Nanogold probe for enzyme-linked immunoassay, kit and application thereof

Abstract

The application belongs to the technical field of detection, and discloses a novel nano-gold probe for enzyme-linked immunoassay, and a synthesis method thereof comprises the following steps: taking a colloidal gold solution, adding 0.2M K2CO3 to change the pH of the solution to 7.5±0.5, and then adding a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase; adding a PEG20000 solution; adding a milk powder solution, centrifuging to obtain a precipitate; re-dissolving; adding acetamiprid antigen, incubating at a constant temperature, coating; blocking, and oscillating; washing, or drying. The method of the application adopts an immunoassay method. Compared with an instrument analysis method, the immunoassay method can realize on-site detection, does not need to be sent for detection, and controls the cost. In addition to saving the cost, the method of the application makes on-site detection possible.

Description

Technical Field

[0001] This invention belongs to the fields of materials and food technology, and in particular to an enzyme-linked immunosorbent assay (ELISA) for detecting acetamiprid, including a gold nanoparticle probe, a reagent kit, and its application. Background Technology

[0002] The existing technology for detecting acetamiprid has the following problems:

[0003] Problem 1: Unsuitable for on-site testing and costly. Currently, many testing personnel still choose instrumental analysis methods such as high performance liquid chromatography and liquid chromatography-mass spectrometry for acetamiprid residue detection. However, these methods require large instruments and samples need to be transferred to specialized laboratories, making on-site testing impossible and very cumbersome. They also incur transportation and labor costs.

[0004] Question 2: Complex operation and time-consuming. The commonly used traditional enzyme-linked immunosorbent assay (ELISA) method involves many steps and requires professional personnel to operate, which is not only inconvenient for the public to operate, but also incurs additional costs.

[0005] Problem 3: Poor specificity and low sensitivity. Currently, the methods and reagents used in existing technologies have limitations, and the influence of interfering substances cannot be eliminated during the detection process, resulting in poor detection effects and inaccurate detection results.

[0006] The search revealed the following patent publications related to this invention's patent application:

[0007] 1. Patent publication 1 (CN202110432287.5) discloses a double-antibody sandwich ELISA kit for detecting acetamiprid and its application. The kit includes: a capture agent composed of a specific monoclonal antibody and / or polyclonal antibody against acetamiprid, an enzyme-labeled secondary antibody, a sample diluent, and a substrate. The sandwich method requires at least two epitopes on the antigen for antibody binding, thus requiring a specific molecular weight of the antigen. Acetamiprid, being a small molecule, is generally not suitable for the sandwich method. Using the sandwich method not only leads to increased reagent consumption and cost but also prolongs the experimental time, and may not necessarily yield optimal detection results.

[0008] 2. Patent publication 2 (CN201811104470.7) discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting acetamiprid and its application, comprising: an ELISA plate coated with acetamiprid-conjugated antigen, acetamiprid-specific antibody, enzyme-labeled anti-antibody, acetamiprid standard solution, substrate chromogenic solution, stop solution, washing solution, and reconstitution solution; sample pretreatment is performed first, followed by detection using the kit, and finally analysis of the detection results. This patent uses a traditional indirect competitive method to detect acetamiprid, but the demand for rapid sample detection is increasing, and traditional methods are too time-consuming. A faster and more sensitive detection method is desired.

[0009] 3. Patent publication 3 (CN201921841647.1) discloses an acetamiprid colloidal gold immunochromatographic test strip, including a shell and a test strip disposed within the shell for detecting acetamiprid. The immunochromatographic test strip method used in this patent can only make a rough judgment on the sample, which is not conducive to the quantitative detection of the sample.

[0010] By comparison, the present invention patent application is fundamentally different from the aforementioned patent publications. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a gold nanoparticle probe, kit, and application for the detection of acetamiprid using an enzyme-linked immunosorbent assay (ELISA).

