Development of novel fluorescent probe- protein protected gold nanoclusters for biomedical applications.

摘要

This dissertation explores photo-physics, fluorescence polarization properties; resonance energy transfer (RET) probes, and cellular imaging applications for novel fluorescent probe BSA Au clusters By Rayleigh scattering subtraction from extinction spectrum, we found that BSA Au clusters shows peak absorption around 360 nm and shoulder near 500 nm. Peak fluorescence emission lies around 650nm. The fluorescence quantum yield was determined to be 0.06%, while it displayed long fluorescence lifetime of 1.8 mus. BSA Au clusters show stable fluorescence properties and resistance to unfolding and quenching with varying pH, temperature, quencher and denaturant concentration. Moreover, BSA Au clusters shows distinct fluorescence polarization behavior as measured in solvents of different viscosity. The BSA Au cluster, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight and developing fluorescence polarization immunoassays. BSA Au clusters suffer from several disadvantages such as low quantum efficiency (typically near 6%) and broad emission spectrum (540nm to 800nm). We describe an approach by developing RET probes to enhance the apparent brightness more than 2 fold of BSA Au clusters by linking it with high extinction donor organic dye pacific blue (PB). Moreover, we prepared another conjugate 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% transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Transferring energy from BSA Au cluster to Dy750 will have a RET probe with narrow emission spectrum and long lifetime component which can be explored in imaging applications. Furthermore, herein I describe the use of these long lived BSA Au clusters in cellular and tissue imaging applications. In first approach, I have shown its utility as FLIM probe as well as a time gated intensity imaging probe. In second approach we have shown the 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 noise ratio at least by 15 times. Furthermore, by applying gated detection strategy to multipulse excitation 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.