Micromolecule immunodetection kit and micromolecule immunodetection method

Abstract

The invention discloses a micromolecule immunodetection kit and a micromolecule immunodetection method, and relates to the technical field of immunodetection. According to the present invention, the coupling reaction between the ELISA plate and the DNA is utilized to directionally fix one end of the DNA on the ELISA plate, and the coupling reaction between the antibody and the DNA is further utilized to directionally fix the antibody on the ELISA plate in a cascade manner; according to the present invention, the antibody is directionally fixed through DNA cascade, such that the Fab region of the antibody is completely exposed, the steric hindrance of the antibody is effectively reduced, the overall binding efficiency of the antibody and the elisa plate is improved, the utilization efficiency of the antibody is significantly improved, and the immunodetection sensitivity is further improved. The invention can be widely used for detecting various micromolecules such as chemical pesticides, animal hormones, plant growth regulators, antibiotics and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular, to an immunoassay kit for small molecules and an immunoassay method for small molecules. Background Art

[0002] Immunoassay is an analytical technique based on the specific binding of antigens and antibodies. Due to its advantages such as high sensitivity, strong specificity, and rapid operation, it has been widely used in the fields of food safety, environmental detection, disease diagnosis, etc. In immunoassay, antibodies, as important recognition elements, are usually immobilized on enzyme-linked immunosorbent assay (ELISA) plates by physical adsorption. However, physical adsorption makes the orientation of antibodies completely random, and the antigen recognition sites (Fab regions) of antibodies may be partially or completely blocked, resulting in a decrease in binding ability and thus affecting the sensitivity of immunoassay. Therefore, in immunoassay, ensuring the correct orientation of antibody immobilization is crucial for improving the sensitivity of immunoassay.

[0003] Currently, researchers have developed various methods for orienting antibodies to improve the sensitivity of immunoassay. For example, Protein A and Protein G are commonly used antibody orientation techniques, which can specifically bind to the Fc region of antibodies, exposing the Fab regions of antibodies and greatly improving the detection sensitivity. In addition, the orientation method based on the high affinity between biotin and avidin has also been widely used. However, when large proteins are immobilized on ELISA plates, there is still a large steric hindrance, and the problem of low detection sensitivity exists.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide an immunoassay kit for small molecules and an immunoassay method for small molecules to solve the above technical problems.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, the present invention provides an immunoassay kit for small molecules, which includes: an ELISA plate, on which a plurality of DNAs are conjugated, and the DNAs are conjugated with small molecule antibodies;

[0008] Each DNA is a nucleotide or nucleotide analog with a length of 4 - 10 nt, and the sequences of the plurality of DNAs on the same ELISA plate are the same; any two DNA molecules are not complementary to each other; the small molecule is selected from chemical pesticides, animal hormones, plant growth regulators, or antibiotics.

[0009] In a second aspect, the present invention also provides an immunoassay method for small molecules, which is not for the purpose of diagnosing diseases, and includes the following steps:

[0010] S1: First, fix DNA on the enzyme-linked immunosorbent assay (ELISA) plate to obtain an ELISA plate conjugated with DNA.

[0011] S2: Incubate the small molecule antibody with the ELISA plate conjugated with DNA in S1, so that the antibody is conjugated with DNA through a chemical bond; obtain an ELISA plate conjugated with antibody and DNA.

[0012] S3: Load the negative control, small molecule standard or sample to be tested onto the ELISA plate conjugated with antibody and DNA respectively, add the antigen labeled with nanozyme, and incubate.

[0013] S4: Add the substrate and incubate.

[0014] S5: Detect the absorbance of the ELISA plate, establish a standard curve based on the absorbance, and realize the detection of small molecules in the sample to be tested.

[0015] The present invention has the following beneficial effects:

[0016] The present invention utilizes the conjugation reaction between the ELISA plate and DNA, so that one end of DNA is directionally fixed on the ELISA plate. Further, through the conjugation reaction between the antibody and DNA, the antibody is cascade directionally fixed on the ELISA plate. The present invention fixes the antibody directionally through DNA cascade, ensuring that the Fab region of the antibody is fully exposed, effectively reducing the steric hindrance of the antibody, improving the overall binding efficiency of the antibody and the ELISA plate, significantly enhancing the utilization efficiency of the antibody, and further improving the sensitivity of immunoassay. The proposed invention can be widely used for the detection of various small molecules such as chemical pesticides, animal hormones, plant growth regulators, antibiotics, etc., especially providing a good technical basis for the rapid detection of residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a schematic diagram of the principle of DNA cascade directional antibody provided by the present invention;

[0019] Figure 2 It is a standard curve graph of acetamiprid solvent provided by an embodiment of the present invention;

[0020] Figure 3 It is a standard curve graph of acetamiprid under the matrix systems of Chinese cabbage, cucumber and zucchini provided by an embodiment of the present invention;

[0021] Figure 4Antibody standard curve established for the present invention;

[0022] Figure 5 Statistical result graph of the antibody conjugation ratio of physical adsorption and DNA cascade orientation provided by the embodiment of the present invention. Detailed implementation manners

[0023] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained by purchasing in the market.

