Cyanine fluorescent dyes based on energy transfer mechanisms enable ratiometric sensing

Cyanine dyes, structurally characterized by two nitrogen containing heterocycles connected by a conjugated polymethine chain, have attracted great attentions in optical imaging. Here, we developed two novel cyanine fluorescent dyes based on energy transfer mechanisms and demonstrated their applications in ratiometric sensing of biological analytes. One is based on Förster resonance energy transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits at ~660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits at ~780 nm). Nitroreductase-catalyzed reduction of the 4-nitrobenzyl group on the Cy7 component induces increase of FRET efficiency, leading to a large increase in Cy7/Cy5 fluorescence ratio. The other one is based on through bond energy transfer (TBET) from a pyrene, tetraphenylethylene, or biphenyl(ethynylene) component as ultraviolet-absorbing energy donor to the covalently attached pentamethine cyanine acceptor dye (Cy5). These Cy5 TBET cassettes, as non-responsive dyes, can be mixed with another ultraviolet light-excited, responsive hydrocarbon sensor (emits at <550 nm) to achieve ratiometric imaging.