An excitation-customized ICT-type turn-on probe for visualization of cysteine fluctuation in diverse drug-induced ferroptosis pathways.

Cysteine (Cys) acts as a key upstream regulator of cellular redox homeostasis and ferroptosis. Distinct ferroptosis inducers exert different effects on Cys metabolism. Erastin disrupts Xc--dependent cystine uptake to deplete cellular Cys and trigger ferroptosis, while RSL3 induces ferroptosis by impairing downstream lipid metabolism without disturbing Cys homeostasis. Thus, real-time monitoring of Cys fluctuations is vital for distinguishing early ferroptosis initiation from downstream cascades. Conventional PET-type turn-on fluorescent probes suffer from high background and poor sensitivity. Herein, we report an excitation-customized strategy to develop ICT-type turn-on probe for Cys sensing. Based on a series of chalcogen-regulated near-infrared hemicyanine dyes, a selenium-substituted fluorophore HDXZ was screened out to construct the Cys-specific probe BXXZ. Under the customized excitation, BXXZ exhibited high fluorescence enhancement (66-fold), a low detection limit (0.23 μM), fast response, and excellent selectivity for Cys over Hcy and GSH. Benefiting from these merits, BXXZ enabled real-time visualization of differential Cys fluctuation in ferroptosis induced by erastin and RSL3, and achieved in situ tracking of intratumoral Cys fluctuation in HepG2 xenograft models. This work provides a feasible design strategy for high-quality ICT-type turn-on probes and a powerful imaging tool to explore the relationship between Cys homeostasis and ferroptosis.