Colloidal gold exhibits remarkable kinetic stability, and under normal conditions, it resists self-aggregation for extended periods—sometimes even years—without significant clumping. The stability of colloidal gold is primarily influenced by factors such as electrolyte concentration, sol concentration, temperature, and the presence of non-electrolytes.
A small amount of electrolyte is essential to maintain the stability of the gold sol, but excessive amounts can destabilize the system. Similarly, high concentrations of hydrophilic non-electrolytes may disrupt the protective hydration layer surrounding the particles, leading to aggregation. On the other hand, certain high-molecular-weight substances like proteins, glucose, and PEG 20000 can enhance the stability of the sol by acting as stabilizers. However, too much of these materials may cause unwanted condensation.
When proteins are adsorbed onto colloidal gold, the stability of the solution becomes dependent on the pH of the environment. This change is closely related to the isoelectric point of the adsorbed protein. For instance, ConA or peroxidase remains stable at low pH but becomes unstable when the pH increases, only regaining stability near its isoelectric point. To ensure long-term storage, a small amount of PEG 20000 (0.2–0.5 mg/ml) is often used as a stabilizer. When stored at 4–10°C, the labeled colloidal gold solution can remain stable for several months. Freezing should be avoided, as it may lead to partial aggregation, which can be removed through centrifugation.
Preparing high-quality colloidal gold requires careful attention to several key steps. First, all glassware must be thoroughly cleaned, preferably using siliconized or pre-stabilized containers. Double-distilled water should be used for rinsing to avoid contamination. Any impurities in the reagents or water can affect the final particle size and stability. Reagents should be prepared with ultra-pure water and filtered through a 0.45 μm pore filter to remove any potential contaminants. Chloroauric acid, which is highly hygroscopic and corrosive, should be handled carefully, avoiding contact with metal tools. A 1% aqueous solution of chloroauric acid can be stored for several months at 4°C.
The pH of the gold solution should not be measured using a standard pH electrode, as gold particles tend to adsorb onto the electrodes, potentially altering the measurement. Instead, a buffer system with sufficient capacity, such as citrate phosphate, Tris-HCl, or sodium borate, should be used. However, care must be taken to avoid excessive buffer concentrations, which could promote aggregation. The ideal pH range for colloidal gold solutions is around neutral (pH 7.2). High-quality chloroauric acid with minimal impurities is recommended, ideally imported from reliable sources.
Colloidal gold has a wide range of applications, especially in immunology. At the electron microscope level, colloidal gold is one of the most widely used markers due to its ability to allow multi-color labeling using different-sized particles. Particles between 3–15 nm are commonly used for detecting single antigens, while larger particles (15 nm) are better suited for identifying infected cells. Colloidal gold is also used in pre-embedding and post-embedding staining, immunonegative staining, double labeling, and in situ hybridization techniques. It offers advantages such as fast detection, high sensitivity, and ease of use, making it valuable in both research and diagnostic settings.
At the light microscope level, colloidal gold serves as an alternative to traditional fluorescent or enzymatic markers. It is used for detecting surface antigens, intracellular antigens, and tissue antigens. Its application helps reduce background interference and enzyme activity issues that are common with other methods. In flow cytometry, colloidal gold can act as a non-fluorescent marker, allowing multiple labels to be detected simultaneously based on changes in laser scattering.
In agglutination tests, colloidal gold provides a visual indication of immune reactions. When specific antigens or antibodies bind to the gold particles, they aggregate, causing a color change that can be used for qualitative or quantitative analysis. Immunoblotting also benefits from colloidal gold, where the signal can be enhanced using silver staining, improving detection sensitivity to as low as 0.1 ng. This technique is simple, rapid, and increasingly used in clinical diagnostics.
At the naked eye level, colloidal gold replaces traditional immunoassay markers. It offers advantages such as low sample volume requirements, no need for expensive equipment, and the absence of harmful chemicals. Results are easy to store and interpret, and the process is significantly faster than conventional methods. The physical adsorption method allows almost any macromolecule to be labeled without affecting its biological activity. Combined with silver staining, colloidal gold-based assays can achieve sensitivity comparable to ELISA.
Overall, colloidal gold technology continues to evolve and find new applications in immunodiagnostic and biomedical fields, offering a versatile, sensitive, and user-friendly alternative to traditional labeling methods.
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