Towards the Development of Techniques to Facilitate the Assembly and Characterization of Quantum Dot— Gold Nanoparticle Complexes for Resonance Energy Transfer Based Assays

Resonance energy transfer between quantum dots and gold nanoparticles represents one of the most popular transduction schemes for biosensors using multi-nanoparticle complexes. This thesis investigates the influence of quantum dot positioning, relative to the gold nanoparticle, on the analytical performance of these assays. Stoichiometric assembly of the quantum dot-gold nanoparticle complexes required accurate knowledge of the quantum dot concentration. A technique was developed to quantify the molar concentration of nanoparticles in solution. This technique relied on the quantitative analysis of the statistical distribution of nanoparticle-ligand conjugates that form when nanoparticles are functionalized with small equivalents of ligand. The technique was successfully validated using gold nanoparticles, and was subsequently applied to determine the concentration of the alloy quantum dots. The precise positioning of the quantum dots relative to the gold nanoparticles required monovalent conjugates of the quantum dots. These were prepared using a magnetic bead based technique where positively charged magnetic beads were used to capture and selectively isolate the monovalent quantum dot-DNA conjugates. Successful isolation of monovalent conjugates was confirmed using single molecule fluorescence spectroscopy and the monovalent conjugates were found to retain their activity as determined using quantitative hybridization assays. Optimizing the quantum dot-gold nanoparticle assay required the investigation of two different configurations of nanoparticle arrangement. The first involved arranging quantum dots around a gold nanoparticle core, while the second involved arranging gold nanoparticles around a quantum dot core. The first configuration was found to have optimal analytical performance, as evaluated using quenching efficiencies and influence of inner filter effect. The ability to arrange multiple quantum dots around a single large gold nanoparticle minimized the negative influence of the inner filter effect, while the high quenching efficiency ensured high responses to target hybridization.Ph.D