A chemiluminescent immunoassay kit for acetamiprid and its preparation method and application

Abstract

The present invention relates to the technical field of chemiluminescent immunoassay for pesticide residues, and more specifically, to a chemiluminescent immunoassay kit for acetamiprid, and a preparation method and application thereof. A chemiluminescent immunoassay kit for acetamiprid comprises a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, liquid A and liquid B; the liquid A comprises 3-amino-phthalhydrazide and a Tris buffer solution; the liquid B comprises a hydrogen peroxide solution. The luminescent kit obtained by the present invention can prolong the continuous luminescence time and avoid problems such as short luminescence time in the natural enzyme catalysis process. The luminescence intensity of the chemiluminescent kit can reach 7×10 4 The luminescence time can last up to 60 minutes, and the chemiluminescence stable platform period can last up to 35 minutes.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide residue chemiluminescence immunoassay, and in particular to an acetamiprid chemiluminescence immunoassay kit and a preparation method and application thereof. Background Art

[0002] Acetamiprid is the most effective insecticide for controlling piercing-sucking mouthparts pests, small lepidoptera and coleoptera pests. It is widely used to prevent pests and diseases in agricultural products such as vegetables, fruits and vegetables, and rice. Improper use of acetamiprid can cause damage to bees and ecosystems such as environmental soil. Therefore, the development of a rapid detection kit for acetamiprid is of great significance.

[0003] Common detection methods for acetamiprid include high performance liquid chromatography and gas chromatography. Traditional detection technologies have disadvantages such as complex operation, high cost, and long time consumption. Chemiluminescent immunoassay not only combines the high sensitivity of chemiluminescent technology and the high specificity of immunoassay, but also has the advantages of simple operation, low cost, and short time consumption.

[0004] In traditional chemiluminescent immunoassays, commonly used catalytic enzymes are HRP natural enzymes, etc. The catalytic activity of natural enzymes changes with changes in ambient temperature, humidity, and pH, and they have poor tolerance to the environment, resulting in poor precision and accuracy of chemiluminescent immunoassay technology. Secondly, the preparation process of natural enzymes is complicated and the cost is relatively high, making it difficult to achieve rapid on-site detection.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One object of the present invention is to provide a chemiluminescent immunoassay kit for acetamiprid to solve the technical problems of existing chemiluminescent kits for pesticide residue detection, such as short luminescence duration and poor environmental tolerance.

[0007] Another object of the present invention is to provide a method for preparing a chemiluminescent immunoassay kit for acetamiprid, which is simple and efficient.

[0008] Another object of the present invention is to provide a chemiluminescent immunoassay method for acetamiprid, which is highly efficient and accurate.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] A chemiluminescent immunoassay kit for acetamiprid, comprising a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, a solution A and a solution B;

[0011] The A solution includes 3-amino-phthalic acid hydrazide and Tris buffer solution;

[0012] The B liquid includes a hydrogen peroxide solution.

[0013] Preferably, in the liquid A, the concentration of 3-amino-phthalic acid hydrazide is 0.005-0.03 mol / L; in the liquid B, the concentration of hydrogen peroxide is 0.015-0.03 mol / L;

[0014] Preferably, the volume ratio of the liquid A to the liquid B is (0.8-1.2): (0.8-1.2);

[0015] Preferably, the concentration of the Tris buffer solution is 0.08-0.12 mol / L, and the pH is 10.5-10.8;

[0016] Preferably, the acetamiprid standard comprises acetamiprid, methanol and water; wherein the concentration of acetamiprid is less than or equal to 200 μg / L.

[0017] Preferably, the nanozyme Au probe comprises the product obtained by labeling the ovalbumin-coupled acetamiprid hapten onto the nanozyme Au particles;

[0018] Preferably, the antibody-labeled microwell plate comprises a product obtained by labeling acetamiprid antibody to a 96-well white microwell plate.

[0019] The preparation method of the acetamiprid chemiluminescent immunoassay kit comprises the following steps:

[0020] Nanozyme Au probes, antibody-labeled microplates, acetamiprid standards, solution A and solution B were prepared respectively.

