A near-infrared fluorescent probe, A, was designed by substituting the carbonyl group of the coumarin dye's lactone with a 4-cyano-1methylpyridinium methylene group and then attaching an electron-withdrawing NADH-sensing methylquinolinium acceptor via a vinyl bond linkage to the coumarin dye at the 4-position. The probe exhibits primary absorption maxima at 603, 428, and 361 nm, and fluoresces weakly at 703 nm. The addition of NAD(P)H results in a significant blue shift in the fluorescence peak from 703 to 670 nm, accompanied by a substantial increase in fluorescence intensity. This spectral shift is attributed to the transformation from an A−π−A−π−D configuration to a D−π− A−π−D pyridinium platform in probe AH, owing to the addition of a hydride from NADH to the electron-accepting quinolinium acceptor producing the electroncontributing 1-methyl-1,4-dihydroquinoline donor in probe AH. This conclusion is supported by theoretical calculations. The probe was utilized to investigate NAD(P) H dynamics under various conditions. In HeLa cells, treatment with glucose or maltose resulted in a substantial elevation in nearinfrared emission intensity, suggesting increased NAD(P)H levels. Chemotherapeutic agents including cisplatin and fludarabine at concentrations of 5, 10, and 20 μM brought about a dose-dependent increase in emission intensity, reflecting heightened NAD(P)H levels due to drug-induced stress and cellular damage. In vivo experiments with hatched, starved Drosophila melanogaster larvae were also conducted. The results showed a clear relationship between emission intensity and the levels of NADH, glucose, and oxaliplatin, confirming that the probe can detect variations in NAD(P)H levels in a living organism. Our investigation also demonstrates that NAD(P)H levels are significantly elevated in the cystic kidneys of ADPKD mouse models and human patients, indicating substantial metabolic alterations associated with the disease. This near-infrared emissive probe offers a highly sensitive and specific method for monitoring NAD(P)H levels across cellular, tissue and whole-organism systems. The ability to detect NAD(P)H variations in reaction to varying stimuli, including nutrient availability and chemotherapeutic stress, underscores its potential as a valuable resource for biomedical research and therapeutic monitoring.
