Method for detecting organophosphorus pesticide based on immobilized plant esterase double-enzyme colorimetric method and application

Abstract

The invention belongs to the technical field of bioengineering and pesticide residue detection, and discloses a method for detecting organophosphorus pesticide by a two-enzyme colorimetric method based on immobilized plant esterase, which comprises the following steps of: (1) respectively mixing every 50 microliters of immobilized plant esterase with final concentration of 1mg / mL with 50 microliters of organophosphorus standard sample solutions with different concentrations, taking distilled water as a blank control, and taking distilled water as a standard control; the preparation method comprises the following steps of: performing incubation at room temperature, sequentially adding HRP (horse radish peroxidase), a substrate 1-NA, TMB (tetramethylbenzidine), H2O2 and an acetic acid buffer solution into a reaction solution, oscillating, incubating, measuring an absorbance value A659, and calculating an inhibition ratio I; and (2) taking a to-be-detected sample, operating according to the step (1), calculating the inhibition ratio I, and substituting the inhibition ratio I to obtain the concentration of the organophosphorus pesticide in the to-be-detected sample. The method is simple and rapid to operate, low in cost, good in stability, high in OPs detection sensitivity and wide in detection range, and has a relatively good application prospect in rapid screening and detection of organophosphorus pesticide detection in fruits and vegetables.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and pesticide residue detection, in particular to a method and application of detecting organophosphorus pesticides by a dual-enzyme colorimetric method based on immobilized plant esterase. Background Art

[0002] Organophosphorus pesticides (OPs) in the ecological environment pose a huge threat to human health through the food chain, forcing the rapid development of OPs analysis methods. At present, there are many gold standard methods for pesticide residues, including gas chromatography, mass spectrometry, and high-pressure liquid chromatography equipped with different detectors. However, these methods have obvious disadvantages, such as high cost, complex pretreatment process, time-consuming, and expensive experimental equipment, which seriously limit the rapid detection of OPs by these methods. In this context, as one of the most promising detection methods, colorimetry based on the principle of enzyme inhibition has attracted attention. This type of method often does not require complex instruments, the results are intuitive, and it is suitable for rapid detection. However, this type of method often has high requirements for the detection enzyme, and often requires the detection enzyme to have high sensitivity and high stability to the detected OPs. At present, acetylcholinesterase (AchE) is the most commonly used detection enzyme for this method. The enzyme can enzymatically hydrolyze thioacetylcholine (ATCI), but the enzyme is expensive and has low stability to the environment (pH, temperature). Therefore, it is necessary to find an ideal detection enzyme with strong stability, easy preparation, high catalytic activity and low cost. Plant-derived α-naphthyl acetate esterase (ANAE, EC 3.1.1.X) can be recognized and inhibited by OPs, and its effect is similar to that of AChE. In addition, the enzyme is abundant in sources (in tubers, woody plants, herbs, cereals and beans) and has low cost, making it a potential substitute for AChE. However, like other enzymes, the enzyme has disadvantages such as instability, easy loss of activity, and low affinity, especially low stability under certain extreme conditions, which limits its application. An effective way to solve this problem is to fix the target enzyme in a polymer or inorganic carrier matrix to enhance the storage and operational stability of the enzyme.

[0003] Chitosan (CTS) is a natural polysaccharide composed of D-GlcN and D-GlcNAc monomers with β1-4 bonds. It has outstanding biocompatibility, plasticity, stability, high affinity for proteins, high loading capacity and non-toxicity, and is widely considered to be an ideal enzyme immobilization matrix. In addition, by modifying its free amino group (-NH2), acetyl group and hydroxyl group (-OH), a multifunctional carrier of CTS can be obtained. Glutaraldehyde (GA) is a well-known aminating agent, which contains reactive aldehyde groups at both ends of its molecule. Through covalent bonds, hydrophobic bonds and physical absorption, the -NH2 groups on the surface of CTS and various chemical groups in the enzyme protein (amino, thiol, phenol and imidazole) can be combined with the aldehyde groups at both ends of GA to achieve the purpose of CTS amination, and CTS / GA has better enzyme immobilization efficiency. Glycine (Gly) contains amino and hydroxyl groups, which can be used to modify the surface of CTS, and can also be combined with GA to form CTS / GA / Gly composite materials. The composite material not only has abundant functional groups, but also can adsorb enzyme proteins through various modes of action, such as ion adsorption, hydrogen bonding, cross-linking adsorption, etc. The composite material has high fixation efficiency and adsorption specificity for enzyme proteins, and helps to improve the hydrophobicity of enzymes. It is an ideal fixation material, especially for plant-extracted enzyme proteins containing impurities, and provides a good strategy for the application of plant esterases in OPs. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method and application of detecting organophosphorus pesticides by a dual enzyme colorimetric method based on immobilized plant esterase.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A method for detecting organophosphorus pesticides by dual enzyme colorimetry based on immobilized plant esterase comprises the following steps:

[0007] (1) A one-step method was used to perform a colorimetric reaction test and a test standard curve was drawn, that is, each 50 μL of a final concentration of 1 mg / mL immobilized plant esterase was mixed with 50 μL of organophosphorus pesticide standard solutions of different concentrations, i.e., 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, and 100 ng / mL, and a blank control was distilled water. After incubation at room temperature for 1 h, 50 μL of 1 μM HRP, 50 μL of 0.5 mM substrate 1-naphthyl acetate, i.e., 1-NA, 50 μL of 1.5 mM TMB, 50 μL of 1 mM H2O2, and 900 μL of 0.2 M, pH 5.0 acetate buffer were added to the reaction solution in sequence, and the mixture was shaken for 5-10 s and incubated at room temperature in the dark for 30 min. The absorbance value A at 659 nm was measured by a UV-visible photometer. 659, the inhibition rate I (%) was calculated according to the following formula:

[0008]

[0009] Where, I is the esterase activity inhibition rate; A0 is the absorbance value of the control reaction solution at 659nm A 659 ; A1 is the absorbance value of the sample reaction solution at 659nm;

[0010] Then, the concentration logarithm lg of the organophosphorus pesticide standard solution was taken as the independent variable X, and the inhibition rate was taken as the dependent variable Y. The Excel software was used to establish a linear regression equation of one variable, Y=aX+b;

[0011] (2) Take 50 μL of the sample treatment solution to be tested, and perform the operation according to step (1) to calculate the inhibition rate I, and substitute it into the concentration linear standard linear equation in the above step (1) to calculate the concentration of the organophosphorus pesticide in the sample to be tested.

