Novel redox indicators for optical detection of NADH

The rapid, sensitive, and selective sensing of nicotinamide adenine dinucleotides NAD(P)H is of significant interest for applications in diagnostic assays, synthesis of chiral compounds and drug discovery. The redox couple NAD(P)+/NAD(P)H plays a crucial role in energy metabolism and has been used as a biomarker to detect altered cancer cell metabolism. Furthermore, NAD(P)H detection is desirable for in vitro analysis of NAD+-dependent enzymes, which is important in the context of high-throughput screening and the development of these enzymes for industrial applications. Moreover, NAD(P)+-dependent glucose dehydrogenase (GDH) has been widely utilized for glucose sensors in point-of-care (POC) testing. Optical techniques proved to be a powerful tool for quantitation of analytes in biological fluids. Both NADH and NADPH absorb light at 340 nm and have intrinsic fluorescence. Direct NAD(P)H detection does not provide adequate sensitivity for most applications. In this regard, colorimetric and fluorescent indicators are particularly attractive, because they are able to change their absorption spectra to a longer wavelength upon reaction with a target analyte. The key considerations in a search for a tailor made NADH indicator include water solubility, rate of reaction with NADH and sensitivity. Up to now, it still remains a challenge to develop an indicator meeting all these demands. The aim of this thesis was to discover a small molecule indicator for NADH detection. Chapter 1 introduces the concepts of test strip development for blood glucose monitoring and gives a brief overview of research activities in this area. Chapter 2 describes, for the first time, that a colorimetric indicator — directly accepting electrons from an enzymatic reaction — can be utilized for glucose sensing on a dry chemistry test strip. The glucose assay reagent included a quinone-functionalised Ru (II) complex, the artificial cofactor cNAD+ and mutated GDH. An absorbance response was observed at 840 nm in proportion to glucose concentration. Chapter 3 presents a new optical NADH indicator to analyze intracellular metabolic pathways at the single-cell level. The design is based on an electron deficient heterocycle (active acceptor) conjugated to a polymethine chain of the cyanine dye scaffold. Such compounds are nearly colourless and nonfluorescent. Upon hydride transfer from NADH to the active acceptor moiety, cyanine chromophore is unmasked leading to a sensitive absorbance and fluorescence response