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 acetylcholine, acetylcholin esterase, choline oxidase, two-dimensional Cu-TCPP (Fe) NSs and a color developing agent capable of generating a hydroxyl color developing group. The acetylcholin esterase can hydrolyze acetylcholine to generate choline, the choline is oxidized by the choline oxidase to generate H2O2, the H2O2 is oxidized by the two-dimensional Cu-TCPP (Fe) NSs to generate hydroxyl free radicals (. OH), and the. OH can oxidize a color developing agent capable of generating hydroxyl color developing groups to generate colored substances. When the organophosphorus pesticide exists in the to-be-detected liquid, the color of the solution is light blue or colorless; otherwise, the color of the solution is dark blue. The organophosphorus pesticide can inhibit the activity of acetylcholin esterase, so that the color of the solution is changed in the whole cascade catalytic amplification reaction, and then quantitative analysis of the organophosphorus pesticide is realized through a concentration-absorbance curve of the solution absorbance and the 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, varieties, and dosages ultimately lead to their residues in agricultural products. In order 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, a variety of methods for detecting organophosphorus pesticides have been 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 enzyme-like 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, 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, which includes acetylcholine, acetylcholinesterase, choline oxidase, two-dimensional Cu-TCPP(Fe)NSs, and a color reagent that can generate a hydroxyl chromogenic group.

[0007] Preferably, it further includes an electrode;

[0008] The two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase, and choline oxidase are loaded on the electrode;

[0009] Alternatively, the two-dimensional Cu-TCPP(Fe) NSs are loaded on the electrode, and the acetylcholinesterase and choline oxidase are loaded on the two-dimensional Cu-TCPP(Fe) NSs.

[0010] Preferably, the method for loading the two-dimensional Cu-TCPP(Fe) NSs, acetylcholinesterase and choline oxidase on the electrode includes the following steps:

[0011] Mix the dispersion of two-dimensional Cu-TCPP(Fe) NSs, the acetylcholinesterase solution and the choline oxidase solution, then drop the mixture onto the electrode and dry it.

[0012] Preferably, the loading amount of acetylcholinesterase on the electrode is 0.09 - 0.4 mU / 7 mm 2 ; the loading amount of two-dimensional Cu-TCPP(Fe) NSs on the electrode is 0.007 - 0.04 mg / 7 mm 2 ;

[0013] The volume ratio of the dispersion of two-dimensional Cu-TCPP(Fe) NSs to the acetylcholinesterase solution is 1:1 - 10;

[0014] The concentration of the dispersion of two-dimensional Cu-TCPP(Fe) NSs is 10 - 50 mg / mL; the concentration of the acetylcholinesterase solution is 50 - 100 mU / mL.

[0015] Preferably, the loading amount of choline oxidase on the electrode is 0.02 - 0.10 mU / 7 mm 2 ;

[0016] The volume ratio of the dispersion of two-dimensional Cu-TCPP(Fe) NSs to the choline oxidase solution is 1 - 5:1;

[0017] The concentration of the choline oxidase solution is 50 - 250 mU / mL.

[0018] The present invention also provides the application of the detection sensor described in the above technical solution in the colorimetric detection of organophosphorus pesticides.

[0019] The present invention also provides the application of the detection sensor described in the above technical solution in the electrochemical detection of organophosphorus pesticides and the colorimetric detection of organophosphorus pesticides.

[0020] Preferably, the electrochemical detection of organophosphorus pesticides includes the following steps:

[0021] Mix the test solution containing organophosphorus with the acetylcholinesterase solution to obtain a mixture;

[0022] The mixture is mixed with a dispersion of two-dimensional Cu-TCPP(Fe) NSs and a choline oxidase solution, and then dropped onto the electrode and dried to obtain the sensor to be detected.

[0023] The sensor to be detected is placed in a DPV test solution containing acetylcholine and a color reagent capable of generating a hydroxyl chromogenic group for DPV testing to obtain a peak current signal value.

[0024] According to 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 organophosphorus in the solution to be detected is obtained.

[0025] Preferably, the colorimetric detection of organophosphorus pesticides includes the following steps: measuring the absorbance of the solution after DPV testing, and then obtaining the concentration of organophosphorus in the solution to be detected according to the concentration-absorbance curve of the obtained absorbance and the standard solution.

[0026] Preferably, the preparation method of the DPV test solution containing acetylcholine and a color reagent capable of generating a hydroxyl chromogenic group includes the following steps:

[0027] Mix the acetylcholine solution and the solution of the color reagent capable of generating a hydroxyl chromogenic group;

[0028] The mass of acetylcholine in the acetylcholine solution is 0.04 g to 0.12 g;

[0029] The concentration of the acetylcholine solution is 1 g / mL, and the volume is 40 to 120 μL.

[0030] Acetylcholinesterase can hydrolyze acetylcholine to generate choline, choline is oxidized by choline oxidase to generate H2O2, H2O2 is oxidized by two-dimensional Cu-TCPP(Fe) NSs to generate hydroxyl radicals (·OH), and ·OH can oxidize the color reagent capable of generating a hydroxyl chromogenic 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, thereby affecting the entire cascade catalytic amplification reaction and changing the solution color, and then realizing the quantitative analysis of organophosphorus pesticides through the concentration-absorbance curve of the solution absorbance and the standard solution.

