Ratiometric Molecular Probes Based on Dual Emission of a Blue Fluorescent Coumarin and a Red Phosphorescent Cationic Iridium(III) Complex for Intracellular Oxygen Sensing

Ratiometric molecular probes RP1 and RP2 consisting of a blue fluorescent coumarin and a red phosphorescent cationic iridium complex connected by a tetra- or octaproline linker, respectively, were designed and synthesized for sensing oxygen levels in living cells. These probes exhibited dual emission with good spectral separation in acetonitrile. The photorelaxation processes, including intramolecular energy transfer, were revealed by emission quantum yield and lifetime measurements. The ratios (R-I = (I-p/I-f) ) between the phosphorescence (I-p) and fluorescence (I-f) intensities showed excellent oxygen responses; the ratio of R-I under degassed and aerated conditions (R-I(0)/R-I) was 20.3 and 19.6 for RP1 and RP2. The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker. The emission spectra of the ratiometric probes internalized into living HeLa or MCF-7 cells could be obtained using a conventional microplate reader. The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R-I value of RP2 under hypoxia (2.5% O-2) to that under normoxia (21% O-2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively. Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.