A Cascade Activation Probe with Double-Enhanced Near-Infrared\nImaging for Monitoring Peroxynitrite Fluctuations in Vivo

Monitoring\nperoxynitrite (ONOO<sup>–</sup>) fluctuations\nis particularly important for assessing pathological progression and\noxidative damage due to their crucial role in maintaining the redox\nbalance of organisms. However, due to the lack of efficient tools\nfor differentially monitoring ONOO<sup>–</sup> fluctuations\nat different concentration ranges in vivo, the precise detection of\nendogenous ONOO<sup>–</sup> fluctuations under pathological\nconditions in living systems remains challenging. Herein, we rationally\ndesigned a double-enhanced emission cascade activatable near-infrared\n(NIR) fluorescent probe (<b>B-TCF</b>) for the measurement of\nONOO<sup>–</sup>, which consists of a borate ester response\ngroup and a malononitrile hemicyanine fluorophore. Especially, after\nsequential oxidative hydrolysis of the borate ester group and xanthene\nskeleton, <b>B-TCF</b> exhibited a sequentially double-enhanced\nNIR emission response at 776 and 625 nm for different ONOO<sup>–</sup> concentration ranges. Moreover, <b>B-TCF</b> revealed excellent\nand promising performance for ONOO<sup>–</sup> in terms of\nhigh selectivity, sensitivity, and reaction rate (<i>k</i> = 28.2 M<sup>–1</sup> s<sup>–1</sup>). Motivated by\nthe two-step emission signal enhancement and large wavelength shift\nin the NIR region, <b>B-TCF</b> enabled discriminative imaging\nof ONOO<sup>–</sup> with the low and high concentrations in\nliving cells. Importantly, <b>B-TCF</b> was successfully applied\nfor assessing the pathological progression of isoniazid and acetaminophen-induced\nliver damage in vivo by detecting the endogenous different ONOO<sup>–</sup> levels. Overall, this study not only demonstrates\nthe first double-enhanced emission cascade activatable NIR fluorescent\nprobe for measuring the dynamic variation of ONOO<sup>–</sup> in related diseases but also shows great potential as an effective\nmolecular tool for evaluating the various stages of drug-induced liver\ndamage.