A colloidal gold card for detecting pesticide residues and its preparation method and application

Abstract

The present invention relates to a colloidal gold card for detecting pesticide residues and a preparation method and application thereof. The colloidal gold card comprises a bottom plate and a sample pad, a gold binding pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on the bottom plate. The nitrocellulose membrane is provided with a detection line coated with a pesticide antigen and a quality control line coated with a secondary antibody. The gold binding pad contains colloidal gold-labeled pesticide antibodies, sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol. In the colloidal gold card, the gold binding pad contains sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol, which can effectively protect antigens and antibodies, eliminate the influence of tea polyphenols, flavonoids, pigments and minerals in tea on color development, and improve the anti-interference property of the colloidal gold card.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide detection, and relates to a colloidal gold card for detecting pesticide residues, a preparation method and an application thereof, and in particular to a colloidal gold card for detecting pesticide residues in tea leaves, a preparation method and an application thereof. Background Art

[0002] Pesticides are widely used in agriculture to control pests and diseases, remove weeds and regulate plant growth, but pesticide residues in crops are also harmful to the human body. Chinese people have a long history of drinking tea for thousands of years and are also a major exporter of tea. Therefore, pesticide residues in tea are highly valued.

[0003] At present, the main means of pesticide residue detection still relies on large-scale modern analytical instruments such as liquid chromatography, gas chromatography, liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry. Although these instruments have high detection sensitivity, they are expensive, cumbersome to operate, and have a long cycle, making it difficult to meet the needs of timely and rapid detection of large quantities of samples or temporary samples. Therefore, people are committed to developing methods that can detect pesticide residues quickly, accurately, conveniently, and at low cost.

[0004] CN101319997 discloses a card suitable for rapid detection of pesticide residues in tea leaves and a detection method thereof. The card is composed of an immobilized enzyme sheet containing a stabilizer and a color developing sheet. It is easy to operate and has a short detection time. It can quickly and effectively identify tea leaves with excessive pesticide residues, but its accuracy and anti-interference performance are poor.

[0005] CN105181949A discloses a heterologous competitive immunochromatographic gold-labeled test strip suitable for rapid detection of multiple residues of three chloronicotinoid pesticides such as imidacloprid in tea leaves. The test strip comprises a backing plate, a sample pad, a gold-labeled primary antibody binding pad, a nitrocellulose membrane, and a water-absorbing pad. The gold-labeled primary antibody binding pad is coated with a colloidal gold-labeled imidacloprid-specific mouse monoclonal antibody, the nitrocellulose membrane is coated with a detection line of chlorothiazide or clothianidin artificial antigens for heterologous competition and a quality control line coated with rabbit anti-mouse IgG. The test strip can quickly detect the results of pesticide residues in tea leaves, but has poor anti-interference performance and the color development is easily affected by tea polyphenols, flavonoids, pigments or minerals in the tea leaves.

[0006] In summary, providing a method with strong anti-interference ability and capable of quickly and accurately detecting pesticide residues in tea is of great significance to the field of pesticide detection technology. Summary of the invention

[0007] In view of the deficiencies in the prior art and actual needs, the present invention provides a colloidal gold card for detecting pesticide residues and a preparation method and application thereof. The colloidal gold card can directly detect various tea matrices and is not affected or is less affected by matrix interference, thereby avoiding the situation where the detection line does not appear or the line is weak.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a colloidal gold card for detecting pesticide residues, the colloidal gold card comprising a bottom plate and a sample pad, a gold conjugation pad, a nitrocellulose membrane and a water-absorbing pad sequentially overlapped on the bottom plate, the nitrocellulose membrane being provided with a detection line coated with a pesticide antigen and a quality control line coated with a secondary antibody, the gold conjugation pad containing colloidal gold-labeled pesticide antibodies, sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol.

[0010] In the present invention, sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol are added to the gold conjugation pad, so as to avoid the influence of complex matrices such as tea polyphenols, flavonoids, pigments and minerals in tea on the colloidal gold-labeled pesticide antibodies and pesticide antigens. Sucrose, trehalose and polyethylene glycol can make the colloidal gold-labeled pesticide antibodies maintain a certain hydrophilicity, and avoid the possibility that the specific binding ability of the antibody is reduced, the non-specific binding probability is increased, and the detection result may be false positive due to the complete exposure of hydrophobic proteins inside the antibody and the change of conformation; disodium hydrogen phosphate dodecahydrate can maintain the salt ion concentration of the whole system, increase the solubility of protein, and promote the antigen-antibody specific reaction; bovine serum albumin and sodium caseinate as non-specific proteins in the conjugation pad can protect the stability of the colloidal gold-labeled pesticide antibodies.

[0011] In summary, the present invention utilizes the mutual coordination among sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol, which can avoid the influence of complex matrices such as tea polyphenols, flavonoids, pigments and minerals in tea on the corresponding antigens and antibodies in the colloidal gold card, thereby improving the anti-interference of the colloidal gold card, being able to directly detect various teas, ensuring the normal color development of the detection line, and improving the stability of the color development. Therefore, the pesticide residues in the tea can be accurately determined according to the color development result of the detection line, and the phenomenon of false positive results occurring after the components in the tea affect the normal color development is eliminated.

