Near-infrared Fluorescent Probes for Sensitive Detection of NADH in Live Cells - The project aims to develop water-soluble near-infrared fluorescent probes for the precise and quantitative analysis of NAD(P)H concentration changes in mitochondria of live cells by incorporating the nicotinamide moiety into different electron-withdrawing fluorophores as electron acceptors such as tetraphenylethylene (TPE), dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and BODIPY. We will develop ratiometric near-infrared fluorescent probes to overcome systematic errors of intensity-based fluorescent probes by introducing near-infrared BODIPY donors to near-infrared rhodamine and rhodol acceptors, respectively. In order to significantly enhance water solubility and biocompatibility, we will introduce branched oligo(ethylene glycol) residues to the probes. The strong electron-withdrawing properties of the fluorophores such as dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and BODIPY dyes will facilitate a quick reduction of the nicotinamide moiety by NAD(P)H into an electron-donating dihydronicotinamide moiety and significantly turn on the probe fluorescence. We will use computation chemistry to guide how to design effective fluorescent probes and study the relationship of the optical properties of the probes and their chemical structures such as HOMO and LUMO levels, pKa values, absorption and emission wavelengths, fluorescence quantum yields, and redox reaction between the probes and NAD(P)H, and assess the through-bond energy transfer efficiency from the donors to the acceptors of ratiometric fluorescent probes in the presence of NAD(P)H. We will investigate the effects of substrate-specific glucose and pyruvate/lactate imbalance on NADH levels in the glycolysis pathway in normal and cancer cells. We will also investigate the co-relationship between GSH/GSSG (glutathione/glutathione disulfide) and the cellular redox regulator NAD+/NADH in maintaining a critical redox balance for proper functioning in the different metabolic processes. Finally, we will investigate the unique significant role of NADH in oxidative phosphorylation in mitochondria, and monitor NADH concentration changes during the mitophagy process caused by starvation and drug treatment.
