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Title: Intracellular analysis of pH and nitric oxide using fluorescence based nanosensors and molecular probes.
Author: Marín Altaba, Maria Jose
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2012
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The main objective of this thesis was to contribute to the optimisation of the biosensing of two species that play an important role in living organisms, i.e., protons (H+) and nitric oxide (NO). The intracellular imaging and quantification of these species was achieved using fluorescence based nanoparticles and fluorescent molecular probes in combination with confocallaser scanning microscopy (CLSM). A novel photoinduced electron transfer (PET) based pH nanosensor was reported to improve the signalling of acidic organelles in cells. A PET-based pH ligand incorporating a thiolated moiety was synthesised and used to stabilise gold nanoparticles. The free ligand and the PET-nanosensor were unambiguously characterised and their fluorescence emissions were recorded in acidic and basic media. The fluorescence emission of the free ligand was quenched in alkaline conditions and enhanced in an acidic environment. The PET- nanosensor afforded a similar ON-OFF switching process in acid and basic environments. The PET-nanosensor was used for intracellular imaging of acidic environments in Chinese hamster ovary (CHO) cells by CLSM. The internalisation of the nanoparticles by the cells was confirmed recording images and fluorescence emission spectra of the PET-nanosensor from within the cells using CLSM. To determine the pH of individual regions within the intracellular environment, a ratiometric PET-based nanosensor (ratiometric pH nanosensor) was synthesised. The PET based pH- sensitive ligand and a synthetic rhodamine derivative ligand were used to stabilise gold nanoparticles yielding the ratiometric pH nanosensor. The fluorescence emission of the ratiometric pH nanosensor was recorded in acidic and basic media using a single excitation wavelength. The variation of the fluorescence signals of the ratiometric pH nanosensor enabled ratiometric fluorescence measurements of pH between 3.5 and 6.5. The ratiometric pH nanosensor was used for intracellular imaging of living CHO cells using CLSM. The fluorescence emission spectrum within CHO cells confirmed the internalisation of the ratiometric pH nanosensor since the spectrum was composed of the characteristic bands of both ligands. Cc-localisation experiments using a known marker of acidic organelles strongly suggested that the ratiometric pH nanosensor accumulated in the acidic organelles within the CHO cells. The ratiometric pH nanosensor was utilised to determine the pH of different regions in live cells. The fluorescence emission spectra of the regions where the pH nanosensor was accumulated within the cell were acquired and the pH of those different regions within the CHO cells was calculated. The estimated values of pH were in agreement with the values of pH reported in the literature for acidic organelles. The chemical reactivity of l,2-diaminoanthraquinone (DAA), a fluorescent probe for NO, when used for intracellular measurements was studied. DAA is an ortho-diamino compound and is postulated to react with NO in an oxygenated medium leading to the formation of a l,2,3-benzotriazole derivative (DAA-TZ). The formation of DAA-TZ when DAA reacts with NOj02 within cells has not been demonstrated previously. The objective of this part of the thesis was to confirm that DAA-TZ is the species formed intracellularly when OM reacts with NO/02. Independent intracellular evaluations of DAA and DAA-TZ were performed. RAW 264.7 macrophage cells were loaded with DAA-TZ and DAA under conditions of unstimulation and stimulation with interferon-y and lipopolysaccharide to induce the production of NO. CLSM was used for the intracellular studies providing images and fluorescence emission spectra of the cells. The results presented confirmed the intracellular production of DAA-TZ when DAA reacts with NO in the biological environment.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available