Autometallographically enhanced Nanogold probes have proved highly sensitive when used in conjunction with Catalyzed Reporter Deposition (CARD), an enzymatic amplification method. In ultrasensitive in situ hybridization, this method detected single copies more reliably than enzymatic detection. More simple and direct amplification methods will be developed, in which suitably functionalized gold cluster labels are deposited catalytically at a site of interest using either enzymatic deposition, or a chemical amplification method in which a free radical polymerization initiator, targeted to the site of interest by an antibody or other targeted biomolecule, will initiate the polymerization and deposition of gold clusters functionalized with monomers which propagate the reaction. The new methods will be robust and simple, and may be used both for the ultrasensitive detection of low copy number antigens in immunocytochemistry, and for in situ hybridization of specific DNA and RNA. The new methods will extend the applications of amplification to electron microscopy while providing an alternative, simpler method for light microscopy and pathology. The new reagents and procedures will combine the high sensitivities of amplification with the high spatial resolution which in only available with gold probes, and the unique ability of Nanogold to access to restricted antigens.
Amplification is widely used in ultrasensitive in situ hybridization detection, and has many other applications for high-resolution, ultrasensitive detection of low copy number antigens in electron microscopy and immunohistochemistry. The new reagents will be as sensitive as those currently available, simpler and robust, and the small size and covalent attachment means that probes may be constructed using any functionalizable probe for the widest possible range of applications.
