Hairpin Nucleic Acid Probes for Metal Nanowire-Based Surface Studies and Biosensing.

Open Access
Cederquist, Kristin Baine
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
November 16, 2010
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor/Co-Advisor
  • Christine Dolan Keating, Committee Chair/Co-Chair
  • Philip C. Bevilacqua, Committee Member
  • Thomas E Mallouk, Committee Member
  • James Hansell Adair, Committee Member
  • surface characterization
  • spectroscopy
  • nanotechnology
  • fluorescence
  • bioconjugate synthesis
  • biochemical interactions
  • bioassays
  • multiplexed sensing
This dissertation details the use of hairpin DNA-nanowire conjugates for biosensing and surface studies. These nanowires, which are typically 6 microns in length and ~300 nm in cross-sectional diameter, can be synthesized with a barcode pattern by modulation of Au/Ag segment length, and have thus been used as solid supports for different types of multiplexed bioassays in previous works. Chapter 2 details characterization of the immobilized hairpin probes for bioassays and provides investigations of optimal loop and stem length. Ionic strength during the hybridization process was found to be of importance due to the mechanism of hairpin-oligonucleotide target binding and signal elucidation. Finally, a successful three-plex assay using barcoded nanowires was demonstrated, and this was built upon in Chapter 3, where a successful five-plex assay was demonstrated. In Chapter 3, hairpin probe surface coverage was found to be a critical parameter for bioassay performance and could be modulated by ionic strength during the nanowire immobilization process. Probe sequence, and therefore extent of folding, also dictated surface coverage to a certain extent, with probes predicted by melting curves to exhibit a higher footprint on the nanowire surface grafting with lower densities, even when challenged with higher concentrations. Kinetics of probe assembly were independent of sequence and almost immeasureably fast due to the particulate nature of the nanowires. Chapter 4 expanded upon the findings of Chapter 3 by investigating the impact of probe density upon hybridization efficiency. Probe accessibility was greatly affected by surface coverage, with hybridization efficiencies maximized at intermediate coverage, but diminishing at coverage extremes. The inclusion of short oligoethylene glycol spacers increased hybridization efficiency somewhat at the lowest coverages evaluated due to a reduction in nonspecific binding. Chapter 5 provided a more in-depth investigation into assay sensitivity, a concept first introduced in Chapter 2, through the investigation of the effects of nanowire population reduction on sensitivity and dynamic range. Nanowire population reduction aided sensitivity to a point, after which thermodynamics of target binding solely dictated performance. These studies were aided by receiver operating characteristic curves and Monte Carlo simulations. As immobilized structured probes gain popularity with the rise of aptamer technology, it has become increasingly important to characterize their behavior at the solid-solution interface. The studies contained herein highlight a number of differences between surface-immobilized structured DNA probes as compared to surface-immobilized linear DNA probes, which are designed not to contain secondary structure, and provide design and fabrication guidelines for future assay design.