Detection sensor and application thereof

Abstract

The invention provides a detection sensor and application thereof, and belongs to the field of pesticide detection. The detection sensor provided by the invention comprises an acetylcholine solution, an acetylcholin esterase solution, a choline oxidase solution, a Cu-BTC dispersion liquid and a solution containing a color developing agent capable of generating a hydroxyl color developing group, or comprises acetylcholine, a color developing agent capable of generating a hydroxyl color developing group and a Cu-BTC-based composite material; the Cu-BTC based composite material is prepared from Cu-BTC, choline oxidase and acetylcholin esterase, wherein the choline oxidase and the acetylcholin esterase are loaded on the Cu-BTC. Acetylcholine is hydrolyzed by acetylcholin esterase to generate choline, the choline is oxidized by choline oxidase to generate H2O2, the H2O2 is oxidized by Cu-BTC to generate. OH, and the. OH can oxidize a color developing agent to generate a colored substance. The organophosphorus pesticide can inhibit the activity of acetylcholin esterase to change the color of the solution, and then quantitative analysis of the organophosphorus pesticide is realized through the absorbance and the absorbance of a standard solution.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and particularly to a detection sensor and its application. Background Art

[0002] Organophosphorus pesticides are inexpensive and have a broad spectrum of efficacy. Their use plays a positive role in increasing the yield of agricultural products, resulting in the widespread use of organophosphorus pesticides. However, unreasonable use time, variety, and dosage ultimately lead to their residues in agricultural products. To correctly manage and regulate the use of pesticides, people need to accurately evaluate their pollution status in food. Therefore, the accurate and sensitive detection of organophosphorus pesticide residues in food has become the key to ensuring the healthy development of the agricultural and food industries.

[0003] Currently, there are already a variety of methods for detecting organophosphorus pesticides that are widely used, such as gas chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, etc., but they all have certain limitations. For example, the equipment is expensive and requires specialized technical personnel to operate, and the sample pretreatment is relatively complex. In contrast, the colorimetric method does not require expensive instrument equipment and has the advantages of low cost and easy portability in the detection of organophosphorus pesticides.

[0004] Metal-organic frameworks (MOFs) are crystalline materials formed by connecting various metal ions and organic ligands, with characteristics such as high porosity, large specific surface area, high loading capacity, and homogeneous structure. In recent years, a large number of research reports have shown that metal-organic framework materials composed of metal elements such as Fe, Cu, Co, Ni, and Ce have mimetic enzyme catalytic activity. Among them, copper-based metal-organic framework materials have the advantages of low cost, unique morphological characteristics, and excellent physical and chemical properties, and have been widely used in multiple fields such as colorimetry and biochemical analysis of biomolecules. For example, the detection of harmful toxins in milk and the detection of veterinary drug residues in feed and livestock products. However, there are few reports on the colorimetric detection of organophosphorus pesticides by metal-organic frameworks. Summary of the Invention

[0005] The present invention provides a detection sensor and its application. The detection sensor of the present invention can achieve colorimetric detection of organophosphorus pesticides.

[0006] The present invention provides a detection sensor, including an acetylcholine solution, an acetylcholinesterase solution, a choline oxidase solution, a dispersion of Cu-BTC, and a solution containing a color reagent capable of generating a hydroxyl chromogenic group;

[0007] Or including acetylcholine, a color reagent capable of generating a hydroxyl chromogenic group, and a Cu-BTC-based composite material;

[0008] The Cu-BTC based composite material includes Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase loaded on the Cu-BTC.

[0009] The present invention also provides a detection sensor, which includes an electrolyte containing acetylcholine and a color reagent capable of generating a hydroxyl chromogenic group, a Cu-BTC based composite material, and an electrode; the Cu-BTC based composite material is loaded on the electrode;

[0010] The Cu-BTC based composite material includes Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase loaded on the Cu-BTC.

[0011] Preferably, the dosage ratio of acetylcholinesterase in the acetylcholinesterase solution to acetylcholine in the acetylcholine solution is 20-260 mU:1 g;

[0012] The volume ratio of the acetylcholinesterase solution to the acetylcholine solution is 1:1-3;

[0013] The concentration of the acetylcholinesterase solution is 120-300 mU / mL; the concentration of the acetylcholine solution is 0.5-2 g / mL.

[0014] Preferably, the dosage ratio of acetylcholine in the acetylcholine solution to choline oxidase in the choline oxidase solution is 1 g:4-300 mU;

[0015] The volume ratio of the acetylcholine solution to the choline oxidase solution is 2-15:1;

[0016] The concentration of the choline oxidase solution is 120-300 mU / mL.

[0017] Preferably, the mass ratio of acetylcholine in the acetylcholine solution to Cu-BTC in the dispersion of Cu-BTC is 1000:0.1-12;

[0018] The volume ratio of the acetylcholine solution to the dispersion of Cu-BTC is 2-8:1;

[0019] The concentration of the dispersion of Cu-BTC is 3-12 mg / mL.

[0020] The present invention also provides the application of the sensor described in the above technical solution in the colorimetric detection of organophosphorus pesticides.

[0021] The present invention also provides the application of the detection sensor described in the above technical solution in the electrochemical detection of organophosphorus pesticides.

[0022] Preferably, the colorimetric detection of organophosphorus pesticides includes the following steps:

[0023] Mix the detection sensor described in the above technical solution with the solution to be detected for a color reaction to obtain a color solution;

[0024] According to the absorbance of the color solution and the concentration-absorbance curve of the standard solution, obtain the concentration of organophosphorus pesticides in the solution to be detected.