[0012] The colloidal gold card of the present invention can directly detect pesticide residues in a variety of teas, including Tieguanyin, rock tea, Shenhong, Jinjunmei, Baimudan, Puer, Maojian, Yinzhen and Huangya.

[0013] Preferably, the sample pad contains tris(hydroxymethyl)aminomethane, bovine serum albumin, fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone.

[0014] In the present invention, tris(hydroxymethyl)aminomethane, bovine serum albumin, fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone are added to the sample pad, wherein the tris(hydroxymethyl)aminomethane solution ensures the buffering capacity of the entire system, and buffers the influence of pH difference of the detection solution after different samples are extracted on the color development of the colloidal gold card; the bovine serum albumin can preferentially bind to the non-specific binding substances in the detection sample, thereby removing the influence of such interfering substances; the fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone can promote the crawling of the pesticide antibody labeled with colloidal gold, so that the entire detection process can be completed quickly and normally, and the normal color development of the detection line is ensured.

[0015] Preferably, the test line also contains sodium chloride, trehalose and polyoxyethylene lauryl ether.

[0016] In the present invention, sodium chloride, trehalose and polyoxyethylene lauryl ether are added to the detection line, which can improve the stability of the pesticide antigen and ensure the normal color development of the detection line.

[0017] Preferably, the pesticide antibody comprises any one of chlorpyrifos antibody, carbaryl antibody, triazophos antibody, triadimefon antibody, aldicarb antibody, fipronil antibody, paclobutrazol antibody, imidacloprid antibody, pyridabenzyl antibody or thiophanate-methyl antibody.

[0018] Preferably, the pesticide antibody is a mouse pesticide IgG antibody.

[0019] Preferably, the pesticide antigen comprises any one of chlorpyrifos antigen, carbaryl antigen, triazophos antigen, triadimefon antigen, aldicarb antigen, fipronil antigen, paclobutrazol antigen, imidacloprid antigen, pyridabenzyl antigen or thiophanate-methyl antigen.

[0020] Preferably, the method for preparing the pesticide antigen and the pesticide antibody (taking imidacloprid antigen and antibody as an example) comprises the following steps:

[0021] (1') Antigen preparation

[0022] 1) Synthetic Imidacloprid-coupled Antigen Derivatization: Weigh 1 g of imidacloprid standard into a 100 mL single-mouth bottle, add 15 mL of dimethyl sulfoxide and stir to dissolve, then add 0.45 g of KOH, weigh 630 mg of 3-mercaptopropionic acid (dissolved in 5 mL of dimethyl sulfoxide), drop into the reaction solution, react at 100 ° C for 24 h, monitor by TLC until the reaction is completed, add 30 mL of water to the reaction solution, shake well, and then use 6M The pH value was adjusted to 5 with HCl, and the solution was extracted with dichloromethane (30 mL each time, 3 times of extraction), monitored by TLC, the organic phases were combined, washed once with saturated sodium chloride, and then dried by adding an appropriate amount of anhydrous sodium sulfate, the organic phase was rotary evaporated to dryness, the product obtained by rotary drying was dissolved with a small amount of ethyl acetate, an appropriate amount of silica gel was added, and then loaded onto a column, first rinsed with dichloromethane:n-hexane=1:1 (V / V); then the column was flushed with 200 mL of dichloromethane, and finally the column was passed with dichloromethane:methanol=20:1 to obtain imidacloprid hapten;

[0023]

[0024] 2) Activation: Dissolve the imidacloprid hapten with 1000 μL DMF by stirring, add 12 mg NHS and 20 mg EDC, and react at room temperature overnight to obtain an activated product;

[0025] 3) Coupling: Weigh 30 mg BSA (the feed ratio can be adjusted) and dissolve it in 2 mL of boric acid solution with a pH of 9.0, slowly add 0.5 mL of the activated substance, stir at room temperature for 0.5 h, and react at 4°C overnight;

[0026] Weigh 20 mg OVA (the feed ratio can be adjusted) and dissolve it in 2 mL of PH = 9.0 boric acid solution, slowly add 0.5 mL of the activator dropwise, stir at room temperature for 0.5 h, and react at 4 ° C overnight;

[0027] 4) Purification: dialyze with 0.01M pH 7.4 PBS to obtain imidacloprid-coupled antigen I. After dialysis, measure the concentration and prepare for immunization;

[0028] (2') Antibody Preparation

[0029] 1) Immunizing animals: After the imidacloprid-coupled antigen I prepared in the above step is completely emulsified with an equal amount of Freund's complete adjuvant, mice are immunized;

