Method and kit for rapid detection of organophosphorus pesticides in guixi traditional Chinese medicinal materials

Abstract

The application discloses a kind of Gui Shiwei Chinese herbal medicine organophosphorus pesticide rapid detection method and detection kit, by the sample of Gui Shiwei medicine powder to be measured is extracted by acetonitrile solution containing acetic acid, salting-out stratification is taken supernatant;With colloidal gold labeled anti-organophosphorus pesticide small peptide antibody incubation, carry out competition immune response in the microwell plate coated with organophosphorus hapten-protein complex;After washing, the absorbance is determined at 450 to 550nm wavelength, calculate competition inhibition rate and compare with preset first threshold value to determine whether it is over standard;The method is simple and rapid;In addition, for the detection needs of different organophosphorus pesticides, by setting different organophosphorus hapten-protein complex coating area in the microwell plate, the simultaneous detection of multiple organophosphorus pesticides is realized, and the applicability of organophosphorus pesticide residue detection is further improved.

Description

Technical Field

[0001] This invention relates to the technical field of pesticide residue detection, and more specifically, to a rapid detection method and reagent kit for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs. Background Technology

[0002] Currently, the detection of organophosphorus pesticides mainly relies on instrumental analysis methods such as gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography (HPLC). Although these methods are highly sensitive and accurate in quantification, they have obvious shortcomings: (1) The instruments are expensive, the operation is complicated, and professional technicians are required, which cannot meet the needs of rapid screening at the production sites, purchasing stations, and distribution centers of Chinese medicinal materials; (2) The pretreatment steps are cumbersome, such as solid phase extraction, which takes a long time and makes it difficult to achieve rapid initial screening of batch samples. Summary of the Invention

[0003] To address at least one of the aforementioned technical problems, the present invention aims to provide a rapid detection method and reagent kit for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs, which can improve the accuracy and timeliness of organophosphorus pesticide residue detection.

[0004] The first aspect of this invention provides a rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs, comprising: Step 1: Extract the pre-set weight of the powder of the ten medicinal herbs to be tested, add an acetonitrile solution containing 0.5% to 2.0% acetic acid by volume for extraction, then add anhydrous magnesium sulfate and sodium chloride for salting out and separation, centrifuge and take the supernatant to obtain the sample extract; Step 2: Mix the sample extract with colloidal gold-labeled anti-organophosphorus pesticide peptide antibody solution, add it to a microplate coated with organophosphorus hapten-protein complex, and conduct a competitive reaction at 35 to 40°C for 10 to 20 minutes to obtain experimental reaction microwells; Step 3: After washing the micropores of the experimental reaction, measure the absorbance value at a wavelength of 450 to 550 nm, and use it as the experimental absorbance value; Step 4: Extract a pre-set weight of buffer solution without the sample to replace the sample extraction solution, and follow steps 1 to 3 to obtain the comparative absorbance values; Step 5: Determine the competitive inhibition rate based on the experimental absorbance value and the comparative absorbance value; the formula for calculating the competitive inhibition rate R is: ×100%, of which To compare absorbance values, This represents the experimental absorbance value; Step 6: If the competitive inhibition rate is greater than or equal to the preset first threshold, it is determined that the organophosphorus pesticide residues in the tested Gui Shiwei medicinal materials exceed the standard.

[0005] In this scheme, the "Ten Flavors of Cinnamon" refers to any one of the following: cinnamon, monk fruit, star anise, turmeric, spatholobus suberectus, sophora tonkinensis, poria cocos, kudzu root, or lysimachia christinae.

[0006] In this scheme, after extracting the preset weight of the ten medicinal herbs to be tested powder, the method further includes: Obtain the initial moisture content of the powder of the ten medicinal herbs of Guiwei to be tested; If the initial moisture content is greater than the preset target moisture content, a dehydration pretreatment prompt message will be triggered. Based on the dehydration pretreatment prompts, anhydrous sodium sulfate was added to the powder of the tested Gui Shiwei medicinal material for dehydration pretreatment to obtain the dehydrated Gui Shiwei medicinal material powder, and the dehydrated Gui Shiwei medicinal material powder was used for subsequent testing. The mass of the anhydrous sodium sulfate is ,in To determine the mass of anhydrous sodium sulfate added, The initial moisture content of the powder containing the ten medicinal herbs of Guiwei is given. For the target moisture content, This represents the weight of the powdered Gui Shi Wei medicinal herbs to be tested. This is the correction factor.