[0025] Preferably, when the detection sensor includes a solution of acetylcholine, a solution of acetylcholinesterase, a solution of choline oxidase, a dispersion of Cu-BTC, and a solution of a color reagent containing a hydroxyl group-producing chromogenic group, the detection of organophosphorus pesticides includes the following steps:

[0026] Mix the acetylcholinesterase solution with the solution to be detected for the first incubation to obtain a first incubation solution;

[0027] Mix the first incubation solution with the dispersion of Cu-BTC, the acetylcholine solution, and the choline oxidase solution for the second incubation to obtain a second incubation solution containing hydrogen peroxide;

[0028] Mix the second incubation solution with the solution of the color reagent containing a hydroxyl group-producing chromogenic group for a color reaction to obtain a color solution;

[0029] According to the absorbance of the color solution and the concentration-absorbance curve of the standard solution, obtain the concentration of organophosphorus pesticides in the solution to be detected.

[0030] Preferably, the temperature of the first incubation is 30-40 °C and the time is 5-25 min;

[0031] The temperature of the second incubation is 30-40 °C and the time is 20-40 min; the volume ratio of the solution to be detected to the acetylcholinesterase solution is 1-5:1;

[0032] The concentration of organophosphorus in the solution to be detected is 1-100 μg / L.

[0033] Acetylcholinesterase can hydrolyze acetylcholine to produce choline, choline is oxidized by choline oxidase to produce H2O2, H2O2 is oxidized by Cu-BTC to produce hydroxyl radicals (·OH), and ·OH can oxidize the color reagent containing a hydroxyl group-producing chromogenic group to produce a colored substance. When there are organophosphorus pesticides in the test solution, the solution color is light blue or colorless; otherwise, the solution color is dark blue. Organophosphorus pesticides can inhibit the activity of acetylcholinesterase, thus affecting the entire cascade catalytic amplification reaction and causing a change in the solution color. Then, through the concentration-absorbance curve of the solution absorbance and the standard solution, quantitative analysis of organophosphorus pesticides can be achieved. Description of the Drawings

[0034] Figure 1 It is the schematic diagram of the sensor of the present invention;

[0035] Figure 2 It is the SEM image of Cu-BTC in Example 1;

[0036] Figure 3 It is the XRD pattern of Cu-BTC in Example 1;

[0037] Figure 4 It is the result of the ultraviolet spectrophotometer of four groups of solutions in the peroxidase-like activity test of Cu-BTC;

[0038] Figure 5 It is the color development result after shortening the enzyme cascade reaction system in the feasibility analysis of the enzyme cascade system method;

[0039] Figure 6 It is the color development result after extending the enzyme cascade system in the feasibility analysis of the enzyme cascade system method;

[0040] Figure 7 It is the color development result in the enzyme cascade system;

[0041] Figure 8 It is the optimization result of the mass concentration of Cu-BTC, the reaction time of the organophosphorus solution and acetylcholinesterase, the reaction time of the enzyme and substrate and Cu-BTC, the addition amount of acetylcholinesterase, the addition amount of choline oxidase and the addition amount of the substrate acetylcholine;

[0042] Figure 9 It is the curve of methyl parathion concentration and absorbance;

[0043] Figure 10 It is the experimental result diagram of anti-interference;

[0044] Figure 11 It is the experimental result diagram of reproducibility;

[0045] Figure 12 It is the experimental result diagram of stability. Detailed implementation manners

[0046] The present invention provides a detection sensor, including an acetylcholine solution, an acetylcholinesterase solution, a choline oxidase solution, a dispersion of Cu-BTC and a solution containing a color developer capable of generating a hydroxyl color group;

[0047] Or including acetylcholine, a color developer capable of generating a hydroxyl color group and a Cu-BTC-based composite material;

[0048] The Cu-BTC-based composite material includes Cu-BTC, choline oxidase and acetylcholinesterase loaded on the Cu-BTC.

[0049] A detection sensor provided by the present invention includes an acetylcholine solution, an acetylcholinesterase solution, a choline oxidase solution, a dispersion of Cu-BTC, and a solution containing a chromogenic agent capable of generating a hydroxyl chromogenic group.

[0050] In the present invention, the dosage ratio of acetylcholinesterase in the acetylcholinesterase solution to acetylcholine in the acetylcholine solution is preferably 20 - 260 mU:1 g. In specific embodiments of the present invention, the dosage ratio of acetylcholinesterase in the acetylcholinesterase solution to acetylcholine in the acetylcholine solution can be 20 mU:1 g, 60 mU:1 g, 100 mU:1 g, 128 mU:1 g, 900 mU:7 g, 150 mU:1 g, 200 mU:1 g, 240 mU:1 g, or 260 mU:1 g; the volume ratio of the acetylcholinesterase solution to the acetylcholine solution is preferably 1:1 - 3. In specific embodiments of the present invention, the volume ratio of the acetylcholinesterase solution to the acetylcholine solution can be 1:1, 1:1.6, 1:2, 1:2.5, or 1:3.

[0051] The concentration of the acetylcholinesterase solution is preferably 120 - 300 mU / mL. In specific embodiments of the present invention, the concentration of the acetylcholinesterase solution can be 120 mU / mL, 150 mU / mL, 180 mU / mL, 200 mU / mL, 240 mU / mL, 280 mU / mL, or 300 mU / mL; the concentration of the acetylcholine solution is preferably 0.5 - 2 g / mL. In specific embodiments of the present invention, the concentration of the acetylcholine solution can be 0.5 g / mL, 1 g / mL, 1.5 g / mL, or 2 g / mL.

[0052] In the present invention, the dosage ratio of acetylcholine in the acetylcholine solution to choline oxidase in the choline oxidase solution is preferably 1 g:4 - 300 mU. In specific embodiments of the present invention, the dosage ratio of acetylcholine in the acetylcholine solution to choline oxidase in the choline oxidase solution can be 1 g:4 mU, 1 g:37 mU, 260 g:7 mU, 1 g:50 mU, 1 g:100 mU, 1 g:150 mU, 1 g:200 mU, 1 g:250 mU, or 1 g:300 mU.