[0031] Furthermore, the detection sensor further includes an electrode. When the two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase, and choline oxidase are loaded on the electrode, the sensor can be used for dual-mode detection of colorimetric detection and electrochemical detection of organophosphorus pesticides. The dual-mode detection not only exhibits the inherent characteristics of a single signal output mode but also can combine the environmental conditions during analysis, select the analysis instrument, flexibly respond to different scenarios, and at the same time, the output signals of the two modes can be self-calibrated to improve the accuracy and reliability of the analysis results.

[0032] In the present invention, the MOF material two-dimensional Cu-TCPP(Fe)NSs that can both serve as a loading carrier for the enzymes required for the cascade reaction to maintain enzyme catalytic activity and can also serve as a peroxidase mimicking enzyme is selected to load AChE and ChOx, or a mixture of the MOF material two-dimensional Cu-TCPP(Fe)NSs, AChE, and ChOx to construct a novel biosensor for colorimetry and electrochemistry to achieve quantitative analysis of MP. Brief Description of the Drawings

[0033] Figure 1 is the schematic diagram of the sensor of the present invention;

[0034] Figure 2 are the CV test results of the glassy carbon electrode and two-dimensional Cu-TCPP(Fe)NSs / GCE in Example 1;

[0035] Figure 3 are the experimental test results of the DPV test to explore the feasibility of the sensor for the determination of MP;

[0036] Figure 4 is the graph of the absorbance test results;

[0037] Figure 5 are the test results of the concentration of the two-dimensional Cu-TCPP(Fe)NSs dispersion;

[0038] Figure 6 are the test results of the AChE concentration;

[0039] Figure 7 are the test results of the ChOx concentration;

[0040] Figure 8 are the test results of the addition amount of ACh;

[0041] Figure 9 are the peak current signal values corresponding to different standard concentrations of MP;

[0042] Figure 10 are the absorbances recorded corresponding to different standard concentrations of MP;

[0043] Figure 11 are the anti-interference results of the electrochemical detection

[0044] Figure 12 Results of reproducibility for electrochemical tests of five parallel electrodes;

[0045] Figure 13 Stability of electrochemical tests;

[0046] Figure 14 Results of colorimetric anti-interference;

[0047] Figure 15 Results of reproducibility for colorimetric tests;

[0048] Figure 16 Results of stability for colorimetric tests. Detailed implementation manners

[0049] The present invention provides a detection sensor, including acetylcholine, acetylcholinesterase, choline oxidase, two-dimensional Cu-TCPP(Fe) NSs, and a color reagent capable of generating a hydroxyl chromogenic group.

[0050] In the present invention, the detection sensor preferably further includes an electrode; the electrode preferably includes glassy carbon.

[0051] In the present invention, the two-dimensional Cu-TCPP(Fe) NSs, acetylcholinesterase, and choline oxidase are preferably loaded on the electrode.

[0052] In the present invention, the loading amount of acetylcholinesterase on the electrode is preferably 0.09 - 0.4 mU / 7 mm 2 , in a specific embodiment of the present invention, the loading amount of acetylcholinesterase on the electrode can be 0.09 mU / 7 mm 2 , 0.1 mU / 7 mm 2 , 0.2 mU / 7 mm 2 , 0.3 mU / 7 mm 2 or 0.4 mU / 7 mm 2 ;

[0053] The loading amount of two-dimensional Cu-TCPP(Fe) NSs on the electrode is preferably 0.007 - 0.04 mg / 7 mm 2 , in a specific embodiment of the present invention, the loading amount of two-dimensional Cu-TCPP(Fe) NSs on the electrode can be 0.007 mg / 7 mm 2 , 0.01 mg / 7 mm 2 , 0.014 mg / 7 mm 2 , 0.02 mg / 7 mm 2 , 0.03 mg / 7 mm 2 or 0.04 mg / 7 mm 2 ;

[0054] The loading amount of choline oxidase on the electrode is preferably 0.02 - 0.10 mU / 7 mm 2 , in a specific embodiment of the present invention, the loading amount of choline oxidase on the electrode can be 0.02 mU / 7 mm 2 , 0.03 mU / 7 mm 2 , 0.04 mU / 7 mm 2 , 0.05 mU / 7 mm 2 , 0.06 mU / 7 mm 2 , 0.07 mU / 7 mm 2 , 0.08 mU / 7 mm 2 , 0.09 mU / 7 mm 2 or 0.10 mU / 7 mm 2 .

[0055] In the present invention, the loading method preferably includes the following steps:

[0056] Mix the dispersion of two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase solution and choline oxidase solution, then drop the mixture onto the electrode and dry it.

[0057] In the present invention, before the dropping, it is preferable to polish, wash and perform cyclic voltammetry measurement on the electrode.

[0058] The present invention has no special limitation on the cyclic voltammetry measurement, and the potential difference of the electrode not exceeding 100 mV is sufficient (less than 100 mV indicates that the electrode polishing is qualified).

