Matrix interference resistant rapid detection method for pesticide residues in agricultural products

Abstract

The invention discloses an anti-matrix interference rapid detection method for pesticide residues in agricultural products. The method comprises the following steps: (1) extracting a sample: crushing an agricultural product sample to be detected, and extracting with an organic solvent to obtain an extracting solution; (2) dropwise adding the extracting solution onto a sample introduction card, or immersing the sample introduction card into the extracting solution and then taking out the sample introduction card; (3) placing the sample introduction card in the air, and naturally airing; (4) soaking the air-dried sample introduction card in an enzyme reagent, incubating, adding an enzyme activity probe, and incubating for reaction; and (5) carrying out qualitative, quantitative or semi-quantitative detection on the pesticide according to the color change of the solution. According to the method, the interference of an organic solvent in a sample extracting solution on a liquid phase enzyme inhibition method is eliminated, the problem of matrix interference in agricultural product sample detection is solved, and accurate quantification can be carried out without using a matrix matching working curve. The method is easy and convenient to operate, low in cost, high in detection sensitivity and accuracy and suitable for high-sensitivity, rapid and accurate pesticide residue screening of samples in food circulation.

Description

Technical Field

[0001] This invention relates to the field of pesticide detection, specifically to a rapid detection method for pesticide residues in agricultural products that is resistant to matrix interference. Background Technology

[0002] The rapid detection technology for organophosphorus and carbamate pesticide residues based on cholinesterase inhibition is currently the earliest developed rapid pesticide residue detection method on the market and the most widely used rapid detection method in my country's agricultural product circulation sector. However, since the enzyme reagents themselves cannot maintain their activity in organic solvents, traditional enzyme inhibition methods mostly use aqueous solutions for sample extraction. Most pesticides have poor water solubility, and water extraction leads to low extraction rates and poor reliability of detection results for most target pesticides. According to existing literature, under the extraction conditions of traditional enzyme inhibition methods, the extraction rate of most organophosphorus and carbamate pesticides is less than 50%, seriously affecting the sensitivity and reliability of detection. Changing the extraction solvent from aqueous to organic solvents can solve the problem of low extraction rates for lipid-soluble pesticides. However, the activity of the enzyme reagents used for subsequent pesticide detection is easily destroyed by organic solvents, making it difficult to directly use enzyme inhibition methods to detect sample solutions obtained from organic solvent extraction. In previous research, the applicant utilized the rapid autonomous evaporation of organic solvents on paper-based surfaces to achieve enzyme inhibition detection of organic solvent samples on microfluidic paper chips and enzyme inhibition rapid test cards. However, both microfluidic paper chips and enzyme inhibition rapid test cards are solid-phase enzyme inhibition reactions, and can only be analyzed based on the color signal on their surface, resulting in poor detection sensitivity.

[0003] Enzyme inhibition methods in solution systems can be used with spectrophotometers, fluorometers, and other detection equipment for data acquisition. Compared with the aforementioned rapid test cards and paper chips (solid-phase enzyme inhibition methods), they have significant advantages in detection sensitivity and resistance to color / light interference, resulting in more reliable detection results. However, in existing technologies, conventional liquid-phase enzyme inhibition methods are difficult to directly detect organic solvent extracts. Generally, complex solvent removal methods and steps (such as rotary evaporation, nitrogen blowing, etc.) are required, followed by fixed-volume reconstitution of the sample with aqueous solvents such as buffer solutions before subsequent enzyme inhibition detection. This involves many steps and is highly dependent on equipment. Furthermore, for small-volume samples, complex and precise conditions are required to ensure the sample recovery rate during the evaporation-reconstitution process, making the actual operation complex and prone to human error. On the other hand, if the sample volume is increased or large-scale sample processing is carried out, solvent evaporation can easily cause environmental pollution.