[0053] The volume ratio of the acetylcholine solution to the choline oxidase solution is preferably 2 to 15:1. In specific embodiments of the present invention, the volume ratio of the acetylcholine solution to the choline oxidase solution can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1; the concentration of the choline oxidase solution is preferably 120 to 300 mU / mL. In specific embodiments of the present invention, the concentration of the choline oxidase solution can be 120 mU / mL, 150 mU / mL, 180 mU / mL, 200 mU / mL, 240 mU / mL, 280 mU / mL, or 300 mU / mL.

[0054] In the present invention, the mass ratio of acetylcholine in the acetylcholine solution to Cu-BTC in the Cu-BTC dispersion is preferably 1000:0.1 to 12. In specific embodiments of the present invention, the mass ratio of acetylcholine in the acetylcholine solution to Cu-BTC in the Cu-BTC dispersion can be 1000:0.1, 1000:1, 1000:2, 1000:3, 220:7, 1000:4, 1000:5, 1000:6, 1000:7, 1000:8, 1000:9, 1000:10, 1000:11, or 1000:12.

[0055] The volume ratio of the acetylcholine solution to the Cu-BTC dispersion is preferably 2 to 8:1. In specific embodiments of the present invention, the volume ratio of the acetylcholine solution to the Cu-BTC dispersion can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1; the concentration of the Cu-BTC dispersion is preferably 3 to 12 mg / mL. In specific embodiments of the present invention, the concentration of the Cu-BTC dispersion can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, or 12 mg / mL; the structure of Cu-BTC is preferably a regular octahedron, and the particle size is preferably 2 to 3 μm.

[0056] In the present invention, the volume ratio of the acetylcholine solution to the solution containing a color reagent capable of generating a hydroxyl chromogenic group is preferably 4 to 8:1; the concentration of the solution containing a color reagent capable of generating a hydroxyl chromogenic group is preferably 3 to 3.5 mg / mL; the color reagent capable of generating a hydroxyl chromogenic group preferably includes TMB.

[0057] Another detection sensor provided by the present invention includes acetylcholine, a color reagent capable of generating a hydroxyl chromogenic group, and a Cu-BTC-based composite material;

[0058] The Cu-BTC based composite material includes Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase. In the present invention, the loading amount of choline oxidase on Cu-BTC is preferably 200 mU:11 mg; the loading amount of acetylcholinesterase on Cu-BTC is preferably 200 mU:11 mg.

[0059] In the present invention, the mass ratio of acetylcholine to Cu-BTC is preferably 10:1; the mass ratio of acetylcholine to a color-developing agent capable of generating a hydroxyl color-developing group is preferably 3:1.

[0060] In the present invention, the preparation method of the Cu-BTC based composite material preferably includes: dispersing Cu-BTC in a solution containing acetylcholinesterase and choline oxidase, followed by solid-liquid separation and washing.

[0061] In the present invention, the dispersion is preferably carried out under stirring, and the dispersion time is preferably 2 h; the solid-liquid separation preferably includes centrifugation.

[0062] The schematic diagram of the present invention is as Figure 1 shown: Acetylcholinesterase can hydrolyze acetylcholine to generate choline, choline is oxidized by choline oxidase to generate H2O2, H2O2 is oxidized by Cu-BTC to generate hydroxyl radicals (·OH), and ·OH can oxidize a color-developing agent capable of generating a hydroxyl color-developing group to generate a colored substance. When there is an organophosphorus pesticide in the test solution, the solution color is light blue or colorless; otherwise, the solution color is dark blue. Organophosphorus pesticides can inhibit the activity of acetylcholinesterase, thus affecting the entire cascade catalytic amplification reaction to cause the solution to change color, and quantitative analysis of organophosphorus pesticides is achieved through the absorbance of the solution and the concentration-absorbance curve of the standard solution.

[0063] The present invention also provides the application of the detection sensor described in the above technical solution in the colorimetric detection of organophosphorus pesticides.

[0064] In the present invention, the colorimetric detection of organophosphorus pesticides preferably includes the following steps:

[0065] Mixing the detection sensor described in the above technical solution with the test solution for a color reaction to obtain a color solution;

[0066] According to the absorbance of the color solution and the concentration-absorbance curve of the standard solution, the concentration of the organophosphorus pesticide in the test solution is obtained.

[0067] In the present invention, when the detection sensor includes a solution of acetylcholine, a solution of acetylcholinesterase, a solution of choline oxidase, a dispersion of Cu-BTC, and a solution containing a color-developing agent capable of generating a hydroxyl color-developing group, the detection of the organophosphorus pesticide includes the following steps:

[0068] Mix the acetylcholinesterase solution with the solution to be detected for the first incubation to obtain a first incubation solution;

[0069] Mix the first incubation solution with a dispersion of Cu - BTC, an acetylcholine solution, and a choline oxidase solution for the second incubation to obtain a second incubation solution;

[0070] Mix the second incubation solution with a solution containing a color reagent capable of generating a hydroxyl chromogenic group for a color reaction to obtain a color solution;

[0071] According to the absorbance of the color solution and the concentration - absorbance curve of the standard solution, obtain the concentration of the organophosphorus pesticide in the solution to be detected.

[0072] In the present invention, the acetylcholinesterase solution is mixed with the solution to be detected for the first incubation to obtain a first incubation solution.

[0073] In the present invention, the volume ratio of the acetylcholinesterase solution to the solution to be detected is preferably 1:1; the concentration of the organophosphorus in the solution to be detected is preferably 1 - 100 μg / L. In specific embodiments of the present invention, the concentration of the organophosphorus in the solution to be detected can be 1 μg / L, 10 μg / L, 20 μg / L, 30 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L, or 100 μg / L.

