Electrochemical monitoring of living cells

Abstract: This thesis presents the utilization of electroanalytical methods for analysis of dynamic cellular processes such as exocytosis, intracellular redox reactions and cell surface interactions. Microchips have been designed and manufactured with the purpose of monitoring dopamine exocytosis from single or a small ensemble of PC12 cells. The electrode surface was modified with self assembled monolayers of thiols in order to improve the electrochemical detection of dopamine. The first generation microchip relied on manual deposition of cells on top of the electrode whereas the second generation was more advanced and allowed the automatic positioning of cells prior to stimulation and measurements of quantal exocytosis. The metabolic properties of the yeast Saccharomyces cerevisiae have been investigated by monitoring intracellular reduction of the lipophilic mediator menadione. By employing a phosphoglucose isomerase deletion mutant, it was possible to determine the influence of the two major metabolic pathways, i.e. the glycolytic and the pentose phosphate pathway, on intracellular menadione reduction. It was found that cells counteracted the oxidative stress imposed on the cells by the mediators by increasing the rate of NADPH producing reactions. The same strain was utilized for the investigation of enzyme inhibition and mitochondrial uncoupling by the anticoagulant dicoumarol. It was found that dicoumarol mainly affected NADH-dependent processes such as cytosolic mediator reduction and mitochondrial respiration. Electrochemical impedance spectroscopy was used to monitor interactions between yeast and thiol modified gold electrodes. The proliferation of S. cerevisiae could be monitored, up to monolayer coverage, by recording the electrode-solution interface capacitance.