The synthesis and characterisation of novel Ruthenium (II) polypyridyl complexes for use in cellular imaging
Stack, Zoe Joseph (2013) The synthesis and characterisation of novel Ruthenium (II) polypyridyl complexes for use in cellular imaging. Master of Science thesis, Dublin City University.
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Ruthenium polypyridyl complexes have been under extensive investigation for many years across a range of optical applications, from solar energy conversion to sensing due to their unique photophysical properties including intense polarised luminescence, large Stokes shifts, red emission wavelengths, and good photostability. These properties, along with the sensitivity of their luminenscene to molecular oxygen make them potentially invaluable probes for cellular imaging.
This thesis describes the study of cell uptake and luminescence and resonance Raman imaging of a novel Ru(II) polypeptide conjugate and attempts to attach an internal oxygen and pH insensitive reference to a related luminescent ruthenium complex to create a self-referenced probe.
Chapter 1 overviews the photophysics of ruthenium polypyridyl complexes and discusses current state of the art in their application to cellular imaging. In particular, we discuss how conjugation of these complexes to biomolecules has lead to more targeted and improved applications in medical diagnostics, photodynamic therapy and cellular imaging. Chapter 2 describes the experimental methodology used in this thesis.
Chapter 3 described the synthesis and photophysical characterisation of two novel ruthenium probes, [Ru (NO2phen)2 picCOOH]2+ and [Ru (NO2phen)2 picNH2]2+, where NO2phen is 5-nitro-1,10-phenanthraline and the pic ligands are 2-(4-Carboxylphenyl)imidazo [4,5-f]1,10-phenanthroline and 2-(4-Aminophenyl)imidazo[4,5-f]1,10-phenanthroline respectively. Both the carboxyl and amino functionalised nitrophenanthroline complexes behave very similarly and may be characterised by comparable absorbance, emission, Raman profiles and similar pH dependence. These complexes exhibited a Stokes‟ shift of over 150 nm as well as having long lived emission lifetime (over 400 ns) which make them potential candidates for cell imaging. In the latter part of this chapter attempts to conjugate these complexes to a reference probe are described. The ultimate goal of attaching both to a cell penetrating peptide and an internal reference proved unsuccessful despite numerous different attempts, and this chapter concludes with a discussion of a different synthetic approach which may ultimately lead to the target complex.
Chapter 4 described the ability of a novel ruthenium – peptide conjugate to transport passively and irreversibly across the cellular membrane of mammalian SP2 and CHO cells. Using confocal microscopy, by comparison, the parent complex was found not to transport across the cell membrane, confirming it is the peptide which is behaving as a cargo carrier in this instance. Resonance Raman mapping was used to investigate the presence of the complex within both cell types, by analysing individual cells. The most intense Raman signals, corresponding to the greatest concentration of dye, were observed in the center of each cell. The localization of the dye within SP2 cells was not confirmed via co-localization studies, but it was found to localize in the endoplasmatic reticulum of CHO cells.
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