[0059] In the present invention, the volume ratio of the dispersion of two-dimensional Cu-TCPP(Fe)NSs to the acetylcholinesterase solution is preferably 1:1 - 10. In a specific embodiment of the present invention, the volume ratio of the dispersion of two-dimensional Cu-TCPP(Fe)NSs to the acetylcholinesterase solution can be 1:1, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10; the concentration of the dispersion of two-dimensional Cu-TCPP(Fe)NSs is preferably 10 - 50 mg / mL. In a specific embodiment of the present invention, the concentration of the dispersion of two-dimensional Cu-TCPP(Fe)NSs can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL; the concentration of the acetylcholinesterase solution is preferably 50 - 200 mU / mL. In a specific embodiment of the present invention, the concentration of the acetylcholinesterase solution can be 50 mU / mL, 60 mU / mL, 70 mU / mL, 80 mU / mL, 90 mU / mL or 100 mU / mL.

[0060] In the present invention, the volume ratio of the dispersion of the two-dimensional Cu-TCPP(Fe) NSs to the choline oxidase solution is preferably 1 to 5:1. In specific embodiments of the present invention, the volume ratio of the dispersion of the two-dimensional Cu-TCPP(Fe) NSs to the choline oxidase solution can be 1:1, 2:1, 3:1, 4:1, or 5:1. The concentration of the choline oxidase solution is 50 to 250 mU / mL. In specific embodiments of the present invention, the concentration of the choline oxidase solution can be 50 mU / mL, 60 mU / mL, 70 mU / mL, 80 mU / mL, 90 mU / mL, 100 mU / mL, 110 mU / mL, 120 mU / mL, 130 mU / mL, 140 mU / mL, 150 mU / mL, 160 mU / mL, 170 mU / mL, 180 mU / mL, 190 mU / mL, 200 mU / mL, 210 mU / mL, 220 mU / mL, 230 mU / mL, 240 mU / mL, or 250 mU / mL.

[0061] The present invention also provides the application of the detection sensor described in the above technical solution in the colorimetric detection of organophosphorus pesticides.

[0062] The present invention also provides the application of the detection sensor described in the above technical solution in the electrochemical detection of organophosphorus pesticides.

[0063] In the present invention, the application preferably includes the following steps:

[0064] Mix a solution to be detected containing organophosphorus with an acetylcholinesterase solution to obtain a mixture;

[0065] After mixing the mixture with the dispersion of the two-dimensional Cu-TCPP(Fe) NSs and the choline oxidase solution, drop the mixture onto an electrode and then dry it to obtain a sensor to be detected;

[0066] Place the sensor to be detected in a DPV test solution containing acetylcholine and a color developer capable of generating a hydroxyl chromogenic group for DPV testing to obtain a peak current signal value;

[0067] According to the peak current signal value, the peak current signal value of a standard organophosphorus solution measured under the same conditions, and the concentration of organophosphorus in the standard organophosphorus solution, obtain the concentration of organophosphorus in the solution to be detected.

[0068] The present invention mixes a solution to be detected containing organophosphorus with an acetylcholinesterase solution to obtain a mixture.

[0069] In the present invention, the volume ratio of the solution to be detected to the acetylcholinesterase solution is preferably 1:1.

[0070] In the present invention, the temperature of the mixing 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; the mixing is preferably carried out under water bath conditions.

[0071] During the mixing process, the solution to be measured inhibits the activity of acetylcholinesterase.

[0072] After obtaining the mixture, the present invention mixes the mixture with a dispersion of two-dimensional Cu-TCPP(Fe) NSs and a choline oxidase solution, then drops the mixture onto an electrode and dries it to obtain the sensor to be detected.

[0073] In the present invention, the temperature of the mixing 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; the mixing is preferably carried out under water bath conditions.

[0074] After obtaining the sensor to be detected, the present invention places the sensor to be detected in a DPV test solution containing acetylcholine and a color reagent capable of generating a hydroxyl chromogenic group for DPV testing to obtain a peak current signal value.

[0075] In the present invention, the preparation method of the DPV test solution includes the following steps:

[0076] Mix a solution of acetylcholine and a solution of a color reagent capable of generating a hydroxyl chromogenic group.

[0077] In the present invention, the mass of acetylcholine in the acetylcholine solution is preferably 0.04 g to 0.12 g. In specific embodiments of the present invention, the mass of acetylcholine in the acetylcholine solution can be 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.11 g or 0.12 g;

[0078] The volume ratio of the acetylcholine solution to the solution of the chromogenic agent capable of generating a hydroxyl chromogenic group is preferably 1:7.5 to 37.5. In specific embodiments of the present invention, the volume ratio may be 1:7.5, 1:10, 1:15, 1:17.5, 1:20, 1:25, 1:27.5, 1:30, 1:35 or 1:37.5; the concentration of the acetylcholine solution is preferably 1 g / mL; the volume of the acetylcholine solution is preferably 40 to 120 μL. In specific embodiments of the present invention, the volume of the acetylcholine solution may be 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL or 200 μL; the concentration of the solution of the chromogenic agent capable of generating a hydroxyl chromogenic group is preferably 0.4 mol / L; the chromogenic agent capable of generating a hydroxyl chromogenic group preferably includes TMB.

[0079] In the present invention, the DPV test solution preferably further includes: a buffer solution; when the DPV test solution includes a buffer solution, the preparation method of the DPV test solution preferably includes the following steps:

[0080] Mix the solution of acetylcholine and the solution of the chromogenic agent capable of generating a hydroxyl chromogenic group with the buffer solution.

