A DNA tetrahedron space-based immunoassay method and its application in detection of imidacloprid residues

Abstract

The application discloses a DNA tetrahedron space-mediated immunoassay method and application thereof in detection of imidacloprid residues, relates to the field of pesticide detection, and realizes spatial ordered arrangement of target antibodies by regulating the spatial density of target antibodies through coupling target antibodies on magnetic beads in the form of DNA tetrahedrons, so that a detection probe of MB@TDN@Ab is formed, trace detection of a to-be-detected substance can be realized, and high sensitivity and accuracy are achieved. Compared with a traditional magnetic separation immunoassay method, the detection sensitivity of the application is improved by about 2 times, and the linear range is improved by 1 order of magnitude. When the application is applied to detection of pesticides such as imidacloprid, it has important significance for guaranteeing food safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pesticide detection, and in particular, to a DNA tetrahedron space-mediated immunoassay method and application thereof in imidacloprid residue detection. BACKGROUND

[0002] Imidacloprid pesticide is widely used in agricultural production for pest control, ensuring the yield and quality of vegetables. Although the control efficiency of imidacloprid pesticide on pests is high, the abuse of imidacloprid pesticide can seriously threaten the environment and public health, and even seriously damage the ecosystem. Therefore, it is crucial to establish an efficient, high-sensitivity, convenient imidacloprid detection and analysis method.

[0003] Immunomagnetic separation method has the advantages of rapidity, convenience, simplicity and economy, and has been widely used in pesticide residue detection. However, in the traditional magnetic separation immunization method, the orientation of the antibody on the surface of the magnetic bead is random and disordered, which leads to the shielding of the active site of the antibody and reduces the recognition efficiency.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The present application aims to provide a DNA tetrahedron space-mediated immunoassay method and application thereof in imidacloprid residue detection, so as to solve the problems of random and disordered immunization antibody of the existing immunomagnetic bead, deep buried antigen recognition site, and improve the sensitivity, effectiveness and accuracy of detection.

[0006] The present application is implemented as follows:

[0007] In a first aspect, the present application provides a detection probe, comprising: a magnetic bead, a DNA tetrahedron and a target antibody; the target antibody is connected to the magnetic bead through the DNA tetrahedron.

[0008] In a second aspect, the present application provides a preparation method of the detection probe as described in the preceding embodiments, comprising the following steps: coupling the DNA tetrahedron with the magnetic bead and the target antibody.

[0009] In a third aspect, the present application provides a kit, comprising: the detection probe as described in the preceding embodiments.

[0010] In a fourth aspect, the present application provides application of the detection probe as described in the preceding embodiments, or the detection probe prepared by the preparation method as described in the preceding embodiments, or the kit as described in the preceding embodiments in detection of pesticide.

[0011] The present application has the following beneficial effects:

[0012] (1) The embodiment of the present application utilizes the rigid structure of DNA tetrahedron, couples DNA tetrahedron on magnetic beads, arranges monoclonal antibodies in space on the surface of magnetic beads, forms the detection probe of MB@TDN@Ab, and improves the exposure area of the active site of the antibody.

[0013] (2) Compared with the traditional magnetic bead immune probe, the sensitivity IC50 of the MB@TDN@Ab detection probe provided by the embodiment of the present application is as low as 1.40 ng / mL under the same antibody concentration and coupling conditions, and the linear range is 0.05-50 ng / mL, while the traditional MB@Ab magnetic bead immune probe is only 2.66 ng / mL, and the linear range is 0.05-5 ng / mL; the sensitivity is improved by about 2 times, and the linear range is increased by 1 order of magnitude. 50

[0014] (3) The detection method of the present application does not need to rely on precise instruments for detection, and has the advantages of low cost, rapidness, simplicity, sensitivity and good specificity. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 TDN different characterization results;

[0017] Figure 2 Zeta potential diagram of MB, MB@TDN, MB@TDN@Ab;

[0018] Figure 3 Verification diagram of optimization of different concentrations of antibodies;

[0019] Figure 4 Sensitivity comparison of MB@Ab and MB@TDN@Ab probes;

[0020] Figure 5 Solvent standard curve of MB@Ab and MB@TDN@Ab;

[0021] Figure 6 Standard curve of MB@TDN@Ab in Chinese cabbage, cucumber and zucchini matrix;

[0022] Figure 7 Specificity verification of MB@TDN@Ab probe. DETAILED DESCRIPTION

[0023] ​In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased in the market are adopted.