[0012] The technical solution adopted by this invention to solve its technical problem is:

[0013] A gold nanoparticle probe for enzyme-linked immunosorbent assay (ELISA), wherein the synthesis method of the gold nanoparticle probe includes the following steps:

[0014] (1) Take a 40 nm colloidal gold solution with a mass concentration of 0.02% (wt%), add 0.2M K2CO3 to change the pH of the solution to 7.5±0.5, and then add a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase, wherein the mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1:2, and react at room temperature for 30 min;

[0015] (2) Add 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles, and react for 30 min;

[0016] (3) Add 4% milk powder solution to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate.

[0017] (4) Dissolve the precipitate in the reconstituted solution and store at 4°C;

[0018] (5) Add 100 μL of 100 ng / mL acetamiprid antigen to a 96-well plate, seal the plate with a sealing film, and incubate it in a 37°C oven for 2 h. Then, incubate it overnight in a -4°C refrigerator for coating.

[0019] (6) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer; add 3% milk powder solution to the washed microplate for sealing, 300 μL per well, then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0020] (7) After sealing, take out the enzyme-labeled plate, pour out the liquid, wash it according to step (6), or dry it in a 37°C oven and then store it in a 4°C refrigerator to obtain the nano gold probe for enzyme-linked immunosorbent assay.

[0021] The ratio of colloidal gold solution: mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase: PEG20000 solution: milk powder solution: reconstituted solution (mL:μg:μL:μL:mL) is 1:5:40:100:1.

[0022] Further, the formulation of the reconstitution solution in step (4) is as follows: 0.01 M PB buffer solution containing 2.5% sucrose, 0.5% BSA, 0.1% PEG20000, and pH=7.5, mixed well.

[0023] The application of the probes described above in the detection of acetamiprid.

[0024] An enzyme-linked immunosorbent assay kit containing the probes described above for acetamiprid.

[0025] The advantages and positive effects of this invention are as follows:

[0026] 1. The method of this invention employs an immunoassay method. Compared to instrumental analysis methods, immunoassay methods enable on-site testing without the need for sample delivery, while also controlling costs. In addition to cost savings, this invention makes on-site testing possible.

[0027] 2. The method of this invention employs the direct method in enzyme-linked immunosorbent assay (ELISA). Typically, for small molecule antigens, indirect competitive methods are chosen to ensure detection effectiveness. However, this invention uses a direct method, which means that only the antibody and sample need to be added to the ELISA plate simultaneously, eliminating the need for a secondary antibody. Besides simplifying the operation, the reaction time in existing technologies generally requires 2.5 hours, while the detection time of this invention is within 1.5 hours, reducing the reaction time by at least 1 hour.

[0028] 3. The method of this invention utilizes colloidal gold and horseradish peroxidase to bind with antibodies for signal amplification. The detection of small molecules using conventional direct methods is limited in effectiveness, exhibiting poor specificity and sensitivity, and a narrow linear range. However, the method of this invention mixes immunoassay monoclonal antibodies and horseradish peroxidase and attaches them to colloidal gold particles. The colloidal gold binds the two together, avoiding cumbersome chemical modifications, thereby achieving signal amplification. This ensures both specificity and detection range while improving sensitivity. The sensitivity (based on my experimental results using traditional methods, IC50) is [value missing]. 50The value is typically 0.164 ng / mL, which is 1.1 times higher than the traditional indirect competitive method. The linear range is 0.05–0.48 ng / mL, and the IC50 value is [missing value]. 50 The value decreased to 0.15 ng / mL.

[0029] 4. The method of this invention uses the direct method in enzyme-linked immunosorbent assay (ELISA), which solves the problems of reagent waste, increased cost, long experimental time, and poor detection results caused by the sandwich method and indirect competitive method respectively used in patent publications 1 and 2. It also solves the problem that patent publication 3 cannot perform quantitative detection of samples. Attached Figure Description

[0030] Figure 1 These are probe diagrams at different pH values ​​in this invention; where 1.5: 1.5 μl of 0.2M K2CO3 was added; 2: 2 μl of 0.2M K2CO3 was added; 2.5: 2.5 μl of 0.2M K2CO3 was added; 3: 3 μl of 0.2M K2CO3 was added; 3.5: 2 μl of 0.2M K2CO3 was added; 4: 4 μl of 0.2M K2CO3 was added; 4.5: 4.5 μl of 0.2M K2CO3 was added.

