A hybrid enzyme cascade pseudo-enzyme modified electrode and preparation method thereof and a detection method for organophosphorus pesticides

Abstract

The present invention discloses a hybrid enzyme cascade pseudo-enzyme modified electrode and a preparation method thereof and a detection method for organophosphorus pesticides, belonging to the field of detection and analysis technology, and solves the problems that the existing methods for detecting organophosphorus pesticide residues are high in cost, small in detection range, and few in detectable pesticide types. The present invention utilizes Burkholderia cepacia lipase to react with a metal salt solution to prepare a hybrid lipase Zn / hNF, and utilizes it to react with a chitosan solution to prepare a modified electrode AuNPs@Zn / hNF@Au; the present invention uses P‑NPP as a hydrolysis substrate, and uses the modified electrode AuNPs@Zn / hNF@Au to detect the electrical signal of the Zn / hNF hydrolysis product P‑NP by differential pulse voltammetry, and the inhibition rate of OPs on Zn / hNF is electrochemically analyzed to determine the concentration of the organophosphorus pesticide. The method of the present invention has the advantages of fast detection speed, wide detection range, low cost, high sensitivity, etc.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical sensors, and in particular relates to a hybrid enzyme cascade pseudo-enzyme modified electrode and a preparation method thereof, and a method for detecting organophosphorus pesticides. Background Art

[0002] Organophosphorus pesticides (OPs) are a type of pesticide with phosphorus as its core structure. They have the advantages of broad spectrum, high efficiency and low cost, and are widely used in the production of agricultural products such as fruits and vegetables. However, in actual applications, there are unscientific usage methods and mixing problems, which lead to serious organophosphorus pesticide residues in agricultural products.

[0003] Nowadays, there are two main types of detection methods for organophosphorus pesticide residues, traditional instrumental detection and analysis technology and rapid detection methods. Conventional instrumental detection methods are mainly concentrated in chromatographic methods such as gas chromatography, chromatography-mass spectrometry and high-performance liquid chromatography, as well as immunological methods and enzyme inhibition methods. Although chromatography has high sensitivity and stability, the technical cost of conventional chromatography-mass spectrometry detection and other methods for analyzing pesticide residues is high, the detection cycle is long, and most of them have expensive equipment and large solvent consumption, making it difficult to screen and detect a large number of samples in a short period of time. Immunological methods have the disadvantages of cumbersome sample pretreatment, complex operation, long detection time and difficulty in achieving on-site detection and analysis. In order to accurately, conveniently, and quickly detect pesticide residues in real time, many rapid detection methods have also emerged. One of them is enzyme inhibition technology detection method, which is a relatively mature method for detecting pesticide residue content. The most commonly used one is colorimetric method, which has relatively high accuracy and sensitivity. However, when the enzyme inhibition method is used to detect pesticide residues, the types of pesticides that can be detected by this method are limited, which greatly limits the application of enzyme inhibition analysis in pesticide residue detection. Therefore, it is urgent to carry out in-depth research in this field to establish a fast and effective method to detect the residues of organophosphorus pesticides. Nowadays, the sensitive detection of organophosphorus pesticides using electrochemical sensors has gradually become a research hotspot. Among them, electrochemical enzyme biosensors combine the interaction between enzymes and substrates with electrochemical analysis, and have attracted much attention due to their high sensitivity, good selectivity, fast reaction time, and simple operation. As the most commonly used biosensor, acetylcholinesterase (AChE) has been developed into a biosensor for detecting organophosphorus pesticides based on the inhibition of the enzyme by organophosphorus pesticides.

