Measuring chemical concentration is vital for understanding normal and disease physiology involving metabolism and signalling, but monitoring chemical concentrations in living systems poses a unique challenge because of biological heterogeneity. The purpose of this work is to develop a system able to monitor chemical concentrations in primary cultured cells, and apply it to the detection of oxygen, a nutritional status marker, and nitric oxide which is a signalling molecule. Both of these electroactive species are involved in angiogenesis which is the growth of new blood vessels, and a hallmark of cancer. The approach used in this study is to grow porcine endothelial cells onto bronectin-coated gold microelectrode arrays with diameter 25 μm and perform electrochemical measurement on them. An experimental protocol is developed for measurements of dissolved oxygen and nitric oxide around cells in their normal cell-culture environment. It includes developing instrumentation like a heating platform and silver reference microelectrode; data processing for automation and normalisation; and optimising voltammetry techniques. Culture medium is found to a ect electrochemical measurements by changing double layer capacitance, reaction rate constant and di usion. The measurement system is used to detect oxygen reduction around cells, and this is used to estimate their oxygen consumption rate. Nitric oxide produced by cells is also measured, and this is used to identify an angiogenic pathway leading to nitric oxide production by endothelial cells. Variability in cell measurements is shown to originate from the biological system rather than from sensor design. A novel electroanalytical technique for determining parameters of reversible redox systems is developed by experimentally testing an analytical solution for the current response to a large-amplitude sinusoidal voltage input. The technique is used to nd estimates for double layer capacitance, half wave potential and di usion coe cients for both potassium ferrocyanide and ruthenium hexaamine.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656672 |
| Date | January 2014 |
| Creators | van Schaik, Tempest |
| Contributors | O'Hare, Danny |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/25054 |
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