[0031] Figure 2 These are probe maps for different amounts of protein mixture used in this invention; where 2: protein mixture amount is 2 μg; 5: protein mixture amount is 2 μg; 10: protein mixture amount is 2.5 μg; 15: protein mixture amount is 15 μg; 20: protein mixture amount is 20 μg.

[0032] Figure 3 The diagram shows probes with different protein mixing ratios in this invention; where 2, 1, 1 / 2, 1 / 4, and 1 / 6 represent the mass ratios of anti-acetamiprid monoclonal antibody to horseradish peroxidase as 2, 1, 1 / 2, 1 / 4, and 1 / 6, respectively.

[0033] Figure 4 The UV spectrum used to verify the catalytic activity of the probe in this invention is shown below. TMB, TMB+H2O2, TMB+H2O2+AuNPs, and TMB+H2O2+synthesized probe represent: TMB chromogenic solution without 1% H2O2, with 50 μL of 0.01M PBS added; TMB chromogenic solution with 50 μL of 0.01M PBS added; TMB chromogenic solution with 50 μL of 0.02% (wt%) 40 nm colloidal gold solution added; and TMB chromogenic solution with 50 μL of the probe synthesized in step 1.5.

[0034] Figure 5The UV spectrum of the probe protein labeling used in this invention is shown below; where AuNPs, AuNPs+HRP, AuNPs+mAb, and AuNPs+HRP+mAb represent: 0.02% (wt%) 40 nm colloidal gold solution; 0.02% (wt%) 40 nm colloidal gold solution with a certain amount of HRP added; 0.02% (wt%) 40 nm colloidal gold solution with a certain amount of antibody added; and 0.02% (wt%) 40 nm colloidal gold solution with a certain amount of HRP and antibody mixed together.

[0035] Figure 6 These are transmission electron microscope (TEM) images from this invention; where a and c represent 0.02% (wt%) 40 nm colloidal gold solution; and b and d represent the synthesized probe.

[0036] Figure 7 This is a particle size distribution chart for the present invention; where a: 0.02% (wt%) 40 nm colloidal gold solution; b: synthetic probe;

[0037] Figure 8 Standard curve for traditional enzyme-linked immunosorbent assay (ELISA) of acetamiprid;

[0038] Figure 9 This is a standard curve diagram of the acetamiprid enzyme-linked immunosorbent assay (ELISA) method in this invention. Detailed Implementation

[0039] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0040] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0041] A gold nanoparticle probe for enzyme-linked immunosorbent assay (ELISA), wherein the synthesis method of the gold nanoparticle probe includes the following steps:

[0042] (1) Take a 40 nm colloidal gold solution with a mass concentration of 0.02% (wt%), add 0.2M K2CO3 to change the pH of the solution to 7.5±0.5, and then add a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase, wherein the mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1:2, and react at room temperature for 30 min;

[0043] (2) Add 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles, and react for 30 min;

[0044] (3) Add 4% milk powder solution to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate.

[0045] (4) Dissolve the precipitate in the reconstituted solution and store at 4°C;

[0046] (5) Add 100 μL of 100 ng / mL acetamiprid antigen to a 96-well plate, seal the plate with a sealing film, and incubate it in a 37°C oven for 2 h. Then, incubate it overnight in a -4°C refrigerator for coating.

[0047] (6) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer; add 3% milk powder solution to the washed microplate for sealing, 300 μL per well, then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0048] (7) After sealing, take out the enzyme-labeled plate, pour out the liquid, wash it according to step (6), or dry it in a 37°C oven and then store it in a 4°C refrigerator to obtain the nano gold probe for enzyme-linked immunosorbent assay.

[0049] The ratio of colloidal gold solution: mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase: PEG20000 solution: milk powder solution: reconstituted solution (mL:μg:μL:μL:mL) is 1:5:40:100:1.

