Method for rapidly detecting dichlorvos on site based on scattering turbidity

Abstract

The invention discloses a method for rapidly detecting dichlorvos on site based on scattering turbidity, and belongs to the technical field of food and agricultural product safety detection. The method comprises the following steps: incubating dichlorvos with different concentrations with candida antarctica lipase, a phosphate buffer solution and L-ascorbyl palmitate, centrifugally incubating to obtain a reaction solution, taking a supernatant to react with a manganese dioxide nanosheet, measuring the turbidity of the reaction solution through a portable turbidimeter, and establishing a linear equation of the concentration and the turbidity of the dichlorvos; by measuring the turbidity of an unknown sample, the concentration of the dichlorvos can be calculated according to a linear equation. According to the method, invisible dichlorvos concentration signals are converted into measurable turbidity signals by utilizing the synergistic effect of enzyme inhibition reaction and turbidity change induction of the nano material, so that rapid and sensitive detection of dichlorvos is realized. The method is easy and convenient to operate, low in cost, high in detection speed, free of large instruments and especially suitable for on-site rapid screening of agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of food and agricultural product safety testing technology, specifically relating to a method for rapid on-site detection of dichlorvos based on scattering turbidity. Background Technology

[0002] Dichlorvos is a broad-spectrum insecticide belonging to the organophosphorus pesticide class. Due to its relatively low cost and high insecticidal efficacy, it is widely used in agriculture and public health. However, its residues in agricultural products seriously endanger food safety. Long-term exposure may cause liver and kidney damage, nervous system disorders, and potential carcinogenic risks. The World Health Organization has clearly listed it as a highly toxic substance. Therefore, simple, rapid, and sensitive methods for detecting dichlorvos are of great significance for ensuring food safety.

[0003] Traditional laboratory detection methods for dichlorvos mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), ultraviolet-visible spectroscopy (UV-Vis spectroscopy), and chromatography-mass spectrometry (GC-MS). These techniques offer high sensitivity, high resolution, and high reliability, enabling precise quantification of dichlorvos. However, they also have limitations, such as requiring large and expensive instruments, specialized technicians, high costs, and lengthy and cumbersome operations, making them unsuitable for rapid on-site detection. Currently, the detection of organophosphorus pesticides primarily relies on chromatographic methods, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC). These methods leverage the differences in partition coefficients between the stationary and mobile phases to achieve efficient separation and accurate detection of organophosphorus pesticides in complex samples. However, the separation of individual components is easily affected by factors such as ambient temperature, humidity, and pH, leading to unstable detection results. Furthermore, quantitative detection using chromatographic instruments requires instruments that are still relatively large and lack portability, making them less than ideal for on-site detection. In conclusion, a simple, economical, accurate, and reliable rapid on-site detection method for dichlorvos pesticides remains to be developed.

[0004] Turbidity is an indicator used to characterize the cloudiness of a liquid. The turbidity value of a liquid is determined by the ability of particles in the liquid to scatter and absorb light. When light passes through a liquid, if the liquid is a true solution, the light will pass through completely, and the turbidity value will be low. If the liquid is turbid, the light will be scattered and absorbed by suspended particles, leading to attenuation of light intensity and changes in the light path, resulting in a high turbidity value. Turbidimeters calculate the turbidity of water by measuring the attenuation of light intensity and changes in the light path as light passes through the liquid, whether it is scattered or transmitted. Portable scattering turbidimeters are small, inexpensive, and easy to carry for rapid on-site turbidity detection. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention discloses a method for rapid on-site detection of dichlorvos based on scattering turbidity, so as to solve the technical problems of existing dichlorvos detection methods being cumbersome to operate, time-consuming, costly and with poor selectivity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for rapid on-site detection of dichlorvos based on scattering turbidity, which includes the following steps: S1: Mix different concentrations of dichlorvos with Candida antarcticis lipase (CALB) solution and phosphate buffer, incubate at 37°C for 5 min, then add L-ascorbate palmitate (L-AP) suspension, and continue incubation at 37°C for 20 min to obtain the incubated reaction solution. S2: After centrifugation and incubation, add manganese dioxide nanosheets (MnO2NSs) solution and phosphate buffer to the supernatant of the reaction solution, react at room temperature for 5 min, and measure the turbidity value T of the reaction solution. Then, establish a linear equation between the dichlorvos concentration C and the turbidity value T with the dichlorvos concentration C as the abscissa and the turbidity value T as the ordinate. S3: Measure the turbidity value T of the unknown sample according to the methods in S1~S2. Substitute the obtained turbidity value T into the linear equation to calculate the concentration of dichlorvos in the unknown sample.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, in step S1, the concentration of dichlorvos is 10~100 ng / mL; the concentration of Candida antarcticis lipase solution is 7.2 mg / mL; the concentration of phosphate buffer is 1 mM; and the concentration of L-ascorbate palmitate suspension is 1.44 mM.

