Colloidal gold solution refers to the dispersed phase particles diameter between l-150 nm gold sol, belong to heterogeneous system, the color is orange-red to fuchsia. Colloidal gold as a marker for immunohistochemistry began in 1971, Faulk et al. applied electron microscopy immunocolloid gold staining (IGS) to observe Salmonella, after which they combined colloidal gold with multiple proteins. In 1974 Romano et al. labeled colloidal gold on a second antibody (horse anti-human IgG) and established an indirect immune colloidal gold staining method. In 1978 geoghega discovered the application of colloidal gold markers at the optical level. The application of colloidal gold in immunochemistry is also called immunogold. After that, many scholars have further confirmed that colloidal gold can adsorb proteins stably and rapidly, but the biological activity of proteins has not changed significantly. It can be used as a probe for the precise localization of cell surface and intracellular polysaccharides, proteins, skin, antigen, hormone, nucleic acid and other biomolecules. And spot immunogold staining technology has become a powerful tool for scientific research and clinical diagnosis.
1. The basic principle of immune colloidal gold technique
HAuCl4 can be polymerized into a certain size of gold particles under the action of reducing agent to form a negatively charged hydrophobic glue solution. because of the electrostatic action to become a stable colloidal state, so called colloidal gold. Colloidal gold particles are composed of a basal gold core (atomic gold Au) and a biionic layer surrounded by an inner negative ion (AuC12-) attached to the surface of the gold core, and the outer layer H is dispersed in an intercolloid solution to maintain the suspension state of colloidal gold free from the sol.
The base gold core of colloidal gold particles is not an ideal orb. the smaller colloidal gold particles are basically spherical, and the larger colloidal gold particles (generally referred to as larger than 30 nm) are mostly elliptical. The particle morphology of colloidal gold can be observed under electron microscope. colloidal gold labeling, in essence, is the coating process in which polymers such as proteins are adsorbed on the surface of colloidal gold particles. the adsorption mechanism may be the colloidal gold particle surface negative charge, and the positive charge groups of the protein form a firm binding due to electrostatic adsorption. Various colloidal gold particles of different sizes and colors can be easily prepared from chloric acid by reduction. This spherical grain it has a strong adsorption function to proteins and can be noncovalently conjugated with staphylococcus a protein, immunoglobulin, toxin, glycoprotein, enzyme, antibiotic, hormone, bovine serum albumin polypeptide conjugate and so become very useful tools in basic research and clinical experiments.
Immunogold labeling technique mainly utilizes the high electron density properties of gold particles, which can be seen under the microscope at the junction of gold standard proteins, and red or pink spots are visible to the naked eye when these markers are massed at the corresponding ligands point, and therefore used in qualitative or semi-quantitative rapid immune detection methods, this reaction can also be amplified by the deposition of silver particles, called immunogold and silver staining.
2. The characteristics of the colloidal gold
2.1 The colloidal gold particles are mostly of 1~100 nm in colloidal nature, and the tiny gold particles are suspended in liquid in a stable, uniform and single dispersed state and become colloidal gold solution. Colloidal gold thus has a variety of colloidal properties, especially sensitivity to electrolytes. the electrolyte can destroy the peripheral permanent hydration layer of the colloidal gold particles, thus breaking the stable state of the colloid and condensing the dispersed single gold particles into large particles while settling down from the liquid. Some macromolecular substances such as proteins have the function of protecting colloidal gold and enhancing its stability.
2.2 The micro-particle colloids are red in color, but there are some differences in color in the colloids of different sizes. the smallest colloidal gold (2–5 nm) is orange-yellow, the medium-sized colloidal gold (10–20 nm) is wine-red, and the larger particles are fuchsia-red. According to this characteristic, the color of colloidal gold can be roughly estimated with the naked eye. 3。 In recent 10 years, colloidal gold has been developed as an important immunolabeling technology. The study of colloidal gold immunoassay has been developed in many fields, such as drug detection, biomedicine and so on. attention in related research fields. the light-absorbing colloidal gold has a single light absorption peak in the visible range. the wavelength (955; max) of this light absorption peak varies with the colloidal gold particle size in the range of 510–550 nm.955; max of the large particle colloidal gold is biased toward the long wavelength, whereas 955; max of the small particle colloidal gold is biased toward the short wavelength. table 1 lists the partial colloidal gold 955; max.
|Colloidal gold diameter（nm）||1% sodium citrate（ml）*||Colloidal gold properties（colour）||λmax|
3. The preparation of colloidal gold
Colloidal gold has a variety of preparation methods, commonly used are: trisodium citrate reduction, trisodium citrate-tanning acid mixed reduction, white phosphorus reduction, ascorbic acid reduction, ethanol ultrasonic reduction, and so on. However, some of the details in the preparation have a great impact on the quality of colloidal gold, which should be noted:
(1) Chloruric acid is easily deliquescent and should be kept dry and light-free.
