A Rhodamine-BasedRatiometric Fluorescent Sensor forDual-Channel Visible and Near-Infrared Emission Detection of NAD(P)Hin Living Cells and Fruit Fly Larvae

The detection and dynamic monitoring of intracellular NAD­(P)H concentrations are crucial for comprehending cellular metabolism, redox biology, and their roles in various physiological and pathological processes. To address this need, we introduce sensor <b>A</b>, a near-infrared ratiometric fluorescent sensor for real-time, quantitative imaging of NAD­(P)H fluctuations in live cells. Sensor <b>A</b> combines a 3-quinolinium electron-deficient acceptor with a near-infrared rhodamine dye, offering high sensitivity and specificity for NAD­(P)H with superior photophysical properties. In its unbound state, sensor <b>A</b> emits strongly at 650 nm and weakly at 465 nm upon 400 nm excitation. Upon binding to NAD­(P)­H, it shows a fluorescence increase at 465 nm and a decrease at 650 nm, enabling accurate ratiometric measurements. Sensor <b>A</b> also exhibits ratiometric upconversion fluorescence when excited at 800 or 810 nm, offering additional flexibility for different experimental setups. The sensor’s response relies on the reduction of the 3-quinolinium acceptor by NAD­(P)­H, forming a 1,4-dihydroquinoline donor that enhances fluorescence at 465 nm and quenches the near-infrared emission at 650 nm through photoinduced electron transfer. This mechanism ensures high sensitivity and reliable quantification of NAD­(P)H levels while minimizing interference from sensor concentration, excitation intensity, or environmental factors. Sensor <b>A</b> was validated in HeLa and MD-MB453 cells under various metabolic and pharmacological conditions, including glucose and maltose stimulation and treatments with chemotherapeutic agents. Co-localization with mitochondrial-specific dyes confirmed its mitochondrial targeting, enabling precise tracking of NAD­(P)H fluctuations. <i>In vivo</i> imaging of <i>Drosophila</i> larvae under nutrient starvation or chemotherapeutic exposure revealed dose-dependent fluorescence responses, highlighting its potential for tracking NAD­(P)­H changes in live organisms. Sensor <b>A</b> represents a significant advancement in NAD­(P)H imaging, providing a powerful tool for exploring cellular metabolism and redox biology in biomedical research.