Montgomery, Craig (2009) Luminescent lanthanide complexes as cellular imaging agents or HTRF Assay Components. Doctoral thesis, Durham University.
A series of new azaxanthone chromophores has been established as sensitisers of europium and terbium emission in aerated aqueous media. Chromophores incorporating either a pyridyl or pyrazoyi moiety have been devised with large molar extinction coefficients and long excitation wavelengths. The pyridyl and pyrazoyi-1-azaxanthones have been incorporated into an array of functionalised macrocycles, to yield a series of emissive europium and terbium complexes, including examples suitable for conjugation. The complexes possess high emissive quantum yields (Φ (_Eu) = 25 %, Φ (_Tb) = 64 %) and long emissive lifetimes (up to 2.3 ms for Tb) in aqueous media. These properties arise from exclusion of coordinated water molecules, as a result of bidentate chelation of the heterocyclic chromophores. Quenching studies of the lanthanide excited states indicate that non-covalent protein association shields the complexes from quenching by endogenous electron rich species, such as urate and ascorbate. Under simulated cellular conditions, (0.4 mM serum albumin, 0.13 mM urate) the emissive lifetimes remain within 10 % of that observed in water, indicating protein binding does not quench the lanthanide excited states. Cellular uptake experiments have been performed with CHO and NIH-3T3 cell lines. Examination using fluorescence microscopy reveals cellular uptake, with complexes exhibiting a time-dependent localisation profile partitioning between mitochondrial and lysosomal regions. Following establishment of an efficient and reproducible method of conjugation, complexes have been covalently attached to benzyl guanine vectors to examine their performance as components in homogeneous time resolved fluorescence (HTRF) assays.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:25|