040] (5) Products for detecting reducing active substances or substances related to reducing active substances;

[0041] (6) Products for detecting the presence of related substances in bionic nanozymes containing iron units modulated with molybdenum disulfide;

[0042] (7) Products for pesticide testing;

[0043] (8) Products for testing anticholinesterase drugs.

[0044] The bionic nanozyme with iron units modulated by molybdenum disulfide is present with related substances such as pesticides and anticholinesterase drugs, and the qualitative and / or quantitative detection of pesticides and anticholinesterase drugs is carried out through the qualitative and / or quantitative detection of the redox reaction of acetylcholinesterase and iron single-atom nanozyme converted into a color indicator (TMB).

[0045] The present invention provides a detection method for a bionic nanozyme based on molybdenum disulfide modulating an iron unit, the steps comprising: subjecting a sample to be tested, a bionic nanozyme with molybdenum disulfide modulating an iron unit provided by the present invention, and a color indicator to a color reaction in a reaction system, and performing qualitative and / or quantitative determination by measuring the absorbance value of an ultraviolet-visible absorption spectrum or extracting the RGB value of an image.

[0046] The sample to be tested contains oxidatively active substances, or reductively active substances, or substances associated with oxidatively active substances, or substances associated with reductively active substances, or substances associated with the bionic nanozyme, or pesticides, or anticholinesterase drugs.

[0047] When the sample to be tested contains substances associated with the oxidatively active substance, or substances associated with the reductively active substance, or substances associated with the bionic nanozyme with iron units modulated by molybdenum disulfide, the sample to be tested first undergoes an associated reaction, and then undergoes a color development reaction in the reaction system with the bionic nanozyme with iron units modulated by molybdenum disulfide and a color indicator of the present invention.

[0048] A buffer is also added to the color development reaction and the associated reaction system, and the buffer includes a phosphate buffer and an acetic acid-sodium acetate buffer. In the color development reaction system, the pH value of the buffer is 3-5, preferably 3.5-5, preferably pH=4. The buffer is preferably an acetic acid-sodium acetate buffer. The buffer in the associated reaction system is a phosphate buffer.

[0049] The final concentration of the bionic nanozyme modulated with iron units provided by the present invention is 5-40 μg mL -1 , preferably 15-30 μg mL -1 , preferably 20 μg mL -1 .

[0050] The temperature for the qualitative and / or quantitative determination is not less than 25°C, preferably 25°C-50°C, more preferably 25°C-30°C, with 25°C being the most optimal.

[0051] The tools or instruments for the color development reaction and the associated reaction meet the requirements of optical detection, do not affect the absorbance detection, and do not interfere with the color observation. They can be colorimetric dishes, well plates, test papers, etc., and can be made of glass, plastic, or paper.

[0052] When used for the detection of pesticide residues or anticholinesterase drugs, the pesticide or anticholinesterase drug is first incubated with acetylcholinesterase, then acetylcholine is added for incubation, and then the bionic nanozyme of the iron unit modulated by molybdenum disulfide provided by the present invention, buffer, and color indicator are added for incubation, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

[0053] When a paper substrate is used for colorimetric detection, the incubation solution obtained after the pesticide or anticholinesterase drug is incubated with acetylcholinesterase and acetylcholine in sequence is dropped onto the paper substrate loaded with the bionic nanozyme with molybdenum disulfide modulated iron units provided by the present invention, and then a color indicator is added to carry out a color reaction, and the RGB value of the image is extracted for qualitative and / or quantitative determination.

[0054] The pH value of the buffer solution is 3-5, preferably 3.5-5, preferably pH=4; the buffer solution is acetic acid-sodium acetate buffer solution.

[0055] The final concentration of the bionic nanozyme modulated with iron units provided by the present invention is 5-40 μg mL -1 , preferably 15-30 μg mL -1 , preferably 20 μg mL -1 .

[0056] The color indicator is TMB.

[0057] The absorbance value is the absorbance value at 625 nm.

[0058] The pesticide or anticholinesterase drug hinders the hydrolysis reaction of acetylcholine, either by inhibiting the activity of cholinesterase or by binding to acetylcholine to hinder its hydrolysis. The anticholinesterase drug is an alkaloid, including neostigmine bromide, berberine hydrochloride, physostigmine, matrine, evodiamine, etc. The pesticide is acetamiprid.