[0081] In the present invention, the volume ratio of the acetylcholine solution to the buffer solution is preferably 1 to 4:25. In specific embodiments of the present invention, the volume ratio of the acetylcholine solution to the buffer solution may be 1:25, 2:25, 3:25 or 4:25; the buffer solution preferably includes PBS buffer; the pH value of the buffer solution is preferably 6.86; the concentration of the buffer solution is preferably 0.025 mol / L.

[0082] In the present invention, the voltage of the DPV test is preferably 0.8 to -0.4 V, the pulse amplitude is preferably 0.05 V, the pulse width is preferably 0.05 s, and the pulse period is preferably 0.5 s.

[0083] After obtaining the peak current signal value, the present invention obtains the concentration of the organophosphorus in the solution to be detected based on the peak current signal value, the peak current signal value of the standard organophosphorus solution measured under the same conditions, and the concentration of the organophosphorus in the standard organophosphorus solution.

[0084] The colorimetric detection of organophosphorus pesticides includes the following steps: measuring the absorbance of the solution after the DPV test, and then comparing the obtained absorbance with the concentration-absorbance curve of the standard solution to obtain the concentration of the organophosphorus pesticide in the solution to be detected.

[0085] In the present invention, the wavelength for measuring the absorbance of the solution after DPV testing is preferably 625 nm 。

[0086] The detection sensor and its application provided by the present invention will be described in detail below with reference to embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0087] (4-Carboxyphenyl)porphyrin iron chloride (TCPP(Fe)), copper nitrate, trifluoroacetic acid (CF3COOH), acetylcholine chloride (ACh), acetylcholinesterase (AChE), choline oxidase (ChOx), 3,3’,5,5’-tetramethylbenzidine (TMB), and polyvinylpyrrolidone (PVP) were purchased from Shanghai Macklin Biochemical Co., Ltd.; absolute ethanol (EtOH) and N,N-dimethylformamide (DMF) were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.;

[0088] Phosphate buffer solution (PBS) was purchased from Shanghai Yidian Scientific Instrument Co., Ltd.;

[0089] Methyl parathion solution standard substance (MP) in acetone was purchased from Beijing Coast Hongmeng Standard Substance Technology Co., Ltd.;

[0090] The actual sample of lettuce was purchased from a nearby supermarket.

[0091] Electrochemical workstation CHI 660E (Shanghai Chenhua Instrument Co., Ltd.);

[0092] Centrifuge JW-2018H (Anhui Jiawen Instrument and Equipment Co., Ltd.);

[0093] Desktop ultrasonic cleaner KH-2500B (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.);

[0094] Analytical balance Fa2204b (Sartorius Scientific Instruments Co., Ltd.);

[0095] Vacuum drying oven DZF-6050 (Shanghai Boxun Industry Co., Ltd.);

[0096] Microplate reader PL-9602G (Beijing Purong New Technology Co., Ltd.);

[0097] Digital display constant temperature water bath HH·S21-4 (Changzhou Jintan Jingda Instrument Manufacturing Co., Ltd.);

[0098] The model of the glassy carbon is CHI104.

[0099] Example 1

[0100] Synthesis of two-dimensional Cu-TCPP(Fe) NSs

[0101] Dissolve 9.6 mg of Cu(NO3)2·3H2O, 40 μL of 1 mol / L C F3COOH solution, and 40.0 mg of PVP in 48 mL of a mixed solution of DMF and EtOH (the volume ratio of DMF to EtOH is 3:1). Under stirring, dissolve 17.6 mg of TCPP(Fe) in the mixed solution of DMF and EtOH (the volume ratio of DMF to EtOH is 3:1). Put these two prepared mixed solutions into a sealed bottle and ultrasonicate for 15 min, then heat in a water bath at 80 °C for 4 h. After cooling to room temperature, wash twice with EtOH, centrifuge at 8000 r / min for 20 min, and finally place the obtained sample in a vacuum drying oven and dry at 80 °C for 8 h to obtain two-dimensional Cu-TCPP(Fe) NSs.

[0102] Put the GCE (glassy carbon electrode) and the two-dimensional Cu-TCPP(Fe) NSs / GCE into a 0.025 mol / L PBS buffer solution with pH = 6.86 for CV testing, and the results are as Figure 2 shown.

[0103] As Figure 2 can be seen, no obvious peak appears on the GCE, but an anodic peak close to -0.27 V is observed on the two-dimensional Cu-TCPP(Fe) NSs / GCE. This is because the two-dimensional Cu-TCPP(Fe) NSs have good conductivity, ensuring the transfer of electrons. Therefore, the two-dimensional Cu-TCPP(Fe) NSs / GCE shows a stronger current response than the GCE.