[0050] Preferably, the formulation of the reconstitution solution in step (4) is: 0.01 M PB buffer solution containing 2.5% sucrose, 0.5% BSA, 0.1% PEG20000, and pH=7.5, which is then mixed.

[0051] The application of the probes described above in the detection of acetamiprid.

[0052] An enzyme-linked immunosorbent assay kit containing the probes described above for acetamiprid.

[0053] Specifically, the relevant preparation and testing methods are as follows:

[0054] Process steps and parameters

[0055] 1. Synthesis and Characterization of Gold Nanoprobes

[0056] 1.1 Investigating the pH of the probe system:

[0057] (1) Take 1 mL of 0.02% (wt%) 40 nm colloidal gold solution and add 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 μL of 0.2M K2CO3 to change the pH of the solution. Then add 2.5 μg of anti-acetamiprid monoclonal antibody and 2.5 μg of horseradish peroxidase mixture and react at room temperature for 30 min.

[0058] (2) Add 40 μL of 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles and react for 30 min.

[0059] (3) Add 100 μL of 4% milk powder solution to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate.

[0060] (4) Dissolve the precipitate in 1 mL of reconstitution solution and store at 4 °C. The reconstitution solution is prepared as follows: 0.01 M PB buffer solution containing 2.5% sucrose, 0.5% BSA, 0.1% PEG20000, and pH 7.5. Mix well.

[0061] (5) Add 100 μL of 100 ng / mL acetamiprid antigen to a 96-well plate, seal the plate with a sealing film, and incubate at 37°C for 2 h, then incubate at -4°C overnight for coating.

[0062] (6) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer. Add 3% milk powder solution to the washed microplate for sealing, 300 μL per well, then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0063] (7) After sealing, remove the enzyme-labeled plate, pour out the liquid, wash it according to step (6), or dry it in a 37°C oven and then store it in a 4°C refrigerator.

[0064] (8) Dilute the probe reconstituted in step (4) 5 times and add it to each well of the microplate, 50 μL per well, two wells in total. Then add 50 μL of 0.01M PBS buffer to one well and 50 μL of 100 ng / mL acetamiprid standard to the other well. Shake at room temperature for 1 hour. Set up 3 replicates.

[0065] (9) After the reaction, remove the microplate, wash it 5 times with a plate washer, and prepare the chromogenic solution (TMB chromogenic solution: consists of three parts, namely substrate buffer, TMB stock solution and 1% H2O2. Preparation of substrate buffer (pH 3.8): Dissolve 46.04 g potassium dihydrogen citrate hydrate (99% dry basis, Alfa Aesar, Cat. No. 44415) and 0.10 g potassium sorbate (Sigma-Aldrich, 85520) in 1 mL of ultrapure water. No pH adjustment is required, and store at room temperature. Heating, such as microwave, may be necessary to promote dissolution. Preparation of TMB stock solution: Dissolve 375 mg (or 562.5 mg) 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich, T2885) in 30 mL (or 45 mL) dimethyl sulfoxide (DMSO, EMD). In Millipore (MX1458-3), store in the dark. To prepare 1% H2O2: Add 1 mL of 30% H2O2 (Fisher Scientific, H325-500) to 29 mL of ultrapure water to obtain 1% H2O2, and store in the dark at 4℃. Prepare the TMB chromogenic solution for ELISA fresh before use: Add 101 μL of 1% H2O2 and 200 μL of stock TMB solution to 11 mL of substrate buffer. (The TMB chromogenic solution formulation is the same in subsequent steps). Add 100 μL to each well of the microplate and react at room temperature for 15 min.

[0066] (10) After the color development is complete, take out the microplate, add 100 μL of 1M H2SO4 to each well to stop the reaction, and then immediately put it into the microplate reader for detection at a wavelength of 450 nm.

[0067] (11) Based on the measured absorbance value, select the pH with the best absorbance and inhibition effect, which is the optimal pH of the probe system.

