UBC Theses and Dissertations
Development and characterization of quantum dots and other nanoparticles for applications in bioanalysis Kim, Hyungki
Quantum dots (QDs) are fluorescent nanoparticles with a typical diameter of 1-10 nm that boast up to 1000-fold greater brightness than fluorescent dyes, broad absorption, high resistance to photobleaching, size-tunable and narrow emission, and an inorganic surface that could be biofunctionalized with numerous peptides or other biomolecules. One of their applications is concentric Förster resonance energy transfer (cFRET), which takes advantage of their excellent photoluminescence and physical properties. Previous QD-based cFRET configurations have been comprised of a central QD conjugated with multiple copies of two distinct peptide sequences, each labeled with a different fluorescent dye. The activities of two different proteases can be spectroscopically analyzed concurrently with these single vectors through ratiometric three colour analysis of the QD and each dye. Due to the ability of cFRET probes to analyze the activity of multiple proteases in real-time simultaneously, they have promising potential for biological applications, such as improving the understanding of complex biological mechanisms like cell cascades that depend on many classes of proteases. In this thesis, a three-acceptor configuration was developed and the activity of three distinct proteases (trypsin, chymotrypsin, enterokinase) was successfully analyzed for the first time in a proof-of-concept study. A central green QD was conjugated with three distinct peptides, each of which behaved as an exclusive target for trypsin, chymotrypsin and enterokinase, labeled with Alexa Fluor 555, Cyanine 3.5 or Atto 594, and Alexa Fluor 647 dyes. This thesis also provides an overview of intracellular microinjections of various fluorescent materials, including QDs, QD-dye conjugates and single-chain polymer nanoparticles. In addition, this thesis investigates the development of a novel “stubby” capillary gel electrophoresis method that enabled rapid characterization of QDs relative to slab poly(acrylamide) gel electrophoresis or agarose gel electrophoresis. Lastly, characterization of multiple modification chemistries for cellulose paper substrates for affinity-based immobilized of QDs, gold and platinum nanoparticles were investigated using on-paper immobilization of colour-tuned mixtures of QDs, and post-immobilization enhancement of protease activity in model assays. Overall, this thesis contributes towards development of better nanoparticle-based probes and methods for biological and characterization applications.
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