Method for rapidly detecting thiophanate-methyl pesticide on site

Abstract

The invention belongs to the technical field of safety detection of food and agricultural products, and particularly relates to a method for rapidly detecting a thiophanate-methyl pesticide on site. According to the method, a copper-zinc metal organic framework nano material is used for identifying thiophanate-methyl, a smart phone is used for photographing, and red-green-blue (RGB) chromatic values are analyzed to serve as detection signals for quantification. The CuZn-MOF has peroxidase-like activity, and the combination of the CuZn-MOF and hydrogen peroxide can rapidly catalyze a colorless substrate TMB to be oxidized so as to be light blue. A to-be-detected sample reacts with a solution containing CuZn-MOF, due to specific interaction, thiophanate-methyl can be adsorbed to the surface of CuZn-MOF, the peroxidase-like activity of CuZn-MOF is effectively improved, then catalytic oxidation of TMB is enhanced, and at the moment, the blue color of the solution is deepened. A linear relation related to the concentration of the thiophanate-methyl is established through the change of the chromatic value of the solution color, so that the thiophanate-methyl pesticide can be rapidly detected on site.

Description

Technical Field

[0001] The invention belongs to the technical field of food and agricultural product safety detection, and particularly relates to a method for rapid on-site detection of thiophanate-methyl pesticide. Background Art

[0002] Methyl thiophanate is a broad-spectrum benzimidazole fungicide that is widely used to control various fruit and vegetable pathogens before and after harvest due to its relatively low cost and high antifungal efficiency. However, methyl thiophanate degrades slowly in the natural environment. After application, it not only remains on plants or fruits, but is also washed into the aquatic environment by rainwater, causing spread and contamination. Methyl thiophanate is easily converted into carbendazim, which has low acute toxicity but is carcinogenic to mammals and may affect reproduction. Therefore, a simple, rapid, and sensitive method for detecting methyl thiophanate is of great significance to ensure food safety.

[0003] Traditional laboratory detection methods for methyl thiophanate mainly include high performance liquid chromatography, gas chromatography and chromatography-mass spectrometry. These technologies have high sensitivity, high resolution and high reliability, and can accurately quantify methyl thiophanate pesticides. However, they also have some limitations, such as the need for large and expensive instruments and equipment, professional technicians, high costs and long and cumbersome operations, which make it impossible to promote them in laboratories with limited conditions and cannot meet the needs of instant detection. At present, the simple and rapid detection of benzimidazole pesticides is mainly through enzyme-linked immunosorbent assay, which depends on antigens, antibodies and biological enzymes. However, this type of natural macromolecule is easily affected by factors such as ambient temperature, humidity and pH value, resulting in false negative or false positive results, resulting in unstable test results. In addition, enzyme-linked immunosorbent assay often needs to be used in conjunction with a spectrophotometer. The current spectrophotometer is still relatively large and not portable enough, making it difficult to use for on-site detection. In short, a simple, economical, accurate and reliable on-site rapid detection method for methyl thiophanate pesticides remains to be developed.

[0004] Smartphone-based digital image colorimetric analysis has been applied to instant rapid testing in the fields of food, medicine, and environment. By using the mobile phone camera to capture sample images, analyzing the RGB colorimetric values ​​of the images, and establishing a linear relationship between sample concentration and colorimetric values, quantitative detection of analytes in unknown samples can be achieved. Compared with traditional spectrophotometric methods, smartphone-based digital image colorimetric detection is simple, economical, and more popular, making it possible to conduct on-site, accurate, and rapid testing of food safety.

