Pesticide residue detection method based on colloidal gold recognition

Abstract

The invention belongs to the technical field of pesticide detection, and particularly relates to a pesticide residue detection method based on colloidal gold recognition, which comprises the following steps: taking edible parts of agricultural products to be detected, chopping, and homogenizing; adding an extraction solvent with the volume 3-5 times that of the homogenate into the homogenate, fully mixing, standing, and taking supernate; taking 10 g of 5 parts of the supernate, respectively adding 10 times of diluting solvent, 50 times of diluting solvent, 100 times of diluting solvent, 500 times of diluting solvent and 1000 times of diluting solvent, and uniformly mixing to obtain five groups of diluents; respectively adding 80 microliters of the five groups of diluents into detection ports of nano colloidal gold detection cards, and transferring the five groups of nano colloidal gold detection cards into a cassette with an annular LED light source after the five groups of diluents react for 15-20 minutes; shooting a color development result of the nano colloidal gold detection card through a camera, transmitting an image back to a display terminal, and judging the pesticide residue by checking the image; the defects in the prior art are overcome, and agricultural product extracts with different dilution degrees are detected by utilizing the nano colloidal gold detection card, so that the pesticide residue is judged.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide detection, and in particular relates to a pesticide residue detection method based on colloidal gold recognition. Background Art

[0002] Pesticide detection currently mainly relies on large modern analytical instruments such as liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-tandem mass spectrometry (HPLC / MS / MS), and gas chromatography-mass spectrometry (GC / MS). Although these instruments have high detection sensitivity, they are expensive and require strictly trained professional technicians to operate and use. At the same time, it takes several hours or even overnight from sample processing to the final result. The operation process during this period requires several to more than ten steps. It is technically and economically impossible to meet the timely and rapid detection of large quantities of samples or temporary samples, and thus it is impossible to guarantee people's requirements for food quality and safety in daily life. Therefore, the study of rapid detection technology for pesticide residues in vegetables and food has become a concern of competent departments at all levels and the general public. Therefore, it is of great significance to develop equipment and methods that can detect organophosphorus sensitively, quickly and accurately as soon as possible to detect organophosphorus residues in samples such as vegetables, fruits, and tissues. Summary of the invention

[0003] The purpose of the present invention is to provide a method for detecting pesticide residues based on colloidal gold recognition, which overcomes the shortcomings of the prior art and uses nano-colloidal gold detection cards to detect agricultural product extracts of different dilutions to determine the amount of pesticide residues.

[0004] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0005] A method for detecting pesticide residues based on colloidal gold recognition comprises the following steps:

[0006] S1. Take the agricultural products to be tested, wash them and dry them naturally;

[0007] S2, taking the edible part of the dried agricultural products, chopping them, adding them into a homogenizer for homogenization, and obtaining a homogenate;

[0008] S3, add 3-5 times the volume of extraction solvent to the homogenate, mix thoroughly and let stand, and take the supernatant;

[0009] S4, take 5 portions (10 g) of the supernatant, add 10 times, 50 times, 100 times, 500 times and 1000 times of dilution solvent respectively, mix well to obtain five groups of dilution solutions;

[0010] S5. Take 80ul of each of the five dilutions and add them to the detection port of the nanocolloidal gold detection card. After reacting for 15-20 minutes, transfer the five sets of nanocolloidal gold detection cards to a dark box with a ring-shaped LED light source.

[0011] S6. The color development result of the nano-colloidal gold detection card is photographed by the camera on the top of the dark box, and the image is transmitted back to the display terminal, and the pesticide residue is determined by viewing the image.

[0012] Furthermore, the extraction solvent in S3 is any one of acetonitrile, methanol and acetone.

[0013] Furthermore, the dilution solvent in S4 is any one of petroleum ether, n-hexane and benzene.

[0014] Furthermore, the nano colloidal gold detection card in S5 includes a sample pad, a binding pad, a chromatography pad, and an absorption pad, and each component is overlapped by 2 mm in sequence and pasted on the bottom plate; on the binding pad, conventional antibodies with color labels are adsorbed, and the color labels include nano gold balls and colored microspheres; on the chromatography pad, two parallel lines are set, the detection line T line and the quality control line C line; the T line is fixed with pesticide-coupled protein, and the C line is fixed with nano antibodies.

[0015] Furthermore, the surface of the gold nanosphere is modified by Raman signal molecules, and the modification method includes:

[0016] Take 1-2 ml of gold nanospheres, add 1 μL of 1 mmol / L 4-mercaptobenzoic acid, shake and react at room temperature, then centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain Au-MBA solution;

[0017] The pH of the Au-MBA solution was adjusted, and the pesticide-coupled protein was added after standing. After mixing, the solution was allowed to react at room temperature for 30 minutes. The solution was then centrifuged at 4°C for 20 minutes, the supernatant was removed, and the bottom precipitate was taken and resuspended with 200 μL of resuspension solution to obtain the Raman signal molecule-modified gold nanospheres.