[0024] The embodiments of the present application label a first group on three apices of a DNA tetrahedron, the surface of a magnetic bead is modified with a third group capable of mutual combination with the first group, the DNA tetrahedron nanostructure is modified on the surface of the magnetic bead through the combination of the first group and the third group, the fourth apex of the DNA tetrahedron is modified with a second group, and the DNA tetrahedron and the target antibody are coupled through the second group, so as to form a complex of magnetic bead-DNA tetrahedron-target antibody as a detection probe, also recorded as MB@TDN@Ab. According to the principle of specific recognition of antigen and antibody, the unknown concentration of the measured substance (for example, imidacloprid) in the sample competes with the known concentration of enzyme-labeled hapten for the active site, so as to detect the measured substance, which has the advantages of high sensitivity, high accuracy, high throughput and high efficiency. Although the ordinary immunological probe such as MB@Ab can achieve the function of fixing the antibody, since the antibody is tightly coupled on the surface of the magnetic bead, it cannot achieve the spatially ordered arrangement as the DNA tetrahedron, and the active site is deeply buried. The detection probe of the present application has important significance for food safety protection when applied to pesticide detection in vegetables.

[0025] In one aspect, the embodiments of the present application provide a detection probe, comprising: a magnetic bead, a DNA tetrahedron and a target antibody; the target antibody is connected to the magnetic bead through the DNA tetrahedron.

[0026] In some embodiments, the DNA tetrahedron comprises four apices, three of which are connected to the magnetic bead, and one of which is connected to the target antibody.

[0027] In some embodiments, the ratio of the magnetic bead and the DNA tetrahedron is 1 mg: 0.1-10 nmol, which can be specifically any one or a range between any two of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 nmol.

[0028] In some embodiments, the ratio of the DNA tetrahedron and the target antibody is 0.1 nmol: 4-10 μg, which can be specifically any one or a range between any two of 4, 5, 6, 7, 8, 9 and 10 μg.

[0029] In some embodiments, the DNA tetrahedron is formed by self-assembly of four DNA single strands.

[0030] In some embodiments, three of the four DNA single strands are modified with a first group, and one of the four DNA single strands is modified with a second group; the DNA tetrahedron is coupled with the magnetic beads through the first group, and coupled with the target antibody through the second group.

[0031] In some embodiments, the first group and the second group are modified at the 5' end of the DNA single strand.

[0032] In some embodiments, the first group comprises an NH2 group.

[0033] In some embodiments, the second group comprises a DBCO group.

[0034] In some embodiments, the DNA tetrahedron is modified on the surface of the magnetic beads by covalent bond of amide at three vertices of the DNA tetrahedron. Then, the DBCO group at the fourth vertex of the DNA tetrahedron is reacted with the NHS-PEG4-azide solution to form an NHS ester, and the NHS ester can be coupled with the target antibody to form a detection probe.

[0035] In some embodiments, the four DNA single strands comprise S1-first group, S2-first group, S3-second group, and S4-first group.

[0036] In some embodiments, the base sequences of the S1, the S2, the S3, and the S4 are sequentially shown in SEQ ID NOs: 1-4.

[0037] In some embodiments, the target antibody comprises an antibody against imidacloprid.

[0038] In some embodiments, the antibody comprises a monoclonal antibody and / or a polyclonal antibody.

[0039] In some embodiments, the magnetic beads have a size of 50-300 nm, specifically, any one of 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 nm or a range between any two of them.

[0040] In another aspect, the embodiments of the present application also provide a preparation method of the detection probe as described in any of the foregoing embodiments, comprising the following steps: coupling the DNA tetrahedron with the magnetic beads and the target antibody.

[0041] In some embodiments, before the coupling, the preparation method further comprises synthesizing the DNA tetrahedron.

