Fluorescence Resonance Energy Transfer (FRET) systems for biomedical sensor applications

This thesis investigates the use of Fluorescence Resonance Energy Transfer (FRET) for biomedical sensor applications. FRET is a process by which energy is transferred, via long range dipole-dipole interactions, from a donor molecule (D) in an excited electronic state to an acceptor molecule (A). The emission band of D must overlap the absorption band of A in order for FRET to occur. FRET is employed in a variety of biomedical applications, including the study of cell biology and protein folding/unfolding and is also used for enhanced optical bioassays. The distance dependence of the FRET interaction enables the technique to be used as a molecular ruler to report, for example, on conformational changes in biomolecules. The �rst phase of this work involved the design and implementation of a model 2-D FRET platform that is compatible with optical biochips. The donor-acceptor pair used was a Ruthenium-complex/Cy5 system where the donor-acceptor separation was controlled using highly reproducible polyelectrolyte spacer layers, which were deposited using a layer-by-layer technique. The FRET process was demonstrated in both uorescence intensity and lifetime mode. The interaction between FRET and the plasmonic enhancement of uorescence in the presence of adjacent metal nanoparticles was also investigated. Dipole-dipole interactions limit the FRET e�ect to donor-acceptor distances of typically less than 10nm. The use of the plasmonic e�ect to increase this distance, which would facilitate the use of FRET in a wider variety of applications, was explored. The size, shape and composition of metal nanoparticles were tailored to give a resonance absorption which optimises the enhancement of the dye uorescence. As well developing a 2-D solid planar platform, the FRET-plasmonic interaction was also investigated in solution phase, by designing a model that incorporated donor and acceptor-labeled oligonucleotides as controlled spacers and spherical gold and silver nanoparticles for plasmonic enhancement. Throughout the work, theoretical calculations were carried out, and, where relevant, theoretical predictions were compared with experimental measurements. Apart from designing two FRETplasmonic investigation models, a key result to emerge from this work is that while individual plasmonic enhancement of the donor and acceptor is occurring in the presence of metal nanoparticles, no plasmonic enhancement of the FRET interaction is observed for the experimental systems. Theoretical modeling confirmed the reduction of the FRET efficiency