PROBING NAD(P)H AVAILABILITY IN LIVING CELLS USING MINIATURIZED ELECTROCHEMICAL SYSTEMS

Abstract: The work presented in this thesis demonstrates the application of electrochemical methods for probing the dynamics of cellular metabolism in prokaryotic and eukaryotic cells. The main part of the work has been done using genetically modified strains of yeast. Employing baker’s yeast, S. cerevisiae, as a model organism, it was demonstrated how an amperometric method can be utilized for real-time in vivo screening of intracellular NAD(P)H availability in relation to redox enzyme activities. NAD(P)H availability was evaluated using the amperometric method based on a menadione-ferricyanide double-mediator system. Mediated amperometry is based on the ability of menadione, a lipophilic quinone, to freely diffuse through the plasma membrane and accept electrons from certain intracellular NAD(P)H-dependent enzymes. The reduced form of menadione is extracellularly oxidized by ferricyanide. The ferrocyanide formed is in turn oxidized at the gold microband electrode, giving rise to a bioelectrocatalytic current proportional to the activity of the menadione reducing enzyme. The bioelectrocatalytic current was measured by immobilizing living S. cerevisiae cells on gold microband electrode chips (Si/SiO2 substrate) in Ca-alginate gel. The microelectrodes (width/length 25/1000 µm) were fabricated using micromachining technology for real-time amperometric probing of NAD(P)H-dependent redoxprocesses and pathways in living cells. The dependence of menadione-reducing enzyme activity on the main metabolic pathways was investigated by introducing different carbon sources (glucose and fructose). It was found that the amplitude of the yeast-catalysed amperometric signal was significantly higher when carbon sources were administered, primarily resulting in the formation of NAD(P)H through the pentose phosphate pathway and glycolytic pathway. The crucial effect of inhibitors on menadione-reducing activity was surveyed using the method. The results obtained during this work demonstrate the significance of enzyme activity determination in living cells as the source of information on cellular responses, reflecting different metabolic pathways and regulatory mechanisms. This gives further support to the potential of mediated amperometry as a tool in metabolic profiling.