Preparation method of weak-charge nano-gold particles and application of weak-charge nano-gold particles in enzyme-linked immunosorbent assay of pesticide

Abstract

The invention discloses a preparation method of weak-charge nano-gold particles and application of the weak-charge nano-gold particles in enzyme-linked immunosorbent assay of pesticides, and belongs to the technical field of biosensing and immunoassay. The weak-charge nano-gold particles are synthesized by taking ascorbic acid and chloroauric acid as raw materials and adopting a chemical reduction one-pot method. The antibody-probe-enzyme complex can be constructed by simultaneously coupling an antibody and an enzyme marker through electrostatic adsorption by utilizing the good dispersity and stability of the antibody-probe-enzyme complex, and the complex can be directly used for competitive immunoreaction, so that the use of an enzyme-labeled secondary antibody in the traditional ELISA (Enzyme-Linked Immunosorbent Assay) is omitted. The compound is applied to pesticide residue detection, shows high sensitivity and specificity, and is low in cross reaction rate with other common pesticides and strong in anti-interference capability. In addition, the preparation method of the weak-charge nano-gold particles and the compound is simple and easy to operate and low in cost. Therefore, the weak-charge gold nanoparticles, the compound and the detection method provided by the invention have good application prospects.

Description

Technical Field

[0001] This invention relates to the fields of biosensing and immunoassay technology, and more specifically, to a method for preparing weakly charged gold nanoparticles and their application in enzyme-linked immunosorbent assay (ELISA) for pesticide detection. Background Technology

[0002] Triazophos is a broad-spectrum organophosphate insecticide widely used in agricultural production. However, its improper use can lead to residues in agricultural products and the environment, which can accumulate through the food chain and harm the human nervous system. Therefore, establishing rapid, sensitive, and reliable methods for detecting triazophos residues is crucial.

[0003] Currently, the main methods for detecting triazophos are chromatographic methods (such as high-performance liquid chromatography and gas chromatography-mass spectrometry). Although these methods are sensitive and accurate, they require expensive instruments, professional operators, and complex sample pretreatment processes, resulting in high costs and difficulty in achieving rapid on-site detection.

[0004] Enzyme-linked immunosorbent assay (ELISA) offers advantages such as ease of operation, low cost, high throughput, and suitability for on-site screening. Traditional competitive ELISA methods typically employ enzyme-labeled secondary antibodies for signal amplification and detection. However, this method requires the separate preparation of enzyme-labeled primary and secondary antibodies, which is cumbersome; the use of secondary antibodies also increases costs; and the sensitivity is sometimes insufficient for detecting trace pesticide residues.

[0005] To overcome the limitations of traditional ELISA, nanomaterials, especially gold nanoparticles, have been introduced to enhance detection performance. However, traditional gold nanoparticles prepared using the sodium citrate reduction method require precise pH control when conjugating biomolecules. Inappropriate pH can cause aggregation of colloidal gold and antibody conjugates, resulting in poor stability and dispersibility. Therefore, conventional gold nanoparticle labeling strategies are often more complex and difficult to control, and the strong surface charge can affect antibody adsorption, leading to limited detection sensitivity. These reasons limit the application of gold nanoparticles in ELISA, preventing them from meeting practical needs.

[0006] Therefore, there is an urgent need in this field to develop a new ELISA detection method that is simpler to operate, lower in cost, and has higher sensitivity.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing weakly charged gold nanoparticles and their application in enzyme-linked immunosorbent assay (ELISA) for pesticide detection. The weakly charged gold nanoparticles prepared by this invention have good stability and dispersibility, and can be coupled with more antibodies. Furthermore, the weakly charged gold nanoparticles are used as the core of an immunoassay probe to prepare an antibody-probe-enzyme complex, which can improve the sensitivity and repeatability of detection in a competitive immunoassay.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides a method for preparing weakly charged gold nanoparticles, which uses ascorbic acid and chloroauric acid as raw materials and synthesizes gold nanoparticles by a one-pot chemical reduction method. The steps include: stirring and heating an aqueous solution of chloroauric acid, adding an ascorbic acid solution, and carrying out a reduction reaction under constant temperature conditions until a wine-red solution is obtained, thereby obtaining weakly charged gold nanoparticles.