[0074] In the present invention, the temperature of the first incubation is preferably 30 - 40 °C. In specific embodiments of the present invention, the temperature of the first incubation can be 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C; the time is preferably 5 - 25 min. In specific embodiments of the present invention, the time of the first incubation can be 5 min, 10 min, 15 min, 20 min, or 25 min.

[0075] During the first incubation, the solution to be detected inhibits the activity of acetylcholinesterase.

[0076] After obtaining the first incubation solution, in the present invention, the first incubation solution is mixed with a dispersion of Cu - BTC, an acetylcholine solution, and a choline oxidase solution for the second incubation to obtain a second incubation solution.

[0077] In the present invention, the temperature of the second incubation is preferably 30 to 40 °C. In specific embodiments of the present invention, the temperature of the second incubation can be 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C; the time is preferably 20 to 40 min. In specific embodiments of the present invention, the time of the first incubation can be 20 min, 25 min, 30 min, 35 min or 40 min.

[0078] The second incubation successfully constructs a cascade system and generates hydrogen peroxide.

[0079] After obtaining the second incubation solution, the present invention mixes the second incubation solution with a solution containing a chromogenic agent capable of generating a hydroxyl chromogenic group to carry out a chromogenic reaction, thereby obtaining a chromogenic solution.

[0080] In the present invention, after mixing, the pH value of the obtained solution is preferably adjusted to 3 to 6 with a pH regulator. Controlling the pH at 3 to 6 can achieve the best color development.

[0081] In the present invention, the pH regulator preferably includes a weak acid and a PBS buffer solution. The weak acid preferably includes acetic acid. The concentration of the PBS buffer solution is preferably 0.025 mol / L, and the pH value is preferably 6.86. The volume ratio of the PBS buffer solution to the weak acid is preferably 1:1, and the volume ratio of the solution containing the chromogenic agent capable of generating a hydroxyl chromogenic group to the PBS buffer solution is preferably 1:1.

[0082] In the present invention, the temperature of the chromogenic reaction is preferably 25 to 40 °C. In specific embodiments of the present invention, the temperature of the chromogenic reaction can be 25 °C, 30 °C, 35 °C or 40 °C; the time is preferably 1 to 5 min. In specific embodiments of the present invention, the time of the chromogenic reaction can be 1 min, 2 min, 3 min, 4 min or 5 min.

[0083] After obtaining the chromogenic solution, the present invention determines the concentration of the organophosphorus pesticide in the solution to be detected according to the absorbance of the chromogenic solution and the concentration-absorbance curve of the standard solution.

[0084] In the present invention, when the detection sensor includes acetylcholine, a chromogenic agent capable of generating a hydroxyl chromogenic group, and a Cu-BTC-based composite material, the detection of the organophosphorus pesticide includes the following steps

[0085] Mix the solution to be detected with the Cu-BTC-based composite material for incubation, then mix the obtained product with the acetylcholine solution and then with the chromogenic agent capable of generating a hydroxyl chromogenic group for a chromogenic reaction to obtain a chromogenic solution;

[0086] According to the absorbance of the chromogenic solution and the concentration-absorbance curve of the standard solution, the concentration of the organophosphorus pesticide in the solution to be detected is obtained.

[0087] In the present invention, the wavelength during the measurement of the absorbance is preferably 625 nm.

[0088] The present invention also provides another detection sensor, comprising acetylcholine, a chromogenic agent capable of generating a hydroxyl chromogenic group, a Cu-BTC-based composite material, and an electrode; the Cu-BTC-based composite material is loaded on the electrode;

[0089] The Cu-BTC-based composite material comprises Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase loaded on the Cu-BTC.

[0090] The present invention also provides the application of the detection sensor described in the above technical solution in the electrochemical detection of organophosphorus pesticides.

[0091] The electrochemical detection of organophosphorus pesticides preferably comprises the following steps:

[0092] Mix a solution to be detected containing organophosphorus with an acetylcholinesterase solution to obtain a mixture;

[0093] Mix the mixture with a dispersion of Cu-BTC and a choline oxidase solution, then drop the mixture onto the electrode and dry it to obtain a detection sensor to be detected;

[0094] Place the detection sensor to be detected into a DPV test solution containing acetylcholine and a chromogenic agent capable of generating a hydroxyl chromogenic group for DPV testing to obtain a peak current signal value;

[0095] According to the peak current signal value of the solution to be detected and the linear relationship between the peak current signal value and the concentration of the standard organophosphorus solution measured under the same test conditions, the concentration of the organophosphorus in the solution to be detected is obtained.

[0096] The following is a detailed description of the detection sensor and its application provided by the present invention in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0097] Zinc oxide (analytical pure), N,N-dimethylformamide (analytical pure), copper nitrate trihydrate (analytical pure), 1,3,5-benzenetricarboxylic acid (analytical pure), absolute ethanol (analytical pure), acetic acid (analytical pure), 3,3',5,5'-tetramethylbenzidine (analytical pure), hydrogen peroxide, sodium acetate, sodium phosphate, acetylcholine, acetylcholinesterase, choline oxidase, choline, sodium chloride, potassium chloride, sodium carbonate, urea, glucose;

[0098] The sample Chinese cabbages and lettuces were all purchased from the local market.

[0099] Reference material: Methyl parathion (MP) with an initial concentration of 1×10 -6 mol / L was diluted to 100 μg / L with absolute ethanol.