[0030] 2) Cell fusion and hybridoma cell screening: Mouse spleen cells immunized with imidacloprid-coupled antigen I were fused with SP2 / 0 myeloma cells and cultured in HAT medium. The culture supernatant was tested to screen and obtain the cell line 14A4-B1-C12 that stably secreted anti-imidacloprid monoclonal antibodies;

[0031] 3) Preparation of ascites: After culturing the cell line 14A4-B1-C12 secreting anti-imidacloprid monoclonal antibodies, the cell line was injected into the peritoneal cavity of BALB / C mice, and the ascites was collected;

[0032] 4) Purification and preparation of anti-imidacloprid monoclonal antibodies: The ascites was dialyzed and filtered using an ammonium sulfate precipitation method, and the supernatant after the dialyzed and filtered treatment was purified using an imidacloprid affinity chromatography column to purify and prepare anti-imidacloprid monoclonal antibodies.

[0033] Preferably, the secondary antibody comprises goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody.

[0034] Preferably, the base plate comprises a PVC base plate.

[0035] In a second aspect, the present invention provides a method for preparing the colloidal gold card for detecting pesticide residues according to the first aspect, the method comprising the following steps:

[0036] (1) streaking a nitrocellulose membrane using a streaking solution containing sodium chloride, trehalose, polyoxyethylene and dodecyl ether to prepare a test line and a quality control line, coating the test line with a pesticide antigen, and coating the quality control line with a secondary antibody to obtain a nitrocellulose membrane having a test line and a quality control line;

[0037] The glass fiber is soaked in a gold conjugation pad treatment solution containing sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol, and then dried, and a colloidal gold-labeled pesticide antibody is sprayed on the glass fiber to obtain a gold conjugation pad;

[0038] Using a sample pad treatment solution containing tris(hydroxymethylaminomethane), bovine serum albumin, fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone to soak the glass fiber, and drying it to obtain a sample pad;

[0039] (2) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0040] Preferably, the stripping solution in step (1) contains 0.05-0.2M sodium chloride (for example, 0.06M, 0.07M, 0.08M, 0.1M, 0.12M, 0.16M or 0.18M), 1%-5% trehalose (for example, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 4.2%, 4.6% or 4.8%) and 0.02%-0.05% polyoxyethylene dodecyl ether (for example, 0.022%, 0.024%, 0.026%, 0.03%, 0.04%, 0.042%, 0.046% or 0.048%).

[0041] Preferably, the gold binding pad treatment solution in step (1) contains 2% to 8% sucrose (for example, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 4%, 5%, 6%, 7%, 7.2%, 7.4%, 7.6% or 7.8%), 0.05 to 0.2M disodium hydrogen phosphate dodecahydrate (for example, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.12M, 0.14M, 0.16M or 0.18M), 0.5% to 5% bovine serum albumin (for example, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 3. 5%, 4%, 4.2%, 4.6% or 4.8%), 0.5% to 5% sodium caseinate (for example, it can be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 3.5%, 4%, 4.2%, 4.6% or 4.8%), 1% to 3% trehalose (for example, it can be 1.1%, 1.2%, 1.4%, 1.6%, 1%, 2%, 2.2%, 2.4%, 2.6% or 2.8%) and 0.5% to 3% polyethylene glycol (for example, it can be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 2.2%, 2.4%, 2.6% or 2.8%).

[0042] Preferably, the sample pad treatment solution in step (1) contains 0.02-0.2M tris(hydroxymethyl)aminomethane (for example, 0.03M, 0.04M, 0.05M, 0.06M, 0.08M, 0.1M, 0.12M, 0.14M, 0.16M or 0.18M), 0.5%-5% bovine serum albumin (for example, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 2%, 3%, 4%, 4.2%, 4.4%, 4.6% or 4.8%), 0.1%-1.5 % fatty alcohol polyoxyethylene (for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 1%, 1.2%, 1.3% or 1.4%), 0.1% to 1.5% Tween (for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 1%, 1.2%, 1.3% or 1.4%) and 0.25% to 1.5% polyvinyl pyrrolidone (for example, it can be 0.26%, 0.3%, 0.4%, 0.5%, 0.6%, 1%, 1.2%, 1.3% or 1.4%).

[0043] Preferably, the overlap in step (2) is 2 to 3 mm.

[0044] Preferably, the method further comprises the step of preparing a colloidal gold solution.

[0045] Preferably, the colloidal gold solution is prepared by reducing chloroauric acid with trisodium citrate.

[0046] Preferably, the particle size of the colloidal gold in the colloidal gold solution is 35 to 45 nm, including but not limited to 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm or 44 nm.

[0047] Preferably, the method further comprises the step of preparing colloidal gold-labeled pesticide antibodies.

[0048] Preferably, the colloidal gold-labeled pesticide antibody is prepared by using a K2CO3 solution to adjust the pH of the colloidal gold solution, and then mixing the colloidal gold solution with the pesticide antibody for labeling to obtain the colloidal gold-labeled pesticide antibody.

