A Quinolinium-Functionalized Hemicyanine Dye for Ratiometric NAD(P)H Sensing in Live Cells, Kidney Tissues, and Drosophila melanogaster

NAD⁺ (Nicotinamide adenine dinucleotide) and NADP⁺ (its phosphorylated variation) are key coenzymes regulating cellular metabolism, biosynthesis, and redox balance. Variations in NAD(P)H activity reflect metabolic activity and are associated with diseases like cancer, metabolic disorders, and neurodegeneration. Live tracking of NAD(P)H is important for comprehending these processes and their dysregulation in disease. However, existing ratiometric emission sensors are hindered by spectral interference from NADH emission, limiting their sensitivity and accuracy in complex biological systems. We present a ratiometric emission sensor based on a 3-quinolinium-hemicyanine dye with amine linkage, designed to address these limitations. By using a longer excitation wavelength (470 nm), the sensor avoids interference from NADH emission, enabling precise monitoring of NAD(P)H activity. Binding to NAD(P)H triggers a photo-induced electron transfer (PET) mechanism, resulting in an increase in visible emission at 519 nm and subtle reduction in near-infrared emission at 711 nm, providing a clear ratiometric signal. We demonstrate the sensor’s effectiveness in live cells, tissues, and whole organisms. In HeLa cells, exposure to glucose, maltose, fludarabine or cisplatin induced dose-dependent ratiometric emission changes, reflecting metabolic shifts and oxidative stress. The sensor also successfully detected NAD(P)H fluctuations in Drosophila melanogaster larvae and mammalian kidney tissues, including disease models like ADPKD (autosomal dominant polycystic kidney disease). Importantly, the sensor can distinguish NAD(P)H dynamics from high NADH levels, overcoming a key limitation of current sensors. Co-localization with a mitochondrial dye confirmed the sensor’s selective targeting to mitochondria, highlighting its suitability for studying Mitochondrial energy processes and redox changes. This ratiometric sensor provides a sensitive, interference-free tool for live tracking of NAD(P)H kinetics in complex biological systems. Its high sensitivity, accuracy, and versatility offer new opportunities for investigating cellular metabolism, disease mechanisms, and therapeutic interventions in both cellular and in vivo models.