Onnicha Khaikate,[a],* Phongsithorn Phudphong,[a] Worakrit Saiyasombat,[b] Bongkot Ouengwanarat,[c] Tanakorn Wonglakhon,[d] Anyanee Kamkaew,[c] Chutima Kuhakarn[e]

[a] Department of Industrial Chemistry, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand

[b] Institute of Nutrition, Mahidol University, Phutthamonthon, Nakhon Pathom 73170, Thailand

[c] School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand

[d] Futuristic Science Research Center, School of Science, Walailak University, Nakhon Si Thammarat, 80160, Thailand

[e] Department of Chemistry and Center of Excellence for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

This present work offers an effective strategy for the development of NIR fluorescent probes capable of selectively detecting cysteine (Cys) and homocysteine (Hcy) in complex biological environments. A NIR fluorescent probe, Cy7-TCF-SPy, was successfully designed and synthesized based on an asymmetric heptamethine cyanine scaffold by incorporation of a 2-mercaptopyridine moiety as the recognition unit and fluorescence quencher. the probe exhibited a fluorescence “turn-on” response by emitting strong signal at 774 nm upon reaction with Cys/Hcy even in the presence of glutathione (GSH). Cy7-TCF-SPy probe demonstrated excellent analytical performance, including high sensitivity with low detection limits, rapid response within 1 min, and low cytotoxicity. Importantly, the probe was successfully applied for fluorescence imaging of exogenous Cys/Hcy in A549 cells, confirming its good membrane permeability and biocompatibility for cellular imaging applications. In addition, Cy7-TCF-SPy probe showed practical applicability for the determination of Cys in various food samples with satisfactory analytical performance.