Method for detecting acetamiprid based on covalent directed antibody

Abstract

The invention discloses an acetamiprid detection method based on a covalent directed antibody, and relates to the technical field of food detection. Carboxyl in an Fc region of the antibody is activated to form an amido bond with amino of the elisa plate, so that the antibody orientation is realized. Then, an acetamiprid standard solution or a sample to be detected and an Au-Pt peroxidase-labeled antigen are added, the acetamiprid standard solution or the sample to be detected and the Au-Pt peroxidase-labeled antigen are competitively combined with an antibody on the elisa plate, a standard curve of the inhibition rate and the acetamiprid concentration is established according to the change of signal intensity, and then high-sensitivity monitoring of acetamiprid in agricultural products is achieved. Compared with a traditional physical adsorption mode, the method has the advantages that the Fc region of the antibody is covalently oriented to the solid-phase carrier, so that the Fab region of the antibody is fully exposed, the steric hindrance of the antibody is reduced, and the sensitivity, stability and specificity of the enzyme-linked immunosorbent assay are effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a detection method of acetamiprid based on covalently directed antibodies. Background Art

[0002] As a broad-spectrum neonicotinoid insecticide, acetamiprid has the characteristics of broad insecticide spectrum, rapid effect and low dosage, and is widely used to control pests such as aphids, thrips and whiteflies. However, the misuse and abuse of acetamiprid often leads to excessive pesticide residues in some agricultural products, drinking water in the food chain, etc. Therefore, rapid detection of acetamiprid residue levels in agricultural products is of great significance to ensure food safety.

[0003] At present, the reported detection methods of acetamiprid mainly include large-scale instrument detection methods such as high-performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, etc. These detection methods have complex pre-treatment procedures, and also have limitations such as dependence on large and expensive instruments and the need for professional technicians, which cannot meet the requirements of rapid on-site detection of acetamiprid.

[0004] Enzyme-linked immunosorbent assay, aptamer method, colloidal gold immunochromatography test strips, etc. have the advantages of being fast and simple, and are widely used in the field of food safety testing. Among them, the enzyme-linked immunosorbent assay is mainly based on the specific binding of antigens and antibodies, and has the advantages of high specificity and speed, which has attracted great attention. However, the traditional enzyme-linked immunosorbent assay mainly uses the simplest physical adsorption method (polar, ionic or hydrophobic interaction) to non-covalently adsorb antibodies to the ELISA plate. This physical adsorption method often causes the antibody to be randomly labeled on the ELISA plate, and the antigen binding site (Fab region) on the antibody may be partially or completely shielded, resulting in reduced signal strength and sensitivity. In addition, the traditional enzyme-linked immunosorbent assay relies on natural enzymes, which have the disadvantages of high preparation cost and poor environmental tolerance.

[0005] Therefore, there is an urgent need to improve the sensitivity and stability of ELISA while improving its environmental tolerance.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a detection method for acetamiprid based on covalently directed antibodies, aiming to improve the sensitivity, stability and specificity of enzyme-linked immunosorbent assay.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a method for detecting acetamiprid based on covalent directed antibodies, comprising:

[0010] Activate the carboxyl group of the Fc region of the antibody and react with the amino-modified ELISA plate to form an amide bond, thereby obtaining an ELISA plate bound with the antibody;

[0011] Add a buffer solution into the wells on the ELISA plate bound with the antibody as a blank control well, add a standard solution of acetamiprid or a sample to be tested into the wells on the ELISA plate bound with the antibody as an inhibition well, and mix and incubate with the antigen labeled with Au@Pt peroxidase mimetic enzyme;

[0012] Add substrate and incubate;

[0013] A standard curve of acetamiprid concentration and inhibition rate was established according to the absorbance, and the content of acetamiprid in the sample to be tested was obtained according to the standard curve.

[0014] In an optional embodiment, the preparation process of the ELISA plate bound with the antibody includes: firstly diluting the antibody with a succinimide solution, then mixing and reacting with the amino-modified ELISA plate and a carbodiimide solution, and then washing to remove the unbound antibody;

[0015] Preferably, in the process of preparing the ELISA plate bound with the antibody, the reaction temperature is controlled to be 25° C.-27° C., and the reaction time is 1 h-3 h.