[0059] The temperature for the qualitative and / or quantitative determination is not less than 25°C, preferably 25°C-50°C, more preferably 25°C-30°C, with 25°C being the most optimal.

[0060] The incubation system of the pesticide or anticholinesterase drug and acetylcholinesterase contains a buffer solution, which is a phosphate buffer solution.

[0061] The present invention provides a method for synthesizing bionic nanozyme FeN5-MoS2 using molybdenum disulfide to modulate single iron atoms and its application. Compared with simple single iron atoms, the introduction of molybdenum disulfide can provide more electrons for the active center of FeN5, accelerate the decomposition of H2O2 and the desorption of the final product water, and is beneficial to the surface reconstruction of the catalytic center, thereby exhibiting better peroxidase-like catalytic activity. At the same time, single iron atoms and sulfhydryl-containing molecules will undergo a complexation reaction. Therefore, when the acetylcholine content is fixed, the content of acetylcholinesterase will affect the catalytic activity of FeN5-MoS2, resulting in a color signal change in the colorimetric sensing system. Furthermore, the FeN5-MoS2 and acetylcholinesterase cascade colorimetric sensing platform can detect that the detection limit of the insecticide acetamiprid can reach 52 ng / mL, with high sensitivity, and a good linear range of 0.1-100 μg / mL, strong anti-interference ability, and good specificity. Furthermore, the colorimetric sensing platform can clearly distinguish different natural alkaloids, and its half-inhibitory concentration IC 50 It is within the scope of other research reports. Finally, the colorimetric sensing platform is combined with the paper base to detect the acetamiprid residue on the surface of vegetables through a mobile phone. Compared with other detection methods, it has the advantages of low equipment cost, simple operation, low environmental requirements, and convenience, and is suitable for convenient real-time detection in complex environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the synthesis steps of FeN5-MoS2 nanozyme.

[0063] Figure 2 This is a transmission electron microscopy image of FeN5-MoS2 nanozyme.

[0064] Figure 3High-angle annular dark-field scanning transmission electron microscopy image of FeN5-MoS2 nanozyme corrected for spherical aberration.

[0065] Figure 4 This is the X-ray diffraction pattern of FeN5-MoS2 nanozyme.

[0066] Figure 5 This is the element distribution map of FeN5-MoS2 nanozyme.

[0067] Figure 6 This is the X-ray photoelectron spectrum of each element of FeN5-MoS2 nanozyme (I).

[0068] Figure 7 This is the X-ray photoelectron spectrum of each element of FeN5-MoS2 nanozyme (II).

[0069] Figure 8 These are the synchrotron radiation results of FeN5-MoS2 nanozyme.

[0070] Fig. 9 This is the free energy calculation of FeN5-MoS2 nanozyme reaction based on density functional theory.

[0071] Fig.10 This is the charge density difference image of FeN5-MoS2 nanozyme.

[0072] Fig.11 It is the peroxidase-like activity of FeN5-MoS2 nanozyme.

[0073] Fig.12 These are the optimal conditions for the peroxidase-like activity of FeN5-MoS2 nanozyme.

[0074] Fig.13 are the steady-state kinetic parameters of FeN5-MoS2 nanozyme.

[0075] Fig.14 Detection of acetylcholinesterase by FeN5-MoS2 nanozyme.

[0076] Fig.15 Anti-interference ability of acetylcholinesterase detection system.

[0077] Fig.16 Colorimetric sensors constructed for acetylcholinesterase and FeN5-MoS2 for the detection of acetamiprid.

[0078] Fig.17 Colorimetric sensors constructed for acetylcholinesterase and FeN5-MoS2 for discrimination of alkaloids.

[0079] Fig.18 Detection of acetamiprid using paper-based colorimetric sensor. DETAILED DESCRIPTION

[0080] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0081] Example 1: Preparation of bionic nanozyme FeN5-MoS2 using molybdenum disulfide to modulate single iron atoms.