[0068] (12) As shown in Table 1, the probe reaction effect is best when the pH is lower, i.e., when the amount of 0.2M K2CO3 added is smaller. However, for overall inhibition, the optimal pH is achieved when the amount of 0.2M K2CO3 added is 2.5 μL and 3 μL. The stability of colloidal gold affects its color; the more purple the color, the greater the degree of aggregation of the colloidal gold, and therefore the less stable the colloidal gold. According to... Figure 1 As can be seen, the less 0.2M K2CO3 added, the more purple the color becomes. Therefore, based on the above results, adding 3 μL of 0.2M K2CO3 to 1 mL of 0.02% (wt%) 40 nm colloidal gold solution, i.e., pH 7.5, is selected as the optimal pH for the probe.

[0069] Table 1 Absorbance values ​​of the probe system at different pH values

[0070]

[0071] 1.2 Calculation of the amount of protein mixture used in the probe system:

[0072] (1) Take 1 mL of 0.02% (wt%) 40 nm colloidal gold solution and add a certain amount of 0.2M K2CO3 to achieve the pH optimized in step 1.1. Then add 2, 5, 10, 15, 20, and 30 μg of anti-acetamiprid monoclonal antibody and horseradish peroxidase protein mixture at a mass ratio of 1:1 and react at room temperature for 30 min. The remaining steps are the same as in 1.1.

[0073] (2) Based on the measured absorbance value, select the amount of protein mixture with the best absorbance and inhibition effect, which is the optimal amount of protein mixture for the probe system.

[0074] (3) According to the data in Table 2, when 5 or 10 μg of protein mixture is added to 1 mL of 0.02% (wt%) 40 nm colloidal gold solution, the probe reaction and inhibition effects are good. Considering the overall protein consumption, the optimal amount of protein mixture is 5 μg. Furthermore, based on... Figure 2 As can be seen, the probe is purple-red when using 5 μg of protein mixture, indicating that the probe has high stability at this amount, proving that this amount is feasible.

[0075] Table 2 Absorbance values ​​of probe systems with different amounts of protein mixture

[0076]

[0077] 1.3 Investigating the protein mixing ratio of the probe system:

[0078] (1) Take 1 mL of 0.02% (wt%) 40 nm colloidal gold solution and add a certain amount of 0.2 M K2CO3 to achieve the pH optimized in step 1.1. Then, mix the anti-acetamiprid monoclonal antibody and horseradish peroxidase at mass ratios of 2:1, 1:1, 1:2, 1:4, and 1:6 and add them to the anti-acetamiprid monoclonal antibody and horseradish peroxidase protein mixture with the optimal protein dosage optimized in step 1.2. React at room temperature for 30 min. The remaining steps are the same as in 1.1.

[0079] (2) Based on the measured absorbance value, select the protein mixing ratio with the best absorbance value and the best inhibition effect, which is the optimal protein mixing ratio of the probe system.

[0080] (3) According to the data in Table 3, when the mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1 / 2 in 1 mL of 0.02% (wt%) 40 nm colloidal gold solution, the probe reaction effect and inhibition effect are better. Considering the overall antibody consumption, the optimal mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1 / 2. Furthermore, based on... Figure 3 As can be seen, at a mass ratio of 1 / 2 anti-acetamiprid monoclonal antibody to horseradish peroxidase, the probe is purple-red, indicating that the probe has high stability at this time, proving that the mass ratio is feasible.

[0081] Table 3 Absorbance values ​​of probe systems with different protein mixing ratios

[0082]

[0083] Among them, mAb / HRP: mass of anti-acetamiprid monoclonal antibody / massradish peroxidase.

[0084] 1.4 Investigating the concentration of the blocking solution in the probe system:

[0085] (1) Take 1 mL of 0.02% (wt%) 40 nm colloidal gold solution and add a certain amount of 0.2M K2CO3 to achieve the pH optimized in step 1.1. Then add the anti-acetamiprid monoclonal antibody and horseradish peroxidase protein mixture at a mass ratio of 1:2 and react at room temperature for 30 min.

[0086] (2) Add 40 μL of 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles and react for 30 min.

