Abstract
Cyanine dyes constructed for NAD(P)H near-infrared sensing utilize extended π-conjugation but often exhibit delayed fluorescence responses to NAD(P)H due to reduced positive charge density in 3-quinolinium acceptors. This study introduces deep-red and near-infrared compact cyanine dyes represented by probes A and B for mitochondrial NAD(P)H detection in live cells. Probes A and B feature a unique structural design with a double bond connection linking 3-quinolinium to strategically positioned 1-methylquinolinium acceptor units at 2- and 4-positions, correspondingly. Probe A absorbs at 359 and 531 nm, while probe B absorbs at 324 and 370 nm, emitting subtle fluorescence at 587 and 628 nm, respectively, with no NADH present. Upon NADH exposure, probes A and B exhibit significant emission enhancements at 612 and 656 nm, correspondingly, attributed to the efficient reduction of 3-quinolinium units to electron-donative 1-methyl-1,4-dihydroquinoline units. Probe B, chosen for its near-infrared emission and fast response to NAD(P)H, effectively monitored dynamic intracellular NAD(P)H levels throughout diverse experimental conditions. In HeLa cells, minimal basal fluorescence increased upon NADH stimulation. It also identified increased NAD(P)H levels following chemical treatments with acesulfame potassium, cisplatin, carboplatin, and temozolomide, CoCl(2)-induced hypoxia, and TLR4 activation in macrophages and in disease models of kidney pathology, where diseased tissues exhibited higher fluorescence than normal tissues. In fruit fly larvae under starvation conditions, probe B tracked NAD(P)H increases triggered by exogenous NADH, demonstrating its in vivo applicability for metabolic studies. These findings highlight probe B's utility in elucidating dynamic NAD(P)H fluctuations in diverse biological contexts, offering insights into mitochondrial function and cellular metabolism.