[0100] HH·S21-4 Digital display constant temperature water bath: Changzhou Jintan Jingda Instrument Manufacturing Co., Ltd.;

[0101] bsa124s Analytical balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.;

[0102] PL-9602G Microplate reader: Beijing PULANG New Technology Co., Ltd.;

[0103] TU-1901 Double-beam ultraviolet spectrophotometer: Beijing Purkinje General Instrument Co., Ltd.;

[0104] H185OR High-speed desktop refrigerated centrifuge: Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

[0105] Example 1

[0106] Preparation of microporous Cu-BTC by rapid synthesis method using crystal defects

[0107] Weigh 0.3 g of ZnO and dissolve it in 8 mL of distilled water. Stir for 30 min, then add 8 mL of DMF dropwise and stir until the solid is evenly dispersed to obtain solution A;

[0108] Weigh 1.32 g of Cu(NO3)2 and dissolve it in 8 mL of distilled water. Stir until the solid is completely dissolved to obtain solution B;

[0109] Weigh 0.84 g of H3BTC and dissolve it in 8 mL of distilled water. Stir until the solid is completely dissolved to obtain solution C;

[0110] Weigh 0.24 mL of acetic acid and dissolve it in 8 mL of absolute ethanol. Stir evenly to obtain solution D;

[0111] Add solution B dropwise to solution A and stir for 30 min until homogeneous to obtain solution E;

[0112] Add solution C and solution D dropwise to solution E in sequence and stir for 10 min. Centrifuge the product, and wash the obtained crystals with absolute ethanol 3 - 4 times. Place the finally purified product in a vacuum drying oven and dry it under vacuum at 150 °C for 12 h to prepare microporous Cu-BTC.

[0113] Perform scanning electron microscopy analysis on the microporous Cu-BTC, and the results are as Figure 2 shown.

[0114] From Figure 2It can be seen that the octahedral structure of microporous Cu-BTC appears to be not very complete, which is a normal phenomenon. Because the large crystal size of Cu-BTC can lead to difficulty in controlling the nucleation centers during the solvothermal synthesis process, the integrity of the final particles is poor, with an edge length of about 2 μm to 3 μm, conforming to the common morphology of Cu-BTC.

[0115] XRD analysis was performed on microporous Cu-BTC, and the results are as Figure 3 shown.

[0116] From Figure 3 it can be seen that a series of sharp diffraction peaks appear at 2θ = 6.7°, 9.5°, 11.6°, 13.4°, 16.5°, 17.4°, and 19.0°. These characteristic peaks correspond to the (200), (220), (222), (400), (422), (511), and (440) crystal planes of Cu-BTC respectively, which match the characteristic peaks in the relevant literature. It can be judged that the Cu-BTC sample was successfully synthesized and has good crystallinity.

[0117] Peroxidase-like activity test of Cu-BTC

[0118] The PBS buffer solution (0.025 mol / L, pH = 6.86) was set as the blank solution by using the ultraviolet spectrophotometer method. The volume of the solution in the cuvette was fixed at 3 mL, and the wavelength parameter was set to 300 - 700 nm.

[0119] First, 3 mL of PBS buffer solution (0.025 mol / L, pH = 6.86) was added to both cuvettes for calibration. After calibration, the cuvette in the outer cell was taken out, rinsed clean, and rinsed with the solution. The following four groups of solutions were successively filled in the other cuvette, and their ratios were as follows:

[0120] ①: 20 μL of H2O2 (0.4 mol / L) + 50 μL of TMB solution (3.33 mg / mL) + 2930 μL of PBS buffer solution (0.025 mol / L, pH = 6.86);

[0121] ②: 20 μL of H2O2 (0.4 mol / L) solution + 100 μL of Cu-BTC (10 mg / mL) solution + 2880 μL of PBS buffer solution (0.025 mol / L, pH = 6.86);

[0122] ③: 50 μL of TMB solution (3.33 mg / mL) + 100 μL of Cu-BTC (10 mg / mL) solution + 2850 μL of PBS buffer solution (0.025 mol / L, pH = 6.86);

[0123] ④: 50 μL of TMB solution (3.33 mg / mL) + 40 μL of H2O2 solution (0.4 mol / L) + 100 μL of Cu-BTC solution (10 mg / mL) + 2810 μL of PBS buffer (0.025 mol / L, pH = 6.86).

[0124] The UV spectrophotometer results of solutions ① to ④ are as Figure 4 shown.

[0125] As Figure 4 can be seen, curves ①, ②, and ③ have no peaks, and only absorption curve ④ has a peak at 652 nm, proving that the synthesized Cu-BTC has peroxidase-like activity.

[0126] Feasibility analysis of the enzyme cascade system method

[0127] Shorten the enzyme cascade reaction system and accurately verify the feasibility of the method: Add 80 μL of choline solution (100 mg / mL), 20 μL of choline oxidase solution (50 mU / mL), and 40 μL of Cu-BTC material dispersion (10 mg / mL) to a centrifuge tube, incubate in a water bath at 30 °C for 30 min, then add 20 μL of TMB solution (3.33 mg / mL) and 10 μL of acetic acid, and observe the color of the solution. The color development results are as Figure 5 shown.

[0128] Secondly, extend the enzyme cascade system: Add 160 μL of acetylcholine solution (1 mg / mL), 100 μL of acetylcholinesterase solution (200 mU / mL), and 20 μL of choline oxidase solution (200 mU / mL) to a centrifuge tube, and mix well. Place the centrifuge tube in a water bath at 37 °C and incubate for 30 min, then add 20 μL of TMB (3.33 mg / mL) and 10 μL of acetic acid, and observe the color of the solution. The results are as Figure 6 shown.

[0129] As Figures 5 - 6 can be seen, the ·OH finally generated in the system oxidizes TMB to form a colored substance, successfully verifying that the method for rapid detection of organophosphorus residues in pesticides based on enzyme cascade catalytic amplification reaction is feasible.

[0130] Enzyme cascade system

[0131] Add 100 μL of PBS buffer (0.025 mol / L, pH = 6.86) and 100 μL of acetylcholinesterase solution (200 mU / mL) to a centrifuge tube as a blank control, and add 100 μL of organophosphorus solution (100 μg / L) and 100 μL of acetylcholinesterase solution (200 mU / mL) to another centrifuge tube. Place both centrifuge tubes in a water bath at 37 °C and incubate for 15 min to allow the organophosphorus solution and AchE to react fully.