[0024] The inventors found that when large proteins are immobilized on an ELISA plate, the large steric hindrance between them may have an adverse effect on the binding efficiency. This steric hindrance effect may hinder the effective interaction between the antibody and the antigen, thereby reducing the overall binding efficiency. Therefore, small biomolecules are developed for the directed antibody to reduce steric hindrance and significantly improve the sensitivity of immunoassay.

[0025] The present invention utilizes the conjugation reaction between an ELISA plate and DNA, so that one end of the DNA is directionally immobilized on the ELISA plate. Further, through the conjugation reaction between the antibody and the DNA, the antibody is cascade directionally immobilized on the ELISA plate. The present invention ensures the full exposure of the Fab region of the antibody by DNA cascade directional immobilization of the antibody, effectively reduces the steric hindrance of the antibody, improves the overall binding efficiency of the antibody and the ELISA plate, significantly improves the utilization efficiency of the antibody, and further improves the sensitivity of immunoassay. The proposal of the present invention can be widely used for the detection of various small molecules such as chemical pesticides, animal hormones, plant growth regulators, and antibiotics, and especially provides a good technical basis for the rapid detection of residues.

[0026] In a first aspect, the present invention provides an immunoassay kit for small molecules, which includes: an ELISA plate, on which a plurality of DNAs are conjugated, and the DNA is conjugated with an antibody against a small molecule;

[0027] Each DNA is a nucleotide or nucleotide analogue with a length of 4-10 nt, and the sequences of the plurality of DNAs on the same ELISA plate are the same; any two DNA molecules are not complementary to each other; the small molecule is selected from chemical pesticides, animal hormones, plant growth regulators or antibiotics.

[0028] When performing immunoassay on chemical pesticides, DNA is conjugated with antibodies of small molecules; when performing immunoassay on animal hormones, DNA is conjugated with antibodies of target animal hormones; when performing immunoassay on plant growth regulators, DNA is conjugated with antibodies of target plant growth regulators; when performing immunoassay on antibiotics, DNA is conjugated with antibodies of target antibiotics.

[0029] Multiple DNAs conjugated on the enzyme-linked immunosorbent assay (ELISA) plate, the number or the number of strands of DNA ≥ 2. For example, 10 strands, 20 strands, 30 strands, 50 strands, 100 strands, 150 strands, 200 strands, 250 strands, 300 strands, 500 strands, 1000 strands, 10000 strands, etc.

[0030] In order to avoid the difference in absorbance between some detection wells caused by the difference in antibody conjugation efficiency due to different DNA sequences among multiple DNA sequences on the same ELISA plate, it is specifically set that multiple DNA sequences on the same ELISA plate are the same. And any two DNA molecules do not complementarily pair with each other, otherwise self-winding of DNA molecules will occur, affecting the binding efficiency with antibodies and further leading to a decrease in detection sensitivity.

[0031] Compared with physically adsorbed antibodies, the present invention improves the conjugation ratio of antibodies with the ELISA plate through DNA-directed antibodies, avoiding waste of antibodies.

[0032] In a preferred embodiment of the application of the present invention, DNA is the same type of nucleotide or nucleotide analog with a length of 4 - 10 nt. For example, it is 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt or 10 nt.

[0033] In a preferred embodiment of the application of the present invention, the bases in the nucleotide or nucleotide analog are selected from natural nucleobases or modified nucleobases.

[0034] In a preferred embodiment of the application of the present invention, the natural nucleobases are selected from any one of adenine, uracil, guanine, hypoxanthine, cytosine, thymine, adenine derivatives, uracil derivatives, guanine derivatives, cytosine derivatives, thymine derivatives.

[0035] In a preferred embodiment of the application of the present invention, the modified nucleobases are selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2'-O-N dimethyladenosine, pseudouracil, N 1 -methylpseudouracil, 5-iodouracil, 4-thiouracil, 2-thiouracil, 5-methyluracil, 5-oxymethyluracil, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 15-methylcytosine, 5-hydroxymethylcytosine, N 1 -methylguanine, or isoguanine.