[0004] On the other hand, traditional liquid-phase enzyme inhibition methods suffer from significant matrix interference when detecting actual agricultural product samples. To reduce matrix interference, traditional methods are forced to employ coarse extraction methods such as "whole plant soaking" or "large-scale cutting" to avoid matrix release caused by homogenized extraction and minimize the matrix in the extract. For example, GB / T 5009.199-2003 "Rapid Determination of Organophosphorus and Carbamate Pesticide Residues in Vegetables" stipulates that "onions, garlic, radishes, leeks, celery, coriander, water chestnuts, mushrooms, and tomato juice contain plant secondary metabolites that affect enzymes, easily producing false positives. When processing these samples, whole plant extraction or surface assay methods can be used. For some vegetables with high chlorophyll content, whole plant extraction can also be used to reduce pigment interference." However, in practical applications, it has been found that, apart from the above-mentioned varieties, most vegetables and fruits exhibit significant matrix interference in detection.

[0005] Meanwhile, the existing standards for extraction, such as "whole plant soaking" or "large-scale cutting," result in extremely low pesticide extraction rates, severely impacting the practical application of enzyme inhibition methods. However, homogenized extraction methods can lead to matrix interference, creating a contradiction between high extraction rates and matrix interference. Furthermore, the complexity and diversity of interfering matrices in different types of samples make developing a universal matrix interference removal method extremely difficult.

[0006] Therefore, it is necessary to develop new liquid-phase enzyme inhibition detection methods to solve the compatibility problem between liquid-phase enzyme inhibition and organic solvent extracts, and to solve the matrix interference problem caused by homogenization extraction methods. Summary of the Invention

[0007] The purpose of this invention is to provide a method for rapid detection of pesticide residues in organic solvent extracts using liquid-phase enzyme inhibition, thereby solving the compatibility problem between liquid-phase enzyme inhibition reaction and organic phase sample pretreatment, and significantly reducing the interference of the sample matrix on the enzyme inhibition method during detection.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A rapid detection method for pesticide residues in agricultural products with resistance to matrix interference, the method comprising the following steps: (1) Sample extraction: The agricultural product sample to be tested is crushed and extracted with an organic solvent to obtain the extract; The agricultural product samples include various vegetables, fruits and their related by-products such as vegetable juice, fruit juice, fruit puree, etc.

[0009] The pulverization method includes one or more of cutting, shearing, stirring, grinding, or homogenizing, preferably homogenizing.

[0010] Before crushing agricultural products, surface impurities such as dried leaves and soil are usually removed.

[0011] The organic solvent is one or more of ethyl acetate, acetonitrile, n-hexane, acetone, petroleum ether, dichloromethane, or chloroform, preferably ethyl acetate.

[0012] The pesticide residues mentioned are mainly organophosphate and carbamate pesticide residues.

[0013] After the agricultural product sample to be tested is pulverized, the mass ratio of the pulverized sample to the volume ratio of the organic solvent is generally 1g:1mL.

[0014] (2) Add the extract to the injection card, or immerse the injection card in the extract and then remove the injection card; The injection card is a porous sheet or plate material that can withstand organic solvents, such as test paper material.

[0015] The shape and area of ​​the injection card are not specifically required and can be changed according to experimental needs. For example, the shape can be square, equilateral polygon, or circle. A circle is preferred as it facilitates the uniform distribution of the extract on the injection card.

[0016] The sample card can be made by cutting paper-based material into the target shape, but the specific cutting method or shape does not affect the use effect.

[0017] When the sample card is immersed in the extraction solution, the immersion time is generally more than 10 seconds, preferably 10 to 20 seconds.

[0018] (3) Place the injection card in the air to air dry naturally; The natural air drying process generally involves placing the injection cartridge in the air for 3 to 5 minutes, during which the organic solvent will evaporate completely. (4) After drying, the sample card is completely immersed in the enzyme reagent. After incubation, the enzyme activity probe is added and the incubation reaction continues.

[0019] Furthermore, the injection card is completely immersed in the enzyme reagent and incubated for 5-15 minutes. Then, the enzyme activity probe is added, and the reaction is continued for another 3-5 minutes.

[0020] The incubation temperature can be from room temperature to 37°C, preferably 37°C.

[0021] (5) Use visual observation to observe the color of the reaction solution for qualitative or semi-quantitative determination, or use photometry to quantitatively detect the enzyme residual activity data of the reaction solution and calculate the pesticide content level in the agricultural product sample to be tested.