[0018] Furthermore, the method for preparing the gold nanospheres comprises the following steps:

[0019] Take 1 ml of colloidal gold and centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain a colloidal gold resuspension;

[0020] The colloidal gold resuspension droplets were applied on a copper grid, dried at 37° C. overnight, and then collected to obtain the gold nanospheres.

[0021] Further, the color development result of the nano-colloidal gold detection card is photographed by the camera at the top of the dark box in S6, and the image is transmitted back to the processing terminal to identify the color development result of the nano-colloidal gold detection card and determine the pesticide residue. The specific steps include:

[0022] Obtaining the RBG color image of the nano-colloidal gold detection card, and gray-processing the RBG color image to obtain a gray-scale image;

[0023] The edge algorithm is used to extract the edge of the target area to be analyzed from the grayscale image and transmit it to the display screen.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention extracts the edible part of agricultural products, dilutes it according to different proportions, and uses a nano-colloidal gold detection card attached with pesticide coupling protein to detect it, and determines the pesticide residue according to the reaction results of different dilutions.

[0026] 2. The colloidal gold detection card strip used in the present invention has the advantages of being fast, convenient, low cost, not relying on professional inspectors, and not requiring expensive instruments and equipment.

[0027] 3. The present invention modifies Raman signal molecules on the surface of nano-colloidal gold and combines immunochromatography technology with SERS technology, which not only retains the advantages of immunochromatography such as rapidity and convenience, but also greatly improves the detection sensitivity and realizes quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a flow chart of a method for detecting pesticide residues based on colloidal gold recognition. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] As shown in the figure, the pesticide residue detection method based on colloidal gold recognition of the present invention comprises the following steps:

[0031] S1. Take the agricultural products to be tested, wash them and let them dry naturally.

[0032] S2. Take the edible part of the dried agricultural products, chop them into pieces, add them into a homogenizer and homogenize them to obtain a homogenate.

[0033] S3, add 3-5 times the volume of extraction solvent to the homogenate, mix thoroughly and let stand, and take the supernatant;

[0034] Wherein, the extraction solvent is any one of acetonitrile, methanol and acetone.

[0035] S4. Take 5 portions (10 g) of the supernatant, add 10-fold, 50-fold, 100-fold, 500-fold and 1000-fold dilution solvents respectively, mix well and obtain five groups of dilution solutions.

[0036] The diluting solvent is any one of petroleum ether, n-hexane and benzene.

[0037] S5. Take 80ul of each of the five groups of dilutions and add them to the detection port of the nanocolloidal gold detection card. After reacting for 15-20 minutes, transfer the five groups of nanocolloidal gold detection cards to a dark box with a ring-shaped LED light source.

[0038] Among them, the nano colloidal gold detection card includes a sample pad, a binding pad, a chromatography pad, and an absorption pad, and each component is overlapped by 2 mm and pasted on the bottom plate; on the binding pad, conventional antibodies with color labels are adsorbed, and the color labels include nano gold balls and colored microspheres; on the chromatography pad, two parallel lines are set, the detection line T line and the quality control line C line; the T line is fixed with pesticide-coupled protein, and the C line is fixed with nano antibodies.

[0039] The surface of the gold nanosphere is modified by Raman signal molecules, and the modification methods include:

[0040] Take 1-2 ml of gold nanospheres, add 1 μL of 1 mmol / L 4-mercaptobenzoic acid, shake and react at room temperature, then centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain Au-MBA solution;

[0041] The pH of the Au-MBA solution was adjusted, and the pesticide-coupled protein was added after standing. After mixing, the solution was allowed to react at room temperature for 30 minutes. The solution was then centrifuged at 4°C for 20 minutes, the supernatant was removed, and the bottom precipitate was taken and resuspended with 200 μL of resuspension solution to obtain the Raman signal molecule-modified gold nanospheres.

[0042] The preparation method of gold nanospheres comprises the following steps:

[0043] Take 1 ml of colloidal gold and centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain a colloidal gold resuspension;

[0044] The colloidal gold resuspension droplets were applied on a copper grid, dried at 37° C. overnight, and then collected to obtain the gold nanospheres.

[0045] S6. The color development result of the nano-colloidal gold detection card is photographed by the camera on the top of the dark box, and the image is transmitted back to the display terminal, and the pesticide residue is determined by viewing the image.

[0046] The specific steps are:

[0047] Obtaining the RBG color image of the nano-colloidal gold detection card, and gray-processing the RBG color image to obtain a gray-scale image;

[0048] The edge algorithm is used to extract the edge of the target area to be analyzed from the grayscale image and transmit it to the display screen.

