Introduction: How does Gold Cluster Labeling Work?

Structure of Monomaleimido Undecagold Gold clusters are coordination complexes. They contain a core of gold atoms in a predetermined arrangement; gold atoms at the surface of this core are coordinated by either a tris (aryl) phosphine ligand, or by a halide ion (chloride in the undecagold cluster shown above) so that the bonding valencies of all the gold atoms are satisfied. As a result, the gold cluster is a stable molecule.

Undecagold and Nanogold®, which is a larger gold cluster containing the same ligands, are uncharged, although other gold clusters such as hexagold (6 gold atoms) and octagold (8 gold atoms) can have an ionic charge. In contrast, colloidal gold is not a molecule with a defined structure, but suspended particles with some of the properties of metallic gold. Colloidal gold in solution adopts a negative charge, thought to arise from [Au^ICl₂] whichmake up the surface of the particle, as shown below:

Colloidal Gold (From: Weiser, H. B.: "Inorganic Colloid Chemistry;" Wiley, New York, NY; Vol. 1, pp. 21-57 (1933).

Because of this charge, colloidal gold has an affinity for many proteins, which tend to be positively charged at neutral or physiological pH. Some proteins, especially antibodies, adsorb strongly to colloidal gold particles: when introduced into a biological specimen which contains binding sites, they carry the colloidal gold particle to this site where it may be visualized. However, this affinity for proteins, or "stickiness," means that the colloidal gold particles can also adsorb to other components of the system, causing false or background labeling; andin addition to the conjugated antibody, colloidal gold conjugates must also be stabilized with an inert macromolecule such as bovine serum albumin (BSA), gelatin, carbowax or polyethylene glycol; this increases the size of the probe and can prevent it from penetrating into specimens, for example to label nuclear features. The adsorbed antibody can also dissociate, and free antibodies compete with the conjugate, resulting in less dense labeling. Below are twolight micrographs which show a direct comparison between colloidal gold and Nanogold®:

Spindle microtubules labeled with GAM colloidal gold (LEFT) and Fab'-Nanogold® (RIGHT) (Light micrograph courtesey of Dr. D. Vandré and Dr. R. Burry, Ohio State University. Original magnification = 1300x).

Gold clusters are not adsorbed, but covalently cross-linked to a specific functional group on the targeted biomolecule. For example, monomaleimido undecagold, shown above, reacts only with thiol (sulfhydryl) groups, and therefore attaches to a protein only at a thiol site. This site-specificity allows it to be conjugated at a position remote from the binding site so that it does not interfere with substrate binding; for example, monomaleimido undecagold labels antibody Fab' fragments specifically at the hinge region (which contains the only thiol), is at the other end of the Fab' to the antigen combining region, as shown below:

Clusters may be prepared with a number of different reactive groups, for labeling different sites on biological molecules. Undecagold and Nanogold® are also available as mono-amines, which may be used in a variety of cross-linking reactions, or as mono-sulfo-NHS esters, which react with amines as shown below (To replay the animation, re-open in a new window or use your browser's "reload" button):

Site-specific covalent conjugation means that clusters canbe used to label a wide variety of biologically active molecules, such as lipids, oligonucleotides, peptides and drugs, which do not adsorb to colloidal gold and hence cannot be colloidal gold-labeled. Reactive forms of Nanogold® and undecagold are supplied lyophilized as labeling reagents, with which our customers can label any molecule with the appropriate chemical group. Once conjugated, they are stable indefinitely, and do not have residual charges which can interact non-specifically with biological systems. Furthermore, the coordinated ligands can be modified to confer desirable properties on the molecule (such as water-solubility).

References

1. Immunogold-Silver Staining: Principles, Methods and Applications," M. A. Hayat (Ed.); CRC Press, Boca Raton, FL, 1995. See chapters by: Burry, R. W.; Hainfeld, J. F., and Furuya, F. R. (pp. 71-96); and Hacker, G.
2. Hainfeld, J.F. Site specific cluster labels. Ultramicroscopy, 46, 135-144 (1992).
3. Hainfeld, J.F. and Furuya, F.R. A 1.4nm Gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177-184 (1992).
4. Takizawa, T. and Robinson, J.M. Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryo- sections. J. Histochem. Cytochem., 42, 1615-1623 (1994).

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