[0021] Preferably, the method for preparing the nanozyme Au probe comprises the following steps:

[0022] The mixture of the ovalbumin-coupled acetamiprid hapten and the nanozyme Au solution is shaken, centrifuged and washed, and the resulting solid phase is collected and re-dissolved with water;

[0023] Preferably, the method for preparing the nanozyme Au solution comprises: performing a first stirring treatment on chloroauric acid and water under heating conditions, then adding a trisodium citrate solution and performing a second stirring treatment, cooling the obtained mixed system and performing a centrifugal treatment, collecting the solid phase and re-dissolving it with water;

[0024] Preferably, the molar ratio of the chloroauric acid to the trisodium citrate is (0.8-1.2): (3.5-4.2);

[0025] Preferably, the temperature of the first stirring treatment is 99-100° C., and the time of the first stirring treatment is 30-60 seconds;

[0026] Preferably, the second stirring treatment lasts for 18 to 25 minutes.

[0027] Preferably, the nanozyme Au solution is filtered through a 0.22 μm filter membrane;

[0028] Preferably, the shaking treatment is carried out at 36.8-37.2°C, and the shaking treatment time is 25-35 min;

[0029] Preferably, the centrifugal speed is 9000-11000 r / min, and the centrifugal time is 12-16 min;

[0030] Preferably, the washing solution used in the washing comprises phosphate-Tween buffer, and the number of washing times is 3 to 4 times.

[0031] Preferably, the method for preparing the antibody-labeled microplate comprises the following steps:

[0032] The acetamiprid antibody and the 96-well plate were mixed and incubated;

[0033] Preferably, the incubation temperature is 3.5-4.5°C, and the incubation time is 11-13h;

[0034] Preferably, the 96-well plate is washed after the incubation, and the washing solution used for the washing includes phosphate-Tween buffer, and the number of washing times is 3 to 4 times.

[0035] The chemiluminescent immunoassay method of acetamiprid is implemented using the chemiluminescent immunoassay kit for acetamiprid as described above, comprising the following steps:

[0036] The nanozyme Au probe, the antibody-labeled microplate, and the acetamiprid standards of different concentrations are respectively mixed for the first time, and different composite microplates are obtained after washing; the different composite microplates are respectively mixed for the second time with the A solution and the B solution to obtain multiple second mixed systems;

[0037] Determining the chemiluminescence intensity of a plurality of the second mixed systems;

[0038] A standard curve was established according to the concentration of the acetamiprid standard and the luminescence intensity inhibition rate corresponding to each concentration, and the concentration of acetamiprid in each sample was calculated according to the standard curve.

[0039] Preferably, the temperature of the first mixing is 36.8-37.2°C, and the time of the first mixing is 55-65min;

[0040] Preferably, the humidity of the first mixture is 70% to 80%;

[0041] Preferably, the second mixing time is 20 to 30 minutes;

[0042] Preferably, the washing is performed 3 to 4 times.

[0043] Preferably, the standard curve is: y=31.793x+4.5925, wherein x is the concentration of the acetamiprid standard, y is the luminescence intensity inhibition rate, and the correlation coefficient R 2 =0.9677.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The present invention uses easy-to-prepare, low-cost, and storage-resistant nanozyme Au as a substitute for natural enzymes to prepare a nanozyme Au acetamiprid chemiluminescent immunoassay kit. By combining the various components, the continuous luminescence time can be extended to avoid the problem of short luminescence time during the catalysis of natural enzymes. The luminescence intensity of the chemiluminescent kit can reach 7×10 4 The luminescence time can last up to 60 minutes, and the chemiluminescence stable platform period can last up to 35 minutes.

[0046] (2) The preparation method of the acetamiprid chemiluminescent immunoassay kit of the present invention is simple and efficient.

[0047] (3) The chemiluminescent immunoassay method for acetamiprid of the present invention is highly efficient and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 This is the kinetic curve of the chemiluminescent immunoassay kit based on nanozyme Au in the present invention;

[0050] Figure 2 This is a transmission electron microscopy image of the nanozyme Au particles in the present invention;

[0051] Figure 3 It is a linear graph of the concentration and inhibition rate of the acetamiprid standard in the present invention. DETAILED DESCRIPTION

[0052] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0053] According to one aspect of the present invention, the present invention relates to a chemiluminescent immunoassay kit for acetamiprid, comprising a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, a solution A and a solution B;

[0054] The A solution includes 3-amino-phthalic acid hydrazide and Tris buffer solution;

[0055] The B liquid includes a hydrogen peroxide solution.

[0056] The present invention adopts the easy-to-prepare, low-cost and storage-resistant nanozyme Au as a substitute for natural enzymes to prepare the nanozyme Au acetamiprid chemiluminescent immunoassay kit. By combining the various components, the continuous luminescence time is extended to avoid the problem of short luminescence time in the natural enzyme catalysis process.

[0057] In one embodiment, the concentration of 3-amino-phthalic acid hydrazide in the liquid A is 0.005-0.03 mol / L. The concentration of 3-amino-phthalic acid hydrazide includes but is not limited to 0.005 mol / L, 0.008 mol / L, 0.01 mol / L, 0.015 mol / L, 0.02 mol / L, 0.025 mol / L or 0.028 mol / L.

[0058] In one embodiment, the concentration of hydrogen peroxide in the liquid B is 0.015-0.03 mol / L. The concentration of hydrogen peroxide includes but is not limited to 0.018 mol / L, 0.02 mol / L, 0.022 mol / L, 0.25 mol / L or 0.028 mol / L.

[0059] In one embodiment, the volume ratio of the liquid A to the liquid B is (0.8-1.2):(0.8-1.2), for example, 1:1, 0.8:1, 1.2:1 or 1:1.2.

[0060] In one embodiment, the concentration of the Tris buffer solution is 0.08 to 0.12 mol / L, and the pH is 10.5 to 10.8. In one embodiment, the concentration of the Tris buffer solution includes but is not limited to 0.09 mol / L, 0.1 mol / L or 0.11 mol / L.

[0061] In one embodiment, the acetamiprid standard comprises acetamiprid, methanol and water; wherein the concentration of acetamiprid is less than or equal to 200 μg / L. The concentration of acetamiprid includes but is not limited to 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 30 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L, 100 μg / L, 110 μg / L, 120 μg / L, 130 μg / L, 150 μg / L, 160 μg / L, 170 μg / L, 180 μg / L or 190 μg / L.

[0062] In one embodiment, the nanozyme Au probe includes ovalbumin (OVA)-coupled acetamiprid hapten labeled on nanozyme Au particles.

[0063] In one embodiment, the antibody-labeled microwell plate includes the product obtained by labeling acetamiprid antibody to a 96-well white microwell plate.

[0064] According to another aspect of the present invention, the present invention also relates to a method for preparing the acetamiprid chemiluminescent immunoassay kit, comprising the following steps:

[0065] Nanozyme Au probes, antibody-labeled microplates, acetamiprid standards, solution A and solution B were prepared respectively.

[0066] In one embodiment, the Tris buffer solution is prepared by dissolving 1.2114 g of tris(hydroxymethyl)aminomethane in 100 mL of ultrapure water to form a 0.1 mol / L Tris buffer solution with a pH of about 10.5 to 10.8.

[0067] In one embodiment, solution A (luminol solution) is prepared by dissolving 0.0354 g of 3-amino-phthalic acid hydrazide in 20 mL of 0.1 mol / L Tris buffer solution to prepare a 0.01 mol / L luminol solution.

[0068] In one embodiment, the H2O2 solution is prepared from a 30% mass fraction H2O2 solution to form a 0.02 mol / L H2O2 solution.

[0069] In one embodiment, the method for preparing the nanozyme Au probe comprises the following steps:

[0070] The mixture of ovalbumin-coupled acetamiprid hapten and nanozyme Au solution was shaken and then centrifuged for washing, and the obtained solid phase was collected and re-dissolved with water.

[0071] In one embodiment, the method for preparing the nanozyme Au solution comprises: subjecting chloroauric acid and water to a first stirring treatment under heating conditions, adding trisodium citrate solution and subjecting the mixture to a second stirring treatment, cooling the obtained mixed system and subjecting the mixed system to a centrifugal treatment, collecting the solid phase and re-dissolving it with water.

[0072] In one embodiment, the molar ratio of the chloroauric acid to the trisodium citrate is (0.8-1.2):(3.5-4.2), for example, 1:4.

[0073] In one embodiment, the temperature of the first stirring treatment is 99-100° C., and the time of the first stirring treatment is 30-60 seconds, for example, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds.

[0074] In one embodiment, the second stirring treatment lasts for 18 to 25 minutes, for example, 10 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes or 24 minutes.

[0075] In one embodiment, the rotation speed of the centrifugal treatment is 9500-10500 r / min, and the time of the centrifugal treatment is 10-20 min.

[0076] The reconstituted nanozyme Au was stored in an environment of 3.8-4.3°C away from light.

[0077] In one embodiment, the preparation method of nanozyme Au specifically comprises the following steps:

[0078] (a) All glassware used in the experiment, including rotors, three-hole round-bottom flasks, beakers, measuring cylinders, glass vials, etc., must be soaked in aqua regia (VHNO3:VHCl=1:3) for 4 h, rinsed 5-6 times with ultrapure water, and dried for later use; the ultrapure water used in the experiment is distilled water;

[0079] (b) Set up the iron stand, electric heating mantle, and reflux condenser, place the three-hole round-bottom flask on the electric heating mantle, add 100 mL of ultrapure water, put in a 2 cm shuttle magnet, rotate at 360 r / min, turn on the heating mantle, set the temperature to 100 °C, and the power to 500 W;

[0080] (c) When the ultrapure water is heated to a slight boil, add 10% HAuCl4 solution into the flask, stir for 30-60 seconds, quickly add 10 mL of 38.8 mmol / L trisodium citrate solution, continue stirring and heating for 20 minutes, and then turn off the heating mantle;

[0081] (d) Cooling to room temperature under reflux conditions, centrifuging the prepared Au nanozyme at 10,000 r / min for 15 min, removing the supernatant, re-dissolving the Au nanozyme with ultrapure water, and storing the re-dissolved Au nanozyme at 4°C in the dark.

[0082] In one embodiment, the nanozyme Au solution is filtered through a 0.22 μm filter membrane.

[0083] In one embodiment, the shaking treatment is carried out at 36.8-37.2°C, and the shaking treatment time is 25-35 min. In one embodiment, the shaking treatment temperature includes but is not limited to 36.9°C, 37°C or 37.1°C. The shaking treatment time includes but is not limited to 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min or 34 min.

[0084] In one embodiment, the centrifugal speed is 9000-11000 r / min, and the centrifugal time is 12-16 min. The centrifugal speed includes but is not limited to 9100 r / min, 9200 r / min, 9300 r / min, 9400 r / min, 9500 r / min, 9600 r / min, 9700 r / min, 9800 r / min, 9900 r / min, 10000 r / min, 10500 r / min or 10700 r / min.

[0085] In one embodiment, the washing solution used for washing includes phosphate-buffered saline-Tween buffer (PBST), and the number of washings is 3 to 4 times. The concentration of PBST is 0.04 to 0.06 mol / L.

[0086] In one embodiment, the method for preparing the antibody-labeled microplate comprises the following steps:

[0087] The acetamiprid antibody was mixed with the 96-well plate and incubated.

[0088] In one embodiment, the incubation temperature is 3.5-4.5°C, and the time is 11-13 hours. The incubation temperature includes but is not limited to 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4°C, 4.1°C, 4.2°C, 4.3°C or 4.4°C. The incubation time is 11.5h, 12h, 12.5h or 12.7h.

[0089] In one embodiment, the 96-well plate is washed after the incubation, and the washing solution used for the washing includes phosphate-Tween buffer, and the number of washing times is 3 to 4 times.

[0090] According to another aspect of the present invention, the present invention also relates to a chemiluminescent immunoassay method for acetamiprid using the acetamiprid chemiluminescent immunoassay kit, comprising the following steps:

[0091] The nanozyme Au probe, the antibody-labeled microplate, and the acetamiprid standards of different concentrations are respectively mixed for the first time, and different composite microplates are obtained after washing; the different composite microplates are respectively mixed for the second time with the A solution and the B solution to obtain multiple second mixed systems;

[0092] Determining the chemiluminescence intensity of a plurality of the second mixed systems;

[0093] A standard curve was established according to the concentration of the acetamiprid standard and the luminescence intensity inhibition rate corresponding to each concentration, and the concentration of acetamiprid in each sample was calculated according to the standard curve.

[0094] This method is efficient and accurate.

[0095] In one embodiment, the first mixing temperature is 36.8-37.2° C., and the time is 55-65 min. The first mixing temperature includes but is not limited to 36.9° C., 37° C., or 37.1° C. The first mixing time is 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, or 64 min.

[0096] In one embodiment, the first mixed humidity is 70% to 80%, and the first mixed humidity includes but is not limited to 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%.

[0097] In one embodiment, the second mixing time is 20 to 30 minutes, and includes but is not limited to 22 minutes, 25 minutes, 26 minutes, 27 minutes or 29 minutes.

[0098] In one embodiment, the washing is performed 3 to 4 times.

[0099] In one embodiment, the standard curve is: y=31.793x+4.5925, wherein x is the concentration of the acetamiprid standard, y is the luminescence intensity inhibition rate corresponding to each concentration of the acetamiprid standard, and the correlation coefficient R 2 =0.9677.

[0100] The present invention will be further explained below with reference to specific embodiments.

[0101] Figure 1This is the kinetic curve of the chemiluminescent immunoassay kit based on nanozyme Au in the present invention. Figure 2 This is a transmission electron microscopy image of the nanozyme Au particles in the present invention. Figure 3 It is a linear graph of the concentration and inhibition rate of the acetamiprid standard in the present invention.

[0102] Example 1

[0103] A chemiluminescent immunoassay kit based on nanozyme Au includes a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, liquid A and liquid B; the liquid A includes 0.01 mol / L 3-amino-phthalhydrazide and 0.1 mol / L Tris buffer solution, with a pH of 10.5; the liquid B includes a hydrogen peroxide solution, and the concentration of the hydrogen peroxide is 0.02 mol / L.

[0104] Among them, the preparation method of the antibody-labeled microplate includes: taking acetamiprid antibody (10 μg / L), adding it to a 96-well plate, and incubating it at 4°C for 12 hours; after incubating for 12 hours, the 96-well plate needs to be washed three times with 0.05 mol / L PBST washing solution, discarding the washing solution, and placing it in a 4°C refrigerator for standby use.

[0105] The preparation method of the nanozyme Au probe includes: first taking 1 mL of the nanozyme Au solution filtered through a 0.22 μm filter membrane, then taking the OVA-coupled acetamiprid hapten and adding it to the nanozyme Au solution, and shaking it at 37°C for 30 minutes; after shaking for 30 minutes, centrifuging it at 10,000 r / min for 15 minutes. Wash it three times with 0.05 mol / L PBST washing solution, discard the washing solution, and then re-dissolve it with ultrapure water; the preparation method of the nanozyme Au solution specifically includes:

[0106] (a) All glass instruments used in the experiment, such as the rotor, three-hole round-bottom flask, beaker, measuring cylinder, glass vial, etc., need to be soaked in aqua regia (VHNO3:VHCl=1:3) for 4 h, rinsed 5-6 times with ultrapure water, and dried for use; the ultrapure water used in the experiment is distilled water; (b) Build the iron stand, electric heating jacket, and reflux condenser, place the three-hole round-bottom flask on the electric heating jacket, add 100 mL of ultrapure water, put in a 2 cm shuttle magnet, rotate at 360 r / min, turn on the heating jacket, set the temperature to 100 °C, and power on. (c) when the ultrapure water is heated to a slight boil, add 10% HAuCl4 solution into the flask, stir for 40 seconds, quickly add 10mL, 38.8mmol / L trisodium citrate solution, continue stirring and heating for 20 minutes, then turn off the heating jacket; (d) cool to room temperature under reflux conditions, centrifuge the prepared nanozyme Au at 10000r / min for 15 minutes, remove the supernatant, re-dissolve the nanozyme Au with ultrapure water, and store the re-dissolved nanozyme Au at 4°C in a dark environment.

[0107] The chemiluminescent immunoassay method of acetamiprid is implemented using the above-mentioned chemiluminescent immunoassay kit, comprising the following steps:

[0108] Preparation of acetamiprid standard: Use 10% methanol solution to prepare a series of pesticide gradient acetamiprid standards (concentration gradient includes: 0μg / L, 1μg / L, 5μg / L, 10μg / L, 20μg / L, 50μg / L, 100μg / L, 200μg / L) for competitive reaction;

[0109] 20 μL of acetamiprid standard and 20 μL of acetamiprid nanozyme Au probe were added to the antibody-labeled microplate to cause a competitive reaction, and the mixture was incubated at 37°C and 75% humidity for 1 hour; the complex after the competitive reaction was washed with ultrapure water for 3 times; liquid A and liquid B were added to the washed competitive reaction complex microplate, and the mixture was mixed at a volume ratio of 1:1, and the liquid A and liquid B were added to react at room temperature for 25 minutes, and then the chemiluminescence intensity was measured with a multifunctional microplate reader;

[0110] The concentration of the acetamiprid standard was used as the horizontal coordinate, and the luminescence intensity inhibition rate corresponding to each concentration was used as the vertical coordinate to establish a standard curve, and the acetamiprid concentration in each sample was calculated according to the standard curve. The standard curve was: y=31.793x+4.5925, wherein x was the concentration of the acetamiprid standard, y was the luminescence intensity inhibition rate corresponding to each concentration of the acetamiprid standard, and the correlation coefficient R 2 =0.9677.

[0111] Example 2

[0112] A chemiluminescent immunoassay kit based on nanozyme Au includes a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, liquid A and liquid B; the liquid A includes 0.03 mol / L 3-amino-phthalhydrazide and 0.15 mol / L Tris buffer solution, with a pH of 10.8; the liquid B includes a hydrogen peroxide solution, and the concentration of the hydrogen peroxide is 0.015 mol / L.

[0113] The preparation method of the nanozyme Au probe includes: first taking 1 mL of the nanozyme Au solution filtered through a 0.22 μm filter membrane, then taking the OVA-coupled acetamiprid hapten and adding it to the nanozyme Au solution, shaking at 36.8°C for 35 minutes; after shaking for 35 minutes, centrifuging at 11000 r / min for 12 minutes. Washing by centrifugation 4 times with 0.05 mol / L PBST washing solution, discarding the washing solution, and re-dissolving with ultrapure water; the preparation method of the nanozyme Au solution is the same as that in Example 1.

[0114] The preparation method of the antibody-labeled microplate comprises: taking acetamiprid antibody (20 μg / L), adding it to a 96-well plate, and incubating it at 4.5°C for 11 hours; after incubating for 11 hours, the 96-well plate needs to be washed 4 times with 0.05 mol / L PBST washing solution, the washing solution is discarded, and it is placed in a 4°C refrigerator for use.

[0115] The chemiluminescent immunoassay method for acetamiprid is the same as that in Example 1.

[0116] Example 3

[0117] A chemiluminescent immunoassay kit based on nanozyme Au includes a nanozyme Au probe, an antibody-labeled microplate, an acetamiprid standard, liquid A and liquid B; the liquid A includes 0.008 mol / L 3-amino-phthalhydrazide and 0.08 mol / L Tris buffer solution, with a pH of 10.6; the liquid B includes a hydrogen peroxide solution, and the concentration of the hydrogen peroxide is 0.025 mol / L.

[0118] Among them, the preparation method of the nanozyme Au probe includes: first taking 1 mL of the nanozyme Au solution filtered through a 0.22 μm filter membrane, then taking the OVA-coupled acetamiprid hapten and adding it to the nanozyme Au solution, and shaking it at 37.2°C for 25 minutes; after shaking for 25 minutes, centrifuging it at 9000r / min for 16 minutes. Wash it three times with 0.05mol / L PBST washing solution by centrifugation, discard the washing solution, and then re-dissolve it with ultrapure water. The preparation method of the nanozyme Au solution is the same as that in Example 1.

[0119] The preparation method of the antibody-labeled microplate comprises: taking acetamiprid antibody (10 μg / L), adding it to a 96-well plate, and incubating it at 4°C for 13 hours; after incubating for 13 hours, the 96-well plate needs to be washed 4 times with 0.05 mol / L PBST washing solution, the washing solution is discarded, and the plate is placed in a 4°C refrigerator for standby use.

[0120] Experimental example

[0121] The measured data of the concentration of the acetamiprid standard product and the luminescence intensity inhibition rate in Example 1 are shown in Table 1. The curve diagram of the concentration of the acetamiprid standard product and the luminescence intensity inhibition rate is shown in Table 1. Figure 3 shown.

