A suspension of gold (Au) spheres is produced by the chemical reduction of yellow gold chloride (chloroauric acid) in solution. This happens in a sequence of stages (5). Initially, the reduction of Au3+ produces a supersaturated molecular Au solution. Nucleation is initiated when the concentration of Au increases and the gold atoms cluster and form nuclei. Particle growth proceeds with the deposition of molecular gold on the nuclei. The theoretical size of the gold is inversely proportional to the cube root of the number of nuclei formed if it is assumed that the conversion of Au3+ to Au is complete and that the concentration of gold remains constant (6).
Properties and Features of Colloidal Gold as a Marker in Microscopy
• Gold probes are widely used in light microscopy transmission elec tron microscopy, scanning electron microscopy, freeze fracture, in situ hybridization, negative staining, and enzyme cytochemistry as are easy and inexpensive to prepare.
• They are readily adsorbed to a variety of macromolecules including antibodies, lectins, enzymes, lipids, high-energy phosphate compounds such as ATP, nucleic acids, and reporter probes such as streptavidin.
• Unbound gold sols and gold-labeled probes both have a long shelf life.
• Gold particles are electron dense and do not generally decay in an electron beam.
• Covalent linking of newer, smaller gold particles to macromolecules is even more stable than conventional adsorption.
• Probes offer high labeling intensity and sensitivity. The smaller the gold particle, the more sites that are labelled.
• There is no diffusion of reaction products as is often experienced in immunoenzyme labeling.
• The smaller the particle, the less size variation occurs; gold clusters in particular have a very specific size.
• Clusters show negligible tendency to aggregate compared with other gold preparations.
• Smaller gold particles in particular have high-resolution properties.
• High resolution of smaller gold clusters can be used for labeling specific sites on single biomolecules.
• The particulate nature of gold markers is especially useful in the subtle delineation of cell structures.
• Gold particles show high penetration into cells and tissues. This is especially so with the newer, smaller probes.
• Gold particles form an efficient nucleus for metallic silver enhance ment—silver enhancement may be used to render gold labeling visible by EM, LM and, in some novel applications, even directly by eye.
• Gold probe labeling can be used with other affinity labeling proce dures for colocalization experiments.
• Gold particles do not interfere with the functional integrity of bio logical compounds after attachment
• Because of the particulate nature of gold particles, they can provide quantitative data at the molecular level.
• Gold labels can be used to provide dynamic information about molecu lar movement and membrane conformational changes in cells.
The use of different reducing agents allows us to produce particles of a prechosen size range. The speed of reduction of the gold chloride determines how many gold nuclei are formed. In a closed system, this will determine the final size of the gold. The aim has always been to work towards monodispersed suspensions, avoiding particle aggregates, and to encourage as little variance as possible in the mean particle diameter of samples. Experience has shown that the desired routine sizes are 5, 10, and 15 nm. All these are useful for transmission electron microscope studies, and the 5 nm of gold is recommended for light microscopy (see Note 5). Gold spheres of 30 nm are occasionally used for scanning electron microscope immunocytochemistry. A 1-nm probe is now readily available commercially (see Notes 6 and 7) and is proving useful as a more efficient nucleus for silver enhancement (see Notes 8-12). The following procedures have been described in the literature as being useful for colloidal gold production:
1. White phosphorus reduction: white phosphorus, and indeed sodium or potassium thiocyanate, are fast reducers. Chloroauric acid is boiled under reflux with white phosphorus (7) dissolved in diethylether. Boiling is continued until the solution turns from a brownish shade to red—this usually takes approx 5 min. This method produces gold particles in the smaller size range (2-12 nm). The accurate production of the particle size range is not easy and, as the procedure is potentially highly dangerous, it is not commonly used.
2. Ascorbate reduction: this method (8) entails the fairly rapid addition of sodium ascorbate to chloroauric acid while stirring. If the reaction is conducted on ice, the gold particle size range is between 6 and 8 nm. Higher temperatures tend to increase the particle size. The method is also really of academic interest currently because it is not easy to control the final sphere diameter.
3. Citrate reduction: chloroauric acid is boiled under reflux with sodium citrate (9) until the solution turns red. Particle sizes achieved are in the range of 15-150 nm, and a particular diameter is chosen for a batch by adjusting the amount of citrate added to the boiling flask. This method has been used, therefore, for producing probes in the larger particle size range.
4. Combined tannic acid-citrate reduction: in 1985, Slot and Geuze (5) described a method of using two reducing agents, tannic acid and sodium citrate, in combination to accurately control the diameter of gold particles yielded. Adjusting the amount of tannic acid in the mixture will control the sphere diameter very precisely at the point within the size range of 3-17 nm. The method produces sols with very little variance in mean particle diameter. Centrifugation to purify the end product is not usually needed as gold particle aggregates are infrequently found.
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