Methylquinolinium-enhanced near-infrared hemicyanine dye for ratiometric NAD(P)H sensing in live cells <i>via</i> carbon–carbon bond conjugation

We introduce a ratiometric sensor designed for sensitive and specific detection of NAD(P)H in living cells, tissues, and whole organisms. The sensor incorporates a methylquinolinium acceptor linked to a near-infrared hemicyanine dye, enabling a dual-emission ratiometric mechanism. Upon binding to NAD(P)H, the near-infrared emission at 684 nm decreases, while visible emission at 517 nm increases. This change results from the reduction of the methylquinolinium acceptor to an electron-giving 1-methyl-1,4-dihydroquinoline donor, which quenches the near-infrared emission through photon-induced electron transfer (PET). This ratiometric behavior ensures precise, live tracking of NAD(P)H levels while overcoming the systematic errors associated with intensity-based measurements. We validate the sensor's performance in several experimental settings. In HeLa cells, treatment with oxaliplatin, fludarabine, and glucose all induced a dose-dependent increase in NAD(P)H, as indicated by the rising visible emission and decreasing near-infrared emission. These treatments reflect changes in cellular NADH levels, demonstrating the sensor's ability to track metabolic shifts in response to pharmacological and nutritional stimuli. Additionally, larvae of the fruit fly <i>Drosophila melanogaster</i> treated with increasing concentrations of NADH showed similar dose-dependent emission responses, confirming the utility of this sensor in live organisms. Finally, we applied the sensor to human and mouse kidney tissue samples, including normal and diseased (autosomal dominant polycystic kidney disease, ADPKD) tissues. Diseased tissues exhibited higher NADH activity and viscosity, as evidenced by stronger visible emission and enhanced near-infrared emission, providing insights into the metabolic alterations in kidney diseases. The ratiometric nature of this near-infrared sensor allows for accurate, spatially resolved measurement of NAD(P)H activity in living systems, offering in-depth understanding of cellular metabolism, oxidative stress, and the pathophysiology of diseases such as cancer, metabolic disorders, and polycystic kidney disease. This innovative tool has great potential for advancing research in cellular bioenergetics, redox regulation, and disease mechanisms.