[0104] DPV test to explore the feasibility experiment of the sensor for MP determination

[0105] (1) First, polish the bare glassy carbon electrode successively with 1.0 μm, 0.3 μm, and 0.05 μm of A12O3 polishing powder into a mirror surface, wash it ultrasonically with ethanol and secondary distilled water, and perform cyclic voltammetry (CV) measurement in a mixed solution of 0.1 mol / L KCl and 5 mmol / L K3[Fe(CN)6] and K4[Fe(CN)6] with a ratio of 1:1 until the potential difference does not exceed 100 mV;

[0106] (2) Mix 25 μL of an organophosphorus solution with an MP concentration of 50 μg / L and 25 μL of an acetylcholinesterase solution with a concentration of 100 mU / mL evenly, incubate in a water bath at 37 °C for 15 min, then add 10 μL of a 2D Cu-TCPP(Fe)NSs dispersion with a concentration of 30 mg / mL and 5 μL of a choline oxidase solution with a concentration of 100 mU / mL and mix evenly. Incubate in a water bath at 37 °C for 30 min, take 5 mL and drop it onto an electrode with a diameter of 3 mm and dry it. Then place the electrode into a test bottom solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.46 mL of PBS buffer solution with a pH of 6.86 and a concentration of 0.025 mol / L, and 120 μL of an ACh solution with a concentration of 1 g / mL. Conduct DPV testing under the conditions of a set voltage of 0.8 to -0.4 V, a pulse amplitude of 0.05 V, a pulse width of 0.05 s, and a pulse period of 0.5 s;

[0107] The only difference from steps (1) to (2) is that the test solution to be detected with an MP concentration of 50 μg / L is replaced with a PBS buffer solution with a pH of 6.86 and a concentration of 0.025 mol / L;

[0108] The experimental test results of the DPV test to explore the feasibility of the sensor for MP determination are as Figure 3 shown.

[0109] As Figure 3 can be seen, there is an obvious peak when MP is not introduced, and the peak drops significantly when MP is introduced, proving the feasibility of the electrochemical experiment.

[0110] Absorbance test

[0111] Take 2.080 mL of PBS buffer solution with a pH of 6.86 and a concentration of 0.025 mol / L, mix it evenly with 40 μL of H2O2 with a mass concentration of 30%, and measure its absorbance at 625 nm and record it as a(Cu-TCPP(Fe)NSs + H2O2);

[0112] Take 2.080 mL of PBS buffer solution with a pH of 6.86 and a concentration of 0.025 mol / L and mix it evenly with 40 μL of TMB solution with a concentration of 0.4 mol / L, and measure its absorbance at 625 nm and record it as b(Cu-TCPP(Fe)NSs + TMB);

[0113] Take 2 mL of 0.025 mol / L PBS buffer solution with pH = 6.86, 40 μL of 0.4 mol / L TMB solution, 40 μL of 30% (mass concentration) H2O2, and 40 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe)NSs dispersion (the two-dimensional Cu-TCPP(Fe)NSs are the two-dimensional Cu-TCPP(Fe)NSs in Example 1). After mixing evenly, measure its absorbance at 625 nm, denoted as c(Cu-TCPP(Fe)NSs + H2O2 + TMB);

[0114] The test results of absorbance are as Figure 4 shown.

[0115] As Figure 4 can be seen, there is no obvious color change and no obvious absorption peak in the Cu-TCPP(Fe)NSs + H2O2 and Cu-TCPP(Fe)NSs + TMB systems, while there is an obvious color change and an obvious absorption peak at 652 nm in the Cu-TCPP(Fe)NSs + H2O2 + TMB system, indicating that the two-dimensional Cu-TCPP(Fe)NSs have prominent peroxidase activity.

[0116] Preparation of MP / AChE / ChOx / Cu-TCPP(Fe)NSs / GCE

[0117] First, polish the bare glassy carbon electrode successively with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder into a mirror surface, wash it ultrasonically with ethanol and secondary distilled water, and perform cyclic voltammetry (CV) measurement in a mixed solution of 0.1 mol / L KCl, 5 mmol / L K3[Fe(CN)6], and K4[Fe(CN)6] with a ratio of 1:1 until the potential difference does not exceed 100 mV;

[0118] Take 25 μL of MP solutions with concentrations of 1 μg / L, 5 μg / L, and 10 μg / L, 25 μL of 100 mU / mL AChE solution respectively, mix evenly, incubate in a water bath at 37 °C for 15 min, then add 10 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe)NSs dispersion and 5 μL of 100 mU / mL ChOx solution, mix evenly, incubate in a water bath at 37 °C for 30 min, take out 5 μL from the mixed solution and drop it onto the polished electrode with a diameter of 3 mm, and after air drying, different MP / AChE / ChOx / Cu-TCPP(Fe)NSs / GCE modified electrodes are obtained using MP solutions with different concentrations.

[0119] As a control experiment, replace MP with 0.025 mol / L PBS solution with pH = 6.86 when preparing the mixed solution.

[0120] The electrode was placed in a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.46 mL of 0.025 mol / L PBS solution with pH = 6.86, and 120 μL of 1 g / mL ACh solution for DPV testing, and the peak current signal value I was recorded. The set voltage was 0.8 to -0.4 V, the pulse amplitude was 0.05 V, the pulse width was 0.05 s, and the pulse period was 0.5 s. After the test, the absorbance of the mixed solution was measured at 652 nm. All determinations were carried out three times under the same conditions.

[0121] In order to make the test more accurate and optimize the performance of the sensor, the concentration of two-dimensional Cu-TCPP(Fe)NSs, the concentration of AChE, the concentration of ChOx, and the addition amount of Ach were explored and optimized in this experiment.