[0016] In an optional embodiment, the amount of succinimide solution and carbodiimide solution is adjusted so that the molar ratio of succinimide to carbodiimide is 1:(4.0-4.5), and the mass ratio of carbodiimide to antibody is 1:(3-4);

[0017] Preferably, the concentration of the succinimide solution is 0.2 mg / mL-0.3 mg / mL, the concentration of the carbodiimide solution is 1.0 mg / mL-2.0 mg / mL, and the volume ratio of the succinimide solution to the carbodiimide solution is 1:(0.5-1.5);

[0018] Preferably, the succinimide is N-hydroxysuccinimide.

[0019] In an optional embodiment, during the incubation with the Au@Pt peroxidase-labeled antigen, the incubation temperature is controlled to be 35° C.-40° C., and the incubation time is 1 h-3 h.

[0020] In an optional embodiment, the preparation process of the Au@Pt peroxidase-mimicking antigen includes:

[0021] Provide Au@Pt peroxidase mimics;

[0022] After the pH value of Au@Pt peroxidase mimetic was adjusted to 8-9, it was mixed with acetamiprid antigen for reaction, and then mixed with bovine serum albumin to block Au@Pt peroxidase. After solid-liquid separation, the obtained solid material was washed and redissolved with phosphate buffer solution;

[0023] Preferably, the reaction temperature of Au@Pt peroxidase mimetic enzyme and acetamiprid antigen is 2°C-6°C, and the reaction time is 10h-15h;

[0024] Preferably, the temperature for blocking with bovine serum albumin is 35° C.-40° C., and the reaction time is 1 h-5 h.

[0025] In an optional embodiment, the preparation process of Au@Pt peroxidase mimetic enzyme includes: heating the chloroauric acid solution to a boil and then mixing it with a sodium citrate solution, boiling it for 20-40 minutes to make the solution wine red, cooling it and separating the solid and liquid to obtain gold nanoparticles, heating the gold nanoparticles and the chloroplatinic acid solution to 70°C-90°C, and then mixing them with an ascorbic acid solution and heating them to keep boiling for 20-40 minutes;

[0026] Preferably, the molar ratio of sodium citrate to chloroauric acid is (6-7):1;

[0027] Preferably, the addition ratio of gold nanoparticles, chloroplatinic acid and ascorbic acid is 1.077×10 -16 g: 0.05mM-0.30mM: 1.15mM-1.35mM.

[0028] In an optional embodiment, the buffer solution is a methanol-phosphate buffer solution;

[0029] Preferably, the volume proportion of methanol in the methanol-phosphate buffer solution is 5%-10%.

[0030] In an optional embodiment, the substrate is selected from at least one of o-phenylenediamine, tetramethylbenzidine and 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt.

[0031] In an optional embodiment, the calculation formula of the inhibition rate I is as follows:

[0032] I(%)=(T 0 -T) / T 0 ×100%;

[0033] Among them, T 0 is the absorbance value corresponding to the control well, and T is the absorbance value corresponding to the acetamiprid standard solution or the sample to be tested;

[0034] Preferably, the detection wavelength of the absorbance value is 650-652 nm.

[0035] In an optional embodiment, the preparation process of the sample to be tested includes: homogenizing the agricultural product and mixing it with acetonitrile and vortexing it, then mixing it with anhydrous magnesium sulfate and sodium chloride and vortexing it, centrifuging and separating it, and taking the supernatant;

[0036] The supernatant is vortexed in a dispersed solid phase extractant, and then centrifuged and filtered; wherein the dispersed solid phase extractant includes ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black, and the mass ratio of ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black is 1: (0.5-1.5): (0.3-0.6);

[0037] Preferably, the agricultural product is selected from at least one of Chinese cabbage, cucumber, carrot, zucchini, apple, barley, pepper, persimmon, potato, rose, pomegranate, lettuce, sorghum, walnut, strawberry, corn, cotton, blueberry, broccoli, fig, citrus, cherry and celery.

[0038] The present invention has the following beneficial effects: by activating the carboxyl group of the Fc region of the antibody, an amide bond is formed with the amino group of the ELISA plate to achieve antibody orientation. Subsequently, a standard solution of acetamiprid or a sample to be tested and an antigen labeled with Au@Pt peroxidase are added, and the two competitively bind to the antibody on the ELISA plate, and a standard curve of inhibition rate and acetamiprid concentration is established according to the change in signal intensity, thereby achieving high-sensitivity monitoring of acetamiprid in agricultural products. Compared with the traditional physical adsorption method, the present invention covalently directs the Fc region of the antibody to the solid phase carrier, fully exposes the Fab region of the antibody, reduces the steric hindrance of the antibody, and effectively improves the sensitivity, stability and specificity of the enzyme-linked immunosorbent assay.