[0049] Preferably, the concentration of the K2CO3 solution is 0.2-0.4M, including but not limited to 0.22M, 0.24M, 0.25M, 0.26M, 0.3M, 0.32M, 0.35M, 0.36M or 0.38M.

[0050] Preferably, the ratio of the colloidal gold solution and the pesticide antibody is 1 mL: (5-8) μg, including but not limited to 1 mL: 5 μg, 1 mL: 5.2 μg, 1 mL: 5.4 μg, 1 mL: 5.6 μg, 1 mL: 5.8 μg, 1 mL: 6 μg, 1 mL: 7 μg, 1 mL: 7.2 μg, 1 mL: 7.4 μg, 1 mL: 7.6 μg or 1 mL: 7.8 μg.

[0051] As a preferred technical solution, the method for preparing the colloidal gold card for detecting pesticide residues comprises the following steps:

[0052] (1) using trisodium citrate to reduce chloroauric acid to prepare a colloidal gold solution so that the particle size of the colloidal gold is 35 to 45 nm;

[0053] (2) adjusting the pH of the colloidal gold solution using a 0.2-0.4 M K2CO3 solution, and then mixing the colloidal gold solution with the pesticide antibody at a ratio of 1 mL: (5-8) μg for labeling to obtain a colloidal gold-labeled pesticide antibody;

[0054] (3) using a streaking solution containing 0.05-0.2M sodium chloride, 1%-5% trehalose and 0.02%-0.05% polyoxyethylene lauryl ether to streak a nitrocellulose membrane, with a streaking volume of 0.75-1.0 μL / cm, preparing a test line and a quality control line, coating the test line with a pesticide antigen, coating the quality control line with a secondary antibody, and drying at 35-40°C for 10-16 hours to obtain a nitrocellulose membrane having a test line and a quality control line;

[0055] (4) soaking the glass fiber for 3 to 7 minutes in a gold conjugation pad treatment solution containing 2% to 8% sucrose, 0.05 to 0.2 M disodium hydrogen phosphate dodecahydrate, 0.5% to 5% bovine serum albumin, 0.5% to 5% sodium caseinate, 1% to 3% trehalose and 0.5% to 3% polyethylene glycol, drying at 35 to 40° C. for 10 to 16 hours, and spraying colloidal gold-labeled pesticide antibodies on the glass fiber to obtain a gold conjugation pad;

[0056] (5) soaking the glass fiber in a sample pad treatment solution containing 0.02-0.2M tris(hydroxymethyl)aminomethane, 0.5%-5% bovine serum albumin, 0.1%-1.5% fatty alcohol polyoxyethylene, 0.1%-1.5% Tween, and 0.25%-1.5% polyvinyl pyrrolidone for 3-7 minutes, and drying at 35-40°C for 10-16 hours to obtain a sample pad;

[0057] (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the absorbent pad are overlapped in sequence by 2 to 3 mm and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0058] In a third aspect, the present invention provides the use of the colloidal gold card for detecting pesticide residues described in the first aspect in detecting pesticide residues in tea.

[0059] The colloidal gold card of the present invention is based on the principle of immune competition, that is, when pesticides exist in the sample, the pesticides can be combined with the pesticide antibodies on the gold binding pad, resulting in the inability of the pesticide antibodies to be combined with the pesticide antigens on the detection line and the inability to develop color. When there is no pesticide residue in the sample or the pesticide residue is lower than the detection limit, the pesticide antibodies on the gold binding pad can be combined with the pesticide antigens on the detection line and develop color, while the binding of the secondary antibody on the quality control line with the antigen is not affected by the pesticide. Therefore, if the quality control line develops color, it indicates that the colloidal gold card is effective, and if it does not develop color, it is invalid. If the detection line is darker than the quality control line or is the same depth, it indicates that the pesticide in the sample is lower than its detection limit and is negative. If the detection line develops color weaker than the quality control line or does not develop color, it indicates that the pesticide residue in the sample is higher than the card detection limit and is positive.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) In the colloidal gold card of the present invention, the gold binding pad contains sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol, which can eliminate the influence of tea polyphenols, flavonoids, pigments and minerals in tea on color development, improve the anti-interference ability of the colloidal gold card, and improve the color development stability;

[0062] (2) The colloidal gold card of the present invention can directly detect pesticide residues in a variety of teas, including Tieguanyin, rock tea, Shenhong, Jinjunmei, Baimudan, Puer, Maojian, Yinzhen and Huangya. The results can be obtained within 10 minutes. The pesticide residues in the tea can be accurately determined according to the color development results of the test line, and the phenomenon of false positive results caused by the components in the tea affecting the normal color development is eliminated. The results are accurate and reliable;

[0063] (3) The colloidal gold detection card of the present invention has high sensitivity, and the detection limit can meet or exceed the limit requirements of national standard 2763-2019. It also has multiple protective antigen and antibody mechanisms, which can greatly improve the binding efficiency of antigen and antibody, thereby saving raw materials and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the colloidal gold card structure in Example 1, wherein 1 is a sample pad, 2 is a gold binding pad, 3 is a nitrocellulose membrane, 4 is a water absorbent pad, and 5 is a base plate;

