My research during this interdisciplinary investigation was to primarily focus on the development of novel single cell sampling and analysis methods. Using micromanipulation, microsampling and mass spectroscopic analysis techniques to determine the concentration of biologically relevant molecules quantitatively from living fungal cells, as well as to study the uptake of externally applied compounds. Evaluation of a range of on-line separation techniques, including Capillary Liquid Chromatography (CapLC), nanoLC and capillary electrophoresis (CE) was made for suitability to be coupled to a range of mass spectrometers for the analysis of fungal cytoplasm. Techniques for microsampling at the single cell level from the model organism, <i>Neurospora crassa</i>, and transfer of the sample from under the microscope for analysis by mass spectrometry, following on-line separation, were developed. Capillary electrophoresis electrospray mass spectrometry (CE-ESI-MS) was used to measure intra cellular concentrations of trehalose, quantitatively, with mechanisms developed for detection of external contamination of the samples taken from viable, actively growing cells of <i>Neurospora crassa</i>, by the use of laser induced fluorescence (LIF). The measured concentration for intra-cellular disaccharide was determined to be 1.3mM by the methods developed and described during this thesis. In addition, topical applications of fungicide were made, with intra-cellular measurements taken by single cell analysis. Following application of 14.8μM azoxystrobin, the fungicide under investigation at saturation concentration in water, intra-cellular concentrations were measured at 9.9μM after 5 minutes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651603 |
| Date | January 2006 |
| Creators | Goodwin, Richard J. A. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12042 |
Page generated in 0.0012 seconds