Tunable NBD platform: high-performance fluorescent monitoring of cysteine fluctuations in ferroptosis.

Ferroptosis represents a novel and well-characterized form of programmed cell death that is mechanistically distinct from canonical cell death modalities such as apoptosis, necrosis, and autophagy, and is characterized by unique pathophysiological features and molecular execution mechanisms. It plays an integral role in a broad range of biological processes, including developmental regulation and disease pathogenesis. This study designed a high-performance nitrobenzoxadiazole (NBD) fluorescent platform and screened a novel NBD-based fluorescent probe, NBD5, capable of specifically detecting key changes of Cysteine (Cys) during the process of ferroptosis based on the principle of CO bond cleavage. By optimizing the type of leaving groups, it achieved a fluorescence enhancement effect of up to 403-fold and was able to respond fast within 12 min. This characteristic enables NBD5 to perform real-time imaging of ferroptosis-related changes in HepG2 cells, HeLa cells and zebrafish models. Further research has demonstrated that NBD5 successfully validates the significant decrease of Cys in ferroptosis models and visually exhibits the therapeutic effects of Traditional Chinese Herb (TCH) in ferroptosis models from a fluorescence perspective. These results not only substantiate the potential of NBD5 as an efficient tool in ferroptosis research but also provide new insights and methodologies for the diagnosis and treatment of ferroptosis-related diseases.