Method and application of chlorpyrifos detection using flower-shaped vanadium disulfide biosensor

Abstract

The present invention relates to the field of pesticide residue detection technology, and specifically to a method and application for detecting chlorpyrifos using a flower-shaped vanadium disulfide-based biosensor. The method of the present invention, based on flower-shaped vanadium disulfide, employs both a colorimetric biosensor and an electrochemical biosensor for quantitative analysis of chlorpyrifos. The dual-mode approach not only broadens the detection range of chlorpyrifos but also enhances the sensor's application flexibility, allowing the selection of appropriate detection methods based on actual testing needs and reducing resource waste. Nanomaterials are also employed to enhance sensor detection performance. In colorimetric detection, the detection range is 0 to 100 μM, with a detection limit as low as 0.52 nM and a linear correlation coefficient of the prediction model reaching 0.989. In electrochemical detection, the detection range is 0 to 1 nM, with a detection limit as low as 5.8 fM and a linear correlation coefficient of the prediction model reaching 0.993, achieving highly sensitive and visual detection and analysis of chlorpyrifos.

Description

Technical Field

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

[0002] Organophosphorus pesticides account for a significant portion of pesticide usage, primarily used to manage plant pests and diseases to increase agricultural yields. Organophosphorus pesticides are highly toxic and are not only associated with hematological cancers and solid tumors, but can 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, hindering the breakdown of acetylcholine. Accumulated acetylcholine can cause paralysis or death in animals. Chlorpyrifos is one of the most important broad-spectrum organophosphorus insecticides, widely used in agriculture, public health, and other systems. It can be continuously transferred 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 for food safety, environmental safety, and human health.

[0003] In recent years, rising awareness of dietary health and the incidence of poisoning due to pesticide residues have significantly driven market demand for food safety testing technologies. Traditional instrumental analytical methods include high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). While these methods offer high accuracy, their widespread adoption is hindered by costly equipment, complex pre-treatment processes, the need for specialized technicians, and time-consuming processes. In contrast, rapid food safety testing technologies offer advantages such as speed, sensitivity, portability, efficiency, and cost-effectiveness, making them more widely applicable. Rapid food safety testing technologies facilitate self-inspection by businesses, helping them meet regulatory requirements and reduce financial losses. They also address issues within the food supply chain by identifying potential risks in food processing and production, ensuring transparency and traceability from production to consumption. They also help improve food processing and production efficiency, shorten inspection times, and expedite food supply.

[0004] Biosensors are a type of rapid detection technology. Depending on the signal type, they can be categorized as electrochemical, fluorescence, colorimetric, and photothermal sensors. Colorimetric methods allow for rapid visual identification of test results. Electrochemistry offers high detection sensitivity. However, these technologies typically rely on single-signal outputs and may have limitations, such as a narrow detection range, limited application flexibility, and poor anti-interference capabilities. In contrast, multimodal biosensors can not only effectively broaden the detection range but also generate multidimensional detection signals, helping to improve detection sensitivity and enhance the accuracy of results. Furthermore, nanomaterials are an effective means of enhancing sensor detection performance, offering advantages such as controllable synthesis and good stability. Metal sulfide nanomaterials, such as MoS2, WS2, and VS2, possess inherent peroxidase-mimicking properties and excellent electrical properties. Therefore, to improve the reliability of chlorpyrifos detection results, it is crucial 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 of traditional detection methods being complicated and time-consuming, as well as the single signal and poor application flexibility of biosensors during detection.

[0006] To achieve the above object, the present invention adopts the following technical solution: 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 reaction was incubated at 37°C;

[0010] S14. Measure the absorbance of the solution after the reaction in S13 is completed to 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 the above-mentioned S14, the absorbance is measured using an ultraviolet-visible spectrophotometer, and the measuring wavelength is 652 nm.

[0012] Furthermore, in the above S14, a standard curve for colorimetric detection of chlorpyrifos is established with the concentration of chlorpyrifos as the horizontal axis and the absorbance ΔA (A0-A) as the vertical axis, 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), with a detection limit of 0.52 nM.

[0013] A method for detecting chlorpyrifos based on a flower-shaped vanadium disulfide biosensor, wherein the biosensor includes an electrochemical sensor, and the detection of the electrochemical sensor includes the following steps:

[0014] S21, constructing an electrochemical sensor;

[0015] S22, adding different concentrations of chlorpyrifos 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 the above-mentioned S21, constructing the electrochemical sensor includes the following steps:

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

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

[0020] S33. Add AChE to the surface of the washed glassy carbon electrode, incubate the reaction at 4°C, and wash with PBS to obtain the constructed electrochemical sensor.

