Abstract
Fluorescence detection is a central component in biological research. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. A single-stranded oligonucleotide was chemically bound to a single 50 nm diameter silver particle and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the particle-bound oligonucleotide. The bound Cy5 molecules on the silver particles were spatially separated from the silver surface by the hybridized DNA duplex chains, which were about 8 nm in length, to reduce the competitive quenching. We use fluorescence lifetime correlation spectroscopy (FLCS) with picosecond time-resolved detection to separate the fluorescence correlation spectroscopy (FCS) contributions from fluorophores and metal-conjugated fluorophores. The single Cy5-labeled 50 nm silver particles displayed a factor of 15-fold increase in emission signal and 5-fold decrease in emission lifetimes in solution relative to the Cy5-DNA in the absence of metal. Lifetime measurements support the near-field interaction mechanism between the fluorophore and silver nanoparticle. In this study, FLCS is being applied to a system where the brightness and the fluorescent lifetime of the emitting species are significantly different. Our measurements suggest that FLCS is a powerful method for investigating the metal-fluorophore interaction at the single molecule level and to separate two different species from a mixture solution emitting at the same wavelength. Additionally, the highly bright Cy5-DNA-Ag molecules offer to be excellent probes in high background biological samples.