[0009] Furthermore, the volume ratio of dichlorvos, Candida antarcticis lipase solution, phosphate buffer, and L-ascorbate palmitate suspension was 1:1:2:1.

[0010] Furthermore, in step S2, the centrifugation time is 5 minutes and the centrifugation speed is 10,000 rpm.

[0011] Furthermore, in step S2, the concentration of the manganese dioxide nanosheet solution is 0.168 mg / mL; the concentration of the phosphate buffer is 1 mM; and the volume ratio of the supernatant, the manganese dioxide nanosheet solution, and the phosphate buffer is 7.5:1.5:9.

[0012] Furthermore, the equation of the linear equation in step S2 is T = 0.1531C + 33.8501.

[0013] The beneficial effects of this invention are: it is very simple to operate, low in cost, requires no specific professional instruments, can be quickly detected on site, has high sensitivity and excellent selectivity, can perform qualitative analysis of dichlorvos through changes in solution turbidity, and can perform quantitative analysis of dichlorvos concentration based on solution turbidity value. Attached Figure Description

[0014] Figure 1 This is the standard curve for detecting dichlorvos in Example 1; Figure 2 The results show the selectivity of the detection method for different pesticides; Figure 3 For; the selective detection results of the detection method for different ions or organic molecules; Figure 4 SEM image of MnO2NSs; Figure 5 SEM image of MnO2NSs after pyrolysis and reduction; Figure 6 Figure showing the feasibility experiment results of rapid detection of dichlorvos based on a portable turbidimeter. Detailed Implementation

[0015] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0016] Example 1 ① Solution preparation Preparation of manganese dioxide nanosheets (MnO2NSs) solution: 2.884 g sodium dodecyl sulfate was dissolved in 85 mL of ultrapure water, 5 mL of 0.5 mol / L sulfuric acid solution and 10 mL of 0.05 mol / L potassium permanganate solution were added, and the mixture was incubated in a water bath at 95 °C for 60 min. The solution was then centrifuged (10000 rpm) to obtain a brown-black precipitate. The brown-black precipitate was washed three times with ultrapure water and then dispersed in 20 mL of ultrapure water to obtain a MnO2NSs solution with a concentration of 2 mg / mL. The solution was then diluted to 0.168 mg / mL for later use.

[0017] Preparation of L-ascorbate palmitate (L-AP) suspension: 11.94 mg L-AP was added to 20% ethanol and sonicated to disperse it evenly. The suspension was then diluted 5 times with ultrapure water to obtain an L-AP suspension with a concentration of 1.44 mM.

[0018] ② A method for rapid on-site detection of dichlorvos based on scattering turbidity 200 μL of different concentrations of dichlorvos (10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, and 120 ng / mL) were mixed with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0). The mixture was incubated at 37°C for 5 min, then 200 μL of L-AP suspension (1.44 mM) was added, and the mixture was incubated at 37°C for 20 min to obtain the incubated reaction solution. The solution was then centrifuged at 10000 rpm for 5 min, and 750 μL of the supernatant was collected. 150 μL of MnO2NSs solution (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0) were added, and the mixture was reacted at room temperature for 5 min. The turbidity of the solution was measured using a portable turbidimeter. Figure 1 As shown, a standard curve can be obtained by plotting the concentration of dichlorvos (C) on the x-axis and the turbidity value on the y-axis. Within the concentration range of 10–100 ng / mL, the linear equation for dichlorvos is T = 0.1531C + 33.8501, indicating a good linear correlation between turbidity value T and dichlorvos concentration (R²). 2 =0.9946).

