Hemicyanine-Based Fluorescent Probes for Cysteine Detection in Cellular Imaging and Food Samples

Cysteine (Cys) is an essential thiol in food and biological systems, yet its selective quantification remains challenging due to interference from structurally related analytes such as homocysteine (Hcy) and glutathione (GSH). Here, we report a hemicyanine-based, turn-off fluorescent probe (PRH) that undergoes Cys-triggered cyclization to release PRH-OH, resulting in fluorescence quenching. PRH exhibits near-infrared emission at 630 nm, enabling low self-absorption and reduced background. The probe affords a broad linear range (0–100 μM) with a detection limit of 0.344 μM, along with high selectivity over Hcy, GSH, and 18 other amino acids. In food matrices (garlic, onion, and dried red pepper), PRH achieved recoveries of 98.8–101.3% with RSD < 2% (n = 3), demonstrating analytical robustness. Live-cell imaging in HeLa cells further verified practical responsiveness: N-ethylmaleimide-mediated thiol depletion increased PRH fluorescence, whereas Cys replenishment decreased it, consistent with the probe’s turn-off behavior. DFT calculations support an intramolecular charge-transfer change upon Cys reaction, correlating with the observed spectral shift. Overall, PRH provides a simple and selective platform for reliable Cys quantification in food samples and for visualizing Cys dynamics in cells.