[0042] In some embodiments, the synthesis of the DNA tetrahedron comprises: mixing four DNA single strands and a buffer (e.g., TM buffer) and then placing the mixture in a PCR instrument for reaction. The molar ratio of any two of the four DNA single strands can be 1:1-3, and specifically can be 1:1. The reaction conditions can be: heating at 93-97°C for 8-15 min, cooling at 0-8°C for 10-30 min, and specifically can be heating at 94-96°C for 8-12 min, cooling at 2-6°C for 8-12 min.

[0043] In some embodiments, the coupling comprises: incubating the DNA tetrahedron with magnetic beads to obtain magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads; incubating the magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads with target antibodies to obtain the detection probe.

[0044] In some embodiments, the mixing ratio of the DNA tetrahedron to the magnetic beads is 0.1-10 nmol: 1 mg.

[0045] In some embodiments, the mixing conditions of the DNA tetrahedron and the magnetic beads comprise: 0-35°C, 3-12 h.

[0046] In some embodiments, the magnetic beads incubated with the DNA tetrahedron are surface-modified with a third group capable of being connected to the first group described in any of the preceding embodiments.

[0047] In some embodiments, the third group comprises a carboxyl group.

[0048] In some embodiments, before incubating the magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads with target antibodies, the preparation method further comprises mixing the magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads with a blocking agent to block the excess sites on the magnetic beads.

[0049] In some embodiments, the blocking agent comprises an ethanolamine solution with a volume fraction of 10%.

[0050] In some embodiments, before incubating the magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads with target antibodies, the preparation method further comprises: mixing the magnetic beads having the DNA tetrahedron coupled to the surface of the magnetic beads with an activating agent to activate the second group in the preceding embodiments to an NHS ester group.

[0051] In some embodiments, the activating agent comprises an NHS-PEG4-azide solution.

[0052] In some embodiments, 0.01-1 μmol of the activating agent is added to 1-5 mg of the magnetic beads to which the DNA tetrahedron is coupled on the surface. The 1-5 mg can be specifically any one of 1, 2, 3, 4 and 5 mg or a range between any two of them. The 0.01-1 μmol can be specifically any one of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 μmol or a range between any two of them.

[0053] In some embodiments, the conditions for mixing the magnetic beads to which the DNA tetrahedron is coupled on the surface and the activating agent include a temperature of 0-35 °C and a time of 1-10 h. The temperature can be specifically any one of 0, 5, 10, 15, 20, 25, 30 and 35 °C or a range between any two of them. The time can be specifically any one of 1, 2, 4, 6, 8 and 10 h or a range between any two of them.

[0054] In some embodiments, the mixing ratio of the DNA tetrahedron to the target antibody is 0.1 nmol: 4-10 μg.

[0055] In some embodiments, the conditions for mixing the DNA tetrahedron and the target antibody include a temperature of 0-35 °C and a time of 2-5 h. The temperature can be specifically any one of 0, 5, 10, 15, 20, 25, 30 and 35 °C or a range between any two of them. The time can be specifically any one of 2, 3, 4 and 5 h or a range between any two of them.

[0056] In another aspect, an embodiment of the present application also provides a kit, which comprises the detection probe of any of the foregoing embodiments.

[0057] In some embodiments, the kit further comprises any one or more of the enzyme-labeled hapten, a combination of the hapten and the enzyme capable of forming the enzyme-labeled hapten, the substrate developing solution, the magnetic bead activating agent, the magnetic bead washing solution, the blocking agent of any of the foregoing embodiments and the activating agent of any of the foregoing embodiments.

[0058] In some embodiments, the enzyme comprises any one or more of horseradish peroxidase (HRP) and alkaline phosphatase.

[0059] In some embodiments, the substrate developing solution comprises tetramethylbenzidine.

[0060] In addition, the embodiments of the present application also provide the use of the detection probe as described in any of the foregoing embodiments or the detection probe prepared by the preparation method as described in any of the foregoing embodiments or the kit as described in any of the foregoing embodiments in detecting a pesticide.