[0007] In this scheme, when the tested medicinal herbs are star anise or cinnamon, it also includes: After extraction with acetonitrile solution, graphitized carbon black adsorbent is added additionally, the amount of which is... ,in ,in and These are the corresponding coefficients. The characteristic value of pigment content in the medicinal material to be tested. This represents the characteristic value of volatile oil content in medicinal materials. The quality of Chinese medicinal powder.

[0008] This plan also includes: A series of standard solutions of organophosphorus pesticides with known concentrations were prepared, and the results were tested according to steps 1 to 3 to obtain the competitive inhibition rate of each standard solution. Based on the preset regression equation, the concentration of organophosphorus pesticides in the sample is determined according to the competitive inhibition rate of the sample extract. If the concentration value is greater than or equal to the preset concentration threshold, it is determined that the organophosphorus pesticide residue in the tested Gui Shiwei medicinal materials exceeds the standard.

[0009] This plan also includes: Different organophosphorus hapten-protein complexes were coated in different wells of the microplate, with each hapten corresponding to an organophosphorus pesticide. The sample extract was simultaneously added to each detection well and competed with the colloidal gold-labeled anti-organophosphorus pesticide antibody corresponding to each well. The competitive inhibition rate of each well was measured separately, and the competitive inhibition rate of each well was substituted into the preset regression equation of the corresponding organophosphorus pesticide to calculate the concentration of the corresponding organophosphorus pesticide in each well; thus realizing the simultaneous quantitative detection of multiple organophosphorus pesticides.

[0010] In this scheme, when the tested Gui Shi Wei Chinese medicinal materials are Luo Han Guo, Ge Gen, or Fu Ling, before adding the acetonitrile solution, the following is also included: First, an enzymatic pretreatment is performed by adding a glycoside hydrolase complex, which contains cellulase, pectinase, and... Glucosidase with an activity of 50 to 200 U / g of medicinal material, an enzymatic hydrolysis temperature of 40 to 50℃, and an enzymatic hydrolysis time of 15 to 30 minutes; After enzymatic pretreatment, polyvinylpyrrolidone is added to the pretreated solution at a mass of 1% to 5% of the medicinal powder mass to adsorb the polypeptides and proteins released by enzymatic hydrolysis.

[0011] In this scheme, the absorbance value measured at a wavelength of 450 to 550 nm in step 3 is completed using a portable colorimeter or a smartphone image colorimetric analysis module; the smartphone image colorimetric analysis module extracts the color feature values ​​of each well by taking a picture of the microplate and converts them into concentration values ​​based on a pre-stored standard curve.

[0012] Another aspect of the present invention provides a detection kit for the detection method, comprising: a microplate pre-coated with an organophosphorus hapten-protein complex, a colloidal gold-labeled anti-organophosphorus pesticide small peptide antibody working solution, a series of organophosphorus pesticide standards of known concentrations, a washing buffer, and sample extraction tubes containing a predetermined mass of anhydrous magnesium sulfate and sodium chloride; each well of the microplate is pre-coated with an organophosphorus hapten-protein complex at a concentration of 1 to 10 μg / mL, and the colloidal gold-labeled antibody is diluted at a concentration of 1:50 to 1:200.

[0013] In this scheme, the microplate is also provided with positive control wells and negative control wells; the positive control wells are coated with an organophosphorus hapten-protein complex of known concentration and a preset standard competitive inhibition rate value, and the negative control wells are coated with a blocking solution without the relevant antigen; each time a test is performed, if the competitive inhibition rate of the positive control well is within a preset range and the absorbance value of the negative control well is higher than a preset threshold, then the test result is valid.

[0014] One or more technical solutions proposed in this application have at least the following technical effects: 1. For the commonalities in the ten herbs of Cinnamomi Ramulus, an organophosphorus residue detection method from Step 1 to Step 6 was designed, which is simple and rapid to operate. For the distinguishing features of different herbs in the ten herbs of Cinnamomi Ramulus, for example, for fresh herbs with high water content such as Desmodii Styracifolii Herba, Puerariae Lobatae Radix, and Momordicae Grosvenori Fructus, dehydration treatment was carried out with anhydrous sodium sulfate. For example, for dark-colored and high-pigment herbs such as Spatholobi Caulis, Sophorae Tonkinensis Radix, and Cinnamomi Cortex, graphitized carbon black adsorbent was added to adsorb pigments and volatile oils in the solution, further improving the accuracy of organophosphorus residue detection. 2. By setting different organophosphorus hapten-protein complex coating areas in the microplate, simultaneous detection of multiple organophosphorus pesticides was achieved, further improving the applicability of organophosphorus pesticide residue detection.

