A coumarin-based dual-channel fluorescent probe for discriminating Hcy and GSH in live cells

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) share similar functional groups, such as thiol (-SH) and amino (-NH₂), but differ in their spatial structures. These structural differences can lead to distinct binding behaviors with fluorescent probes that possess multiple reactive sites, resulting in diverse reaction products and thereby producing distinct color and fluorescence responses. Based on this principle, a phenyl selenide-substituted coumarin probe (PSeC) capable of distinguishing GSH from Cys and Hcy is designed and synthesized in this work. The probe itself is non-fluorescent, but exhibits strong fluorescence upon reacting with biothiols. Within the PSeC structure, both the phenyl selenide and aldehyde groups serve as potential reaction sites for biothiol interaction, yielding characteristic fluorescence emissions-green for Cys and Hcy, and red for GSH. The probe displayed fluorescence enhancement factors of 90-fold for GSH, 142-fold for Hcy, and 29-fold for Cys. Moreover, the response time toward Cys was markedly longer than those toward Hcy and GSH, enabling discrimination of the three biothiols through distinct fluorescence channels and kinetic behaviors. Furthermore, the probe demonstrated the capability to differentiate Hcy and GSH in living cells based on their unique fluorescence signatures.