[0021] Furthermore, in the above S23, a standard curve for electrochemical detection of chlorpyrifos is drawn with the concentration of chlorpyrifos as the horizontal axis and the current ΔI (I0-I) as the vertical axis; wherein I0 represents the current value when chlorpyrifos is not present, and I represents the current value when chlorpyrifos is present; the standard curve is linearly fitted to obtain a regression equation of 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 and continuing the reaction;

[0025] S43. After the reaction is completed, the flower-shaped 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 includes the following steps:

[0028] S51, adding the fruit and vegetable samples to distilled water, sonicating, 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 solutions 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 or an electrochemical biosensor, and calculating the spike recovery rates respectively.

[0031] Beneficial effects of the present invention:

[0032] 1. The present method utilizes the 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 reaches 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 reaches 0.993, achieving highly sensitive and visual detection and analysis of chlorpyrifos.

[0033] 2. The colorimetric / electrochemical dual-mode sensor constructed in 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 testing requirements, reducing resource waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the principle of 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 with reference to 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 properties and conductive properties of flower-shaped VS2 to construct a colorimetric / electrochemical dual-mode sensor for detecting chlorpyrifos.

[0040] like Figure 1 As shown, in colorimetric detection, acetylcholinesterase catalyzes the hydrolysis of acetylcholine to produce choline and acetic acid. The resulting choline, catalyzed by choline oxidase, undergoes oxidation to produce betaine and hydrogen peroxide (H2O2). The resulting H2O2, catalyzed by the VS2 mimetic enzyme, oxidizes the organic color developer TMB into oxTMB, causing the solution color to change from colorless to blue. When the test solution contains a target compound, the target compound inhibits the biological activity of acetylcholinesterase, hindering the decomposition of acetylcholine and reducing the concentration of choline generated. This, in turn, reduces the amount of H2O2 produced by choline decomposition and the amount of TMB oxidized, ultimately resulting in a lighter solution color and a lower absorbance. Therefore, qualitative analysis of the target compound can be achieved by the solution color change, while quantitative analysis of the target compound can be achieved by measuring the absorbance of the solution using an instrument.

[0041] In electrochemical detection, acetylcholinesterase catalyzes the production of thiocholine from acetylthiocholine. Thiocholine undergoes a redox reaction on the electrode surface, generating an electrical signal. Modifying the electrode with VS2 enhances the electron transfer rate and signal sensitivity. When the test solution contains a target substance, the target substance inhibits the biological activity of acetylcholinesterase, thereby hindering the decomposition of acetylthiocholine and reducing the amount of thiocholine generated, resulting in a smaller electrical signal, thus enabling electrochemical detection of the target substance.

[0042] Example 1

[0043] Preparation of flower-shaped VS2:

[0044] 6 mmol of sodium orthovanadate (Na3VO4·12H2O) and 32 mmol of thioacetamide (TAA) were dissolved in 50 mL of deionized water and the solution was vigorously stirred 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] Mix 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) and incubate at 37°C for 30 min.

[0050] 25 μL of choline oxidase (2 U / 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] The standard curve for the detection of chlorpyrifos by this method was drawn with the concentration of chlorpyrifos as the horizontal axis and the absorbance ΔA (A0-A) as the vertical axis ( 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 in the absence of chlorpyrifos, and A represents the absorbance in the presence of 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 washed 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 for electrochemical detection of chlorpyrifos:

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

[0058] The electrode was placed in 1×PBS solution containing 0.5 mM acetylthiocholine to perform the It curve test ( 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; where I0 represents the current value when chlorpyrifos is not present, and I represents the current value when chlorpyrifos is present.

[0059] Example 5

[0060] Detection of chlorpyrifos in actual samples:

[0061] 15 g of apple peel sample was added to 40 mL of distilled water and ultrasonically treated for 60 min.

[0062] The mixed solution was centrifuged at 10,000 rpm for 10 minutes to remove insoluble matter; the supernatant was filtered through a 0.22 μm membrane filter 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 at concentrations of 5 μM, 15 μM, and 45 μM was added to the three test solutions in group A, respectively. Chlorpyrifos at concentrations of 5 pM, 15 pM, and 45 pM was added to the three test solutions in group B, respectively.

