Preparation method of novel electrochemical enzyme sensor for profenofos pesticide detection based on enzyme inhibition action principle

Abstract

The invention relates to the technical field of preparation of electrochemical sensors, and particularly discloses a preparation method of a novel electrochemical enzyme sensor for profenofos pesticide detection based on an enzyme inhibition action principle, and the preparation method comprises the following steps: S1, treating a glassy carbon electrode to obtain a bare glassy carbon electrode GCE; s2, obtaining a composite material modified electrode Au50Ag2 (at) G / GCE; s3, an enzyme-chitosan composite modified electrode KbE-CS / Au50Ag2 (at) G / GCE is obtained; and S4, finally obtaining the profenofos electrochemical enzyme sensor NF / KbE-CS / Au50Ag2 (at) G / GCE based on the enzyme inhibition effect. According to the present invention, the white kidney bean esterase derived from plants is adopted as the detection enzyme source, and the electro-catalysis amplification effect of the Au50Ag2 (at) G composite material is adopted to construct the novel electrochemical enzyme sensor, and the method is the profenofos pesticide detection technology based on the enzyme inhibition effect principle;

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical sensor preparation, and particularly relates to a method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the principle of enzyme inhibition. Background Art

[0002] Pesticides have long played a significant role in protecting forests, agriculture, animal husbandry, and fisheries. However, with the increasing use of pesticides, problems have gradually arisen, such as abuse, misuse, and residues. Pesticide residues are unavoidable on crops consumed daily, posing a potential threat to public health. Therefore, pesticide testing has become a crucial tool for ensuring agricultural product safety, ecological and environmental safety, and public health. Organophosphorus pesticides (OPPs), known for their high toxicity, effectively kill pests in a short period of time, making them highly effective in controlling crop pests and diseases. Furthermore, their enhanced biological activity can improve crop yield and quality. However, less than 1% of applied OPPs enters the bodies of pests. The remaining OPPs or their metabolites in the environmental medium can be transferred to organisms through various pathways. Exposure to OPPs, in particular, can cause adverse effects on humans, such as neurotoxicity, teratogenicity, endocrine regulation, and immunotoxicity, leading to negative impacts on human health. Although other large-scale instrumental analyses in the laboratory can provide accuracy and high sensitivity, sample preparation is tedious, the procedures are complex, and a certain knowledge base and experience in operating the instrument are required.

[0003] In the design of electrochemical sensors, nanomaterials are an important component of sensor construction because of their unique physical and chemical properties, good conductivity, large specific surface area and stability. Among them, clusters are used in signal amplification and the construction of sensitive interfaces due to their excellent catalytic properties and biocompatibility. In particular, when they are fixed on graphene (G) two-dimensional nanomaterials, the composite materials formed, such as Au 50 Ag2@G and Au 25 @G can not only significantly enhance the conductivity of the electrode, but also provide a stable adsorption platform for biomolecules, further improving the performance of the sensor.

[0004] It is based on the above background that we focus on developing Au-based 50 The electrochemical esterase sensor based on Ag2@G composite material can be used to determine bromophos with high sensitivity and selectivity. 50The Ag2@G composite material provides an ideal attachment environment for the loading of plant esterases. The plant esterase (KbE-CS) catalyzes the hydrolysis of the substrate 1-naphthyl acetate (1-NA) to produce the electrochemically active substance 1-naphthol, which then undergoes electrochemical oxidation to generate an electrical signal. In the presence of the pesticide profenofos, KbE enzyme activity is inhibited, resulting in a decrease in the amount of 1-naphthol produced and a corresponding decrease in the electrical signal, enabling accurate and low-cost detection of the organophosphorus pesticide profenofos. This sensor design aims to overcome the shortcomings of traditional methods and provide a more convenient and efficient detection method. It is expected to play an important role in practical applications, such as agricultural product processing sites and environmental monitoring stations, providing strong technical support for rapid screening of pesticide residues and environmental monitoring. By optimizing the sensor structure and testing conditions, it is expected to further improve the detection limit, reduce cross-interference, and ensure the accuracy and reliability of test results, thereby better serving food safety management and environmental protection.

[0005] To address this issue, the inventors proposed a novel electrochemical enzyme sensor preparation method for detecting profenofos pesticide based on the principle of enzyme inhibition to solve the above problems. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the principle of enzyme inhibition, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A novel electrochemical enzyme sensor preparation method for detecting profenofos pesticide based on the principle of enzyme inhibition includes:

[0009] The following steps are involved:

[0010] S1. Polish the glassy carbon electrode with 0.05 μm Al2O3 powder, clean it with ultrapure water, and test it in potassium ferrocyanide (K3[Fe(CN)5]–K4[Fe(CN)5]) electrolyte solution by cyclic voltammetry until the redox peak potential difference ΔEp ≤ 100 mV to obtain a bare glassy carbon electrode (GCE) with a clean surface and qualified electrochemical performance.

[0011] S2, 5 μL Au 50 The Ag2@G composite material suspension was added dropwise to the surface of the pretreated bare glassy carbon electrode and dried at low temperature to form a conductive enhancement layer on the electrode surface to obtain a composite modified electrode Au composed of bimetallic clusters and multilayer graphene. 50 Ag2@G / GCE;

[0012] S3, 10 μL of plant esterase-chitosan KbE-CS solution was added to the surface of the composite material modified electrode, and dried at low temperature to obtain the enzyme-chitosan composite modified electrode KbE-CS / Au. 50 Ag2@G / GCE;

[0013] S4, 4 μL of 0.5% Nafion solution was added to the surface of the enzyme-chitosan composite modified electrode and dried at low temperature to form a selective permeable membrane to prevent enzyme leakage, and finally the electrochemical enzyme sensor NF / KbE-CS / Au based on enzyme inhibition was obtained. 50 Ag2@G / GCE.

[0014] Preferably, the preparation method of the potassium ferricyanide (K3[Fe(CN)5]–K4[Fe(CN)5]) electrolyte solution is:

[0015] Accurately weigh 0.1646 g of K3[Fe(CN)6] and 0.2112 g of K4[Fe(CN)6] on an analytical balance and place them in a clean beaker. Add an appropriate amount of pH 7.0 PBS buffer solution to dissolve them completely. Rinse the beaker and glass rod with pH 7.0 PBS solution 1 to 3 times. Then transfer the solution to a 100 mL volumetric flask, adjust the volume, and store in a dark place.

[0016] Preferably, the Au 50 The preparation method of Ag2@G composite cluster solution is as follows:

[0017] 50 mg Au 25 Dissolve in 20 mL of acetonitrile solution and centrifuge, then take the supernatant to a 50 mL clean, dry flask, weigh 2.3 mg of AgNO3 and dissolve it in 1 mL of methanol solution, then slowly drop the methanol solution into the flask until the color of the solution turns yellow-green, then stop dropping, then stir for 30 minutes, and wait for the reaction to end; wash the product with acetonitrile 2-3 times to obtain Au 50 Ag2(PET) 18 clusters.

[0018] Then, 4 mg of multilayer graphene was accurately weighed using an analytical balance and added to a 10 mL centrifuge tube. 4 mL of N, N-dimethylformamide was then added. The centrifuge tube was ultrasonicated for two hours until the multilayer graphene was evenly dispersed. 1 mL of Au was then added. 50 Ag2(PET) 18 The N, N-dimethylformamide solution (1 mg / mL) of the clusters was added to a centrifuge tube and ultrasonicated for 5 minutes to obtain Au 50 Ag2@G cluster solution.

[0019] Preferably, the preparation method of the KbE-CS solution is:

[0020] Accurately weigh 1 mg of chitosan using an analytical balance, add 5 mL of the prepared KbE crude enzyme solution to dissolve it, and shake well to obtain a 0.2 g / L KbE-CS solution.

[0021] Preferably, the preparation method of the Nafion solution is:

[0022] Use a pipette to accurately draw 10 μL of 5% Nafion solution into a 2 mL centrifuge tube, then add 90 μL of anhydrous ethanol solution to dilute and shake well to obtain a 0.5% Nafion solution.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses white kidney bean esterase from plants as the detection enzyme source, and uses Au 50 The synergistic amplification effect of Ag2@G composite material was used to construct NF / KbE-CS / Au 50 The new electrochemical enzyme sensor Ag2@G / GCE is a technology for detecting profenofos pesticide based on the principle of enzyme inhibition. Cyclic voltammetry was performed in K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution. Calculations show that: Au 50 Ag2@G has a large specific surface area, which can provide a good attachment environment for the loading of clusters and plant esterase. 50 Ag2@G has good electrical conductivity and synergistic electrocatalytic effect, which can effectively promote the transfer of electrons, thus improving the response sensitivity of the sensor. The optimal conditions were obtained by optimizing the experimental conditions for a certain factor through square wave voltammetry: pH of PBS buffer solution = 7.0, Au 50 The loading amount of Ag2@G was 5 μL, and the inhibition time of profenofos was 15 min. Under the optimal experimental conditions, the inhibition rate showed a good linear relationship with the logarithm of the profenofos concentration, and the linear equation was Y=20.7637lgc-20.3616, with a correlation coefficient R 2 =0.9382, the detection limit was 0.094 μg / L (R SN =3).

[0025] (2) NF / KbE-CS / Au of the present invention 50 The Ag2@G / GCE electrochemical biosensor has good stability, repeatability and anti-interference. It has a wide linear range and a low detection limit. No profenofos was detected in the actual samples of lettuce, leek and purple cabbage, indicating that the constructed electrochemical biosensor has certain practicality. 50Preparation of Ag2 nanoclusters, and UV-visible spectrophotometry of Au 50 The Ag2 sample was subjected to UV absorption spectrum analysis. 50 Ag 2的 The characteristic absorption peaks appear at 400nm, 458nm, and 695nm, respectively, and the characteristic peaks are consistent with the reported Au 50 The characteristic ultraviolet absorption peaks of Ag2 nanoclusters correspond one to one.

[0026] (3) The present invention calculates the number of transferred electrons n=1 through the cyclic voltammogram; there is a good linear relationship between the oxidation peak potential Epa and the solution pH, and the linear equation is Epa=2.0248-0.06477pH, R 2 =0.98015, the ratio of the number of protons participating in the reaction to the number of transferred electrons in the oxidation process of 1-NA is 1:1, so the oxidation process of this electrode is an irreversible isoelectronic and isoprotic reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The Au of the present invention 50 Ag2(PET) 36 Cluster structure diagram; (orange is Au atom, green is Ag atom, purple is S atom, gray is C atom, H atom is omitted);

[0028] Figure 2 The Au of the present invention 50 Ag2(PET) 36 UV-visible spectra of clusters;

[0029] Figure 3 The electrode morphology of different electrodes of the present invention;

[0030] Figure 4 The Au of the present invention 50 Energy spectrum analysis of Ag2@G;

[0031] Figure 5 Cyclic voltammograms of various electrodes of the present invention in supporting electrolyte solutions;

[0032] Figure 6 The AC impedance diagrams of various electrodes of the present invention in supporting electrolyte solutions;

[0033] Figure 7 Cyclic voltammograms of different electrodes of the present invention in PBS blank solution and PBS mixed solution containing 1-NA;

[0034] Figure 8 Cyclic voltammograms of different electrodes of the present invention in PBS blank solution and PBS mixed solution containing 1-NA;

[0035] Figure 9 The diagrams are the mechanism of enzyme catalysis (a) and 1-naphthol oxidation (b) of the present invention;

[0036] Figure 10 CV graphs of various electrodes of the present invention reacting in 1-NA solution;

[0037] Figure 11 NF / KbE-CS / Au of the present invention 50 Square wave voltammetry curves of Ag2@G / GCE under different conditions;

[0038] Figure 12 Graphs showing square wave voltammetric response of different modified electrodes of the present invention;

[0039] Figure 13 NF / KbE-CS / Au of the present invention 50 Square wave voltammograms of Ag2@G / GCE in PBS mixed solutions with different pH values;

[0040] Figure 14 is a line graph of different pH values and peak current I of the present invention;

[0041] Figure 15 NF / KbE-CS / Au of the present invention 50 Composite material Au in Ag2@G / GCE electrochemical biosensor 50 Square wave voltammograms with different Ag2@G loading amounts;

[0042] Figure 16 The different composite materials Au of the present invention 50 Line graph of Ag2@G loading and peak current I;

[0043] Figure 17 NF / KbE-CS / Au of the present invention 50 Square wave voltammograms of Ag2@G / GCE in profenofos solution with different inhibition times;

[0044] Figure 18 It is a broken line graph of different inhibition times and inhibition rates of profenofos of the present invention;

