A sequence-activatable dual-locked fluorescent probe for simultaneous detection of hypochlorous acid and peroxynitrite during drug-induced liver injury.

Drug-induced liver injury (DILI) is a crucial factor that poses a significant threat to human health. DILI process leads to the changes of reactive oxygen species and reactive nitrogen species content in cells, which leads to oxidative and nitrosative stress in cells. However, the high reactivity of hypochlorous acid (HOCl) and peroxynitrite (ONOO⁻), combined with a lack of in situ imaging techniques, has hindered a detailed understanding of their roles in DILI. Therefore, this paper reports a novel sequence-activatable dual-locked molecular probe HA-P3 for the identification and imaging of two DILI-related biomarkers. First, HA-P3 selectively reacts with reactive oxygen species HOCl to leave the recognition receptor diethyl thiocarbamate to form HA-P2. Subsequently, HA-P2 reacts with ONOO⁻, liberating the fluorophore 4-hydroxy-1,8-naphthalimide, which emits a strong fluorescence signal. The two-step reaction effectively reduces the probability of false positive in predicting DILI. HA-P3 achieved the sensitive detection of HOCl and ONOO^- in different cells and zebrafish. Furthermore, HA-P3 can distinguish between normal liver cells and hepatoma cells and monitored the elevated levels of HOCl and ONOO⁻ during acetaminophen (APAP)-induced cellular damage. It is worth noting that in the APAP-induced mouse model, the positive correlation between HOCl and ONOO^- and DILI was revealed, providing strong direct evidence for the relationship between oxidative/nitrosative stress and DILI.