[0004] At present, the patent research on the determination of organophosphorus pesticide residues based on electrochemical enzyme biosensor method mainly focuses on inhibiting the activity of acetylcholinesterase (AChE) by organophosphorus pesticides, thereby reducing the content of thiocholine (TCh) generated by the hydrolysis of substrate acetylthiocholine (ATCh), so that the current intensity generated after TCh oxidation is reduced. This method can improve the enzyme fixation efficiency, improve the sensitivity of the sensor, and realize sensitive detection of organophosphorus pesticide residues. However, the cost of AChE used in this method is high, and there are certain limitations in the determination of different types of organophosphorus pesticides, and the detection range is small. At the same time, AChE, as a protein, will hinder the electron transfer between the enzyme and the electrode, affect the sensitivity of the sensor, and there will be a series of problems caused by the impaired stability and selectivity of the AChE biosensor. For the establishment of detection and analysis technology, the existing methods for detecting organophosphorus pesticide residues are high in cost and have a small detection range, and the types of pesticides that can be detected are few. However, it is feasible to use the low-cost and widely available Burkholderia cepacia lipase and prepare hybrid lipase by reacting with a metal salt solution to improve its stability and achieve high-performance determination of organophosphorus pesticides. Summary of the invention

[0005] In view of this, the present invention provides a hybrid enzyme cascade enzyme-mimicking modified electrode and a preparation method thereof, which utilizes enzyme-inorganic hybridization and enzyme-mimicking cascade technology to prepare and assemble modified electrodes, construct an enzyme-based electrochemical sensing analysis system, and realize rapid and sensitive detection of organophosphorus pesticide residues in food.

[0006] The technical solution adopted by the present invention is as follows: a method for preparing a hybrid enzyme cascade pseudo-enzyme modified electrode, the steps are as follows:

[0007] S1: According to the following proportions: take 5 mL of PBS buffer solution with a concentration of 0.01-0.2 mol / L and a pH of 6.0-10.0, add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of ZnSO with a concentration of 0.1-0.4 mol / L. 4 The solution is mixed evenly, and then incubated, centrifuged, washed, and freeze-dried to obtain Zn / hNF hybrid lipase;

[0008] S2: A mixed solution was prepared by mixing a chitosan solution with a concentration of 1.0-3.0 mg / mL and a Zn / hNF hybrid lipase solution with a concentration of 1.0-3.0 mg / mL in a volume ratio of 1:1, and 5-25 μL of the mixed solution was dropped on the surface of the AuNPs@Au electrode and dried at 4°C to obtain an AuNPs@Zn / hNF@Au modified electrode.

[0009] Furthermore, the preferred preparation method of the hybrid enzyme cascade pseudo-enzyme modified electrode as described above comprises the following steps:

[0010] S1: According to the following proportions: take 5 mL of PBS solution with a concentration of 0.1 mol / L and a pH of 8.0, add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of ZnSO with a concentration of 0.3 mol / L. 4 The solution was mixed evenly, then incubated at 4°C, centrifuged, washed three times with distilled water, and freeze-dried to obtain Zn / hNF hybrid lipase;

[0011] S2: A mixed solution was prepared by mixing a chitosan solution with a concentration of 2.0 mg / mL and a Zn / hNF hybrid lipase solution with a concentration of 2.0 mg / mL in a volume ratio of 1:1. 20 μL of the mixed solution was drop-coated on the surface of the AuNPs@Au electrode and dried at 4°C for 12 h to obtain an AuNPs@Zn / hNF@Au modified electrode.

[0012] Furthermore, the preparation method of the AuNPs@Au electrode as described above is as follows: drop-coating the AuNPs solution on the surface of a bare gold electrode, placing it at 4° C. and drying it for 12 hours to prepare the AuNPs@Au electrode.

[0013] Another object of the present invention is to provide a hybrid enzyme cascade enzyme-mimicking modified electrode prepared according to the preparation method of a hybrid enzyme cascade enzyme-mimicking modified electrode as described above.

[0014] Another object of the present invention is to provide a method for detecting organophosphorus pesticides based on the hybrid enzyme cascade pseudo-enzyme modified electrode as described above, the steps of which are as follows:

[0015] S 1 :According to the following proportions: with a volume of 200 μL, concentrations of 1, 2, 3…, 12, unit: ng / L, to establish standard solutions of organophosphorus pesticides of different concentrations, and then respectively with a volume of 3 mL, a concentration of 0.1 mol / L, pH 8.0 PBS buffer solution and AuNPs@Zn / hNF@Au modified electrode placed in a special electrochemical bottle, incubated at room temperature for 20 min, and then added with a volume of 200 μL, a concentration of 2 mmol / L palmitic acid p-nitrophenol solution, placed in a 35°C water bath for 20 min, and then scanned by differential pulse voltammetry. When there is no organophosphorus pesticide, that is, the current response value without inhibition by the organophosphorus pesticide is I pa0 , the current response value of the organophosphorus pesticide inhibition is I pa1 , and then according to the inhibition rate formula: I = (1-I pa1 / I pa0)%, and calculate the inhibition rate of the standard solution of organophosphorus pesticides at different concentrations, so as to construct a detection standard curve of the linear relationship between the inhibition rate and the organophosphorus pesticides at different concentrations: the horizontal axis x is the concentration of the organophosphorus pesticide, and the vertical axis y is the inhibition rate;

[0016] S 2 : According to the following ratio: During the detection, a volume of 200 μL of the sample solution to be tested, a volume of 3 mL of PBS buffer solution with a concentration of 0.1 mol / L and pH 8.0, and an AuNPs@Zn / hNF@Au modified electrode were placed in a special electrochemical bottle and incubated at room temperature for 20 minutes; then a volume of 200 μL of palmitic acid p-nitrophenol solution with a concentration of 2 mmol / L was added and placed in a 35°C water bath for reaction for 20 minutes. The differential pulse voltammetry method was used to scan to detect the oxidation peak current generated on the AuNPs@Zn / hNF@Au modified electrode, and then the concentration of organophosphorus pesticides in the sample solution to be tested was obtained by detecting the standard curve.

[0017] Furthermore, the concentration range of organophosphorus pesticide detection corresponding to the standard curve constructed by the above detection method is 2.63×10 -8 mol / L~3.15×10 -6 mol / L.

[0018] Furthermore, the organophosphorus pesticides described in the above detection method include methyl parathion, dimethoate, dichlorvos and malathion.

[0019] The principle of the present invention is based on the preparation of a new hybrid lipase (Zn / hNF) with simulated peroxidase activity. By increasing the effective area of ​​the enzyme and the peroxidase activity generated by the hybridization, the catalytic efficiency of the lipase is improved, and a cascade reaction occurs in the electrochemical detection system, increasing the electron transfer rate, and using nano-gold (AuNPs) modified electrodes to improve the sensitivity and stability of electrochemical detection. Based on the catalytic hydrolysis activity of the hybrid lipase and the inhibition principle of organophosphorus pesticides on the hydrolysis activity of the lipase, an indirect detection system is constructed for different detection situations in practical applications. With palmitic acid p-nitrophenol (P-NPP) as the hydrolysis substrate, the modified electrode AuNPs@Zn / hNF@Au is used to detect the electrical signal of the hydrolysis product of Zn / hNF p-nitrophenol by differential pulse voltammetry (DPV), and the inhibition rate of organophosphorus pesticides on Zn / hNF is electrochemically analyzed. The present invention can simultaneously realize the detection and analysis of the residues of four organophosphorus pesticides, namely, methyl parathion, dimethoate, dichlorvos and malathion.

[0020] Advantages and beneficial effects of the present invention: The hybrid lipase assembled modified electrode AuNPs@Zn / hNF@Au prepared by the present invention has the advantages of high stability and wide detection range for organophosphorus pesticides. At the same time, its detection method accelerates the detection speed and has the advantages of high sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Fourier transform infrared spectra characterization diagram of free lipase and hybrid lipase;

[0022] Figure 2 is the SEM characterization picture of hybrid lipase;

[0023] Figure 3 Electrochemical behavior diagram of AuNPs@Zn / hNF@Au for detecting MP, (a), PBS, (b), Zn / hNF@Au, (c), AuNPs@free lipase@Au, (d), AuNPs@Zn / hNF@Au, (e), MP-AuNPs@Zn / hNF@Au;

[0024] Figure 4 Optimization diagram for different buffer solutions;

[0025] Figure 5 It is the optimization diagram of different buffer solution concentrations;

[0026] Figure 6 Optimization diagram for different pH;

[0027] Figure 7 Optimization diagram for different substrate concentrations;

[0028] Figure 8 Optimization diagram for different Zn / hNF concentrations;

[0029] Fig. 9 It is the optimization diagram of different hydrolysis time;

[0030] Fig.10 Optimization diagram for different hydrolysis temperatures;

[0031] Fig.11 Optimization diagram for different inhibition times;

[0032] Fig.12 This is the standard curve diagram of AuNPs@Zn / hNF@Au detection, (a), methyl parathion, (b), dimethoate, (c), dichlorvos; (d), malathion. DETAILED DESCRIPTION

[0033] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and use the present invention.