[0087] (3) Add 100 μL of milk powder solution with a mass concentration of 2%, 4%, 6%, 8%, and 10% respectively to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate. The remaining steps are the same as in 1.1.

[0088] (4) Based on the measured absorbance value, select the milk powder concentration with the best absorbance value and the best inhibition effect and the lowest nonspecificity, which is the optimal blocking solution concentration of the probe system.

[0089] (5) According to the data in Table 4, the best sealing and detection effect can be achieved when the mass concentration of milk powder is 4%.

[0090] Table 4 Absorbance values ​​of the probe system at different blocking solution concentrations

[0091]

[0092] Meanwhile, through Tables 1 to 4, Figures 1 to 3 It can be seen that the addition of 0.2M K2CO3 to change the pH of the solution to 7.5±0.5, the addition of a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase at a mass ratio of 1:2, and the addition of 4% milk powder solution to block unbound sites have a synergistic effect, which can synergistically improve the relevant performance of the prepared gold nanoparticle probe.

[0093] 1.5 Probe Synthesis:

[0094] (1) Take 1 mL of 0.02% (wt%) 40 nm colloidal gold solution, add 0.2 M K2CO3 to change the pH of the solution to 7.5±0.5, and then add 5 g of a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase, wherein the mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1:2, and react at room temperature for 30 min;

[0095] (2) Add 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles, and react for 30 min;

[0096] (3) Add 4% milk powder solution to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate.

[0097] (4) Dissolve the precipitate in the reconstituted solution and store at 4°C;

[0098] (5) Add 100 μL of 100 ng / mL acetamiprid antigen to a 96-well plate, seal the plate with a sealing film, and incubate it in a 37°C oven for 2 h. Then, incubate it overnight in a -4°C refrigerator for coating.

[0099] (6) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer; add 3% milk powder solution to the washed microplate for sealing, 300 μL per well, then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0100] (7) After sealing, take out the ELISA plate, pour out the liquid, wash it according to step (6), or dry it in a 37°C oven and then store it in a 4°C refrigerator to obtain the nano gold probe for enzyme-linked immunosorbent assay.

[0101] 2. Probe characterization

[0102] 2.1 Characterization of probe catalytic activity using ultraviolet spectrophotometry:

[0103] (1) Add 1 mL of TMB colorimetric solution to four 1.5 mL centrifuge tubes respectively. One tube does not add 1% H2O2 but adds 50 μL of 0.01M PBS; one tube only adds 50 μL of 0.01M PBS; one tube adds 50 μL of 0.02% (wt%) 40 nm colloidal gold solution; and one tube adds 50 μL of the probe synthesized in step 1.5.

[0104] (2) Transfer the four samples to a quartz cuvette and place it in a UV spectrophotometer to detect the absorbance curve at 400-800 nm.

[0105] (3) Use Origin 2021 to plot the absorbance based on the measured absorbance. The horizontal axis is the wavelength and the vertical axis is the absorbance value.

[0106] (4) Because HRP reacts with TMB colorimetric solution to produce a peak at 652 nm, from Figure 4 It can be seen that only the sample with the added probe produced a peak at 652 nm, showing the catalytic activity of HRP, proving that HRP was successfully labeled on colloidal gold and has a catalytic effect.

[0107] 2.2 Ultraviolet spectrophotometer characterization of protein labels:

[0108] (1) Add 1 mL of 0.02% (wt%) 40 nm colloidal gold solution to four 1.5 mL centrifuge tubes respectively. One tube does not contain HRP and antibody; one tube contains the corresponding amount of HRP from step 1.5 (1); one tube contains the corresponding amount of antibody from step 1.5 (1); and one tube contains the corresponding amount of protein mixture from step 1.5 (1). React at room temperature for 30 min.

[0109] (2) Transfer the four samples to a quartz cuvette and place it in a UV spectrophotometer to detect the absorbance curve at 400-600 nm.

[0110] (3) Use Origin 2021 to plot the absorbance based on the measured absorbance. The horizontal axis is the wavelength and the vertical axis is the absorbance value.

