Novel double nanopore platforms for protein sensing and single-molecule manipulation
File(s)
Author(s)
Campolo, Giulia
Type
Thesis
Abstract
In the last decade, solid-state nanopores have emerged as a powerful single-molecule
detection technique. Their versatility, simple operating principles, and minimal sample
preparation make them promising candidates for a wide range of biophysical studies. However,
the current state-of-the-art suffers from low selectivity, inadequate sensitivity for small
analytes, and a lack of control over molecular transport, limiting their practical applications. In
this work, we exploit the versatility of nanopipettes, an inexpensive sub-set of solid-state
nanopores, to counteract these challenges.
A variety of analytes was used for this study, but particular attention was placed on
alpha-synuclein, a small unfolded protein predominantly found in the brain. In recent years,
abnormal self-aggregation of alpha-synuclein has been identified as a potential biomarker for
Parkison's disese, the second most common neurodegenerative disorder. The heterogeneous
and dynamic system formed by the oligomers, together with the monomers' small size makes
their study and detection under current methodologies particularly challenging, and therefore
a perfect candidate for single-molecule sensing using nanopores.
We present two novel platforms, the nanobridge and the double barrel nanopore, based
on double-barrel nanopipettes. In the nanobridge, enhanced sensitivity was obtained by
confining biomolecules in a zeptolitre (10-21 L) volume. We demonstrate that the molecular
confinement increased temporal resolutions and signal-to-noise ratios, allowing the detection
of analytes that cannot be probed in more conventional nanopore architectures, such as small
proteins.
In the double barrel nanopore, two independently addressable nanopores separated by
a 20 nm gap were used to tune the forces exerted onto a single DNA molecule. This technique
allowed to finely control the transport of molecules between the pore with almost 100 %
efficiency and increase temporal resolutions by two orders of magnitude. Furthermore, we
demonstrate that the platform was capable of obtaining multiple readings of a single protein.
Finally, we introduce an original aptamer-based DNA carrier capable of selective detection of
proteins in solution, which can be used in concomitance with the aforementioned double barrel
architecture.
detection technique. Their versatility, simple operating principles, and minimal sample
preparation make them promising candidates for a wide range of biophysical studies. However,
the current state-of-the-art suffers from low selectivity, inadequate sensitivity for small
analytes, and a lack of control over molecular transport, limiting their practical applications. In
this work, we exploit the versatility of nanopipettes, an inexpensive sub-set of solid-state
nanopores, to counteract these challenges.
A variety of analytes was used for this study, but particular attention was placed on
alpha-synuclein, a small unfolded protein predominantly found in the brain. In recent years,
abnormal self-aggregation of alpha-synuclein has been identified as a potential biomarker for
Parkison's disese, the second most common neurodegenerative disorder. The heterogeneous
and dynamic system formed by the oligomers, together with the monomers' small size makes
their study and detection under current methodologies particularly challenging, and therefore
a perfect candidate for single-molecule sensing using nanopores.
We present two novel platforms, the nanobridge and the double barrel nanopore, based
on double-barrel nanopipettes. In the nanobridge, enhanced sensitivity was obtained by
confining biomolecules in a zeptolitre (10-21 L) volume. We demonstrate that the molecular
confinement increased temporal resolutions and signal-to-noise ratios, allowing the detection
of analytes that cannot be probed in more conventional nanopore architectures, such as small
proteins.
In the double barrel nanopore, two independently addressable nanopores separated by
a 20 nm gap were used to tune the forces exerted onto a single DNA molecule. This technique
allowed to finely control the transport of molecules between the pore with almost 100 %
efficiency and increase temporal resolutions by two orders of magnitude. Furthermore, we
demonstrate that the platform was capable of obtaining multiple readings of a single protein.
Finally, we introduce an original aptamer-based DNA carrier capable of selective detection of
proteins in solution, which can be used in concomitance with the aforementioned double barrel
architecture.
Version
Open Access
Date Issued
2022-01
Date Awarded
2023-02
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Edel, Joshua
Ivanov, Alex
Cass, Tony
Sponsor
UK Research and Innovation
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)