[0132] After the incubation, 40 μL of Cu-BTC solution (10 mg / mL), 160 μL of acetylcholine solution (1 mg / mL), and 20 μL of choline oxidase solution (200 mU / mL) were added to each of the two centrifuge tubes, and the mixture was homogenized. The centrifuge tubes were incubated in a water bath at 37 °C for 30 min, which allowed AchE and ChoX to be loaded onto Cu-BTC and generated an incubation product containing hydrogen peroxide. After the incubation, 20 μL of TMB solution (concentration 3.33 mg / mL) and 10 μL of acetic acid were added, and the mixture was homogenized. The color of the solution was observed, and the color development results were as Figure 7 shown.

[0133] As Figure 7 can be seen, the color of the solution in the centrifuge tube with PBS as the blank control was dark blue, while the color of the solution in the centrifuge tube with the organophosphorus solution became lighter. This is because the organophosphorus inhibited the activity of acetylcholinesterase, thereby weakening the occurrence of the enzyme cascade reaction and resulting in a lighter color of the solution after the reaction. As shown, it is proved that this experiment is feasible.

[0134] Each experimental condition will affect the performance of the enzyme cascade reaction. Therefore, the mass concentration of Cu-BTC, the action time of the organophosphorus solution and acetylcholinesterase, the action time of the enzyme and substrate and Cu-BTC, the addition amount of acetylcholinesterase, the addition amount of choline oxidase, and the addition amount of the substrate acetylcholine were optimized. The results were as Figure 8 shown:

[0135] Optimization experiment process of the mass concentration of Cu-BTC

[0136] 100 μL of PBS buffer solution with 0.025 mol / L and pH = 6.86, 20 μL of TMB solution with a concentration of 3.33 mg / mL, 40 μL of Cu-BTC dispersions with different concentrations (3 mg / mL, 5 mg / mL, 7 mg / mL, 9 mg / mL, 11 mg / mL), and 20 μL of acetic acid were added to the centrifuge tube. After homogenization, the absorbance was measured in an enzyme-linked immunosorbent assay reader. The results were as Figure 8 shown in A.

[0137] As Figure 8 can be seen from A, as the mass concentration of Cu-BTC increased, the absorbance value of the solution also continuously increased. To obtain the best detection effect, 11 mg / mL was selected as the optimal mass concentration of Cu-BTC for subsequent experiments.

[0138] Optimization experiment process of the action time of the organophosphorus solution and acetylcholinesterase

[0139] Add 50 μL of methyl parathion solution with a concentration of 50 μg / L and 50 μL of acetylcholinesterase solution (200 mU / mL) to a centrifuge tube and incubate in a water bath (37 °C) for a certain period of time (5 min, 10 min, 15 min, 20 min, 25 min). After the water bath is completed, add 80 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC (11 mg / mL), and 10 μL of choline oxidase (200 mU / mL), and then incubate in a water bath (37 °C, 25 min). After the water bath is completed, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and measure the absorbance using an enzyme-linked immunosorbent assay reader. The results are as Figure 8 shown in B of

[0140] As can be seen from Figure 8 B in the figure, when the water bath time of the organophosphorus solution and acetylcholinesterase is 15 min, the absorbance value is the highest. However, as the water bath time increases, the inhibitory effect of the organophosphorus on AChE increases, resulting in a decrease in the absorbance value of the solution. Therefore, 15 min is selected as the optimal condition.

[0141] Optimization experiment process of the reaction time of the enzyme, substrate, and Cu-BTC

[0142] Add 50 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 50 μL of acetylcholinesterase solution (200 mU / mL) to a centrifuge tube and incubate in a water bath (37 °C) for 15 min. After the water bath is completed, add 80 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion (11 mg / mL), and 10 μL of choline oxidase solution (200 mU / mL), and then incubate in a water bath (37 °C) for a certain period of time (20 min, 25 min, 30 min, 35 min, 40 min). After the water bath is completed, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and measure the absorbance using an enzyme-linked immunosorbent assay reader. The results are as Figure 8 shown in C of

[0143] Figure 8 As shown in C of the figure, when the reaction time of the enzyme, substrate, and Cu-BTC is 25 min, the absorbance value of the solution is the highest. As the reaction time increases, the activity of the enzyme gradually decreases, and the H2O2 generated during the enzyme cascade reaction decreases, resulting in a lighter solution color and a lower absorbance value. Therefore, 25 min is the optimal reaction time for the enzyme, substrate, and Cu-BTC.

[0144] Optimization experiment process of the addition amount of acetylcholinesterase

[0145] Add 50 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and different volumes (40 μL, 45 μL, 50 μL, 55 μL, 60 μL) of acetylcholinesterase solution (200 mU / mL) into centrifuge tubes, and then perform a water bath at 37 °C for 15 min. After the water bath, add 80 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion (11 mg / mL), and 10 μL of choline oxidase (200 mU / mL), and then perform a water bath at 37 °C for 25 min. After the water bath, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and measure the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as Figure 8 shown in D of

[0146] As can be seen Figure 8 from D, as the addition amount of AChE increases, the absorbance of the solution reaches the highest at 45 μL and then gradually decreases. The reason may be that the more AChE is added, the stronger the inhibitory effect of the organophosphorus solution on it.

[0147] Optimization experiment process of the addition amount of choline oxidase

[0148] Add 50 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 45 μL of acetylcholinesterase solution (200 mU / mL) into centrifuge tubes respectively, and then perform a water bath at 37 °C for 15 min. After the water bath, add 70 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion (11 mg / mL), and add different volumes (4 μL, 7 μL, 10 μL, 13 μL, 16 μL) of choline oxidase solution (200 mU / mL), mix well, and then perform a water bath at 37 °C for 25 min. After the water bath, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and measure the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as Figure 8 shown in E of

[0149] As can be seen from E in 8, the optimal addition amount of choline oxidase solution is 13 μL. As the addition amount increases, the absorbance value gradually increases. The reason for this phenomenon may be that when the addition amount of ChOx is too small, not all the generated choline is reacted, resulting in a relatively low absorbance.

