Two-Acceptor Cyanine-Based Fluorescent Indicator for NAD(P)H in Tumor Cell Models

Sensing of intracellular NAD(P)H is important for numerous applications ranging from diagnostic assays to drug discovery. Up to now, investigation of NAD(P)H-dependent pathways in live cells has been restrained by the lack of efficient tools. Toward this goal, we developed a small molecule indicator, which allows both colorimetric and fluorescent NAD(P)H detection in biological samples. The design is based on a cyanine dye scaffold and utilizes a novel two-acceptor "turn-on" mechanism. Consequently, this indicator features unprecedented sensitivity and rapid response toward NAD(P)H at low micromolar levels under physiological conditions. First, we demonstrated the value of this reagent in a diagnostic assay of glucose, through the enzyme-coupling reaction of NAD(P)+-dependent glucose dehydrogenase (GDH). Second, we showed the utility of our indicator for NAD(P)H imaging in live cells. We confirmed its ability to reflect different NAD(P)H levels using the human colon cancer cell line deficient on mitochondrial respiration. Expanding the use of this indicator to advanced tissue models, we demonstrated its ability to visualize different metabolic states in the hypoxic core of tumor spheroids. This study demonstrates that small molecule indicators could serve as a valuable tool for the specific analysis of redox states at the single-cell level and may be well suitable for high-throughput metabolic screening of antitumor agents.