Ternary solid nonmetal co-doped diatomic nano enzyme-based fluoropyrazole furanone electrochemical sensor and preparation method thereof

Abstract

The invention relates to a ternary solid non-metal co-doped diatomic nano-enzyme-based fluoropyrazole furanone electrochemical sensor and a preparation method thereof, and the preparation method comprises the following steps: preparing a ternary solid non-metal doped diatomic nano-enzyme with a hierarchical porous hollow structure and multiple atomic active sites by a confinement pyrolysis method; forming a nano enzyme-hapten probe by the nano enzyme and a hapten through directional covalent coupling; based on a competitive immune recognition mechanism, a target object is preferentially combined with an electrode immobilized antibody, probe enrichment is hindered, and then catalytic reduction current of the nano-enzyme to a substrate is influenced. The preparation method has the technical advantages that (1) diatomic active sites and ternary doping cooperate to enhance sensitivity; (2) a competitive signal attenuation mode effectively inhibits background interference; and (3) nano-enzyme catalytic amplification and electrochemical detection are integrated, and rapid and sensitive detection of flupyradifurone in agricultural products is realized. Compared with traditional methods such as chromatography, the method has the advantages of simplicity and convenience in operation, high sensitivity, good accuracy and the like.

Description

Technical Field

[0001] The present invention relates to a ternary solid-state non-metal co-doped diatomic nanozyme-based fluoropyranone electrochemical sensor and a preparation method thereof, and in particular to a preparation method of a ternary solid-state non-metal co-doped diatomic nanozyme-hapten complex derived from a multi-level composite structure. Background Art

[0002] Flupyralidone is a new butyrolactone insecticide officially registered and marketed in China in 2018. Flupyralidone acts on the central nervous system of target pests and is characterized by rapid action and long-lasting effects. However, flupyralidone and its metabolites exhibit cytotoxic and genetic effects on human lymphocytes, and many countries have established maximum residue limits (MRLs) for flupyralidone in food. Therefore, determining flupyralidone residues is crucial for risk assessment. Currently, flupyralidone is primarily determined through instrumental analytical methods, including liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography. However, these methods are complex, require expensive equipment, are time-consuming, and labor-intensive, making them unsuitable for the determination of flupyralidone in large quantities of real-world samples. Electrochemical sensors offer advantages such as portability, rapid response, low cost, and high sensitivity, and are currently widely used in pesticide detection. However, there is currently a gap in the electrochemical sensor analysis method for fluoropyrafuranone, so the development of a fast, convenient and sensitive electrochemical sensor detection method for fluoropyrafuranone is of great practical significance. Summary of the Invention