[0036] In a preferred embodiment of the application of the present invention, the chemical pesticide is selected from herbicides, insecticides or antimicrobials;

[0037] In a preferred embodiment of the application of the present invention, the insecticide is selected from nematicides or insecticides;

[0038] In a preferred embodiment of the application of the present invention, the insecticide is selected from acetamiprid, imidacloprid, fenthion, chlorpyrifos, forchlorfenuron or thiamethoxam;

[0039] In a preferred embodiment of the application of the present invention, the chemical pesticide is selected from acetyl-CoA carboxylase inhibitors, phenoxy phenoxy propionate, clodinafop-propargyl or clodinafop; protoporphyrinogen IX oxidase inhibitors, diphenyl ether, acifluorfen or acifluorfen sodium; acetolactate synthase inhibitors, preferably sulfonylureas, more preferably pyriftalid; lipid biosynthesis inhibitors, preferably thioureas, more preferably thiobencarb; photosynthesis inhibitors, preferably propanil; carotenoid biosynthesis inhibitors, preferably isoxazolone, more preferably clomazone; cyclohexanedione, preferably clethodim; aryloxyphenoxypropionate, preferably cyhalofop-butyl; enolpyruvylshikimate-3-phosphate synthase inhibitors, preferably glyphosate (N-(phosphonomethyl)glycine) or bilanafos.

[0040] The chemical pesticides are selected from the group consisting of: acetyl-CoA carboxylase inhibitors, such as cyclohexenone oxime ethers, such as alloxydim, clethodim, sethoxydim, thiencarbazone-methyl, quizalofop-p-ethyl, tralkoxydim, butroxydim, profoxydim or pyrazoxyfen; aryloxyphenoxypropionate esters, such as clodinafop-propargyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-p-ethyl, fenoxaprop-P-ethyl, fenthiaprop-ethyl, fluazifop-butyl, fluazifop-P-butyl, fluroxypr-butyl-methyl, haloxyfop-P-methyl, haloxyfop-R-methyl, imazethapyr, imazamox, imazapic, imazapyr or imazaquin; or arylaminopropionic acids, such as flamprop-methyl or flamprop-isopropyl; acetolactate synthase inhibitors, such as imidazolinones, such as imazapyr, imazaquin, imazapic, imazamox, imazethapyr or imazaquin; pyrimidinyl ethers, such as pyrithiobac-sodium, pyrithiobac, bispyribac-sodium, KIH-6127 or pyraflufen-ethyl; sulfonamides, such as florasulam, flumetsulam or metosulam; or sulfonylureas, such as amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, halosulfuron-methyl, halosulfuron, imazosulfuron, nicosulfuron, rimsulfuron, sulfometuron-methyl, thifensulfuron-methyl, tribenuron-methyl, trifloxysulfuron-sodium, triflusulfuron-methyl, sulfosulfuron, mesosulfuron-methyl or iodosulfuron-methyl-sodium; amides, such as dipropetryn, desmedipham, bromobutide, chlorthiamid, diphenamid, etobenzanid, flufenacet, glyphosine or heptanophos.

[0041] Auxin herbicides, such as picolinic acids, such as clopyralid or aminopyralid; or 2,4-D or benazolin; auxin transport inhibitors, such as napropamide or flucarbazone; carotenoid biosynthesis inhibitors, such as pyrazolate, clomazone, diflufenican, flurochloridone, fluridone, pyrazolynate, benzobicyclon, isoxazoline, isoxachlortole, mesotrione, sulcotrione, ketospiradox, furanone, norflurazon or amitrole; enolpyruvylshikimate-3-phosphate synthase inhibitors, such as glyphosate or glufosinate; glutamine synthetase inhibitors, such as bilanafos (bialaphos) or glufosinate-ammonium; lipid biosynthesis inhibitors, such as acetanilides, such as anilofos or pretilachlor; chloroacetanilides, such as dimethachlor, S-dimethachlor, acetochlor, alachlor, butachlor, butenachlor, acetochlor-methyl, dimethachlor-ethyl, pyrazochlor, metolachlor, S-metolachlor, propachlor, propanil, propyzamide, terbutachlor, thenylchlor or dimethanamid; thioureas, such as butylate, cycloate, vernolate, piperophos, EPTC, pebulate, molinate, diallate, pyributicarb, thiobencarb (benthiocarb), triallate or pebulate; or flurtamone or bensulide; mitosis inhibitors, such as carbamates, such as sulfentrazone, carbetamide, chlorpropham, dimepiperate (pyributicarb), anilofos or dimepiperate; dinitroanilines, such as fluchloralin, butralin, trifluralin, ethalfluralin, chlorthal-dimethyl, oryzalin, pendimethalin, diflufenican or trifluralin; pyridines, such as fluthiacet-methyl or thiazopyr; or bilanafos, phthalide or maleic hydrazide; protoporphyrinogen IX oxidase inhibitors, such as diphenyl ethers, such as acifluorfen, acifluorfen-sodium, ethoxyfen, methylarsonate, chlomethoxynil, fluorodifen, nitrofluorfen, oxyfluorfen, fomesafen, flumiclorac-pentyl, lactofen, trifluoromethoxyfen or ethoxyfluorofen; oxadiazoles, such as oxyfluorfen or oxadiazon; cyclic imides, such as carfentrazone-ethyl, flumioxazin, sulfentrazone, indanofan, flumiclorac-pentyl, propanil, flupropacil, fluthiacet-methyl, sulfentrazone, thidiazimin or thidiazolethione; or pyrazoles, such as ET-751, JV 485 or flufenacet; photosynthesis inhibitors, such as propanil, pyridafol or pyridazinol; benzothiadiazinones, such as bentazone; dinitrophenols, such as bromofenoxim, dinoseb, dinoseb acetate, dinoterb or DNOC; bipyridyliums, such as diquat-chloride, desmedipham, diquat or paraquat dichloride; ureas, such as chlorbromuron, chlorotoluron, cumyluron, flucarbazone, diuron, sulfometuron-methyl, monuron, fluometuron, isoproturon, isoxuron, linuron, methylthidiazuron, methazole, pyrazolynate, methoxuron, chloroxuron, bromacil, cycluron or buthiuron; phenols, such as bromoxynil or ioxynil; chloridazon;Triazines, such as ametryn, atrazine, cyanazine, desmetryn, dimethametryn, hexazinone, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbutryn, terbuthylazine or trietazine; triazinones, such as metamitron or metamitram; uracils, such as bromacil, lenacil or terbacil; or bis-carbamates, such as desmedipham or phenmedipham; synergists, such as ethylene oxides, such as bromoxynil; cell wall synthesis inhibitors, such as clomazone or dichlobenil; various other herbicides, such as dalapon, such as dalapon; benzofurans, such as ethofumesate; phenylacetic acids, such as flurochloridone; or aziprotryne, barban, bensulide, bentazone, flurochloridone, butamifos, buthiuron, butralin, benzofenap, chlorthal-dimethyl, clomeprop, cycloxydim, cinmethylin, cyclosulfamuron, cyprazine, cycloxydim, dimepiperate, dimethametryn, dimefuron, ethidimuron, flucarbazone, fluorbentranil, flumioxazin, butamifos, isopropalin, carbetamide, flazasulfuron, chloroxuron, napropamide, methalaxyl, oxaciclomefone, mefluidide, piperophos, cyprocyanazine, cyprofluridon, pyributicarb, secbumeton, ethofumesate, terbucarb, triaziflam, triazofenamid or trimeturon; and their environmentally compatible salts, and combinations thereof.

