Monodisperse colloidal gold for immunodetection and preparation and application thereof
By adjusting the pH value and reaction temperature of the aqueous chloroatric acid solution and using sodium citrate as the reducing agent, colloidal gold with good stability, spherical monodispersed and high particle concentration was prepared, which solved the preparation problems in the prior art and was suitable for large-scale production and biomarker applications.
Patent Information
- Application Number
- CN202311764866.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-21
- Publication Date
- 2025-07-01
AI Technical Summary
It is difficult to prepare colloidal gold with a particle size of 20-60 nm, which not only meets the problems of good stability, spherical monodispersion and high particle concentration, but also has complex operation and high cost.
By adjusting the pH value and reaction temperature of the aqueous chloroatric acid solution, the growth path of gold nanoparticles is controlled, and sodium citrate is used as the reducing agent to prepare colloidal gold with good stability, spherical monodispersed and high particle concentration.
It realizes large-scale production with simple operation, good repeatability and low cost. The prepared colloidal gold has good stability, spherical monodispersion and high particle concentration, which is suitable for the needs of biomarking and immunochromatography technology.
Smart Images
Figure CN120233085A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of colloidal gold preparation, and particularly relates to a colloidal gold for monodisperse immunoassay, its preparation and application. Background Art
[0002] Colloidal gold is a colloidal solution with stable dispersion properties in solution. Its main component is gold nanoparticles with a certain morphology and size and a negatively charged surface. These gold nanoparticles are formed by the reduction of chloroauric acid into gold atoms and then aggregation under the action of reducing agents (such as white phosphorus, ascorbic acid, hydrogen peroxide, sodium borohydride, sodium citrate, and tannic acid, etc.). It has a strong adsorption to proteins and can firmly bind proteins on the particle surface. Moreover, whether in liquid or solid form, it can long-term stably maintain the inherent biological activity of proteins. At the same time, colloidal gold has obvious color development, so colloidal gold is widely used in the rapid diagnosis field of medical tests.
[0003] The morphology and size of colloidal gold will affect the interaction between colloidal gold and proteins, and thus affect the performance of immunoassay products. Small-sized colloidal gold has a large specific surface area and a high coupling efficiency with biomolecules, but the color development effect is poor. Large-sized colloidal gold has a good color development effect, but the coupling efficiency is relatively low. And colloidal gold with a size between 20 - 60 nm can take into account the detection requirements of both coupling efficiency and color development effect. In addition, if the morphology of colloidal gold is not uniform or the particle size is not uniform, it will cause the colloidal gold complex labeled with proteins to not be stably preserved, and then result in incomplete chromatography, too high background, or false positive results.
[0004] Currently, the common method for preparing colloidal gold is the one-step reduction method. By using different types of reducing agents, colloidal gold with a particle size of 1 - 200 nm can be prepared. Among numerous reducing agents, the most commonly used reducing agent is sodium citrate. Frens et al. kept the amount of chloroauric acid unchanged and adjusted the amount of sodium citrate, that is, changed the molar ratio of sodium citrate to Au 3+ to obtain colloidal gold with different particle sizes. The one-step reduction method has simple operation and is suitable for large-scale production and preparation. It is currently the most commonly used method for preparing colloidal gold. Generally speaking, by controlling the molar ratio of sodium citrate to Au 3+ between 2.8 - 1:5, colloidal gold with a particle size of 10 - 60 nm can be prepared, especially suitable for preparing spherical monodisperse colloidal gold with a particle size of 15 - 30 nm. However, this method has several problems. First, as the molar ratio of sodium citrate to Au 3+When the molar ratio decreases, the morphology of the prepared colloidal gold tends to be irregular (ellipse, triangle, etc.) and the monodispersity of gold nanoparticles gradually decreases. Secondly, when synthesizing large-sized colloidal gold, as the amount of sodium citrate decreases, the pH value of the reaction system gradually tends to be acidic, resulting in a decrease in the surface charge of gold nanoparticles, and further leading to poor stability of colloidal gold. Finally, in the Frens method, the concentration of chloroauric acid is usually low (~0.25 mM), resulting in a relatively low concentration of gold nanoparticles in colloidal gold. To increase the yield and expand the reaction scale, a large amount of manpower and material resources are required.