[0111] (4) Since the colloidal gold solution produces a peak at approximately 528 nm, Figure 5 It can be seen that the peak of the probe with added protein is red-shifted compared to that of colloidal gold alone, and the red shift is most obvious when the colloidal gold with added protein mixture is added, proving that the protein was successfully labeled on the colloidal gold.

[0112] 2.3 Scanning electron microscopy analysis

[0113] (1) Drop 0.02% (wt%) 40 nm colloidal gold solution and the synthetic probe prepared in step 1.5 onto a copper grid, dry it under a baking lamp, and observe it under a transmission electron microscope.

[0114] (2) The obtained transmission electron microscopy images were used to count the particle size in ImageJ software.

[0115] (3) From Figure 6 and Figure 7 It can be seen that after protein labeling, there is an indistinct white film on the surface of colloidal gold, and the particle size increases slightly, which verifies the successful labeling of protein on colloidal gold.

[0116] 3. Drawing the standard curve

[0117] 3.1 Determination of the working point:

[0118] (1) First, dilute the acetamiprid antigen with a concentration of 4.5 mg / mL to 5 μg / mL with 0.05M CBS (carbonate buffer), then dilute it in half with 0.05M CBS to six concentrations, and then mix the diluted solution evenly with a micro vortex mixer.

[0119] (2) Add the mixed solution to the microplate using a multi-well pipette, 100 μL / well, 2 columns for each concentration. After sealing the microplate with film, incubate it in a 37℃ oven for 2 h, and then incubate it overnight in a -4℃ refrigerator for coating.

[0120] (3) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer.

[0121] (4) After washing the microplate, add 3% milk powder solution to block it, 300 μL per well. Then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0122] (5) After sealing, take out the enzyme-labeled plate, pour out the liquid, wash it according to step (3), or dry it in a 37°C oven and then store it in a 4°C refrigerator.

[0123] (6) Dilute the probe solution synthesized according to step 1.5 with antibody diluent (0.01M PBS buffer) by 1, 2, 4, 8, and 10 times. Take the mixed diluent and add 50 μL of 0.01M PBS buffer / 100ng / ml acetamiprid standard and 50 μL of probe diluent to each well of the microplate using a pipette, one dilution factor per row, decreasing from top to bottom. Then seal the microplate with film and shake at room temperature for 1 hour.

[0124] (7) After the reaction is complete, take out the microplate, wash it 5 times with a plate washer, prepare TMB colorimetric solution, add 100 μL to each well of the microplate, and react at room temperature for 15 min.

[0125] (8) After the color development is complete, take out the microplate, add 100 μL of 1M H2SO4 to each well to stop the reaction, and then immediately put it into the microplate reader for detection at a wavelength of 450 nm.

[0126] (9) Based on the absorbance values ​​in Table 5, the probe dilution factor is determined to be 5 and the coating antigen concentration is 156.25 ng / mL as the working concentration.

[0127] Table 5 Absorbance values ​​at different coating antigen concentrations and probe dilution factors

[0128]

[0129] 3.2 Establishing a standard curve

[0130] (1) Dilute the 1 mg / mL acetamiprid-OVA antigen solution with 0.05M CBS (carbonate buffer) to the selected optimal working antigen coating concentration of 156.25 ng / mL, and then mix the diluted solution evenly with a micro vortex mixer.

[0131] (2) Add the mixed solution to the microplate using a multi-well pipette, 100 μL / well, coat 3 columns, seal the microplate with film, and incubate in a 37℃ oven for 2 h, then incubate overnight in a -4℃ refrigerator for coating.

[0132] (3) After the antigen coating is completed, take out the enzyme-labeled plate, pour out the solution in the plate, and wash it 3 times with a plate washer.

[0133] (4) After washing the microplate, add 3% milk powder solution to block it, 300 μL per well. Then cover the microplate with sealing film and shake at room temperature for 1 hour.