[0042] In a preferred embodiment of the application of the present invention, the plant growth regulator is selected from auxin, gibberellin, cytokinin, abscisic acid, ethylene or brassinolide;

[0043] In a preferred embodiment of the application of the present invention, the kit further comprises at least one of the following components: an antigen labeled with a nanozyme, a small molecule standard and a diluent.

[0044] The antigen labeled with a nanozyme refers to: a biomolecule labeled with a nanomaterial having peroxidase-like activity. The substrate undergoes an oxidation reaction under the catalytic action of the nanozyme to produce a visible product with a specific color. The color change is related to the content of the small molecule in the analyte, so the content of the small molecule in the analyte can be determined by detecting the color change.

[0045] In a preferred embodiment of the application of the present invention, the nanozyme refers to Au@Pt, Fe 3 O 4 , Ag@Pt and CeO 2 and other nanomaterials with peroxidase activity.

[0046] In a second aspect, the present invention also provides a method for immunoassay of small molecules, which is not for the purpose of diagnosing diseases, and comprises the following steps:

[0047] S1: First, fix DNA on an enzyme-linked immunosorbent assay (ELISA) plate to obtain an ELISA plate conjugated with DNA;

[0048] S2: Incubate the enzyme-labeled plate with DNA conjugated in S1 and small molecule antibodies, so that the antibodies are conjugated to the DNA through chemical bonds; obtain an enzyme-labeled plate conjugated with antibodies and DNA.

[0049] S3: Load the negative control and small molecule standards onto the enzyme-labeled plate conjugated with antibodies and DNA respectively, add the antigen labeled with nanozyme, and incubate.

[0050] S4: Add the substrate and incubate.

[0051] S5: Detect the absorbance of the enzyme-labeled plate, establish a standard curve based on the absorbance, and realize the detection of small molecules in the measured sample.

[0052] The target small molecule in the small molecule standard or the sample to be measured can competitively bind to the antibody on the enzyme-labeled plate with the antigen labeled with nanozyme. The higher the content of the target small molecule in the sample to be measured, the less antigen labeled with nanozyme can bind to the enzyme-labeled plate, and the smaller the absorbance (or the higher the inhibition rate). The standard curve can be drawn according to the inhibition rate of the absorbance and the concentration of the small molecule standard, and the content of the target small molecule in the sample to be measured can be obtained based on the standard curve.

[0053] In a preferred embodiment of the application of the present invention, step S1 includes:

[0054] Add DNA to the enzyme-labeled plate, add a crosslinking agent and an activator, and incubate together; wash after incubation; then add casein for blocking. The mass percentage concentration of casein is 6-10%.