[0065] Figure 2 Schematic diagram of the top view of the colloidal gold card in Example 1, wherein 1 is the sample pad, 2 is the gold binding pad, 3 is the nitrocellulose membrane, 4 is the water absorption pad, 6 is the detection line, and 7 is the quality control line;

[0066] Figure 3 The results of the colloidal gold card prepared in Example 1 for detecting pesticide residues in tea;

[0067] Figure 4 The result of the colloidal gold card detection of pesticide residues in tea in Example 2;

[0068] Figure 5 The results of the colloidal gold card detection of pesticide residues in tea in Example 3;

[0069] Figure 6 The results of the colloidal gold card detection of pesticide residues in tea in Example 4;

[0070] Figure 7 The results of the colloidal gold card detection of pesticide residues in tea in Comparative Example 1;

[0071] Figure 8 The results of the colloidal gold card detection of pesticide residues in tea in Comparative Example 2;

[0072] Fig. 9 The results of the colloidal gold card detection of pesticide residues in tea in Comparative Example 3;

[0073] Fig.10 The results of the colloidal gold card detection of pesticide residues in tea in Comparative Example 4;

[0074] Fig.11This is the result of using colloidal gold card to detect pesticide residues in tea in Comparative Example 5. DETAILED DESCRIPTION

[0075] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0076] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0077] Example 1

[0078] This embodiment provides a colloidal gold card for detecting pesticides. The structural schematic diagram of the colloidal gold card is as follows Figure 1 and Figure 2 shown.

[0079] The preparation method of the colloidal gold card comprises the following steps:

[0080] (1) using trisodium citrate to reduce chloroauric acid to prepare colloidal gold solution;

[0081] (2) adjusting the pH of the colloidal gold solution using a 0.2 M K2CO3 solution, and then mixing the colloidal gold solution with the imidacloprid antibody at a ratio of 1 mL:6 μg for labeling to obtain a colloidal gold-labeled imidacloprid antibody;

[0082] (3) Using a stripping solution containing 0.01 M pH 7.4 phosphate buffer, 3% trehalose and 0.035% polyoxyethylene lauryl ether (Brij58) (the balance is water) to strip a nitrocellulose membrane, the stripping volume is 0.9 μL / cm, and a test line and a quality control line are prepared. The test line is coated with imidacloprid antigen, and the quality control line is coated with goat anti-mouse IgG. The membrane is dried at 38° C. for 13 h to obtain a nitrocellulose membrane with a test line and a quality control line.

[0083] (4) soaking the glass fiber for 5 min in a gold conjugate pad treatment solution containing 5% sucrose, 0.12 M sodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate, 2% trehalose and 2% polyethylene glycol (PEG4000) (the balance is water), drying at 38° C. for 13 h, and spraying colloidal gold-labeled imidacloprid antibody on the glass fiber to obtain a gold conjugate pad;

[0084] (5) soaking the glass fiber in a sample pad treatment solution containing 0.1 M tris(hydroxymethyl)aminomethane, 3% bovine serum albumin, 0.8% fatty alcohol polyoxyethylene, 0.8% Tween 20, and 0.9% polyvinyl pyrrolidone (PVP-K30) (the balance is water) for 5 min, and drying at 38° C. for 13 h to obtain a sample pad;

[0085] (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0086] Example 2

[0087] This embodiment provides a colloidal gold card for detecting pesticides. The preparation method of the colloidal gold card comprises the following steps:

[0088] (1) using trisodium citrate to reduce chloroauric acid to prepare colloidal gold solution;

[0089] (2) adjusting the pH of the colloidal gold solution using a 0.2 M K2CO3 solution, and then mixing the colloidal gold solution with the imidacloprid antibody at a ratio of 1 mL:5 μg for labeling to obtain a colloidal gold-labeled imidacloprid antibody;

[0090] (3) using 0.01 M phosphate buffer with a pH of 7.4 to streak a nitrocellulose membrane at a streaking volume of 0.75 μL / cm to prepare a test line and a quality control line, coating the test line with imidacloprid antigen, coating the quality control line with goat anti-mouse IgG, and drying at 38° C. for 13 h to obtain a nitrocellulose membrane with a test line and a quality control line;

[0091] (4) soaking the glass fiber for 5 min in a gold conjugate pad treatment solution containing 5% sucrose, 0.12 M sodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate, 2% trehalose and 2% polyethylene glycol (PEG4000) (the balance is water), drying at 38° C. for 13 h, and spraying colloidal gold-labeled imidacloprid antibody on the glass fiber to obtain a gold conjugate pad;

[0092] (5) soaking the glass fiber in a sample pad treatment solution containing 0.1 M tris(hydroxymethyl)aminomethane, 3% bovine serum albumin, 0.8% fatty alcohol polyoxyethylene, 0.8% Tween 20, and 0.9% polyvinyl pyrrolidone (PVP-K30) (the balance is water) for 5 min, and drying at 38° C. for 13 h to obtain a sample pad;