[0022] In step (1), the agricultural product sample to be tested is extracted with an organic solvent. The main target substances extracted are organophosphorus and carbamate pesticides. Since organophosphorus and carbamate pesticides have poor water solubility and are easily soluble in organic solvents, the extraction rate of the sample is too low when using water phase solution to extract the sample. Only by using organic solvent to extract the sample can the extraction efficiency be improved and the recovery rate can reach more than 90%, thereby improving the accuracy and reliability of the detection.

[0023] In step (4), the sample card, after the solvent has evaporated, is immersed in the enzyme solution. The sample card and the solution together form a heterogeneous enzyme reactor to carry out an enzyme inhibition reaction and detect the enzyme activity signal in the solution, thereby achieving rapid detection of pesticide residues. The enzyme reagent is a conventional esterase reagent that can be used for rapid pesticide detection by enzyme inhibition method. The esterase reagent can be acetylcholinesterase, butyrylcholinesterase, plant esterase, etc., with acetylcholinesterase being preferred. The enzyme reagent is generally a liquid enzyme reagent, prepared by mixing various esterase reagents with PBS or other types of buffer solutions to a suitable concentration.

[0024] The concentration of the enzyme reagent is between 0.01 and 1 U / ml, preferably 0.02 U / ml; The enzyme activity probe can be a reagent that can be used to indicate the activity of the enzyme reagent used, such as commercially available indoleacetic acid ester, indophenol acetate ester, Ellman reaction reagent, etc., or non-commercial laboratory-developed reagents can be used without affecting the effectiveness of the method of the present invention.

[0025] The concentration of enzyme activity probes is generally 0.01~5mM, preferably 0.1mM.

[0026] In step (5), the photometric method can be performed using a spectrophotometer or a fluorescence spectrophotometer.

[0027] Furthermore, when using photometry for quantitative detection, the reaction solution from step (4) is generally taken and added to a cuvette for photometric detection.

[0028] In a preferred embodiment, the method of the present invention is carried out according to the following steps: (1) Sample extraction: The agricultural product sample to be tested was homogenized and extracted with ethyl acetate to obtain the extract; The mass ratio of the pulverized sample to the volume ratio of the organic solvent was 1 g: 1 mL. (2) Add 50 μL of extract to the injection card, which is a round test paper; (3) Place the injection card in the air for 3-5 minutes to air dry naturally; (4) After drying, add 250 μL of acetylcholinesterase solution with a concentration of 0.02 U / ml to the injection card, incubate at 37℃ for 10 min, then add 10 μL of acetylcholinesterase fluorescent probe solution with a concentration of 0.1 mM, and continue the reaction at 37℃ for 5 min. (5) Take the solution other than the injection card and add it to the cuvette, and use a fluorometer to measure the fluorescence intensity of the solution; By comparing fluorescence intensity with the standard curve, the pesticide content level in the agricultural product sample to be tested can be calculated. The standard curve can be obtained by the following method: Standard solutions of different concentrations of the pesticide to be tested were prepared using ethyl acetate. The fluorescence intensity of the standard solutions of different concentrations was measured according to steps (2), (3), (4), and (5). A standard curve was plotted with concentration as the abscissa and fluorescence intensity as the ordinate.