[0055] In a preferred embodiment of the application of the present invention, the activator is selected from carboxyl activators, amino activators, hydroxyl activators or thiol activators; the sample loading wells of the enzyme-labeled plate are modified with carboxyl, amino, hydroxyl or thiol.

[0056] In a preferred embodiment of the application of the present invention, the sample loading wells of the enzyme-labeled plate are modified with amino.

[0057] In a preferred embodiment of the application of the present invention, the molar ratio of the crosslinking agent to the activator is 1:(1-3). For example, the molar ratio is 1:1, 1:2 or 1:3, and a relatively high DNA immobilization efficiency can be obtained under the above molar ratios.

[0058] In a preferred embodiment of the application of the present invention, the activator is a carbodiimide, and the carbodiimide is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DCI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) or a combination thereof.

[0059] In a preferred embodiment of the application of the present invention, the crosslinking agent is selected from 1-methylimidazole.

[0060] For example, the concentration of 1-methylimidazole is 0.08 - 0.12 M, and the pH is 6.5 - 7.5. The concentration of carbodiimide is 0.1 - 0.3 M.

[0061] In a preferred embodiment of the application of the present invention, when DNA is immobilized in step S1, the final concentration of the DNA solution is 1 - 3 μM.

[0062] In a preferred embodiment of the application of the present invention, the conditions for co-incubation in step S1 are 45 - 55 °C; the time is 4 - 6 h. According to different DNAs, those skilled in the art can adaptively adjust the reaction conditions to achieve higher coupling efficiency. For example, the conditions for co-incubation are 45 - 50 °C, 50 - 55 °C.

[0063] In a preferred embodiment of the application of the present invention, the 5'-terminal phosphate group of DNA is bound to the enzyme-linked immunosorbent assay (ELISA) plate through a phosphoramide bond and washed with PBST.

[0064] In a preferred embodiment of the application of the present invention, the structure of DNA is: 3'NH 2 -TTTTTTTTTT-5'P.

[0065] In a preferred embodiment of the application of the present invention, step S2 includes: adding the activated small molecule antibody to the ELISA plate, and making the antibody coupled with the DNA on the ELISA plate through a chemical reaction; the antibody is activated by an activator; after the coupling reaction, the unbound antibody is removed by washing.

[0066] In a preferred embodiment of the application of the present invention, the activator is selected from succinimide solution and carbodiimide; in a preferred embodiment of the application of the present invention, the molar ratio of succinimide to carbodiimide is 1:(4.0 - 4.5). At the above concentrations, a good coupling effect is achieved. For example, the molar ratio of succinimide to carbodiimide is 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4 or 1:4.5.

[0067] The concentration of carbodiimide is 1 - 2 mg / mL, and the concentration of succinimide solution is 0.3 - 0.5 mg / mL.

[0068] The succinimide is N-hydroxysuccinimide.

[0069] In a preferred embodiment of the application of the present invention, the acetamiprid antibody activated by carbodiimide and succinimide solution is added to the ELISA plate, and the amino group on the ELISA plate reacts with the carboxyl group of the antibody to form an amide bond, and the unbound antibody is removed by washing.

[0070] In a preferred embodiment of the application of the present invention, the nanozyme-labeled antigen in step S3 refers to: a biomolecule labeled with a nanomaterial having peroxidase activity;

[0071] In a preferred embodiment of the application of the present invention, the peroxidase nanozyme is a nanomaterial with peroxidase activity.

[0072] In a preferred embodiment of the application of the present invention, the peroxidase nanozyme is selected from Au@Pt, Fe 3 O 4 , Ag@Pt, and CeO 2 and other nanomaterials with peroxidase activity.

[0073] In a preferred embodiment of the application of the present invention, the incubation reaction temperatures in steps S2, S3, and S4 are all 36 - 38°C. Under the above incubation conditions, good detection effects can be obtained.

[0074] In a preferred embodiment of the application of the present invention, the substrates include, but are not limited to, 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, diaminobenzidine, or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). As long as it can be catalyzed by the peroxidase nanozyme with peroxidase activity to cause a color reaction, it is feasible.

[0075] In a preferred embodiment of the application of the present invention, the detection wavelength for detecting the absorbance of the enzyme-linked immunosorbent assay (ELISA) plate is 650 - 652 nm.

[0076] In an alternative embodiment, the materials of the ELISA plates are all polystyrene and are all modified with amino groups.

[0077] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0078] Example 1

[0079] This example provides an immunoassay method for acetamiprid, which includes the following steps:

[0080] (1) Fixing DNA: Add 75 μL of DNA (1 μM) solution to the ELISA plate (Thermo Fisher Scientific, model 478042), then add 7.5 μL of 1-methylimidazole (0.1 M, pH 7.0) and 25 μL of carbodiimide solution (0.2 M). The above steps are all carried out on ice. Finally, after the mixed solution is gently shaken at 50°C for 5 hours, a stable phosphoramide bond is formed between the phosphate group at the 5' end of DNA and the amino group of the ELISA plate, and the unbound antibody is washed away with PBST. The structure of DNA is: 3'NH 2 -TTTTTTTTTT-5'P.

