Development of a fluorescein modified ruthenium(II) complex probe for lysosome-targeted ratiometric luminescence detection and imaging of peroxynitrite in living cells

Ratiometric luminescence (fluorescence/phosphorescence) probes have attracted widespread attention of researchers in the field of biological detection and noninvasive imaging of bioactive molecules in living systems. However, most of them suffer from some defects such as small emission shift, different excitation wavelength and spectral overlap, which eventually affect the luminescence ratio, thus leading to limitations in ratiometric bioimaging applications. In this paper, we present a novel "ruthenium(II) complex-fluorescein" scaffold probe (Ru-FL-ONOO) for ratiometric luminescence detection of peroxynitrite (ONOO-), in which a Ru(II) complex was conjugated to fluorescein serving as the dual-emissive moiety and the spirocyclic structure of fluorescein-phenylhydrazine was used as the specifically-reactive moiety for recognizing ONOO-. The probe possesses not only favourable specificity but also high sensitivity for responding to ONOO-, exhibiting a large emission shift (Δλem > 120 nm) at a single excitation wavelength. After being transferred into living cells, the probe localized within lysosomes, allowing ONOO- therein to be imaged at ratiometric mode. The imaging results reveal that the ratiometric probe bearing the Ru(II) complex-fluorescein scaffold could be a useful approach for overcoming the drawback of spectral overlap of dual-emissive moiety under single-wavelength excitation so as to improve the signal-to-noise ratio, thus benefiting the development of ratiometric bioimaging.