[0029] The beneficial effects of this invention are as follows: This invention breaks away from the homogeneous reaction mode of conventional liquid-phase enzyme inhibition methods, employing a heterogeneous reaction mode combining solid-phase and liquid-phase reactions for the inhibition reaction of pesticides on enzyme reagents and the catalytic hydrolysis of probes by enzyme reagents. This heterogeneous reaction mode avoids the necessity of direct contact between the test solution and the enzyme reagent, making it possible for the injection card to act as an intermediate carrier for analyte transfer. This invention also changes the sample addition method; after the organic solvent in the sample rapidly evaporates and separates on the injection card, the solid injection card is completely immersed in the enzyme reagent, eliminating the involvement and interference of organic solvents in the enzyme inhibition reaction process. Furthermore, compared with the sample processing in traditional liquid-phase enzyme inhibition methods... Unlike conventional solvent-sample reconstitution methods, this invention involves directly immersing the solid injection card in the enzyme reagent solution. The pesticide components on the injection card do not require specialized elution and directly participate in the enzyme inhibition reaction. The resulting solution is then detected using conventional spectrophotometers or fluorometers. The solid injection card can be directly removed from the solution without affecting the spectrophotometric detection process and without requiring additional steps. Most importantly, using this method for sample extraction-injection-detection not only significantly improves the extraction rate of the target pesticide in the sample extraction step but also significantly reduces the matrix effect during detection. These features are not present in conventional liquid-phase enzyme inhibition detection methods. In existing technologies, conventional liquid-phase enzyme inhibition methods struggle to directly detect organic solvent extracts. They typically require complex solvent removal methods and steps (such as rotary evaporation and nitrogen blowing) followed by fixed-volume reconstitution of the sample using aqueous solvents like buffer solutions before the injection reaction can proceed. This involves numerous steps and high equipment dependence. Furthermore, it requires evaporating organic solvents from milliliters of sample, leading to significant environmental pollution during large-scale sample processing. However, if organic solvents are not used for sample extraction, the extraction rate of pesticides will be poor, and false negatives are very likely to occur, resulting in unreliable results. At the same time, the matrix effect has a very serious impact on the detection of existing enzyme inhibition methods. Most samples cannot be detected after homogenization due to the severe matrix effect. For common samples such as oranges, tea, and peppers, no sample juice should be mixed into the extract during extraction. Even a small amount of sample matrix can cause serious false positive and false negative test results, which seriously restricts the practical application of the method.

[0030] This invention creatively integrates solvent separation, sample introduction, and enzyme reactor functions using a sample introduction card. Utilizing the porous nature of the paper material, it accelerates the online evaporation of organic solvents in the sample introduction zone. After the sample introduction card is directly immersed in the enzyme reagent solution, the paper material itself acts as a porous solid-phase carrier, increasing the contact area between pesticides on the solid-phase surface and the solution system. This facilitates efficient enzyme inhibition reactions and detection, meeting the dual requirements of combining sample organic phase pretreatment with enzyme inhibition and zero contact between organic solvents and the liquid-phase detection system. This allows pesticide components in the organic solvent to react simply, rapidly, and cost-effectively with acetylcholinesterase reagents in the solution, thereby improving the sensitivity and accuracy of rapid detection of pesticide residues in actual samples. It enables rapid, interference-resistant screening and detection of target pesticides using liquid-phase enzyme inhibition.

[0031] Most importantly and unexpectedly, the experiment revealed that the method of this invention can significantly reduce matrix interference in liquid-phase enzyme inhibition detection. Compared with the traditional liquid-phase enzyme inhibition method, this invention shows excellent resistance to matrix interference in actual sample detection. After thorough homogenization and extraction of common fruit, vegetable, and tea test samples, the enzyme activity in the extract can be accurately detected. This not only significantly improves the pesticide extraction rate through homogenization and organic solvent extraction, but also significantly reduces matrix interference in the detection. Accurate quantitative detection of pesticides can be performed without the use of matrix-matched working curves, which is difficult to achieve or even unimaginable in previous enzyme inhibition detection methods.

[0032] The method of this invention is simple to operate, low in cost, short in detection time, easy to promote and use, and has high sensitivity and accuracy. It can be applied to high-sensitivity, rapid and accurate screening of large batches of samples in the food circulation process. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the steps of the method of the present invention.

[0034] Figure 2 This is a comparison chart of the matrix effect between the method of this invention and the conventional liquid-phase enzyme inhibition method in the detection of nine kinds of vegetable and fruit samples.

[0035] Figure 3 This is the working curve of the method of the present invention for detecting different pesticides in organic solvents.

[0036] Figure 4 This is the working curve for the detection of dichlorvos and chlorpyrifos using the traditional enzyme inhibition method.

[0037] Figure 5 This is a statistical chart showing the distribution of pesticide spike detection data for different vegetable and fruit samples using the method of this invention.

[0038] Figure 6This is a comparison chart showing the matching results of pesticide spiked in actual samples with the standard curve. Detailed Implementation

[0039] The multifunctional chip of the present invention and its usage method are further described below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0040] Example 1

[0041] The matrix effects of nine common vegetable and fruit samples were tested using both the method of this invention and the traditional enzyme inhibition method. The specific experiments are as follows: (1-1) Sample preparation: Common fruits and vegetables were purchased from local supermarkets, including: oranges, onions, carrots, broccoli, lettuce, potatoes, bell peppers, chili peppers (small red chilies), and tomatoes, a total of 9 kinds. Liquid chromatography-mass spectrometry was used to confirm that the selected samples did not contain organophosphorus and carbamate pesticides, for later use.

