Colloidal gold solution refers to a gold sol with a dispersed phase particle diameter between 1 and 150 nm, belonging to a heterogeneous multiphase system. Its color ranges from orange-red to purple-red. The use of colloidal gold as a marker in immunohistochemistry began in 1971 when Faulk and others applied the immunogold staining method (IGS) using electron microscopy to observe Salmonella. Subsequently, they combined colloidal gold with various proteins. In 1974, Romano and others labeled colloidal gold on the second antibody (horse anti-human IgG) and established the indirect immunogold staining method. In 1978, Geoghega discovered the application of colloidal gold markers at the light microscopy level.
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This application of colloidal gold in immunohistochemistry is also known as immunogold. Later, many scholars further confirmed that colloidal gold can rapidly and stably adsorb proteins without significant changes in their biological activity. It can be used as a probe for precise localization of cell surface and intracellular macromolecules such as polysaccharides, proteins, polysaccharides, antigens, hormones, nucleic acids, etc. It can also be used for routine immunodiagnosis and immunohistochemical localization, and as a result, its application in clinical diagnosis and drug testing has received widespread attention. Currently, immunogold staining at the electron microscopy level (IGS), immunogold-silver staining at the light microscopy level (IGSS), and spot immunogold staining technology at the macroscopic level are increasingly becoming powerful tools for scientific research and clinical diagnosis.
Basic Principles of Immunogold Technology
Chloroauric acid (HAuCl4), under the action of a reducing agent, can aggregate into gold particles of a certain size, forming a negatively charged hydrophobic sol. Due to electrostatic forces, it stabilizes into a colloidal state, hence the term "colloidal gold." The colloidal gold particles consist of a basic gold nucleus (atomic gold Au) surrounded by a double-ion layer. The inner layer is composed of negative ions (AuCl2−) tightly bound to the gold nucleus surface, while the outer ion layer (H+) disperses in the solution between colloids to maintain the suspended state of colloidal gold.
The basic gold nucleus of colloidal gold particles is not an ideal spherical core; smaller colloidal gold particles are generally spherical, while larger particles (usually larger than 30nm) often exhibit an elliptical shape. The particle morphology of colloidal gold can be observed under an electron microscope.
Colloidal gold labeling essentially involves the adsorption process of high-molecular-weight substances like proteins onto the surface of colloidal gold particles. The adsorption mechanism may involve the negatively charged surface of colloidal gold particles forming a strong bond with positively charged groups of proteins through electrostatic adsorption. Various-sized colloidal gold particles, and thus different colors, can be conveniently prepared from chloroauric acid using a reduction method. These spherical particles possess a strong adsorption capacity for proteins, making them useful tools in basic research and clinical experiments for non-covalent binding with Staphylococcus aureus protein A, immunoglobulins, toxins, glycoproteins, enzymes, antibiotics, hormones, bovine serum albumin peptides, and other compounds.
Immunogold labeling technology primarily leverages the high electron density characteristics of gold particles. At the binding sites of gold-labeled proteins, dark brown particles can be observed under a microscope. When these markers aggregate in large quantities at corresponding ligand sites, visible red or pink spots can be seen with the naked eye. Therefore, this reaction is used in qualitative or semi-quantitative rapid immunodiagnostic methods. This reaction can also be amplified through the deposition of silver particles, known as immunogold-silver staining.
Characteristics of Colloidal Gold
Colloidal gold particles typically range in size from 1 to 100 nm. Tiny gold particles remain stably, uniformly, and individually dispersed in a liquid, forming a colloidal gold solution. Colloidal gold exhibits various colloidal properties, especially sensitivity to electrolytes. Electrolytes can disrupt the peripheral hydration layer of colloidal gold particles, breaking the colloidal stability. This leads to the aggregation of dispersed individual gold particles into larger particles, precipitating from the liquid. Certain large molecules like proteins play a protective role, enhancing the stability of colloidal gold.