[0045] Figure 19 CV graphs of the electrochemical sensor of the present invention in PBS substrate solutions with different pH values;

[0046] Figure 20 The effect of pH value on the oxidation peak potential of 1-NA in the present invention;

[0047] Figure 21 SWV diagram after inhibition of different mass concentrations of profenofos according to the present invention;

[0048] Figure 22 It is a linear relationship diagram of different profenofos mass concentrations and inhibition rates of the present invention;

[0049] Figure 23 The SWV test plots were repeated for the same electrode of the present invention. DETAILED DESCRIPTION

[0050] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0051] Example 1:

[0052] First, rinse the abrasive cloth under the tap three times, then rinse it three times with ultrapure water. Pour an appropriate amount of abrasive powder (0.05μm Al2O3 powder) on the abrasive cloth, and then wet it with ultrapure water until the abrasive powder covers the surface of the abrasive cloth without accumulating. Next, hold the electrode perpendicular to the abrasive cloth and gently make circles in the shape of an "8" until the electrode surface is clean and smooth. Rinse the electrode surface with ultrapure water and wipe off excess water stains and Al2O3 powder on the outside of the electrode with filter paper. Use an ear bulb to blow dry the electrode surface and cover the electrode with a cap to prevent dust from falling.

[0053] Add 7mL of a 5mmol / L K3[Fe(CN)6]–K4[Fe(CN)6] electrolyte solution to a 20mL unsealed electrolytic cell and place the freshly ground bare electrode in it for cyclic voltammetry. Once the correct parameters are set, the test can officially begin. Only when ΔEp ≤ 100mV does the glassy carbon electrode have been successfully polished to the point where it can proceed to the next step. Otherwise, repeat the electrode pretreatment steps after gently circling the "8" until ΔEp ≤ 100mV before proceeding to the next step.

[0054] NF / KbE-CS / Au 50 Ag2@G / GCE electrochemical biosensor

[0055] First, the composite material was thoroughly vortexed on a vortex oscillator. Then, 5 μL of the composite cluster was dropped onto the pretreated bare glassy carbon electrode surface using a pipette and dried under an infrared lamp at low temperature. After the composite cluster was dried, 10 μL of KbE-CS was added and dried under an infrared lamp at low temperature. After the KbE-CS was dried, 4 μL of 0.5% Nafion solution was added onto the surface of the modified two-layer glassy carbon electrode using a pipette. The solution was then dried under an infrared lamp at low temperature. Finally, the NF / KbE-CS / Au composite was rinsed with ultrapure water, the water stains were wiped off with filter paper, and the composite was dried with an ear bulb. 50 Ag2@G / GCE electrochemical enzyme sensor

[0056] All experimental reagents and instruments used in the present invention are shown in Tables 1 and 2 below.

[0057] Table 1 shows the composition of the experimental reagents:

[0058]

[0059]

[0060] Ultrapure water was used to prepare the experimental solutions.

[0061] The experimental instruments used in the present invention are shown in Table 2:

[0062] Table 2 Experimental instruments

[0063] instrument model factory Electrochemical workstation CHI660e Shanghai Chenhua Instrument Co., Ltd. Ultrasonic cleaning machine F-009SD Kunshan Ultrasonic Instrument Co., Ltd. Field emission scanning electron microscopy S-4800 Hefei Guoyi Quantum Technology Co., Ltd. centrifuge TG16-WS Shenzhen Bailai Co., Ltd. analytical balance JC-TP1204 Jinan Bokun Scientific Instrument Co., Ltd. Vortex mixer SI-T246 Hangzhou Ruicheng Instrument Co., Ltd. Written test acidity meter pH-20B Guangzhou Beize Instrument Technology Co., Ltd. Magnetic stirrer DF-101S Jinan Bohang Biotechnology Co., Ltd. grinding mill 304 stainless steel Jinhua Murphy Household Appliances Co., Ltd. pipette 10-1000μL German Eppendorf single-channel pipette

[0064] Specifically, the preparation of the solution

[0065] Preparation of PBS buffer solutions with different pH values

[0066] Table 3 shows the mass of reagents required to prepare PBS buffer solutions of different pH values.

[0067]

[0068] Based on the data in Table 3, weigh the required solid sodium hydrogen phosphate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4·2H2O), and potassium chloride (KCl) for different pH values on an analytical balance. Dissolve these solids in ultrapure water in a beaker and transfer the resulting solution to a 100mL volumetric flask, bringing the volume to 100mL. This yields 100mL of 0.2mol / L PBS buffer solutions of varying pH values. After preparation, test the solutions with a pH meter.

[0069] Specifically, a 5 mmol / L balanced electrode solution of potassium ferrocyanide and potassium ferrocyanide was prepared;

[0070] Accurately weigh 0.1646 g of K3[Fe(CN)6] and 0.2112 g of K4[Fe(CN)6] on an analytical balance, place them in a clean beaker, and add an appropriate amount of pH = 7.0 PBS buffer solution to dissolve them completely. Rinse the beaker and glass rod with pH = 7.0 PBS solution 1 to 3 times, then transfer the solution to a 100 mL volumetric flask, make up to volume, and then refrigerate in the dark.

[0071] Specifically, Au 50 Ag2(PET) 18 Synthesis of clusters

[0072] 50 mg Au 25 Dissolve in 20 mL of acetonitrile and centrifuge. Transfer the supernatant to a clean, dry 50 mL flask. Weigh 2.3 mg of AgNO₃ and dissolve in 1 mL of methanol. Slowly add the methanol dropwise to the flask until the solution turns yellow-green. Stir for 30 minutes and allow the reaction to complete. Wash the product 2-3 times with acetonitrile.

[0073] Specifically, Au 50 Synthesis of Ag2@G composites

[0074] 4 mg of multilayer graphene was accurately weighed with an analytical balance and added to a 10 mL centrifuge tube. 4 mL of N, N-dimethylformamide was then added. The centrifuge tube was ultrasonicated for two hours until the multilayer graphene was evenly dispersed. 1 mL of Au was then added. 50 Ag2(PET) 18 The N, N-dimethylformamide solution (1 mg / mL) of the clusters was added to a centrifuge tube and ultrasonicated for 5 minutes to obtain Au. 50 Ag2@G cluster solution.