[0005] The present invention provides a method for rapid on-site detection of methyl thiophanate pesticide by using nanomaterials with peroxidase-like activity and a smart phone to analyze RGB values ​​of images. Summary of the invention

[0006] In order to realize the rapid detection of the residue of thiophanate-methyl in agricultural products, the object of the present invention is to provide an economical and sensitive method for rapid on-site detection of the concentration of thiophanate-methyl.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] A method for rapid on-site detection of thiophanate-methyl pesticide, the method comprising the following steps:

[0009] (1) Preparation of CuZn-MOF nanomaterials;

[0010] Dissolve copper nitrate trihydrate and zinc nitrate hexahydrate in ultrapure water, and then add sodium hydroxide solution to obtain liquid A; dissolve terephthalic acid and aminoterephthalic acid in N,N-dimethylformamide to obtain liquid B; mix the liquids A and B, mix them by ultrasonic mixing, and then centrifuge to obtain a green precipitate, which is washed with ethanol; finally, dry the green precipitate to obtain the CuZn-MOF nanomaterial;

[0011] (2) Preparation of detection standard curve:

[0012] With the concentration of methyl thiophanate pesticide as the horizontal coordinate and the color difference ratio (GR) / R as the vertical coordinate, the standard curve can be obtained as a linear equation (GR) / R=0.2868[methyl thiophanate](ng / mL)+0.0004586;

[0013] (3) Detection:

[0014] The sample to be tested was mixed with CuZn-MOF nanomaterials diluted with phosphate buffer, reacted at 30°C for 10 min, then H2O2 solution and TMB solution were added, reacted at 35°C for 20 min, and photographed. Red Green The colorimetric value can be obtained by substituting the obtained colorimetric difference ratio (GR) / R into the standard curve to calculate the concentration of methyl thiophanate residue in the sample to be tested.

[0015] Preferably, the specific method for preparing the CuZn-MOF nanomaterial in step (1) is:

[0016] 747.5 mg of copper nitrate trihydrate and 321.7 mg of zinc nitrate hexahydrate were dissolved in 7.5 mL of ultrapure water, and 8.25 mL of 1 M sodium hydroxide solution was added to obtain solution A; then, 332 mg of terephthalic acid and 360 mg of aminoterephthalic acid were dissolved in 19 mL of N, N-dimethylformamide to obtain solution B; the A and B solutions were mixed and ultrasonicated for 20 min to obtain a green solution, the green solution was centrifuged at 8000 rpm for 10 min, and then washed three times with ethanol to obtain a green precipitate, and finally the green precipitate was dried in a vacuum drying oven at 60° C. to obtain the CuZn-MOF nanomaterial.

[0017] In step (3), the sample to be tested is pretreated, and the pretreatment method is as follows: first, the sample is chopped, mixed with acetonitrile by ultrasonic mixing, and then allowed to stand, and then centrifuged at 8000 rpm for 10 minutes, the supernatant is collected, concentrated in a water bath, filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, and then the filtrate is diluted to obtain the sample to be tested.

[0018] First, 5 g of the sample was chopped, mixed with 15 mL of acetonitrile, ultrasonicated for 5 minutes, and then allowed to stand for 30 minutes. The mixture was then centrifuged at 8000 rpm for 10 minutes, the supernatant was collected, concentrated in a water bath, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate. The filtrate was then supplemented to 10 mL with methanol and diluted 300 times as the sample to be tested.

[0019] Beneficial effects of the present invention:

[0020] The method is very simple to operate, low in cost, does not require specific professional instruments, can be detected on site, and has excellent selectivity. It can perform qualitative analysis on methyl thiophanate through changes in solution color and perform quantitative analysis on methyl thiophanate concentration according to the solution chromaticity value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The schematic diagram of the method for rapid detection of methylthiophanate based on RGB analysis of smartphone photography is shown.

[0022] Figure 2 Scanning electron microscopy (SEM) images of CuZn-MOF without (A) and with methyl-thiophanate (B).

[0023] Figure 3 This is the catalytic kinetic diagram of CuZn-MOF enzyme.

[0024] Figure 4 This is a feasibility diagram of rapid detection of methylthiophanate based on smartphone RGB method.

