A Red-Emissive TCF-Based Fluorescent Probe for Selective Cysteine Detection in Food Samples and Living Cells

Accurate determination of cysteine (Cys) remains challenging because of the structural similarity and comparable nucleophilicity of biological thiols. Herein, a red-emissive turn-on fluorescent probe, TCF-C1, was developed for selective Cys detection by coupling an acrylate recognition unit with a thiophene-extended TCF fluorophore. Reaction with Cys triggered a conjugate addition-cyclization process, resulting in the release of highly fluorescent TCF-OH and a pronounced fluorescence enhancement at 664 nm, accompanied by a visible color change from pink to purple. TCF-C1 exhibited a rapid response within 10 min and high selectivity for Cys over homocysteine, glutathione, amino acids, and common inorganic ions, together with a large Stokes shift of approximately 160 nm. The fluorescence intensity showed a linear dependence on Cys concentration from 0 to 35 µM, with a detection limit of 32.91 nM. The proposed sensing mechanism was supported by ESI-MS analysis and theoretical calculations. In addition, TCF-C1 exhibited low cytotoxicity and enabled fluorescence imaging of endogenous and exogenous Cys in living cells. Its analytical applicability was further demonstrated by the determination of Cys in samples of spinach, broccoli, snap beans, and carrots. These results establish TCF-C1 as a simple and sensitive fluorescent probe for Cys determination in food samples and for cellular imaging.