[0010] Secondly, the present invention provides weakly charged gold nanoparticles prepared by the above preparation method.

[0011] Thirdly, the present invention provides an antibody-probe-enzyme complex, which uses the aforementioned weakly charged gold nanoparticles as the core and conjugates an antibody and an enzyme label.

[0012] Fourthly, the present invention provides the application of the above-mentioned weakly charged gold nanoparticles or antibody-probe-enzyme complexes in any one of the following (1) to (2): (1) Preparation of competitive enzyme-linked immunosorbent assay (ELISA) products; (2) Competitive enzyme-linked immunosorbent assay (ELISA) for pesticides.

[0013] Fifthly, the present invention provides a competitive enzyme-linked immunosorbent assay kit comprising the above-described antibody-probe-enzyme complex.

[0014] In a sixth aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) method for detecting pesticide residues, which uses the aforementioned weakly charged gold nanoparticles, antibody-probe-enzyme complexes, or kits for ELISA detection.

[0015] The present invention has the following beneficial effects: This invention synthesizes weakly charged gold nanoparticles using a one-pot method with ascorbic acid as a mild reducing agent and stabilizer, exhibiting good dispersibility and stability. When prepared as an antibody-probe-enzyme complex, these weakly charged gold nanoparticles can adsorb more antibodies, thereby enhancing detection sensitivity. Furthermore, no pH adjustment is required, avoiding the problems of pH adjustment and easy aggregation during the labeling process of traditional gold nanoparticles, ensuring the reliability and reproducibility of detection results. When used in pesticide residue detection, this complex demonstrates high specificity, low cross-reactivity with other common pesticides, and strong anti-interference ability. In addition, the preparation method of the weakly charged gold nanoparticles and complex of this invention is simple and easy to operate, and eliminates the use of enzyme-labeled secondary antibodies in the entire detection process, simplifying the operation steps, shortening the detection time, and significantly reducing costs. Therefore, the weakly charged gold nanoparticles, complex, and detection method of this invention have promising application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a transmission electron microscope image of the weakly charged gold nanoparticles AA-AuNPs prepared in Example 1. Figure 2 The image shows the HRTEM-EDS image of the weakly charged gold nanoparticles AA-AuNPs prepared in Example 1. Figure 3 The image shows the HAADF pattern of the weakly charged gold nanoparticles AA-AuNPs prepared in Example 1 after conjugation with the antibody. Figure 4 The image shows the HAADF of gold nanoparticles (Cit-AuNPs) prepared with conventional sodium citrate ligands in Comparative Example 1 after conjugation with the antibody. Figure 5 The image shows the UV-Vis pattern of the weakly charged gold nanoparticles AA-AuNPs prepared in Example 1. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Although gold nanoparticles have been used in enzyme-linked immunosorbent assay (ELISA), the conventional chemical preparation method uses chloroauric acid as a raw material and sodium citrate as a reducing agent and stabilizer. The resulting gold nanoparticles have a strongly negatively charged surface. When coupled with antibodies (whose isoelectric point pI is typically higher than 7), which also have a negative charge, the pH needs to be adjusted to a specific value slightly higher than the antibody's pI. The nanoparticles carry a weak positive charge, making this process cumbersome and prone to causing nanoparticle aggregation. Furthermore, the strong charge affects the amount of antibody adsorbed, thus impacting the detection sensitivity. Based on these shortcomings, the inventors of this invention have optimized the gold nanoparticles and their preparation method.

[0020] The present invention provides a weakly charged gold nanoparticle (AA-AuNPs). The "weak charge" characteristic of the gold nanoparticle is reflected in its zeta potential of -7mV to -10mV. This characteristic can significantly reduce the electrostatic repulsion between it and the antibody.