[0061] In some embodiments, the pesticide comprises imidacloprid.

[0062] In some embodiments, the sample to be detected comprises a vegetable sample, which can be one or more of Chinese cabbage, zucchini and cucumber.

[0063] In some embodiments, the detection comprises the following steps: mixing the detection probe, the enzyme-labeled hapten and the sample solution to be detected to perform a reaction; mixing the magnetic beads after the reaction and the substrate color developing solution to obtain a detection signal; and performing qualitative and / or quantitative analysis on the sample to be detected according to the detection signal.

[0064] In some embodiments, the reaction conditions comprise 20-45℃ and 0.1-2h. The temperature can be any one or a range between any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 45℃. The time can be any one or a range between any two of 0.1, 0.5, 1, 1.5 and 2h.

[0065] In some embodiments, the enzyme-labeled hapten has an action concentration of 1-10mg / L, which can be any one or a range between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10mg / L.

[0066] The features and performances of the present application are further described in detail below in combination with embodiments.

[0067] Embodiment 1

[0068] Reagents and instruments used in the experiment:

[0069] Nucleic acid sequences were synthesized and purified by Sangon Co., Ltd. (Shanghai, China). MBs containing 150 μmol / g-COOH (10 mg / mL, 180 nm) were provided by Allrun Co. (Shanghai, China). Monoclonal antibody of imidacloprid was provided by Zhejiang University (Hangzhou). Hapten (imidacloprid) was synthesized by the Institute of Chemistry, Chinese Academy of Sciences (Beijing, China). Imidacloprid standard (100 mg / kg) was provided by Beijing Manhai Gene Biotechnology Co., Ltd. (Beijing, China). TMB (3,3,5,5-tetramethylbenzidine) one-component substrate solution, phosphate buffered saline tablets, horseradish peroxidase (HRP), and tris(hydroxymethyl)aminomethane were purchased from Beijing Solabio Biotech Co., Ltd. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), bovine serum albumin (BSA), and ovalbumin (OVA) were from Shanghai Yuenye Biotech Co., Ltd. Tween 20 was purchased from Shanghai Melin Biological Technology Co., Ltd. Animmuno transparent flat-bottom 96-well plates (part number 439454#), NHS-PEG4-azide, and 10x TBE electrophoresis buffer were purchased from Thermo Fisher Scientific. Methanol was purchased from National Pharmaceutical Group. The polyacrylamide kit was purchased from Jiangsu Kangwei Century Company.

[0070] The sequence information used in the examples is shown in Table 1.

[0071] Table 1 Sequence information

[0072]

[0073] Example 1: Synthesis of DNA tetrahedron

[0074] The nucleic acids (S1-NH2, S2-NH2, S3-DBCO, and S4-NH2, sequence shown in Table 1) were diluted to 100 μM with TE buffer, respectively. EP tubes were taken, 1 μL of nucleic acid solution (100 μM S1-NH2, S2-NH2, S3-DBCO, S4-NH2) was added, 96 μL of TM buffer (20 mM Tris, pH = 8.0, 50 mM MgCl2) was added, and after mixing, it was placed in a PCR at 95 °C for 10 min, cooled at 4 °C for 20 min, and used at 4 °C.

[0075] The synthesized DNA tetrahedron (TDN) was characterized by TEM, AFM, DLS, and PAGE, respectively. The transmission electron micrograph is shown in (a) of FIG. 1, and the morphology is in the shape of a regular tetrahedron. The PAGE image is shown in (b) of FIG. 1, and as the number of nucleic acid chains increases, the migration distance of the DNA gel band becomes shorter, proving the synthesis of the DNA tetrahedron. The DLS image is shown in FIG. 2, and the average particle size of the DNA tetrahedron is about 180 nm. Figure 1 Figure 1 Figure 1 ​​(c) in FIG. 1, which demonstrates the size of DNA tetrahedron is about 8 nm. AFM image (d) in FIG. 1 demonstrates the successful synthesis of DNA tetrahedron. Figure 1 AFM image (d) in FIG. 1 demonstrates the successful synthesis of DNA tetrahedron.