[0049] After obtaining the grayscale image, the operator first observes the titration test results of the 10-fold diluted solution. If the T line is obviously colored, it means that pesticide residues exist. The operator further observes the titration test results of the 50-fold, 100-fold, 500-fold and 1000-fold diluted solutions. If the T line is obviously colored, it means that the pesticide residues obviously exceed the standard. If the T line no longer shows color in the titration test results of the 500-fold diluted solution, it means that there are pesticide residues, but they do not exceed the standard; if the T line does not show color or the color is not obvious in the titration test results of the 50-fold diluted solution, there is no pesticide residue.

[0050] In summary, the pesticide residue detection method based on colloidal gold recognition described in the present invention is to extract the edible part of agricultural products, dilute them according to different proportions, and detect them using a nano-colloidal gold detection card attached with pesticide-coupled protein, and determine the pesticide residue according to the reaction results of different dilutions.

[0051] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for detecting pesticide residues based on colloidal gold recognition, characterized in that: The following steps are involved: S1. Take the agricultural products to be tested, wash them and dry them naturally; S2, taking the edible part of the dried agricultural products, chopping them, adding them into a homogenizer for homogenization, and obtaining a homogenate; S3, add 3-5 times the volume of extraction solvent to the homogenate, mix thoroughly and let stand, and take the supernatant; S4, take 5 portions (10 g) of the supernatant, add 10 times, 50 times, 100 times, 500 times and 1000 times of dilution solvent respectively, mix well to obtain five groups of dilution solutions; S5. Take 80ul of each of the five dilutions and add them to the detection port of the nanocolloidal gold detection card. After reacting for 15-20 minutes, transfer the five sets of nanocolloidal gold detection cards to a dark box with a ring-shaped LED light source. S6. The color development result of the nano-colloidal gold detection card is photographed by the camera on the top of the dark box, and the image is transmitted back to the display terminal, and the pesticide residue is determined by viewing the image.

2. A method for detecting pesticide residues based on colloidal gold recognition according to claim 1, characterized in that: The extraction solvent in S3 is any one of acetonitrile, methanol and acetone.

3. A method for detecting pesticide residues based on colloidal gold recognition according to claim 1, characterized in that: The dilution solvent in S4 is any one of petroleum ether, n-hexane and benzene.

4. The method for detecting pesticide residues based on colloidal gold recognition according to claim 1, characterized in that: The nano colloidal gold detection card in S5 includes a sample pad, a binding pad, a chromatography pad, and an absorption pad, and each component is overlapped by 2 mm and pasted on the bottom plate; the binding pad is adsorbed with a color-labeled conventional antibody, and the color label includes nano gold balls and color microspheres; two parallel lines are set on the chromatography pad, the detection line T line and the quality control line C line; the T line is fixed with pesticide-coupled protein, and the C line is fixed with nano antibodies.

5. A method for detecting pesticide residues based on colloidal gold recognition according to claim 4, characterized in that: The surface of the gold nanosphere is modified by Raman signal molecules, and the modification method includes: Take 1-2 ml of gold nanospheres, add 1 μL of 1 mmol / L 4-mercaptobenzoic acid, shake and react at room temperature, then centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain Au-MBA solution; The pH of the Au-MBA solution was adjusted, and the pesticide-coupled protein was added after standing. After mixing, the solution was allowed to react at room temperature for 30 minutes. The solution was then centrifuged at 4°C for 20 minutes, the supernatant was removed, and the bottom precipitate was taken and resuspended with 200 μL of resuspension solution to obtain the Raman signal molecule-modified gold nanospheres.

6. A method for detecting pesticide residues based on colloidal gold recognition according to claim 4 or 5, characterized in that: The preparation method of the gold nanospheres comprises the following steps: Take 1 ml of colloidal gold and centrifuge at high speed, remove the supernatant and take the bottom precipitate, resuspend it with 1 mL of deionized water to obtain a colloidal gold resuspension; The colloidal gold resuspension droplets were applied on a copper grid, dried at 37° C. overnight, and then collected to obtain the gold nanospheres.

7. The method for detecting pesticide residues based on colloidal gold recognition according to claim 1, characterized in that: S6 describes that the color development result of the nano-colloidal gold detection card is photographed by the camera at the top of the dark box, and the image is transmitted back to the processing terminal to identify the color development result of the nano-colloidal gold detection card and determine the pesticide residue. The specific steps include: Obtaining the RBG color image of the nano-colloidal gold detection card, and gray-processing the RBG color image to obtain a gray-scale image; The edge algorithm is used to extract the edge of the target area to be analyzed from the grayscale image and transmit it to the display screen.

Images