[0075] Specifically, prepare the crude enzyme solution of kidney bean esterase (KbE):

[0076] Grind a certain amount of white kidney beans in a grinder for 5 minutes and pass through a 100-mesh sieve. Then, add ultrapure water at a ratio of 1:5 (g / mL) and stir thoroughly in a magnetic stirrer for 30 minutes. Refrigerate at 4°C overnight. Centrifuge at 5000 rpm for 10 minutes. Remove the supernatant to obtain the crude KbE enzyme solution. Refrigerate and replace the crude KbE enzyme solution every three days.

[0077] Specifically, 0.2 g / L enzyme (KbE)-chitosan (CS) solution:

[0078] Accurately weigh 1 mg of chitosan using an analytical balance, add 5 mL of the prepared KbE crude enzyme solution to dissolve it, and shake well to obtain a 0.2 g / L KbE-CS solution.

[0079] Specifically, the preparation of 0.08 mol / L α-naphthyl acetate (1-NA):

[0080] Accurately weigh 0.0745 g of α-naphthyl acetate (1-NA) on an analytical balance and place it in a 10 mL centrifuge tube. Add 5 mL of anhydrous ethanol solution to dilute it. Then, use a pipette to accurately draw 50 μL of 0.08 mol / L α-naphthyl acetate (1-NA) solution, and then add 5 mL of pH = 7.0 PBS buffer solution to obtain a 0.8 mmol / L α-naphthyl acetate (1-NA) solution.

[0081] Specifically, the preparation of 0.5% Nafion solution:

[0082] Use a pipette to accurately draw 10 μL of 5% Nafion solution into a 2 mL centrifuge tube, then add 90 μL of anhydrous ethanol solution to dilute and shake well to obtain a 0.5% Nafion solution.

[0083] Specifically, the preparation of 1 mol / L hydrochloric acid solution:

[0084] Accurately measure 8.30 mL of concentrated hydrochloric acid and place it in a beaker. Add an appropriate amount of ultrapure water to make the total volume of the solution 100 mL. Oscillate and shake well.

[0085] Specifically, prepare different concentrations of profenofos solution

[0086] (1) Preparation of profenofos stock solution:

[0087] Use a pipette to accurately measure 3.37 μL of commercially available profenofos into a 10 mL centrifuge tube, and then use the same method to accurately measure 5 mL of anhydrous ethanol to mix it to obtain 1 g / L of profenofos. Then, dilute the 1 g / L profenofos with anhydrous ethanol step by step to 10 4 μg / L

[0088] (2) Preparation of different concentrations of profenofos solution:

[0089] Preparation of different concentrations of bromophos solutions: using the stepwise dilution method. First, starting from a 1g / L bromophos solution, accurately pipette 1mL of the solution each time into a container containing 9mL of anhydrous ethanol. After thorough mixing, solutions of 100ug / L, 10ug / L, and 1ug / L are prepared. Then, to obtain a 200ug / L solution, take 1mL from the 1000ug / L solution, add 4mL of anhydrous ethanol thereto, and mix thoroughly. By taking 1mL of the 100ug / L solution and mixing it evenly with 1mL of anhydrous ethanol, a 50ug / L bromophos solution is generated. Finally, in order to obtain a low-concentration solution of 5ug / L, 1mL of the 50ug / L solution is combined with 9mL of anhydrous ethanol to complete the entire concentration sequence. The entire configuration process emphasizes precise measurement in each step and thorough shaking after mixing. As shown in Table 4 below:

[0090] Table 4 Preparation of different concentrations of profenofos

[0091]

[0092] Example 2:

[0093] refer to Figure 1 As shown, Au 50 Ag2(PET) 36 Cluster structure analysis

[0094] Gold nanoclusters have excellent catalytic and luminescent properties, and their uniform size and precise structure are conducive to interpreting structure-activity relationships. 50 Ag2(PET) 36 The cluster is composed of two Au 25 (PET) 18 The unit is formed by bridging two Ag atoms, Au 50 Ag2(PET) 36 The structure of the cluster is similar to that of two Au 25 The clusters are connected hand in hand.

[0095] Example 3:

[0096] Au 50 Ag2(PET) 36 UV-visible spectrum of the cluster

[0097] In order to determine the Au 50 Preparation of Ag2 nanoclusters, and UV-visible spectrophotometry of Au 50 The Ag2 sample was subjected to UV absorption spectrum analysis, such as Figure 2 It can be found that the characteristic absorption peaks of the sample appear at 400nm, 458nm, and 695nm respectively. The characteristic peak positions are consistent with the reported Au 50The UV absorption characteristic peaks of Ag2 nanoclusters completely correspond to those of Au 50 Ag2 nanoclusters were successfully prepared.

[0098] Example 4:

[0099] Electrode morphology characterization and elemental analysis of different modified electrodes

[0100] The surface of different modified electrodes was analyzed using scanning electron microscopy. Figure 3 As shown in the figure, the bare glassy carbon electrode exhibits a uniform and smooth surface (a). However, after the electrode surface is modified with graphene, the layered graphene is clearly visible, along with a small number of small particles scattered across the surface (b). After modification with the composite material, the clusters within the composite material make the multilayer graphene more evenly dispersed, but since the clusters are less than 1nm in diameter, the majority of the visible multilayer graphene fragments are fragments (c). Finally, after the composite clusters are modified layer by layer on the electrode surface, followed by KbE-CS and 0.5% Nafion, a star-like, evenly dispersed patch of dots forms on the electrode surface (d).

[0101] The elements of the composite material were analyzed using EDS spectrum, and the results are as follows: Figure 4 , Au 50 C, O, Au and Ag elements coexist in Ag2@G composite materials, and the content of Au is significantly greater than that of Ag, indicating that Au 50 Ag2 clusters are uniformly dispersed in graphene, further confirming that Au 50 Ag2@G / GCE electrode was successfully prepared.