[0076] Example 2: Preparation of MB@TDN@Ab detection probe

[0077] Take 100 μL of 180 nm surface carboxyl modified magnetic beads (1 mg) MB, wash twice with 500 μL of MEST buffer (0.01 M, 0.05% Tween 20, pH 6.0), then add 200 microliters of EDC (5 mg / mL, MES 0.01 M, pH=6.0) and 200 microliters of NHS (5 mg / mL, MES 0.01 M, pH=6.0), and rotate for 30 min at 25°C. After magnetic separation, 100 μL of 1 μmol / L TDN (PBS 0.01 M, pH=7.4) is mixed with MB and incubated at room temperature overnight. After magnetic separation, the supernatant is discarded, 500 μL of 10% ethanolamine (PBS 0.01 M, pH=7.4) is added and incubated at room temperature for 1 h to block the excess sites. The supernatant is discarded after magnetic separation, washed with PBS for 3 times, 10 μL of NHS-PEG4-azide (10 mM, DMSO) is added and incubated at room temperature for 3 h to activate the NHS ester group at the top of TDN, obtaining MB@TDN-NHS ester. The supernatant is discarded after magnetic separation, washed with PBS for 3 times, 10 μg of imidacloprid (PB0.1 M, pH=8.0) mAbs (monoclonal antibody) is mixed with the sediment and incubated at 25°C for 2 hours. After magnetic separation, wash three times, resuspend with BSA 0.5%, 1% PEG (PBS 0.01 M, pH=7.4), and store at 4°C. Finally, MB@TDN@Ab is obtained.

[0078] Principle verification: Zeta potential is used to characterize the surface potential of MB, MB@TDN and MB@TDN@Ab. It can be seen that the surface potential of MB after modification with TDN decreases sharply, which is caused by the negative charge of the phosphate backbone of TDN, indicating the successful loading of TDN on the surface of MB. The potential of MB@TDN@Ab increases sharply compared with that of MB@TDN, which is caused by the combination of antibody and TDN. The results are shown in FIG. 2. Figure 2 (MB, MB@TDN, MB@TDN@Ab).

[0079] Example 3: Optimization of antibody concentration of MB@TDN conjugate

[0080] 0.5, 1, 2, 5, 8, 10 μg of the antibody was added to the MB@TDN-NHS ester obtained in Example 2, respectively, and dissolved in PB solution to 500 μL, and incubated for 2 h. The blocked microplate was taken, 50 μL of MB@TDN@Ab probe was added to each well, and 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (containing imidacloprid at a concentration of 50 ng / mL) were added. The three times were repeated, and the reaction was carried out at 37°C for 1 h. The PBST buffer was washed three times by magnetic separation, 50 μL of PBS was added for reconstitution, and 100 μL of TMB was added for color development for 15 min, and the absorbance value at 650 nm was read. Figure 3 The absorbance value and inhibition rate of MB@TDN@Ab at different antibody concentrations under the same HRP-hapten and imidacloprid concentrations are shown. With the increase of the antibody concentration, the absorbance value and the inhibition rate both show an increasing trend.

[0081] Example 4: Preparation of MB@Ab detection probe

[0082] Take 100 μL of 180 nm surface carboxyl modified magnetic beads (1 mg) MB, disperse in 500 μL of MEST buffer (0.01 M, 0.05% Tween 20, pH 6.0) and wash twice, then add 200 microliters of EDC (5 mg / mL, MES 0.01 M, pH=6.0) and 200 microliters of NHS (5 mg / mL, MES 0.01 M, pH=6.0), and rotate for 30 min at 25°C. After magnetic separation, 10 μg of imidacloprid (PB 0.1 M, pH=8.0) mAbs (monoclonal antibodies) are mixed with the sediment and incubated at 25°C for 2 hours. Wash three times by magnetic separation, discard the supernatant, add 500 μL of 10% ethanolamine (PBS 0.01 M, pH=7.4) and incubate at room temperature for 1 h to block the excess sites. Magnetic separation, discard the supernatant, wash with PBS three times, reconstitute with 0.5%, 1% BSA, 1% PEG (PBS 0.01 M, pH=7.4), and use at 4°C. Finally, MB@Ab is obtained.

