Ratiometric oxygen probes with a cell-penetrating peptide for imaging oxygen levels in living cells

Abstract

We investigated the photophysical and intracellular properties of two newly designed molecule-based ratiometric oxygen probes, 7DEAC-R8-BTQphen (R8) and 7DEAC-R12-BTQphen (R12), to evaluate their suitability for intracellular O2 measurements. R8 and R12 consist of a blue fluorescent coumarin derivative (7DEAC) and a red phosphorescent iridium(III) complex (BTQphen) connected respectively by an octa- or dodecaarginine linker to promote intracellular delivery of the probes. The probes showed oxygen-sensitive dual emissions in acetonitrile, lipid bilayer membrane, and in living cells. Energy state diagrams showing the relaxation processes of R8 and R12 in the excited state were constructed using emission spectra, quantum yield, and lifetime data. Efficient photon absorption at 405 nm due to the 7DEAC chromophore is followed by intramolecular energy transfer to the BTQphen moiety, resulting in 7DEAC fluorescence and BTQphen phosphorescence with an intensity ratio and spectral separation suitable for ratiometric measurements. The validities of O2 concentrations derived from the ratios between the phosphorescence and fluorescence intensities of R8 and R12 were verified in lipid membranes, as well as in solution by phosphorescence lifetime measurements of the BTQphen moiety. The cellular uptake efficiencies of R8 and R12 were significantly enhanced due to their oligoarginine linkers compared to previous probes with oligoproline linkers. These ratiometric oxygen probes allowed visualization of the O2 gradient produced by placing a thin coverslip onto a HeLa cell monolayer.