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Title: Single-molecule DNA detection in nanopipettes using high-speed measurements and surface modifications
Author: Fraccari, Raquel Leh-na
ISNI:       0000 0004 6059 3151
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Inspired by transmembrane pores found in cell membranes and the operating principle of the Coulter counter used for cell counting, nanopore biosensors have emerged as a tool for single-molecule detection. This thesis describes single-molecule DNA detection through resistive pulse sensing using nanopipettes, a novel subclass of solid-state nanopores. In the first part of this thesis, double-stranded (ds) DNA-nanopipette surface interactions were probed in 1 M KCl electrolyte using DNA molecules with lengths ranging from 48.5 to 4 kilobase pair (kbp). A custom-built current amplifier was employed for low-noise and high-bandwidth measurements. Results from these experiments were used to theoretically rationalise DNA-surface interactions and suggest that dsDNA adsorption to the nanopipette surface prior to translocation through the pore is likely to be an important factor in the process. Subsequently, initial investigations to probe DNA-surface interactions were carried out by modifying the surface charge of nanopipettes using silanes. Additionally, experiments were performed to detect shorter dsDNA lengths. In 1 M KCl electrolyte, 200 base pair (bp) long dsDNA was successfully detected using the low-noise and high-bandwidth current amplifier. However detection of 100 bp long dsDNA required the use of 2 or 4 M LiCl electrolyte. Attention was finally shifted to the detection of 100 bp dsDNA in 1 M KCl electrolyte using functionalised lipid bilayer coated nanopipettes. Additional techniques were employed to prepare and characterise the lipid bilayers, including atomic force microscopy (AFM) and dynamic light scattering (DLS). The promising preliminary results provide a framework for further experiments using functionalised lipid bilayers to coat nanopipettes. Overall, results of the aforementioned research presented in this thesis demonstrate high-speed single-molecule detection of DNA and provide novel insights into the translocation dynamics of DNA molecules in nanopipettes and the sensing capabilities of nanopipettes.
Supervisor: Albrecht, Tim Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral