Space-mediated immunoassay method based on DNA tetrahedron and application of space-mediated immunoassay method in imidacloprid residue detection

Abstract

The invention discloses a DNA tetrahedron-based space-mediated immunoassay method and application thereof in imidacloprid residue detection, and relates to the field of pesticide detection.The DNA tetrahedron-based space-mediated immunoassay method is characterized in that a target antibody is coupled to a magnetic bead through a DNA tetrahedron, so that the space density of the target antibody is regulated, spatial ordered arrangement of the target antibody is achieved, and a detection probe MB (at) TDN (at) Ab is formed; according to the present invention, the trace detection of the to-be-detected object can be achieved, the high sensitivity and the high accuracy are provided, the detection sensitivity is improved by about 2 times and the linear range is improved by 1 order of magnitude compared with the traditional magnetic immunoassay method, and the application of the magnetic immunoassay method in the detection of pesticides such as imidacloprid has important significance in food safety guarantee.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and in particular to a DNA tetrahedron-mediated immunoassay method and its application in the detection of imidacloprid residues. Background Art

[0002] Imidacloprid pesticides are widely used in agricultural production for pest control, ensuring vegetable yield and quality. Although highly effective in pest control, their overuse poses a serious threat to the environment and public health, and can even severely damage ecosystems. Therefore, establishing efficient, sensitive, and convenient methods for detecting and analyzing imidacloprid is crucial.

[0003] Immunomagnetic separation has been widely used in the detection of pesticide residues due to its advantages of speed, convenience, simplicity, and economy. However, in traditional magnetic separation immunoassays, the orientation of antibodies on the magnetic bead surface is random and disordered, resulting in the shielding of antibody active sites and reduced recognition efficiency.

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

[0005] The purpose of the present invention is to provide a DNA tetrahedron space-mediated immunoassay method and its application in the detection of imidacloprid residues, so as to solve the problems of random disorder of existing immunomagnetic beads and deep buried antigen recognition sites, and improve the sensitivity, effectiveness and accuracy of detection.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a detection probe comprising: magnetic beads, DNA tetrahedrons, and target antibodies; the target antibodies are connected to the magnetic beads via the DNA tetrahedrons.

[0008] In a second aspect, an embodiment of the present invention provides a method for preparing the detection probe as described in the above embodiment, which comprises the following steps: coupling DNA tetrahedrons with magnetic beads and target antibodies.

[0009] In a third aspect, an embodiment of the present invention provides a kit comprising: the detection probe described in the aforementioned embodiment.

[0010] In a fourth aspect, embodiments of the present invention provide use of the detection probe described in the preceding embodiments, or the detection probe prepared by the preparation method described in the preceding embodiments, or the kit described in the preceding embodiments in detecting pesticides.

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

[0012] (1) The embodiments of the present invention utilize the rigid structure of DNA tetrahedrons to couple DNA tetrahedrons to magnetic beads, spatially arrange monoclonal antibodies on the surface of the magnetic beads in an orderly manner, forming the detection probe MB@TDN@Ab, thereby increasing the exposed area of ​​the antibody active site.

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

[0014] (3) The detection method of the present invention does not require the aid of precision instruments for detection and has the advantages of being low cost, rapid, simple, sensitive and specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Different characterization results of TDN;

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

[0018] Figure 3 Validation plots for optimization of different antibody concentrations;

[0019] Figure 4 Comparison of the sensitivity of MB@Ab and MB@TDN@Ab probes;

[0020] Figure 5 is the solvent standard curve of MB@Ab and MB@TDN@Ab;

[0021] Figure 6 This is the standard curve of MB@TDN@Ab in Chinese cabbage, cucumber, and zucchini matrices;

[0022] Figure 7 This is the specificity verification of the MB@TDN@Ab probe. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0024] The embodiment of the present invention marks the first group on the three tops of the DNA tetrahedron, and the surface of the magnetic bead is modified with a third group that can bind to the first group. The DNA tetrahedron nanostructure is modified on the surface of the magnetic bead by combining the first group and the third group. The fourth top of the DNA tetrahedron is modified with a second group. Through the second group, the DNA tetrahedron and the target antibody are coupled to form a complex of magnetic beads-DNA tetrahedron-target antibody as a detection probe, which is also recorded as MB@TDN@Ab. Utilizing the principle of antigen-antibody specific recognition, the unknown concentration of the analyte (such as imidacloprid) in the sample competes with the enzyme-labeled hapten of known concentration for the active site, thereby detecting the detection of the analyte, with the advantages of high sensitivity, high accuracy, high throughput and high efficiency. Although ordinary immune probes such as MB@Ab can achieve the effect of fixing antibodies, since the antibodies are tightly coupled to the surface of the magnetic beads, they cannot be arranged in the same space as the DNA tetrahedron, and the active sites are deeply buried. The application of the detection probe of the present application to pesticide detection in vegetables is of great significance for ensuring food safety.

[0025] On the one hand, an embodiment of the present invention provides a detection probe, which includes: magnetic beads, DNA tetrahedrons and target antibodies; the target antibodies are connected to the magnetic beads through the DNA tetrahedrons.

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

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

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

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

[0030] In some embodiments, 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 beads through the first group, and is connected to 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 includes an NH2 group;

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

[0034] Three apexes of the DNA tetrahedron are labeled with amino groups, and the DNA tetrahedron nanostructures are modified on the surface of magnetic beads through amide covalent bonds. Then, an NHS-PEG4-azide solution is added to react the DBCO group at the fourth apex of the DNA tetrahedron to form an NHS ester. This NHS ester can be coupled to the target antibody, forming a detection probe.

[0035] In some embodiments, the four single-stranded DNAs include S1-first group, S2-first group, S3-second group, and S4-first group.

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

[0037] In some embodiments, the antibody of interest comprises an anti-imidacloprid antibody.

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

[0039] In some embodiments, the size of the magnetic beads is 50 to 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] On the other hand, an embodiment of the present invention further provides a method for preparing the detection probe as described in any of the above embodiments, comprising the following steps: coupling DNA tetrahedrons with magnetic beads and target antibodies;

[0041] In some embodiments, before performing 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 with a buffer (e.g., TM buffer) and reacting in a PCR instrument. The molar ratio of any two of the four DNA single strands can be 1:1 to 3, specifically 1:1. The reaction conditions can be: heating at 93-97°C for 8-15 minutes, cooling at 0-8°C for 10-30 minutes, specifically heating at 94-96°C for 8-12 minutes, and cooling at 2-6°C for 8-12 minutes.

[0043] In some embodiments, the coupling comprises: mixing and incubating the DNA tetrahedrons with magnetic beads to obtain magnetic beads with the DNA tetrahedrons coupled to their surfaces; mixing and incubating the magnetic beads with the DNA tetrahedrons coupled to their surfaces with target antibodies to obtain the detection probes;

[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 include: 0-35° C., 3-12 h;

[0046] In some embodiments, the surface of the magnetic beads mixed and incubated with the DNA tetrahedrons is modified with a third group that can be connected to the first group described in any of the aforementioned embodiments.

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

[0048] In some embodiments, before the magnetic beads having the DNA tetrahedrons coupled to their surfaces are mixed and incubated with the target antibodies, the preparation method further comprises mixing the magnetic beads having the DNA tetrahedrons coupled to their surfaces with a blocking agent to block redundant sites on the magnetic beads.

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

[0050] In some embodiments, before the magnetic beads having the DNA tetrahedrons coupled to their surfaces are mixed and incubated with the target antibody, the preparation method further comprises: mixing the magnetic beads having the DNA tetrahedrons coupled to their surfaces with an activator to activate the second group in the aforementioned embodiment into an NHS ester group.

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

[0052] In some embodiments, 0.01 to 1 μmol of the activator is added per 1 to 5 mg of the magnetic beads having the DNA tetrahedrons coupled to their surfaces. The 1 to 5 mg amount may specifically be in the range of any one or any two of 1, 2, 3, 4, and 5 mg. The 0.01 to 1 μmol amount may specifically be in the range of any one or any two 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.

[0053] In some embodiments, the conditions for mixing the magnetic beads having the DNA tetrahedrons coupled to their surfaces and the activating agent include: a temperature of 0 to 35° C. and a time of 1 to 10 hours. The temperature can be any one of 0, 5, 10, 15, 20, 25, 30, and 35° C., or a range between any two thereof. The time can be any one of 1, 2, 4, 6, 8, and 10 hours, or a range between any two thereof.

[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 mixing conditions of the DNA tetrahedron and the target antibody include: a temperature of 0 to 35° C. and a time of 2 to 5 hours. The temperature can be any one of 0, 5, 10, 15, 20, 25, 30, and 35° C., or a range between any two thereof. The time can be any one of 2, 3, 4, and 5 hours, or a range between any two thereof.

[0056] On the other hand, an embodiment of the present invention further provides a kit, which includes: the detection probe described in any of the above embodiments.

[0057] In some embodiments, the kit further comprises: an enzyme-labeled hapten or a combination of a hapten and an enzyme capable of forming the enzyme-labeled hapten, a substrate color development solution, a magnetic bead activator, a magnetic bead washing solution, any one or more of the blocking agent described in any of the foregoing embodiments, and the activator described in any of the foregoing embodiments.

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

[0059] In some embodiments, the substrate color developing solution includes: tetramethylaniline.

[0060] In addition, embodiments of the present invention further provide use of the detection probe described in any of the foregoing embodiments, or the detection probe prepared by the preparation method described in any of the foregoing embodiments, or the kit described in any of the foregoing embodiments in detecting pesticides.

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

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

[0063] In some embodiments, the detection includes the following steps: mixing the detection probe, enzyme-labeled hapten and the sample solution to be tested to react; mixing the magnetic beads and substrate color development solution after the reaction to obtain a detection signal; and performing qualitative and / or quantitative analysis on the test object based on the detection signal.

[0064] In some embodiments, the reaction conditions include: 20-45° C., 0.1-2 h. 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° C. The reaction time can be any one or a range between any two of 0.1, 0.5, 1, 1.5, and 2 h.

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

[0066] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0067] Example 1

[0068] Reagents and instruments used in this experiment:

[0069] The nucleic acid sequences are shown in the table below and were synthesized and purified by Sangon Co., Ltd. (Shanghai, China). MBs (10 mg / mL, 180 nm) containing 150 μmol / g-COOH were provided by Allrun Co., Ltd. (Shanghai, China). Imidacloprid monoclonal antibody was provided by Zhejiang University (Hangzhou). Imidacloprid hapten was synthesized by the Institute of Chemistry, Chinese Academy of Sciences (Beijing, China). Imidacloprid standard (100 mg / kg) was provided by Beijing Manhaeger Biotechnology Co., Ltd. (Beijing, China). TMB (3,3,5,5-tetramethylbenzidine) single-component substrate solution, phosphate-buffered saline tablets, horseradish peroxidase (HRP), and tris(hydroxymethyl)aminomethane were purchased from Beijing Solaibao Biotechnology Co., Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), bovine serum albumin (BSA), and ovalbumin (OVA) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. Tween 20 was purchased from Shanghai Mailin Biochemical Co., Ltd. Animmuno clear flat-bottom 96-well plates (Cat. No. 439454), NHS-PEG4-azide, and 10× TBE electrophoresis buffer were purchased from Thermo Fisher Scientific. Methanol was purchased from Sinopharm Group. The polyacrylamide kit was purchased from Jiangsu Kangwei Century Co., Ltd.

[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] Dilute the nucleic acids (S1-NH2, S2-NH2, S3-DBCO, and S4-NH2, sequences shown in Table 1) to 100 μM in TE buffer. Add 1 μL each of the nucleic acid solutions (100 μM S1-NH2, S2-NH2, S3-DBCO, and S4-NH2) to an EP tube, along with 96 μL of TM buffer (20 mM Tris, pH 8.0, 50 mM MgCl2). Mix well and heat at 95°C for 10 minutes in a PCR reaction. Cool at 4°C for 20 minutes and set aside at 4°C.

[0075] The synthesized DNA tetrahedron (TDN) was characterized by TEM, AFM, DLS and PAGE. Figure 1 As shown in (a), its morphology is a regular tetrahedron. Figure 1 As shown in (b), as the number of nucleic acid chains increases, the migration distance of the DNA gel band becomes shorter, proving the synthesis of DNA tetrahedrons. Figure 1As shown in (c), it is proved that the size of DNA tetrahedron is about 8nm. AFM image ( Figure 1 (d)) in the figure demonstrates the successful synthesis of DNA tetrahedron.

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

[0077] 180 nm surface carboxyl-modified magnetic beads (1 mg) of MB were dispersed in 500 μL of MEST buffer (0.01 M, 0.05% Tween 20, pH 6.0) and washed twice. Then, 200 μL of EDC (5 mg / mL, MES 0.01 M, pH 6.0) and 200 μL of NHS (5 mg / mL, MES 0.01 M, pH 6.0) were added and activated with rotation at 25°C for 30 min. After magnetic separation, 100 μL of 1 μmol / L TDN (0.01 M in PBS, pH 7.4) was mixed with the MB and incubated overnight at room temperature. After magnetic separation, the supernatant was discarded, and 500 μL of 10% ethanolamine (0.01 M in PBS, pH 7.4) was added and incubated with rotation at room temperature for 1 h to block excess sites. After magnetic separation, the supernatant was discarded and the pellet was washed three times with PBS. 10 μL of 10 mM NHS-PEG4-azide (DMSO) was added and incubated with rotation at room temperature for 3 hours to activate the NHS ester group on the TDN tip, yielding MB@TDN-NHS ester. After magnetic separation, the pellet was discarded and washed three times with PBS. 10 μg of imidacloprid (0.1 M PBS, pH 8.0) and mAbs (monoclonal antibodies) were added to the pellet, mixed with rotation, and incubated at 25°C for 2 hours. After magnetic separation and washing three times, the pellet was reconstituted with 0.5% BSA and 1% PEG (0.01 M PBS, pH 7.4) and incubated at 4°C until ready for use. MB@TDN@Ab was obtained.

[0078] Proof of principle: Zeta potential was used to characterize the surface potential of MB, MB@TDN, and MB@TDN@Ab. It can be seen that the surface potential of MB modified with TDN dropped sharply, which is caused by the negative charge of the phosphate backbone of TDN, indicating that TDN was successfully loaded on the MB surface. The potential of MB@TDN@Ab increased sharply compared to MB@TDN, which is due to the increase in potential caused by the combination of antibody and TDN. Figure 2 (MB, MB@TDN, MB@TDN@Ab).

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

[0080] To the MB@TDN-NHS ester obtained in Example 2, 0.5, 1, 2, 5, 8, and 10 μg of antibody were added, respectively, and the mixture was reconstituted with PB solution to 500 μL. The mixture was incubated for 2 h. A sealed microplate was placed, and 50 μL of MB@TDN@Ab probe was added to each well. Then, 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (containing 50 ng / mL imidacloprid) were added three times. The mixture was reacted at 37°C for 1 h. The plate was washed three times with PBST buffer, and 50 μL of PBS was added to reconstitute the mixture. 100 μL of TMB was added for color development for 15 min, and the absorbance at 650 nm was read. Figure 3 The absorbance values ​​and inhibition rates of MB@TDN@Ab with different antibody concentrations at the same HRP-hapten and imidacloprid concentrations are shown. With the increase of antibody concentration, the absorbance value and inhibition rate both show an increasing trend.

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

[0082] 100 μL (1 mg) of 180 nm surface carboxyl-modified magnetic beads were dispersed in 500 μL of MEST buffer (0.01 M, 0.05% Tween 20, pH 6.0) and washed twice. Then, 200 μL of EDC (5 mg / mL, MES 0.01 M, pH 6.0) and 200 μL of NHS (5 mg / mL, MES 0.01 M, pH 6.0) were added and activated with rotation at 25°C for 30 min. After magnetic separation, 10 μg of imidacloprid (0.1 M PBS, pH 8.0) and mAbs (monoclonal antibodies) were added to the pellet, mixed with rotation, and incubated at 25°C for 2 hours. After three magnetic washes, the supernatant was discarded and 500 μL of 10% ethanolamine (0.01 M PBS, pH 7.4) was added and incubated with rotation at room temperature for 1 hour to block excess sites. After magnetic separation, the supernatant was discarded, the solution was washed with PBS for 3 times, and re-dissolved with 0.5% BSA and 1% PEG (PBS 0.01M, pH=7.4) and kept at 4°C for use to finally obtain MB@Ab.

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

[0084] A sealed 96-well microplate was added with 50 μL of a 0.3 mg / mL solution of the MB@TDN@Ab probe (Example 2), followed by 50 μL of a 1 mg / L HRP-hapten and different concentrations of imidacloprid (10% methanol solution) at 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 8, 10, 15, 20, 25, 50, and 100 ng / mL. The plates were reacted at 37°C for 1 h, washed three times by magnetic separation, redissolved in 50 μL PBS, and developed with 100 μL TMB for 15 min. The absorbance at 650 nm was read.

[0085] A sealed 96-well microplate was added with 50 μL of a 0.3 mg / mL solution of the MB@Ab probe (Example 4), followed by 50 μL of a 1 mg / L HRP-hapten and different concentrations of imidacloprid (10% methanol solution) at 0, 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, and 100 ng / mL. The plates were reacted at 37°C for 1 h, washed three times by magnetic separation, and reconstituted with 50 μL of PBS and developed with 100 μL of TMB for 15 min. The absorbance at 650 nm was read.

[0086] See the results Figure 4 , Figure 5 Under the same magnetic bead concentration, antibody concentration and coupling conditions, MB@TDN@Ab sensitivity IC 50 The sensitivity of MB@TDN@Ab was approximately 2-fold higher, with a linear range of 0.05-50 ng / mL, compared to 2.66 ng / mL for MB@Ab, with a linear range of 0.05-5 ng / mL. This resulted in an order of magnitude increase in linear range and sensitivity, suggesting that MB@TDN@Ab has an order of magnitude advantage over MB@Ab in terms of sensitivity.

[0087] Example 6: Establishing an Imidacloprid Standard Curve

[0088] 1. Preparation of Imidacloprid Standards: Use 10% methanol phosphate buffer to prepare a series of imidacloprid standards (the concentration gradient includes: 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).

[0089] 2. Establish a standard curve

[0090] (1) Plate coating: Add 350 μL of Thermo blocking solution to each well of a 96-well microplate and incubate in a 37°C incubator for 2 hours. Wash each well three times with PBST buffer and set aside.

[0091] (2) Immunocompetitive reaction: 50 μL of MB@TDN@Ab probe (Example 2) was added to each well, followed by 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (the solution containing imidacloprid was used for the inhibition well). This reaction was repeated three times and allowed to react at 37°C for 1 h. The cells were then magnetically separated and washed three times with PBST buffer for later use.

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

[0093] (4) Based on the above measurement results, the inhibition rate was calculated using the following formula: I (%) = (B0-B) / B0 × 100%; where B0 is the absorbance value corresponding to the blank control well and B is the absorbance value of the inhibition well.

[0094] (5) A standard curve was established with the logarithm of the imidacloprid concentration as the horizontal axis and the inhibition rate as the vertical axis (e.g. Figure 5 b) Y = 0.223X + 0.4674, R 2 =0.9703, and the linear range is 0.05μg / L-50μg / L.

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

[0096] The matrix standard curve was drawn as follows:

[0097] 1. Sample pretreatment

[0098] Chinese cabbage, cucumber, and zucchini (purchased from a local supermarket) were selected as spiked samples and homogenized using a homogenizer. 5 g of the homogenized sample was weighed into a 15 mL centrifuge tube and imidacloprid standard solution was added 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. 5 mL of methanol was added, the mixture was manually shaken for 10 seconds, extracted for 30 minutes, and then centrifuged at 8824 × g (4°C) for 15 minutes. The entire supernatant was transferred to a 50 mL plastic centrifuge tube and made up to 20 mL with methanol. The sample was diluted 5 times with methanol and finally diluted with PBS buffer solution (10 mM, pH = 7.4) to ensure that the methanol ratio was 10%.

[0099] 2. Establish a standard curve

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

[0101] (2) Immunocompetitive reaction: 50 μL of MB@TDN@Ab probe (Example 2) was added to each well, followed by 50 μL of 1 mg / L HRP-hapten and 50 μL of 10% methanol solution (the solution containing imidacloprid was used for the inhibition well). This reaction was repeated three times and allowed to react at 37°C for 1 h. The cells were then magnetically separated and washed three times with PBST buffer for later use.

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

[0103] (4) Calculate the inhibition rate based on the above measurement results. The inhibition rate (I) is calculated as follows: I (%) = (B0 - B) / B0 × 100% where B0 is the absorbance value corresponding to the blank control well and B is the absorbance value corresponding to the inhibition well.

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

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

[0106] According to the steps in Example 3, 50 μL of 50 μg / L other pesticides such as acetamiprid, fenthion, thiamethoxam, chlorfenuron, triazophos, malathion, and isocarbophos, and 50 μL of 50 μg / L imidacloprid were coated on an ELISA plate, and the coating, competitive reaction, and color development were carried out according to the steps in Example 3.

[0107] Figure 7 The results showed that the method had good selectivity for imidacloprid.

[0108] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A detection probe, characterized in that It includes: Magnetic beads, DNA tetrahedrons, and target antibodies; The target antibody is connected to the magnetic beads through the DNA tetrahedron.

2. The detection probe according to claim 1, characterized in that The DNA tetrahedron includes four tops, three of which are connected to the magnetic beads and one is connected to the target antibody; Optionally, the ratio of the magnetic beads to the DNA tetrahedron is 1 mg: 0.1-10 nmol; Optionally, the ratio of the DNA tetrahedron to the target antibody is 0.1 nmol: 4-10 μg.

3. The detection probe according to claim 1, wherein The DNA tetrahedron is formed by self-assembly of four DNA single strands; Optionally, 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 beads via the first group, and is connected to the target antibody via the second group; Optionally, the first group and the second group are modified at the 5' end of the DNA single strand; Optionally, the first group includes an NH2 group; Optionally, the second group includes a DBCO group; Optionally, the four DNA single strands include S1-first group, S2-first group, S3-second group and S4-first group; Optionally, the base sequences of the S1, S2, S3 and S4 are shown in SEQ ID NOs: 1 to 4, respectively.

4. The detection probe according to any one of claims 1 to 3, characterized in that The target antibodies include antibodies against imidacloprid; Optionally, the antibody comprises a monoclonal antibody and / or a polyclonal antibody; Optionally, the size of the magnetic beads is 50 to 300 nm.

5. The method for preparing a detection probe according to any one of claims 1 to 4, wherein: It includes the following steps: Couple the DNA tetrahedron to magnetic beads and target antibodies; Optionally, before performing the coupling, the preparation method further comprises synthesizing the DNA tetrahedron.

6. The preparation method according to claim 5, characterized in that The coupling comprises: mixing and incubating the DNA tetrahedrons with magnetic beads to obtain magnetic beads with the DNA tetrahedrons coupled to the surface; mixing and incubating the magnetic beads with the DNA tetrahedrons coupled to the surface with target antibodies to obtain the detection probes; Optionally, the mixing ratio of the DNA tetrahedron and the magnetic beads is 0.1-10 nmol:1 mg; Optionally, the mixing conditions of the DNA tetrahedron and the magnetic beads include: 0-35° C., 3-12 h; Optionally, the surface of the magnetic beads mixed and incubated with the DNA tetrahedron is modified with a third group capable of connecting to the first group described in claim 3; Optionally, the third group includes a carboxyl group; Optionally, before the magnetic beads having the DNA tetrahedrons coupled to their surfaces are mixed and incubated with the target antibodies, the preparation method further comprises mixing the magnetic beads having the DNA tetrahedrons coupled to their surfaces with a blocking agent to block redundant sites on the magnetic beads; Optionally, the blocking agent comprises a 10% volume fraction ethanolamine solution; Optionally, before the magnetic beads having the DNA tetrahedrons coupled to their surfaces are mixed and incubated with the target antibody, the preparation method further comprises: mixing the magnetic beads having the DNA tetrahedrons coupled to their surfaces with an activator to activate the second group in claim 3 into an NHS ester group; Optionally, the activating agent comprises NHS-PEG4-azide; Optionally, 0.01 to 1 μmol of the activator is added per 1 mg of the magnetic beads having the DNA tetrahedrons coupled to the surface; Optionally, the conditions for mixing the magnetic beads having the DNA tetrahedron coupled to the surface and the activator include: a temperature of 0 to 35° C. and a time of 1 to 10 h; Optionally, the mixing ratio of the DNA tetrahedron and the target antibody is 0.1 nmol: 4-10 μg; Optionally, the mixing conditions of the DNA tetrahedron and the target antibody include: temperature of 0 to 35° C. and time of 2 to 5 hours.

7. A kit, characterized in that It includes: The detection probe according to any one of claims 1 to 4.

8. The kit according to claim 7, characterized in that The kit further comprises: an enzyme-labeled hapten or a combination of a hapten and an enzyme capable of forming the enzyme-labeled hapten, a substrate color development solution, a magnetic bead activator, a magnetic bead washing solution, any one or more of the blocking agent described in claim 6 and the activator described in claim 6; Optionally, the enzyme includes: any one or more of horseradish peroxidase and alkaline phosphatase; Optionally, the substrate color developing solution includes: tetramethylaniline.

9. Use of the detection probe according to any one of claims 1 to 4, or the detection probe prepared by the preparation method according to claim 5 or 6, or the kit according to claim 7 or 8 in detecting pesticides.

10. The use according to claim 9, characterized in that The pesticides include imidacloprid; Optionally, the sample to be tested includes a vegetable sample; Optionally, the vegetable sample includes any one or more of Chinese cabbage, cucumber, and zucchini; Optionally, the detection comprises the following steps: mixing the detection probe, enzyme-labeled hapten and a sample solution to be tested to react; mixing the reacted magnetic beads with a substrate color development solution to obtain a detection signal; and performing qualitative and / or quantitative analysis on the test substance based on the detection signal; Optionally, the reaction conditions include: 20-45° C., 1-2 h.

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