[0122] Table 1 Luminescence intensity inhibition rate corresponding to different concentrations of acetamiprid standard

[0123] Concentration of acetamiprid standard (μg / L) Luminescence intensity suppression rate (%) 10 31.16 20 44.28 50 63.89 100 72.76 200 74.32

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chemiluminescent immunoassay kit for acetamiprid, characterized in that: Includes nanozyme Au probe, antibody-labeled microplate, acetamiprid standard, solution A and solution B; The A solution includes 3-amino-phthalic acid hydrazide and Tris buffer solution; the concentration of the Tris buffer solution is 0.08-0.12 mol / L, and the pH is 10.5-10.8; The B liquid includes a hydrogen peroxide solution; The nanozyme Au probe includes the product obtained by labeling the ovalbumin-coupled acetamiprid hapten onto the nanozyme Au particles; The antibody-labeled microplate includes the product obtained by labeling acetamiprid antibody to a 96-well white microplate; The preparation method of the acetamiprid chemiluminescent immunoassay kit comprises the following steps: Prepare nanozyme Au probe, antibody-labeled microplate, acetamiprid standard, solution A and solution B respectively; The preparation method of the nanozyme Au probe comprises the following steps: The mixture of the ovalbumin-coupled acetamiprid hapten and the nanozyme Au solution is subjected to an oscillation treatment, and then centrifuged and washed, and the obtained solid phase is collected and redissolved with water; the preparation method of the nanozyme Au solution comprises: performing a first stirring treatment on chloroauric acid and water under heating conditions, and then adding a trisodium citrate solution and performing a second stirring treatment, cooling the obtained mixed system, centrifuging, collecting the solid phase and redissolving with water; The method for preparing the antibody-labeled microplate comprises the following steps: The acetamiprid antibody and the 96-well plate are mixed and incubated; the incubation temperature is 3.5-4.5° C., and the incubation time is 11-13 hours.

2. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: In the liquid A, the concentration of 3-amino-phthalic acid hydrazide is 0.005-0.03 mol / L; in the liquid B, the concentration of hydrogen peroxide is 0.015-0.03 mol / L.

3. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The volume ratio of the A liquid to the B liquid is (0.8~1.2):(0.8~1.2).

4. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The acetamiprid standard comprises acetamiprid, methanol and water; wherein the concentration of acetamiprid is less than or equal to 200 μg / L.

5. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The molar ratio of the chloroauric acid to the trisodium citrate is (0.8-1.2):(3.5-4.2).

6. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The temperature of the first stirring treatment is 99-100° C., and the time of the first stirring treatment is 30-60 seconds.

7. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The second stirring treatment time is 18~25min.

8. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that The nanozyme Au solution was filtered through a 0.22 μm filter membrane.

9. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The shaking treatment is carried out at 36.8-37.2° C., and the shaking treatment time is 25-35 min.

10. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The rotation speed of the centrifugation during the further centrifugation and washing is 9000-11000 r / min, and the time of the centrifugation during the further centrifugation and washing is 12-16 min.

11. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The washing solution used in the washing includes phosphate-Tween buffer, and the washing times are 3 to 4 times.

12. The acetamiprid chemiluminescent immunoassay kit according to claim 1, characterized in that: The incubated 96-well plate is washed with a washing solution comprising phosphate-Tween buffer for 3 to 4 times.

13. A chemiluminescent immunoassay method for acetamiprid is carried out using the acetamiprid chemiluminescent immunoassay kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: The nanozyme Au probe, the antibody-labeled microplate, and the acetamiprid standards of different concentrations are respectively mixed for the first time, and different composite microplates are obtained after washing; the different composite microplates are respectively mixed for the second time with the A solution and the B solution to obtain multiple second mixed systems; Determining the chemiluminescence intensity of a plurality of the second mixed systems; A standard curve was established according to the concentration of the acetamiprid standard and the luminescence intensity inhibition rate corresponding to each concentration, and the concentration of acetamiprid in each sample was calculated according to the standard curve.

14. The chemiluminescent immunoassay method of acetamiprid according to claim 13, characterized in that: The temperature of the first mixing is 36.8-37.2° C., and the time of the first mixing is 55-65 min.

15. The chemiluminescent immunoassay method of acetamiprid according to claim 13, characterized in that: The humidity of the first mixture is 70% to 80%.

16. The chemiluminescent immunoassay method of acetamiprid according to claim 13, characterized in that: The second mixing time is 20 to 30 minutes.

17. The chemiluminescent immunoassay method for acetamiprid according to claim 13, characterized in that: The number of times of washing is 3 to 4 times.

18. The chemiluminescent immunoassay method for acetamiprid according to claim 13, characterized in that: The standard curve is: y=31.793x+4.5925, where x is the concentration of the acetamiprid standard, y is the luminescence intensity inhibition rate, and the correlation coefficient R 2 =0.9677.

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