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.