[0015] 3. By integrating the detection method into a ready-to-use detection kit and introducing a portable colorimeter or a smartphone image colorimetric analysis module, rapid screening can be completed on-site at the production areas, purchasing stations, distribution centers, etc. of Chinese herbal medicines without large professional equipment, significantly reducing the detection threshold and operation difficulty. At the same time, through the quality control system with built-in positive control wells and negative control wells, the validity of the results can be verified by itself each time, further improving the reliability of on-site detection.

[0016] In summary, the present invention sets different detection steps for the commonalities and differences of the ten herbs of Cinnamomi Ramulus, which is simple and rapid to operate. Brief Description of the Drawings

[0017] The following further describes the present invention in detail with reference to the drawings and specific implementation methods.

[0018] Figure 1 Shows the flowchart of a rapid detection method for organophosphorus pesticides in ten herbs of Cinnamomi Ramulus according to the present invention; Figure 2 Shows the schematic diagram of the detection standard curve of parathion according to the present invention; Figure 3 Shows the schematic diagram of the stability change curve of the kit under different temperature conditions according to the present invention; Figure 4 Shows the schematic diagram of the analysis results of the detection limit of organophosphorus according to the present invention; Figure 5 Shows the schematic diagram of the spiked recovery results of five herbs of Cinnamomi Ramulus according to the present invention; Figure 6 Shows the schematic diagram of the comparison of the detection results between the present invention and the enzyme inhibition rate method; Figure 7 Shows the schematic diagram of the analysis results of the correlation of the portable detection according to the present invention; Figure 8 Shows the schematic diagram of the verification results of the quality control system according to the present invention. Detailed Description of the Invention

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] Figure 1 The flowchart of a rapid detection method for organophosphorus pesticides in Gui Shiwei Chinese medicinal materials according to the present invention is shown.

[0021] like Figure 1 As shown, this invention discloses a rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs, comprising: Step 1: Extract the pre-set weight of the powder of the ten medicinal herbs to be tested, add an acetonitrile solution containing 0.5% to 2.0% acetic acid by volume for extraction, then add anhydrous magnesium sulfate and sodium chloride for salting out and separation, centrifuge and take the supernatant to obtain the sample extract; Step 2: Mix the sample extract with colloidal gold-labeled anti-organophosphorus pesticide peptide antibody solution, add it to a microplate coated with organophosphorus hapten-protein complex, and conduct a competitive reaction at 35 to 40°C for 10 to 20 minutes to obtain experimental reaction microwells; Step 3: After washing the micropores of the experimental reaction, measure the absorbance value at a wavelength of 450 to 550 nm, and use it as the experimental absorbance value; Step 4: Extract a pre-set weight of buffer solution without the sample to replace the sample extraction solution, and follow steps 1 to 3 to obtain the comparative absorbance values; Step 5: Determine the competitive inhibition rate based on the experimental absorbance value and the comparative absorbance value; the formula for calculating the competitive inhibition rate R is: ×100%, of which To compare absorbance values, This represents the experimental absorbance value; Step 6: If the competitive inhibition rate is greater than or equal to the preset first threshold, it is determined that the organophosphorus pesticide residues in the tested Gui Shiwei medicinal materials exceed the standard.

[0022] According to an embodiment of the present invention, the colloidal gold-labeled anti-organophosphorus pesticide small peptide antibody solution is diluted from 1:50 to 1:200 to specifically recognize organophosphorus pesticide hapten-protein complexes; the concentration of organophosphorus hapten-protein complexes coated in the microplate is 1 to 10 μg / mL.

[0023] It should be noted that, for example, 5g of the powder of the ten medicinal herbs to be tested is added to 20mL of acetonitrile solution containing 1% acetic acid, and the mixture is shaken and extracted for 5min. Then, 2g of anhydrous magnesium sulfate and 1g of sodium chloride are added, and the mixture is centrifuged for 5min (9000rpm). 1mL of the supernatant is taken. 0.5mL of a 1:100 diluted colloidal gold-labeled anti-organophosphorus pesticide peptide antibody solution is added and mixed. The mixture is then added to a microplate coated with a 5μg / mL organophosphorus hapten-protein complex. The mixture is subjected to a competitive reaction at 37℃ for 15min. After washing, the absorbance is measured at 450 to 550nm. For example, if the preset first threshold is 30%, then when the competitive inhibition rate is greater than or equal to 30%, the organophosphorus pesticide residue in the ten medicinal herbs to be tested is determined to be excessive.

[0024] It should be noted that the centrifugation conditions are: a rotation speed of 8,000 to 10,000 rpm and a time of 2 to 5 minutes.