[0093] (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0094] Example 3

[0095] This embodiment provides a colloidal gold card for detecting pesticides. The preparation method of the colloidal gold card comprises the following steps:

[0096] (1) using trisodium citrate to reduce chloroauric acid to prepare colloidal gold solution;

[0097] (2) adjusting the pH of the colloidal gold solution using a 0.2 M K2CO3 solution, and then mixing the colloidal gold solution with the imidacloprid antibody at a ratio of 1 mL:5 μg for labeling to obtain a colloidal gold-labeled imidacloprid antibody;

[0098] (3) Using a stripping solution containing 0.01 M pH 7.4 phosphate buffer, 3% trehalose and 0.035% polyoxyethylene lauryl ether (Brij58) (the balance is water) to strip a nitrocellulose membrane, the stripping volume is 0.9 μL / cm, and a test line and a quality control line are prepared. The test line is coated with imidacloprid antigen, and the quality control line is coated with goat anti-mouse IgG. The membrane is dried at 38° C. for 13 h to obtain a nitrocellulose membrane with a test line and a quality control line.

[0099] (4) soaking the glass fiber for 5 min in a gold conjugate pad treatment solution containing 5% sucrose, 0.12 M sodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate, 2% trehalose and 2% polyethylene glycol (PEG4000) (the balance is water), drying at 38° C. for 13 h, and spraying colloidal gold-labeled imidacloprid antibody on the glass fiber to obtain a gold conjugate pad;

[0100] (5) soaking the glass fiber in a treatment solution containing 0.1 M phosphate buffer solution with a pH of 7.4, 1% bovine serum albumin, and 0.5% Tween 20 (the balance is water) for 5 min, and drying at 38° C. for 13 h to obtain a sample pad;

[0101] (6) Overlapping the sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad in sequence, and gluing them on a bottom plate, to obtain the colloidal gold card for detecting pesticide residues. (6) Overlapping the sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad in sequence, and gluing them on a bottom plate, to obtain the colloidal gold card for detecting pesticide residues.

[0102] Example 4

[0103] This embodiment provides a colloidal gold card for detecting pesticides. The preparation method of the colloidal gold card comprises the following steps:

[0104] (1) using trisodium citrate to reduce chloroauric acid to prepare colloidal gold solution;

[0105] (2) adjusting the pH of the colloidal gold solution using a 0.2 M K2CO3 solution, and then mixing the colloidal gold solution with the imidacloprid antibody at a ratio of 1 mL:5 μg for labeling to obtain a colloidal gold-labeled imidacloprid antibody;

[0106] (3) using a 0.01 M phosphate buffer solution at pH 7.4 to streak a nitrocellulose membrane at a streaking volume of 0.75 μL / cm to prepare a test line and a quality control line, coating the test line with imidacloprid antigen, coating the quality control line with goat anti-mouse IgG, and drying at 38° C. for 13 h to obtain a nitrocellulose membrane having a test line and a quality control line;

[0107] (4) soaking the glass fiber for 5 min in a gold conjugate pad treatment solution containing 5% sucrose, 0.12 M sodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate, 2% trehalose and 2% polyethylene glycol (PEG4000) (the balance is water), drying at 38° C. for 13 h, and spraying colloidal gold-labeled imidacloprid antibody on the glass fiber to obtain a gold conjugate pad;

[0108] (5) soaking the glass fiber in a treatment solution containing 0.1 M phosphate buffer solution with a pH of 7.4, 1% bovine serum albumin, and 0.5% Tween 20 (the balance is water) for 5 min, and drying at 38° C. for 13 h to obtain a sample pad;

[0109] (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0110] Comparative Example 1

[0111] A colloidal gold card prepared by a conventional process, the conventional process comprising the following steps:

[0112] (1) using trisodium citrate to reduce chloroauric acid to prepare colloidal gold solution;

[0113] (2) adjusting the pH of the colloidal gold solution using a 0.2 M K2CO3 solution, and then mixing the colloidal gold solution with the imidacloprid antibody at a ratio of 1 mL:5 μg for labeling to obtain a colloidal gold-labeled imidacloprid antibody;

[0114] (3) using 0.01 M phosphate buffer with a pH of 7.4 to streak a nitrocellulose membrane at a streaking volume of 0.75 μL / cm to prepare a test line and a quality control line, coating the test line with imidacloprid antigen, coating the quality control line with goat anti-mouse IgG, and drying at 38° C. for 13 h to obtain a nitrocellulose membrane with a test line and a quality control line;

[0115] (4) soaking the glass fiber in a 6% sucrose aqueous solution for 5 min, drying at 38° C. for 13 h, and spraying the imidacloprid antibody labeled with colloidal gold on the glass fiber to obtain a gold binding pad;

[0116] (5) soaking the glass fiber in a treatment solution containing 0.1 M phosphate buffer solution with a pH of 7.4, 1% bovine serum albumin, and 0.5% Tween 20 (the balance is water) for 5 min, and drying at 38° C. for 13 h to obtain a sample pad;

[0117] (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

[0118] Comparative Example 2

[0119] Compared with Example 1, the only difference is that the gold binding pad treatment solution in step (4) contains 0.12M disodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate, 2% trehalose and 2% polyethylene glycol (PEG4000), and the balance is water. The rest is the same as Example 1.