[0034] Example 1

[0035] A method for preparing a hybrid enzyme cascade pseudo-enzyme modified electrode, the steps are as follows:

[0036] S1: According to the following proportions: take 5 mL of PBS buffer solution with a concentration of 0.01-0.2 mol / L and a pH of 6.0-10.0, add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of ZnSO with a concentration of 0.1-0.4 mol / L. 4 The solution is mixed evenly, and then incubated, centrifuged, washed, and freeze-dried to obtain Zn / hNF hybrid lipase;

[0037] S2: 20 μL of AuNPs solution was drop-coated on the surface of the bare gold electrode, and then placed in a 4°C refrigerator for drying for 12 hours to prepare a drop-coated AuNPs@Au electrode. A chitosan solution with a concentration of 1.0-3.0 mg / mL and a Zn / hNF hybrid lipase solution with a concentration of 1.0-3.0 mg / mL were prepared in a volume ratio of 1:1 to prepare a mixed solution, and 5-25 μL of the mixed solution was drop-coated on the surface of the AuNPs@Au electrode, and then placed at 4°C for drying to obtain an AuNPs@Zn / hNF@Au modified electrode.

[0038] like Figure 1 It can be seen that free lipase is at 1657~1415cm -1 The three characteristic peaks at 3000-3200 cm -1 The characteristic peak at is attributed to -CH 2 and -CH 3 ; Zn 2+ Compared with free lipase, the lipase hybrid nanoflower (Zn / hNF) has a wavelength range of 928-1091 cm -1 Multiple PO vibration stretching characteristic peaks were observed, and a relatively small band was observed at 554-643 cm -1 Yes PO 4 3- Weak absorption peaks of antisymmetric and symmetric stretching vibrations.

[0039] Depend on Figure 2It can be seen that the SEM image of the prepared hybrid lipase Zn / hNF formed a nanoflower with a structure similar to that of natural flowers, with a particle size range of 20 to 40 μm, and the morphology changed from stacked spheres to flower-like nanoparticles, whose petal structure has a hierarchical lamellar structure and a large specific surface area.

[0040] Depend on Figure 3 It can be seen that in the absence of substrate (curve a), the electrode has no reaction and only a weak background current is observed. Curve b is the current response value of P-NP when Zn / hNF@Au is not added with MP (1.11×10 -6 A), curve c is AuNPs@free lipase@Au without adding MP. The current response value of P-NP is 9.88×10 -7 A, curve d is the current response value of P-NP when AuNPs@Zn / hNF@Au is not added with MP (1.81×10 -6 A), the above results show that the current response value of AuNPs@Zn / hNF@Au is significantly higher than that of the other two modified electrodes. Among them, curve e is the current response value of AuNPs@Zn / hNF@Au detected after adding MP, which is 1.66×10 -6 A, and the current value of P-NP decreased significantly after the addition of MP, indicating that the addition of MP inhibited the activity of Zn / hNF.

[0041] Depend on Figure 4 It can be seen that among the four buffer solutions, obvious oxidation peaks can be observed in PBS-1, PBS-3, and PBS-4, but their current response values ​​are affected to varying degrees. The DPV in PBS-2 is a smooth curve, no oxidation peak is observed, and only a weak background current can be observed at the peak potential of 0.1V. 2 HPO 4 +NaH 2 PO 4 ) obtained the highest current response of 1.38×10 -6 A, significantly higher than PBS-3 and PBS-4.

[0042] Depend on Figure 5 It can be seen that with the increase of buffer solution concentration, the peak current of DPV oxidation increases first, and the peak current increases from 6.62×10 -7 A increased significantly to 1.49×10 -6 A. When the concentration of PBS buffer solution continued to increase, in the concentration range of 0.10mol / L to 0.20mol / L, the oxidation peak current increased from 1.49×10 -6 A is reduced to 1.18×10 -6 A.