[0003] The present invention relates to a ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and a preparation method thereof The ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and its preparation method comprise the following steps: Preparation of the ternary solid-state non-metallic co-doped diatomic nanozyme: ① Preparation of the hierarchical composite nanomaterial: Under inert atmosphere, 0.7-1.5 g of a reducing agent containing a solid non-metallic element and 5.0-8.0 g of organic ligand A were weighed and uniformly mixed in a sealed reactor. The mixture was then heated to 200-400°C for 2-4 hours for thermal decomposition. The resulting product was centrifuged at 8000-10000 rpm for 5-20 minutes, washed 2-5 times with an organic solvent, and dried at 50-80°C for 12-24 hours. Subsequently, 1.0-3.5 g of a transition metal salt solution, 0.5-1.5 g of organic ligand B, and 0.1-0.3 g of the resulting product were dispersed in 25-45 mL of organic solvent. After ultrasonic treatment for 10-30 minutes, the coordination reaction was carried out at 70-150°C with continuous stirring for 2-4 hours. The resulting product was centrifuged at 8,000–10,000 rpm for 5–20 minutes, washed 2–5 times with an organic solvent, and dried at 50–80°C to obtain the base framework material. 50–150 mg of organic ligand C and 100–200 mg of organic ligand D were dissolved in 100–150 mL of organic solvent, respectively, to form two precursor solutions. The base framework material and the two precursor solutions were mixed, ultrasonically dispersed for 20–40 minutes, and stirred for 12–24 hours. The resulting hierarchical composite nanostructured material was centrifuged at 8,000–10,000 rpm for 5–20 minutes, washed 2–5 times with an organic solvent, and dried at 50–80°C for 12–24 hours. ② Preparation of ternary solid-state non-metallic co-doped diatomic nanozymes derived from a hierarchical composite structure: The hierarchical composite nanomaterial was placed in a tube furnace and heated at a rate of 2–10°C / min to 800–1000°C under an inert gas atmosphere for 1–3 h to complete the primary carbonization treatment. 50–200 mg of the carbonized product was dispersed in 5–20 mL of organic solvent and sonicated for 5–20 min to form a homogeneous dispersion. 20–80 mg of the two solid non-metallic organic compounds were dissolved in 10–50 mL of organic solvent and slowly added dropwise to the reaction system. 0.1–0.5 mL of stabilizer was then added, and the reaction was stirred at 20–40°C for 12–24 h. The resulting product was centrifuged at 8000–10000 rpm for 5–20 min, washed 2–5 times with organic solvent, and dried under vacuum at 50–80°C for 12–24 h before undergoing a secondary carbonization treatment. Disperse 5–20 mg of the two transition metal salt solutions and 50–200 mg of the secondary carbonization product in 20–50 mL of an organic solvent. Ultrasonic dispersion is performed for 5–10 minutes, followed by stirring at 20–40°C for 12–24 hours. The resulting product is centrifuged at 8,000–10,000 rpm for 5–20 minutes, washed two to five times with an organic solvent, and dried at 50–80°C for 12–24 hours.Then it was placed in a tube furnace and heated to 850~1200℃ at a heating rate of 2~10℃ / min under an inert gas atmosphere for heat treatment for 1~3 hours, finally obtaining a ternary solid-state non-metal co-doped double single-atom nanozyme derived from a multi-level composite structure;. Preparation of the nanozyme-hapten complex: ① Preparation of the fluoropyrrolone hapten: In a light-proof reactor, dissolve 80-120 mg of fluoropyrrolone in 0.5-2 mL of organic solvent and add 30-50 mg of an alkali metal hydroxide as a catalyst to form a preliminary reaction system. Dissolve 0.3-0.5 mmol of a thiol-containing carboxylic acid monomer in 0.5-2 mL of organic solvent and add the resulting mixture dropwise. Nucleophilic substitution reaction proceeds in an oil bath at 80-120°C for 1-3 hours. The reaction is terminated by adding 3-8 mL of a protonating agent (0.005-0.02 mol / L). The product is extracted with an organic solvent and then dried and concentrated to obtain a viscous liquid. After recrystallization, crystals were obtained, namely the carboxylated fluoropyranone hapten; ② The nanozyme-hapten complex was prepared using the active ester method: 10-30 mg of the prepared ternary solid non-metal co-doped diatomic nanozyme was dispersed in 10-30 mL of deionized water, ultrasonicated for 5-15 minutes to make it uniformly dispersed, and then 1-3 mL of amination reagent (3%) was added and slowly stirred for 1-2 hours. The resulting product was centrifuged at 8000-10000 rpm for 5-20 minutes, washed with deionized water 2-5 times, and dried at 50-80°C for 8-12 hours to obtain the amination nanozyme. 10-25 mg of the fluoropyranone hapten was dissolved in 1.0-2.5 mL of organic solvent, 5-25 mg of organic coupling reagent was slowly added, and pre-activated under ice bath conditions for 30-60 minutes. Then 1-2 mg of the amination nanozyme was added to the pre-activated system and reacted in an ice bath for 8-12 hours to finally obtain the nanozyme-hapten complex; The electrochemical immunosensor was prepared by using magnetron sputtering technology to prepare a thin-film gold electrode. The reference electrode was coated with silver / silver chloride paste using screen printing technology. 3-8 μL of anti-flupyrrolidone antibody was drop-coated on the working electrode surface using a micropipette and incubated at 4°C for 8-12 hours. Nonspecific sites were then blocked with a blocking agent. After incubation at room temperature for 1-3 hours, the electrode was washed three times with distilled water. 5-10 μL of a mixture containing varying concentrations of flupyrrolidone and a nanozyme-hapten complex was pipetted onto the gold electrode surface and incubated at 30-60°C for 1-3 hours. The mixture was then washed three times with distilled water to remove unbound nanozyme-hapten competitors. The electrode is then immersed in a detection solution containing hydrogen peroxide and a chromogenic substrate. The target fluoropyraclostrobin competes with the nanozyme-hapten complex for binding to the antibody fixed on the electrode surface. The change in the signal generated by the multi-level composite structure ternary solid-state non-metal co-doped double single-atom nanozyme in the nanozyme-hapten complex catalyzing hydrogen peroxide and the chromogenic substrate is used to reflect the content of the target fluoropyraclostrobin, thereby obtaining a signal-reducing electrochemical immunosensor for fluoropyraclostrobin detection.

