A dicyanoisophorone-based highly selective fluorescent probe with a large stokes shift for cysteine detection in lipid droplets

Cysteine (Cys), a pivotal biothiol involved in protein synthesis, metabolism, and detoxification, plays essential roles in various physiological and pathological processes. Dysregulation of Cys levels is associated with diseases such as Parkinson's, Alzheimer's, cardiovascular conditions, and hepatic pathologies. Despite advancements in Cys detection, probes specifically designed for lipid droplet (LD)-targeted Cys monitoring remain scarce. Here, we report the development of a fluorescent probe, DCI-HBT-Cys , for the selective detection of Cys within LDs. The probe features an acrylate-based recognition unit for specific nucleophilic cyclization with Cys and a hydrophobic DCI-HBT-OH fluorophore enabling efficient LD localization. DCI-HBT-Cys exhibits remarkable properties, including high selectivity, a large Stokes shift (240 nm), excellent photostability, and pH stability in the physiological range (pH 6–9). In vitro and cellular assays demonstrate the probe's ability to sensitively detect endogenous and exogenous Cys with minimal interference from related biothiols (e.g., Hcy, GSH). Furthermore, DCI-HBT-Cys enables real-time imaging of Cys upregulation in various cell lines, including HeLa and HepG2 cells, in response to ginseng polysaccharide stimulation. Co-localization studies confirm the probe’s LD-targeting specificity, while quantitative fluorescence analysis highlights its potential for precise Cys monitoring. These findings underscore the utility of DCI-HBT-Cys as a powerful tool for elucidating the intricate interplay between Cys metabolism, oxidative stress, and LD-associated cellular processes, paving the way for novel diagnostic and therapeutic strategies.