TBET-based ratiometric fluorescent probe for Hg2+ with large pseudo-Stokes shift and emission shift in aqueous media and intracellular colorimetric imaging in live Hela cells

The detection of Hg2+ in biological systems and its imaging is of highly importance. In this work, a novel ratiometric fluorescence probe is developed based on through-bond energy transfer (TBET) with a 2-(2-hydroxyphenyl)benzoxazole (HBO) as donor and a Rhodamine derivative-Hg conjugate (RDM-Hg) as acceptor. Hg2+ weakens the fluorescence of HBO at 430 nm and meanwhile interacts with Rhodamine B derivative to form a fluorescent conjugate (RDM-Hg) giving rise to emission at 597 nm with a 167 nm red-shift. Further, the difference 282 nm between the donor absorption (315 nm) and the accepter emission (597 nm) for 1 + Hg2+ is comparable to the highest value of the Stokes shift (282 nm) reported earlier for other reported TBET-based cassette. Through-bond energy transfer from HBO to RDM-Hg is triggered by Hg2+ resulting in concentration dependent variation of fluorescence ratio I-597/I-430. A linear calibration of I-597/I-430 versus Hg2+ concentration is obtained within 0-5 mu M, along with the lowest detection limit being found to be as low as 1.31 x 10(-9) mol center dot l(-1) (similar to 0.26 ppb) for Hg2+. This feature is further demonstrated by colorimetric imaging of test strip and intracellular Hg2+. On the other hand, the HBO/RDM TBET sensing system is characterized by a combination of high sensitivity and selectivity. The present study provides an approach for further development of ratiometric probes dedicated to selective in vitro or in vivo sensing some species of biologically interest.