[0012] Furthermore, the method uses glutaraldehyde GA to aminize the surface of chitosan CTS, and then cross-links with glycine Gly to prepare a new CTS / GA / Gly composite material. The plant esterase is fixed by simple physical adsorption principle, and the fixed plant esterase is used as the detection enzyme. The substrate 1-naphthyl acetate 1-NA generates 1-N with reduction activity under the catalysis of the enzyme, which can inhibit the color development / colorimetric change of the TMB / H2O2 system induced by HRP. According to the principle of enzyme inhibition, OPs can inhibit this color development reaction, and it is proportional to the amount of OPs; accordingly, a dual enzyme colorimetric method based on fixed plant esterase is successfully invented, which is a dual enzyme colorimetric method for detecting OPs.

[0013] Furthermore, the plant esterase is derived from mung beans, and the preparation steps are as follows:

[0014] Grind 40g of mung bean into powder, add 200mL of deionized water DIW and stir for 40min, centrifuge at 3000rpm at room temperature for 20min, collect 20.0mL of supernatant, add different saturation, i.e. 20-90% ammonium sulfate gradient purification at 4℃, let stand for 12h, centrifuge at 8000rpm for 10min, collect precipitate, dissolve with 0.02mol / L, pH7.5 PBS buffer, purify with diethylaminoethyl (DEAE)-cellulose anion exchange column (length×diameter: 50mm×16mm) equilibrated with 0.02mol / L, pH7.5 PBS buffer 4 times the volume of the purification column, elute with the same buffer at a flow rate of 30mL / h, collect fractions, centrifuge at 5℃, 1645g for 25min, ultrafilter with 30kDa ultrafiltration tube, take concentrated enzymatic hydrolysate and filter through sephadex G-25 gel column chromatography, eluted with buffer, flow rate of 30mL / h, collected 3mL fractions, combined the fractions with higher absorbance at 595nm, concentrated in vacuo to obtain purified plant esterase, and stored at 4℃ for future use.

[0015] Furthermore, the preparation steps of the immobilized plant esterase are as follows:

[0016] 1) Preparation of chitosan GTS gel: Chitosan powder was immersed in a 5% by volume glacial acetic acid solution at a ratio of 1:5 g:mL, and mechanically stirred at 25°C and 1000 rpm for 4 hours, and a 0.1M NaOH solution was added dropwise at a volume ratio of chitosan solution to NaOH solution of 1:10, and stirring was continued at 1000 rpm overnight to prepare a crude chitosan gel; filtered, rinsed with distilled water to pH = 7.0, and chitosan gel, i.e., CTS gel, was obtained, and placed in a 70% by volume ethanol solution and stored at 4°C;

[0017] 2) Preparation of chitosan gel GTS / GA activated by glutaraldehyde GA: Prepare glutaraldehyde working solution: Mix pH 7.0, 0.2M sodium dihydrogen phosphate solution and 25% glutaraldehyde solution in a volume ratio of 11.2:16.8 to obtain a newly prepared glutaraldehyde working solution. Then, add chitosan gel in a ratio of g:mL of 10:28 to the newly prepared glutaraldehyde working solution, and continuously stir mechanically at 25°C and 200rpm for 1h to prepare a crude chitosan gel activated by glutaraldehyde. Wash the chitosan gel thoroughly with deionized water, and filter with filter paper to remove excess water to obtain a glutaraldehyde-activated chitosan gel, namely CTS / GA gel;

[0018] 3) Preparation of CTS / GA / Gly composite immobilization material: CTS / GA gel was placed in a 0.5M, pH 8.0 glycine Gly solution, the ratio of CTS / GA gel: glycine Gly solution was 10:90 g:mL, and the mixture was stirred continuously at 25°C and 200 rpm for 48 h. The obtained CTS / GA gel was thoroughly soaked in deionized water for 48 h, filtered, and rinsed with distilled water to remove free Gly. The prepared CTS / GA / Gly composite immobilization material was stored in a 70% volume concentration ethanol solution at 4°C for future use;

[0019] 4) Immobilization of plant esterase: The purified plant esterase was dissolved in 0.02 mol / L, pH 7.5 PBS buffer at a ratio of 20:10 (mg:mL) to obtain an enzyme solution. The initial loading amount of the CTS / GA / Gly composite immobilized material was 10 mg / g. The immobilized material and the enzyme solution were mixed at a ratio of 1:20 (g:mL) of immobilized material: enzyme solution. The mixture was stirred continuously at 25°C and 200 rpm for 12 h, filtered, washed with distilled water, and the prepared immobilized plant esterase was stored at 4°C.

[0020] Furthermore, the dual enzymes are immobilized mung bean esterase (I-mANAE) and horseradish peroxidase (HRP).

[0021] The reaction system of the method also includes: the color developing substrate is TMB, 1-NA, H2O2, and the buffer is acetate buffer.

[0022] Furthermore, the preparation method of the sample treatment solution is as follows: the fruit and vegetable samples are washed and cut into small pieces, acetone is added, and homogenized; the sample solution is centrifuged at 8000 rpm for 20 minutes at 37°C, the supernatant is collected, and filtered through a 0.2 μm polyvinylidene fluoride (PVDF) membrane, the filtrate is collected, and it is diluted 10 times with deionized water to obtain a sample treatment solution for organophosphorus pesticide analysis.

[0023] Furthermore, the organophosphorus pesticide is dichlorvos.

[0024] Furthermore, the detection range of the method is 10 to 100 ng / mL.

[0025] Application of the method described above in the detection of organophosphorus pesticides.

[0026] The above-mentioned method is applied to the detection of OPs residues in fruits and vegetables.