[0083] Example 5: Sensitivity of MB@Ab and MB@TDN@Ab method

[0084] Take the blocked 96-well microplate, add 50 μL of MB@TDN@Ab probe (Example 2) 0.3 mg / mL solution, then add 50 μL of 1 mg / L HRP-hapten and 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 8, 10, 15, 20, 25, 50, 100 ng / mL of imidacloprid at different concentrations (10% methanol solution), and react at 37°C for 1 h. After washing three times by magnetic separation, add 50 μL of PBS for reconstitution and 100 μL of TMB for color development for 15 min, and read the absorbance value at 650 nm.

[0085] Take the closed 96-well plate, add MB@Ab probe (Example 4) 0.3 mg / mL solution 50 μL, and then add 1 mg / L HRP-hapten 50 μL and different concentrations of imidacloprid (10% methanol solution) 0, 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100 ng / mL, react at 37°C for 1 h, then wash three times with magnetic separation, add 50 μL PBS and 100 μL TMB for color development for 15 min, and read the absorbance at 650 nm.

[0086] The results are shown in Table 1. Figure 4 , Figure 5 Under the same magnetic bead concentration, antibody concentration and coupling conditions, the sensitivity IC 50 of MB@TDN@Ab is as low as 1.40 ng / mL, with a linear range of 0.05-50 ng / mL, while the sensitivity of MB@Ab is only 2.66 ng / mL, with a linear range of 0.05-5 ng / mL; the sensitivity is improved by about 2 times, and the linear range is increased by 1 order of magnitude. Thus, it is determined that the linear range of MB@TDN@Ab is 1 order of magnitude better than that of MB@Ab, and the sensitivity is higher.

[0087] Example 6: Establishment of imidacloprid standard curve

[0088] 1. Preparation of imidacloprid standard: A series of concentration gradients of imidacloprid standard (concentration gradients include: 0 μg / L, 0.01 μg / L, 0.05 μg / L, 0.1 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 8 μg / L, 10 μg / L, 15 μg / L, 20 μg / L, 25 μg / L, 50 μg / L, 100 μg / L) are prepared using 10% methanol phosphate buffer solution.

[0089] 2. Establishment of standard curve

[0090] (1) Plate sealing: 350 μL of Thermo sealing liquid is added to each well of the 96-well plate, and incubated in a 37°C incubator for 2 hours. Each well is washed 3 times with PBST buffer for standby.

[0091] (2) Immune competition reaction: 50 μL of MB@TDN@Ab probe (Example 2) is added to each well, followed by the addition of 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (which contains the imidacloprid solution as the inhibition well), three times, 37°C for 1 h. Wash with PBST buffer three times with magnetic separation, and standby.

[0092] (3) Color development and reading: add 50 μL PBS reconstitution and 100 μL TMB color development for 15 min, read the absorbance value at 650 nm.

[0093] (4) According to the determination results, the inhibition rate is calculated. The inhibition rate is calculated by the following formula: I (%) = (B0-B) / B0x100%; wherein, B0 is the absorbance value corresponding to the blank control hole, and B is the absorbance value of the inhibition hole.

[0094] (5) The standard curve is established with the logarithm of imidacloprid concentration as the abscissa and the inhibition rate as the ordinate (such as Figure 5 b): Y = 0.223X + 0.4674, R 2 = 0.9703, the linear range is 0.05 μg / L-50 μg / L.

[0095] Example 7: A method for detecting imidacloprid in agricultural product samples is provided.

[0096] The method for drawing the matrix standard curve is as follows:

[0097] 1. Sample pretreatment

[0098] Select Chinese cabbage, cucumber and zucchini (purchased from a local supermarket) as the spiked samples, and homogenize the samples with a homogenizer. Weigh 5 g of the homogenized sample into a 15 mL centrifuge tube, add imidacloprid standard solution to the centrifuge tube to obtain spiked samples with concentrations of 0 μg / L, 0.05 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L and 25 μg / L. Add 5 mL of methanol, manually shake for 10 s, extract for 30 min, and then centrifuge at 8824xg (4°C) for 15 min. Transfer all the supernatant to a 50 mL plastic centrifuge tube, and dilute with methanol to 20 mL. Continue to dilute 5 times with methanol, and finally dilute with PBS buffer solution (10 mM, pH = 7.4) to ensure that the methanol proportion is 10%.