[0042] (1-2) Sample preparation: Remove inedible parts such as soil, yellow leaves, vegetable roots, and fruit stalks from the sample surface, and homogenize the remaining part for later use.

[0043] (1-3) Extraction: Place 2g of homogenized sample into each test tube, weigh 6 tubes for each sample, and weigh a total of 54 tubes for 9 samples. Divide all centrifuge tubes into two identical portions, each containing 9 samples, with 3 tubes for each sample.

[0044] Two sets of sample tubes were extracted using PBS buffer (pH 7.4, conventional method) and ethyl acetate (method of this invention), respectively, with a sample-to-solvent ratio of 1:1 (m:V). After centrifugation, the supernatant was used as the sample extraction solution for later use. Specifically, after extraction with ethyl acetate, solid-liquid separation and liquid phase separation were achieved by centrifugation, and the upper organic phase was used as the extraction solution.

[0045] (1-4) Determination: The enzyme inhibition blank detection of PBS extract and ethyl acetate extract was performed using the traditional liquid phase enzyme inhibition method and the method of the present invention, respectively. The detection data were compared with the detection results of the blank control group (blank PBS buffer and blank ethyl acetate) to evaluate the matrix effect of the detection.

[0046] The steps of the traditional liquid-phase enzyme inhibition method are as follows: Add 250 μL of sample extract to a centrifuge tube, add 10 μL of acetylcholinesterase solution (0.6 U / ml), incubate at 37℃ for 10 min, then add 10 μL of 0.1 mM enzyme activity probe, and continue the reaction at 37℃ for 5 min; aspirate the solution from the centrifuge tube after the reaction and measure the fluorescence intensity of the solution.

[0047] The steps of the method of the present invention are as follows Figure 1 As shown. Figure 1 In Figure A, the actual sample to be tested is shown, with vegetables as a typical example. The actual sample to be tested can be any solid or liquid agricultural product sample. Figure B shows the extraction of the sample after it has been pulverized or homogenized and added to a centrifuge tube containing an organic solvent. Commonly used solvents include ethyl acetate and acetonitrile. Figure C shows the extraction solution obtained in step B being added to the injection card; Figure D shows the sample cartridge being left in the air for 3-5 minutes to air dry naturally; Figure E shows the process of immersing the entire injection card in the enzyme reagent, incubating it at a suitable temperature, adding the enzyme activity probe, and continuing incubation to allow the probe to react with the enzyme reagent.

[0048] Figure F shows the results of colorimetric analysis using visual methods for qualitative / semi-quantitative judgment, or the data of enzyme activity measured quantitatively using a spectrophotometer or fluorophotometer to calculate pesticide residue levels.

[0049] The specific steps in this embodiment are as follows: A 1 cm diameter circular test strip was used as the injection card. 50 μL of extraction solution was pipetted onto the surface of the injection card, and the card was left to air dry for 3 minutes. After drying, the injection card was completely immersed in 250 μL of enzyme reagent. The enzyme reagent tube containing the injection card was incubated at 37°C for 10 minutes, followed by the addition of 10 μL of 0.1 mM acetylcholinesterase activity probe solution, and the reaction was continued at 37°C for 5 minutes. The resulting solution was then transferred to a cuvette, and the fluorescence intensity was measured using a fluorometer. The enzyme reagent was an acetylcholinesterase solution prepared with pH 7.4 PBS buffer at a concentration of 0.02 U / ml; the acetylcholinesterase activity probe was indoleacetic acid.

[0050] Fluorescence intensity of different samples detected by two methods, such as Figure 2 As shown.