[0081] (2) Blocking: After washing three times with PBST, 300 μL of 8% casein was added to each well, and the wells were blocked at 37° C. for 1 hour, followed by washing with PBST.

[0082] (3) Cascade Directed Immobilization Antibody: Schematic diagram of Cascade Directed Immobilization Antibody Figure 1 As shown. 50 μL of anti-acetamiprid antibody (diluted with 0.4 mg / mL succinimide solution) and 50 μL of carbodiimide solution were added to the above DNA-fixed ELISA plate and reacted at 37°C for 2 hours. The 3'-end modified amino group of DNA formed an amide bond with the carboxyl group of the antibody to achieve cascade orientation of the antibody, and finally PBST was used to wash and remove unbound antibodies. Antibodies were provided by Zhejiang University and can be purchased from Hangzhou Baisheng Huixing Biotechnology Co., Ltd.

[0083] (4) Incubation with antigen: Add phosphate buffer solution (containing 10% methanol) or acetamiprid standard solution (Beijing Manhag Biotechnology Co., Ltd., BePure 20021XM) to the cascade-directed antibody ELISA plate, and then add Au@Pt nanozyme-labeled acetamiprid antigen (provided by Zhejiang University and available for purchase at Hangzhou Biosun Huixing Biotechnology Co., Ltd.) (Au@Pt-Ag) and incubate at 37°C for 2 h.

[0084] (5) Color development: Add 100 μL TMB to each well and react at room temperature for 15 minutes.

[0085] (6) Reading: Use an ELISA plate to detect the absorbance at a wavelength of 650 nm.

[0086] The logarithm of the acetamiprid concentration was plotted on the X-axis, and the inhibition rate was plotted on the Y-axis to establish a standard curve ( Figure 2 ), Y = 0.2355 + 0.2199, the correlation coefficient is: R 2 =0.9862, detection limit: 0.31μg / L, sensitivity: 15.46μg / L.

[0087] The inhibition rate was calculated as follows: (negative wells-positive wells) / negative wells*100%.

[0088] The negative wells were phosphate buffer solution (containing 10% methanol) and the positive wells were acetamiprid standard solution.

[0089] Reference patent for the preparation of Au@Pt nanozyme: A detection method for acetamiprid based on Au@Pt peroxidase mimetic enzyme.

[0090] Example 2

[0091] This embodiment provides a method for detecting acetamiprid residues in vegetable samples, and establishes a corresponding matrix standard curve ( Figure 3), the specific operation steps are as follows:

[0092] 1. Sample pretreatment

[0093] First, select Chinese cabbage, zucchini, and cucumber as the detection objects. After cleaning them, make them into homogenates. Then, accurately weigh 5 g of the sample homogenate into a 50-mL centrifuge tube, and add 10 mL of acetonitrile. After vortex mixing for 1 minute, add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, and vortex mix again for 1 minute. Subsequently, centrifuge at a speed of 6000 revolutions per minute for 5 minutes, take 2 mL of the supernatant, and add it to a mixture containing 260 μL of a dispersive solid-phase extraction agent (which contains 104 μL of ethylenediamine-N-propylsilane, 104 μL of octadecylsilane, and 52 μL of graphitized carbon black), and vortex mix for 30 seconds. After that, centrifuge at a speed of 6000 revolutions per minute for 2 minutes. Finally, filter through a 0.22-μm filter membrane to obtain the sample for use.

[0094] 2. Establish a matrix calibration curve (that is, different vegetable samples to be tested of acetamiprid diluted with the extraction solutions of different vegetables are used to establish calibration curves respectively).

[0095] (1) Fix DNA: Add 75 μL of DNA (1 μM) solution to the enzyme-linked immunosorbent assay (ELISA) plate (Thermo Fisher Scientific, model number 478042). Subsequently, add 7.5 μL of 1-methylimidazole (0.1 M, pH 7.0) and 25 μL of carbodiimide solution (0.2 M). The above steps are all carried out on ice. Finally, after gently shaking the mixed solution at 50 °C for 5 hours, a stable phosphoramide bond is formed between the phosphate group at the 5'-end of DNA and the amino group of the ELISA plate, and the unbound DNA is washed away using PBST.

[0096] (2) Blocking: After washing three times with PBST, add 300 μL of 8% (mass fraction) casein to each well. After blocking at 37 °C for one hour, wash with PBST.

[0097] (3) Cascade directional fixation of antibodies: Add 50 μL of antibody (diluted with a 0.4 mg / mL succinimide solution) and 50 μL of carbodiimide solution to the above DNA-fixed ELISA plate, and react at 37 °C for 2 hours. An amide bond is formed between the amino group modified at the 3'-end of DNA and the carboxyl group of the antibody to achieve the cascade direction of the antibody. Finally, wash with PBST to remove the unbound antibody.

