Solid-state nanopore sensors are an emerging platform for performing single-molecule characterization of biomolecules such as DNA. With the advent of Controlled Breakdown (CBD), creating a simple, tunable, ultra-low concentration sensing device in situ has enabled their direct integration with a host of platforms. The simplicity and sensitivity in performing measurements allows nanopore-based technologies to find uses which enhance existing methods.
One such promising avenue for nanopore-based sensing is in the detection of infectious diseases, where early and accurate identification of the causative pathogen is essential for successful patient outcomes. Conventional assays, while effective, often have limitations in speed, cost, or target sensitivity, and may benefit from nanopore sensing approaches. However, solid-state nanopores currently lack the ability to discriminate between biomarkers sharing identical size and charge densities, such as sequentially-heterogeneous strands of DNA.
Addressing the weakness of both conventional assays and nanopores could come from combining nanopore sensing with the polymerase chain reaction (PCR), a well-established and highly-selective nucleic acid amplification scheme. PCR is designed to produce large quantities of identical fragments of DNA, known as amplicons, if a user-defined parent copy is present. After the PCR process has finished, the signals produced by this population of amplicons on a nanopore sensor should therefore be indicative to the presence of a DNA biomarker in the starting sample.
As PCR reactions can use a mix of different proteins, detergents, and other molecules, the challenge lies in ensuring the compatibility of these reagents with a nanopore, and determining whether the background signal they produce can be discriminated from an amplicon signal. To this end, this thesis investigates PCR-nanopore compatibility, and experimentally demonstrates a nanopore signal-classification technique to successfully identify the presence of chromosomal DNA from Group A Streptococcus (GAS), an infectious bacterium responsible for strep throat, in samples derived from clinical extracts.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/43385 |
| Date | 15 March 2022 |
| Creators | King, Simon |
| Contributors | Tabard-Cossa, Vincent |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution-NonCommercial-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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