A Hemicyanine‐Embedded Diphenylselenide‐Containing Probe “HemiSe” in which SePh2 Stays Reduced for Selective Detection of Superoxide in Living Cells

: A simple one-step synthesis of fluorescent probe HemiSe has been developed for the detection of superoxide (O2.- ). The probe undergoes reaction specifically with O2.- when in the presence of other competitive ROS/RNS/metal ions. The diphenylselenide was incorporated to completely quench the fluorescence of the hemicyanine unit through the action of a photoinduced electron transfer (PET) photomechanism. However, after the addition of O2.- , the latent fluorophore regains its fluorescence owing to the reaction at the C=C bond of the hemicyanine with O2.- through nucleophilic attack; the increase in blue emission is due to a reaction of the double bond within HemiSe followed by an increase in fluorescence quantum yield (Φ) up to 0.45; the limit of detection (LOD) is 11.9 nm. A time-dependent study shows that HemiSe can detect superoxide within 13 min with high sensitivity, high selectivity, over a wide pH range, and through confirmation with a xanthine/xanthine oxidase biochemical assay (λem =439 nm). A study in the RAW 264.7 macrophage living cells also shows that HemiSe is not toxic, cell permeable (experimental log P=2.11); confocal imaging results show that HemiSe can detect O2.- in endogenous and exogeneous systems.