[0004] The solid non-metallic reducing agent is one or more of sodium borohydride, sodium thiosulfate, and sodium hypophosphite; The organic ligand A and the organic ligand B are one or more of 2-methylimidazole, 2,2'-bipyridine, and 4,4'-benzidine; The inert gas is one or more of argon, nitrogen, and helium; The organic solvent is one or more of methanol, ethanol, acetone, N,N'-dimethylformamide, and tetrahydrofuran; The transition metal salt is one or more of zinc nitrate, nickel nitrate, iron phthalocyanine, and trichlorotris(ethylenediamine)cobalt; The organic ligand C is one or more of 2,4,6-triamino-1,3,5-triazine, melamine, and cyanuric chloride; The organic ligand D is one or more of [2,2'-bipyridine]-5,5'-diamine, 6,6'-dinitro-[2,2'-bipyridine]-5,5'-diamine, and 2,6-pyridinedicarboxylic acid; The solid non-metallic organic compound is one or more of bis(4-hydroxyphenyl)sulfone, tripolyphosphazene chloride, and sodium tetraphenylborate; The stabilizer is one or more of triethylamine, polyvinyl pyrrolidone, and sodium lauryl sulfate; The organic solvent is one or more of dimethyl sulfoxide, N,N'-dimethylformamide, methanol, ethanol, and acetone; The alkali metal hydroxide is one or more of potassium hydroxide, sodium hydroxide and lithium hydroxide; The mercaptocarboxylic acid monomer is one or more of 3-mercaptopropionic acid, 2-mercaptoacetic acid, and 4-mercaptobenzoic acid; The acidic solution is one or more of hydrochloric acid, sulfuric acid, and acetic acid; The amination agent is one or more of polyethyleneimine, 3-aminopropyltriethoxysilane, and polylysine; The organic coupling reagent is one or more of N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The blocking agent is one or more of bovine serum albumin, casein, and polyethylene glycol; The color developing substrate is one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

[0005] Beneficial effects of the present invention (1) The present invention makes full use of the multi-level composite structure of the diatomic nanozyme and, through high-temperature calcination, makes it have a hierarchical porous hollow structure, which effectively promotes the electron transmission efficiency of the sensing interface.

[0006] (2) This paper adopts the strategy of ternary solid-state non-metal co-doped diatomic nanozymes to construct single-atom nanozymes with multiple atomic-level active sites. By utilizing the electronic structure tuning effect of two transition metal diatomic sites co-doped with ternary heteroatoms, a multi-active center synergistic catalytic system is formed, further enhancing the catalytic activity of the nanozyme and significantly improving the detection sensitivity.

[0007] (3) The present invention uses an amination reagent to aminate the diatomic nanozyme, and then uses the active ester method to couple it with the carboxylated hapten, which not only significantly improves the coupling efficiency but also further enhances the stability of the resulting complex.

[0008] (4) The present invention adopts a competitive signal attenuation mode to collect electrochemical signals, effectively avoiding the background interference problem of traditional immunosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0010] Figure 1 This is the preparation route of B, S, and P co-doped diatomic nanozymes derived from the metal organic framework @ covalent organic framework in Example 1 of the present invention.

[0011] Figure 2Scanning electron microscopy (a) and transmission electron microscopy (b) images of the B, S, and P co-doped diatomic nanozyme derived from the metal-organic framework @ covalent organic framework in Example 1 of the present invention.

[0012] Figure 3 This is a linear relationship diagram of the detection of fluoropyraclostrobin by the nanozyme-hapten complex / fluoropyraclostrobin / antibody / thin film gold electrode in Example 1 of the present invention. DETAILED DESCRIPTION

[0013] The present invention will be described below in conjunction with specific embodiments: Example

[0014] The specific steps are as follows: Preparation of B-, S-, and P-codoped Co and Ni diatomic nanozymes derived from metal-organic frameworks (MOFs) and covalent organic frameworks: ① Preparation of the B-doped MOF-covalent organic framework nanocomposite: In an argon-protected three-necked flask, 1.0 g of sodium borohydride and 6.5 g of 2-methylimidazole were thermolyzed at 300°C for 3 h. The resulting product was centrifuged at 8000 rpm for 5 min, washed three times with methanol, and dried at 65°C for 12 h. 1.5 g of zinc nitrate, 0.6 g of 4,4'-benzidine, and 0.25 g of the above product were dispersed in 30 mL of N,N'-dimethylformamide, sonicated for 20 min, and then refluxed in an oil bath at 90°C for 3 h. The resulting product was centrifuged at 8000 rpm for 5 min, washed three times with N,N'-dimethylformamide, and dried at 65°C for 12 h to obtain the B-doped MOF-based framework. 100 mg of 2,4,6-triamino-1,3,5-triazine and 150 mg of [2,2'-bipyridyl]-5,5'-diamine were dissolved in 100 mL of tetrahydrofuran to obtain two precursor solutions. Subsequently, the boron-doped metal-organic framework (MOF) substrate and the two precursor solutions were mixed, ultrasonicated for 30 minutes, and magnetically stirred at room temperature for 24 hours. The resulting product was centrifuged at 8000 rpm for 5 minutes, washed three times with tetrahydrofuran, and dried at 65°C for 12 hours. (2) Preparation of B-, S-, and P-codoped Co and Ni double single-atom nanozymes derived from MOF@CFO: The B-doped MOF@CFO nanocomposite was placed in a tube furnace and heated to 900°C under argon at 5°C / min for carbonization for 3 hours. 50 mg of the resulting product was dissolved in 10 mL of methanol, and 32 mg of bis(4-hydroxyphenyl)sulfone and 12 mg of triphosphazene chloride were added sequentially. 0.1 mL of triethylamine was then added and the reaction was stirred at room temperature for 12 h. The resulting product was centrifuged at 8000 rpm for 5 min, washed three times with methanol, and dried in vacuo at 65°C for 12 h. The resulting product was placed in a tube furnace and heat-treated at 900°C for 3 h at a heating rate of 5°C / min under an argon atmosphere. 5.2 mg of nickel nitrate, 8.7 mg of trichlorotris(ethylenediamine)cobalt, and 54 mg of the above-mentioned product were dissolved in 25 mL of methanol, ultrasonically dispersed for 5 min, and magnetically stirred at room temperature for 12 h. The resulting product was centrifuged at 8000 rpm for 5 min, washed three times with methanol, and dried at 65°C for 12 h. It was then placed in a tube furnace and heat-treated at 900°C for 3 h at a heating rate of 5°C / min under an argon atmosphere, ultimately obtaining a metal-organic framework@covalent organic framework-derived B, S, and P co-doped Co and Ni double single-atom nanozyme. (2) Preparation of metal-organic framework @ covalent organic framework-derived B, S, P co-doped Co, Ni diatomic nanozyme-hapten complex: ① Preparation of fluoropyrrolidone hapten: In a light-proof reactor, 100 mg of fluoropyrrolidone was dissolved in 1 mL of N,N'-dimethylformamide, and 30 mg of sodium hydroxide was added as a catalyst to form a preliminary reaction system. 0.3 mmol of 3-mercaptopropionic acid was dissolved in 1 mL of N,N'-dimethylformamide and added dropwise to the reaction system under stirring. After nucleophilic substitution reaction was carried out in an oil bath at 100°C for 2 h, 5 mL of hydrochloric acid solution (0.01 M) was added for protonation. The product was extracted with chloroform, dried and concentrated to obtain a viscous liquid. After recrystallization, crystals were obtained, namely the carboxylated fluoropyrrolidone hapten. ② Preparation of nanozyme-hapten complex using active ester method: 10 mg of metal organic framework @ covalent organic framework derived B, S, P co-doped Co, Ni double single atom nanozyme was dissolved in 1 mL of deionized water, ultrasonicated for 5 minutes to disperse it evenly, 1 mL of 3% polyethyleneimine was added, and slowly stirred for 2 hours. The resulting product was centrifuged at 8000 rpm for 5 minutes, washed three times with deionized water, and dried at 65 ° C for 8 hours to obtain amino nanozyme. 10 mg of carboxylated fluoropyranone hapten was dissolved in 1 mL of methanol, 12 mg of N,N'-dicyclohexylcarbodiimide and 15 mg of N-hydroxysuccinimide were slowly added, and the reaction was carried out in an ice bath for 60 minutes. Then 1 mg of amino nanozyme was added and the reaction was carried out in an ice bath for 12 hours to obtain nanozyme-hapten complex; (3) Preparation of nanozyme-hapten complex / fluoropyrrolidone / antibody / thin film gold electrode: Thin film gold electrode was prepared by magnetron sputtering coating technology. The reference electrode was coated with silver / silver chloride paste by screen printing technology. 5 μL of anti-fluoropyrrolidone antibody was drop-coated on the surface of the working electrode using a micropipette and incubated at 4°C for 12 h. Nonspecific sites were then blocked with bovine serum albumin (0.25%). After incubation at room temperature for 2 h, the electrode was washed three times with distilled water. 10 μL of a mixture containing different concentrations of fluoropyrrolidone and 5 μL of nanozyme-hapten complex was pipetted onto the surface of the gold electrode and incubated at 37°C for 2 h. The mixture was then washed three times with distilled water to remove unbound nanozyme competitors. The electrode was then immersed in a detection solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine. The change in the catalytic signal generated by the competition between the target fluoropyraclostrobin and the nanozyme-hapten complex for binding to the antibody fixed on the electrode surface was used to reflect the content of the target fluoropyraclostrobin, thereby obtaining a signal-reducing electrochemical immunosensor for fluoropyraclostrobin detection.

[0015] The prepared electrochemical sensor has high accuracy and wide linear range (1×10-1 ~1×10 4 ng / mL), with a low detection limit (2.7×10 -2 ng / mL). At the same time, the detection results of actual samples (spinach) show that the prepared sensor has very good practical application value.

[0016] The above examples are intended to illustrate the present invention, not to limit the present invention. Numerous improvements and modifications may be made to the present invention based on the above description. Within the scope of the appended claims, the present invention may have other implementations different from those described above, and methods such as selecting other reagent materials and adjusting the incubation time are all within the scope of the present invention.

Claims

1. Ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and its preparation method, characterized in that: The following steps are involved: (1) Preparation of ternary solid non-metal co-doped diatomic nanozymes derived from multi-level composite structures: ① Preparation of multi-level composite structure nanomaterials: Under inert atmosphere, weigh 0.7-1.5 g of a reducing agent containing a solid non-metallic element and 5.0-8.0 g of an organic ligand A, mix them evenly in a closed reactor, and heat them to 200-400 °C for pyrolysis reaction for 2-4 h. The obtained product is centrifuged at 8000-10000 rpm for 5-20 min, washed 2-5 times with an organic solvent, and dried at 50-80 °C for 12-24 h. Then, 1.0-3.5 g of a transition metal salt solution, 0.5-1.5 g of an organic ligand B, and 0.1-0.3 g of the above-obtained product are dispersed in 25-45 mL of an organic solvent, ultrasonically treated for 10-30 min, and then subjected to coordination reaction at 70-150 °C for 2-4 h under continuous stirring. The resulting product was centrifuged at 8,000–10,000 rpm for 5–20 minutes, washed 2–5 times with an organic solvent, and dried at 50–80°C to obtain the base framework material. 50–150 mg of organic ligand C and 100–200 mg of organic ligand D were dissolved in 100–150 mL of organic solvent, respectively, to form two precursor solutions. The base framework material and the two precursor solutions were mixed, ultrasonically dispersed for 20–40 minutes, and stirred for 12–24 hours. The resulting hierarchical composite nanostructured material was centrifuged at 8,000–10,000 rpm for 5–20 minutes, washed 2–5 times with an organic solvent, and dried at 50–80°C for 12–24 hours. ② Preparation of ternary solid-state non-metallic co-doped diatomic nanozymes derived from a hierarchical composite structure: The hierarchical composite nanomaterial was placed in a tube furnace and heated at a rate of 2–10°C / min to 800–1000°C under an inert gas atmosphere for 1–3 h to complete the primary carbonization treatment. 50–200 mg of the carbonized product was dispersed in 5–20 mL of organic solvent and sonicated for 5–20 min to form a homogeneous dispersion. 20–80 mg of the two solid non-metallic organic compounds were dissolved in 10–50 mL of organic solvent and slowly added dropwise to the reaction system. 0.1–0.5 mL of stabilizer was then added, and the reaction was stirred at 20–40°C for 12–24 h. The resulting product was centrifuged at 8000–10000 rpm for 5–20 min, washed 2–5 times with organic solvent, and dried under vacuum at 50–80°C for 12–24 h before undergoing a secondary carbonization treatment. Disperse 5-20 mg of the two transition metal salt solutions and 50-200 mg of the secondary carbonization product in 20-50 mL of an organic solvent. After ultrasonic dispersion for 5-10 min, stir and react at 20-40°C for 12-24 h.The resulting product was centrifuged at 8,000-10,000 rpm for 5-20 minutes, washed 2-5 times with an organic solvent, and dried at 50-80°C for 12-24 hours. It was then placed in a tube furnace and heat-treated at a rate of 2-10°C / min to 850-1200°C for 1-3 hours under an inert gas atmosphere, ultimately yielding a ternary solid-state non-metallic co-doped diatomic nanozyme derived from a hierarchical composite structure. (2) Preparation of nanozyme-hapten complex: ① Preparation of fluoropyrafuranone hapten: In a light-proof reactor, 80-120 mg of fluoropyrafuranone was dissolved in 0.5-2 mL of organic solvent, and 30-50 mg of alkali metal hydroxide was added as a catalyst to form a preliminary reaction system. 0.3-0.5 mmol of carboxylic acid monomer containing a thiol functional group was dissolved in 0.5-2 mL of organic solvent, and then added dropwise to the above reaction system. The nucleophilic substitution reaction was carried out in an oil bath at 80-120 °C for 1-3 h. 3-8 mL of a protonating agent (0.005-0.02 mol / L) was added to terminate the reaction. The product was extracted with an organic solvent, dried, and concentrated to obtain a viscous liquid. After recrystallization, crystals were obtained, namely the carboxylated fluoropyranone hapten; ② The nanozyme-hapten complex was prepared using the active ester method: 10-30 mg of the prepared multi-level composite structure ternary solid non-metal co-doped diatomic nanozyme was dispersed in 10-30 mL of deionized water, ultrasonicated for 5-15 minutes to make it uniformly dispersed, and then 1-3 mL of amination reagent (3%) was added and slowly stirred for 1-2 hours. The resulting product was centrifuged at 8000-10000 rpm for 5-20 minutes, washed with deionized water 2-5 times, and dried at 50-80°C for 8-12 hours to obtain the amination nanozyme. 10-25 mg of the fluoropyranone hapten was dissolved in 1.0-2.5 mL of organic solvent, 5-25 mg of organic coupling reagent was slowly added, and pre-activated under ice bath conditions for 30-60 minutes. Then 1-2 mg of the amination nanozyme was added to the pre-activated system and reacted in an ice bath for 8-12 hours to finally obtain the nanozyme-hapten complex; (3) Preparation of electrochemical immunosensor: Thin-film gold electrodes were prepared using magnetron sputtering technology. The reference electrode was coated with silver / silver chloride paste using screen printing technology. 3-8 μL of anti-fluoropyrrolidone antibody was drop-coated on the surface of the working electrode using a micropipette and incubated at 4°C for 8-12 h. Nonspecific sites were then blocked using a blocking agent. After incubation at room temperature for 1-3 h, the electrode was washed three times with distilled water. 5-10 μL of a mixture containing different concentrations of fluoropyrrolidone and nanozyme-hapten complex was pipetted onto the surface of the gold electrode and incubated at 30-60°C for 1-3 h. The mixture was then washed three times with distilled water to remove unbound nanozyme-hapten competitors. The electrode is then immersed in a detection solution containing hydrogen peroxide and a chromogenic substrate. The target fluoropyraclostrobin competes with the nanozyme-hapten complex for binding to the antibody fixed on the electrode surface. The change in the signal generated by the ternary solid-state non-metal co-doped diatomic nanozyme in the nanozyme-hapten complex catalyzing hydrogen peroxide and the chromogenic substrate is used to reflect the content of the target fluoropyraclostrobin, thereby obtaining a signal-reducing electrochemical immunosensor for fluoropyraclostrobin detection.

