Use this URL to cite or link to this record in EThOS:
Title: Advances in microfluidics and DNA nanotechnology for biomolecular analysis
Author: Arter, William
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
The sensing and characterisation of biomolecules is crucial for medical diagnostics and an understanding of biological systems. Proteins play a leading role in biology, by performing the majority of biochemical and biophysical processes that maintain homeostasis. As such, they and their interactions are probed as biomarkers and molecular machineries key to the analysis of system state and function. However, the tools predominantly employed for the solution-phase study of biomolecules often operate under non-physiological conditions, and are poorly suited to the analysis of analytes present in and interacting with heterogeneous environments typical of biological reality. In this thesis, I describe the development of new methods that address many of the principal challenges associated with biomolecular sensing and analysis. I employ a combination of microfluidic techniques and DNA nanotechnology to facilitate protein detection and characterisation purely in the solution phase. Through these approaches, targets are probed in free solution under conditions closer to the native state than allowed by conventional methods, which commonly rely upon surface-mediated manipulation of analytes. To begin, these fields are combined in a synergistic approach, with microscale electrophoresis utilised for the isolation of specific protein analytes on the basis of electrophoretic mobility, combined with isothermal signal amplification and quantitation by a DNA nanocircuit. Micro-electrophoresis is then applied to the characterisation of oligomeric protein complexes, which are implicated in the pathogenesis of misfolding diseases. In this study, micro-electrophoresis enables the fractionation of heterogeneous protein mixtures in free solution, an operation that is combined with single-molecule microscopy for high-resolution analysis. In a complementary study, DNA nano-switches are employed as in-situ molecular beacons for the continuous analysis of protein oligomerisation. Furthermore, the signal amplification from nano-switch protein recognition by isothermal, enzyme-free DNA circuitry is explored as a means to sensitively detect protein analytes in a mix-and-read process. Finally, the inherent advantages of small-volume compartmentalisation afforded by droplet microfluidics are combined with DNA circuitry, to achieve isothermal and enzyme-free single-molecule DNA-sensing in a digital droplet assay. This approach is applied to the field of molecular computing, where the assay functions as a highly sensitive transducer for the operation of simple logic circuits.
Supervisor: Keyser, Ulrich ; Knowles, Tuomas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: microfluidics ; DNA nanotechnology ; biophysics ; protein ; misfolding disease ; single molecule