[0019] ③ Detection of unknown samples To detect dichlorvos residue in cucumbers: First, wipe the surface of the cucumber clean and peel it. Weigh 10g of peel and chop it. Mix it with 50mL of ultrapure water and sonicate for 5 minutes. Then filter it through a 0.45μm microporous membrane and dilute it 10 times as the sample to be tested. 200 μL of the sample to be tested was mixed with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0), and incubated at 37 °C for 5 min. Then, 200 μL of L-AP suspension (1.44 mM) was added, and the mixture was incubated at 37 °C for 20 min to obtain the reaction solution. The solution was then centrifuged at 10000 rpm for 5 min, and 750 μL of the supernatant was collected. 150 μL of MnO2NSs solution (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0) were added, and the mixture was reacted at room temperature for 5 min. The turbidity T of the solution was measured using a portable turbidimeter. The concentration of dichlorvos in the cucumber sample was calculated by substituting the obtained turbidity T into the linear equation. The results are shown in Table 1.

[0020] To detect dichlorvos residue in tomatoes: First, wipe the surface of the tomatoes clean and peel them. Weigh 10g of the peel and chop it. Mix it with 50mL of ultrapure water and sonicate for 5 minutes. Then filter it through a 0.45μm microporous membrane and dilute it 10 times as the sample to be tested. 200 μL of the sample to be tested was mixed with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0), and incubated at 37 °C for 5 min. Then, 200 μL of L-AP suspension (1.44 mM) was added, and the mixture was incubated at 37 °C for 20 min to obtain the incubated reaction solution. The solution was then centrifuged at 10000 rpm for 5 min, and 750 μL of the supernatant was collected. 150 μL of MnO2NSs solution (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0) were added, and the mixture was reacted at room temperature for 5 min. The turbidity T of the solution was measured using a portable turbidimeter. The concentration of dichlorvos in the tomato sample was calculated by substituting the obtained turbidity T into the linear equation. The results are shown in Table 1.

[0021] Table 1

[0022] As shown in Table 1, the spiked recovery rate of dichlorvos detected by the present invention is 91.02-110.74%, and the relative standard deviation is 3.87-8.48%, indicating that the present invention has reliable accuracy and stability.

[0023] Comparative Example 1 200 μL of other pesticides (profenofos, dimethoate, demeton-methyl, chlorpyrifos, acephate, carbofuran, dinotefuran, and DDT) at a concentration of 12 μg / mL and dichlorvos (1.2 μg / mL) were mixed with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0), respectively. The mixtures were incubated at 37°C for 5 min, followed by the addition of 200 μL of L-AP suspension (1.44 mM). The mixtures were then incubated at 37°C for 20 min to obtain the incubated reaction solution. The solution was centrifuged at 10000 rpm for 5 min, and 750 μL of the supernatant was collected. This supernatant was then added to 150 μL of MnO2NSs solution (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0). The reaction was carried out at room temperature for 5 min, and the turbidity of the solution was measured using a portable turbidimeter. Figure 2 It can be seen that the reaction system has good specificity for dichlorvos.

[0024] Comparative Example 2 200 μL of a solution of ions or organic molecules with a concentration of 12 μg / mL (Na+) + Mg 2+ K + Cl- ,Br - CO3 2- SO4 2- Glucose, tartaric acid, histidine, cysteine, oxalic acid, and citric acid, along with dichlorvos (1.2 μg / mL), were mixed with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0), respectively. The mixtures were incubated at 37°C for 5 min, followed by the addition of 200 μL of L-AP suspension (1.44 mM). The mixtures were then incubated at 37°C for 20 min to obtain the incubated reaction solution. The solution was centrifuged at 10000 rpm for 5 min, and 750 μL of the supernatant was collected. This supernatant was then added to 150 μL of MnO2NSs solution (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0). The reaction was carried out at room temperature for 5 min, and the turbidity of the solution was measured using a portable turbidimeter. Figure 3 It can be seen that the reaction system has good selectivity for dichlorvos.