[0051] The results clearly show that in the traditional liquid-phase enzyme inhibition method, all samples exhibited significant matrix effects, with detection signals increasing by 194% to 1221% compared to the blank control. This indicates that even with thorough sample pulverization, the matrix of different samples has a significant impact on the detection results in the traditional method, making it impossible to detect any of the nine tested samples using the enzyme inhibition method. This is also the reason why the traditional enzyme inhibition method requires extremely low sample pulverization (sometimes even whole-plant extraction). Obviously, insufficient pulverization results in extremely poor pesticide extraction from the samples, leading to low detection sensitivity and poor result reliability. In contrast, using the method of this invention, no significant matrix effects were observed in any of the nine tested samples. The difference in sample detection signals compared to the blank control was only -5.4% to +0.2%, and the detection results of all samples in three repeated measurements showed no statistically significant difference from the control group data. This invention completely eliminates the matrix interference problem in the liquid-phase enzyme inhibition method for the first time, and has significant and important application value for the enzyme inhibition method itself.

[0052] Example 2

[0053] Using the method of this invention, standard curves for four typical organophosphorus and carbamate pesticides in different solvents (including water and organic solvents) were determined. Specific experiments are as follows: (2-1) Prepare single-component standard solutions of different concentrations of methamidophos, parathion, dichlorvos and chlorpyrifos with ethyl acetate; prepare acetylcholinesterase solution with pH 7.4 PBS buffer and the enzyme solution concentration is 0.02 U / ml; prepare acetylcholinesterase fluorescent probe solution with a concentration of 0.1 mM with water; prepare circular paper base with a diameter of 1 cm as sample card.

[0054] (2-2) Use a pipette to transfer 50 μL of methamidophos pesticide solution and drop it onto the surface of the injection card. Place the injection card in the air for 3 min to allow it to air dry naturally. Immerse the chip in a centrifuge tube containing 250 μL of acetylcholinesterase solution and incubate at 37°C for 10 min. Then add 10 μL of enzyme activity probe and continue the reaction at 37°C for 5 min. After the reaction, remove the solution from the centrifuge tube except for the injection card and add it to a cuvette. Use a fluorometer to measure the fluorescence intensity of the solution.

[0055] (2-3) Following the steps in (2-2), repeat the measurement of data for different concentrations of methamidophos solutions. Plot the data using Origin software with methamidophos concentration as the abscissa and fluorescence intensity as the ordinate. Fit the data using the Hill equation to plot the standard curve of the pesticide response. (2-4) Following the method in (2-3), standard curves for parathion, dichlorvos, and chlorpyrifos were determined and plotted respectively. A summary of the specific results can be found in [link to results]. Figure 3 .

[0056] (2-5) Using water, n-hexane, acetonitrile, and water respectively instead of ethyl acetate, standard solutions were prepared according to steps (1) to (4) and tested. Working curves were plotted, and it was found that the working curves obtained by different solvents were basically consistent. That is to say, when using the method of the present invention, not only is the destructive effect of organic solvents on enzyme inhibition method avoided, but the type of solvent also has no effect on the detection effect. Therefore, in actual use, using different solvents to extract pesticides will not affect the detection effect. Even if the extraction solvent and the standard solution solvent are different, the working curve can still be used for the quantification of pesticides in the sample. Therefore, it is not necessary to use matrix extract to prepare standard solutions to create matrix standard curves.

[0057] Example 3

[0058] As a control, the traditional enzyme inhibition method was used to detect the spiked recovery of dichlorvos and chlorpyrifos pesticides in oranges, lettuce, and chili peppers. The specific experimental methods are as follows: (3-1) Plotting the working curves: Using PBS buffer as the solvent, standard solutions of dichlorvos and chlorpyrifos were prepared. The working curves were determined according to the traditional enzyme inhibition method. The specific steps were as follows: 250 μL of standard solution was added to a centrifuge tube, followed by 10 μL of acetylcholinesterase solution (0.6 U / ml). After incubation at 37°C for 10 min, 10 μL of enzyme activity probe was added, and the reaction was continued at 37°C for 5 min. The solution in the centrifuge tube after the reaction was aspirated, and its fluorescence signal was measured. The working curves of dichlorvos and chlorpyrifos were plotted with pesticide concentration as the abscissa and fluorescence signal value as the ordinate. The results are shown in the figure. Figure 4 .

[0059] (3-2) Sample preparation: Remove inedible parts such as soil, yellow leaves, vegetable roots, and fruit stalks from the sample surface, and homogenize the remaining part for later use.