[0122] First, 50 μL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, 50 μL of acetylcholinesterase solution with 200 mU / mL were added to a centrifuge tube and incubated in a water bath at 37 °C for 15 min. Secondly, 20 μL of two-dimensional Cu-TCPP(Fe)NSs dispersion solutions with different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL) and 10 μL of choline oxidase solution with 200 mU / mL were added to the centrifuge tube, and incubated in a water bath at 37 °C for 30 min. Then, 5 μL was taken out and dropped onto the polished electrode with a diameter of 3 mm and dried. The prepared electrode was placed in a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 120 μL of 1 g / mL ACh solution for DPV testing. The test results of the concentration of two-dimensional Cu-TCPP(Fe)NSs dispersion solution are as Figure 5 shown.

[0123] As Figure 5 can be seen, when two-dimensional Cu-TCPP(Fe)NSs with different concentrations were immobilized on the electrode, the peak current signal increased with the increase of its concentration. When the concentration reached 30 mg / mL, the peak current value was the largest. When its concentration further increased, the peak current value began to decline. Therefore, 30 mg / mL was selected as the optimal concentration.

[0124] First, add 50 μL of PBS buffer solution with pH = 6.86 and 0.025 mol / L to the centrifuge tube. Then add 50 μL of acetylcholinesterase solutions with different concentrations (50 mU / mL, 100 mU / mL, 150 mU / mL, 200 mU / mL) to the centrifuge tube and incubate in a water bath at 37 °C for 15 min. Next, add 20 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe) NSs and 10 μL of 200 mU / mL choline oxidase solution to the centrifuge tube, incubate in a water bath at 37 °C for 30 min, take out 5 μL of the above solution, drop it onto the polished electrode with a diameter of 3 mm, and then dry. Place the fabricated electrode into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, and 120 μL of 1 g / mL ACh solution for DPV testing. The test results of AChE concentration are as Figure 6 shown.

[0125] As can be seen from Figure 6 , when the concentration is 50 - 100 mU / mL, the peak current signal shows an upward trend. When the concentration is 100 - 200 mU / mL, the peak current signal shows a downward trend. Therefore, 100 mU / mL is selected as the optimal concentration of AChE.

[0126] First, add 50 μL of PBS buffer solution with pH = 6.86 and 0.025 mol / L and 50 μL of 200 mU / mL acetylcholinesterase solution to the centrifuge tube, and incubate in a water bath at 37 °C for 15 min. Then add 20 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe) NSs dispersion and 10 μL of choline oxidase solutions with different concentrations (50 mU / mL, 100 mU / mL, 150 mU / mL, 200 mU / mL, 250 mU / mL), incubate in a water bath at 37 °C for 30 min, take out 5 μL of the above solution, drop it onto the polished electrode with a diameter of 3 mm, and then dry. Place the fabricated electrode into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, and 120 μL of 1 g / mL ACh solution for DPV testing. The test results of ChOx concentration are as Figure 7 shown.

[0127] As can be seen from Figure 7 , when the concentration of ChOx is 50 - 100 mU / mL, the peak current signal increases with the increase of ChOx concentration. After 100 mU / mL, the value of I decreases with the increase of ChOx concentration. Therefore, the optimal concentration selected is 100 mU / mL.

[0128] First, add 50 μL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, 50 μL of acetylcholinesterase at 200 mU / mL into a centrifuge tube, and incubate in a water bath at 37 °C for 15 min. Then add 20 μL of the 2D Cu-TCPP(Fe)NSs dispersion at 30 mg / mL and 10 μL of choline oxidase solution at 100 mU / mL, incubate in a water bath at 37 °C for 30 min. Take out 5 μL of the above solution, drop it onto the polished electrode with a diameter of 3 mm, and dry it. Place the fabricated electrode into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, and different volumes (40 μL, 80 μL, 120 μL, 160 μL, 200 μL) of ACh solution at 1 g / mL for DPV testing. The test results of the added amount of ACh are as Figure 8 shown.

[0129] It can be Figure 8 seen that when the added amount of ACh in the test bottom solution is 40 - 120 μL, the peak current signal value increases with the increase of the added amount of ACh. When the added amount of ACh in the test bottom solution is 40 - 120 μL, the peak current signal value decreases with the increase of the added amount of ACh. Therefore, the optimal added amount of ACh is 120 μL.

[0130] Establishment of the standard curve

[0131] First, add 50 μL of MP with different standard concentrations, 50 μL of acetylcholinesterase solution at 200 mU / mL into a centrifuge tube, and incubate in a water bath at 37 °C for 15 min. Then add 20 μL of the 2D Cu-TCPP(Fe)NSs dispersion at 30 mg / mL and 10 μL of choline oxidase solution at 100 mU / mL, incubate in a water bath at 37 °C for 30 min. Take out 5 μL of the above solution, drop it onto the polished electrode with a diameter of 3 mm, and dry it. Place the fabricated electrode into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of PBS buffer solution with pH = 6.86 and 0.025 mol / L, and 120 μL of ACh solution at 1 g / mL for DPV testing, and record the peak current signal values corresponding to different standard concentrations of MP. The results are as Figure 9 shown.

[0132] After the DPV testing, use a microplate reader to detect the absorbance of the test bottom solution, and record the absorbance corresponding to different standard concentrations of MP. The results are as Figure 10 shown.