[0150] Optimization experiment process of the addition amount of substrate acetylcholine

[0151] Add 50 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 45 μL of acetylcholinesterase solution (200 mU / mL) into centrifuge tubes respectively, and incubate in a water bath (at a temperature of 37 °C) for 15 min. After the water bath, add several volumes (65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL) of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion (11 mg / mL) and 13 μL of choline oxidase (200 mU / mL) in sequence, mix well and incubate in a water bath (at a temperature of 37 °C) for 25 min. After the water bath, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 10 μL of acetic acid, and measure the absorbance using an enzyme-linked immunosorbent assay reader. The results are as Figure 8 shown in Figure F.

[0152] As Figure 8 can be seen from Figure F, the absorbance value increases from 65 μL to 75 μL, which may be due to too little substrate added. In addition, the absorbance values are all lower than the absorbance value when the substrate addition amount is 75 μL, which may be due to substrate excess, resulting in a decrease in the absorbance value.

[0153] Under the above optimal conditions, using methyl parathion (MP) as an OPs mimic, evaluate the analytical performance of the constructed enzyme cascade system for OPs. The steps are as follows:

[0154] First, add 50 μL of 100 μg / L methyl parathion solution into a centrifuge tube, then add 45 μL of acetylcholinesterase solution (200 mU / mL), mix well and place it in a water bath at 37 °C for 15 min. After the water bath, add 70 μL of acetylcholine (1 g / mL), 20 μL of Cu-BTC dispersion (11 mg / mL) and 13 μL of choline oxidase (200 mU / mL), mix well and incubate in a water bath at 37 °C for 25 min. Finally, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 10 μL of acetic acid. Then, use a pipette to transfer 200 μL of this solution into a 96-well plate and measure the absorbance at a peak wavelength of 652 nm using an enzyme-linked immunosorbent assay reader.

[0155] As Figure 9 the concentration of methyl parathion increases, the absorbance value of the solution gradually decreases. This is because methyl parathion inhibits the activity of AChE, thereby weakening the production of choline, and finally leading to a decrease in ·OH, resulting in a decrease in the absorbance value of the solution;

[0156] The absorbance of the solution shows a linear correlation with the mass concentration of methyl parathion in the range of 1 - 100 μg / L (Y = -0.0090X + 0.9455, R 2＝0.993), and the detection limit was 7.28 ng / mL. Therefore, the enzyme cascade catalytic system constructed based on Cu-BTC is expected to achieve highly sensitive detection of OPs.

[0157] Anti-interference

[0158] First, add 50 μL of 100 μg / L parathion-methyl, 50 μL of 5 μg / mL interference ion solution (the interference ions are sodium ions, potassium ions, chloride ions, bicarbonate ions, urea, or glucose, and only one interference ion is added in each anti-interference experiment), and 45 μL of acetylcholinesterase solution (200 mU / mL) into a centrifuge tube. After mixing evenly, place it in a water bath at 37 °C for 15 min. After the water bath, add 70 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion, and 13 μL of choline oxidase (200 mU / mL), mix evenly, and then place it in a water bath at 37 °C for 25 min. Finally, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and mix evenly. Then, use a pipette to transfer 200 μL of this solution into a 96-well plate and measure its absorbance with an enzyme-labeled instrument at the peak of 652 nm.

[0159] Figure 10 It can be seen that when the volume of the enzyme cascade catalytic amplification reaction system is kept constant, after adding interfering substances 100 times the concentration of the organophosphorus solution, the absorbance is not much different from that without adding the interference ion solution, both around 0.7. This indicates that adding sodium ions, potassium ions, chloride ions, bicarbonate ions, urea, and glucose to the entire enzyme cascade system will not affect the system. Therefore, the entire enzyme cascade catalytic amplification reaction system has a certain anti-interference ability.

[0160] Six experiments were carried out under the optimal conditions to verify the reproducibility:

[0161] Add 100 μL of 50 μg / L parathion-methyl solution and 45 μL of 200 mU / mL acetylcholinesterase solution to each solution (a total of 6 portions), place it in a water bath at 37 °C for 15 min, then add 70 μL of acetylcholine solution (1 mg / mL), 20 μL of Cu-BTC dispersion (11 mg / mL), and 13 μL of choline oxidase solution (200 mU / mL), mix them, and then place it in a water bath at 37 °C for 25 min. Finally, add 10 μL of TMB solution (concentration: 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 10 μL of acetic acid, and mix evenly. Measure its absorbance with an enzyme-labeled instrument at the peak of 652 nm. The results Figure 11 are shown as follows.

[0162] As Figure 11It can be seen that the relative standard deviation of the obtained absorbance value is 1.87%, indicating that the enzyme cascade catalytic amplification reaction system has good reproducibility.

[0163] The solution after measuring the absorbance in the reproducibility experiment was left for 5 days and then the absorbance was measured again. The results are as Figure 12 shown.

[0164] As Figure 12 can be seen, the absorbance value of the solution after five days is 83% of the original value, indicating that the enzyme cascade system has good stability.

[0165] To meet the actual detection needs, fresh Chinese cabbages and lettuces were bought from the market for the spike recovery experiment (the edible parts of the fresh Chinese cabbages and lettuces were cut into pieces, 2.0000 g was weighed respectively, put into 5 mL of water and soaked for 1 h, covered with plastic wrap, and finally the actual sample solution was obtained by filtration. Then methyl parathion (MP) was added to obtain test solutions with methyl parathion (MP) concentrations of 30 μg / L, 50 μg / L and 70 μg / L.