[0043] Depend on Figure 6 It can be seen that at pH 6.0, the DPV is a smooth curve without peaks. At pH 7.0-9.0, there are obvious current response values, and with the increase of pH value, the current response value shows a trend of first rising and then falling. At pH 8.0, the highest current response is 1.64×10 -6 A, then with the increase of pH value, the current response value decreased, and when the pH value was 9.0, the current response value was 1.15×10 -6 A, the oxidation value at this time is significantly lower than pH 8.0.

[0044] Depend on Figure 7 It can be seen that when the substrate concentration increases from 1.0mmol / L to 2.0mmol / L, the current response value increases significantly with the increase of substrate concentration. When the substrate concentration is 2.0mmol / L, the current response value is 1.77×10 -6 A, when the substrate concentration continued to increase from 2.0mmol / L to 3.0mmol / L, the current response value decreased to 1.64×10 -6 A.

[0045] Depend on Figure 8 It can be seen that when the concentration of Zn / hNF is within the range of 1.0 mg / mL to 1.5 mg / mL, the concentration of the hydrolysis product P-NP increases due to the increase in hydrolysis capacity. The peak current response gradually increases with the increase in P-NP concentration and reaches the maximum value when the Zn / hNF concentration is 1.5 mg / mL. At this time, the current response value is 1.76×10 -6 A, When the Zn / hNF concentration continues to increase, the oxidation peak current shows a downward trend.

[0046] Depend on Fig. 9 It can be seen that within the reaction time of 5min to 15min, the oxidation peak current shows an upward trend with the increase of time. When the reaction reaches 15min, the peak current value is 1.80×10 -6 A is significantly higher than the current response value at 5min to 10min. As time goes by, the current response value tends to be flat.

[0047] Depend on Fig.10 It can be seen that with the increase of reaction temperature, the oxidation peak current of the hydrolysis product P-NP shows a trend of first rising and then falling. When the temperature is in the reaction range of 25℃~35℃, the response value is the best at 35℃, which is significantly higher than the current response value at 25℃. The oxidation peak current at this time is 1.81×10 -6 A. When the reaction temperature continues to increase within the range of 40℃~50℃, the oxidation peak value decreases accordingly.

[0048] Depend on Fig.11It can be seen that as the incubation time increased from 5 min to 20 min, the hydrolysis activity of Zn / hNF decreased significantly. As the incubation time further increased, the peak current reached a stable level without obvious changes after 20 min.

[0049] Example 2

[0050] A method for preparing a hybrid enzyme cascade pseudo-enzyme modified electrode, comprising the following steps:

[0051] S1: Add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of 0.3 mol / L ZnSO to 5 mL of 0.1 mol / L PBS solution with a pH of 8.0. 4 The solution was mixed evenly, incubated at 4°C, centrifuged, washed three times with distilled water, and freeze-dried to obtain Zn / hNF hybrid lipase.

[0052] S2: 20 μL of AuNPs solution was drop-coated on the surface of the bare gold electrode, and then placed in a 4°C refrigerator to dry for 12 hours to prepare a drop-coated AuNPs@Au electrode. A mixed solution of 2.0 mg / mL chitosan solution and 2.0 mg / mL Zn / hNF hybrid lipase solution was prepared in a volume ratio of 1:1, and 20 μL of the mixed solution was drop-coated on the surface of the AuNPs@Au electrode, and then placed in a 4°C refrigerator to dry for 12 hours to obtain an AuNPs@Zn / hNF@Au modified electrode.

[0053] Example 3

[0054] A method for detecting organophosphorus pesticides using a hybrid enzyme cascade pseudo-enzyme modified electrode prepared in Example 2 comprises the following steps:

[0055] S 1 :With a volume of 200 μL and a concentration of 1, 2, 3…, 12, unit: ng / L, a standard solution of organophosphorus pesticides of different concentrations was established, and then it was placed in a special electrochemical bottle with a volume of 3 mL, a concentration of 0.1 mol / L, pH 8.0 PBS buffer solution and AuNPs@Zn / hNF@Au modified electrode, incubated at room temperature for 20 min, and then a volume of 200 μL, a concentration of 2 mmol / L palmitic acid p-nitrophenol solution was added, and placed in a 35°C water bath for 20 min. After scanning by differential pulse voltammetry, when there was no organophosphorus pesticide, that is, the current response value without the inhibition of organophosphorus pesticide was I pa0 , the current response value of the organophosphorus pesticide inhibition is I pa1 , and then according to the inhibition rate formula: I = (1-I pa1 / I pa0)%, and calculate the inhibition rate of the standard solution of organophosphorus pesticides at different concentrations, so as to construct a detection standard curve of the linear relationship between the inhibition rate and the organophosphorus pesticides at different concentrations: the horizontal axis x is the concentration of the organophosphorus pesticide, and the vertical axis y is the inhibition rate;

[0056] S 2 :According to the following ratio: During the test, a 200 μL sample solution and a 3 mL PBS buffer solution with a concentration of 0.1 mol / L and pH 8.0 and an AuNPs@Zn / hNF@Au modified electrode were placed in an electrochemical bottle and incubated at room temperature for 20 min; then a 200 μL palmitic acid p-nitrophenol solution with a concentration of 2 mmol / L was added and placed in a 35°C water bath for 20 min. The oxidation peak current generated on the AuNPs@Zn / hNF@Au modified electrode was detected by differential pulse voltammetry scanning, and then the concentration of organophosphorus pesticides in the sample solution was obtained by detecting the standard curve. Among them, the concentration range of organophosphorus pesticide detection corresponding to the constructed standard curve is 2.63×10 -8 mol / L~3.15×10 -6 mol / L.

[0057] Example 4

[0058] (1) Based on the optimal conditions screened out from the experimental results of Examples 1-2, an AuNPs@Zn / hNF@Au electrochemical detection system was assembled and used for the detection and analysis of methyl parathion, dimethoate, dichlorvos and malathion residues. The DPV method was used to detect a series of organophosphorus pesticides at different concentrations. The results are shown in Figure 2. Fig.12 As shown, the standard curves corresponding to the four standard solutions are: y=7.0559x+3.8115, y=5.2036x+3.7659, y=6.62947x+5.1628, y=5.9600x+3.4239;

[0059] (2) The prepared sensor was tested for analytical application in tap water, vegetable and fruit samples using the standard addition method. Methyl parathion stock solution was added to tap water; for fruit and vegetable samples, 50 g of grapes, apples, spinach and baby cabbage were weighed and placed in 50 mL of 0.10 mol / L pH 7.5 PBS buffer solution, centrifuged for 10 min, and the supernatant was collected as the actual sample. The supernatant was diluted 100 times with PBS and used as the actual sample.

[0060] The pretreated samples were placed in a special electrochemical bottle, and different concentrations (0.1ng / L, 5.0ng / L, 10.0ng / L) of methyl parathion (MP), 3mL, 0.1mol / L, pH 8.0 PBS buffer solution and AuNPs@Zn / hNF@Au modified electrode were added to the actual samples and incubated at room temperature for 20min; then 200μL of palmitic acid p-nitrophenol solution with a concentration of 2mmol / L was added and placed in a 35℃ water bath for 20min, and differential pulse voltammetry was used for scanning. The potential practicality of the developed electrochemical detection system was evaluated by a recovery test, and the results are shown in Table 1;

[0061] Table 1 Spike recovery experiment of indirect detection of MP by AuNPs@Zn / hNF@Au electrode

[0062]

[0063]

[0064] No methyl parathion residues were detected in the actual samples. Different concentrations of methyl parathion were added to the samples. The standard curve was used to calculate the measured concentration and analyze the spiked recoveries of different samples. The spiked methyl parathion recoveries observed in tap water, grapes, apples, spinach and baby cabbage samples were between 97.83% and 122.01%, with RSD less than 5%. The experimental results show that the prepared AuNPs@Zn / hNF@Au electrochemical detection system has the potential to sensitively and accurately analyze OPs in food.