[0133] It can be Figures 9 - 10It can be seen that the concentration of MP, current I, and absorbance A show a linear relationship in the range of 1 μg / L to 100 μg / L. The linear regression equation for electrochemistry is I = -0.1089×C + 55.908, R2 = 0.9917, and the detection limit LOD = 0.2534 μg / L; the linear regression equation for colorimetry is A = -0.0007×C + 0.2153, R2 = 0.9911, and the detection limit LOD = 0.1722 μg / L.

[0134] To verify the performance of the fabricated sensor for electrochemical and colorimetric detection, the present invention investigated its reproducibility, stability, and anti-interference ability.

[0135] Anti-interference ability

[0136] Add 50 μL of 50 μg / L MP and 50 μL of interference ion solution (the interference ions are sodium chloride, urea, copper sulfate, or glucose, and one interference ion is added for each anti-interference experiment) with a concentration of 8 mg / L to a centrifuge tube, and then add 50 μL of acetylcholinesterase solution with a concentration of 200 mU / mL. Incubate in a water bath at 37 °C for 15 min. Then add 20 μL of 2D Cu-TCPP(Fe)NSs dispersion with a concentration of 30 mg / mL and 10 μL of choline oxidase solution with a concentration of 100 mU / mL, and incubate in a water bath at 37 °C for 30 min. Take out 5 μL of the above solution and drop it onto the polished electrode with a diameter of 3 mm, and then dry it. Place the fabricated electrode into a mixture composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of PBS buffer solution with a pH of 6.86 and a concentration of 0.025 mol / L, and 120 μL of ACh solution with a concentration of 1 g / mL for DPV testing. Then use a pipette to transfer 200 μL of this solution into a 96-well plate and measure its absorbance at a peak wavelength of 652 nm using a microplate reader.

[0137] To verify its anti-interference ability, sodium chloride, urea, copper sulfate, and glucose with a concentration of 8 mg / L were selected as interferents in this experiment. The anti-interference results of electrochemical detection are as Figure 11 shown, and the anti-interference results of colorimetry are as Figure 14 shown.

[0138] From Figure 10 and 14 it can be seen that the sensor has good anti-interference ability.

[0139] The reproducibility was verified by conducting 5 experiments under the optimal conditions:

[0140] Add 50 μL of 50 μg / L MP and 50 μL of 200 mU / mL acetylcholinesterase solution into a centrifuge tube, and incubate in a water bath at 37 °C for 15 min. Then add 20 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe)NSs dispersion and 10 μL of 100 mU / mL choline oxidase solution, incubate in a water bath at 37 °C for 30 min. Take out 5 μL of the above solution and drop it onto the polished electrode with a diameter of 3 mm, and then dry it (repeat the experiment for 5 groups to fabricate 5 electrodes). Put the fabricated electrodes into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of 0.025 mol / L PBS buffer solution with pH = 6.86 and 120 μL of 1 g / mL ACh solution for DPV test, record the peak current signal values corresponding to different standard concentrations of MP, and then use a pipette to transfer 200 μL of this solution into a 96-well plate and measure its absorbance with a microplate reader at the peak wavelength of 652 nm.

[0141] Figure 12 Figure shows the reproducibility results of electrochemical tests for five parallel electrodes, and the RSD is 1.66%.

[0142] Figure 15 Figure shows the reproducibility results of colorimetric tests, and the RSD is 1.14%.

[0143] From Figure 12 and Figure 15 it can be seen that the reproducibility of this sensor is good.

[0144] After the solutions for measuring current values and absorbance in the reproducibility experiment are placed for 5 days, measure the current values and absorbance again, and the results are shown in Figure 13 and Figure 16 as shown.

[0145] Figure 13 Figure shows the stability of electrochemical tests, Figure 16 Figure shows the stability of colorimetric tests.

[0146] From Figure 13 and Figure 16 it can be seen that the stability of this sensor is good

[0147] Preparation of MP solutions with different concentrations

[0148] Purchase lettuce samples from the supermarket. First, rinse the soil on the surface of the lettuce with water, chop it up and put it into a juicer to extract juice. Secondly, pour the juice into a beaker and ultrasonicate it for 5 min to make it evenly mixed. Take three portions of the above solution (2.5 g) and mix them with MP dilutions of different concentrations (5 mL of 0, 1 μg / L, 5 μg / L, 10 μg / L), dilute to 25 mL with absolute ethanol, then ultrasonicate for 15 min and filter with a 0.22 μm organic filter membrane. Place the filtrate in a 60 °C water bath and concentrate for 30 min. Finally, dilute the sample to 5 mL. At this time, the concentrations of MP in the lettuce samples are 1 μg / L, 5 μg / L, and 10 μg / L respectively.

[0149] Since MP was not detected in the lettuce samples, MP with concentrations of 0, 1 μg / L, 5 μg / L, and 10 μg / L was added to the lettuce samples, and it was incubated with 50 μL of 200 mU / mL acetylcholinesterase in a 37 °C water bath for 15 min. Then add 20 μL of 30 mg / mL two-dimensional Cu-TCPP(Fe)NSs dispersion and 10 μL of 100 mU / mL choline oxidase solution, and incubate in a 37 °C water bath for 30 min. Take out 5 μL of the above solution and drop it onto the polished electrode with a diameter of 3 mm and dry it. Place the prepared electrode into a mixed solution composed of 1.5 mL of 0.4 mol / L TMB solution, 1.5 mL of 0.025 mol / L PBS buffer solution with pH = 6.86, and 120 μL of 1 g / mL ACh solution for DPV testing. After the testing, measure the absorbance of the bottom solution with an enzyme-labeled instrument. Each group is repeated three times. The electrochemical results are shown in Table 1, and the colorimetric results are shown in Table 2.