[0021] Regarding the preparation method of the aforementioned weakly charged gold nanoparticles, this invention uses ascorbic acid and chloroauric acid as raw materials and employs a one-pot chemical reduction method to synthesize gold nanoparticles. The specific preparation steps are as follows: S1. Soak all glassware used in the synthesis process in aqua regia overnight and rinse thoroughly with ultrapure water.

[0022] Soaking in aqua regia is used to remove all impurities from the glassware. A clean reaction environment can improve the reproducibility of the synthesis and the purity of the product.

[0023] S2. Heat the aqueous chloroauric acid solution with continuous stirring, and quickly add ascorbic acid solution. React at a constant temperature until the solution turns wine red.

[0024] In some embodiments, the aqueous chloroauric acid solution is heated to 90-105°C.

[0025] In some embodiments, the ratio of chloroauric acid to ascorbic acid in the reaction system is 0.5~0.6:1.

[0026] In this invention, ascorbic acid, as a mild reducing agent and stabilizer, reacts with chloroauric acid to synthesize weakly charged gold nanoparticles. The surface charge density of these nanoparticles is lower than that of traditional gold nanoparticles synthesized using sodium citrate as a reducing agent.

[0027] S3. Cool the wine-red solution to room temperature, then filter it through a microporous membrane. Centrifuge the filtrate, discard the supernatant, and redissolve the precipitate in ultrapure water to restore it to its original volume to obtain a stock solution of weakly charged gold nanoparticles.

[0028] In some embodiments, the pore size of the microporous filter membrane is 0.22 μm.

[0029] In some embodiments, the conditions for centrifuging the filtrate are: temperature of 2~7℃, rotation speed of 8000~10000 rpm / min, and time of 20~30 min.

[0030] In S3, filtration is used to remove potential macro-aggregates, while centrifugation is used to remove unreacted reagents.

[0031] Based on the weak charge properties of the aforementioned gold nanoparticles, they can be applied to enzyme-linked immunosorbent assays (ELISA). Specifically, they can be prepared as ELISA reagents. More specifically, this invention applies them to the preparation of antibody-probe-enzyme complexes. These complexes use the aforementioned weakly charged gold nanoparticles as the core, utilizing their good dispersibility and stability to simultaneously couple antibodies and enzyme markers through electrostatic adsorption.

[0032] In this invention, the antibodies coupled to the antibody-probe-enzyme complex can be adjusted and selected according to the detection purpose, and there is no limitation thereto. Any antibody that can be coupled to the weakly charged gold nanoparticles by electrostatic adsorption is within the protection scope of this invention.

[0033] Of course, the antibody in the complex can also be other proteins, peptides, etc. that can specifically bind to the detection target. Those skilled in the art can make conventional substitutions or adjustments. Any scheme that utilizes the complex design concept of this invention is within the protection scope of this invention.

[0034] In some embodiments, the antibody is a triazophos antibody, and the complex prepared by conjugating the triazophos antibody can be used for the detection of triazophos pesticide residues.

[0035] The enzyme markers in the above-mentioned complexes include, but are not limited to, horseradish peroxidase (HRP), and may also be other enzyme markers with similar functions in the art, such as alkaline phosphatase (AP) and β-galactosidase.

[0036] In some embodiments, the enzyme marker is horseradish peroxidase. By conjugating a triazophos antibody and horseradish peroxidase, the present invention also provides an antibody-probe-enzyme complex for detecting triazophos pesticide residues, denoted as Ab-AuNPs-HRP.

[0037] Meanwhile, this invention also provides a method for preparing the above-mentioned antibody-probe-enzyme complex, which involves sequentially binding weakly charged gold nanoparticles via a stepwise incubation method. The specific steps are as follows: S1. The purified weakly charged gold nanoparticle solution is mixed with the antibody solution and incubated for the first time under suitable conditions to allow the antibody to adsorb onto the surface of the weakly charged gold nanoparticles.

[0038] In some embodiments, after the weakly charged gold nanoparticles are mixed with the antibody, the concentration of the antibody in the mixed solution is 6~10 μg / mL.

[0039] In some embodiments, the conditions for the first incubation are: incubation at room temperature for 0.5 to 2 hours.