[0102] Embodiment 5:

[0103] Electrochemical behavior of the sensor

[0104] Electrochemical behavior of different electrodes in supporting electrolyte solutions

[0105] In order to characterize the construction process of electrochemical enzyme sensors, bare GCE, G / GCE, Au 50 Ag2@G / GCE、KbE-CS / Au 50 Ag2@G / GCE、NF / KbE-CS / Au 50 Ag2@G / GCE, these electrodes were placed in K3[Fe(CN)6] / K4[Fe(CN)6] (containing 0.1mol / L KCl) solution and cyclic voltammetry (CV) was performed ( Figure 5 In the range of -0.2V to 0.8V, all curves show a pair of reversible redox peaks. At this time, the reaction process in the electrolytic cell is [Fe(CN)6] 3－ +e－ =[Fe(CN)6] 4－ The results show that the peak value of the bare electrode (black curve) is the largest, while the Au-modified 50 After Ag2@G (pink curve), the oxidation peak current value showed a significant decrease compared to the bare electrode (black curve), and the difference in peak potential increased significantly, indicating that the irreversibility of the electrode reaction increased. After KbE-CS (green curve) was modified on it, the response current showed a slight decrease. Because enzymes are proteins, their conductivity is poor, thus hindering the transfer of electrons. After the final modification with 0.5% Nafion (yellow curve), the response current dropped significantly. This effect is because the Nafion film is negatively charged, which limits the [Fe(CN)6] 3－ / 4－ Diffusion of ions to the electrode surface

[0106] The electrochemical characteristics of the modified electrode were studied under the same conditions. Figure 6 As shown in the figure, the diameter of the semicircle in the high-frequency region represents the size of the charge transfer resistance of the electrode. A smaller radius reflects a smaller charge transfer resistance, while a larger radius indicates a smaller charge transfer resistance. From this, it can be concluded that the size of the semicircle diameter is proportional to the charge transfer resistance of the electrode. The smaller the radius, the smaller the charge transfer resistance, and the larger the radius, the larger the charge transfer resistance. It can be seen from the figure that the radius of the semicircle is: black curve < red curve < green curve < pink curve < blue curve < dark blue curve, and the corresponding resistance values Ret are 38.50Ω, 486.8Ω, 3155Ω, 3474Ω, 1.0581×10 4 Ω, 1.875×10 4 Ω. It can be seen that the semicircle diameter of the bare electrode is smaller, that is, the charge transfer resistance is the smallest. After modification with KbE-CS and 0.5% Nafion, it can be seen that the radius of the semicircle increases significantly, indicating that NF / KbE-CS / Au 50 The charge transfer resistance of Ag2@G / GCE electrode (dark blue curve) increases, and the electron transfer ability is the worst. Figure 6 The AC impedance curve and Figure 5 The cyclic voltammetry curves of NF / KbE-CS / Au showed the same results. 50 Ag2@G / GCE electrochemical biosensor has been successfully constructed.

[0107] Electrochemical reaction of 1-NA on enzyme-catalyzed modified electrode

[0108] The electrochemical reaction of enzyme catalysis to 1-NA was studied by sensing electrode. 0.8mmol / L 1-NA and 0.2mol / L PBS with pH=7.0 were used as the base solution. 50Ag2@G / GCE modified electrode was subjected to cyclic voltammetry scanning to obtain the red curve. Then, the constructed NF / KbE / Au was used in PBS solution containing 0.8mmol / L 1-NA, pH=7.00.2mol / L and PBS solution without 0.8mmol / L 1-NA, pH=7.00.2mol / L, respectively. 50 Ag2@G / GCE modified electrode was used to measure cyclic voltammetry, and the green and blue curves were obtained as follows: Figure 7 Finally, NF / KbE-CS / Au was used in PBS solution containing 0.8mmol / L 1-NA, pH=7.00.2mol / L and PBS solution without 0.8mmol / L 1-NA, pH=7.00.2mol / L. 50 Ag2@G / GCE modified electrode was used to measure cyclic voltammetry, and the red and blue curves were obtained. Figure 7 .

[0109] The catalytic effect of KbE on 1-NA was studied by cyclic voltammetry. Figure 8 The electrode modified with KbE showed no peak in the solution without 1-NA as substrate (blue curve). Figure 7 After adding 1-NA, a clear oxidation peak (green curve) appears near the voltage of 0.5 V. 1-NA in the substrate is hydrolyzed by the enzyme to produce an electrochemically active substance (1-naphthol), which is then electrochemically oxidized, so a corresponding oxidation peak will appear in the electrochemical workstation. The reaction process is as follows: Figure 9 As shown. Figure 7 The peak in the first half of the red curve is the redox peak of water, which further proves that the oxidation peaks in the red and green curves are caused by KbE catalyzing 1-NA. The above phenomenon shows that KbE has been successfully modified on the electrode and the enzyme activity has been maintained. Figure 7 Green curve and upper Figure 8 The red curve shows that the oxidation peak of the red curve is higher, indicating that the addition of CS to the modified electrode can increase the current response and has a certain antibacterial effect, which can protect the enzyme and prevent it from being contaminated by bacteria.

[0110] Electrochemical behavior of different enzyme electrodes towards 1-NA

[0111] The electrochemical responses of four different modified material electrodes at the same concentration of 1-NA were investigated ( Figure 10 NF / KbE-CS / GCE, NF / KbE-CS / G / GCE, NF / KbE-CS / Au were used in a 0.2 mol / L, pH 7.0 PBS solution containing 1-NA as substrate. 50 Ag2 / GCE、NF / KbE-CS / Au50 Ag2@G / GCE modified electrode was measured for cyclic voltammetry, and red, blue, purple and green curves were obtained. The results showed that when only KbE-CS and CS were modified on the electrode, there was basically no peak (red and blue curves), and when Au was modified 50 After Ag2, oxidation peak (purple curve) begins to appear. 50 After Ag2@G, the oxidation peak current increased significantly (green curve). This is speculated to be due to the 50 Ag2 and multilayer graphene have a synergistic electrocatalytic effect, which can effectively promote electron transfer, thereby enhancing the sensitization effect on the biocatalytic electrochemical response of KbE.

[0112] Profenofos inhibition of enzymes

[0113] The effect of profenofos on the biocatalytic activity of KbE on the modified electrode was discussed. Figure 11 It can be seen that the electrode modified with KbE-CS did not show an oxidation peak in a blank PBS solution without the substrate 1-NA (blue curve). After an appropriate amount of 1-NA was added to the substrate, a clear electrochemical oxidation peak appeared in the figure (green curve). When treated with 100 μg / L profenofos for a certain period of time, the response value of the oxidation peak current was observed to decrease significantly (red curve). Based on the above experimental phenomena, it can be concluded that profenofos can inhibit the activity of kidney bean esterase, thereby reducing the amount of 1-naphthol generated in the system, and ultimately resulting in a decrease in the 1-naphthol oxidation peak response current value.

