Molecular probe to visualize the effect of a glycolytic inhibitor on reducing NADH levels in a cellular system

The reduced form of nicotinamide adenine dinucleotide, commonly known as NADH, is an essential coenzyme existing in living organisms. Due to its involvement in various biological process, fluorescence imaging of intracellular NADH levels in different pathological conditions has emerged as an interesting area of research. We report here the exploration of a fluorescent probe, MQ-CN-BTZ, as a dual-channel NADH imaging agent (green and red channels) for cellular systems. Interestingly, depending on the ratio between the probe and NADH concentration in the solution phase, the probe showed emission at ∼529 nm and ∼656 nm when excited at 475 nm. It should be noted that the probe showed a very large Stokes shift of ∼180 nm with respect to the longer-wavelength emission with a good fluorescence response towards NADH. In general, such a large Stokes shift is highly beneficial for imaging applications, largely due to the better separation between the emission and excitation spectra and reduced spectral overlap. Finally, the probe was utilized to image a glycolysis pathway event by employing 3-bromopyruvic acid (3-BrPA) as a glycolytic inhibitor that significantly inhibits the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is involved in a crucial step of glycolysis. As the depletion of the NADH levels corresponds to the inactivity of GADPH upon treatment with the inhibitor, we attempted to image the modulation of the NADH concentration in the cellular system in the presence of the inhibitor 3-BrPA, indicating the importance of the glycolysis step in elevating NADH levels. Overall, the present study attempts to demonstrate the importance of a molecular probe for fluorescence imaging of intracellular NADH in the presence of a glycolytic inhibitor.