[0098] (4) Incubate the antibody: Add the vegetable sample dilution (containing 10% methanol) or the acetamiprid standard solution (different concentrations of acetamiprid standard solutions obtained by diluting acetamiprid of analytical purity with different vegetable samples in step 1) to the enzyme-linked immunosorbent assay (ELISA) plate of the cascade-directed antibody, and then add the acetamiprid antigen labeled with Au@Pt nanozyme (Au@Pt-Ag), and incubate at 37 °C for 2 h.

[0099] (5) Color development: Add 100 μL of TMB to each well and react at room temperature for 15 minutes.

[0100] (6) Reading: Measure the absorbance at a wavelength of 650 nm using an ELISA plate reader.

[0101] Establish a standard curve with the acetamiprid concentration on the X-axis and the inhibition rate on the Y-axis (as Figure 3 ), the standard curve for Chinese cabbage is: Y = 0.2834X + 0.0305, and the correlation coefficient is: R 2 = 0.9896, and the detection limit is 1.76 μg / L; the standard curve for cucumber is: Y = 0.1277X + 0.2102, and the correlation coefficient is: R 2 = 0.9684, and the detection limit is, and the linear range is: 0.14 μg / L; the standard curve for zucchini is: Y = 0.1651X + 0.2731, and the correlation coefficient is: R 2 = 0.9645, and the detection limit is 0.09 μg / L.

[0102] Example 3

[0103] To explore the conjugation ratio of the antibody, in this example, the antibody conjugation rates of physically immobilized antibody and DNA-directed antibody were compared. Collect the antibody that was not bound to the ELISA plate, and use the standard curve of the antibody ( Figure 4 ) to calculate the antibody conjugation ratio of different immobilization methods. The method for establishing the antibody standard curve is the same as that for physically immobilized antibody, but the acetamiprid standard should be modified to acetamiprid antibody. The calculation formula is as follows:

[0104] C(%) = (B 0 - B) / B 0

[0105] where C is the conjugation ratio, B 0 is the total concentration of the antibody, and B is the concentration of the unbound antibody.

[0106] The steps for physically immobilizing the antibody are as follows:

[0107] (1) Coating the antibody: Coat 100 μL of acetamiprid antibody on the ELISA plate, gently shake at 37 °C for 2 hours, and then wash away the unbound antibody with PBST. The antibody is provided by Zhejiang University and can be purchased from Hangzhou Baisheng Huixing Biotechnology Co., Ltd.

[0108] (2) Blocking: After washing three times with PBST, 300 μL of 2% bovine serum albumin was added to each well, and the wells were blocked at 37° C. for 1 hour, followed by washing with PBST.

[0109] (3) Antigen incubation: Add phosphate buffer solution (containing 10% methanol) or acetamiprid standard solution (Beijing Manhag Biotechnology Co., Ltd., BePure20021XM) to the above-mentioned ELISA plate, and then add Au@Pt nanozyme-labeled acetamiprid antigen (provided by Zhejiang University and can be purchased from Hangzhou Baisheng Huixing Biotechnology Co., Ltd.) (Au@Pt-Ag) and incubate at 37°C for 2 h.

[0110] (4) Color development: Add 100 μL TMB to each well and react at room temperature for 15 minutes.

[0111] (5) Reading: Use an ELISA plate to detect the absorbance at a wavelength of 650 nm.

[0112] like Figure 5 As shown, the coupling ratio of the physically adsorbed antibody to the plate was 45%, and the coupling ratio of the ssDNA cascade directed antibody to the ELISA plate was 52%. Figure 5 This indicates that DNA cascade directed antibodies can bind more antibodies, thus avoiding the waste of antibodies.

[0113] The above description is only a preferred embodiment of the present invention and is 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 small molecule immunoassay kit, characterized in that: It comprises: an ELISA plate, on which a plurality of DNAs are coupled, and the DNAs are coupled with small molecule antibodies; Each of the DNAs is a nucleotide or nucleotide analog with a length of 4 to 10 nt, and multiple DNA sequences on the same ELISA plate are identical; any two of the DNA molecules are not complementary; and the small molecules are selected from chemical pesticides, animal hormones, plant growth regulators or antibiotics.

2. The small molecule immunoassay kit according to claim 1, characterized in that: The DNA is a nucleotide or nucleotide analog of the same type with a length of 4 to 10 nt; Preferably, the base in the nucleotide or nucleotide analog is selected from a natural nucleoside base or a modified nucleoside base; Preferably, the natural nucleoside base is selected from any one of adenine, uraine, guanine, hypoxanthine, cytosine, thymine, adenine derivatives, uraine derivatives, guanine derivatives, cytosine derivatives, and thymine derivatives; Preferably, the modified nucleoside base is selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2'-ON dimethyladenosine, pseudouracil, N 1 -methyl pseudouracil, 5-iodouracil, 4-thiouracil, 2-thiouracil, 5-methyluracil, 5-oxymethyluracil, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 1 -methylcytosine, 5-hydroxymethylcytosine, N 1 -Methylguanine, isoguanine.