[0005] In summary, it is difficult to prepare colloidal gold with a particle size of 20 - 60 nm that has good stability, spherical monodispersity, and a high particle concentration using existing technologies, and a new method for synthesizing colloidal gold needs to be proposed. Summary of the Invention
[0006] To solve the above problems, the purpose of the present invention is to provide a colloidal gold for monodisperse immunoassay, its preparation method, and its application. The present invention uses sodium citrate as a reducing agent, and regulates the growth path of gold nanoparticles by adjusting the pH value and reaction temperature of an aqueous chloroauric acid solution, thereby obtaining colloidal gold with good stability, spherical monodispersity, and a high concentration of gold nanoparticles.
[0007] The purpose of the present invention can be achieved by the following technical solutions:
[0008] The first purpose of the present invention is to provide a preparation method for colloidal gold for monodisperse immunoassay, comprising the following steps:
[0009] (S1) Mix chloroauric acid and deionized water, and perform post-treatment to obtain a first mixed solution;
[0010] (S2) Heat up the first mixed solution prepared in step (S1), add sodium citrate, and perform post-treatment after the reaction to obtain spherical colloidal gold for monodisperse immunoassay.
[0011] In an embodiment of the present invention, in step (S1), the dosage ratio of chloroauric acid to deionized water is 0.05 - 0.25 mmol: 100 mL.
[0012] In an embodiment of the present invention, in step (S1), the post-treatment is to adjust the pH value to 2.5 - 5 using a pH regulator.
[0013] In an embodiment of the present invention, the pH regulator is selected from one of hydrochloric acid, sodium hydroxide, or tris(hydroxymethyl)aminomethane.
[0014] In an embodiment of the present invention, in step (S2), the molar ratio of sodium citrate to chloroauric acid is 2 - 10: 1.
[0015] In one embodiment of the present invention, in step (S2), during the reaction, the temperature is 75 - 95 °C, and the reaction proceeds until the color of the solution remains unchanged.
[0016] In one embodiment of the present invention, in step (S2), the post-treatment is to stir and cool to room temperature and then store at low temperature in the dark.
[0017] In one embodiment of the present invention, the colloidal gold for monodisperse immunoassay is stored in the dark at 2 - 8 °C.
[0018] The second object of the present invention is to provide a colloidal gold for monodisperse immunoassay prepared by the above method.
[0019] The third object of the present invention is to provide an application of the colloidal gold for monodisperse immunoassay in immunoassay.
[0020] In the present invention, the pH value of the reaction solution system is adjusted by regulating the reaction temperature and pH regulator instead of changing the concentration of sodium citrate to control the particle size of colloidal gold. Basically, the chemical reaction rate depends to a large extent on the temperature, so a relatively low reaction temperature can reduce the growth rate of gold nanoparticles. The change of the pH value of the reaction system will cause the change of the reaction rate of chloroauric acid in the reaction solution and the structure of the hydrolysis product of sodium citrate, and then can significantly improve the monodispersity of the particle size of colloidal gold and the stability of gold nanoparticles. When the pH value is lower than 6.2, there is only one OH 3+ ligand in the Au - complex, and its reaction activity is very high. At this time, the formation of gold nanoparticles includes three consecutive processes, namely nucleation, non-directional adsorption (or aggregation) and particle internal ripening, resulting in the morphology of colloidal gold mostly presenting an elliptical or irregular shape. As the pH value of the solution increases, the number of OH- ligands in the Au 3+ complex increases to 2 or 3, and its corresponding species activity also decreases accordingly. At this time, the formation mechanism of gold nanoparticles follows the nucleation-growth route, which is conducive to obtaining monodisperse colloidal gold products with uniform particle size. In addition to the influence of the reaction activity of the Au 3+ complex, the change of the pH value also has an impact on the carboxyl structure of sodium citrate. A lower pH value of the reaction solution means a lower surface charge of gold nanoparticles, resulting in a weakened repulsive force between particles, and colloidal gold is prone to aggregation. As the pH value increases to be higher than its third ionization point (pH = 6.4), the main existence form of sodium citrate is citrate, the surface charge of the gold nanonuclei generated by the reaction increases, and the aggregation of particles is weakened, which is conducive to obtaining colloidal gold with excellent stability and high particle concentration. The present invention reasonably adjusts the pH value and reaction temperature of the reaction system to ensure both the monodispersity of the particle size of colloidal gold and the significant improvement of the stability and particle concentration of colloidal gold.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The preparation method of the monodisperse colloidal gold for immunoassay of the present invention has the characteristics of simple operation, good repeatability, low cost, and suitability for large-scale production. The prepared colloidal gold has the advantages of good stability, spherical monodispersity, and high concentration of gold nanoparticles, making it more suitable for the needs of biological labeling and immunochromatography technology. Description of the Drawings
[0023] Figure 1 TEM image of the colloidal gold prepared in Comparative Example 1; wherein, the scale bar is 100 nm.