[0027] The advantages and positive effects achieved by the present invention are:

[0028] 1. The method of the present invention uses chitosan as a matrix, functionalizes it with glutaraldehyde and glycine in turn, and uses a simple physical adsorption principle to immobilize the purified mung bean esterase to construct a new type of immobilized plant esterase. Compared with the free enzyme, the pH, temperature, substrate naphthyl acetate (1-NA) affinity and storage stability of the immobilized plant esterase are significantly improved. Based on the enzyme inhibition principle, the enzymatic hydrolysis product 1-naphthol (1-N) of the substrate 1-NA is coupled to the immobilized plant esterase / 1-NA reaction system and the tetramethylbenzidine / hydrogen peroxide / horseradish peroxidase (TMB / H2O2 / HRP) color development system, and the organophosphorus pesticide residues are quickly qualitatively or quantitatively detected by the change of reaction color / colorimetry, and a "one-step" dual enzyme colorimetric method for determining organophosphorus pesticides is designed. The method is simple to operate, fast, low in cost, good in stability, high in detection sensitivity to OPs, and wide in detection range. It has a good application prospect in the rapid screening and detection of organophosphorus pesticides in fruits and vegetables.

[0029] 2. The method of the present invention uses mung bean esterase as the enzyme source, is sensitive to OPs, has a wide range of sources, is relatively cheap, is easy to obtain, has universal applicability, and is easy to mass produce.

[0030] 3. The method of the present invention sequentially modifies CTS with GA and Gly to prepare an enzyme-fixed composite material. The material preparation method is simple, the synthesis conditions are mild, and it has multiple functional groups. It can fix the enzyme efficiently and retain the original structure of the enzyme to the maximum extent. Compared with free esterase, the immobilized plant esterase has significantly improved pH, temperature, and storage stability, and has enhanced affinity for the substrate 1-NA.

[0031] 4. The method of the present invention is a new dual-enzyme colorimetric method for OPs detection, with fixed plant esterase as the detection enzyme, based on the principle of OPs inhibition of esterase, combined with the color / colorimetric changes of the reaction system, to establish a new dual-enzyme colorimetric method. This method does not require complex instruments, has low cost, simple operation, provides color / colorimetric dual response, short test time, low detection limit, can qualitatively and quantitatively detect OPs residues in fruits and vegetables, and has good application value.

[0032] 5. The present invention adopts a cross-linking method, uses GA to aminize the CTS surface, and then cross-links with Gly to prepare a new CTS / GA / Gly composite material. The plant esterase is fixed by a simple physical adsorption principle, and the fixed plant esterase is used as the detection enzyme. The substrate 1-NA generates 1-N with reduction activity under the catalysis of the enzyme, which can inhibit the color development / colorimetric changes of the TMB / H2O2 system induced by HRP. According to the principle of enzyme inhibition, OPs can inhibit this color development reaction, and it is proportional to the amount of OPs. Based on this, we have successfully invented a dual enzyme colorimetric method based on fixed plant esterase, which is used to detect OPs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the principle of a method for detecting organophosphorus pesticides by a dual-enzyme colorimetric method based on immobilized plant esterase in the present invention;

[0034] Figure 2 is an SDS-PAGE diagram of the plant esterase in Example 1 of the present invention; wherein M represents the molecular weight of the standard protein, a represents the molecular weight distribution of the crude plant esterase, and b represents the molecular weight distribution of the plant esterase after purification by column chromatography;

[0035] Figure 3 This is the optimal temperature diagram of the enzyme in Example 3 of the present invention;

[0036] Figure 4 This is the optimal pH diagram of the enzyme in Example 4 of the present invention;

[0037] Figure 5 This is a diagram of the storage stability of the enzyme in Example 5 of the present invention;

[0038] Figure 6 This is the affinity diagram of the enzyme for the substrate 1-NA in Example 6 of the present invention;

[0039] Figure 7 It is an analysis diagram of the detection principle of the dual enzyme colorimetric method in Example 7 of the present invention; wherein, a. absorbance scan of the reaction system (esterase; 1-NA; 1-N; esterase + 1-NA) in the range of 200-500nm; b. absorbance change at 325nm within 10min of the reaction system (esterase; 1-NA; 1-N; esterase + 1-NA); c. absorbance scan of the reaction system (TMB; TMB + H2O2; TMB + H2O2 + HRP; TMB + H2O2 + HRP + 1-N) in the range of 400-800nm; d. absorbance change at 659nm within 10min of the reaction system (TMB; TMB + H2O2; TMB + H2O2 + HRP; TMB + H2O2 + HRP + 1-N);

[0040] Figure 8 The figure is a graph of the detection of organophosphorus pesticides (DDVP) by the dual enzyme colorimetric method and the establishment of a standard curve in Example 8 of the present invention; wherein a represents the ultraviolet scanning graph of the reaction solution at 200-800nm, and b represents the linear regression fitting graph of the enzyme activity inhibition rate-pesticide concentration logarithmic value. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0043] A method for detecting organophosphorus pesticides by dual enzyme colorimetry based on immobilized plant esterase comprises the following steps:

[0044] (1) A one-step method was used to perform a colorimetric reaction test and a test standard curve was drawn, that is, each 50 μL of a final concentration of 1 mg / mL immobilized plant esterase was mixed with 50 μL of organophosphorus pesticide standard solutions of different concentrations, i.e., 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, and 100 ng / mL, and a blank control was distilled water. After incubation at room temperature for 1 h, 50 μL of 1 μM HRP, 50 μL of 0.5 mM substrate 1-naphthyl acetate, i.e., 1-NA, 50 μL of 1.5 mM TMB, 50 μL of 1 mM H2O2, and 900 μL of 0.2 M, pH 5.0 acetate buffer were added to the reaction solution in sequence, and the mixture was shaken for 5-10 s and incubated at room temperature in the dark for 30 min. The absorbance value A at 659 nm was measured by a UV-visible photometer. 659 , the inhibition rate I (%) was calculated according to the following formula:

[0045]

[0046] Where, I is the esterase activity inhibition rate; A0 is the absorbance value of the control reaction solution at 659nm A 659 ; A1 is the absorbance value of the sample reaction solution at 659nm;

[0047] Then, the concentration logarithm lg of the organophosphorus pesticide standard solution was taken as the independent variable X, and the inhibition rate was taken as the dependent variable Y. The Excel software was used to establish a linear regression equation of one variable, Y=aX+b;

[0048] (2) Take 50 μL of the sample treatment solution to be tested, and perform the operation according to step (1) to calculate the inhibition rate I, and substitute it into the concentration linear standard linear equation in the above step (1) to calculate the concentration of the organophosphorus pesticide in the sample to be tested.

