One of the most crucial steps towards nanopore-based nucleic acid analysis is extending the dwell time of DNA molecules within the sensing region of the nanopore. I address this issue by interfacing solid-state nanopores with gel media, which sterically hinders translocating DNA molecules, increasing dwell times. Specifically, my experimental results focus on two reptation regimes: when the DNA molecule is flexible on the length scale of the gel pore, and when the DNA molecule is inflexible on the length scale of the gel pore. The first regime is achieved through the use of agarose gel and 5 kbp dsDNA fragments, and produces a wide distribution of translocation times, spanning roughly three orders of magnitude. The second regime is achieved through the use of polyacrylamide gel and 100 bp dsDNA fragments, and displays a shift in translocation times by an order of magnitude while maintaining a tight distribution.

Broadly speaking, the work herein discussed encompasses the development of biomolecular assays for biomarker detection. Specific to the assays in this thesis is the design of reaction schemes that consider the unique requirements of one class of single-molecule sensors in particular: solid-state nanopores formed using a novel fabrication and conditioning technique discovered during this research at the University of Ottawa. We present three unique assays for the detection of different biomolecular targets. The first uses a class of DNA origami structures termed nanoswitches to translate the presence of a short segment of single-stranded DNA Zika virus biomarker to a large configurational change in a double-stranded DNA scaffold. The signal amplification inherent in this topological change allowed us to a achieve a high degree of specificity for detecting a small nucleic acid target by requiring two separate binding events. Furthermore, through careful design of the configurational change, the number of topological states that a solid-state nanopore can sense is limited, providing unambiguous signals in ionic current recordings. Quantification of the Zika gene was performed by sensing the relative amounts of nanoswitches in looped and linear configurations from only hundreds of individual molecules. We then explored the sensitivity of solid-state nanopores for detecting small molecular features along short DNA scaffolds. Leveraging the ability of our nanopores to detect the presence of these protrusions, we present results in which ATP, a molecule significantly too small to be directly detected by the nanopore sensor, initiated an aptamer-based DNA displacement reaction to form a protrusion along scaffolds, producing measurable changes in ionic current signatures in nanopore recordings. Finally, we present an assay in which a microRNA, a biomarker linked to various cancers, was detected through the conjugation of two probes, each of which contained a binding site to different segments of the microRNA. In addition to examining different probe set structures for optimal performance, our two-probe design aimed to improve specificity over conventional single-probe-based assays which only require one recognition step, while still providing unambiguous signals due to the greater-than-doubling in molecular complex size upon conjugation. Furthermore, the use of two individual small probes, rather than one large nanoswitch, increased the resolution with which we could differentiate microRNA concentrations. The assay enabled the quantification of six concentrations of microRNA spanning a single order of magnitude, in only several hundred events, and allowed us to take advantage of the reduced cost, material and labour, as well as increased nanopore capture rates, associated with small assembled molecules.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

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.

Solid-state nanopores are a versatile class single-molecule sensors to electrically characterize a range of biological molecules. Nanopores operate on the simple premise that when a voltage is applied across a pore immersed in a salt solution, the passage of a biomolecule results in a transient blockage in the ionic current that provide information about the translocating molecule. This thesis presents studies employing various DNA nanostructures with solid-state nanopore electrical readout for the development of high sensitivity digital single-molecule assays to detect low-abundance biomarkers. Toward this ultimate goal, work presented in this thesis use nanopores to probe DNA nanostructures, their assembly, mechanical properties, and monitor their dynamics with time and temperature. DNA nanostructures are self-assembled via specific base pairing of DNA, their programmability make them particularly useful for applications including drug delivery, molecular computation and biosensing. Here, I first show results of translocation profiles and discuss folding characteristics, mobility, and molecular configuration during passage for different DNA nanostructures such as the short star-shaped DNA nanostructures and large helix-bundle DNA origami structures under various experimental conditions in an effort to understand the passage characteristics through nanopores of these structures before using them in biological assays. I conclude by presenting a magnetic bead-based immunoassay scheme using a digital solid-state nanopore readout. Nanopore has the ability to count molecules one at a time, this allows accurate and precise determination of the concentration of a biomarker in solution. Coupled with the use of specific choice of DNA nanostructures, as proxy labels for proteins of interest, I establish that nanopores sensors can reliably quantify the concentration of a protein biomarker from complex biofluids and overcome the traditional challenges associated with nanopore-based protein sensing, such as specificity, sensitivity, and consistency. I demonstrate the quantification of thyroid stimulating hormone (TSH) with a high degree of precision down to the femtomolar range by using a nanoparticle-based signal amplification strategy. The proposed assay scheme is generalizable to a framework for the detection and quantification of a wide range of target proteins, and given that its performance can further be improved with the use of parallelization, preconcentration, or miniaturization, it opens up exciting opportunities for the development of ultra-sensitive digital assay in a format that is compatible for point-of-care.