Integrated free radical sensor systems for investigation of cellular models of disease

Abstract

Reactive oxygen species (ROS) including superoxide (O2̄), nitric oxide (NO•) and hydrogen peroxide (H2O2) are thought to play a central role in many disease processes. This thesis details the development of novel optical and electrochemical sensor platforms for the analysis of ROS. These technologies were established in response to the current limitations of existing techniques to enable greater understanding of the role of ROS in cellular pathology. The overproduction of O2̄ by mitochondria has been linked to the initiation of disease processes. Specifically, defects in the mitochondrial electron transport chain (mETC) can result in electron leakage and subsequent ROS generation. Using a gold electrode, surface-modified with cytochrome c, the amperometric detection of real-time O2̄ production from isolated mitochondria was enabled. Specific transport proteins within the mETC were chemically inhibited and the change in O2̄ flux was observed, allowing the contribution to ROS production of inhibition of mETC Complex I and Complex III to be observed. ROS-sensitive nanosensors, based on the entrapment of the fluorophore dihydrorhodamine-123 (DHR123) in a porous polyacrylamide shell, were developed. These sensors were successfully introduced into the macrophage cell line NR8383, which facilitated the analysis of intracellular ROS fluctuations following stimulation with phorbol-12-myristate 13-acetate (PMA). Nanosensors containing the pH responsive fluorophore fluorescein isothiocyanate (FITC) were also used to measure intracellular pH (pHi) in primary myoblasts derived from patients with Chronic Fatigue Syndrome (CFS). These sensors have provided new insight into the role of intracellular acidosis in this disease. Intracellular ROS-sensitive nanosensor technology was combined with custom fabricated gold microelectrode arrays to produce a novel integrated cell monitoring platform capable of reporting real-time ROS flux in both the intra- and extracellular environment. Rat macrophage cells loaded with ROS-sensitive nanosensors were seeded into wells containing functionalised, ROS-responsive, gold ring electrodes. Following stimulation of the cells with PMA it was possible to measure intracellular ROS generation using fluorescence spectroscopy. External ROS flux as a consequence of PMA stimulation was simultaneously measured amperometrically.