[0120] Comparative Example 3

[0121] Compared with Example 1, the only difference is that the gold binding pad treatment solution in step (4) contains 5% sucrose, 0.12M disodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 2% trehalose and 2% polyethylene glycol (PEG4000), and the balance is water. The rest is the same as Example 1.

[0122] Comparative Example 4

[0123] Compared with Example 1, the only difference is that the gold binding pad treatment solution in step (4) contains 5% sucrose, 0.12M disodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate and 2% polyethylene glycol (PEG4000), and the balance is water. The rest is the same as Example 1.

[0124] Comparative Example 5

[0125] Compared with Example 1, the only difference is that the gold binding pad treatment solution in step (4) contains 5% sucrose, 0.12M disodium hydrogen phosphate dodecahydrate, 3% bovine serum albumin, 3% sodium caseinate and 2% trehalose, and the balance is water. The rest is the same as Example 1.

[0126] Test Example 1

[0127] The colloidal gold cards prepared in Examples 1-4 and the colloidal gold cards in Comparative Examples 1-5 were used to test Tieguanyin, rock tea, Shenhong, Jinjunmei, white peony, Puer, Maojian, Yinzhen and Huangya tea, respectively. The results are as follows: Figure 3-Figure 11shown.

[0128] Depend on Figure 3-Figure 6 It can be seen that the colloidal gold card test line and the quality control line prepared in Examples 1-4 are colored normally, indicating that the colloidal gold card prepared in Examples 1-4 has strong anti-interference ability and can effectively detect pesticide residues in tea; Figure 7-Figure 11 It can be seen that Comparative Example 1 is a colloidal gold card prepared by a conventional process, and in Comparative Examples 2-5, the components in the gold binding pad are not controlled according to the present invention. When the same tea is detected, the detection line is darker or almost no color is observed, indicating that the colloidal gold card has poor anti-interference ability and it is difficult to accurately detect pesticide residues in tea. This shows that the present invention controls the effective ingredients of the gold binding pad, which can improve the anti-interference ability of the colloidal gold card, effectively detect pesticide residues in tea, and accurately determine the pesticide residues in tea according to the color development results of the detection line, eliminating the phenomenon of false positive results caused by the components in the tea affecting the normal color development.

[0129] In addition, compared with the color development results of the colloidal gold card prepared in Example 1, the color development of the colloidal gold cards prepared in Examples 2-4 is lighter, and the color development of the colloidal gold card prepared in Example 4 is the lightest, indicating that the present invention simultaneously processes the sample pad, the gold binding pad and the detection line so that they contain corresponding effective ingredients, which can further improve the anti-interference ability of the colloidal gold card.

[0130] In summary, the present invention processes the sample pad, the gold binding pad and the detection line so that they contain corresponding effective ingredients, thereby eliminating the influence of different tea matrices on the color development of the colloidal gold card, improving the anti-interference property of the colloidal gold card, and being able to accurately detect pesticide residues in tea.

[0131] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A colloidal gold card for detecting pesticide residues, characterized in that: The colloidal gold card comprises a bottom plate and a sample pad, a gold binding pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on the bottom plate; The nitrocellulose membrane is provided with a detection line coated with the pesticide antigen and a quality control line coated with the secondary antibody; The gold conjugate pad contains colloidal gold-labeled pesticide antibody, sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol; The sample pad contains tris(hydroxymethyl)aminomethane, bovine serum albumin, fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone; The test line also contains sodium chloride, trehalose and polyoxyethylene lauryl ether.

2. The colloidal gold card according to claim 1, characterized in that: The pesticide antibody includes any one of chlorpyrifos antibody, carbaryl antibody, triazophos antibody, triadimefon antibody, aldicarb antibody, fipronil antibody, paclobutrazol antibody, imidacloprid antibody, pyridabenzyl antibody or thiophanate-methyl antibody.

3. The colloidal gold card according to claim 1, characterized in that: The pesticide antibody is a mouse-derived pesticide IgG antibody.

4. The colloidal gold card according to claim 1, characterized in that: The pesticide antigen includes any one of chlorpyrifos antigen, carbaryl antigen, triazophos antigen, triadimefon antigen, aldicarb antigen, fipronil antigen, paclobutrazol antigen, imidacloprid antigen, pyridabenzyl antigen or thiophanate-methyl antigen.

5. The colloidal gold card according to claim 1, characterized in that: The secondary antibody includes goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody.