[0039] In addition, the present invention uses Au@Pt peroxidase mimics to replace natural enzymes, which effectively solves the problems of poor environmental tolerance of natural enzymes and complicated preparation steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 A schematic diagram of the chemically directed labeled antibody of the present invention;

[0042] Figure 2 A transmission electron microscope image of the Au@Pt nanozyme provided in an embodiment of the present invention;

[0043] Figure 3A standard curve of acetamiprid solvent for chemical covalent orientation of antibodies provided in an embodiment of the present invention;

[0044] Figure 4 A standard curve of the acetamiprid matrix with chemical covalent orientation of antibodies provided in an embodiment of the present invention;

[0045] Figure 5 A standard curve of acetamiprid solvent physically adsorbed by antibodies provided in an embodiment of the present invention;

[0046] Figure 6 A graph showing the antigen-antibody affinity determination of the antibody chemical covalent orientation provided in an embodiment of the present invention;

[0047] Figure 7 A graph showing the antigen-antibody affinity determination of antibody physical adsorption provided in an embodiment of the present invention;

[0048] Figure 8 This is a comparison chart of the effects of the activated antibodies provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] The embodiment of the present invention provides a method for detecting acetamiprid based on covalent directional antibodies, comprising the following steps:

[0051] S1. Antibody-directed binding

[0052] The carboxyl group of the Fc region of the activated antibody forms an amide bond with the exposed carboxyl group and an amino-modified ELISA plate (Thermo Fisher Scientific, Inc., Model No. 478042) to obtain an ELISA plate bound with the antibody, such as Figure 1 shown.

[0053] In some embodiments, the preparation process of the ELISA plate bound with the antibody includes: mixing and diluting the antibody with a succinimide solution (such as N-hydroxysuccinimide (NHS) solution), and then mixing and reacting with an amino-modified ELISA plate and a carbodiimide (EDC) solution, and then washing to remove the unbound antibody. The activation principle of EDC and NHS is: EDC reacts with the carboxyl group of the antibody Fc to generate an unstable amine-reactive O-acylisourea intermediate. In the presence of NHS, EDC can convert the carboxyl group into an amine-reactive sulfo-NHS ester, which reacts rapidly with the amino group on the ELISA plate to form an amide bond.

[0054] In some embodiments, during the preparation of the ELISA plate bound with the antibody, the reaction temperature is controlled to be 25°C-27°C, such as 25°C, 26°C, 27°C, etc.; the reaction time is 1h-3h, such as 1h, 2h, 3h, etc. Acetamiprid antibodies can be provided by Zhejiang University or purchased from Beijing Qinbang Biotechnology Co., Ltd.

[0055] In some embodiments, by adjusting the amount of succinimide solution and carbodiimide solution, the molar ratio of succinimide to carbodiimide (EDC) is 1: (4.0-4.5), such as 1: 4.0, 1: 4.2, 1: 4.5, etc.; the mass ratio of carbodiimide to antibody is 1: (3-4), such as 1: 3.0, 1: 3.5, 1: 4.0, etc. The amount of succinimide and carbodiimide (EDC) is preferably within the above range, which can better activate the carboxyl group of the Fc region of the antibody.

[0056] Furthermore, the concentration of the succinimide solution is 0.2 mg / mL-0.3 mg / mL, such as 0.2 mg / mL, 0.24 mg / mL, 0.26 mg / mL, 0.3 mg / mL, etc.; the concentration of the carbodiimide solution is 1.0 mg / mL-2.0 mg / mL, such as 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.8 mg / mL, 2.0 mg / mL, etc.; the volume ratio of the succinimide solution to the carbodiimide solution is 1:(0.5-1.5), such as 1:0.5, 1:1.0, 1:1.5, etc.

[0057] S2. Preparation of Au@Pt peroxidase-labeled antigen

[0058] Au@Pt peroxidase mimic was first prepared and then reacted with the antigen.

[0059] The specific preparation process of Au@Pt peroxidase mimetic enzyme is not limited. Gold nanoparticles can be prepared first, and then platinum reduced by platinum-containing reagents such as chloroplatinic acid is combined with the gold nanoparticles.

