Simultaneous dual-channel fluorescent probe for Cys and ONOO− enables rapid monitoring of dynamic redox balance changes during ferroptosis

Ferroptosis is a programmed cell death process caused by the accumulation of intracellular lipid peroxides due to disorders of iron metabolism and redox imbalance. Currently, its specific molecular mechanism has not been fully elucidated, therefore detecting the changes in the dynamic balance of intracellular redox is of great significance for revealing this mechanism. In living organisms, the balance of redox is mainly regulated by reactive oxygen species (ROS) and active sulfur species (RSS). Here, we are investigating the reactive oxygen species peroxynitrite (ONOO−) and the biological thiol molecule cysteine (Cys), participate in the ferroptosis process by regulating redox balance. In this study, a novel coumarin-based fluorescent probe was designed and synthesized, which contains dual responsive sites for Cys and ONOO−. This probe enables rapid, efficient, and specific detection of Cys (with an emission wavelength of 498 nm) and ONOO− (with an emission wavelength of 580 nm) through green and red fluorescence channels, respectively. The experimental results demonstrate that this probe can dynamically monitor the changes in Cys and ONOO− levels during Erastin-induced ferroptosis: after erastin stimulation, the level of ONOO− significantly increased, while the intracellular Cys content significantly decreased and remained at a low level. Compared with the previously developed LXB probe by our research group, the novel probe constructed in this study has a faster response speed, enabling real-time tracking of dynamic redox homeostasis imbalance during ferroptosis with high spatiotemporal resolution. This establishes a single-probe bimodal tool to decipher the spatiotemporal coupling between cysteine depletion and nitrosative stress during ferroptosis.