[0060] (3-3) Spiking: Place 2g of homogenized sample into each test tube and add 100μL of dichlorvos standard solution of different concentrations to make the dichlorvos concentration in the centrifuge tube 0, 1.0, 2.0 and 10.0 ppb respectively. Vortex mix and let stand for 30min to allow the pesticide to fully contact the sample.

[0061] (3-4) Detection: Add 2 mL of PBS buffer (pH 7.4) as the sample extraction solution to each centrifuge tube described in (3-3), vortex to mix, centrifuge for 2 min, take 250 μL of the supernatant and add it to the centrifuge tube, add 10 μL of acetylcholinesterase solution (0.6 U / ml) to the centrifuge tube, incubate at 37℃ for 10 min, add 10 μL of enzyme activity probe, and continue to react at 37℃ for 5 min; aspirate the solution from the centrifuge tube after the reaction and measure the fluorescence intensity of the solution.

[0062] Replace the dichlorvos pesticide in step (3-3) with different concentrations of chlorpyrifos pesticide, and repeat step (3-4) to determine the fluorescence values ​​of different chlorpyrifos-labeled samples. The measured values ​​for all samples are shown in Table 1. Compare the data in the table with the working curve (…). Figure 4 The comparison revealed that although the pesticide spiked concentrations in all samples were within the quantitative range of the working curve, the actual sample detection data significantly exceeded the range of the vertical axis of the working curve, making qualitative or quantitative detection impossible. Furthermore, the detection signals that significantly deviated from the signal range of the working curve in actual detection indicated that the traditional enzyme inhibition method produced a significant matrix effect in the detection of homogenized samples, severely affecting the detection signal and leading to the failure of pesticide residue detection.

[0063] Table 1. Detection results of spiked samples

[0064] Example 4

[0065] The method of this invention was used to detect the spiked recovery of dichlorvos and chlorpyrifos pesticides in oranges, lettuce, and chili peppers. The specific experimental method is as follows: (4-1) Sample preparation: Remove inedible parts such as soil, yellow leaves, vegetable roots, and fruit stalks from the sample surface, and homogenize the remaining part for later use.

[0066] (4-2) Spiking: Place 2g of homogenized sample into each test tube and add 100μL of dichlorvos standard solution of different concentrations to make the dichlorvos concentration in the centrifuge tube 0, 1.0, 2.0 and 10.0 ppb respectively. Vortex mix and let stand for 30min to allow the pesticide to fully contact the sample.

[0067] (4-3) Detection: Add 2 mL of ethyl acetate as sample extraction solution to each centrifuge tube described in (4-2), vortex to mix, and let stand for 5 min or centrifuge for 2 min to allow the solvent to separate into layers; take 50 μL of the supernatant and drop it onto the surface of the injection card, and place it in the air for 3 min to allow the injection card to air dry naturally; immerse the injection card in a centrifuge tube containing 250 μL of acetylcholinesterase solution, incubate at 37°C for 10 min, add 10 μL of enzyme activity probe, and continue the reaction at 37°C for 5 min; aspirate the solution in the centrifuge tube excluding the injection card after the reaction, and use a fluorometer to measure the fluorescence intensity of the solution; substitute the fluorescence intensity value into the dichlorvos working curve equation drawn in Example 2, calculate the concentration of dichlorvos pesticide in each sample under the 4 spiking levels, and calculate the spiked recovery rate of each sample.

[0068] The spiked recoveries of chlorpyrifos in different samples were determined by replacing the dichlorvos pesticide in steps (4-2) and (4-3) with different concentrations of chlorpyrifos pesticide.

[0069] Tables 2 and 3 show the spiked recovery experimental results of the method of this invention. The comparison and analysis of the spiked recovery experimental data are shown in [Tables 2 and 3]. Figure 5 and Figure 6 : Table 2. Spiked recoveries of dichlorvos pesticide in different samples

[0070] Table 3. Spiked recoveries of chlorpyrifos in different samples

[0071] The three types of fruits and vegetables selected in this experiment are typical representatives of fruits and vegetables with different acidity, alkalinity, and irritation. Dichlorvos and chlorpyrifos are two typical pesticides commonly used in agricultural planting in my country, and these two pesticides are representative in terms of water solubility and pesticide structure.

