Improving droplet interface platforms for nanopore sensing
File(s)
Author(s)
Rogers-Simmonds, Daisy
Type
Thesis
Abstract
Nanopore sensors have emerged since their conception 3 decades ago as a versatile, bio-
inspired tool by which to conduct single-molecule investigations of DNA, and more recently
proteins, RNA, methylated DNA and other polymers. Stable membrane materials are
required for the longevity of these devices, and to this end steps towards making and
characterising droplet interface bilayers from amphiphilic block copolymers are presented
here. The functional impact of nanopore reconstitution in these membranes compared to
a lipid bilayer is investigated through electrophysiological measurements and by single-
channel fluorescent imaging. In order to design nanopore sensing devices for detection
of a wider range of analytes, a detailed understanding of their mechanism of interaction
with nanopores is needed. To gain single-molecule insight into the mechanism by which
DNA interacts with a nanopore during a translocation event, simultaneous single-molecule
dual-colour TIRF microscopy is used to image a surface-tethered G-quadruplex containing
DNA construct as it is captured and unzipped by a nanopore in a lipid DIB. Membranes
rarely act only as passive hosts to biological molecules, playing an active role in regulating
processes. Ultimately, the unification of the two projects described here would give insight
into the impact of synthetic membrane materials on the performance of nanopore sensing
devices.
This thesis is split into 3 sections. Chapter 1 provides background theo-
retical concepts and contextualises further chapters. Chapter 2 provides practical instruction on techniques described in the work.
Chapter 3 describes work done towards understanding the kinetics of DNA capture and by a nanopore. From events collected via TIRFM, a tentative step-by-step mechanism is presented.
Chapter 4 describes efforts to form droplet interface bilayers from PMOXA-b-PDMS-b-PMOXA triblock copolymers. A proprietary
material from Oxford Nanopore Technologies is used as a springboard to compare the behaviour of such systems with their lipid counterparts.
inspired tool by which to conduct single-molecule investigations of DNA, and more recently
proteins, RNA, methylated DNA and other polymers. Stable membrane materials are
required for the longevity of these devices, and to this end steps towards making and
characterising droplet interface bilayers from amphiphilic block copolymers are presented
here. The functional impact of nanopore reconstitution in these membranes compared to
a lipid bilayer is investigated through electrophysiological measurements and by single-
channel fluorescent imaging. In order to design nanopore sensing devices for detection
of a wider range of analytes, a detailed understanding of their mechanism of interaction
with nanopores is needed. To gain single-molecule insight into the mechanism by which
DNA interacts with a nanopore during a translocation event, simultaneous single-molecule
dual-colour TIRF microscopy is used to image a surface-tethered G-quadruplex containing
DNA construct as it is captured and unzipped by a nanopore in a lipid DIB. Membranes
rarely act only as passive hosts to biological molecules, playing an active role in regulating
processes. Ultimately, the unification of the two projects described here would give insight
into the impact of synthetic membrane materials on the performance of nanopore sensing
devices.
This thesis is split into 3 sections. Chapter 1 provides background theo-
retical concepts and contextualises further chapters. Chapter 2 provides practical instruction on techniques described in the work.
Chapter 3 describes work done towards understanding the kinetics of DNA capture and by a nanopore. From events collected via TIRFM, a tentative step-by-step mechanism is presented.
Chapter 4 describes efforts to form droplet interface bilayers from PMOXA-b-PDMS-b-PMOXA triblock copolymers. A proprietary
material from Oxford Nanopore Technologies is used as a springboard to compare the behaviour of such systems with their lipid counterparts.
Version
Open Access
Date Issued
2023-08
Date Awarded
2024-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Ces, Oscar
Seddon, John
Wallace, Mark
Sponsor
Engineering and Physical Sciences Research Council
Oxford Nanopore Technologies
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)