[0082] (1) 5 mL of nitric acid (65 wt%) was added to a mixture containing 1 g of (NH4)6Mo7O 24 ·4H2O in 20mL aqueous solution, then transfer the above solution to a 50mL Teflon-lined autoclave for reaction at 180°C for 20 hours, centrifuge and wash the product, and dry it to obtain MoO3;

[0083] (2) MoO3 was dispersed in a mixed solution of deionized water and ethanol in a volume ratio of 9:1. A 50 mM NH4Fe(SO4)2·12H2O solution was slowly injected into a 20 mM MoO3 solution. The volume ratio of NH4Fe(SO4)2·12H2O solution to MoO3 solution was 1:1.25. The mixture was then transferred to a 70 °C water bath for 3 h. After cooling, MoO3@FeOOH was obtained by centrifugal washing.

[0084] (3) Add 3 mL of 25% ammonia water to 20 mL of an aqueous solution containing 100 mg of MoO3@FeOOH, stir the suspension at room temperature for 3 hours, wash and dry to obtain HoMoO3@FeOOH;

[0085] (4) Under stirring, 10 mL of methanol solution containing 70 mg of hexachlorotripolyphosphinate (HCCP) and 158 mg of bisphenol sulfide (BPS) was added dropwise to 40 mL of methanol solution containing 50 mg of HoMoO3@FeOOH, and then triethylamine (TEA) was added dropwise. The concentration of triethylamine dissolved in the methanol solution was 1.7 mM. The reaction was carried out at room temperature for 12 hours. After centrifugal washing, the mixture was vacuum dried for 12 hours to obtain HoMoO3@FeOOH@PZS.

[0086] (5) The collected HoMoO3@FeOOH@PZS was calcined in a tube furnace for 2 h at a heating rate of 2 °C min -1 The calcination temperature is 750°C, and then the carbonized black powder is dispersed in a 0.5M hydrochloric acid solution and stirred for 12 hours before centrifugal washing to obtain FeN5-MoS2.

[0087] The present invention uses the confined synthesis method to restrict the diffusion of iron and molybdenum elements through PZS, and obtains a monolayer of molybdenum disulfide and FeN5 single atoms after acid washing. The synthesis steps are shown in Figure 1 .

[0088] Transmission electron microscopy images of FeN5-MoS2 materials are shown in Figure 2 , Figure 2 The material morphology is shown to be hollow nanorods without obvious particles.

[0089] The spherical aberration corrected high-angle annular dark-field scanning transmission electron microscopy image of FeN5-MoS2 material is shown in Figure 3 , Figure 3 The lattice spacing of 0.25 nm was measured, corresponding to the (102) crystal plane of MoS2. In addition, isolated bright spots (highlighted by yellow circles) were observed, indicating that the iron was dispersed in the form of single atoms.

[0090] The X-ray diffraction pattern of FeN5-MoS2 material is shown in Figure 4 , Figure 4 The crystal structure of MoS2 was demonstrated to be widely spaced thin layers with iron doped in amorphous carbon.

[0091] The element distribution diagram of FeN5-MoS2 material is shown in Figure 5 , Figure 5 It shows that due to the confinement effect of PZS, each element is evenly distributed under high-temperature pyrolysis conditions.

[0092] The mass fractions of Mo and Fe in FeN5-MoS2 material are 4.03% and 0.28%, respectively.

[0093] The X-ray photoelectron spectrum of each element of FeN5-MoS2 material is shown in Figure 6 and Figure 7 , where the valence state of iron is between +2 and +3, the valence state of molybdenum is mainly +4, and molybdenum disulfide combined with sulfur element exists in the form of 1T phase.

[0094] The synchrotron radiation results of FeN5-MoS2 materials are shown in Figure 8 . Figure 8 It shows that Fe is mainly coordinated with N with a coordination number of 5, and Mo is mainly coordinated with S with a coordination number of 2, which proves the active center structure of the bionic nanozyme FeN5-MoS2 modulated by molybdenum disulfide single iron atom.

[0095] The calculation of the reaction free energy of materials based on density functional theory is shown in Fig. 9 , Fig. 9 This indicates that the introduction of MoS2 can provide more electrons to the FeN5 active center, accelerating the decomposition of H2O2 and the desorption of the final product water.

[0096] The charge density difference image of FeN5-MoS2 material is shown in Fig.10 , Fig.10 Similar to natural enzymes, MoS2 acts as an electron donor and FeN5 acts as an electron acceptor.