[0024] Figure 2 TEM photos of the colloidal gold prepared in Examples 1 to 4; wherein, Figure 2 (a) is the colloidal gold prepared in Example 1; Figure 2 (b) is the colloidal gold prepared in Example 2; Figure 2 (c) is the colloidal gold prepared in Example 3; Figure 2 (d) is the colloidal gold prepared in Example 4; wherein, the scale bar is 100 nm.
[0025] Figure 3 Ultraviolet absorption spectra of the colloidal gold prepared in Examples 1 to 4 and Comparative Example 1. Detailed Embodiments
[0026] The present invention provides a method for preparing monodisperse colloidal gold for immunoassay, comprising the following steps:
[0027] (S1) Mix chloroauric acid with deionized water, and perform post-treatment to obtain a first mixed solution;
[0028] (S2) Heat the first mixed solution prepared in step (S1), add sodium citrate, and perform post-treatment after the reaction to obtain spherical monodisperse colloidal gold for immunoassay.
[0029] Further, in step (S1), the dosage ratio of chloroauric acid to deionized water is 0.05 - 0.25 mmol: 100 mL.
[0030] Further, in step (S1), the post-treatment is to adjust the pH value to 2.5 - 5 using a pH regulator.
[0031] Further, the pH regulator is selected from one of hydrochloric acid, sodium hydroxide, or tris(hydroxymethyl)aminomethane.
[0032] Further, in step (S2), the molar ratio of sodium citrate to chloroauric acid is 2 - 10: 1.
[0033] Further, in step (S2), during the reaction, the temperature is 75-95°C, and the reaction is continued until the color of the solution remains unchanged.
[0034] Further, in step (S2), the post-treatment is to stir and cool to room temperature and then store in the dark at a low temperature.
[0035] Further, the monodisperse colloidal gold for immunoassay is stored in the dark at 2-8°C.
[0036] The present invention provides a monodisperse colloidal gold for immunoassay prepared by the above method.
[0037] The present invention provides an application of the monodisperse colloidal gold for immunoassay in immunoassay.
[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific examples.
[0039] In the following examples, unless otherwise specified, the reagents used are commercially available reagents, and the detection means and methods used are conventional detection means and methods in the art.
[0040] Prepare an aqueous solution of chloroauric acid: Dissolve chloroauric acid in deionized water to prepare a 25 mM aqueous solution, and store it at 4°C.
[0041] The glass containers used in the following examples and comparative examples are soaked in aqua regia overnight before the experiment, rinsed with deionized water, and dried.
[0042] Comparative Example 1
[0043] This example provides a method for preparing colloidal gold by the traditional Frens method.
[0044] Take 1 mL of the aqueous chloroauric acid solution (25 mM) and add it to 99 mL of deionized water. Stir and heat to boiling, keep boiling for 5 min, then quickly add 0.4 mL of the aqueous sodium citrate solution (170 mM) to the reaction flask, keep boiling and reacting for 10 min, stop heating, continue to stir for 20 min, and then cool to room temperature naturally to obtain colloidal gold. Its TEM image is as Figure 1 shown.
[0045] Example 1
[0046] This example provides a monodisperse colloidal gold for immunoassay and its preparation method.
[0047] 10 mL of chloroauric acid aqueous solution (25 mM) was added to 90 mL of deionized water. The pH value of the chloroauric acid aqueous solution was adjusted to 5.0 using sodium hydroxide aqueous solution (0.1 M). The reaction solution was heated to 70 °C with stirring and maintained for 5 min. Subsequently, 2.95 mL of sodium citrate aqueous solution (170 mM) was quickly added to the reaction flask, and the reaction was carried out at 75 °C for 20 min. The heating was stopped, and stirring was continued while naturally cooling to room temperature. The obtained colloidal gold had an average particle size of approximately 21.5 nm and a polydispersity coefficient of 8.5%; its TEM image was as shown in Figure 2 (a).
[0048] Example 2
[0049] This example provides a colloidal gold for monodisperse immunoassay and its preparation method.
