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

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