3. The small molecule immunoassay kit according to claim 1, characterized in that: The chemical pesticide is selected from herbicides, insecticides or antimicrobial agents; Preferably, the pesticide is selected from a nematicide or an insecticide; Preferably, the insecticide is selected from acetamiprid, imidacloprid, fenthion, chlorpyrifos, chlorpyrifos or thiamethoxam; Preferably, the chemical pesticide is selected from acetyl-CoA carboxylase inhibitors, phenoxyphenoxypropionate, clodinafop or clodinafop-butyl; protoporphyrinogen IX oxidase inhibitors, diphenyl ether, trifluorfen or trifluorfen sodium salt; acetolactate synthase inhibitors, preferably sulfonylureas, more preferably pyraclostrobin; lipid biosynthesis inhibitors, preferably thioureas, more preferably cypermethrin; photosynthesis inhibitors, preferably propanil; carotenoid biosynthesis inhibitors, preferably isoxazolidinones, more preferably isoxadiazolone; cyclohexanedione, preferably cypermethrin; aromatic oxygen phenoxy propionate esters, preferably cyhalofop-butyl; enolpyruvylshikimate-3-phosphate synthase inhibitors, preferably glyphosate (N-(phosphonomethyl)glycine) or glufosinate.

4. The small molecule immunoassay kit according to claim 1, characterized in that: The plant growth regulator is selected from auxin, gibberellin, cytokinin, abscisic acid, ethylene or brassinosteroid; Preferably, the kit further comprises at least one of the following components: a nanozyme-labeled antigen, a small molecule standard, and a diluent.

5. A small molecule immunoassay method, characterized in that: The method is not intended for the diagnosis of a disease and comprises the following steps: S1: First fix the DNA on the ELISA plate to obtain the DNA-coupled ELISA plate; S2: incubating the small molecule antibody with the DNA-coupled ELISA plate described in S1, so that the antibody is coupled to the DNA through a chemical bond; Obtaining an ELISA plate coupled with antibodies and DNA; S3: Load the negative control, small molecule standard and the sample to be tested onto the ELISA plate coupled with antibody and DNA, add the nanozyme-labeled antigen and incubate; S4: Add substrate and incubate; S5: Detect the absorbance of the ELISA plate, establish a standard curve based on the absorbance, and detect small molecules in the sample.

6. The small molecule immunoassay method according to claim 5, characterized in that: The step S1 comprises: Add DNA to the ELISA plate, add a cross-linking agent and an activator, and incubate together; wash after incubation; Preferably, the activator is selected from a carboxyl activator, an amino activator, a hydroxyl activator or a sulfhydryl activator; the loading wells of the ELISA plate are modified with carboxyl, amino, hydroxyl or sulfhydryl groups; Preferably, the sample loading wells of the ELISA plate are modified with amino groups; Preferably, the molar ratio of the crosslinking agent to the activator is 1:(1-3); Preferably, the activator is a carbodiimide, and the carbodiimide is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DCI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (EDCI) or a combination thereof; Preferably, the cross-linking agent is selected from 1-methylimidazole.

7. The small molecule immunoassay method according to claim 6, characterized in that: When DNA is fixed in step S1, the final concentration of the DNA solution is 1-3 μM; Preferably, the co-incubation condition in step S1 is 45-55° C. and the time is 4-6 h.

8. The small molecule immunoassay method according to claim 5, characterized in that: The step S2 comprises: adding the activated antibody to the ELISA plate, coupling the antibody to the DNA of the ELISA plate through a chemical reaction; the antibody is activated by an activator; after the coupling reaction, washing to remove the unbound antibody; Preferably, the activator is selected from succinimide solution and carbodiimide; preferably, the molar ratio of succinimide to carbodiimide is 1:(4.0-4.5).

9. The small molecule immunoassay method according to claim 5, characterized in that: The nanozyme-labeled antigen in step S3 refers to: a biological molecule labeled by a peroxidase nanozyme; Preferably, the peroxidase nanozyme is a nanomaterial having peroxidase activity; Preferably, the peroxidase nanozyme is selected from Au@Pt, Fe3O4, Ag@Pt or CeO2.

10. The small molecule immunoassay method according to claim 5, characterized in that: The incubation reaction temperature in steps S2, S3 and S4 is 36-38°C; Preferably, the substrate is 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, diaminobenzidine or 2,2'-azine-(3-acetylphenylthiazolesulfonic acid)-6; Preferably, the detection wavelength for detecting the absorbance of the ELISA plate is 650-652 nm.

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