6. A method for preparing a colloidal gold card for detecting pesticide residues according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) streaking a nitrocellulose membrane using a streaking solution containing sodium chloride, trehalose, polyoxyethylene and dodecyl ether to prepare a test line and a quality control line, coating the test line with a pesticide antigen, and coating the quality control line with a secondary antibody to obtain a nitrocellulose membrane having a test line and a quality control line; The glass fiber is soaked in a gold conjugation pad treatment solution containing sucrose, disodium hydrogen phosphate dodecahydrate, bovine serum albumin, sodium caseinate, trehalose and polyethylene glycol, and then dried, and a colloidal gold-labeled pesticide antibody is sprayed on the glass fiber to obtain a gold conjugation pad; Using a sample pad treatment solution containing tris(hydroxymethylaminomethane), bovine serum albumin, fatty alcohol polyoxyethylene, Tween and polyvinyl pyrrolidone to soak the glass fiber, and drying it to obtain a sample pad; (2) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the water-absorbing pad are overlapped in sequence and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

7. The method according to claim 6, characterized in that The stripping solution in step (1) contains 0.05-0.2M sodium chloride, 1%-5% trehalose and 0.02%-0.05% polyoxyethylene lauryl ether; The gold binding pad treatment solution in step (1) contains 2% to 8% sucrose, 0.05 to 0.2M disodium hydrogen phosphate dodecahydrate, 0.5% to 5% bovine serum albumin, 0.5% to 5% sodium caseinate, 1% to 3% trehalose and 0.5% to 3% polyethylene glycol; The sample pad treatment solution in step (1) contains 0.02-0.2M tris(hydroxymethyl)aminomethane, 0.5%-5% bovine serum albumin, 0.1%-1.5% fatty alcohol polyoxyethylene, 0.1%-1.5% Tween and 0.25%-1.5% polyvinyl pyrrolidone.

8. The method according to claim 7, characterized in that The overlap in step (2) is 2 to 3 mm.

9. The method according to any one of claims 6 to 8, characterized in that: The method further comprises the steps of preparing a colloidal gold solution; The colloidal gold solution is prepared by reducing chloroauric acid with trisodium citrate; The particle size of the colloidal gold in the colloidal gold solution is 35 to 45 nm.

10. The method according to any one of claims 6 to 8, characterized in that: The method further comprises the steps of preparing colloidal gold-labeled pesticide antibodies; The preparation method of the colloidal gold-labeled pesticide antibody is to use a K2CO3 solution to adjust the pH of the colloidal gold solution, and then mix the colloidal gold solution with the pesticide antibody for labeling to obtain the colloidal gold-labeled pesticide antibody; The concentration of the K2CO3 solution is 0.2-0.4M; The ratio of the colloidal gold solution to the pesticide antibody is 1 mL: (5-8) μg.

11. The method according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: (1) using trisodium citrate to reduce chloroauric acid to prepare a colloidal gold solution so that the particle size of the colloidal gold is 35 to 45 nm; (2) adjusting the pH of the colloidal gold solution using a 0.2-0.4 M K2CO3 solution, and then mixing the colloidal gold solution with the pesticide antibody at a ratio of 1 mL: (5-8) μg for labeling to obtain a colloidal gold-labeled pesticide antibody; (3) using a streaking solution containing 0.05-0.2M sodium chloride, 1%-5% trehalose and 0.02%-0.05% polyoxyethylene lauryl ether to streak a nitrocellulose membrane, with a streaking volume of 0.75-1.0 μL / cm, preparing a test line and a quality control line, coating the test line with a pesticide antigen, coating the quality control line with a secondary antibody, and drying at 35-40°C for 10-16 hours to obtain a nitrocellulose membrane having a test line and a quality control line; (4) soaking the glass fiber for 3 to 7 minutes in a gold conjugation pad treatment solution containing 2% to 8% sucrose, 0.05 to 0.2 M disodium hydrogen phosphate dodecahydrate, 0.5% to 5% bovine serum albumin, 0.5% to 5% sodium caseinate, 1% to 3% trehalose and 0.5% to 3% polyethylene glycol, drying at 35 to 40° C. for 10 to 16 hours, and spraying colloidal gold-labeled pesticide antibodies on the glass fiber to obtain a gold conjugation pad; (5) soaking the glass fiber in a sample pad treatment solution containing 0.02-0.2M tris(hydroxymethyl)aminomethane, 0.5%-5% bovine serum albumin, 0.1%-1.5% fatty alcohol polyoxyethylene, 0.1%-1.5% Tween, and 0.25%-1.5% polyvinyl pyrrolidone for 3-7 minutes, and drying at 35-40°C for 10-16 hours to obtain a sample pad; (6) The sample pad, the gold binding pad, the nitrocellulose membrane with the detection line and the quality control line, and the absorbent pad are overlapped in sequence by 2 to 3 mm and bonded to the bottom plate to obtain the colloidal gold card for detecting pesticide residues.

12. Use of the colloidal gold card for detecting pesticide residues as described in any one of claims 1 to 5 in detecting pesticide residues in tea.

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