[0025] According to an embodiment of the present invention, the "Ten Flavors of Cinnamon" is any one of cinnamon, monk fruit, star anise, turmeric, spatholobus suberectus, sophora tonkinensis, poria cocos, kudzu root, or lysimachia christinae.

[0026] According to an embodiment of the present invention, after extracting a preset weight of the powder of the ten medicinal herbs to be tested, the method further includes: Obtain the initial moisture content of the powder of the ten medicinal herbs of Guiwei to be tested; If the initial moisture content is greater than the preset target moisture content, a dehydration pretreatment prompt message will be triggered. Based on the dehydration pretreatment prompts, anhydrous sodium sulfate was added to the powder of the tested Gui Shiwei medicinal material for dehydration pretreatment to obtain the dehydrated Gui Shiwei medicinal material powder, and the dehydrated Gui Shiwei medicinal material powder was used for subsequent testing. The mass of the anhydrous sodium sulfate is ,in To determine the mass of anhydrous sodium sulfate added, The initial moisture content of the powder containing the ten medicinal herbs of Guiwei is given. For the target moisture content, This represents the weight of the powdered Gui Shi Wei medicinal herbs to be tested. This is the correction factor.

[0027] It should be noted that fresh medicinal herbs such as Lysimachia christinae, Pueraria lobata, and Siraitia grosvenorii have a high water content, which can reach 70% to 90%. When these herbs are directly added to acetonitrile solution for extraction, the sample will dilute the acetonitrile solution, reducing its extraction efficiency for organophosphorus pesticides and affecting subsequent salting-out separation. For example, the preset target water content range is 40% to 50%, and the correction coefficient range is 1.2 to 1.5.

[0028] According to an embodiment of the present invention, when the tested medicinal herb is star anise or cinnamon, it further includes: After extraction with acetonitrile solution, graphitized carbon black adsorbent is added additionally, the amount of which is... ,in ,in and These are the corresponding coefficients. The characteristic value of pigment content in the medicinal material to be tested. This represents the characteristic value of volatile oil content in medicinal materials. The quality of Chinese medicinal powder.

[0029] It should be noted that the graphitized carbon black adsorbent is a non-polar porous adsorbent material with strong adsorption capacity for interfering substances such as chlorophyll, carotenoids, and volatile oils (e.g., anethole and cinnamaldehyde), while it does not adsorb organophosphorus pesticides; The characteristic value of pigment content in the medicinal material to be tested is used, for example, cinnamon is set to 8 and star anise to 6; These are characteristic values ​​for the volatile oil content of medicinal materials; for example, cinnamon has a value of 7, and star anise has a value of 9.

[0030] According to an embodiment of the present invention, it further includes: A series of standard solutions of organophosphorus pesticides with known concentrations were prepared, and the results were tested according to steps 1 to 3 to obtain the competitive inhibition rate of each standard solution. Based on the preset regression equation, the concentration of organophosphorus pesticides in the sample is determined according to the competitive inhibition rate of the sample extract. If the concentration value is greater than or equal to the preset concentration threshold, it is determined that the organophosphorus pesticide residue in the tested Gui Shiwei medicinal materials exceeds the standard.

[0031] It should be noted that the values ​​are logarithmic of the concentrations of a series of known organophosphorus pesticide standard solutions. A linear regression was performed with the x-axis representing the competition inhibition rate and the y-axis representing the corresponding competition inhibition rate, resulting in the regression equation. Where a and b are constants, the competitive inhibition rate of the sample extract is substituted into the regression equation, and the concentration of organophosphorus pesticides in the sample is calculated by reverse calculation. .

[0032] According to embodiments of the present invention, the method further includes simultaneous detection of multiple residues of organophosphorus pesticides, specifically: Different organophosphorus hapten-protein complexes were coated in different wells of the microplate, with each hapten corresponding to an organophosphorus pesticide. The sample extract was simultaneously added to each detection well and competed with the colloidal gold-labeled anti-organophosphorus pesticide antibody corresponding to each well. The competitive inhibition rate of each well was measured separately. The competitive inhibition rate of each well was then substituted into the preset regression equation for the corresponding organophosphorus pesticide in that well to calculate the concentration of the corresponding organophosphorus pesticide in each well, thus achieving simultaneous quantitative detection of multiple organophosphorus pesticides.

[0033] According to an embodiment of the present invention, when a portable detector is used, the absorbance value measurement at wavelengths of 450 to 550 nm in step 3 can be completed by a portable colorimeter, whose built-in 450 nm and 520 nm dual-wavelength LED light source and photoelectric detection module can directly read the absorbance value of each well; or it can be completed by image colorimetric analysis using a smartphone camera, which takes a picture of the microplate in a standard light source box, and establishes a standard curve of color feature values ​​and organophosphorus pesticide concentration through a preset color space conversion algorithm, and automatically calculates the competitive inhibition rate and concentration value.