[0049] Preferably, the method uses glutaraldehyde GA to aminize the surface of chitosan CTS, and then cross-links with glycine Gly to prepare a new CTS / GA / Gly composite material. The plant esterase is immobilized using the principle of simple physical adsorption, and the immobilized plant esterase is used as the detection enzyme. The substrate 1-naphthyl acetate 1-NA generates 1-N with reduction activity under the catalysis of the enzyme, which can inhibit the color development / colorimetric changes of the HRP-induced TMB / H2O2 system. According to the principle of enzyme inhibition, OPs can inhibit this color development reaction, and it is proportional to the amount of OPs; accordingly, a dual-enzyme colorimetric method based on immobilized plant esterase was successfully invented for the detection of OPs. Figure 1 shown.

[0050] Preferably, the plant esterase is derived from mung beans, and the preparation steps are as follows:

[0051] Grind 40g of mung bean into powder, add 200mL of deionized water DIW and stir for 40min, centrifuge at 3000rpm at room temperature for 20min, collect 20.0mL of supernatant, add different saturation, i.e. 20-90% ammonium sulfate gradient purification at 4℃, let stand for 12h, centrifuge at 8000rpm for 10min, collect precipitate, dissolve with 0.02mol / L, pH7.5 PBS buffer, purify with diethylaminoethyl (DEAE)-cellulose anion exchange column (length×diameter: 50mm×16mm) equilibrated with 0.02mol / L, pH7.5 PBS buffer 4 times the volume of the purification column, elute with the same buffer at a flow rate of 30mL / h, collect fractions, centrifuge at 5℃, 1645g for 25min, ultrafilter with 30kDa ultrafiltration tube, take concentrated enzymatic hydrolysate and filter through sephadex G-25 gel column chromatography, eluted with buffer, flow rate of 30mL / h, collected 3mL fractions, combined the fractions with higher absorbance at 595nm, concentrated in vacuo to obtain purified plant esterase, and stored at 4℃ for future use.

[0052] Preferably, the preparation steps of the immobilized plant esterase are as follows:

[0053] 1) Preparation of chitosan GTS gel: Chitosan powder was immersed in a 5% by volume glacial acetic acid solution at a ratio of 1:5 g:mL, and mechanically stirred at 25°C and 1000 rpm for 4 hours, and a 0.1M NaOH solution was added dropwise at a volume ratio of chitosan solution to NaOH solution of 1:10, and stirring was continued at 1000 rpm overnight to prepare a crude chitosan gel; filtered, rinsed with distilled water to pH = 7.0, and chitosan gel, i.e., CTS gel, was obtained, and placed in a 70% by volume ethanol solution and stored at 4°C;

[0054] 2) Preparation of chitosan gel GTS / GA activated by glutaraldehyde GA: Prepare glutaraldehyde working solution: Mix pH 7.0, 0.2M sodium dihydrogen phosphate solution and 25% glutaraldehyde solution in a volume ratio of 11.2:16.8 to obtain a newly prepared glutaraldehyde working solution. Then, add chitosan gel in a ratio of g:mL of 10:28 to the newly prepared glutaraldehyde working solution, and continuously stir mechanically at 25°C and 200rpm for 1h to prepare a crude chitosan gel activated by glutaraldehyde. Wash the chitosan gel thoroughly with deionized water, and filter with filter paper to remove excess water to obtain a glutaraldehyde-activated chitosan gel, namely CTS / GA gel;

[0055] 3) Preparation of CTS / GA / Gly composite immobilization material: CTS / GA gel was placed in a 0.5M, pH 8.0 glycine Gly solution, the ratio of CTS / GA gel: glycine Gly solution was 10:90 g:mL, and the mixture was stirred continuously at 25°C and 200 rpm for 48 h. The obtained CTS / GA gel was thoroughly soaked in deionized water for 48 h, filtered, and rinsed with distilled water to remove free Gly. The prepared CTS / GA / Gly composite immobilization material was stored in a 70% volume concentration ethanol solution at 4°C for future use;

[0056] 4) Immobilization of plant esterase: The purified plant esterase was dissolved in 0.02 mol / L, pH 7.5 PBS buffer at a ratio of 20:10 (mg:mL) to obtain an enzyme solution. The initial loading amount of the CTS / GA / Gly composite immobilized material was 10 mg / g. The immobilized material and the enzyme solution were mixed at a ratio of 1:20 (g:mL) of immobilized material: enzyme solution. The mixture was stirred continuously at 25°C and 200 rpm for 12 h, filtered, washed with distilled water, and the prepared immobilized plant esterase was stored at 4°C.

[0057] Preferably, the dual enzymes are immobilized mung bean esterase (I-mANAE) and horseradish peroxidase (HRP).

[0058] The reaction system of the method also includes: the color developing substrate is TMB, 1-NA, H2O2, and the buffer is acetate buffer.

[0059] Preferably, the sample treatment solution is prepared by washing the fruit and vegetable samples, cutting them into small pieces, adding acetone, and homogenizing; the sample solution is centrifuged at 8000 rpm for 20 min at 37°C, the supernatant is collected, and filtered through a 0.2 μm polyvinylidene fluoride (PVDF) membrane, the filtrate is collected, and diluted 10 times with deionized water to obtain a sample treatment solution for organophosphorus pesticide analysis.

[0060] Preferably, the organophosphorus pesticide is dichlorvos.

[0061] Preferably, the detection range of the method is 10-100 ng / mL.

[0062] Application of the method described above in the detection of organophosphorus pesticides.

[0063] The above-mentioned method is applied to the detection of OPs residues in fruits and vegetables.

[0064] Specifically, the relevant preparation and detection are as follows:

[0065] Example 1 Preparation of plant esterase

[0066] 40 g of mung bean was ground into powder, 200 mL of deionized water (DIW) was added and stirred for 40 min, centrifuged at room temperature (3000 rpm) for 20 min, 20.0 mL of supernatant was collected, and different saturations (20, 30, 40, 50, 60, 70, 80, 90%) of ammonium sulfate were added at 4 °C for gradient purification, allowed to stand for 12 h, centrifuged (8000 rpm, 10 min), the precipitate was collected, dissolved in PBS buffer (0.02 mol / L, pH 7.5), and passed through a DEAE-cellulose anion exchange column (length × diameter: 50 mm × 16 mm) equilibrated with PBS buffer (0.02 mol / L, pH 7.5) 4 times the volume of the purification column, eluted with the same buffer at a flow rate of 30 mL / h, the fractions were collected, centrifuged (1645 g, 5 °C) for 25 min, ultrafiltered with a 30 kDa cellulose membrane ultrafiltration tube, and the concentrated enzymatic hydrolysate was filtered through sephadex G-25 gel column chromatography, eluted with buffer, flow rate of 30mL / h, collected 3mL fractions, combined with the fractions with higher absorbance at 595nm, concentrated in vacuo, and the molecular weight (MW) of the enzyme was determined by SDS-PAGE. Figure 2 As shown, there are multiple protein impurity bands with molecular weights of 25-100 kDa in the crude enzyme slurry (band a), and there is only one clear protein band in band b of the purified mANAE, indicating that high-purity ANAE was prepared using mung beans. Based on the relative mobility (Rf) of different protein markers, the molecular weight of plant esterase is estimated to be about 34.5 kDa.

