Fluorescent Probes Based on π-Conjugation Modulation betweenHemicyanine and Coumarin Moieties for Ratiometric Detection of pH Changes inLive Cells with Visible and Near-infrared Channels

摘要

We report two ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine moieties for sensitive ratiometric detection of pH alterations in live cells by monitoring visible and near-infrared fluorescence changes. In a π-conjugation modulation strategy, a coumarin dye was conjugated to a near-infrared hemicyanine dye via a vinyl connection while lysosome-targeting morpholine ligand and o-phenylenediamine residue were introduced to the hemicyanine dye to form closed spirolactam ring structures in probes >A and >B, respectively. The probes show only visible fluorescence of the coumarin moiety under physiological and basic conditions because the hemicyanine moieties retain their closed spirolactam ring structures. However, decrease of pH to acidic condition causes spirolactam ring opening, and significantly enhances π-conjugation within the probes, thus generating new near-infrared fluorescence peaks of the hemicyanine at 755 nm and 740 nm for probes >A and >B, respectively. Moreover, the probes display ratiometric fluorescence response to pH with decreases of the coumarin fluorescence and increases of the hemicyanine fluorescence when pH changes from7.4 to 2.5. The probes are fully capable of imaging pH changes in live cellswith good ratiometric responses in visible and near-infrared channels, andeffectively avoid fluorescence blind spots under neutral and basic pH conditions- an issue that typical intensity-based pH fluorescent probes run into. Theprobe design platform reported herein can be easily applied to prepare a varietyof ratiometric fluorescent probes for detection of biological thiols, metalions, reactive oxygen and nitrogen species by introducing appropriate functionalgroups to hemicyanine moiety.