[0097] Example 2: Investigation of the peroxidase-like activity of FeN5-MoS2.

[0098] First, 3 μL of 10 mM 3,3',5,5'-tetramethylbenzidine (TMB) and 3 μL of 50 mM H2O2 were added to 288 μL of acetic acid-sodium acetate buffer (20 mM, pH 4.0). -1 FeN5-MoS2 was added to the solution and mixed evenly. After incubation at room temperature for 10 minutes, the solution was centrifuged and the supernatant was transferred to a UV-visible spectrometer to record the absorption spectrum.

[0099] The peroxidase-like activity of FeN5-MoS2 materials is shown in Fig.11 , Fig.11 This reflects that the material is mainly characterized by peroxidase activity.

[0100] Example 3: Investigation of the optimal conditions for the peroxidase-like activity of FeN5-MoS2.

[0101] 3μL 10mM 3,3',5,5'-tetramethylbenzidine (TMB) and 3μL 50mM H2O2 were added to 288μL buffer in sequence. Then 6μL FeN5-MoS2 was added to the solution and mixed evenly. After incubation for 10 minutes, the solution was centrifuged and the supernatant was transferred to a UV-visible spectrometer to record the absorption spectrum. The optimal conditions for peroxidase-like activity were explored by changing the buffer pH, incubation temperature, and FeN5-MoS2 concentration.

[0102] The optimal conditions for the peroxidase-like activity of FeN5-MoS2 are shown in Fig.12 , Fig.12 The optimal catalytic conditions were pH = 4, incubation temperature was 25 °C, and the final concentration of FeN5-MoS2 material was 20 μg mL -1 .

[0103] Example 4: Steady-state kinetic study of FeN5-MoS2.

[0104] First, 3 μL of 10 mM 3,3',5,5'-tetramethylbenzidine (TMB) and 3 μL of 50 mM H2O2 were added to 288 μL of acetic acid-sodium acetate buffer (20 mM, pH 4.0). -1FeN5-MoS2 was added to the solution and mixed evenly. After incubation at room temperature for 10 minutes, the solution was centrifuged and the supernatant was transferred to a UV-visible spectrometer to record the absorption spectrum. The steady-state kinetic study of FeN5-MoS2 was performed by changing the concentration of H2O2 (5, 10, 20, 50, 100, 250 and 500 μM) or TMB (5, 10, 25, 50, 75, 100, 200 and 400 μM).

[0105] The steady-state kinetic parameters of FeN5-MoS2 are shown in Fig.13 , Fig.13 This reflects that the material has good affinity for the substrate and a high catalytic rate.

[0106] Example 5: Before constructing the biosensor, acetylcholinesterase was quantitatively detected.

[0107] First, 3 μL of 30 mM acetylcholine was added to 15 μL of phosphate buffer containing different concentrations of acetylcholinesterase, and the solution was immediately mixed. Then, the mixture was incubated at 37 °C for 30 min in preparation for measurement. Subsequently, 270 μL of acetic acid-sodium acetate buffer, 3 μL of 10 mM TMB, 3 μL of 50 mM H2O2, and 6 μL of 1 mg mL -1 FeN5-MoS2. Finally, the resulting solution was stored at room temperature for 10 minutes, and the ultraviolet absorbance of the solution at 652 nm was recorded.

[0108] Acetylcholinesterase detection Fig.14 , Fig.14 The minimum detection limit of acetylcholinesterase was 0.045 mUmL -1 The linear detection range is 1-75mU mL -1 .

[0109] Example 6: Anti-interference ability of acetylcholinesterase detection system.

[0110] 3 μL of 30 mM acetylcholine was added to 15 μL of different concentrations of interfering substances (papain, lysozyme, glucose oxidase, trypsin, glutamate, serine, bovine serum albumin, Ca 2+ , K + ) in phosphate buffer and immediately mixed the solution. The interfering substance was 50-100 times the concentration of acetylcholinesterase. Then, the mixture was incubated at 37°C for 30 minutes in preparation for measurement. Subsequently, 270 μL of acetic acid-sodium acetate buffer, 3 μL of 10 mM TMB, 3 μL of 50 mM H2O2, and 6 μL of 1 mg mL -1FeN5-MoS2. Finally, the resulting solution was stored at room temperature for 10 minutes, and the ultraviolet absorbance of the solution at 652 nm was recorded.