[0034] It should be noted that the construction process of the color space conversion algorithm is as follows: First, a series of microplate images after the reaction of standard solutions of known concentrations are captured using a smartphone camera in a standard light source box, and the RGB average value of the region of interest (ROI) of each well is extracted; then, the RGB values ​​are processed into grayscale values ​​to obtain the corresponding grayscale values; a standard curve is fitted with the logarithm of the standard solution concentration as the abscissa and the corresponding grayscale values ​​as the ordinate to construct a linear equation. During automatic calculation, the color feature values ​​(grayscale values) extracted from the image of the sample to be tested are substituted into the linear equation, and the corresponding concentration value or competitive inhibition rate is automatically read.

[0035] According to an embodiment of the present invention, when the tested medicinal materials are Luo Han Guo (monk fruit), Ge Gen (kudzu root), or Fu Ling (poria), the method further includes the following steps before adding the acetonitrile solution: First, an enzymatic pretreatment is performed by adding a glycoside hydrolase complex, which contains cellulase, pectinase, and... Glucosidase with an activity of 50 to 200 U / g of medicinal material, an enzymatic hydrolysis temperature of 40 to 50℃, and an enzymatic hydrolysis time of 15 to 30 minutes; After enzymatic pretreatment, polyvinylpyrrolidone is added to the pretreated solution at a mass of 1% to 5% of the medicinal powder mass to adsorb the polypeptides and proteins released by enzymatic hydrolysis.

[0036] It should be noted that monk fruit, kudzu root, or poria cocos have high sugar / high viscosity. Through enzymatic pretreatment, the cell walls of the medicinal materials are broken down, the viscosity is reduced, and organophosphorus pesticides that were originally wrapped in starch, pectin, or glycosides are released, significantly improving the extraction efficiency. At the same time, polyvinylpyrrolidone is added to adsorb the polyphenols and proteins released by enzymatic hydrolysis, preventing them from non-specifically binding to or precipitating with colloidal gold-labeled antibodies.

[0037] A second aspect of the present invention provides a detection kit for the detection method of claim 1, comprising: a microplate pre-coated with an organophosphorus hapten-protein complex, a colloidal gold-labeled anti-organophosphorus pesticide small peptide antibody working solution, a series of organophosphorus pesticide standards of known concentrations, a washing buffer, and sample extraction tubes containing a predetermined mass of anhydrous magnesium sulfate and sodium chloride; each well of the microplate is pre-coated with an organophosphorus hapten-protein complex at a concentration of 1 to 10 μg / mL, and the colloidal gold-labeled antibody is diluted at a concentration of 1:50 to 1:200.

[0038] It should be noted that the microplate is pre-coated with a concentration of 1 to 10 μg / mL of organophosphorus hapten-protein complex, a 1:100 diluted colloidal gold-labeled anti-organophosphorus pesticide peptide antibody working solution, a series of organophosphorus pesticide standards of known concentrations (e.g., 0.01, 0.1, 1.0, 10.0 mg / kg), a phosphate wash buffer at pH 7.4, and sample extraction tubes containing 2 g of anhydrous magnesium sulfate and 1 g of sodium chloride; wherein, the microplate is a 96-well detachable ELISA plate, which is convenient for flexible use according to the detection batch.

[0039] The method of using the test kit is as follows: Step 2-1: Take 1 to 5 g of the powder of the ten herbs to be tested, place it in the sample extraction tube, add 10 to 20 mL of acetonitrile solution containing 1% acetic acid, and extract by vigorous shaking for 5 minutes; Step 2-2: Add 2g of anhydrous magnesium sulfate and 1g of sodium chloride to the sample extraction tube, tighten the cap and shake for 1 minute, centrifuge at 9000rpm for 5 minutes, and take the supernatant for later use. Steps 2-3: Take 0.5 to 1 mL of the supernatant, add an equal volume of 1:100 diluted colloidal gold-labeled anti-organophosphorus pesticide peptide antibody working solution, mix well, and add 100 to 200 μL to each well of a microplate pre-coated with organophosphorus hapten-protein complex. Steps 2-4: Incubate the microplate in a 37°C incubator for 15 minutes. After removing it, wash it 3 to 5 times with washing buffer, adding 200 to 300 μL of washing buffer to each well each time, and pat dry. Steps 2-5: Place the washed microplate in a portable colorimeter or microplate reader and measure the absorbance of each well at a wavelength of 450 nm; or use a smartphone image colorimetric analysis module for detection. Steps 2-6: Calculate the competitive inhibition rate based on the measured absorbance value or color characteristic value, and calculate the concentration of organophosphorus pesticides in the sample to be tested from the standard curve or regression equation. If the competitive inhibition rate is greater than or equal to the preset first threshold, it is determined that the standard has been exceeded.

