Method for detecting chlorpyrifos through biosensor based on flower-shaped vanadium disulfide and application

Abstract

The invention relates to the technical field of pesticide residue detection, in particular to a method for detecting chlorpyrifos based on a flower-like vanadium disulfide biosensor and application. According to the method, based on flower-shaped vanadium disulfide, a colorimetric biosensor and an electrochemical biosensor are adopted for quantitative analysis of chlorpyrifos; by adopting a dual-mode method, the detection range of chlorpyrifos can be widened, the application flexibility of the sensor can be enhanced, a proper detection method can be selected according to actual test requirements, and resource waste is reduced; meanwhile, nano materials are adopted to improve the detection performance of the sensor, in colorimetric detection, the detection range is 0-100 [mu] M, the detection limit is as low as 0.52 nM, and the linear correlation coefficient of a prediction model reaches 0.989; in electrochemical detection, the detection range is 0-1nM, the detection limit is as low as 5.8 fM, the linear correlation coefficient of a prediction model reaches 0.993, and high-sensitivity and visual detection analysis on chlorpyrifos is realized.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide residue detection, and in particular to a method and application of detecting chlorpyrifos using a biosensor based on flower-shaped vanadium disulfide. Background Art

[0002] Organophosphorus pesticides are one of the pesticides with a high proportion of pesticide use, mainly used to manage plant pests and diseases to increase agricultural production. Organophosphorus pesticides are highly toxic and are not only associated with hematological cancers and solid tumors, but also damage the human nervous system, leading to neurological diseases such as Parkinson's disease. In the nervous system, organophosphorus pesticides can inhibit the activity of acetylcholinesterase, which hinders the decomposition of acetylcholine, and the accumulated acetylcholine can cause paralysis or death in animals. Chlorpyrifos is one of the most important broad-spectrum organophosphorus insecticides, widely used in agricultural farming, public health and other systems, and can be continuously migrated into the ecological food chain through human activities such as agriculture and industry. Chlorpyrifos can cause DNA damage, gene mutations and chromosomal aberrations, increasing the risk of cancer, especially lung cancer. Therefore, the detection of chlorpyrifos is crucial to food safety, environmental safety and human health.

[0003] In recent years, with the improvement of people's awareness of dietary health and the poisoning incidents caused by pesticide residues, the market demand for food safety testing technology has been greatly promoted. Traditional instrumental analysis methods include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), etc. Although these instrumental analysis methods have high accuracy, they have disadvantages such as expensive instruments and equipment, complex pretreatment processes, the need for professional technicians and time-consuming, which hinder their widespread application in the market. In contrast, food safety rapid testing technology has the advantages of being fast, sensitive, portable, efficient and economical, and is more universal in market applications. Food safety rapid testing technology helps companies to conduct self-inspections, help companies meet regulatory requirements and reduce economic losses; it helps to solve problems in the food supply chain, is used to identify potential risks in the food processing and production process, and ensures that the process from food production to consumption is transparent and traceable; it helps to improve the efficiency of food processing and production, shorten inspection time, and accelerate food supply.

[0004] Biosensors are one of the rapid detection technologies. According to the signal type, they can be divided into electrochemical, fluorescent, colorimetric and photothermal sensors. Colorimetric methods can quickly identify the test results with the naked eye. Electrochemistry has a high detection sensitivity. However, these technologies are usually based on single signal output and may have some limitations, such as narrow detection range, poor application flexibility, and poor anti-interference ability. In contrast, multimodal biosensors can not only effectively broaden the detection range, but also generate multi-dimensional detection signals, which helps to improve the detection sensitivity and enhance the accuracy of the results. In addition, nanomaterials are an effective means to improve the detection performance of sensors, with the advantages of controllable synthesis and good stability. Metal sulfide nanomaterials, such as MoS2, WS2 and VS2, have inherent peroxidase mimetic properties and excellent electrical properties. Therefore, in order to improve the reliability of chlorpyrifos detection results, it is very important to develop a colorimetric / electrochemical dual-mode detection method based on nanomaterials such as metal sulfides. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of a biosensor for detecting chlorpyrifos based on flower-shaped vanadium disulfide, so as to solve the problems that traditional detection methods are complicated and time-consuming, and the signal of the biosensor is single during detection and the application flexibility is poor.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a method for detecting chlorpyrifos based on a flower-shaped vanadium disulfide biosensor, wherein the biosensor comprises a colorimetric biosensor, and the detection of the colorimetric biosensor comprises the following steps:

[0007] S11, mixing different concentrations of chlorpyrifos, acetylcholinesterase and PBS buffer, and incubating the reaction at 37°C;

[0008] After the S12 and S11 reactions were completed, choline oxidase and acetylcholine were added and the reaction was incubated at 37°C;

[0009] After the S13 and S12 reactions were completed, flower-shaped VS2, organic color developer TMB, and acetate buffer were added and the reactions were incubated at 37°C;

[0010] S14. Measure the absorbance of the solution after the reaction in S13 is completed, and establish a standard curve for colorimetric detection of chlorpyrifos, which can be used for quantitative analysis of the sample to be tested.

[0011] Furthermore, in S14, an ultraviolet-visible spectrophotometer is used to measure the absorbance, and the measuring wavelength is 652 nm.

[0012] Furthermore, in the S14, a standard curve for colorimetric detection of chlorpyrifos is established with the concentration of chlorpyrifos as the horizontal coordinate and the absorbance ΔA (A0-A) as the vertical coordinate, wherein A0 represents the absorbance when chlorpyrifos is not present and A represents the absorbance when chlorpyrifos is present; the regression equation obtained by linear fitting of the standard curve is y=0.0752x-0.0594(R 2 =0.989), and the detection limit was 0.52 nM.

[0013] Furthermore, the biosensor also includes an electrochemical sensor, and the detection of the electrochemical sensor includes the following steps:

[0014] S21, constructing an electrochemical sensor;

[0015] S22, adding chlorpyrifos of different concentrations to the surface of the electrochemical sensor constructed in S21, incubating at room temperature, and washing with ultrapure water;

[0016] S23. Place the washed glassy carbon electrode in a PBS solution containing acetylthiocholine to perform an It curve test; draw a standard curve for electrochemical detection of chlorpyrifos, which can be used for quantitative analysis of the sample to be tested.

[0017] Furthermore, in S21, constructing the electrochemical sensor comprises the following steps:

[0018] S31, drop the flower-shaped VS2 onto the surface of the glassy carbon electrode and dry it at room temperature;

[0019] S32, adding glutaraldehyde solution to the surface of the dried glassy carbon electrode, and washing the surface of the glassy carbon electrode with ultrapure water after the reaction is completed;

[0020] S33. AChE was added dropwise to the surface of the washed glassy carbon electrode, incubated at 4°C for reaction, and washed with PBS to obtain the constructed electrochemical sensor.

[0021] Further, in the S23, the standard curve of electrochemical detection of chlorpyrifos is drawn with the concentration of chlorpyrifos as the horizontal coordinate and the current ΔI (I0-I) as the vertical coordinate; wherein I0 represents the current value when chlorpyrifos is not present, and I represents the current value when chlorpyrifos is present; the regression equation of the standard curve obtained by linear fitting is y=8.85×10 -7 x+2.27×10 -6 (R 2 =0.993), with a detection limit of 5.8 fM.

[0022] Furthermore, the preparation of the flower-shaped VS2 comprises the following steps:

[0023] S41, taking ammonium metavanadate and thioacetamide and dissolving them in deionized water to obtain a mixed solution;

[0024] S42, transferring the mixed solution obtained in S41 to a hydrothermal reactor to continue the reaction;

[0025] S43. After the reaction is completed, the flower-like VS2 can be obtained by natural cooling, solid-liquid separation, washing and freeze-drying.

[0026] Application of the above method in the detection of pesticide residues in fruits and vegetables.

[0027] Furthermore, the application in the detection of pesticide residues in fruits and vegetables comprises the following steps:

[0028] S51, adding the fruit and vegetable samples into distilled water, ultrasonicating, centrifuging, and filtering to obtain a solution to be tested;

[0029] S52, dividing the solution to be tested obtained in S51 into two groups, and adding different concentrations of spikes to the solution in each group;

[0030] S53, respectively detecting the two groups of solutions to be detected after the addition of the spikes by a colorimetric biosensor and an electrochemical biosensor, and calculating the spike recovery rates respectively.