[0067] Example 2 Preparation of immobilized plant esterase

[0068] 10 g of chitosan powder was immersed in 50 mL of glacial acetic acid solution (5%, v / v, volume percentage), mechanically stirred at 25° C. and 1000 rpm for 4 h, and a 0.1 M NaOH solution was added dropwise according to a volume ratio of chitosan solution to NaOH solution of 1:10, and stirring was continued at 1000 rpm overnight to prepare a crude chitosan gel; filtered, and rinsed with distilled water until pH = 7.0 to obtain a chitosan gel, namely, CTS gel;

[0069] A pH 7.0, 0.2M sodium dihydrogen phosphate solution and a 25% glutaraldehyde solution were mixed in a ratio of 11.2:16.8 (v / v, volume ratio) to obtain a newly configured glutaraldehyde working solution. Subsequently, chitosan gel was added to the newly configured glutaraldehyde working solution in a ratio of 10:28 (w / v, g: mL), and mechanical stirring was continued at 25° C. and 200 rpm for 1 hour to prepare a crude chitosan gel activated by glutaraldehyde. The chitosan gel was thoroughly washed with deionized water, and excess water was removed by filtering with filter paper to obtain a glutaraldehyde-activated chitosan gel, namely, CTS / GA gel;

[0070] The CTS / GA gel was placed in a 0.5 M, pH 8.0 glycine Gly solution, the ratio of CTS / GA gel to glycine Gly solution was 10:90 g:mL, and the mixture was stirred continuously for 48 h at 25°C and 200 rpm. The obtained CTS / GA gel was thoroughly soaked in deionized water for 48 h, filtered, and rinsed with distilled water to remove free Gly, thereby preparing a CTS / GA / Gly composite immobilization material.

[0071] The purified plant esterase was dissolved in PBS buffer (0.02 mol / L, pH 7.5) at a ratio of 20:10 (mg:mL) to obtain an enzyme solution. The initial loading amount of the CTS / GA / Gly composite immobilized material was 10 mg / g. The immobilized material and the enzyme solution were mixed at a ratio of 1:20 (g:mL) of immobilized material: enzyme solution. The mixture was stirred continuously for 12 h at 25°C and 200 rpm, filtered, and washed with distilled water to prepare the immobilized plant esterase.

[0072] Example 3 Effect of immobilization on enzyme optimal temperature

[0073] Temperature is one of the important factors affecting enzyme activity. Take 50 μL of the plant esterase prepared in Example 1 (2 mg / mL) and the immobilized plant esterase prepared in Example 2 (2 mg / mL), respectively, mix with 50 μL of 1-naphthyl acetate 1-NA (1 mM), 50 μL of Fast Blue B salt (mass concentration 0.08%) and 1850 μL of acetate buffer (0.2 M, pH 3.0), vortex oscillation for 10 seconds, and incubate at 20-90°C for 30 minutes. The absorbance of the reaction solution at 595 nm was determined by spectrophotometry. The enzyme activity was determined according to the 1-N standard analysis curve (y=10.27x, where y is the absorbance value and x is the α-naphthol concentration, μM), and the enzyme activity was expressed by the 1-N generation amount (μmol / min·g).

[0074] Figure 3The relative activities of free and immobilized enzymes with respect to temperature are shown. The optimum temperature for the free enzyme is 40°C. There was no significant change in the optimum temperature (40°C) after immobilization. Immobilized plant esterases showed higher relative activity at higher temperatures (50-80°C) compared to the free enzyme. These results highlight the effect of the immobilization material on the thermal stability of plant esterases. This may be due to the improved rigidity of the enzyme structure even when exposed to high temperatures.

[0075] Example 4 Effect of immobilization on the optimal pH of the enzyme

[0076] pH is another important factor affecting enzyme activity. Take 50 μL of the plant esterase prepared in Example 1 (2 mg / mL) and the immobilized plant esterase prepared in Example 2 (2 mg / mL), respectively, mix with 50 μL 1-NA (1 mM), 50 μL Fast Blue B salt (mass concentration 0.08%) and 1850 μL acetate buffer (0.2M, pH 3.0-10), vortex oscillation for 10 seconds, and incubate at 20-90°C for 30 minutes. The absorbance of the reaction solution at 595 nm was determined by spectrophotometry. The enzyme activity was determined according to the 1-N standard analysis curve (y=10.27x, where y is the absorbance value and x is the α-naphthol concentration, μM), and the 1-N generation amount (HA, μmol / min·g) was used to represent the enzyme activity.

[0077] Figure 4 The results showed that the relative activities of free enzyme and immobilized enzyme were the highest at pH 5 and 6, respectively, and the enzyme activity was significantly improved after immobilization at pH 6.0-10.0. The change of the optimal pH value and the wider pH curve after immobilization may be caused by various interactions between the enzyme and the supporting matrix, such as ionic interactions and hydrogen bonds.

[0078] Example 5 Effect of immobilization on enzyme storage stability

[0079] The storage stability of the enzyme is one of the main indicators for evaluating its performance. Take 50 μL of the non-immobilized plant esterase (2 mg / mL) prepared in Example 1 and the immobilized plant esterase (2 mg / mL) prepared in Example 2, respectively, mix with 50 μL 1-NA (1 mM), 50 μL Fast Blue B salt (mass concentration 0.08%) and 1850 μL acetate buffer (0.2 M, pH 3.0-10), vortex oscillate for 10 seconds, and incubate at 25°C for 30 minutes. The absorbance of the reaction solution at 595 nm was determined by spectrophotometry. The enzyme activity was determined according to the 1-N standard analysis curve (y=10.27x, where y is the absorbance value and x is the α-naphthol concentration, μM), and the enzyme activity was expressed by the 1-N generation amount (μmol / min·g).

