BIOPHYSICAL CHARACTERIZATION OF FLUORESCENT BSA PROTECTED GOLD NANOCLUSTERS FOR POTENTIAL APPLICATION IN CELLULAR IMAGING AND STUDYING MACROMOLECULAR COMPLEXES.

Purpose: Spectroscopic characterization of novel fluorescent probe BSA Au25 nanoclusters for potential biophysical and biomedical applications Methods: In this study, we synthesized the BSA protected Au25 nanoclusters and studied their one photon and two photon steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. UV-Vis absorption and fluorescence measurements were obtained using a Cary 50 bio UV-visible Spectrophotometer (Varian Inc.) and Cary Eclipse Spectrofluorometer (Varian Inc.) respectively. Two photon excitation was achieved with Origami-10, 1038 nm pulsed laser (40 MHz repetition rate, 180 mW average power and 140 fs pulse duration) and 10x objective, mounted on a horizontal positioned and emission was collected using FT300 spectrofluorometer. Results: The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of the BSA Au25 cluster is long and heterogeneous with an average value of 1.84 µs. In glycerol at -200C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 µs. In propylene glycol the measured correlation time is longer and initial anisotropy depends on the excitation wavelength. Our two photon experiment results show a quadratic relation between excitation power and emission intensity whereas with one photon excitation shows a linear dependence. The emission spectrum of BSA Au25 nanoclusters with one photon and two photon excitation shows no appreciable change. Conclusions: The BSA Au25 cluster, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight. The major observation of our two photon experiments is the 2PE ability of BSA Au25 clusters. The presence of 2PE properties among these clusters opens up a door to many exciting applications in microscopy and time resolved fluorescence. Moreover, being in the NIR region together with 2PE capability will make them ideal imaging candidate.