[0060] In some embodiments, the preparation process of Au@Pt peroxidase mimetic enzyme includes: heating the chloroauric acid solution to a boil and then mixing it with a sodium citrate solution, boiling for 20 min-40 min (such as 20 min, 30 min, 40 min, etc.) to make the solution wine red, gold nanoparticles (AuNPs) are formed, and cooling to room temperature to obtain gold nanoparticles by solid-liquid separation; heating the gold nanoparticles and chloroplatinic acid solution to 70°C-90°C (such as 70°C, 80°C, 90°C, etc.), and then mixing with ascorbic acid solution and heating to keep boiling for 20 min-40 min (such as 20 min, 30 min, 40 min, etc.), the solution is brown-red, indicating that Au@Pt peroxidase is synthesized, such as Figure 2 shown.

[0061] Specifically, the chloroauric acid solution can be heated and boiled for 5-10 minutes, and then the sodium citrate solution is added and boiled. The method of removing large-size nanoparticles is not limited, and 0.22 μm nitrocellulose membrane filtration can be used, but it is not limited thereto.

[0062] In some embodiments, the molar ratio of sodium citrate to chloroauric acid is (6-7):1, such as 6.0:1, 6.5:1, 7.0:1, etc. The addition ratio of gold nanoparticles, chloroplatinic acid, and ascorbic acid is 1.077×10 -16 g: 0.05mM-0.30mM: 1.15mM-1.35mM, that is, the mass of gold nanoparticles is 1.077×10 -16 When g, the molar amount of chloroplatinic acid can be 0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.25mM, 0.30mM, etc., and the molar amount of ascorbic acid can be 1.15mM, 1.20mM, 1.25mM, 1.30mM, 1.35mM, etc.

[0063] The preparation process of the Au@Pt peroxidase-labeled antigen includes: adjusting the pH value of the Au@Pt peroxidase to 8-9, mixing with the acetamiprid antigen, and then mixing with bovine serum albumin to block the Au@Pt peroxidase, and then after solid-liquid separation, washing the obtained solid material and redissolving it with a phosphate buffer solution. The method of solid-liquid separation is not limited, and can be a centrifugal method, but is not limited to this. Specifically, the acetamiprid antigen can be provided by Zhejiang University or purchased from Beijing Qinbang Biotechnology Co., Ltd.

[0064] Furthermore, the reaction temperature of Au@Pt peroxidase mimetic enzyme and acetamiprid antigen is 2°C-6°C, such as 2°C, 3°C, 4°C, 5°C, 6°C, etc.; the reaction time is 10h-15h, such as 10h, 11h, 12h, 13h, 14h, 15h, etc.

[0065] Furthermore, the temperature for blocking with bovine serum albumin is 35°C-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.; the reaction time is 1h-5h, such as 1h, 2h, 3h, 4h, 5h, etc.

[0066] S3. Draw standard curve for quantitative analysis

[0067] Add buffer solution to the wells on the ELISA plate bound with antibodies as blank control wells, add acetamiprid standard solution or the sample to be tested to the wells on the ELISA plate bound with antibodies as inhibition wells, and then mix and incubate with the antigen labeled with Au@Pt peroxidase mimic enzyme; then add substrate for incubation and color change. According to the absorbance test results, establish a standard curve of acetamiprid concentration and inhibition rate, and obtain the content of acetamiprid in the sample to be tested according to the standard curve.

[0068] In some embodiments, the buffer solution is a methanol-phosphate buffer solution obtained by mixing methanol and a phosphate buffer solution, wherein the volume proportion of methanol in the methanol-phosphate buffer solution is 5%-10%, such as 5%, 8%, 10%, etc.

[0069] In some embodiments, during incubation with the antigen labeled with Au@Pt peroxidase mimetic enzyme, the incubation temperature is controlled to be 35°C-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.; the incubation time is 1h-3h, such as 1h, 2h, 3h, etc.

[0070] In some embodiments, the substrate is selected from at least one of o-phenylenediamine, tetramethylbenzidine and 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, and the substrate may be any one or more of the above.

[0071] Furthermore, the calculation formula of the inhibition rate I is as follows:

[0072] I(%)=(T 0 -T) / T 0 ×100%;

[0073] Among them, T 0 is the absorbance value corresponding to the control well, and T is the absorbance value corresponding to the acetamiprid standard solution or the sample to be tested. The standard curve is drawn after testing with multiple concentrations of acetamiprid standard solution, and the content of acetamiprid in the sample to be tested is calculated based on the test results of the absorbance of the sample to be tested and the standard curve. The content of acetamiprid in the agricultural product can be further obtained by conversion.

[0074] When detecting the absorbance value, adjusting the detection wavelength to 650-652nm can more accurately detect the content of acetamiprid.