[0040] S2. Add the enzyme-labeled solution to the mixture and perform a second incubation to fix the enzyme-labeled material onto the weakly charged gold nanoparticles through electrostatic interaction.

[0041] In some embodiments, the final concentration of the enzyme label in the mixed system is 10-15 μg / mL.

[0042] In some embodiments, the second incubation time is 0.5 to 2 hours.

[0043] S3. After adding the blocking agent bovine serum albumin solution, perform a third incubation, centrifuge, collect the precipitate and resuspend it to obtain the antibody-probe-enzyme complex.

[0044] In some embodiments, the third incubation period is 0.5 to 2 hours.

[0045] In some embodiments, the centrifugation conditions are: temperature of 4~7℃, rotation speed of 9000~12000 rpm / min, and time of 10~20 min.

[0046] Using the aforementioned weakly charged gold nanoparticles and preparation method, an antibody-probe-enzyme complex can be obtained. Under near-neutral (pH 7.0-7.4) physiological buffer conditions, antibody molecules can be directly and stably adsorbed onto the surface of AA-AuNPs through various mechanisms such as hydrophobic interactions, van der Waals forces, and possible ligand exchange, without any pH pre-adjustment steps. This simplifies the process and improves the success rate and probe stability.

[0047] This complex can be directly used in competitive immune reactions, eliminating the need for enzyme-labeled secondary antibodies in traditional ELISA. During detection, the sample to be tested competes with the antigen coated on the ELISA plate for binding to the complex. The substrate is then catalyzed by HRP for color development, enabling quantitative detection of the sample. Therefore, this invention can also provide a competitive enzyme-linked immunosorbent assay (ELISA) product, such as a kit, containing the aforementioned antibody-probe-enzyme complex.

[0048] In some embodiments, the antibody in the antibody-probe-enzyme complex is a triazophos antibody, and the enzyme marker includes horseradish peroxidase. This kit can be used for competitive enzyme-linked immunosorbent assay (ELISA) detection of triazophos residues.

[0049] Based on the above antibody-probe-enzyme complex, a competitive enzyme-linked immunosorbent assay (ELISA) method for pesticide detection can also be provided. This method uses the above-mentioned weakly charged gold nanoparticles, antibody-probe-enzyme complex, or kit to perform ELISA detection.

[0050] When the target substance is triazophos, the enzyme-linked immunosorbent assay (ELISA) method includes: adding triazophos standard or the sample to be tested, antibody-probe-enzyme complex into the microwells of an ELISA plate coated with triazophos antigen, adding horseradish peroxidase substrate solution after the reaction, detecting absorbance after the colorimetric reaction is completed, and establishing a standard curve to calculate the content of triazophos in the sample.

[0051] Specifically, the steps of the competitive enzyme-linked immunosorbent assay (ELISA) for detecting triazophos based on the above-mentioned complex are as follows: S1. Coating: Add the triazophos-coated antigen solution to each well of the ELISA plate, incubate at 37°C, and then wash.

[0052] S2. Blocking: Add blocking solution (such as 2% BSA) to each well to block the non-specific binding sites on the ELISA plate.

[0053] S3. Competitive reaction: The sample to be tested (or a series of triazophos standard solutions) and the Ab-AuNPs-HRP complex prepared above are added to each well simultaneously.

[0054] S4. Washing: After the reaction, wash thoroughly to remove any complexes that have not specifically bound.

[0055] S5. Color development: Add the substrate solution of horseradish peroxidase and carry out the color development reaction under light-protected conditions.

[0056] S6. Determination and Analysis: The absorbance value of each well was measured using a microplate reader. This value is negatively correlated with the concentration of triazophos in the sample. A standard curve was plotted by comparing the concentration of triazophos standard with the corresponding absorbance value, allowing for accurate quantification of triazophos in unknown samples.

