A long-wavelength turn-on fluorescent probe for intracellular nanomolar level peroxynitrite sensing with second-level response.

As a typical kind of endogenous reactive nitrogen species, peroxynitrite (ONOO) is believed heavily involved in the pathogenesis of many diseases such as inflammation, neurodegenerative conditions, and cardiovascular disorders. Precisely estimating ONOO level in cell compartments is crucial for unraveling the biological relevance of ONOO and enabling effective control of ONOO-associated pathogenicity but suffers from serious difficulty owing to the daunting elusive features of ONOO, namely nanomolar level physiological concentration and millisecond level biological half-life. A new fluorescent probe capable of detecting ONOO with limit of detection down to 1.2 nM, response time less than 1s, and high recognition specificity over other similarly interfering species was developed in this work. For the probe constructed by conjugating an isatin moiety with an electron-withdrawing tricyanofuran (TCF) moiety, the former enabled a highly selective ONOO-mediated oxidative decarbonylation reaction while the latter significantly improved the electrophilicity of the 3-position carbonyl group of isatin moiety and therefore accelerate the ONOO-mediated nucleophilic attack, which eventually enabled prompt and efficient recognition reaction. For the decarbonylated product featured with a released primary aniline moiety, TCF acted as an acceptor for enabling an intramolecular charge transfer (ICT) process and the remarkable change in electronic feature upon reaction with ONOO, which generated turn-on fluorescence with large contrast and therefore the basis for ONOO sensing. The cell fluorescence imaging performed in this work definitely verified the capability of the probe for mapping intracellular ONOO, despite the daunting elusive features of such physiological species.