[0111] The anti-interference ability of the acetylcholinesterase detection system is shown in Fig.15 ,Depend on Fig.15 It can be seen that the color of the solution containing acetylcholinesterase is significantly different from that of the solution containing other interfering substances, and the signal intensity of the ultraviolet absorption peak is significantly reduced, showing good anti-interference ability and good specificity.

[0112] Example 7: FeN5-MoS2 is applied to the detection of acetamiprid and the differentiation of natural alkaloids.

[0113] (1) Add 30 μL of pesticide or alkaloid and 15 μL of 200 mU mL -1 Acetylcholinesterase, incubated at 37°C for 15 min;

[0114] Wherein, the alkaloids include: neostigmine bromide, berberine hydrochloride, physostigmine, matrine, evodiamine, and neostigmine bromide is used as a positive control group to evaluate the maximum inhibition rate of natural alkaloids (berberine hydrochloride, physostigmine, matrine, evodiamine). At this time, 30 μL 1 mmol of different alkaloids need to be added to the test substance;

[0115] (2) Add acetylcholine to the system and incubate at 37°C for 30 min;

[0116] (3) Add 240 μL (pH = 4) of acetic acid-sodium acetate buffer, 3 μL of 10 mM 3,3',5,5'-tetramethylbenzidine (TMB), 3 μL of 50 mM H2O2, and 6 μL of 1 mg mL -1 The FeN5-MoS2 nanozyme material was incubated at 37°C for 10 min;

[0117] (4) According to step (1) to step (3), but without adding the target compound to be tested, a solution A is prepared that does not contain the target compound to be tested; According to step (1) to step (3), but without adding the target compound to be tested and acetylcholine, a solution B is prepared;

[0118] (5) Use an ultraviolet spectrophotometer to record the absorbance of the solution in step (3) at 652 nm, denoted as A i ; Record the absorbance values ​​of solutions A and B at 652 nm in step (4), denoted as A and A0 respectively;

[0119] According to the measured data, according to the formula inhibition rate (%) = (A i-A) / (A0-A)×100, and draw a fitting curve.

[0120] Acetamiprid test Fig.16 From the figure, it can be seen that the detection limit of acetamiprid is 6ng mL -1 , linear range 0.01-100 μg mL -1 For the distinction between alkaloids, see Fig.17 It can be seen that for different alkaloids, the sensors constructed by acetylcholinesterase and FeN5-MoS2 detected different half-inhibitory concentrations IC 50 .

[0121] Example 8: Application of FeN5-MoS2 in the detection of acetamiprid using a paper-based colorimetric sensor.

[0122] (1) Cut the filter paper into discs with a diameter of 1 cm and -1 Soak in FeN5-MoS2 ethanol solution for 1 min;

[0123] (2) drying the soaked filter paper to obtain a paper-based colorimetric sensor;

[0124] (3) 15 μL containing 200 mU mL -1 AChE and different concentrations of acetamiprid were incubated at 37°C for 10 min, followed by the addition of 5 μL of 4 mM acetylcholine. After incubation at 37°C for 30 min, the mixture was dropped onto a paper-based colorimetric sensor and incubated again at 37°C for 30 min.

[0125] (4) Drop 30 μL of acetic acid-sodium acetate buffer containing 2 mM TMB and 10 mM H2O2 onto the paper-based colorimetric sensor and wait for 10 min for color development;

[0126] (5) Use your mobile phone to take photos and compare colors through the WeChat mini program.

[0127] Application of FeN5-MoS2 in paper-based colorimetric sensor for detection of acetamiprid Fig.18 , and its detection limit was 52 ng mL -1 , linear range 0.1-100 μg mL -1 , which can meet the sensitivity detection of acetamiprid in simple environments under actual conditions.

[0128] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A bionic nanozyme of molybdenum disulfide modulating iron single atom, characterized in that: The carbon base anchored with single iron atoms is uniformly doped with the molybdenum disulfide flakes to form a hollow nanorod shape. The single iron atoms are anchored by the nitrogen uniformly doped on the carbon base. The molybdenum disulfide flakes are composed of molybdenum disulfide in the 1T phase, forming a FeN5-MoS2 active center.