Claims

1. A method for preparing a hybrid enzyme cascade enzyme-modified electrode, characterized in that: Here are the steps: S1: According to the following proportions: take 5 mL of PBS buffer solution with a concentration of 0.01-0.2 mol / L and pH=6.0-10.0, add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of ZnSO4 solution with a concentration of 0.1-0.4 mol / L, mix well, incubate, centrifuge, wash, and freeze-dry to obtain Zn / hNF hybrid lipase; S2: A mixed solution was prepared by mixing a chitosan solution with a concentration of 1.0-3.0 mg / mL and a Zn / hNF hybrid lipase solution with a concentration of 1.0-3.0 mg / mL in a volume ratio of 1:1, and 5-25 μL of the mixed solution was dropped on the surface of the AuNPs@Au electrode and dried at 4°C to obtain an AuNPs@Zn / hNF@Au modified electrode.

2. The method for preparing a hybrid enzyme cascade pseudo-enzyme modified electrode according to claim 1, characterized in that: Here are the steps: S1: According to the following proportions: take 5 mL of PBS solution with a concentration of 0.1 mol / L and pH = 8.0, add 1 mL of Burkholderia cepacia lipase with an enzyme activity of 23 U / mg and 200 μL of ZnSO4 solution with a concentration of 0.3 mol / L, mix well, incubate at 4 ° C, centrifuge, wash with distilled water 3 times, and freeze-dry to obtain Zn / hNF hybrid lipase; S2: A mixed solution was prepared by mixing a chitosan solution with a concentration of 2.0 mg / mL and a Zn / hNF hybrid lipase solution with a concentration of 2.0 mg / mL in a volume ratio of 1:

1. 20 μL of the mixed solution was drop-coated on the surface of the AuNPs@Au electrode and dried at 4°C for 12 h to obtain an AuNPs@Zn / hNF@Au modified electrode.

3. The method for preparing a hybrid enzyme cascade enzyme-modified electrode according to claim 2, characterized in that: The preparation method of the AuNPs@Au electrode is as follows: drop-coating the AuNPs solution on the surface of a bare gold electrode, placing it at 4° C. and drying it for 12 hours to prepare the AuNPs@Au electrode.

4. A hybrid enzyme cascade enzyme-modified electrode prepared according to the method for preparing a hybrid enzyme cascade enzyme-modified electrode according to any one of claims 1 to 3.

5. A method for detecting organophosphorus pesticides using a hybrid enzyme cascade pseudo-enzyme modified electrode according to claim 4, comprising the following steps: S1: Prepare standard solutions of organophosphorus pesticides at different concentrations in the following proportions: 200 μL with concentrations of 1, 2, 3…, 12, unit: ng / L, and then place them in a dedicated electrochemical bottle with 3 mL of PBS buffer solution with a concentration of 0.1 mol / L and pH=8.0 and AuNPs@Zn / hNF@Au modified electrode, incubate at room temperature for 20 min, then add 200 μL of palmitic acid p-nitrophenol solution with a concentration of 2 mmol / L and place in a 35°C water bath for 20 min, and scan by differential pulse voltammetry. When there is no organophosphorus pesticide, that is, the current response value without inhibition by organophosphorus pesticide is I pa0 , the current response value of the organophosphorus pesticide inhibition is I pa1 , and then according to the inhibition rate formula: I = (1-I pa1 / I pa0 )%, and calculate the inhibition rate of the standard solution of organophosphorus pesticides at different concentrations, so as to construct a detection standard curve of the linear relationship between the inhibition rate and the organophosphorus pesticides at different concentrations: the horizontal axis x is the concentration of the organophosphorus pesticide, and the vertical axis y is the inhibition rate; S2: According to the following ratio: During the detection, a volume of 200 μL of the sample solution to be tested, a volume of 3 mL, a concentration of 0.1 mol / L, pH = 8.0 PBS buffer solution and an AuNPs@Zn / hNF@Au modified electrode were placed in a special electrochemical bottle and incubated at room temperature for 20 minutes; then a volume of 200 μL, a concentration of 2 mmol / L palmitic acid p-nitrophenol solution was added and placed in a 35°C water bath for reaction for 20 minutes. The oxidation peak current generated on the AuNPs@Zn / hNF@Au modified electrode was detected by differential pulse voltammetry scanning, and then the concentration of organophosphorus pesticides in the sample solution to be tested was obtained by detecting the standard curve.

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