[0040] The sample volume and reagent ratio can be adjusted according to actual testing needs. All operations are performed at room temperature and do not require large professional equipment.

[0041] According to an embodiment of the present invention, the microplate is further provided with positive control wells and negative control wells; the positive control wells are coated with an organophosphorus hapten-protein complex of known concentration and a preset standard competitive inhibition rate value, and the negative control wells are coated with a blocking solution without the relevant antigen; each time a test is performed, if the competitive inhibition rate of the positive control well is within a preset range and the absorbance value of the negative control well is higher than a preset threshold, then the test result is valid.

[0042] Figure 2 A schematic diagram of the standard curve for sulfur and phosphorus detection according to the present invention is shown.

[0043] like Figure 2 As shown, parathion standard solutions with concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 mg / kg were prepared and tested according to steps 1 to 3. The competitive inhibition rates corresponding to each concentration were 8.2%, 18.5%, 29.7%, 48.3%, 62.1%, 78.6%, and 85.4%, respectively. A linear regression was performed with the logarithm of the parathion concentration as the x-axis and the competitive inhibition rate as the y-axis to obtain the regression equation. R² = 0.989, linear range: 0.01 to 10.0 mg / kg; limit of detection (LOD) calculated using 3 times the blank standard deviation is 0.005 mg / kg (e.g., ...). Figure 4 It meets the detection requirements of GB 2763-2021 for the residue limits of organophosphorus pesticides in Chinese medicinal materials (generally 0.05 to 0.5 mg / kg); the thiophosphorus is a type of organophosphorus.

[0044] Figure 5 A schematic diagram showing the spiked recovery rate results of five kinds of cinnamon-based medicinal materials according to the present invention is presented.

[0045] like Figure 5As shown, five representative medicinal herbs, cinnamon, monk fruit, star anise, chicken blood vine, and Lysimachia christinae, were selected. Parathion standard was added at three concentration levels (0.1, 0.5, and 2.0 mg / kg), and the results were analyzed according to the method of this invention, with three replicates for each concentration. The results showed that the recovery rate of cinnamon spiked with parathion was 86.3% to 94.7%, with a relative standard deviation (RSD) of 4.2% to 6.8%; the recovery rate of monk fruit spiked with parathion was 88.1% to 96.2%, with an RSD of 3.5% to 5.9%; the recovery rate of star anise spiked with parathion was 85.6% to 92.3%, with an RSD of 4.8% to 7.2%; the recovery rate of chicken blood vine spiked with parathion was 87.4% to 95.1%, with an RSD of 4.5% to 6.3%; and the recovery rate of Lysimachia christinae spiked with parasimion was 89.2% to 97.5%, with an RSD of 3.8% to 5.4%. The overall average recovery rate was 90.8%, and the average RSD was 5.1%, indicating that the method has good accuracy and precision.

[0046] Figure 6 A schematic diagram showing the comparison between the detection results of the present invention and the enzyme inhibition rate method is presented.

[0047] like Figure 6 As shown, 50 batches of *Cinnamomum cassia* medicinal materials from different origins were randomly selected. Organophosphorus pesticide residues were detected using the method of this invention and the enzyme inhibition rate method (rapid test card method) of GB / T5009.199-2003, with GC-MS as the reference method. Based on samples that tested positive by GC-MS, the positive detection rate of the method of this invention was 96.7% (29 / 30), the negative concordance rate was 100% (20 / 20), and the overall concordance rate was 98.0% (e.g., ...). Figure 5 The traditional enzyme inhibition rate method had a positive detection rate of only 80.0% (24 / 30), and a false negative rate of 25.0% for dark-colored medicinal materials such as *Spatholobus suberectus* and *Sophora tonkinensis*. Further analysis of 18 samples confirmed by GC-MS to contain multiple organophosphates revealed that the method of this invention correctly identified mixed residues in 17 samples through simultaneous multi-well detection, achieving a recognition rate of 94.4%.

[0048] Figure 3 A schematic diagram showing the stability variation curves of the reagent kit of the present invention under different temperature conditions is shown.