[0025] Figure 5 Selectivity for thiophanate-methyl detection: influence of different pesticides (A) and different ions or organic molecules (B).

[0026] Figure 6 The standard curve is used to detect methylthiophanate. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1

[0029] Preparation of CuZn-MOF nanomaterials. First, dissolve 747.5 mg of copper nitrate trihydrate and 321.7 mg of zinc nitrate hexahydrate in 7.5 mL of ultrapure water, and then add 8.25 mL of sodium hydroxide solution (1 M) to prepare liquid A. Next, dissolve 332 mg of terephthalic acid and 360 mg of aminoterephthalic acid in 19 mL of N, N-dimethylformamide to prepare liquid B. Mix liquids A and B, and ultrasonicate for 20 minutes to obtain a green solution. Centrifuge (8000 rpm, 10 min) to obtain a green precipitate, which is washed three times with ethanol. Finally, the precipitate after washing with ethanol is dried in a vacuum drying oven at 60°C to obtain the CuZn-MOF nanomaterial.

[0030] CuZn-MOF can efficiently catalyze the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H2O2. The color of the solution changes from colorless to light blue. The chromaticity value is measured by taking a photo with a smartphone. At this time, the chromaticity difference ratio (GR) / R of the solution system is low. If there are methyl thiophanate pesticide molecules in the solution, they will be adsorbed to the surface of CuZn-MOF through the Cu-N interaction, improving the catalytic activity of CuZn-MOF. At this time, the solution turns dark blue and the system chromaticity difference ratio (GR) / R is high. Figure 1 Schematic diagram of the detection principle.

[0031] exist Figure 2 In A, the surface morphology of CuZn-MOF can be seen. Figure 2 B shows that after CuZn-MOF combines with methylthiophanate, layered wrinkles appear on its surface, and the specific surface area increases, thereby enhancing the enzyme-like activity.

[0032] Example 2

[0033] 100 μL CuZn-MOF (75 mg / mL) diluted with phosphate buffer (10 mM, pH 6.0), 50 μL H2O2 (100 mM), and 50 μL TMB solutions of different concentrations (concentrations: 0, 0.2, 0.5, 0.8, 1.1, 1.4, 1.7, and 2.0 mM) were added to the microplate in sequence, and the absorbance was measured every 30 s. Figure 3 As shown, the Michaelis constant (K m ) was 0.441 mM, indicating that CuZn-MOF has excellent peroxidase-like activity.

[0034] Example 3

[0035] 50 μL of ultrapure water was mixed with 100 μL of CuZn-MOF (75 mg / mL) diluted with phosphate buffer (10 mM, pH 6.0), and the mixture was reacted at 30 °C for 10 min. Then, 50 μL of H2O2 solution (100 mM) and TMB solution (2 mM) were added, and the mixture was reacted at 35 °C for 20 min. The colorimetric value was taken by taking a photo ( Figure 4 a). Replace ultrapure water with methyl thiophanate solution (1000 ng / mL), and the rest is the same as above. Figure 4 b. Figure 4 It can be seen that when methyl thiophanate pesticide molecules are present, the chromaticity difference ratio of the solution increases significantly.

[0036] Example 4

[0037] 50 μL of other pesticides (1000 ng / mL of methyl parathion, dichlorvos, dimethoate, glyphosate, acephate, profenofos and DDT), ultrapure water and methyl thiophanate (1000 ng / mL) were mixed with 100 μL of CuZn-MOF (75 mg / mL) diluted with phosphate buffer (10 mM, pH 6.0), reacted at 30 °C for 10 min, and then 50 μL of H2O2 solution (100 mM) and TMB solution (2 mM) were added, reacted at 35 °C for 20 min, and the colorimetric value was obtained by taking pictures. Figure 5 A shows that the reaction system has good specificity for methyl-thiophanate pesticide.

