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.