[0114] Catalytic performance of electrochemical sensors with different electrodes

[0115] Depend on Figure 12 It can be seen that in the PBS solution containing 1-NA, NF / KbE-CS / Au 50 The Ag2@G / GCE electrochemical biosensor showed an obvious oxidation peak with the highest peak value. Compared with the NF / KbE-CS / GCE electrode, the NF / KbE-CS / G / GCE electrode and the NF / KbE-CS / Au 50 Ag2 / GCE electrode, NF / KbE-CS / Au 50 The oxidation peak current generated on the Ag2@G / GCE modified electrodes is the largest. 50 The Ag2@G composite material has good synergistic electrocatalytic effect and conductivity, which can effectively promote the transfer of electrons, enhance the oxidation current signal of 1-naphthol, and improve the response sensitivity of this sensor.

[0116] Example 6:

[0117] Optimization of experimental conditions

[0118] Effect of solution pH

[0119] At α-naphthyl acetate concentration of 0.8 mmol / L and Au 50 Under the condition of Ag2@G loading of 5μL, NF / KbE-CS / Au 50 The square wave voltammetric curves of Ag2@G / GCE electrochemical biosensor in PBS solutions with different pH values ( Figure 13 ), and the broken line between pH value and peak current (I) is obtained ( Figure 14 The experimental results show that as the pH value gradually increases, the oxidation peak current first increases and then decreases. At pH = 7.0, the oxidation peak current is the largest and has the highest peak shape. Therefore, PBS buffer solution with a pH of 7.0 was selected as the optimal condition for subsequent experiments.

[0120] Au 50 Effect of Ag2@G cluster loading

[0121] As shown in the figure, different amounts of NF / KbE-CS / Au were modified in the presence of 0.8 mmol / L 1-naphthyl acetate and PBS pH = 7.0. 50 The square wave voltammetric curve of Ag2@G / GCE electrochemical biosensor was detected in PBS solution with pH=7.0 ( Figure 15 ), get different Au 50 The line graph between the loading amount of Ag2@G and the peak current I ( Figure 16 ). As can be seen from the figure, Au 50 When the modification amount of Ag2@G increases from 4μL to 8μL, the oxidation peak current gradually increases, and when it increases to 5μL, the oxidation peak current decreases. It can be seen that when the modification amount is 5μL, the oxidation peak current is the largest, the peak shape is the highest, and the response is the best. Therefore, Au is selected 50 The loading amount of Ag2@G was 5 μL.

[0122] Embodiment seven:

[0123] Effect of profenofos inhibition time

[0124] At 0.8 mmol / L 1-naphthyl acetate concentration and Au 50 Under the conditions of Ag2@G loading amount 5μL, buffer solution pH=7.0, and profenofos concentration 100μg / L, the constructed NF / KbE-CS / Au 50The Ag2@G / GCE electrochemical biosensor was immersed in a 0.1 mol / L PBS buffer containing 1-NA and subjected to SWV scanning to obtain the current response value I0. The electrode was cleaned with a pH = 7.0 PBS solution, blown dry with an ear bulb, and then immersed in different concentrations of profenofos solution for a certain period of time. The electrode was cleaned with a pH = 7.0 PBS solution, blown dry with an ear bulb, and then immersed in a 0.1 mol / L PBS buffer containing 1-NA again. SWV scanning was performed to obtain the current response value I1, and the square wave voltammetry curve of different inhibition times in the same concentration of profenofos solution was obtained ( Figure 17 ), the line graph between profenofos inhibition time and inhibition rate ( Figure 18 As the inhibition time in the profenofos solution increases, it can be detected that the inhibition rate reaches the maximum at 15 minutes, indicating that the enzyme is basically inactivated at 15 minutes. Therefore, a profenofos inhibition time of 15 minutes is selected as the optimal condition.

[0125] The inhibition rate of profenofos on the enzyme was calculated according to the following formula:

[0126]

[0127] Where: I0 is the initial current value; I1 is the current value after inhibition by profenofos.

[0128] Embodiment 8:

[0129] Calculation of electrochemical parameters

[0130] Linear relationship between pH and peak potential

[0131] NF / KbE-CS / Au 50 Ag2@G / GCE was subjected to cyclic voltammetry scanning under optimal conditions in PBS buffer solutions with different pH values containing 1-NA, and the curves of the oxidation peak potential at different pH values were observed. The enzyme system of plant esterase is complex, and the enzyme activity is very sensitive to changes in pH value. The enzyme can maintain stability within a specific pH range, but once this range is exceeded, the structure of the enzyme protein will change, which is called denaturation. In extremely acidic or alkaline environments, the electrostatic bonds, hydrogen bonds, and other bonds within the enzyme molecules that maintain their natural structure will be destroyed, and the dissociation state of the enzyme molecules and the way they interact with the substrate will also change significantly. Plant esterases will quickly lose their activity under strong acid or strong alkaline conditions, and strong acid environments will destroy their activity more quickly. In order to gain a deeper understanding of the effect of pH on the activity of plant esterases, the relationship between the peak potential Epa of the oxidation peak and the pH value of the solution was studied in the pH range of 5.0 to 8.0. The experimental results show (such as Figure 19As shown in Figure 2, the oxidation peak potential Epa of 1-NA showed a slight downward trend with the increase of pH value. In the range of pH 5.0 to 8.0, there was a good linear correlation between Epa value and pH value (as shown in Figure 2). Figure 20 This provides an important basis for further understanding the behavior of plant esterases under different pH environments. The linear regression equation is: Epa = 0.86385-0.06477pH, where R 2 =0.98015, the slope is -64.77 mV / pH, which is close to 59 mV / pH, indicating that the ratio of the number of protons participating in the reaction to the number of transferred electrons in the oxidation process of 1-naphthol is 1:1. Therefore, the oxidation process of this electrode is an irreversible isoelectronic and isoprotic reaction.

[0132] Calculation of the number of transferred electrons

[0133] NF / KbE-CS / Au 50 Ag2@G / GCE electrochemical sensor was subjected to cyclic voltammetry scanning in PBS buffer solution containing 1-NA pH = 7.00.2mol / L under optimal conditions to obtain the peak potential value and half-wave potential value. According to the formula: |E P -E h |=47.7 / nα(mV), Ep and Eh are the peak potential and half-wave potential in the cyclic voltammogram respectively. Among them, the irreversible process α=0.5, and the number of transferred electrons n=0.95, which is approximately 1.