[0038] 50 μL of different ions or organic molecules (K + , Ca 2+ 、Na + Mg 2+ 、Zn 2+ , Cl - 、SO4 2- 、CO3 2- , glucose, glycine, threonine, lysine, tartaric acid and citric acid, 10 μg / mL) and methyl thiophanate (1 μg / mL) were mixed with 100 μL CuZn-MOF (75 mg / mL) diluted with phosphate buffer (10 mM, pH 6.0), reacted at 30 ° C for 10 min, and then 50 μL of H2O2 solution (100 mM) and TMB solution (2 mM) were added, reacted at 35 ° C for 20 min, and the color value was taken by taking pictures. Figure 5 B shows that the reaction system has good selectivity for methylthiophanate.

[0039] Example 5

[0040] 50 μL of different concentrations of methyl thiophanate (8-2400 ng / mL) was mixed with 100 μL of CuZn-MOF (75 mg / mL) diluted with phosphate buffer (10 mM, pH 6.0), reacted at 30°C for 10 min, then 50 μL of H2O2 solution (100 mM) and TMB solution (2 mM) were added, reacted at 35°C for 20 min, and the colorimetric value was obtained by taking a photo. Figure 6 As shown, the standard curve can be obtained with the concentration of methyl thiophanate pesticide as the horizontal coordinate and the color difference ratio (GR) / R as the vertical coordinate. The linear equation of methyl thiophanate in the concentration range of 8-2400 ng / mL is (GR) / R=0.2868[methyl thiophanate](ng / mL)+0.0004586. The detection limit is calculated by 3σ / k to be 0.079 ng / mL (σ is the standard deviation of the background signal, and k is the slope of the fitting curve).

[0041] Example 6

[0042] When this rapid detection method is applied to the detection of methyl thiophanate residues in tea, first wipe the stains on the surface of the tea and weigh 5g of it, chop it, mix it with 15mL acetonitrile, sonicate it for 5min, and then let it stand for 30min. Then centrifuge (8000rpm, 10min) to collect the supernatant, concentrate it in a water bath, filter it with a 0.45μm microporous filter membrane, and add methanol to the filtrate to 10mL. Because there are more pigments in tea, this stock solution is diluted 900 times as the sample to be tested. 50μL of the sample to be tested is mixed with 100μL CuZn-MOF (75mg / mL) diluted with phosphate buffer (10mM, pH 6.0), reacted at 30℃ for 10min, and then 50μL of H2O2 solution (100mM) and TMB solution (2mM) were added, reacted at 35℃ for 20min, and the red and green values ​​were taken by taking pictures. Substituting the obtained chromaticity difference ratio (GR) / R into the linear equation obtained above, the concentration of methyl thiophanate residue in the tea sample can be calculated.

[0043] Example 7

[0044] When this rapid detection method is applied to the detection of methyl thiophanate residues in cucumbers, first wipe the stains on the surface of the cucumber and weigh 5g of it, chop it, mix it with 15mL of acetonitrile, sonicate it for 5min, and then let it stand for 30min. Then centrifuge (8000rpm, 10min) to collect the supernatant, concentrate it in a water bath, filter it with a 0.45μm microporous filter membrane, and add methanol to the filtrate to 10mL. This stock solution is diluted 300 times as the sample to be tested. 50μL of the sample to be tested is mixed with 100μL CuZn-MOF (75mg / mL) diluted with phosphate buffer (10mM, pH 6.0), reacted at 30℃ for 10min, and then 50μL of H2O2 solution (100mM) and TMB solution (2mM) were added, reacted at 35℃ for 20min, and the red and green values ​​were taken by taking pictures. The obtained chromaticity difference ratio (GR) / R is substituted into the linear equation obtained above to calculate the concentration of methyl thiophanate residues in the cucumber sample.

