Abstract
Monitoring the dynamic fluctuations of NADH and NADPH, key coenzymes in intracellular redox homeostasis and bioenergetics, is essential for understanding both normal physiology and disease pathology. However, precise, real-time imaging of NAD(P)H levels in complex biological systems remains a major challenge due to interference from endogenous fluorescence and the limitations of intensity-based sensors. We present a newly engineered dual-emission fluorescent sensor, sensor A, which overcomes these obstacles by offering ratiometric detection with high specificity and sensitivity. The sensor features a 3-quinolinium moiety conjugated to a cyanine dye scaffold via an amine linker, enabling selective reactivity toward NAD(P)H. Upon reduction, the sensor displays a pronounced fluorescence shift with visible emission enhancement at 511 nm and a simultaneous near-infrared emission decrease at 711 nm. This ratiometric response provides a self-calibrating readout, minimizing errors from sensor concentration or excitation variability. Sensor A was validated in diverse biological contexts. In live cancer cell lines (HeLa and MD-MB453), it sensitively tracked NAD(P)H alterations induced by metabolic stimulation and chemotherapy. In Drosophila melanogaster larvae, the sensor enabled in vivo imaging of redox changes under nutrient deprivation and drug exposure. Remarkably, sensor A also distinguished between healthy and diseased human kidney tissues, revealing elevated NADH levels in polycystic kidney disease samples. These results demonstrate the broad utility of sensor A for investigating NAD(P)H-related processes in both research and clinical diagnostics. Its dual-emission, ratiometric output, and compatibility with live cell and in vivo imaging make it a valuable platform for probing redox biology across multiple model systems.