2. The bionic nanozyme according to claim 1, characterized in that: The width of the bionic nanozyme is 80-220nm.

3. A method for preparing a bionic nanozyme of molybdenum disulfide modulating a single iron atom, comprising the steps of: (1) mixing nitric acid and ammonium molybdate in a solution and performing a hydrothermal reaction to generate MoO3; (2) adding ammonium ferric sulfate to a solution containing MoO3 to react and generate MoO3@FeOOH; (3) adding ammonia water to the solution containing MoO3@FeOOH and suspending it at room temperature to generate HoMoO3@FeOOH; (4) mixing the methanol solution containing hexachlorotripolyphosphinate, bisphenol sulfur and HoMoO3@FeOOH, and then adding triethylamine to react and generate HoMoO3@FeOOH@PZS; (5) After calcination, HoMoO3@FeOOH@PZS was acid washed to obtain FeN5-MoS2.

4. The biomimetic nanozyme according to claim 1 or 2 has at least one of the following uses: (1) preparing a catalase-like enzyme or a catalase mimetic enzyme; (2) preparing peroxidase-like enzymes or peroxidase-mimicking enzymes; (3) preparing oxidase-like enzymes or oxidase-mimicking enzymes; (4) Preparing products for detecting oxidatively active substances or substances associated with oxidatively active substances; (5) Preparing products for detecting reduction-active substances or substances related to reduction-active substances; (6) preparing a product for detecting the presence of related substances of the bionic nanozyme according to claim 1 or 2; (7) Preparation of products for pesticide detection; (8) Preparation of products for anticholinesterase drug testing; (9) Detecting oxidatively active substances or substances associated with oxidatively active substances; (10) Detecting reduction-active substances or substances associated with reduction-active substances; (11) detecting the presence of related substances to the bionic nanozyme according to claim 1 or 2; (12) Detection of pesticides; (13) Detection of anticholinesterase drugs.

5. The product prepared by the bionic nanozyme according to claim 1 or 2, comprising at least one of the following: (1) Catalase-like enzymes or catalase-mimicking enzymes; (2) Peroxidase-like or peroxidase-mimicking enzymes; (3) Oxidase-like or oxidase-mimicking enzymes; (4) Products for detecting oxidatively active substances or substances related to oxidatively active substances; (5) Products for detecting reducing active substances or substances related to reducing active substances; (6) Products for detecting the presence of related substances in bionic nanozymes containing iron units modulated with molybdenum disulfide; (7) Products for pesticide testing; (8) Products for testing anticholinesterase drugs.

6. The product according to claim 5, characterized in that Products include reagents, test kits, materials, consumables, sensing platforms, sensing systems, and sensors.

7. A detection method of bionic nanozyme based on molybdenum disulfide modulated iron unit, characterized in that the steps include: The sample to be tested, the bionic nanozyme according to claim 1 or 2, and the color indicator are placed in a reaction system for color reaction, and qualitative and / or quantitative determination is performed by measuring the absorbance value of the ultraviolet-visible absorption spectrum or extracting the RGB value of the image.

8. The detection method according to claim 7, characterized in that: The sample to be tested contains oxidatively active substances, or reductively active substances, or substances associated with oxidatively active substances, or substances associated with reductively active substances, or substances associated with the bionic nanozyme, or pesticides, or anticholinesterase drugs.

9. The detection method according to claim 8, characterized in that: When the sample to be tested contains substances associated with the oxidatively active substance, or substances associated with the reductively active substance, or substances associated with the bionic nanozyme with molybdenum disulfide modulated iron units, the sample to be tested first undergoes an associated reaction, and then undergoes a color reaction in the reaction system with the bionic nanozyme and the color indicator as described in claim 1.

10. The detection method according to claim 8, characterized in that: When used for the detection of pesticide residues or anticholinesterase drugs, the pesticide or anticholinesterase drug is first incubated with acetylcholinesterase, then acetylcholine is added for incubation, and then a buffer solution and a color indicator are added for incubation. The qualitative and / or quantitative determination is performed by measuring the absorbance value of the UV-visible absorption spectrum or extracting the RGB value of the image.

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