[0134] (5) After sealing, remove the ELISA plate, pour out the liquid inside the plate, wash it as in step (3), pat it dry and it can be used immediately, or dry it in a 37°C oven and then store it in a 4°C refrigerator.

[0135] (6) Take out 1 mg / mL acetamiprid standard and dilute it with standard diluent to seven concentrations: 3.33 ng / mL, 0.83 ng / mL, 0.21 ng / mL, 0.10 ng / mL, 0.05 ng / mL, 0.03 ng / mL, and 0.01 ng / mL. Then mix the diluents evenly with a micro vortex mixer.

[0136] (7) Use a pipette to add the mixed standard dilution to the last 7 rows of the microplate. The first row is used as a control and 0.01M PBS is added. The amount of PBS is reduced from top to bottom, and the amount of PBS is 50 μL / well. Set up 3 replicates for each solution.

[0137] (8) Next, add the optimal probe dilution factor, i.e., 50 μL of probe dilution solution diluted 5 times, and then seal the plate with the ELISA plate and shake at room temperature for 1 hour.

[0138] (9) After the reaction is complete, take out the microplate, wash it 5 times with a plate washer, prepare TMB colorimetric solution, add 100 μL to each well of the microplate, and react at room temperature for 15 min.

[0139] (10) After the color development is complete, add 100 μL of 1M H2SO4 to each well to stop the reaction, and then immediately place the well in a microplate reader for detection at a wavelength of 450 nm.

[0140] Using Origin 2021, we plotted and calculated the absorbance based on the measured absorbance, with the standard concentration as the x-axis and the average absorbance of the three parallel samples as the y-axis. We also calculated the standard deviation and fitted a standard curve.

[0141] The results are as follows Figure 8 , Figure 9 As shown, from Figure 8 , Figure 9 It can be seen that the sensitivity of the signal amplification enzyme-linked immunosorbent assay method established in this invention is slightly improved, and the IC50 value is reduced by about 1.1 times.

[0142] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A gold nanoparticle probe for enzyme-linked immunosorbent assay (ELISA), characterized in that: The method for synthesizing the gold nanoprobe includes the following steps: (1) Take a 0.02% (w / w) 40 nm colloidal gold solution, add 0.2 M K2CO3 to change the pH of the solution to 7.5 ± 0.5, then add a mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase, wherein the mass ratio of anti-acetamiprid monoclonal antibody to horseradish peroxidase is 1:2, and react at room temperature for 30 min; (2) Add 0.5% PEG20000 solution to stabilize and passivate gold nanoparticles, and react for 30 min; (3) Add 4% milk powder solution to block unbound sites. After reacting for 30 min, centrifuge at 9000 rpm for 20 min and collect the precipitate. (4) Dissolve the precipitate in the reconstituted solution and store at 4°C; (5) Add 100 μL of 100 ng / mL acetamiprid antigen to a 96-well plate, seal the plate with a sealing film, and incubate it in a 37°C oven for 2 h. Then, incubate it overnight in a -4°C refrigerator for coating. (6) After coating, remove the microplate, pour out the liquid, and wash it 3 times with a plate washer; add 3% milk powder solution to the washed microplate for sealing, 300 μL per well, then cover the microplate with sealing film and shake at room temperature for 1 hour. (7) After sealing, take out the enzyme-labeled plate, pour out the liquid, wash it according to step (6), or dry it in a 37°C oven and then store it in a 4°C refrigerator to obtain the nano gold probe for enzyme-linked immunosorbent assay. The ratio of colloidal gold solution: mixture of anti-acetamiprid monoclonal antibody and horseradish peroxidase: PEG20000 solution: milk powder solution: reconstituted solution (mL:μg:μL:μL:mL) is 1:5:40:100:

1. The formulation of the reconstitution solution in step (4) is as follows: 0.01 M PB buffer solution containing 2.5% sucrose, 0.5% BSA, 0.1% PEG20000, and pH=7.5, mixed well.

2. The application of the probe as described in claim 1 in the detection of acetamiprid.

3. An enzyme-linked immunosorbent assay kit containing the probe as described in claim 1 or 2 for acetamiprid.

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