Single molecule and single nanoparticle spectroscopic studies using Forster resonance energy transfer and nanometal surface energy transfer in biological applications

Nanobiotechnology is a young and exciting field. There has been an explosion of growth in this area within the past ten years. The availability of new strategies for synthesizing nanomaterials is partially responsible. New materials used in this field constitute a link between single molecules and bulk systems. The purpose of this dissertation is to examine novel materials and cutting edge technologies employed in spectroscopic studies for biological applications. The dynamic optical and electronic properties of metals and semiconductor materials are explored in terms of biosensors. Single molecule studies employing Forster Resonance Energy Transfer (FRET) and Nanometal Surface Energy Transfer (NSET) are evaluated using total internal reflection fluorescence microscopy. Chapter 1 of this thesis gives a brief introduction to theories and concepts that are used in experimental chapters (3, 4, and 5). It is intended to provide a basic understanding of materials used in current nanotechnology. Chapter 2 describes detailed information in regard to sample preparations and instrumentation used in our single molecule studies. In Chapter 3 steady state fluorescence experiments explore the fluorescent and optical properties of dye-labeled DNA constructs in the presence of gold nanoparticles, also dynamic and static fluorescence quenching processes are examined. Chapter 4 describes a molecular beacon that is designed using two novel hybrid nanobiomaterials; quantum dots and gold nanoparticles. This "smart" biosensor incorporates a disulfide cleavage site allowing for "on and off" states to be monitored at the single molecule level. And finally, a combined FRET and reverse transcription PCR approach is used for in vitro monitoring of pre-mRNA splicing at the single molecule level is discussed in Chapter 5 of this thesis.