[0080] like Figure 5The results showed that after being stored at room temperature for 30 days, the enzyme activity of the immobilized plant esterase could be maintained at more than 80% of the original value, which was significantly higher than the 41% of the free plant esterase. The storage stability was significantly improved.

[0081] Example 6 Effect of immobilization on enzyme affinity

[0082] The affinity of the enzyme for the substrate affects the specificity and sensitivity of the test. Take 50 μL of the non-immobilized plant esterase (2 mg / mL) prepared in Example 1 and the immobilized plant esterase (2 mg / mL) prepared in Example 2, respectively, and mix with 50 μL of different concentrations of 1-NA (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mmol / L), 50 μL of Fast Blue B salt (mass concentration 0.08%) and 1850 μL of acetate buffer (0.2M, pH 3.0-10), vortex oscillation for 10 seconds, and incubate at 25°C for 30 minutes. The absorbance of the reaction solution at 595 nm was measured by spectrophotometry. The reaction rates of fixed esterase and free esterase (i.e., the amount of 1-N produced per unit time, μmol / min) were determined based on the 1-N standard analysis curve (y = 10.27x, where y is the absorbance value and x is the α-naphthol concentration, μM). The Lineweaver-Burk double reciprocal method was used to calculate K according to the following formula: m and V max value.

[0083]

[0084] Where: V-reaction rate, μmol / min; S-substrate concentration, mmol / L; Km-Michaelis constant, mmol / L; V max -Maximum reaction rate, mol / min·L.

[0085] Michaelis-Menten(K m ) value is an indicator of the affinity of the enzyme for the substrate, K m Lower values ​​indicate higher affinity for the enzyme. Figure 6 The typical Michaelis-Menten curve of 1-NA enzymatic hydrolysis in the concentration range of 0.05-1.4mmol / L was shown. The constant Km value of the immobilized plant esterase with 1-NA as substrate was 0.049mM, which was lower than the Km value of the free plant esterase (0.067mM), indicating that the affinity of the immobilized plant ester for 1-NA was higher than that of the free plant esterase. The reduction of the immobilized enzyme Km may be due to conformational changes, steric effects, or enhanced accessibility of the substrate to the immobilized enzyme.

[0086] Example 7 Test Principle Analysis

[0087] In order to analyze the feasibility of the method of the present invention, the spectra of different reaction systems were detected in the range of 200-500 nm using a UV-visible spectrophotometer, and the absorbance value (A 326 ) changes within 10 minutes. The reaction system includes: 150μL immobilized esterase (3mg / mL) + 2850μL acetate buffer (0.2M, pH 5.0) reaction solution, 150μL 1-NA (0.5mM) + 2850μL acetate buffer (0.2M, pH 5.0) reaction solution, 150μL 1-N (0.5mM) + 2850μL acetate buffer (0.2M, pH5.0) reaction solution, 150μL immobilized esterase (3mg / mL) + 150μL 1-NA (0.5mM) + 2700μL acetate buffer (0.2M, pH5.0) mixed solution. In addition, the absorbance value changes of the color reaction system at 300-800nm ​​and the absorbance value at 659nm (A 659 ) changes within 10 min of reaction, the color reaction system includes: 150μLTMB (1mM) + 2850μL acetate buffer (0.2M, pH 5.0); 150μLTMB (1mM) + 150μL H2O2 (1mM) + 2700μL acetate buffer (0.2M, pH 5.0) reaction solution; 150μLTMB (1mM) + 150μL H2O2 (1mM) + 150μL HRP (1μM) + 2550μL acetate buffer (0.2M, pH 5.0) mixed solution; 150μLTMB (1mM) + 150μL H2O2 (1mM) + 150μL HRP (1μM) + 150μL 1-N (0.5 mM) + 2400 μL acetate buffer (0.2 M, pH 5.0) mixed solution.

[0088] The experimental principle of the present invention is as follows: The absorbance of different systems at wavelengths of 400-800nm ​​was investigated. Figure 7. The results showed that: TMB and TMB / H2O2 reaction systems had no obvious absorption peaks at 659nm, while the TMB / H2O2 / HRP reaction system showed an obvious characteristic absorption peak at 659nm, which was the absorption peak of oxidized TMB, and the reaction system changed from colorless to blue. The 659nm absorption peak of the TMB / H2O2 / HRP / 1-N reaction system was significantly reduced, and the reaction system faded, indicating that 1-N can inhibit the production of oxTMB. In addition, the immobilized esterase / 1-NA showed a characteristic peak at 329nm, which coincided with the position of 1-N, indicating that the immobilized esterase hydrolyzed 1-NA to generate 1-N. In summary, 1-N can couple the plant esterase / 1-NA system and the TMB / H2O2 / HRP reaction system. Based on this, combined with the principle of enzyme inhibition, when organophosphorus pesticides OPs are added to the reaction system, plant esterase is inhibited, resulting in a decrease in 1-N production, causing the color of the reaction system to turn blue and the absorbance to increase. Through visual observation and software fitting, a standard equation for the absorbance value and the concentration of organophosphorus pesticides is established to quickly qualitatively or quantitatively detect organophosphorus pesticides.

[0089] Example 8: Double enzyme colorimetric method for detecting dichlorvos (DDVP)

[0090] DDVP was used as an OPs model. 50 μL of fixed esterase solution (final concentration 1 mg / mL) was incubated with 50 μL of 0.01-100 ng / mL DDVP at room temperature for 1 hour, and then 50 μL of HRP (1 μM), 50 μL of TMB (1.5 mM), 50 μL of 1-NA (0.5 mM), 50 μL of H2O2 (1 mM), and 900 μL of acetate buffer (0.2 M, pH 5.0) were added in sequence. Distilled water was used instead of DDVP as the control reaction solution. All reaction solutions were shaken for 5-10 seconds and incubated in the dark for 30 minutes at room temperature; the absorbance value A at 659 nm was measured by UV-visible photometer. 659 , the inhibition rate I (%) was calculated according to the following formula.

[0091]

[0092] Where, I is the esterase activity inhibition rate; A0 is the absorbance value of the control reaction solution at 659nm (A 659 ); A1 is the absorbance value of the sample reaction solution at 659 nm.