[0099] 2. Establish a standard curve

[0100] (1) Plate coating: use Thermo blocking solution, add 350 μL per well to the microplate, and incubate in a 37°C incubator for 2 hours.

[0101] (2) Immune competition reaction: add 50 μL of MB@TDN@Ab probe (Example 2) per well, then add 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (the solution containing imidacloprid is the inhibition hole), repeat three times, and react at 37°C for 1 h. Wash with PBST buffer solution three times, and reserve.

[0102] (3) Color development and reading: add 50 μL PBS reconstitution and 100 μL TMB color development for 15 min, read the absorbance at 650 nm.

[0103] (4) According to the determination results, calculate the inhibition rate. Inhibition rate (I), formula as follows: I (%) = (B0-B) / B0x100% In the formula, B0 is the absorbance value corresponding to the blank control hole, B is the absorbance value corresponding to the inhibition hole.

[0104] The standard curve is established with the logarithm of imidacloprid concentration as the abscissa and the inhibition rate as the ordinate (such as Figure 6 ), Chinese cabbage: Y = 0.3594X + 0.3285, R 2 = 0.9807, the detection limit is 0.232 μg / L, the linear range is: 0.1-10.0 μg / L; cucumber: Y = 0.3723X + 0.5198, R 2 = 0.9579; the detection limit is 0.074 μg / L, the linear range is: 0.05-10.0 μg / L; zucchini: Y = 0.3642X + 0.3770, R 2 = 0.9990, the detection limit is 0.173 μg / L, the linear range is: 0.2-8 μg / L.

[0105] Example 8: Cross-specificity experiment of MB@TDN@Ab detection method

[0106] According to the steps in Example 3, 50 μL of other pesticides such as acetamiprid, sumithrin, thiamethoxam, forchlorfenuron, triazophos, malathion, and amiram at a concentration of 50 μg / L were coated on the enzyme-labeled plate, and the steps of Example 3 were followed for plate coating, competition reaction, and color development.

[0107] Figure 7 It shows that the method has good selectivity for imidacloprid.

[0108] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A detection probe, characterized in that, It includes: The components include magnetic beads, DNA tetrahedrons, and a target antibody; the target antibody is linked to the magnetic beads via the DNA tetrahedrons; the DNA tetrahedrons are formed by the self-assembly of four single-stranded DNA molecules. Three of the four DNA single strands are modified with a first group, and one DNA single strand is modified with a second group; the DNA tetrahedron is connected to the magnetic bead through the first group and to the target antibody through the second group; the first group and the second group are modified at the 5' end of the DNA single strand; the first group is an NH2 group; the second group is a DBCO group; the four DNA single strands include S1-first group, S2-first group, S3-second group, and S4-first group; The base sequences of S1, S2, S3 and S4 are as shown in SEQ ID NO: 1 to 4.

2. The detection probe according to claim 1, characterized in that, The ratio of the magnetic beads to the DNA tetrahedrons is 1 mg: 0.1~10 nmol.

3. The detection probe according to claim 1, characterized in that, The ratio of the DNA tetrahedron to the target antibody is 0.1 nmol: 4–10 μg.

4. The detection probe according to any one of claims 1 to 3, characterized in that, The target antibody includes an antibody against imidacloprid.

5. The detection probe according to claim 4, characterized in that, The antibodies include monoclonal antibodies and / or polyclonal antibodies.

6. The detection probe according to any one of claims 1 to 3, characterized in that, The size of the magnetic beads is 50–300 nm.

7. The method for preparing the detection probe according to any one of claims 1 to 6, characterized in that, It includes the following steps: DNA tetrahedrons are coupled with magnetic beads and target antibodies.

8. The method for preparing the detection probe according to claim 7, characterized in that, Prior to the coupling, the preparation method further includes synthesizing the DNA tetrahedron.

