Release of the self-quenching of fluorescence near silver metallic surfaces

Fluorescein is one of the most widely used fluorescent probes in microscopy, biotechnology, and clinical assays. One difficulty with fluorescein is its self-quenching, which results in decreased intensities with increasing labeling density. In this study we examined human serum albumin (HSA), which contained one to nine covalently linked fluorescein molecules per molecule of HSA. The occurrence of homo resonance energy transfer for labeling ratios greater than 1 were confirmed by decreases in the relative quantum yields, anisotropies, and lifetimes. We found that most of the self-quenching can be partially eliminated by proximity of the labeled protein to metallic silver particles. These results suggest the use of heavily labeled proteins and metallic colloids to obtain ultrabright reagents for use in immunoassays, imaging, and other applications.