[0134] Embodiment 9:

[0135] Establishment of the standard curve of profenofos

[0136] NF / KbE-CS / Au 50 After Ag2@G / GCE was inhibited in profenofos solution with different mass concentrations for 15 minutes, it was then subjected to square wave voltammetry scanning in PBS buffer solution with pH = 7.0 and 0.2 mol / L containing 1-NA. A series of concentration SWV curves were superimposed. Figure 21 It shows that NF / KbE-CS / Au 50 The peak current value of the Ag2 / GCE sensor gradually decreased with the increase of the mass concentration of profenofos, and the inhibition of profenofos on the enzyme activity gradually increased. Figure 22 The results showed that in the range of 0.1 to 1000 μg / L, there was a good linear relationship between the mass concentration of profenofos (logC) and the inhibition rate (Y), and the linear equation was Y = 20.7637logC-20.3616, with a correlation coefficient R 2 =0.9382, the detection limit is 0.094μg / L. Therefore, NF / KbE-CS / Au 50The Ag2@G / GCE electrochemical sensor has better performance and can effectively detect different mass concentrations of bromophos.

[0137] Embodiment 10:

[0138] Repeatability, stability, and anti-interference

[0139] Use the same NF / KbE-CS / Au 50 After the Ag2@G / GCE modified electrode was inhibited in the profenofos solution for 15 min, the square wave voltammetry was continuously measured 5 times in the PBS mixed solution containing 1-NA pH = 7.0. The results are as follows Figure 23 As shown, the relative standard deviation RSD is 7.58%; then seven NF / KbE-CS / Au 50 After the Ag2@G / GCE modified electrode was inhibited in bromophos solution for 15 minutes, square wave voltammetry was measured in a 1-NA pH=7.0 PBS mixed solution. The relative standard deviation (RSD) was 6.67%. 50 Ag2@G / GCE has good reproducibility.

[0140] Take out a NF / KbE-CS / Au 50 The Ag2@G / GCE modified electrode was inhibited in a 100μg / L profenofos solution for 15 minutes, and then the peak current value was measured by square wave voltammetry in a 1-NA pH = 7.0 0.2mol / L PBS mixed solution. Then, it was sealed and stored in a refrigerator at 4°C. After one, three, and five days, the electrode was measured by square wave voltammetry in a 1-NA pH = 7.0 PBS mixed solution. The results are shown in the figure. It was found that the peak current value after three days and the peak current value after five days decreased very little. This proves that NF / KbE-CS / Au 50 Ag2@G / GCE has good stability.

[0141] The effects of common inorganic ions, organic matter and other pesticides that may appear in the sample on the detection were explored. 50 The Ag2@G / GCE modified electrode was incubated in a 1000 μg / L profenofos solution for 20 minutes. A square-wave pulse voltammetry scan was then performed in a 0.2 mol / L PBS buffer solution (pH 7.0) containing 0.8 mmol / L 1-NA to obtain the initial peak current value, I0. The electrode was then rinsed with a pH 7.0 PBS solution and dried with an ear bulb. The electrode was then placed in a profenofos solution containing interfering ions to examine its effect on detection.

[0142] The organic group chose sucrose, and the inorganic group chose Cu 2+ 、Fe3+ 、Zn 2+ 、SO4 2- 、NO3 - 、Cl - Plasma, heavy metal group selected Cd 2+ . Take 100mg / L Cu 2+ 、Fe 3+ 、Zn 2+ 、SO4 2- 、NO3 - and sucrose and 300 mg / L Cl - The samples were mixed with 100 μg / L profenofos and inhibited in the mixed solution for 15 min. Then, the peak current value I1 was obtained by square wave voltammetry scanning in a mixed solution of PBS (pH = 7.00 0.2 mol / L) containing 1-NA. After the addition of sucrose in the organic group, the response signal was 96.77% of the original value and the current change was 3.23% of the original peak current. The peak current value I1 of the inorganic group (Cu 2+ 、Fe 3+ 、Zn 2+ 、SO4 2- 、NO3 - 、Cl - ) After adding interfering ions, the response signals were 83.28%, 79.97%, 61.19%, 61.19%, 82.80%, and 79.97% of the original values, and the current changes were 16.72%, 20.03%, 38.81%, 38.81%, 17.20%, and 20.03% of the original peak currents, respectively. The results show that NF / KbE-CS / Au 50 Ag2@G / GCE electrochemical enzyme sensor has a certain anti-interference ability. 2+ ), the response signal was 89.38% of the original value, and the current change was 10.62% of the original peak current, indicating that heavy metals (Cd 2 + ) has a certain inhibitory effect on the enzyme, which will affect the accuracy of the test results.

[0143] Example 11:

[0144] NF / KbE-CS / Au 50 Detection of Profenofos in Real Samples by Ag2@G / GCE Electrochemical Biosensor

[0145] Evaluation of NF / KbE-CS / Au 50The practicality of the Ag2@G / GCE sensor in actual samples was used to detect the content of bromophos in millet, leek, purple cabbage and cabbage. The purple cabbage and cabbage samples (12.5 g, accurate to 0.0001 g) were accurately weighed and ground, and 50 mL of PBS (0.2 mol / L, pH = 7.0) solution was added and mixed evenly, and then centrifuged at 8000 r / min for 10 minutes. Thereafter, 1 mL of supernatant was extracted and diluted 20 times as purple cabbage juice and cabbage juice samples, respectively. For millet, 0.03 g (accurate to 0.0001 g) of rice flour was ground and then mixed with 100 mL of PBS (0.2 mol / L, pH 7.0). After centrifugation at 8000 r / min for 30 minutes, 0.2 mL of supernatant was aspirated and the millet sample was diluted with 20 mL of ultrapure water. Subsequently, under the optimized optimal conditions, the prepared NF / KbE-CS / Au 50 Ag2@G / GCE sensor was used to detect the content of bromophos in the above samples.

[0146] The modified electrode was subjected to square wave voltammetry scanning in a pH = 7.00.2 mol / L PBS buffer solution containing 0.8 mmol / L 1-NA to obtain I0, and then the NF / KbE-CS / Au 50 The Ag2@G / GCE modified electrode was inhibited for 15 minutes in PBS solutions containing purple cabbage, Chinese cabbage, and millet, respectively, and square wave voltammetry scanning was performed in pH 7.0 PBS buffer solution containing 1-NA to obtain I1. Calculation showed that no residual bromophos was detected in the actual sample.