[0064] The chlorpyrifos standard solution in Example 2 was replaced with the test sample from Group A. The reaction was carried out according to the same steps as in Example 2, and the absorbance of the detection system was measured using a UV-visible spectrophotometer. The obtained absorbance value was substituted into the linear regression equation of the standard working curve to calculate the chlorpyrifos concentration in the test sample, and the spiked recovery was also calculated (Table 1).

[0065] The chlorpyrifos standard solution in Example 4 was replaced with the test samples in Group B, and the reaction was carried out according to the steps in Example 4. The obtained electrical signal was substituting into the linear regression equation of the standard working curve to calculate the concentration of chlorpyrifos in the test samples, and the spiked recovery was calculated (Table 2).

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

[0067]

[0068] Table 2 Electrochemical analysis of chlorpyrifos in actual samples

[0069]

[0070] As shown in Table 1, the colorimetric method had a spiked recovery rate of 86.0% to 109.6% for chlorpyrifos in actual samples, with a relative standard deviation (RSD) of 2.76 to 4.11%. The electrochemical method (Table 2) had a spiked recovery rate of 82.1% to 112.2% for chlorpyrifos in actual samples, with a relative standard deviation (RSD) of 2.83 to 3.92%. These results indicate 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 forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

Claims

1. A method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor, characterized in that: The biosensor includes a colorimetric biosensor, and the detection of the colorimetric biosensor includes 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 reaction was incubated at 37°C; Sodium orthovanadate and thioacetamide were dissolved in deionized water and the solution was vigorously stirred. The prepared solution was transferred to a polytetrafluoroethylene-lined autoclave for reaction. After the reaction was completed, the system was slowly cooled to room temperature and centrifuged to obtain a black precipitate. The black precipitate was rinsed three times with deionized water and anhydrous ethanol, collected by vacuum filtration, and freeze-dried to obtain a black powder solid, which was the flower-like VS2. S14. Measure the absorbance of the solution after the reaction in S13 is completed to 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, wherein: In the step S14, the absorbance is measured using an ultraviolet-visible spectrophotometer at a wavelength of 652 nm.

3. The method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor according to claim 1, wherein: In the above S14, a standard curve for colorimetric detection of chlorpyrifos is established with the concentration of chlorpyrifos as the horizontal axis and the absorbance ΔA (A0-A) as the vertical axis, 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), with a detection limit of 0.52 nM.

4. A method for detecting chlorpyrifos using a flower-shaped vanadium disulfide biosensor, characterized in that: The biosensor includes an electrochemical sensor, and the detection of the electrochemical sensor includes the following steps: S21, constructing an electrochemical sensor; Flower-shaped VS2 was dropped onto the surface of a glassy carbon electrode and allowed to dry at room temperature. Glutaraldehyde solution was added to the dried surface of the glassy carbon electrode, and after the reaction was complete, the surface of the glassy carbon electrode was washed with ultrapure water. AChE was added to the washed surface of the glassy carbon electrode, the reaction was incubated at 4°C, and then washed with PBS to obtain the constructed electrochemical sensor. Sodium orthovanadate and thioacetamide were dissolved in deionized water and the solution was vigorously stirred. The prepared solution was transferred to a polytetrafluoroethylene-lined autoclave for reaction. After the reaction was completed, the system was slowly cooled to room temperature and centrifuged to obtain a black precipitate. The black precipitate was rinsed three times with deionized water and anhydrous ethanol, collected by vacuum filtration, and freeze-dried to obtain a black powder solid, which was the flower-like VS2. S22, adding different concentrations of chlorpyrifos 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, wherein: In the above S23, a standard curve for electrochemical detection of chlorpyrifos is drawn with the concentration of chlorpyrifos as the horizontal axis and the current ΔI (I0-I) as the vertical axis; wherein I0 represents the current value when chlorpyrifos is not present, and I represents the current value when chlorpyrifos is present; the standard curve is linearly fitted to obtain a regression equation of y=8.85×10 -7 x+2.27×10 -6 (R 2 =0.993), with a detection limit of 5.8 fM.

6. Use of the method according to any one of claims 1 to 5 in the detection of pesticide residues in fruits and vegetables.

7. The use according to claim 6, 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 to distilled water, sonicating, 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 solutions in each group; S53, respectively detecting the two groups of solutions to be detected after the addition of the spikes by a colorimetric biosensor or an electrochemical biosensor, and calculating the spike recovery rates respectively.

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