(2) Chloruric acid is strongly corrosive to metals and should not be used in the preparation of chloruric acid aqueous solutions.
(3) The distilled water used in the preparation of colloidal gold should be either double or triple distilled water or high-quality deionized water. (4) Glass containers for preparing colloidal gold must be absolutely clean and washed with distilled water before use. It is best to be treated with siliconization, which can be soaked in chloroform solution of 5% dichloromethane for several minutes, washed with distilled water and dried.
4. Identification of colloidal gold
The preparation of colloidal gold is not difficult, but the preparation of high-quality colloidal gold is not easy. Therefore, each time the colloidal gold should be verified, the main inspection indicators are particle size, particle size uniformity and whether there are agglutinated particles.
Naked eye observation is the most basic and simple and convenient verification method, but it needs some experience. Good colloidal gold should be clear and transparent, if the prepared colloidal gold turbid or liquid surface floating, suggesting that the preparation of colloidal gold has more agglutination particles. By observing the color of colloidal gold carefully in the sun, the size of the gold particles can be roughly estimated Small. of course, the particle size of the gold particles can be estimated by spectrophotometer scanning 955; max. The average particle size of colloidal gold can be measured accurately by microscopy and microscopy.
5.Stability of colloidal gold and storage of immune colloidal gold
Colloidal fittings have high kinetic stability, and their own condensation is very slow when the stabilizing factors are not destroyed, and can be placed for several years without condensation. The main factors affecting stability are electrolyte, sol concentration, temperature, non-electrolyte and so on.
Gold sol must have a small amount of electrolyte as stabilizer, but the concentration should not be too high. The high concentration of hydrophilic non-electrolyte can remove the hydration film outside the colloidal particles to make it agglomerate. A small amount of polymer contributes to sol condensation, but a certain amount of polymer can increase sol stability, such as the addition of protein, glucose and PEG20000 with good stability effect.
when the gold sol adsorbs the protein, the stability of the sol changes with the solution ph, and this change in turn depends on the isoelectric point of the adsorbed protein, such as cona, peroxidase and so on. when the ph is low, it remains stable, and the increase of ph appears unstable, close to the isoelectric point or slightly high and becomes stable again. the marked colloidal gold solution can be used from 0.2 to 0.5 mg/ml peg20000 as a stabilizer. Storage at 4 to 10°C for several months is not suitable for freezing. Storage may occur in different degrees of condensation, can be removed by centrifugation.
6.Application of Colloidal Gold
6.1 Application of colloidal gold at electron microscope level
Colloidal gold applied electron microscope level research is the earliest, the most rapid development, the most widely used. the greatest advantage is that it can be double or multiple labeled by applying different size particles or binding enzyme markers. colloidal gold with a diameter of 3 to 15 nm can be used as a marker for electron microscope level. colloidal gold of 3 to 15 nm is mostly used for the detection of single antigen particles, while the diameter of 15 nm is mostly used for the detection of infected cells with more quantities.
Colloidal gold is used for the study of electron microscope level, mainly including: observation of cell surface antigen in cell suspension or monolayer culture. Single layer detection of intracellular antigens in culture. tissue antigen detection.
6.2 Application of colloidal gold in light mirror level
Colloidal gold can also be used as a light mirror-level marker to replace traditional fluorescein, enzymes, etc. All kinds of cell smears and sections can be used. Mainly used for detection of membrane surface antigen of cell suspension or cultured monolayer cells with monogram antibodies or antiserum. detection of cultured monolayer intracellular antigens, detection of antigens in tissues or subthin sections.
6.3 Agglutination test: The monodisperse immunogold sol is a clear and transparent solution, the color of which varies with the size of the sol particles and is specific to the corresponding antigen or antibody The principle can be applied qualitatively or quantitatively to the immune reaction when the sol particles increase extremely, the light scattering changes and the particles settle, and the color of the solution becomes pale or even colorless.
In addition, colloidal gold is widely used in flow cytometry, immunoimprint technology and immunochromatographic rapid diagnosis technology.
In the past 10 years, colloidal gold marking has been developed as an important immunolabeling technology. Colloidal gold immunoassay has been well developed in many fields, such as drug detection, biomedicine and so on. Predictably, with the development of nanotechnology, glue Body gold will be paid more and more attention by relevant research fields.