A Red‐Emissive Fluorescent Probe for Selective Detection and In Vivo Imaging of Biothiols

ABSTRACT Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are key biothiols that regulate critical physiological processes, such as cellular redox homeostasis and signal transduction. Their abnormal levels are closely associated with various pathological conditions, including cardiovascular diseases, neurodegenerative disorders, and cancer, underscoring the need for analytical tools that enable real‐time, highly selective detection. In this study, a red‐emissive fluorescent probe ( Probe N ) was designed and synthesized using 2‐thiophenecarbonyl as the recognition moiety and 2‐hydroxy Nile red as the fluorophore. Operating through a thiol‐triggered ester bond cleavage mechanism, Probe N exhibited high selectivity toward Cys, Hcy, and GSH. Spectroscopic assays revealed a pronounced fluorescence quenching response to the three biothiols, with detection limits of 13.02 µM for Cys, 35.04 µM for Hcy, and 36.04 µM for GSH. The fluorescence intensity reached a plateau at approximately 35 min for Cys and 45 min for Hcy and GSH. Furthermore, the probe demonstrated low cytotoxicity and excellent biocompatibility, enabling successful application in fluorescence imaging of exogenous biothiols in living mice. Collectively, this work provides a useful tool for biothiol detection and in vivo visualization.