An ESIPT-based ratiometric fluorescent probe for cysteine and its application in complex matrices: milk and fetal bovine serum.

Cysteine (Cys) was an essential biothiol whose dysregulation is linked to neurotoxicity, liver injury, childhood growth retardation, and cardiovascular diseases, making its accurate detection critical for clinical diagnosis and biochemical research. Although excited-state intramolecular proton transfer (ESIPT)-based fluorescent probes offer advantages such as a large Stokes shift and low background interference, conventional ESIPT probes struggled to distinguish Cys from structurally similar homocysteine (Hcy) and glutathione (GSH). Herein, we rationally designed a novel ESIPT probe named LYB-Cys via Knoevenagel condensation, using 2-(2'-hydroxyphenyl)benzoxazole (HBO) as the electron donor and 2-(benzo[d]thiazol-2-yl)acetonitrile (BTAN) as the electron acceptor. The probe specifically responded to Cys through a conjugate addition-cyclization reaction, producing a ratiometric fluorescent signal with excellent selectivity over Hcy, GSH, and other interferents. Owing to this performance, LYB-Cys enabled quantitative Cys detection in milk and fetal bovine serum, superior to Ellman's method for thiol detection. It also provided a robust molecular platform that bridged fundamental probe design with practical applications in food quality control and biochemical analysis.