[0166] 50 μL of different test solutions and 45 μL of acetylcholinesterase solution (200 mU / mL) were taken, mixed evenly and then put into a water bath at 37 °C for 15 min. After the water bath, 70 μL of acetylcholine solution (1 g / mL), 20 μL of Cu-BTC dispersion and 13 μL of choline oxidase (200 mU / mL) were added, mixed evenly and then put into a water bath at 37 °C for 25 min. Finally, 10 μL of TMB solution (concentration 3.33 mg / mL), 10 μL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 10 μL of acetic acid were added and mixed evenly. Then 200 μL of this solution was pipetted into a 96-well plate and its absorbance was measured with an enzyme-linked immunosorbent assay analyzer at the peak value of 652 nm.

[0167] The sample recovery rate was calculated according to the absorbance and the standard curve. The results are shown in Table 1.

[0168] Table 1 Sample recovery rate

[0169]

[0170] As can be seen from Table 1, the recovery rate range of the enzyme cascade system for Chinese cabbage samples is from 108.10% to 120.09%; the recovery rate range of the enzyme cascade system for lettuce samples is from 95.71% to 119.89%. In summary, the present invention can achieve accurate and reliable detection of OPs and has the potential for application in the detection of agricultural product quality and safety.

[0171] The present invention synthesizes Cu-BTC with catalase-like activity by a rapid synthesis method, constructs an enzyme cascade catalytic reaction system based on the enzyme inhibition mechanism, and realizes highly sensitive and highly selective detection of organophosphorus pesticides at a methyl parathion mass concentration of 1-100 μg / L. Moreover, this method has been successfully applied to the detection of organophosphorus pesticides in cabbage and lettuce samples, with good recovery rates and stability. The results show that the method for detecting pesticides established in this study has the advantages of rapidity, sensitivity, and simplicity of operation, and has good application potential in the rapid detection of OPs, providing strong technical support for the detection of organophosphorus drug residues in vegetables.

[0172] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A detection sensor, characterized in that: The method comprises an acetylcholine solution, an acetylcholinesterase solution, a choline oxidase solution, a dispersion of Cu-BTC and a solution containing a color developer capable of generating a hydroxyl color developing group; or comprising acetylcholine, a color developing agent capable of generating a hydroxyl color developing group, and a Cu-BTC based composite material; The Cu-BTC-based composite material comprises Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase loaded on the Cu-BTC.

2. A detection sensor, characterized in that: The invention comprises an electrolyte containing acetylcholine and a developer capable of generating a hydroxyl color-developing group, a Cu-BTC-based composite material and an electrode; the Cu-BTC-based composite material is loaded on the electrode; The Cu-BTC-based composite material comprises Cu-BTC, choline oxidase loaded on the Cu-BTC, and acetylcholinesterase loaded on the Cu-BTC.

3. The detection sensor according to claim 1, characterized in that: The ratio of acetylcholinesterase in the acetylcholinesterase solution to acetylcholine in the acetylcholine solution is 20-260mU:1g; The volume ratio of the acetylcholinesterase solution to the acetylcholine solution is 1:1-3; The concentration of the acetylcholinesterase solution is 120-300 mU / mL; the concentration of the acetylcholine solution is 0.5-2 g / mL.

4. The detection sensor according to claim 1 or 3, characterized in that: The ratio of acetylcholine in the acetylcholine solution to choline oxidase in the choline oxidase solution is 1g:4-300mU; The volume ratio of the acetylcholine solution to the choline oxidase solution is 2 to 15:1; The concentration of the choline oxidase solution is 120-300 mU / mL.

5. The detection sensor according to claim 1 or 3, characterized in that: The mass ratio of acetylcholine in the acetylcholine solution to Cu-BTC in the Cu-BTC dispersion is 1000:0.1-12; The volume ratio of the acetylcholine solution to the Cu-BTC dispersion is 2 to 8:1; The concentration of the Cu-BTC dispersion is 3-12 mg / mL.

6. Use of the sensor according to any one of claims 1, 3 to 5 in colorimetric detection of organophosphorus pesticides.

7. Use of the detection sensor described in claim 2 in electrochemical detection of organophosphorus pesticides.

8. The use according to claim 6, characterized in that: The colorimetric detection of organophosphorus pesticides comprises the following steps: Mixing the detection sensor according to any one of claims 1 and 3 to 5 with a solution to be detected to perform a color development reaction to obtain a color development solution; The concentration of the organophosphorus pesticide in the solution to be detected is obtained according to the absorbance of the color developing solution and the concentration-absorbance curve of the standard solution.

9. The use according to claim 6 or 8, characterized in that: When the detection sensor comprises an acetylcholine solution, an acetylcholinesterase solution, a choline oxidase solution, a dispersion of Cu-BTC and a solution containing a developer capable of generating a hydroxyl color developing group, the detection of the colorimetric organophosphorus pesticide comprises the following steps: Mixing the acetylcholinesterase solution with the solution to be detected for a first incubation to obtain a first incubation solution; The first incubation solution is mixed with a Cu-BTC dispersion, an acetylcholine solution, and a choline oxidase solution for a second incubation to obtain a second incubation solution containing hydrogen peroxide; The second incubation solution is mixed with a solution containing a color developer capable of generating a hydroxyl color-developing group to perform a color development reaction to obtain a color-developing solution; The concentration of the organophosphorus pesticide in the solution to be detected is obtained according to the absorbance of the color developing solution and the concentration-absorbance curve of the standard solution.

10. The use according to claim 9, characterized in that: The first incubation temperature is 30-40°C and the time is 5-25 minutes; The temperature of the second incubation is 30-40° C., and the time is 20-40 min; the volume ratio of the test solution to the acetylcholinesterase solution is 1-5:1; The concentration of the organic phosphorus in the test solution is 1-100 μg / L.

Images