[0049] like Figure 3As shown, the test kit assembled according to the present invention was stored at 4℃, 25℃, and 40℃, respectively. A batch of samples was taken out every 7 days to detect the parathion standard curve and spiked samples. The results showed that after storage at 4℃ for 56 days, the standard curve slope drift was less than 5%, and the spiked recovery rate remained between 88.5% and 94.2%; after storage at 25℃ for 28 days, the standard curve slope drift was less than 8%, and the spiked recovery rate remained between 85.6% and 91.8%; after storage at 40℃ for 14 days, all indicators began to exceed the acceptable range. Therefore, the recommended storage conditions for the kit are 4℃ protected from light, with a shelf life of not less than 56 days.

[0050] Figure 7 A schematic diagram of the correlation analysis results of the portable detection method of the present invention is shown.

[0051] like Figure 7 As shown, 20 simulated samples with known parathion concentrations were selected and detected using both an ELISA reader (450nm wavelength) and a portable colorimeter (built-in 450nm LED light source). The results showed a correlation coefficient of 0.986 (P < 0.001) between the absorbance values ​​measured by the two methods, and the quantitative deviation of the portable colorimeter was within ±12%. Another 15 samples were detected using smartphone image colorimetric analysis, and the correlation coefficient between the results and the ELISA reader results was 0.973, with a quantitative deviation within ±15%, meeting the quantitative requirements for rapid on-site screening.

[0052] Figure 8 A schematic diagram showing the verification results of the quality control system of the present invention is illustrated.

[0053] like Figure 8 As shown, analysis of 50 batches of test data revealed that when the competitive inhibition rate of the positive control well was between 60% and 80% and the OD value of the negative control well was >0.8, the spiked recovery rate of that batch was between 85% and 105%. When the positive or negative control exceeded these ranges (e.g., due to reagent aging or operational errors), the abnormal spiked recovery rate (<80% or >120%) of the corresponding batch reached 42.9%. Therefore, the built-in quality control system can effectively identify invalid test batches, ensuring the reliability of the test results.

[0054] The present invention discloses a rapid detection method and a detection kit for organophosphorus pesticides in ten traditional Chinese medicines of Cinnamomum cassia. The method includes: extracting a sample of the powder of the ten traditional Chinese medicines of Cinnamomum cassia to be detected with an acetonitrile solution containing acetic acid, performing salting-out and layering, and then taking the supernatant; incubating with a colloidal gold-labeled anti-organophosphorus pesticide small peptide antibody; performing a competitive immunoreaction in a microplate coated with an organophosphorus hapten-protein complex; washing and then measuring the absorbance at a wavelength of 450 to 550 nm, calculating the competitive inhibition rate, and comparing it with a preset first threshold to determine whether it exceeds the standard; this method is simple and rapid to operate; in addition, to meet the detection requirements for different organophosphorus pesticides, by setting different regions coated with organophosphorus hapten-protein complexes in the microplate, simultaneous detection of multiple organophosphorus pesticides is achieved, further improving the applicability of the detection of organophosphorus pesticide residues.

[0055] The above are only preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed technical content to form equivalent embodiments with equivalent changes, but as long as they do not depart from the technical content of the present invention, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs, characterized in that, include: Step 1: Extract the pre-set weight of the powder of the ten medicinal herbs to be tested, add an acetonitrile solution containing 0.5% to 2.0% acetic acid by volume for extraction, then add anhydrous magnesium sulfate and sodium chloride for salting out and separation, centrifuge and take the supernatant to obtain the sample extract; Step 2: Mix the sample extract with colloidal gold-labeled anti-organophosphorus pesticide peptide antibody solution, add it to a microplate coated with organophosphorus hapten-protein complex, and conduct a competitive reaction at 35 to 40°C for 10 to 20 minutes to obtain experimental reaction microwells; Step 3: After washing the micropores of the experimental reaction, measure the absorbance value at a wavelength of 450 to 550 nm, and use it as the experimental absorbance value; Step 4: Extract a pre-set weight of buffer solution without the sample to replace the sample extraction solution, and follow steps 1 to 3 to obtain the comparative absorbance values; Step 5: Determine the competitive inhibition rate based on the experimental absorbance value and the comparative absorbance value; the formula for calculating the competitive inhibition rate R is: ×100%, of which To compare absorbance values, This represents the experimental absorbance value; Step 6: If the competitive inhibition rate is greater than or equal to the preset first threshold, it is determined that the organophosphorus pesticide residues in the tested Gui Shiwei medicinal materials exceed the standard.

2. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 1, characterized in that, The "Ten Flavors of Cinnamon" refers to any one of the following: cinnamon, monk fruit, star anise, turmeric, spatholobus, sophora root, poria, kudzu root, or lysimachia.

3. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 1, characterized in that, After extracting a preset weight of the powder of the ten medicinal herbs to be tested, the method further includes: Obtain the initial moisture content of the powder of the ten medicinal herbs of Guiwei to be tested; If the initial moisture content is greater than the preset target moisture content, a dehydration pretreatment prompt message will be triggered. Based on the dehydration pretreatment prompts, anhydrous sodium sulfate was added to the powder of the tested Gui Shiwei medicinal material for dehydration pretreatment to obtain the dehydrated Gui Shiwei medicinal material powder, and the dehydrated Gui Shiwei medicinal material powder was used for subsequent testing. The mass of the anhydrous sodium sulfate is ,in To determine the mass of anhydrous sodium sulfate added, The initial moisture content of the powder containing the ten medicinal herbs of Guiwei is given. For the target moisture content, This represents the weight of the powdered Gui Shi Wei medicinal herbs to be tested. This is the correction factor.

4. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal herbs according to claim 1, characterized in that, When the tested medicinal materials are star anise or cinnamon, the following are also included: After extraction with acetonitrile solution, graphitized carbon black adsorbent is added additionally, the amount of which is... ,in ,in and These are the corresponding coefficients. The characteristic value of pigment content in the medicinal material to be tested. This represents the characteristic value of volatile oil content in medicinal materials. The quality of Chinese medicinal powder.

5. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 1, characterized in that, Also includes: A series of standard solutions of organophosphorus pesticides with known concentrations were prepared, and the results were tested according to steps 1 to 3 to obtain the competitive inhibition rate of each standard solution. Based on the preset regression equation, the concentration of organophosphorus pesticides in the sample is determined according to the competitive inhibition rate of the sample extract. If the concentration value is greater than or equal to the preset concentration threshold, it is determined that the organophosphorus pesticide residue in the tested Gui Shiwei medicinal materials exceeds the standard.

6. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 5, characterized in that, Also includes: Different organophosphorus hapten-protein complexes were coated in different wells of the microplate, with each hapten corresponding to an organophosphorus pesticide. The sample extract was simultaneously added to each detection well and competed with the colloidal gold-labeled anti-organophosphorus pesticide antibody corresponding to each well. The competitive inhibition rate of each well was measured, and the competitive inhibition rate of each well was substituted into the preset regression equation of the corresponding organophosphorus pesticide for that well to calculate the concentration of the corresponding organophosphorus pesticide for each well. Simultaneous quantitative detection of multiple organophosphorus pesticides.

7. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 1, characterized in that, When the tested Gui Shi Wei Chinese medicinal materials are Luo Han Guo, Ge Gen, or Fu Ling, the following steps are included before adding the acetonitrile solution: First, an enzymatic pretreatment is performed by adding a glycoside hydrolase complex, which contains cellulase, pectinase, and... Glucosidase with an activity of 50 to 200 U / g of medicinal material, an enzymatic hydrolysis temperature of 40 to 50℃, and an enzymatic hydrolysis time of 15 to 30 minutes; After enzymatic pretreatment, polyvinylpyrrolidone is added to the pretreated solution at a mass of 1% to 5% of the medicinal powder mass to adsorb the polypeptides and proteins released by enzymatic hydrolysis.

8. The rapid detection method for organophosphorus pesticides in Gui Shi Wei Chinese medicinal materials according to claim 1, characterized in that, The absorbance value measured at a wavelength of 450 to 550 nm in step 3 is performed using a portable colorimeter or a smartphone image colorimetric analysis module. The smartphone image colorimetric analysis module extracts the color feature values ​​of each well by taking a picture of the microplate and converts them into concentration values ​​based on a pre-stored standard curve.

9. A detection kit for use in the detection method of claim 1, characterized in that, include: The microplate is pre-coated with an organophosphorus hapten-protein complex, a colloidal gold-labeled anti-organophosphorus pesticide peptide antibody working solution, a series of organophosphorus pesticide standards of known concentrations, a washing buffer, and sample extraction tubes containing pre-defined masses of anhydrous magnesium sulfate and sodium chloride; each well of the microplate is pre-coated with an organophosphorus hapten-protein complex at a concentration of 1 to 10 μg / mL, and the colloidal gold-labeled antibody is diluted at a concentration of 1:50 to 1:

200.

10. The detection kit according to claim 9, characterized in that, The microplate also includes positive control wells and negative control wells; the positive control wells are coated with an organophosphorus hapten-protein complex of known concentration and a preset standard competitive inhibition rate value, and the negative control wells are coated with a blocking solution without the relevant antigen; each time a test is performed, if the competitive inhibition rate of the positive control well is within a preset range and the absorbance value of the negative control well is higher than a preset threshold, then the test result is valid.

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