[0147] From the above, we can see that the white kidney bean esterase from plants is used as the detection enzyme source, and the cluster Au 50 The synergistic electrocatalytic amplification of Ag2 and multilayer graphene was used to construct NF / KbE-CS / Au 50 The new electrochemical enzyme sensor Ag2@G / GCE is a technology for detecting the pesticide profenofos based on the principle of enzyme inhibition.

[0148] (1) Cyclic voltammetry was performed in K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution. It was calculated that: Au 50 Ag2@G composite material has a large effective area, which can provide a good attachment environment for the loading of clusters and plant esterase. 50 Ag2 composite materials and multilayer graphene have good conductivity and synergistic electrocatalytic effect, which can effectively promote the transfer of electrons, and they improve the response sensitivity of the sensor.

[0149] (2) The experimental conditions were optimized through single-factor experiments, and the optimal conditions were: pH of PBS buffer solution = 7.0, Au 50The loading amount of Ag2@G was 5 μL, and the inhibition time of profenofos was 15 min. Under the optimal experimental conditions, in the range of 0.1 to 1000 μg / L, the inhibition rate showed a good linear relationship with the logarithm of the profenofos concentration, and the linear equation was Y = 20.7637lgc-20.3616, with a correlation coefficient R 2 =0.9382, and the detection limit was 0.094 μg / L (RSN=3).

[0150] (3)NF / KbE-CS / Au 50 The Ag2@G / GCE electrochemical enzyme sensor exhibited excellent stability, reproducibility, and anti-interference properties. It also exhibited a wide linear range and a low detection limit. Profenofos was not detected in the actual samples of lettuce, leek, and purple cabbage, demonstrating the practicality of the constructed electrochemical biosensor.

[0151] (4) In order to determine the Au 50 Preparation of Ag2 nanoclusters, and UV-visible spectrophotometry of Au 50 The Ag2 sample was subjected to UV absorption spectrum analysis, and the experiment obtained Au 50 Ag 2的 The characteristic absorption peaks appear at 400nm, 458nm, and 695nm, respectively, and the characteristic peaks are consistent with the reported Au 50 The characteristic ultraviolet absorption peaks of Ag2 nanoclusters correspond one to one.

[0152] (5) The number of transferred electrons n = 1 was calculated from the cyclic voltammogram; there was a good linear relationship between the oxidation peak potential Epa and the solution pH, and the linear equation was Epa = 2.0248-0.06477pH, R 2 =0.98015, the ratio of the number of protons participating in the reaction to the number of transferred electrons in the oxidation process of 1-NA is 1:1, so the oxidation process of this electrode is an irreversible isoelectronic and isoprotic reaction.

[0153] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A novel electrochemical enzyme sensor for detecting profenofos pesticide based on the principle of enzyme inhibition, characterized in that: The following steps are involved: S1. The glassy carbon electrode was polished with 0.05 μm Al2O3 powder, cleaned with ultrapure water, and tested by cyclic voltammetry in potassium ferrocyanide (K3[Fe(CN)5]–K4[Fe(CN)5]) electrolyte solution until the redox peak potential difference ΔEp≤100 mV to obtain a bare glassy carbon electrode (GCE). S2, 5 μL Au 50 The Ag2@G composite material suspension was added dropwise to the surface of the pretreated bare glassy carbon electrode and dried at low temperature to form a conductive enhancement layer on the electrode surface, fixed enzyme active sites and a biorecognition layer to obtain a composite modified electrode Au composed of bimetallic clusters and multilayer graphene. 50 Ag2@G / GCE; S3, 10 μL of plant esterase-chitosan KbE-CS solution was added to the surface of the composite material modified electrode, and dried at low temperature to obtain the enzyme-chitosan composite modified electrode KbE-CS / Au. 50 Ag2@G / GCE; S4, 4 μL of 0.5% Nafion solution was added to the surface of the enzyme-chitosan composite modified electrode and dried at low temperature to form a selective permeable membrane to prevent enzyme leakage, and finally the electrochemical enzyme sensor NF / KbE-CS / Au based on enzyme inhibition was obtained. 50 Ag2@G / GCE.

2. The method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the enzyme inhibition principle according to claim 1, characterized in that: The preparation method of the potassium ferricyanide (K3[Fe(CN)5]–K4[Fe(CN)5]) electrolyte solution is as follows: Accurately weigh 0.1646 g of K3[Fe(CN)6] and 0.2112 g of K4[Fe(CN)6] on an analytical balance and place them in a clean beaker. Add an appropriate amount of pH = 7.0 PBS buffer solution to dissolve them completely. Rinse the beaker and glass rod with pH = 7.0 PBS solution 1 to 3 times, then transfer the solution to a 100 mL volumetric flask, make up to volume, and store refrigerated in the dark.

3. The method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the enzyme inhibition principle according to claim 1, characterized in that: The Au 50 The preparation method of Ag2@G composite material suspension is as follows: 50 mg Au 25 Dissolve in 20 mL of acetonitrile solution and centrifuge, then take the supernatant to a 50 mL clean, dry flask, weigh 2.3 mg of AgNO3 and dissolve it in 1 mL of methanol solution, then slowly drop the methanol solution into the flask until the color of the solution turns yellow-green, then stop dropping, then stir for 30 minutes, wait for the reaction to end; wash the product with acetonitrile 2-3 times; obtain Au 50 Ag2(PET) 18 clusters; Then, 4 mg of multilayer graphene was accurately weighed using an analytical balance and added to a 10 mL centrifuge tube. 4 mL of N, N-dimethylformamide was then added. The centrifuge tube was ultrasonicated for two hours until the multilayer graphene was evenly dispersed. 1 mL of Au was then added. 50 Ag2(PET) 18 The N, N-dimethylformamide solution of the cluster was added to a centrifuge tube and ultrasonicated for 5 minutes to obtain Au 50 Ag2@G composite suspension.

4. The method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the enzyme inhibition principle according to claim 1, characterized in that: The preparation method of the KbE-CS solution is as follows: Accurately weigh 1 mg of chitosan using an analytical balance, add 5 mL of the prepared KbE crude enzyme solution to dissolve it, and shake well to obtain a 0.2 g / L KbE-CS solution.

5. The method for preparing a novel electrochemical enzyme sensor for detecting profenofos pesticide based on the enzyme inhibition principle according to claim 1, characterized in that: The preparation method of the Nafion solution is: Accurately pipette 10 μL of 5% Nafion solution into a 2 mL centrifuge tube, then add 90 μL of anhydrous ethanol solution to dilute and shake well to obtain a 0.5% Nafion solution.

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