A Rhodamine-Based Ratiometric Fluorescent Sensor for Dual-Channel Visible and Near-Infrared Emission Detection of NAD(P)H in 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 A, a near-infrared ratiometric fluorescent sensor for real-time, quantitative imaging of NAD(P)H fluctuations in live cells. Sensor A 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 A 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 A 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 A 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. In vivo imaging of Drosophila 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 A represents a significant advancement in NAD(P)H imaging, providing a powerful tool for exploring cellular metabolism and redox biology in biomedical research.