9. The preparation method according to claim 7, characterized in that, The conjugation includes: mixing and incubating the DNA tetrahedron with magnetic beads to obtain magnetic beads with the DNA tetrahedron conjugated on their surface; and mixing and incubating the magnetic beads with the DNA tetrahedron conjugated on their surface with a target antibody to obtain the detection probe.

10. The preparation method according to claim 9, characterized in that, The mixing ratio of the DNA tetrahedron to the magnetic beads is 0.1~10 nmol:1 mg.

11. The preparation method according to claim 9, characterized in that, The mixing conditions for the DNA tetrahedron and the magnetic beads include: 0–35°C, 3–12 h.

12. The preparation method according to claim 9, characterized in that, The surface of the magnetic beads incubated with the DNA tetrahedron is modified with a third group that can be linked to the first group as described in claim 1; the third group is a carboxyl group.

13. The preparation method according to claim 9, characterized in that, Before incubating the magnetic beads with the DNA tetrahedrons coupled to their surface with the target antibody, the preparation method further includes mixing the magnetic beads with the DNA tetrahedrons coupled to their surface with an inhibitor to block excess sites on the magnetic beads.

14. The preparation method according to claim 13, characterized in that, The blocking agent comprises a 10% (v / v) solution of ethanolamine.

15. The preparation method according to claim 13, characterized in that, Before incubating the magnetic beads with the DNA tetrahedrons coupled to their surface with the target antibody, the preparation method further includes: mixing the magnetic beads with the DNA tetrahedrons coupled to their surface with an activator to activate the second group in claim 1 into an NHS ester group.

16. The preparation method according to claim 15, characterized in that, The activator includes NHS-PEG4-azide.

17. The preparation method according to claim 15, characterized in that, For every 1 mg of magnetic beads with the DNA tetrahedrons coupled to their surface, 0.01–1 μmol of the activator is added.

18. The preparation method according to claim 15, characterized in that, The conditions for mixing the magnetic beads with the DNA tetrahedrons coupled to their surfaces and the activator include: a temperature of 0–35°C and a time of 1–10 h.

19. The preparation method according to claim 9, characterized in that, The mixing ratio of the DNA tetrahedron to the target antibody is 0.1 nmol: 4–10 μg.

20. The preparation method according to claim 9, characterized in that, The mixing conditions for the DNA tetrahedron and the target antibody include: a temperature of 0–35°C and a time of 2–5 hours.

21. A reagent kit, characterized in that, It includes: The detection probe according to any one of claims 1 to 6.

22. The kit according to claim 21, characterized in that, The kit further includes: an enzyme-labeled hapten or a combination of a hapten and an enzyme capable of forming the enzyme-labeled hapten, a substrate chromogenic solution, a magnetic bead activator, a magnetic bead washing solution, any one or more of the blocking agent as described in claim 14 and the activator as described in claim 16.

23. The reagent kit according to claim 22, characterized in that, The enzymes include any one or more of horseradish peroxidase and alkaline phosphatase.

24. The kit according to claim 22, characterized in that, The substrate colorimetric solution includes tetramethylaniline.

25. The use of the detection probe according to any one of claims 1 to 6, or the detection probe prepared by the preparation method according to any one of claims 7 to 20, or the kit according to any one of claims 21 to 24, in the detection of pesticides.

26. The application according to claim 25, characterized in that, The pesticides include imidacloprid.

27. The application according to claim 25, characterized in that, The samples to be tested include vegetable samples.

28. The application according to claim 27, characterized in that, The vegetable samples include any one or more of Chinese cabbage, cucumber, and zucchini.

29. The application according to claim 25, characterized in that, The detection includes the following steps: mixing the detection probe, enzyme-labeled hapten, and sample solution to be tested, and reacting them; mixing the reacted magnetic beads and substrate colorimetric solution to obtain a detection signal; and performing qualitative and / or quantitative analysis on the analyte based on the detection signal.

30. The application according to claim 29, characterized in that, The reaction conditions include: 20–45°C, 1–2 h.

Images