[0057] The principle of triazophos detection in this invention is as follows: free triazophos molecules in the sample competitively bind to a limited number of antibodies in the triazophos antigen-coated complex on the plate. The target substance is quantitatively detected through an inverse relationship between the intensity of the colorimetric reaction. The detection of triazophos using the weakly charged gold nanoparticles, Ab-AuNPs-HRP complex, and enzyme-linked immunosorbent assay (ELISA) kit provided by this invention has the advantages of simple operation, low cost, high sensitivity, and good specificity, making it suitable for highly sensitive detection of triazophos residues in agricultural products.

[0058] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0059] Example 1 This embodiment describes the synthesis of weakly charged gold nanoparticles (AA-AuNPs), and the specific steps are as follows: (1) Soak all glassware used in the synthesis process in aqua regia overnight and rinse thoroughly with ultrapure water.

[0060] (2) Add 50 mL of deionized water containing HAuCl4 (1 mM) to a 100 mL round-bottom flask, heat to 90°C, then add 3 mL of ascorbic acid (AA) solution with a mass-volume ratio of 1% (g / mL), and continue the reaction for 20 min.

[0061] (3) After the reaction is complete, a wine-red solution is obtained. It is naturally cooled to room temperature and filtered through a 0.22 μm filter membrane.

[0062] (4) Centrifuge at 8000 rpm for 10 min at 4°C to remove unreacted residual reagents, and reconstitute with deionized water to the original volume.

[0063] To comprehensively evaluate the physicochemical properties of the synthesized AA-AuNPs, a series of characterizations were performed in this embodiment: their morphology was observed using field emission transmission electron microscopy (HRTEM), transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS), and high-angle annular dark field imaging (HAADF).

[0064] The observation results of field emission transmission electron microscopy are as follows: Figure 1 As shown, the transmission electron microscope-energy dispersive spectroscopy elemental map is as follows: Figure 2 As shown, the result of the high-angle ring-shaped dark field image is as follows: Figure 3 As shown. From Figures 1-3 As can be seen, the synthesized AA-AuNPs are regular spherical or near-spherical in shape and uniformly distributed. Dynamic light scattering (DLS) characterization showed that their average particle size was 40 nm.

[0065] Scanning was performed using a UV-Vis spectrophotometer, and the results are as follows: Figure 5 As shown, AA-AuNPs exhibit a sharp and symmetrical plasmon resonance absorption peak at a wavelength of 525 nm, which is a typical characteristic of their nanoscale size and good dispersibility.

[0066] The hydrated particle size and surface potential were determined using dynamic light scattering (DLS) and Zeta potential analysis. DLS results showed that the hydration kinetic diameter was consistent with TEM results. Crucially, its Zeta potential value was -8.43 mV. This "weak charge" characteristic, falling between strong electrostatic stability and instability, ensures the stability of the colloid during storage while significantly reducing electrostatic repulsion with negatively charged biomolecules (such as antibodies), laying the foundation for efficient bioconjugation without the need for precise pH control.

[0067] Example 2 This embodiment describes the preparation of an antibody-probe-enzyme complex (Ab-AuNPs-HRP). This embodiment uses AA-AuNPs as the core, simultaneously coupling antibody and enzyme to construct a "three-in-one" detection probe. The specific steps are as follows: (1) Triazole phosphate antibody was added to the AA-AuNPs obtained in Example 1 to make the final concentration of antibody 6 μg / mL.

[0068] (2) Place the mixture in a shaker and incubate for 0.5 h. Then add 5 μL of 1000 μg / mL horseradish peroxidase solution to the mixture, incubate in a shaker for 30 min, add 2% bovine serum albumin (BSA), and incubate for another 0.5 h to block the non-specific binding sites on the surface of AA-AuNP.

[0069] (3) After incubation, centrifuge at 10,000 rpm for 10 min (4°C) and collect the precipitated Ab@AA-AuNPs@HRP. After discarding the supernatant, resuspend the precipitate in 0.01 M PBS buffer and concentrate it 10 times.

[0070] Example 3 This embodiment establishes a competitive ELISA detection method for triazophos based on the Ab-AuNPs-HRP complex prepared in Example 2, as detailed below: (1) The 96-well electrostatic adsorption plate was incubated with the coated antigen (100 μL per well) in phosphate buffer at 37°C for 2 h.