[0045] Example 8

[0046] When this rapid detection method is applied to the detection of methyl thiophanate residues in apples, first wipe the stains on the surface of the apple and weigh 5g of it, chop it, mix it with 15mL acetonitrile, sonicate it for 5min, and then let it stand for 30min. Then centrifuge (8000rpm, 10min) to collect the supernatant, concentrate it in a water bath, filter it with a 0.45μm microporous filter membrane, and add methanol to the filtrate to 10mL. This stock solution is diluted 300 times as the sample to be tested. 50μL of the sample to be tested is mixed with 100μL CuZn-MOF (75mg / mL) diluted with phosphate buffer (10mM, pH 6.0), reacted at 30℃ for 10min, and then 50μL of H2O2 solution (100mM) and TMB solution (2mM) were added, reacted at 35℃ for 20min, and the red and green values ​​were taken by taking pictures. The obtained chromaticity difference ratio (GR) / R is substituted into the linear equation obtained above to calculate the concentration of methyl thiophanate residues in the apple sample.

Claims

1. A method for rapid on-site detection of thiophanate-methyl pesticide, characterized in that: The method comprises the following steps: (1) Preparation of CuZn-MOF nanomaterials; Dissolve copper nitrate trihydrate and zinc nitrate hexahydrate in ultrapure water, and then add sodium hydroxide solution to obtain liquid A; dissolve terephthalic acid and aminoterephthalic acid in N,N-dimethylformamide to obtain liquid B; mix the A and B solutions, mix them by ultrasonic mixing, and then centrifuge to obtain a green precipitate, and then wash them with ethanol to obtain a green precipitate; finally, dry the green precipitate to obtain the CuZn-MOF nanomaterial; (2) Preparation of detection standard curve: With the concentration of methyl thiophanate pesticide as the horizontal coordinate and the color difference ratio (GR) / R as the vertical coordinate, the standard curve can be obtained as a linear equation (GR) / R=0.2868[methyl thiophanate](ng / mL)+0.0004586; (3) Detection: The sample to be tested is mixed with the CuZn-MOF nanomaterial in step (1) diluted with phosphate buffer, reacted at 30°C for 10 minutes, and then H2O2 solution and TMB solution are added, reacted at 35°C for 20 minutes, and the red and green values ​​are obtained by taking a photo. The obtained color difference ratio (GR) / R is substituted into the standard curve described in step (2), and the concentration of methyl thiophanate residue in the sample to be tested can be calculated.

2. The method according to claim 1, characterized in that The specific method for preparing the CuZn-MOF nanomaterial in step (1) is: 747.5 mg of copper nitrate trihydrate and 321.7 mg of zinc nitrate hexahydrate were dissolved in 7.5 mL of ultrapure water, and then 8.25 mL of 1 M sodium hydroxide solution was added to prepare liquid A; then, 332 mg of terephthalic acid and 360 mg of aminoterephthalic acid were dissolved in 19 mL of N, N-dimethylformamide to prepare liquid B; the A and B solutions were mixed and ultrasonicated for 20 minutes to obtain a green solution, the green solution was centrifuged at 8000 rpm for 10 minutes, and then washed three times with ethanol to obtain a green precipitate, and finally the green precipitate was dried in a vacuum drying oven at 60°C to obtain the CuZn-MOF nanomaterial.

3. The method according to claim 1, characterized in that In step (3), the sample to be tested is pretreated, and the pretreatment method is as follows: first, the sample is chopped, mixed with acetonitrile by ultrasonic mixing, and then allowed to stand, and then centrifuged at 8000 rpm for 10 minutes, the supernatant is collected, concentrated in a water bath, filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, and then the filtrate is diluted to obtain the sample to be tested.

4. The method according to claim 3, characterized in that First, 5 g of the sample was chopped, mixed with 15 mL of acetonitrile, ultrasonicated for 5 minutes, and then allowed to stand for 30 minutes. The mixture was then centrifuged at 8000 rpm for 10 minutes, the supernatant was collected, concentrated in a water bath, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate. The filtrate was then supplemented to 10 mL with methanol and diluted 300 times as the sample to be tested.

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