[0093] The logarithmic value (lg) of DDVP concentration was used as the independent variable (X) and the inhibition rate was used as the dependent variable (Y). The detection limit (LOD) of DDVP in the detection method was calculated according to the following formula to evaluate the performance of the invention method. Where a represents the sensitivity of the method, S y / x represents the standard deviation of the fit.

[0094] Figure 8 a shows that in the range of 0.01 to 100 ng / mL, with the increase of DDVP concentration, A 659 The values ​​increased dramatically, revealing the dose-dependent accumulation of oxTMB by DDVP. Due to the inhibition of plant esterase by DDVP, the production of 1-N was reduced, leading to enhanced TMB oxidation and color development. Figure 8 b shows that within the range of 10 to 100 ng / mL, the established linear equation is Y = 48.69X-22.76, where Y is the enzyme inhibition rate (%), X is the logarithmic value of DDVP concentration (lgmM), and the correlation coefficient is (R 2 =0.9946), and the detection limit of DDVP was 1.32 ng / mL. The comparison of the detection performance with the previous report is shown in Table 1. The LOD of dichlorvos determined by this method is lower than or equivalent to the similar TMB colorimetric method reported previously. The experimental results show that this method has good quantitative analysis effect. This is related to the large surface area of ​​the CTS carrier, the increase in the fixed amount of plant esterase, the recognition ability of OPs, and the catalytic effect on 1-NA.

[0095] Table 1 Comparison between the present invention and the colorimetric method based on TMB color development

[0096]

[0097] Among them: [1] LJHuang, DWSun, HBPu, QYWei, LPLuo, JLWang, Acolorimetric paper sensor based on the domino reaction of acetylcholinesterase and degradableγ-MnOOH nanozyme for sensitive detection of organophosphoruspesticides, Sens. Actuators, B.290 (2019) 573-580.

[0098] [2]X.Zou,L.Huang,Y.Y.Liu,Q.M.Chen,X.K.Zheng,M.K.Fan,Z.J.Gong,Metal-organic framework-derivedFe / C / Bi2O3 / as peroxidase-like nanozymes for thedetection of organophosphorus pesticides,Sens.Actuators,B.393(2023).

[0099] [3]S.G.Liu,H.Y.Wang,Q.Zhao,W.L.Gao,X.B.Shi,Z.H.Liu,Aportablecolorimetric sensing platform for rapid and sensitive quantificationofdichlorvos pesticide based on Fe-Mn bimetallic oxide nanozyme-participatedhighly efficient chromogenic catalysis,Anal.Chim.Acta.1292(2024).

[0100] [4]B.Y.Li,W.L.Wu,J.M.Lin,T.Wang,Q.Z.Hu,L.Yu,Water in liquid crystalemulsion-based sensing platform for colorimetric detection oforganophosphoruspesticide,Food Chemistry.436(2024).

[0101] [5]G.Q. Deng, H.Y. Chen, Q. Shi, L.X. Ren, K. Liang, W.J. Long, W. Lan, X.L. Han, Y.B. She, H.Y. Fu, Colorimetric assay based on peroxidase-like activity of dodecyltrimethylammonium bromide-tetramethyl zinc(4-pyridinyl)porphyrin for detection of organophosphorus pesticides, Microchim. Acta. 189(2022).

[0102] [6]J. Cao, M. Wang, Y.X. She, A.M. Abd El-Aty, A. Hacimüftüoglu, J. Wang, M.M. Yan, S.H. Hong, S.B. Lao, Y.L. Wang, Rapid colorimetric determination of the pesticides carbofuran and dichlorvos by exploiting their inhibitory effect on the aggregation of peroxidase-mimicking platinum nanoparticles, Microchim. Acta. 186(2019).

[0103] [7]Z.N. Wei, H.Q. Li, J. Wu, Y.L. Dong, H.Y. Zhang, H.L. Chen, C.L. Ren, 3D RGO-NiFe2O4 / NiO nanoparticles for fast and simple detection of organophosphorus pesticides, Chin. Chem. Lett. 31(2020)177-180.

[0104] Example 9 Applicability Test of Double Enzyme Colorimetry

[0105] The applicability of the designed method was evaluated by a standard addition recovery test. Fruit and vegetable samples (cabbage, tomato, jujube) were washed and cut into small pieces, and 1.2 mL of DDVP solution (acetone as solvent) of different concentrations (50, 100, 300, 600 μg / L) was sprayed on the sample surface. After the solvent evaporated, the sample was chopped and homogenized. The sample solution was centrifuged (8000 rpm) at 37°C for 20 min, and the collected supernatant was filtered with a 0.2 μm PVDF filter and diluted 10 times with deionized water. The diluent was used as the sample test solution, and the absorbance value of each test solution at 659 nm was determined by the method described in Example 8, and the DDVP test value of each sample test solution was calculated according to the standard curve equation drawn in Example 8. The results are shown in Table 2. DDVP was not detected in the original sample, and in the sample test solution with DDVP added, the sample recovery of each sample was in the range of 91.17% to 106.50%, and the RSD was lower than 5.48% (n=3). The results showed that the designed colorimetric method had high accuracy and applicability for OPs detection in actual samples.

[0106] Table 2 Detection of organophosphorus pesticides sprayed in fruit and vegetable samples by dual enzyme detection method (n=3)

[0107]

[0108]

[0109] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for detecting organophosphorus pesticides by dual enzyme colorimetry based on immobilized plant esterase, characterized in that: The steps include: (1) A one-step method was used to perform a colorimetric reaction test and a test standard curve was drawn, that is, each 50 μL of a final concentration of 1 mg / mL immobilized plant esterase was mixed with 50 μL of organophosphorus pesticide standard solutions of different concentrations, i.e., 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, and 100 ng / mL, and a blank control was distilled water. After incubation at room temperature for 1 h, 50 μL of 1 μM HRP, 50 μL of 0.5 mM substrate 1-naphthyl acetate, i.e., 1-NA, 50 μL of 1.5 mM TMB, 50 μL of 1 mM H2O2, and 900 μL of 0.2 M, pH 5.0 acetate buffer were added to the reaction solution in sequence, and the mixture was shaken for 5-10 s and incubated at room temperature in the dark for 30 min. The absorbance value A at 659 nm was measured by a UV-visible photometer. 659 , the inhibition rate I (%) was calculated according to the following formula: Where, I is the esterase activity inhibition rate; A0 is the absorbance value of the control reaction solution at 659nm A 659 ; A1 is the absorbance value of the sample reaction solution at 659nm; Then, the concentration logarithm lg of the organophosphorus pesticide standard solution was taken as the independent variable X, and the inhibition rate was taken as the dependent variable Y. The Excel software was used to establish a linear regression equation, Y=aX+b; (2) Take 50 μL of the sample treatment solution to be tested, and perform the operation according to step (1) to calculate the inhibition rate I, and substitute it into the concentration linear standard linear equation in the above step (1) to calculate the concentration of the organophosphorus pesticide in the sample to be tested.