[0050] 6 mL of chloroauric acid aqueous solution (25 mM) was added to 94 mL of deionized water. The pH value of the chloroauric acid aqueous solution was adjusted to 4.5 using tris(hydroxymethyl)aminomethane aqueous solution (0.1 M). The reaction solution was heated to 80 °C with stirring and maintained for 5 min. Subsequently, 2.92 mL of sodium citrate aqueous solution (170 mM) was quickly added to the reaction flask, and the reaction was carried out at 80 °C for 20 min. The heating was stopped, and stirring was continued while naturally cooling to room temperature. The obtained colloidal gold had an average particle size of approximately 32.6 nm and a polydispersity coefficient of 7.3%; its TEM image was as shown in Figure 2 (b).
[0051] Example 3
[0052] This example provides a colloidal gold for monodisperse immunoassay and its preparation method.
[0053] 4 mL of chloroauric acid aqueous solution (25 mM) was added to 96 mL of deionized water. The pH value of the chloroauric acid aqueous solution was adjusted to 3.4 using tris(hydroxymethyl)aminomethane aqueous solution (0.1 M). The reaction solution was heated to 80 °C with stirring and maintained for 5 min. Subsequently, 2.94 mL of sodium citrate aqueous solution (170 mM) was quickly added to the reaction flask, and the reaction was carried out at 80 °C for 20 min. The heating was stopped, and stirring was continued while naturally cooling to room temperature. The obtained colloidal gold had an average particle size of approximately 29.7 nm and a polydispersity coefficient of 6.7%; its TEM image was as shown in Figure 2 (c).
[0054] Example 4
[0055] This example provides a colloidal gold for monodisperse immunoassay and its preparation method.
[0056] 2 mL of chloroauric acid aqueous solution (25 mM) was added to 98 mL of deionized water, and the pH value of the chloroauric acid aqueous solution was adjusted to 2.5 with hydrochloric acid aqueous solution (0.1 M). The reaction solution was heated to 90 °C with stirring and maintained for 5 min. Subsequently, 2.94 mL of sodium citrate aqueous solution (170 mM) was quickly added to the reaction flask, and the reaction was carried out at 95 °C for 20 min. The heating was stopped, and the mixture was continuously stirred and naturally cooled to room temperature. The obtained colloidal gold had an average particle size of about 37.3 nm and a polydispersity coefficient of 9.4%; its TEM image was as shown in Figure 2 (d).
[0057] The ultraviolet absorption spectra of the colloidal gold prepared in Examples 1 to 4 and Comparative Example 1 were as shown in Figure 3 .
[0058] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the interpretation of the present invention should be within the protection scope of the present invention.
Claims
1. A preparation method of colloidal gold for monodisperse immunoassay, characterized in that, It includes the following steps: (S1) Mix chloroauric acid with deionized water, and perform post-treatment to obtain a first mixed solution; (S2) Heat up the first mixed solution prepared in step (S1), add sodium citrate, and after reaction, perform post-treatment to obtain spherical monodisperse colloidal gold for immunoassay.
2. The preparation method of colloidal gold for monodisperse immunoassay according to claim 1, characterized in that, In step (S1), the dosage ratio of chloroauric acid to deionized water is 0.05 - 0.25 mmol: 100 mL.
3. The preparation method of colloidal gold for monodisperse immunoassay according to claim 1, characterized in that, In step (S1), the post-treatment is to adjust the pH value to 2.5 - 5 using a pH regulator.
4. The preparation method of the colloidal gold for monodisperse immunoassay according to claim 3, wherein, The pH regulator is selected from one of hydrochloric acid, sodium hydroxide, or tris(hydroxymethyl)aminomethane.
5. The preparation method of colloidal gold for monodisperse immunoassay according to claim 1, characterized in that, In step (S2), the molar ratio of sodium citrate to chloroauric acid is 2 - 10:
1.
6. The preparation method of colloidal gold for monodisperse immunoassay according to claim 1, characterized in that, In step (S2), during the reaction process, the temperature is 75 - 95 °C, and the reaction is carried out until the color of the solution remains unchanged.
7. The preparation method of a monodisperse colloidal gold for immunoassay according to claim 1, wherein, In step (S2), the post-treatment is to stir and cool to room temperature and then store in the dark at low temperature.
8. The preparation method of a monodisperse colloidal gold for immunoassay according to claim 7, characterized in that, The monodisperse colloidal gold for immunoassay is stored in the dark at 2 - 8 °C.
9. A monodisperse colloidal gold for immunoassay prepared by the method according to any one of claims 1 - 8.
10. An application of the monodisperse colloidal gold for immunoassay according to claim 9 in immunoassay.