[0075] In some embodiments, the preparation process of the sample to be tested includes: mixing and vortexing the agricultural product with acetonitrile after homogenization, then mixing and vortexing with anhydrous magnesium sulfate and sodium chloride, then centrifuging and taking the supernatant; taking the supernatant and vortexing it in a dispersed solid phase extractant, then centrifuging and filtering. Acetamiprid in the agricultural product can be fully extracted by acetonitrile (to extract acetonitrile in the sample), anhydrous magnesium sulfate (to promote acetonitrile and water separation by absorbing water) and sodium chloride (to promote acetonitrile and water separation by salting out), thereby improving the accuracy of the detection.

[0076] Among them, the dispersed solid phase extractant includes ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black, and the mass ratio of ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black is 1: (0.5-1.5): (0.3-0.6), for example, ethylenediamine-N-propylsilane (50-60 mg), octadecylsilane (50-60 mg) and graphitized carbon black (20-30 mg).

[0077] Furthermore, the agricultural product is selected from at least one of cabbage, cucumber, carrot, zucchini, apple, barley, pepper, persimmon, potato, rose, pomegranate, lettuce, sorghum, walnut, strawberry, corn, cotton, blueberry, broccoli, fig, citrus, cherry and celery, and the agricultural product can be any one or more of the above.

[0078] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0079] Example 1

[0080] This example provides a method for activating acetamiprid antibody (provided by Zhejiang University), and the specific steps are as follows:

[0081] Take 25 μL of acetamiprid antibody and 25 μL of NHS (0.25 mg / mL) aqueous solution, mix and dilute, add to amino-modified ELISA plate (Thermo Fisher Scientific, Shi Er Technology Co., Ltd., model 478042), then add 50 μL of an equal volume of EDC aqueous solution (1.5 mg / mL), shake gently at room temperature (25°C, the same below) for 2±0.5h, wash off unbound antibodies (such as Figure 1 ).

[0082] Example 2

[0083] This example provides a method for preparing Au@Pt peroxidase labeled acetamiprid antigen (provided by Zhejiang University) (Au@Pt-Ag), and the specific steps are as follows:

[0084] (1) Preparation of Au@Pt peroxidase

[0085] Take 99mL ultrapure water and 1mL chloroauric acid solution (1%) (provided by Sigma) and mix them into a three-necked flask. Heat and boil in a water bath and keep it for 6 minutes. Then add 6mL of 1% sodium citrate solution (provided by Sigma) and keep boiling for 30 minutes. The solution turns wine red and AuNPs are formed. After cooling to room temperature, filter it with a 0.22μm nitrocellulose membrane. Subsequently, 10mL of 0.1% chloroplatinic acid (provided by Sigma) (5mM) and 30mL of AuNPs are heated to 80°C, and 10mL of ascorbic acid (5mM) (provided by Sigma) is added and kept boiling for 30 minutes. The solution turns brownish red, indicating that Au@Pt peroxidase is synthesized (such as Figure 2 ).

[0086] (2) Preparation of Au@Pt-Ag

[0087] Take 1 mL of Au@Pt peroxidase solution and use K 2 CO 3 (0.2M) solution was adjusted to pH 8. Subsequently, 80 μL of 100 μg / mL acetamiprid antigen was added, and after reacting at 4°C for 12 h, 108 μL of bovine serum albumin aqueous solution (mass fraction 2%) was added to block Au@Pt, and incubated at 37°C for 3 h. Finally, after centrifugation, 900 μL of 0.01M phosphate buffer solution was used for re-dissolution, and stored at 4°C for later use.

[0088] Example 3

[0089] In this example, an antibody-directed acetamiprid standard curve was established, and the specific steps were as follows:

[0090] 1. Preparation of Acetamiprid Standard

[0091] A series of pesticide gradient acetamiprid standards (concentration gradients include: 0, 1, 2.5, 5, 10, 25, 50, 100, 200, 300 μg / L) were prepared using 10% methanol solution for subsequent competitive reactions.

[0092] 2. Establish a standard curve

[0093] (1) Activation: Take 25 μL of acetamiprid antibody and 25 μL of NHS (0.25 mg / mL) aqueous solution, mix and dilute, add to amino-modified ELISA plate (Thermo Fisher Scientific, Shi Er Technology Co., Ltd., model number 478042), then add 50 μL of EDC aqueous solution (1.5 mg / mL), gently shake at room temperature (25°C, the same below) for 2 h to wash away unbound antibodies.

[0094] (2) Blocking: After washing with PBST, the cells were blocked with an 8% by mass casein aqueous solution for 1 h.