[0072] Tables 2 and 3 and Figure 5 , Figure 6 The data results show that the injection card in the method of the present invention can indeed perform multiple functions simultaneously, including solvent separation, pesticide injection, and heterogeneous enzyme reactor, in actual use. It has shown good performance in the detection of different samples and different pesticides. The comparison between Examples 3 and 4 shows that the method of the present invention solves the long-standing problem of incompatibility between liquid phase enzyme inhibition method and organic solvents. At the same time, it also solves the matrix interference problem that has long plagued the rapid detection of pesticide residues by enzyme inhibition method. The performance of liquid phase enzyme inhibition method in terms of sensitivity, accuracy and reliability of pesticide residue detection has been significantly improved. It has great significance in actual sample detection and provides solid technical support for ensuring the quality and safety level of edible agricultural products such as vegetables and fruits in my country.

Claims

1. A rapid detection method for pesticide residues in agricultural products with resistance to matrix interference, characterized in that... The method includes the following steps: (1) Sample extraction: The agricultural product sample to be tested is crushed and extracted with an organic solvent to obtain the extract; (2) Add the extract to the injection card, or immerse the injection card in the extract and then remove the injection card; (3) Place the injection card in the air to air dry naturally; (4) After drying, the sample card is completely immersed in the enzyme reagent, and after incubation, the enzyme activity probe is added and the incubation reaction continues; (5) Use visual observation to observe the color of the reaction solution for qualitative or semi-quantitative determination, or use photometry to quantitatively detect the enzyme residual activity data of the reaction solution and calculate the pesticide content level in the agricultural product sample to be tested.

2. The method as described in claim 1, characterized in that... In step (1), the organic solvent is one or more of ethyl acetate, acetonitrile, n-hexane, acetone, petroleum ether, dichloromethane, or chloroform.

3. The method as described in claim 1, characterized in that... In step (1), the crushing method includes one or more of cutting, shearing, stirring, grinding or homogenizing.

4. The method as described in claim 1, characterized in that... In step (1), after the agricultural product sample to be tested is pulverized, the mass ratio of the pulverized sample to the volume ratio of the organic solvent is 1g:1mL.

5. The method as described in claim 1, characterized in that... In step (2), the injection card is a porous sheet or plate material that can withstand organic solvents.

6. The method as described in claim 1, characterized in that... In step (3), the injection card is left to stand in the air for 3-5 minutes to allow the organic solvent to evaporate completely.

7. The method as described in claim 1, characterized in that... In step (4), the dried sample card is completely immersed in the enzyme reagent and incubated for 5 to 15 minutes. Then, the enzyme activity probe is added and the incubation reaction continues for 3 to 5 minutes. The incubation temperature is room temperature to 37°C.

8. The method as described in claim 1, characterized in that... In step (5), the photometric method is performed using a spectrophotometer or a fluorescence spectrophotometer.

9. The method as described in claim 1, characterized in that... The method is performed according to the following steps: (1) Sample extraction: The agricultural product sample to be tested was homogenized and extracted with ethyl acetate to obtain the extract; The mass ratio of the pulverized sample to the volume ratio of the organic solvent was 1 g: 1 mL. (2) Add 50 μL of extract to the injection card, which is a round test paper; (3) Place the injection card in the air for 3-5 minutes to air dry naturally; (4) After drying, add 250 μL of acetylcholinesterase solution with a concentration of 0.02 U / ml to the injection card, incubate at 37℃ for 10 min, then add 10 μL of acetylcholinesterase fluorescent probe solution with a concentration of 0.1 mM, and continue the reaction at 37℃ for 5 min. (5) Take the solution other than the injection card and add it to the cuvette, and use a fluorometer to measure the fluorescence intensity of the solution; By comparing the fluorescence intensity with the standard curve, the pesticide content level in the agricultural product sample to be tested can be calculated.

10. The method as described in claim 9, characterized in that... The standard curve is prepared by the following method: Standard solutions of different concentrations of the pesticide to be tested were prepared using ethyl acetate. The fluorescence intensity of the standard solutions of different concentrations was measured according to steps (2), (3), (4), and (5). A standard curve was plotted with concentration as the abscissa and fluorescence intensity as the ordinate.

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