Colloidal gold appears red, with different-sized colloids exhibiting distinct colors. The smallest colloidal gold (2-5 nm) is orange-yellow, medium-sized colloidal gold (10-20 nm) is wine-red, and larger colloidal gold particles (30-80 nm) appear purple-red. Observing the color of colloidal gold with the naked eye allows for a rough estimation of the size of gold particles. Over the past decade, colloidal gold labeling has evolved into a crucial immunolabeling technique. Colloidal gold immunological analysis has developed significantly in fields such as drug testing and biomedical research, garnering increasing attention in related research domains. Optical absorption colloidal gold has a single absorption peak in the visible light range, with the wavelength (λmax) changing within the 510–550 nm range with colloidal gold particle size. Larger colloidal gold particles have a λmax biased towards longer wavelengths, while smaller colloidal gold particles have a λmax biased towards shorter wavelengths.
Colloidal gold exhibits high dynamic stability, undergoing very slow self-coagulation when stability factors remain intact, allowing it to be stored for several years without significant coagulation. Factors affecting stability include electrolytes, sol concentration, temperature, and non-electrolytes. Gold sol must have a small amount of electrolyte as a stabilizer, but the concentration should not be excessively high. High concentrations of hydrophilic non-electrolytes can strip away the hydration layer from colloidal particles, causing coagulation. Small amounts of high-molecular-weight substances induce sol coagulation, but a certain quantity of such substances can enhance sol stability. Additives like proteins, glucose, PEG20000 show good stabilizing effects.
Applications of Immunogold
Application at the Electron Microscopy Level
Colloidal gold was initially and extensively applied at the electron microscopy level, showcasing rapid development. Its significant advantage lies in the ability to achieve dual or multiple labeling through the use of particles of different sizes or enzyme conjugates. Colloidal gold with diameters ranging from 3 to 15 nm serves as an effective marker at the electron microscopy level. Smaller colloidal gold (3-15 nm) is often used for the detection of single antigen particles, while larger particles (15 nm and above) are employed for detecting infected cells with a higher quantity of markers. This method requires minimal sample volume, offers fast detection, provides clear contrasts, is operationally simple, and exhibits high sensitivity and specificity. It is applicable for both antigen and antibody detection, making it suitable for both research and diagnostics.
Application at the Light Microscopy Level
Colloidal gold is also utilized as a marker at the light microscopy level, replacing traditional markers like fluorescent and enzymatic labels. It can be applied to various cell smears and slices. Mainly used for detecting membrane surface antigens of cell suspensions or monolayer cultured cells using single monoclonal antibodies or antisera, it can also be used for detecting intracellular antigens in monolayer cultured cells or antigens in tissues or sub-thin slices. The use of colloidal gold in light microscopy research can overcome the unavoidable drawbacks of high background and internal enzyme activity interference associated with other markers.
Application in Flow Cytometry
In immunological studies, counting and analyzing cell surface antigens using antibodies labeled with fluorochromes through flow cytometry is a crucial technique. However, due to spectral overlap among different fluorochromes, distinguishing between various markers becomes challenging. Therefore, a non-fluorescent marker is needed for flow cytometry counting. Colloidal gold, by significantly changing the red laser scatter angle, serves as one of the markers in flow cytometry.
Application in Immunoblotting Technique
Immunoblotting is a relatively new immunological technique. It involves separating proteins through polyacrylamide gel electrophoresis, transferring the obtained bands to nitrocellulose membrane, and then quantifying using enzyme immunoassay (or immunofluorescence, RIA). Immunogold can also be used for quantification in this technique. After incubating the transferred nitrocellulose membrane with specific antibodies, incubating with colloidal gold sensitized by Staphylococcus aureus protein A, and thoroughly washing away excess colloidal gold, the color depth of the colloidal gold particles on the membrane reveals the presence of specific antigens in the sample. Due to its simplicity, speed, and relatively high sensitivity, immunogold immunoblotting has significant potential applications in clinical immunodiagnosis.
In conclusion, colloidal gold, particularly in immunohistochemistry through immunogold staining, has evolved into a powerful tool for scientific research and clinical diagnosis. Its applications span from electron microscopy to light microscopy, flow cytometry, and immunoblotting. The stability, dynamic nature, and diverse applications of colloidal gold make it an invaluable resource in the precise localization of macromolecules, contributing significantly to advancements in medical diagnostics and scientific inquiry.
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