[0095] (3) Antigen incubation: 50 μL of acetamiprid standard solution (Beijing Manhag Biotechnology Co., Ltd., BePure20021XM) or the sample to be tested and 50 μL of Au@Pt peroxidase-labeled antigen were added to the antibody-labeled ELISA plate and incubated at 25°C for 2 h.

[0096] Color development: Add 100 μL substrate to each well and incubate at room temperature for 15 min.

[0097] Reading: Use a microplate reader to read the absorbance at 650 nm.

[0098] Establish a standard curve with acetamiprid concentration as X-axis and inhibition rate as Y-axis (e.g. Figure 3 ), its standard curve is: Y = 0.1992X + 0.3069, and the correlation coefficient is: R 2 =0.9854, the detection limit is 0.09μg / L, the linear range is: 0.29-266.49μg / L, and the sensitivity is 9.33μg / L.

[0099] Example 4

[0100] This example provides a method for detecting acetamiprid in vegetable samples, and establishes a matrix standard curve. The specific steps are as follows:

[0101] 1. Sample Pretreatment

[0102] Select Chinese cabbage, zucchini and cucumber as test samples, wash and homogenize. Weigh 5g sample homogenate in a 50mL centrifuge tube, add 10mL acetonitrile, vortex for 1min, add 4g anhydrous magnesium sulfate and 1g sodium chloride, vortex again for 1min, and centrifuge at 6000r / min for 5min. Then, take 9mL supernatant in 90mg dispersed solid phase extractant (52mg ethylenediamine-N-propylsilane, 52mg octadecylsilane, 26mg graphitized carbon black), vortex for 30s, centrifuge at 6000r / min for 2min, and finally filter through a 0.22μm membrane for use.

[0103] 2. Establishment of matrix standard curve

[0104] (1) Activation: The acetamiprid antibody was mixed and diluted with a 0.25 mg / mL NHS aqueous solution to obtain a 10 μg / mL antibody solution. 50 μL of the antibody solution was added to an amino-modified ELISA plate (Thermo Fisher Scientific, Model 478042), followed by an equal volume of a 1.5 mg / mL EDC aqueous solution (equal volume to the NHS aqueous solution). The plate was gently shaken at room temperature (25°C, the same below) for 2 h to wash away unbound antibodies.

[0105] (2) Blocking: After washing with PBST, the cells were blocked with 8% by mass casein solution (300 μL) for 1 h.

[0106] (3) Incubation of antigen: 50 μL of the sample to be tested and 50 μL of the antigen labeled with Au@Pt peroxidase mimetic enzyme were added to the antibody-labeled ELISA plate and incubated at 25 °C for 2 h;

[0107] Color development: add 100 μL substrate to each well and incubate at room temperature for 15 min;

[0108] Reading: Use a microplate reader to read the absorbance at 650nm;

[0109] Establish a standard curve with acetamiprid concentration as X-axis and inhibition rate as Y-axis (e.g. Figure 4 ), the standard curve of Chinese cabbage is: Y = 0.3537X-0.14006, and the correlation coefficient is: R 2 =0.9846, the detection limit is 4.77μg / L, the linear range is: 9.15-288.28μg / L; the standard curve of cucumber is: Y=0.3388X-0.1123, the correlation coefficient is: R 2 =0.9734, the detection limit is 4.23μg / L, the linear range is: 4.23-306.08μg / L; the standard curve of zucchini is: Y=0.3498X-0.1172, and the correlation coefficient is: R 2 =0.9773, the detection limit is 4.18μg / L, and the linear range is: 8.07-247.49μg / L.

[0110] Example 5

[0111] In order to further evaluate the superiority of the acetamiprid detection method based on chemical covalent directed antibodies, this example compares the detection limit, sensitivity and linear range of physical adsorption and covalent directed antibodies.

[0112] To establish a physical adsorption standard curve, the specific steps are as follows:

[0113] 1. Preparation of acetamiprid standard: Use 10% methanol solution to prepare a series of pesticide gradient acetamiprid standards (concentration gradient includes: 0, 1, 2.5, 5, 10, 25, 50, 100, 200, 300 μg / L) for subsequent competitive reactions.