[0071] (2) Wash the plate three times with phosphate buffer (pH 7.4) containing 0.1% Tween-20, then block with 2% BSA (300 μL per well) and block at 37°C for 1 h.

[0072] (3) After washing the plate three times, add 50 μL of triazophos pesticide standard to each well (the pesticide standard is serially diluted to 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 ng / ml), and then add 50 μL of reconstituted Ab-AuNPs-HRP to each well.

[0073] (4) The reaction was carried out at 37°C for 2 h, followed by washing three times with PBST. Then, 100 μL of TMB was added to each well, and the absorbance was measured at 650 nm. The sensitivity of the detection was evaluated by plotting a standard curve by calculating the inhibition rate of pesticide on antigen-antibody binding.

[0074] The half-inhibition concentration (IC50) of this embodiment was calculated using the standard curve. 50The concentration was 4.93 ng / mL. The limit of detection (LOD) of the detection method in this embodiment was 0.06 ng / mL, and the linear detection range was 1.08 ng / mL to 40 ng / mL.

[0075] Example 4 This embodiment is a cross-reactivity experiment to verify the specificity of the detection method in Example 3. To verify the specificity of the method of the present invention for the detection of triazophos, this embodiment tested the cross-reactivity rates of several common pesticides. Chlorpyrifos, parathion, diazinon, carbofuran, malathion, and thiamethoxam were selected as interfering agents. The response signals of triazophos and each interfering agent were detected under the same conditions according to the detection method of Example 3.

[0076] The results showed that the cross-reactivity rates of all interfering substances were less than 0.5%. These results demonstrate that the immunoassay method constructed in this invention has extremely high recognition specificity for triazophos and can effectively avoid interference from other components in complex sample matrices.

[0077] Example 5 This example demonstrates an experiment for determining the recovery rate of spiked samples in actual fruits and vegetables. (1) Weigh the fully homogenized sample, add acetonitrile as the extraction solvent, vortex and then add anhydrous magnesium sulfate and sodium chloride to the mixture, vortex and centrifuge.

[0078] (2) Transfer the supernatant to a purified centrifuge tube containing dispersed solid-phase extraction packing material (PSA, C18, graphitized carbon black), vortex, and then centrifuge. Finally, filter the supernatant through a 0.22 μm nitrocellulose filter membrane and transfer it to a centrifuge tube, and store it at 4°C for later use.

[0079] (3) Triazophos was added to three substrates: Chinese cabbage, cucumber, and cabbage. The recovery rate was determined at three different spiking levels: 5 μg / L, 10 μg / L, and 20 μg / L. The results are shown in Table 1. Table 1 Results of recovery rate determination

[0080] The average recoveries for the three different spiking levels ranged from 76.82% to 115.83%, all within the acceptable range of 70%–120%; the intra-batch relative standard deviation (RSD) ranged from 0.95% to 5.69%.

[0081] Therefore, this method has excellent accuracy and precision, fully meeting the reliability requirements of pesticide residue analysis, and confirming that the AuNPs-ELISA method established in this invention has high practical value in actual sample detection.

[0082] Comparative Example 1 In this comparative example, gold nanoparticles were prepared using conventional citrate (reference: Liu, ZW, Hua, QC, Wang, J., Liang, ZQ, Li, JH, Wu, JX, Shen, X., Lei, HT, & Li, XM (2020). As a smartphone-based dual detection mode device integrated with two lateral flow immunoassays for multiplex mycotoxins in cereals. Biosensors & Bioelectronics, 158, 112178. https: / / doi.org / 10.1016 / j.bios.2020.112178). The obtained gold nanoparticles were then prepared into an antibody-probe-enzyme complex using the preparation method of Example 2, and this complex was then used for triazophos detection.

[0083] The antibody-probe-enzyme complex prepared using this comparative ratio was used for the same detection, and its half-maximal inhibitory concentration (IC50) was 124.2 ng / mL, its limit of detection (LOD) was 25.2 ng / mL, and its linear detection range was 37.6 ng / mL to 411 ng / mL.

