Photophysics, RET Probes and Imaging Applications for Fluorescent BSA Au Nanoclusters

Abstract

Protein protected fluorescent metal nanoclusters are attracting great deal of attention from fluorescence community. This work explores photo-physics, fluorescence polarization properties; resonance energy transfer (RET) probes, and cellular imaging applications of novel fluorescent probe bovine serum albumin protected gold (BSA Au) nanoclusters. Important findings were long fluorescence lifetime (> 1µs) and polarized fluorescence emission studied in solvents of different viscosity. The BSA Au cluster, due to long lifetime and high polarization, can potentially be used in developing fluorescence polarization immunoassays. Moreover, we describe an approach by developing RET of BSA Au clusters with the near infra-red (NIR) dye Dylight 750 (Dy750), where BSA Au cluster act as a donor to Dy750, showing 46% energy transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Such RET probe with narrow emission spectrum and long lifetime component can be explored in microscopy imaging applications. Furthermore, we describe the use of these long lived BSA Au clusters in cellular and tissue imaging applications. We have shown using multiple pulse excitation (MPE) scheme that one can increase the intensity of long lived BSA Au cluster in cells without increasing the short lived auto-fluorescence background thereby increasing signal to background ratio at least by 15 times. Moreover, by applying time gated detection strategy to MPE imaging experiment we increased the signal to noise ratio to 30, a dramatic improvement in contrast for low fluorescence quantum yield dye. In summary, BSA Au clusters can be used as a fluorescent cellular stain advancing the bioimaging technology via their long fluorescence lifetime.