[0114] 2. Establish a standard curve

[0115] (1) Plate coating: Add 100 μL of 5 μg / mL acetamiprid antibody solution to the ELISA plate and shake gently at 37°C for 2 h;

[0116] (2) Blocking: After washing with PBST, block with 2% bovine serum albumin (300 μL) for 1 h;

[0117] (3) Incubation of antigen: 50 μL of acetamiprid standard solution or the sample to be tested and 50 μL of Au@Pt peroxidase-labeled antigen were added to the antibody-labeled ELISA plate and incubated at 25 °C for 2 h;

[0118] Color development: Add 100 μL substrate to each well and incubate at room temperature for 15 min;

[0119] Reading: Use a microplate reader to read the absorbance at 650nm;

[0120] Establish a standard curve with acetamiprid concentration as X-axis and inhibition rate as Y-axis (e.g. Figure 5 ), the standard curve is: Y = 0.3727X-0.0454, the correlation coefficient is: R 2 =0.9946, the detection limit is 2.46μg / L, the linear range is: 4.56-120.36μg / L, and the sensitivity is 29.07μg / L.

[0121] It can be seen that compared with physical adsorption, the detection limit of antibody covalent orientation is reduced by two orders of magnitude and the sensitivity is increased by one order of magnitude.

[0122] Example 6 - Affinity Determination

[0123] The affinity of physical adsorption and chemical covalent orientation methods was determined by enzyme-linked immunosorbent assay. According to the steps of Example 3, activated antibodies of different concentrations were coated on the ELISA plate. After blocking, different concentrations of Au@Pt-Ag were added respectively, and incubation, color development and reading were performed according to the steps of Example 3.

[0124] Finally, according to the formula Ka = (n-1) / 2(n[Ab] 1 -[Ab] 2 ) to calculate the affinity constant. The affinity constant of the chemical covalent directed method is 1.410×10 7 L / moL, such as Figure 6 ; The affinity constant of the physical adsorption method is 9.92×10 6 L / moL, such as Figure 7 As can be seen, the affinity of the chemical covalent targeting method is significantly higher.

[0125] Example 7 - Specificity Experiment

[0126] The pesticides (imidacloprid, fenthion, chlorpyrifos, chlorpyrifos and thiamethoxam) that may be present in agricultural products were specifically detected. According to the steps of Example 3: 50 μL of different concentrations of acetamiprid, imidacloprid, fenthion, chlorpyrifos, chlorpyrifos and thiamethoxam were added to the antibody-coated ELISA plate, and incubated, developed and read according to the steps of Example 3. Finally, the cross-reaction rate was calculated, and the formula was: Cross-reaction rate (CR) = IC 50 (Acetamiprid) / IC 50 (Other pesticides)×100%.

[0127] Table 1 Cross-reaction rates of different pesticides

[0128] pesticide <![CDATA[IC 50 (μg / L)]]> CR(%) Acetamiprid 9.33 100 Imidacloprid <![CDATA[4×10 36 ]]> ＜0.5 Fenthion <![CDATA[7×10 9 ]]> ＜0.5 Chlorpyrifos 5709.08 ＜0.5 Forchlorfenuron 1318.95 0.71 Thiamethoxam 665.83 1.40

[0129] Table 1 shows that the cross-reaction rate to thiamethoxam is 1.4%, the cross-reaction rate to chlorfenapyr is 0.71%, and the cross-reaction rates of other pesticides are all less than 0.5%. Generally, the cross-reaction rate less than 5% is ignored and considered to be non-cross-reaction, which shows that the method has good specificity.

[0130] Example 8

[0131] The difference between the antibody and the inactivated antibody was compared by enzyme-linked immunosorbent assay. According to the steps of Example 3: the activated antibody and the inactivated antibody were coated on the ELISA plate respectively, and after blocking, the same concentration of Au@Pt-Ag was added respectively, and the steps of Example 3 were followed for incubation, color development, and reading. Figure 8 , it can be seen that the absorbance value is significantly higher after the antibody is activated, proving that more antibodies are bound to the ELISA plate after activation.

[0132] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting acetamiprid based on covalent directed antibodies, It is characterized in that include: Activate the carboxyl group of the Fc region of the antibody and form an amide bond with the amino-modified ELISA plate to obtain an ELISA plate bound with the antibody; Adding a buffer solution into the wells on the ELISA plate combined with the antibody as blank control wells, adding a standard solution of acetamiprid or a sample to be tested into the wells on the ELISA plate combined with the antibody as inhibition wells, and incubating with the antigen labeled with Au@Pt peroxidase mimetic enzyme; Add substrate and incubate; A standard curve of acetamiprid concentration and inhibition rate is established according to the absorbance, and the content of acetamiprid in the sample to be tested is obtained according to the standard curve.