[0031] Beneficial effects of the present invention:

[0032] 1. The method of the present invention uses flower-shaped VS2 for the first time to construct a colorimetric / electrochemical dual-mode sensor. In colorimetric detection, the detection range is 0-100 μM, the detection limit is as low as 0.52 nM, and the linear correlation coefficient of the prediction model is 0.989; in electrochemical detection, the detection range is 0-1 nM, the detection limit is as low as 5.8 fM, and the linear correlation coefficient of the prediction model is 0.993, achieving high sensitivity and visual detection and analysis of chlorpyrifos;

[0033] 2. The colorimetric / electrochemical dual-mode sensor constructed by the present invention can not only broaden the detection range of chlorpyrifos, but also enhance the application flexibility of the sensor. The appropriate detection method can be selected according to actual test requirements to reduce resource waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the colorimetric / electrochemical dual-mode detection of chlorpyrifos based on the flower-shaped VS2 of the present invention;

[0035] Figure 2 This is a standard working curve diagram for colorimetric detection of chlorpyrifos in Example 2 of the present invention;

[0036] Figure 3 This is a standard working curve diagram of electrochemical detection of chlorpyrifos in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0038] The principle of the present invention is:

[0039] The present invention utilizes the enzyme-mimicking property and the conductive property of the flower-shaped VS2 to construct a colorimetric / electrochemical dual-mode chlorpyrifos detection sensor.

[0040] like Figure 1 As shown, in colorimetric detection, acetylcholinesterase can catalyze the hydrolysis of acetylcholine to generate choline and acetic acid. The generated choline undergoes an oxidation reaction under the catalytic action of choline oxidase to generate betaine and hydrogen peroxide (H2O2). The generated H2O2 oxidizes the organic color developer TMB into oxTMB under the catalytic action of VS2 mimetic enzyme, thereby changing the color of the solution from colorless to blue; when the target substance is contained in the test solution, since the target substance can inhibit the biological activity of acetylcholinesterase, the decomposition of acetylcholine is hindered, the concentration of generated choline is reduced, and the content of H2O2 generated by choline decomposition is reduced, and the amount of TMB oxidized is reduced, which ultimately causes the solution color to become lighter and the absorbance value of the solution to decrease. Therefore, the qualitative analysis of the target object can be achieved through the change of solution color, and the quantitative analysis of the target object can be achieved by measuring the absorbance value of the solution through an instrument.

[0041] In electrochemical detection, acetylcholinesterase can catalyze acetylthiocholine to produce thiocholine, and thiocholine can undergo redox reaction on the electrode surface to generate electrical signals. Modifying the electrode with VS2 can enhance the electron transfer rate and signal sensitivity. When the test liquid contains the target, the target can inhibit the biological activity of acetylcholinesterase, thereby hindering the decomposition of acetylthiocholine, reducing the content of generated thiocholine, and causing the electrical signal to become smaller, thereby achieving electrochemical detection of the target.

[0042] Example 1

[0043] Preparation of flower-shaped VS2:

[0044] Take 6 mmol of sodium orthovanadate (Na3VO4·12H2O) and 32 mmol of thioacetamide (TAA) and dissolve them in 50 mL of deionized water. Stir the solution vigorously for 1 h.

[0045] The prepared solution was transferred into a 100 mL polytetrafluoroethylene-lined autoclave and reacted at 60 °C for 24 h.

[0046] The system was slowly cooled to room temperature and centrifuged to obtain a black precipitate. The black precipitate was washed three times with deionized water and anhydrous ethanol, collected by vacuum filtration, and freeze-dried for 12 hours to obtain a black powder solid, which was the flower-like VS2.

[0047] Example 2

[0048] Drawing of the working curve for colorimetric detection of chlorpyrifos:

[0049] 25 μL of different concentrations of chlorpyrifos (0, 10 nM, 100 nM, 200 nM, 1 μM, 10 μM, 50 μM, 100 μM), 15 μL of acetylcholinesterase (2 U / mL) and 60 μL of PBS buffer (pH 7.2-7.4, 0.01 M) were mixed and incubated at 37°C for 30 min.