[0084] Figure 3 Here is an electron micrograph of the Ab@AA-AuNP complex. Figure 4 This is an electron micrograph of the Ab@Cit-AuNP complex synthesized using conventional sodium citrate. The comparison shows that the weakly charged gold nanoparticles of this invention can significantly couple more antibodies.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing weakly charged gold nanoparticles, characterized in that, Gold nanoparticles were synthesized using ascorbic acid and chloroauric acid as raw materials via a one-pot chemical reduction method. The steps included: After stirring and heating the aqueous solution of chloroauric acid, ascorbic acid solution is added, and a reduction reaction is carried out under constant temperature conditions until a wine-red solution is obtained, thus obtaining the weakly charged gold nanoparticles.

2. The preparation method according to claim 1, characterized in that, The mass ratio of chloroauric acid to ascorbic acid in the reduction reaction is 0.5~0.6:1; the chloroauric acid aqueous solution is heated to 90~105℃ before the reaction.

3. The preparation method according to claim 1, characterized in that, The preparation method further includes pretreatment of the glass container used in the reaction before preparation, and separation and purification of the weakly charged gold nanoparticles in the wine-red solution after the reduction reaction is completed. Preferably, the pretreatment includes: immersing the glass container in aqua regia and then rinsing it with ultrapure water; Preferably, the separation and purification includes: cooling the wine-red solution to room temperature, then filtering it through a microporous membrane, centrifuging the filtrate, discarding the supernatant, and the precipitate being the purified weakly charged gold nanoparticles.

4. The weakly charged gold nanoparticles obtained by the preparation method according to any one of claims 1-3, characterized in that, The zeta potential of the weakly charged gold nanoparticles is -7mV to -10mV.

5. An antibody-probe-enzyme complex, characterized in that, The antibody-probe-enzyme complex uses the weakly charged gold nanoparticles as described in claim 4 as the core, coupled with an antibody and an enzyme label.

6. The antibody-probe-enzyme complex according to claim 5, characterized in that, The preparation method of the antibody-probe-enzyme complex includes: The purified weakly charged gold nanoparticle solution was mixed with the antibody solution and incubated for the first time under suitable conditions to allow the antibody to adsorb onto the surface of the weakly charged gold nanoparticles. An enzyme-labeled solution was added to the mixture, and a second incubation was performed to fix the enzyme-labeled substance onto the weakly charged gold nanoparticles through electrostatic interaction. After adding the blocking agent, a third incubation was performed, followed by centrifugation, collection of the precipitate, and resuspending to obtain the antibody-probe-enzyme complex.

7. The use of the weakly charged gold nanoparticles as described in claim 4 or the antibody-probe-enzyme complex as described in claim 5 or 6 in any one of the following (1) to (2): (1) Preparation of competitive enzyme-linked immunosorbent assay (ELISA) products; (2) Competitive enzyme-linked immunosorbent assay (ELISA) for pesticides.

8. A competitive enzyme-linked immunosorbent assay (ELISA) kit, characterized in that, It comprises the weakly charged gold nanoparticles of claim 4 or the antibody-probe-enzyme complex of claim 5 or 6.

9. The competitive enzyme-linked immunosorbent assay kit according to claim 8, characterized in that, The antibody in the antibody-probe-enzyme complex includes triazole phosphorus antibody, and the enzyme marker includes horseradish peroxidase.

10. An enzyme-linked immunosorbent assay (ELISA) method for detecting pesticide residues, characterized in that, Enzyme-linked immunosorbent assay (ELISA) was performed using the weakly charged gold nanoparticles of claim 4, the antibody-probe-enzyme complex of claim 5 or 6, or the kit of claim 8 or 9. Preferably, the pesticide includes triazophos; Preferably, the enzyme-linked immunosorbent assay (ELISA) method includes: adding triazophos standard or the sample to be tested, the antibody-probe-enzyme complex, and microwells of an ELISA plate coated with triazophos antigen; adding horseradish peroxidase substrate solution after the reaction; detecting absorbance after the colorimetric reaction is completed; and establishing a standard curve to calculate the triazophos content in the sample.

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