2. The detection method according to claim 1, It is characterized in that The preparation process of the ELISA plate combined with the antibody comprises: mixing and diluting the antibody with a succinimide solution, then mixing and reacting with the amino-modified ELISA plate and a carbodiimide solution, and then washing to remove the unbound antibody; Preferably, in the process of preparing the ELISA plate combined with the antibody, the reaction temperature is controlled to be 25° C.-27° C., and the reaction time is 1 h-3 h.

3. The detection method according to claim 2, It is characterized in that By adjusting the dosage of the succinimide solution and the carbodiimide solution, the molar ratio of succinimide to carbodiimide is 1:(4.0-4.5), and the mass ratio of the carbodiimide to the antibody is 1:(3-4); Preferably, the concentration of the succinimide solution is 0.2 mg / mL-0.3 mg / mL, the concentration of the carbodiimide solution is 1.0 mg / mL-2.0 mg / mL, and the volume ratio of the succinimide solution to the carbodiimide solution is 1:(0.5-1.5); Preferably, the succinimide is N-hydroxysuccinimide.

4. The detection method according to claim 1, It is characterized in that During the incubation process with the Au@Pt peroxidase-labeled antigen, the incubation temperature is controlled to be 35° C.-40° C., and the incubation time is 1 h-3 h.

5. The detection method according to claim 1 or 3, It is characterized in that The preparation process of the Au@Pt peroxidase-mimicking antigen includes: Provide Au@Pt peroxidase mimics; After adjusting the pH value of the Au@Pt peroxidase mimetic enzyme to 8-9, the Au@Pt peroxidase mimetic enzyme was mixed with acetamiprid antigen for reaction, and then mixed with bovine serum albumin to block the Au@Pt peroxidase. After solid-liquid separation, the obtained solid material was washed and redissolved with a phosphate buffer solution; Preferably, the reaction temperature of the Au@Pt peroxidase mimetic enzyme and the acetamiprid antigen is 2°C-6°C, and the reaction time is 10h-15h; Preferably, the temperature for blocking with the bovine serum albumin is 35° C.-40° C., and the reaction time is 1 h-5 h.

6. The detection method according to claim 5, It is characterized in that The preparation process of the Au@Pt peroxidase mimetic enzyme comprises: heating a chloroauric acid solution to a boil and then mixing it with a sodium citrate solution, boiling it for 20 minutes to 40 minutes to make the solution wine red, cooling it and performing solid-liquid separation to obtain gold nanoparticles, heating the gold nanoparticles and the chloroplatinic acid solution to 70°C to 90°C, and then mixing them with an ascorbic acid solution and heating them to keep boiling for 20 minutes to 40 minutes; Preferably, the molar ratio of sodium citrate to chloroauric acid is (6-7):1; Preferably, the addition ratio of gold nanoparticles, chloroplatinic acid and ascorbic acid is 1.077×10 -16 g: 0.05mM-0.30mM: 1.15mM-1.35mM.

7. The detection method according to claim 1, It is characterized in that The buffer solution is a methanol-phosphate buffer solution; Preferably, the volume fraction of methanol in the methanol-phosphate buffer solution is 5%-10%.

8. The detection method according to claim 1, It is characterized in that The substrate is at least one selected from o-phenylenediamine, tetramethylbenzidine and 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt.

9. The detection method according to claim 1, It is characterized in that The calculation formula of inhibition rate I is as follows: I(%)=(T 0 -T) / T 0 ×100%; Among them, T 0 is the absorbance value corresponding to the control well, and T is the absorbance value corresponding to the acetamiprid standard solution or the sample to be tested; Preferably, the detection wavelength of the absorbance value is 650-652 nm.

10. The detection method according to claim 1, It is characterized in that The preparation process of the sample to be tested includes: homogenizing the agricultural product and mixing it with acetonitrile and vortexing it, then mixing it with anhydrous magnesium sulfate and sodium chloride and vortexing it, then centrifuging and separating it, and taking the supernatant; The supernatant is vortexed in a dispersed solid phase extractant, and then centrifuged and filtered; wherein the dispersed solid phase extractant comprises ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black, and the mass ratio of ethylenediamine-N-propylsilane, octadecylsilane and graphitized carbon black is 1:(0.5-1.5):(0.3-0.6); Preferably, the agricultural product is selected from at least one of Chinese cabbage, cucumber, carrot, zucchini, apple, barley, pepper, persimmon, potato, rose, pomegranate, lettuce, sorghum, walnut, strawberry, corn, cotton, blueberry, broccoli, fig, citrus, cherry and celery.

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