[0150] Table 1 Electrochemical Detection of MP in Lettuce Samples

[0151]

[0152] Table 2 Colorimetric Detection of MP in Lettuce Samples

[0153]

[0154] As can be seen from Tables 1 - 2, in the electrochemical test, the recovery rate of MP is 91% - 102.7%, and RSD ≤ 2.13%; in the colorimetric test, the recovery rate of MP is 93% - 103.4%, and RSD ≤ 2.42%.

[0155] The present invention realizes the dual-mode detection of organophosphorus by constructing a novel sensor. The dual-mode detection of colorimetry and electrochemistry can play a role of mutual calibration, greatly improving the accuracy of detection. The experimental results show that the sensor has good anti-interference ability, reproducibility and stability, and there is a good linear relationship between I and A and the MP concentration, with the linear correlation coefficients being R2 = 0.9917 and R2 = 0.9911 respectively, and the detection limits being LOD = 0.2534 μg / L and LOD = 0.1722 μg / L respectively. Therefore, the dual-mode detection of colorimetry and electrochemistry can realize the detection of actual samples, and has good future application prospects.

[0156] 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 invention comprises acetylcholine, acetylcholinesterase, choline oxidase, two-dimensional Cu-TCPP(Fe)NSs and a color developing agent capable of generating a hydroxyl color developing group.

2. The detection sensor according to claim 1, characterized in that: Also included are electrodes; The two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase and choline oxidase are loaded on the electrode; Alternatively, the two-dimensional Cu-TCPP(Fe)NSs is loaded on the electrode and the acetylcholinesterase and choline oxidase are loaded on the two-dimensional Cu-TCPP(Fe)NSs.

3. The detection sensor according to claim 2, characterized in that: The method for loading the two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase and choline oxidase on the electrode comprises the following steps: The dispersion of two-dimensional Cu-TCPP(Fe)NSs, acetylcholinesterase solution and choline oxidase solution were mixed and dropped onto the electrode and then dried.

4. The detection sensor according to claim 3, characterized in that: The loading amount of acetylcholinesterase on the electrode is 0.09-0.4 mU / 7 mm 2 The loading amount of the two-dimensional Cu-TCPP(Fe)NSs on the electrode is 0.007-0.04 mg / 7 mm 2 ; The volume ratio of the two-dimensional Cu-TCPP(Fe)NSs dispersion to the acetylcholinesterase solution is 1:1-10; The concentration of the two-dimensional Cu-TCPP(Fe)NSs dispersion is 10-50 mg / mL; the concentration of the acetylcholinesterase solution is 50-100 mU / mL.

5. The detection sensor according to claim 3 or 4, characterized in that: The loading amount of choline oxidase on the electrode is 0.02-0.10 mU / 7 mm 2 ; The volume ratio of the two-dimensional Cu-TCPP(Fe)NSs dispersion to the choline oxidase solution is 1 to 5:1; The concentration of the choline oxidase solution is 50-250 mU / mL.

6. Use of the detection sensor according to claim 1 in colorimetric detection of organophosphorus pesticides.

7. Use of the detection sensor according to any one of claims 2 to 5 in electrochemical detection of organophosphorus pesticides and colorimetric detection of organophosphorus pesticides.

8. The use according to claim 7, characterized in that: The electrochemical detection of organophosphorus pesticides comprises the following steps: mixing a solution to be tested containing an organophosphorus with an acetylcholinesterase solution to obtain a mixture; The mixture is mixed with a dispersion of two-dimensional Cu-TCPP(Fe)NSs and a choline oxidase solution, and then dripped onto an electrode and dried to obtain a sensor to be detected; The sensor to be tested is placed in a DPV test solution containing acetylcholine and a color developer that can generate a hydroxyl color developing group to perform a DPV test to obtain a peak current signal value; The concentration of the organic phosphorus in the solution to be tested is obtained according to the linear relationship between the peak current signal value and the peak current signal value and concentration of a standard organic phosphorus solution measured under the same test conditions.

9. The use according to claim 8, characterized in that: The colorimetric detection of organophosphorus pesticides comprises the following steps: testing the absorbance of the solution after the DPV test, and then obtaining the concentration of the organophosphorus in the solution to be detected based on the obtained absorbance and the concentration-absorbance curve of the standard solution.

10. The use according to claim 8 or 9, characterized in that: The method for preparing the DPV test solution containing acetylcholine and a color developer capable of generating a hydroxyl color developing group comprises the following steps: Mixing an acetylcholine solution and a solution of a color developing agent capable of generating a hydroxyl color developing group; The mass of acetylcholine in the acetylcholine solution is 0.04 g to 0.12 g; The concentration of the acetylcholine solution is 1 g / mL, and the volume is 40 to 120 μL.

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