2. The method according to claim 1, characterized in that: The method uses glutaraldehyde GA to aminize the surface of chitosan CTS, and then cross-links with glycine Gly to prepare a new CTS / GA / Gly composite material. The plant esterase is fixed by a simple physical adsorption principle, and the fixed plant esterase is used as the detection enzyme. The substrate 1-naphthyl acetate 1-NA generates 1-N with reduction activity under the catalysis of the enzyme, which can inhibit the color development / colorimetric change of the TMB / H2O2 system induced by HRP. According to the principle of enzyme inhibition, OPs can inhibit this color development reaction, and it is proportional to the amount of OPs; accordingly, a dual enzyme colorimetric method based on fixed plant esterase is successfully invented, which is a dual enzyme colorimetric method for detecting OPs.

3. The method according to claim 1, characterized in that: The plant esterase is derived from mung beans, and the preparation steps are as follows: Grind 40g of mung bean into powder, add 200mL of deionized water DIW and stir for 40min, centrifuge at 3000rpm at room temperature for 20min, collect 20.0mL of supernatant, add different saturations, i.e. 20-90% ammonium sulfate gradient purification at 4℃, let stand for 12h, centrifuge at 8000rpm for 10min, collect precipitate, dissolve with 0.02mol / L, pH7.5 PBS buffer, use diethylaminoethyl DEAE-cellulose anion exchange column equilibrated with 0.02mol / L, pH7.5 PBS buffer 4 times the volume of the purification column in advance, elute with the same buffer at a flow rate of 30mL / h, collect fractions, centrifuge at 5℃, 1645g for 25min, ultrafilter with a 30kDa ultrafiltration tube, and take the concentrated enzymatic hydrolysate to pass through sephadex G-25 gel column chromatography, eluted with buffer, flow rate of 30mL / h, collected 3mL fractions, combined the fractions with higher absorbance at 595nm, concentrated in vacuo to obtain purified plant esterase, and stored at 4℃ for future use.

4. The method according to claim 1, characterized in that: The preparation steps of the immobilized plant esterase are as follows: 1) Preparation of chitosan GTS gel: Chitosan powder was immersed in a 5% by volume glacial acetic acid solution at a ratio of 1:5 g:mL, and mechanically stirred at 25°C and 1000 rpm for 4 hours, and a 0.1M NaOH solution was added dropwise at a volume ratio of chitosan solution to NaOH solution of 1:10, and stirring was continued at 1000 rpm overnight to prepare a crude chitosan gel; filtered, rinsed with distilled water to pH = 7.0, and chitosan gel, i.e., CTS gel, was obtained, and placed in a 70% by volume ethanol solution and stored at 4°C; 2) Preparation of chitosan gel GTS / GA activated by glutaraldehyde GA: Prepare glutaraldehyde working solution: Mix pH 7.0, 0.2M sodium dihydrogen phosphate solution and 25% glutaraldehyde solution in a volume ratio of 11.2:16.8 to obtain a newly prepared glutaraldehyde working solution. Then, add chitosan gel in a ratio of g:mL of 10:28 to the newly prepared glutaraldehyde working solution, and continuously stir mechanically at 25°C and 200rpm for 1h to prepare a crude chitosan gel activated by glutaraldehyde. Wash the chitosan gel thoroughly with deionized water, and filter with filter paper to remove excess water to obtain a glutaraldehyde-activated chitosan gel, namely CTS / GA gel; 3) Preparation of CTS / GA / Gly composite immobilization material: CTS / GA gel was placed in a 0.5M, pH 8.0 glycine Gly solution, the ratio of CTS / GA gel: glycine Gly solution was 10:90 g:mL, and the mixture was stirred continuously at 25°C and 200 rpm for 48 h. The obtained CTS / GA gel was thoroughly soaked in deionized water for 48 h, filtered, and rinsed with distilled water to remove free Gly. The prepared CTS / GA / Gly composite immobilization material was stored in a 70% volume concentration ethanol solution at 4°C for future use; 4) Immobilization of plant esterase: The purified plant esterase was dissolved in 0.02 mol / L, pH 7.5 PBS buffer at a ratio of 20:10 mg:mL to obtain an enzyme solution. The initial loading amount of the CTS / GA / Gly composite immobilized material was 10 mg / g. The immobilized material and the enzyme solution were mixed at a ratio of 1:20 g:mL of the immobilized material: enzyme solution. The mixture was stirred continuously for 12 h at 25°C and 200 rpm, filtered, washed with distilled water, and the prepared immobilized plant esterase was stored at 4°C.

5. The method according to claim 1, characterized in that: The double enzymes are immobilized mung bean esterase and horseradish peroxidase. The reaction system of the method also includes: the color developing substrate is TMB, 1-NA, H2O2, and the buffer is acetate buffer.

6. The method according to claim 1, characterized in that: The preparation method of the sample treatment solution comprises the following steps: washing the fruit and vegetable samples, cutting them into small pieces, adding acetone, and homogenizing; centrifuging the sample solution at 8000 rpm for 20 minutes at 37° C., collecting the supernatant, filtering through a 0.2 μm polyvinylidene fluoride (PVDF) membrane, collecting the filtrate, and diluting it 10 times with deionized water to obtain the sample treatment solution for organophosphorus pesticide analysis.

7. The method according to any one of claims 1 to 6, characterized in that: The organophosphorus pesticide is dichlorvos.

8. The method according to any one of claims 1 to 7, characterized in that: The detection range of the method is 10 to 100 ng / mL.

9. Use of the method according to any one of claims 1 to 8 in the detection of organophosphorus pesticides.

10. Use of the method according to any one of claims 1 to 8 in the detection of OPs residues in fruits and vegetables.

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