[0050] 25 μL of choline oxidase (2U / mL) and 40 μL of acetylcholine (20 mM) were added to the solution and incubated at 37°C for 20 min. After the reaction was completed, 50 μL of flower-shaped VS2 (50 μL, 0.2 mg / mL), 50 μL of organic color developer TMB (50 μL, 5 mM) and 735 μL of acetate buffer (pH 4.0, 0.1 M) were added to the solution. After incubation at 37°C for 20 min, the absorbance of the solution at 652 nm was measured using a UV-visible spectrophotometer. Figure 2 A).

[0051] With the concentration of chlorpyrifos as the horizontal axis and the absorbance ΔA (A0-A) as the vertical axis, the standard curve for the detection of chlorpyrifos by this method was drawn ( Figure 2 B), the regression equation obtained by linear fitting is y=0.0752x-0.0594(R 2 =0.989), the detection limit of this method was calculated to be 0.52 nM; wherein A0 represents the absorbance when there is no chlorpyrifos, and A represents the absorbance when there is chlorpyrifos.

[0052] Example 3

[0053] Preparation of electrochemical sensor:

[0054] 8 μL of 5 mg / mL flower-shaped VS2 was added to the electrode surface (glassy carbon electrode) and dried at room temperature; 5 μL of glutaraldehyde (2% aqueous solution) was added to the electrode surface, reacted for 4 hours, and then the electrode surface was cleaned with ultrapure water; 8 μL of 200 mU / mL AChE was continued to be added to the electrode surface, incubated in a refrigerator at 4°C for 6 hours, and washed with 0.01 M PBS to obtain an electrochemical sensor.

[0055] Example 4

[0056] Drawing of the working curve of electrochemical detection of chlorpyrifos:

[0057] Different concentrations of chlorpyrifos (0, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM) were added dropwise to the sensor electrode surface prepared in Example 4, incubated at room temperature for 1 h, and then the electrode surface was washed with ultrapure water.

[0058] The electrode was placed in a 1×PBS solution containing 0.5 mM acetylthiocholine for It curve testing ( Figure 3 A); with the concentration of chlorpyrifos as the horizontal axis and the current ΔI (I0-I) as the vertical axis, the standard curve for the detection of chlorpyrifos by this method was drawn ( Figure 3 B), the regression equation obtained by linear fitting is y = 8.85 × 10 -7 x+2.27×10 -6 (R 2 =0.993), the detection limit of this method was calculated to be 5.8 fM; wherein I0 represents the current value when there is no chlorpyrifos, and I represents the current value when there is chlorpyrifos.

[0059] Example 5

[0060] Detection of chlorpyrifos in actual samples:

[0061] Take 15 g of apple peel sample and add it to 40 mL of distilled water and treat it with ultrasound for 60 min.

[0062] The mixed solution was centrifuged at 10000 rpm for 10 min to remove insoluble matter; the supernatant was filtered through a 0.22 μm filter membrane to obtain the corresponding solution to be tested.

[0063] The test solution was divided into two groups, A and B, and each group was divided into three parts. Chlorpyrifos with concentrations of 5 μM, 15 μM, and 45 μM were added to the three test solutions in group A, and chlorpyrifos with concentrations of 5 pM, 15 pM, and 45 pM were added to the three test solutions in group B.

[0064] The sample of group A was used to replace the chlorpyrifos standard solution in Example 2, and the reaction was carried out according to the steps in Example 2, and finally the absorbance of the detection system was tested by a UV-visible spectrophotometer. The obtained absorbance value was substituted into the linear regression equation of the standard working curve to calculate the concentration of chlorpyrifos in the sample to be tested, and the spiked recovery rate was calculated (Table 1).

[0065] The test samples of group B were used to replace the chlorpyrifos standard solution in Example 4, and the reaction was carried out according to the steps in Example 4. The obtained electrical signals were brought into the linear regression equation of the standard working curve to calculate the concentration of chlorpyrifos in the test samples, and the spiked recovery rate was calculated (Table 2).

[0066] Table 1 Analysis of chlorpyrifos in actual samples by colorimetric method

[0067]

[0068] Table 2 Analysis of chlorpyrifos in actual samples by electrochemical method

[0069]

[0070] As shown in Table 1, the recovery rate of chlorpyrifos detected by the colorimetric method in actual samples is 86.0% to 109.6%, and the relative standard deviation (RSD) is 2.76 to 4.11%; the recovery rate of chlorpyrifos detected by the electrochemical method (Table 2) in actual samples is 82.1% to 112.2%, and the relative standard deviation (RSD) is 2.83 to 3.92%; these results show that the present invention has good accuracy in detecting chlorpyrifos in fruit samples.