[0025] Experimental Example ①MnO2NSs can be reduced by AA generated by L-AP hydrolysis via CALB. Due to the weakening of light scattering and light absorption effects, the turbidity of the solution decreases. If dichlorvos molecules are present in the solution, they will inhibit CALB activity through specific interactions, reduce L-AP hydrolysis, and thus keep the solution in a high turbidity state.

[0026] Transmission electron microscope image of MnO2NSs as shown below Figure 4 As shown in the figure, MnO2NSs exhibits a nanoscale sheet-like structure, which gives it a strong scattering ability. Figure 5 The image shows a transmission electron microscope image of MnO2NSs after it has been cleaved and reduced by AA following the addition of CALB and L-AP. The image shows the disappearance of the lamellar structure, which proves that MnO2NSs was cleaved.

[0027] ② Mix 200 μL of ultrapure water with 200 μL of CALB solution (7.2 mg / mL) and 400 μL of phosphate buffer (1 mM, pH=8.0), and incubate at 37℃ for 5 min. Then add 200 μL of L-AP suspension (1.44 mM) and incubate at 37℃ for 20 min to obtain the incubated reaction solution. Centrifuge at 10000 rpm for 5 min, take 750 μL of supernatant, add 150 μL of LmO2NSs (0.168 mg / mL) and 900 μL of phosphate buffer (1 mM, pH=6.0), and react at room temperature for 5 min. Measure the turbidity of the solution using a portable turbidimeter. Then replace the ultrapure water with dichlorvos solution (2 μg / mL), and follow the same procedure as above. The test results are as follows. Figure 6As shown.

[0028] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A method for rapid on-site detection of dichlorvos based on scattering turbidity, characterized in that, Includes the following steps: S1: Mix different concentrations of dichlorvos with Candida antarcticis lipase solution and phosphate buffer, incubate at 37°C for 5 min, then add L-ascorbate palmitate suspension, and continue incubation at 37°C for 20 min to obtain the incubated reaction solution. S2: Centrifuge the incubated reaction solution, add manganese dioxide nanosheet solution and phosphate buffer to the supernatant, react at room temperature for 5 min, measure the turbidity value T of the reaction solution, and then establish a linear equation between the dichlorvos concentration C and the turbidity value T with different dichlorvos concentrations C as the abscissa and the turbidity value T as the ordinate. S3: Measure the turbidity value T of the unknown sample according to the methods in S1~S2. Substitute the obtained turbidity value T into the linear equation to calculate the concentration of dichlorvos in the unknown sample.

2. The method for rapid on-site detection of dichlorvos based on scattering turbidity according to claim 1, characterized in that: In step S1, the concentration of dichlorvos was 10-100 ng / mL; the concentration of Candida antarctica lipase solution was 7.2 mg / mL; the concentration of phosphate buffer was 1 mM; and the concentration of L-ascorbate palmitate suspension was 1.44 mM.

3. The method for rapid on-site detection of dichlorvos based on scattering turbidity according to claim 2, characterized in that: The volume ratio of dichlorvos, Candida antarcticis lipase solution, phosphate buffer, and L-ascorbate palmitate suspension was 1:1:2:

1.

4. The method for rapid on-site detection of dichlorvos based on scattering turbidity according to claim 1, characterized in that: In step S2, the centrifugation time is 5 minutes and the centrifugation speed is 10,000 rpm.

5. The method for rapid on-site detection of dichlorvos based on scattering turbidity according to claim 1, characterized in that: In step S2, the concentration of the manganese dioxide nanosheet solution was 0.168 mg / mL; the concentration of the phosphate buffer was 1 mM; and the volume ratio of the supernatant, manganese dioxide nanosheet solution, and phosphate buffer was 7.5:1.5:

9.

6. The method for rapid on-site detection of dichlorvos based on scattering turbidity according to claim 1, characterized in that: The linear equation in step S2 is T = 0.1531C + 33.8501.

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