UBC Theses and Dissertations
Development of non-traditional FRET-based fluorescent sensors Higgins, Rehan
Fluorescence is a powerful tool for probing changes in structure on the nanometer scale. Förster resonance energy transfer (FRET) between fluorophores has a strong distance dependence in the 1-10 nm range, enabling indirect observation of minute positional changes. FRET has been used extensively for investigation of biomolecular systems and for the design of nanostructures for sensing biomolecules. This thesis describes the design of several FRET based nanostructures for fluorescence-based sensing of biomolecules. Molecular logic devices (MLDs) have the potential to make efficient biosensors which deliver complex information with a single output signal. These devices take multiple inputs and apply Boolean logic operations to produce a single binary (i.e. TRUE/FALSE) output. The unique molecular recognition abilities of DNA make it an ideal material for the construction of MLDs. Several DNA-based MLDs using FRET sensitized fluorescent emissions as output signals were devised. One of these devices was a protease biosensor designed to detect the presence and activity of a target protease, providing useful information about the target while reducing the rate of false-positive results. Though a working sensor was not ultimately achieved, important limitations on the design of this type of device were revealed. Two other devices were designed for the amplification of output signals from MLDs, an important aim to allow for more sensitive devices and more efficient logic circuits. Both devices were designed and tested using cascading DNA hybridization reactions to produce amplification of fluorescent output signals. Chemical ‘nose’ arrays utilize a panel of sensor elements which have non-specific but differing responses to targets to generate a unique ‘fingerprint’ pattern for each analyte. FRET between quantum dots (QDs) and dyes on peptides conjugated to QD surfaces can be used to determine rates of proteolysis. QDs with different surface chemistries can be used as individual sensor elements for the construction of a chemical ‘nose’ array to differentiate proteases, where the pattern of initial rates for the different surface chemistries represents the unique ‘fingerprint’ for a given protease. Three ionic QD surface ligands were synthesized, characterized, and tested for their effects on proteolysis and ability to differentiate between a panel of proteases.
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