[0071] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A method for detecting chlorpyrifos based on a flower-shaped vanadium disulfide biosensor, characterized in that: The biosensor comprises a colorimetric biosensor, and the detection of the colorimetric biosensor comprises the following steps: S11, mixing different concentrations of chlorpyrifos, acetylcholinesterase and PBS buffer, and incubating the reaction at 37°C; After the S12 and S11 reactions were completed, choline oxidase and acetylcholine were added and the reaction was incubated at 37°C; After the S13 and S12 reactions were completed, flower-shaped VS2, organic color developer TMB, and acetate buffer were added and the reactions were incubated at 37°C; S14. Measure the absorbance of the solution after the reaction in S13 is completed, and establish a standard curve for colorimetric detection of chlorpyrifos, which can be used for quantitative analysis of the sample to be tested.

2. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 1, characterized in that: In the step S14, an ultraviolet-visible spectrophotometer is used to measure absorbance, and the measuring wavelength is 652 nm.

3. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 1, characterized in that: In the above S14, a standard curve for colorimetric detection of chlorpyrifos is established with the concentration of chlorpyrifos as the horizontal coordinate and the absorbance ΔA (A0-A) as the vertical coordinate, wherein A0 represents the absorbance when chlorpyrifos is not present and A represents the absorbance when chlorpyrifos is present; the regression equation obtained by linear fitting of the standard curve is y=0.0752x-0.0594(R 2 =0.989), and the detection limit was 0.52 nM.

4. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 1, characterized in that: The biosensor also includes an electrochemical sensor, and the detection of the electrochemical sensor includes the following steps: S21, constructing an electrochemical sensor; S22, adding chlorpyrifos of different concentrations to the surface of the electrochemical sensor constructed in S21, incubating at room temperature, and washing with ultrapure water; S23. Place the washed glassy carbon electrode in a PBS solution containing acetylthiocholine to perform an It curve test; draw a standard curve for electrochemical detection of chlorpyrifos, which can be used for quantitative analysis of the sample to be tested.

5. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 4, characterized in that: In the above-mentioned S21, constructing the electrochemical sensor comprises the following steps: S31, drop the flower-shaped VS2 onto the surface of the glassy carbon electrode and dry it at room temperature; S32, adding glutaraldehyde solution to the surface of the dried glassy carbon electrode, and washing the surface of the glassy carbon electrode with ultrapure water after the reaction is completed; S33. AChE was added dropwise to the surface of the washed glassy carbon electrode, incubated at 4°C for reaction, and washed with PBS to obtain the constructed electrochemical sensor.

6. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 4, characterized in that: In the S23, the standard curve of electrochemical detection of chlorpyrifos is drawn with the concentration of chlorpyrifos as the horizontal coordinate and the current ΔI (I0-I) as the vertical coordinate; wherein I0 represents the current value when chlorpyrifos is not present, and I represents the current value when chlorpyrifos is present; the regression equation of the standard curve obtained by linear fitting is y=8.85×10 -7 x+2.27×10 -6 (R 2 =0.993), with a detection limit of 5.8 fM.

7. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to any one of claims 1 to 6, characterized in that: The preparation of the flower-shaped VS2 comprises the following steps: S41, taking ammonium metavanadate and thioacetamide and dissolving them in deionized water to obtain a mixed solution; S42, transferring the mixed solution obtained in S41 to a hydrothermal reactor to continue the reaction; S43. After the reaction is completed, the flower-like VS2 can be obtained by natural cooling, solid-liquid separation, washing and freeze-drying.

8. Use of the method according to any one of claims 1 to 7 in the detection of pesticide residues in fruits and vegetables.

9. The use according to claim 8, characterized in that: The application in the detection of pesticide residues in fruits and vegetables includes the following steps: S51, adding the fruit and vegetable samples into distilled water, ultrasonicating, centrifuging, and filtering to obtain a solution to be tested; S52, dividing the solution to be tested obtained in S51 into two groups, and adding different concentrations of spikes to the solution in each group; S53, respectively detecting the two groups of solutions to be detected after the addition of the spikes by a colorimetric biosensor and an electrochemical biosensor, and calculating the spike recovery rates respectively.

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