This thesis describes the development and characterisation of fluorescent optical nanosensors for pH, molecular oxygen and temperature for measurement in biological systems. Ratiometric pH-sensitive nanosensors were fabricated by entrapping a combination of two different pH-sensitive fluorophores and a pH-insensitive reference fluorophore into a polyacrylamide matrix. The combination of the two pH-sensitive fluorophores overcame the current limitations in pH sensing with fluorescent nanosensors by demonstrating a tuneable pKa and an extended dynamic range of pH measurement. Molecular oxygen nanosensors were synthesised by surveying a library of novel oxygen-sensitive metalloporphyrins. Through careful selection of fluorophores and a platinum cationic metalloporphyrin, hybrid quadruple fluorophore labelled nanosensors capable of simultaneously making ratiometric pH and oxygen measurements were fabricated. In addition, a custom designed calibration tool was engineered so that oxygen-sensitive nanoparticles can be continually imaged, whilst the oxygen concentration is being changed and monitored at the same time. Temperature-sensitive nanosensors were synthesised by conjugating a temperature-sensitive fluorophore to a photo and thermo-sta ble silica sol-gel matrix. These nanosensors were found to operate over a wide temperature range and can be calibrated through the use of custom designed microelectromechanical systems (MEMS) micro-hotplate. The MEMS micro-hotplate can be mounted onto virtually all fluorescence microscopes, due to its: (1) relatively small size, (2) high temperature operation and (3) uniformity in temperature distribution. Extended dynamic range pH-sensitive nanosensors were successfully delivered to the model organism Caenorhabditis e/egans and used to map the pH of the pharynx and intestine in real-time using an automated image analysis system. The automated image analysis system and the pH-sensitive nanosensors were validated against the gold standard of pH measurements, the pH electrode, to demonstrate high concordance and measurement resolution.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663241 |
| Date | January 2014 |
| Creators | Chauhan, Veeren M. |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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