2. The ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and its preparation method according to claim 1, characterized in that: In step (1), the solid non-metallic reducing agent is one or more of sodium borohydride, sodium thiosulfate, and sodium hypophosphite; the organic ligand A and the organic ligand B are one or more of 2-methylimidazole, 2,2'-bipyridine, and 4,4'-benzidine; the inert gas is one or more of argon, nitrogen, and helium; the organic solvent is one or more of methanol, ethanol, acetone, N,N'-dimethylformamide, and tetrahydrofuran; the transition metal salt is one or more of zinc nitrate, nickel nitrate, iron phthalocyanine, and trichlorotris(ethylenediamine)cobalt; The organic ligand C is one or more of 2,4,6-triamino-1,3,5-triazine, melamine, and cyanuric chloride; the organic ligand D is one or more of [2,2'-bipyridine]-5,5'-diamine, 6,6'-dinitro-[2,2'-bipyridine]-5,5'-diamine, and 2,6-pyridinedicarboxylic acid; the solid non-metallic organic matter is one or more of bis(4-hydroxyphenyl)sulfone, tripolyphosphazene chloride, and sodium tetraphenylborate; and the stabilizer is one or more of triethylamine, polyvinylpyrrolidone, and sodium dodecyl sulfate.

3. The ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and its preparation method according to claim 1, characterized in that: In step (2), the organic solvent is one or more of dimethyl sulfoxide, N,N'-dimethylformamide, methanol, ethanol, and acetone; the alkali metal hydroxide is one or more of potassium hydroxide, sodium hydroxide, and lithium hydroxide; the mercaptocarboxylic acid monomer is one or more of 3-mercaptopropionic acid, 2-mercaptoacetic acid, and 4-mercaptobenzoic acid; the acidic solution is one or more of hydrochloric acid, sulfuric acid, and acetic acid; the amination reagent is one or more of polyethyleneimine, 3-aminopropyltriethoxysilane, and polylysine; and the organic coupling reagent is one or more of N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

4. The ternary solid-state non-metal co-doped diatomic nanoenzyme-based fluoropyranone electrochemical sensor and its preparation method according to claim 1, characterized in that: In step (3), the blocking agent is one or more of bovine serum albumin, casein, and polyethylene glycol; and the chromogenic substrate is one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

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