In recent years the application of single-molecule techniques to probe biomolecules and intermolecular interactions at single-molecule resolution has expanded rapidly. Here, I investigate a series of peptides and proteins in an attempt to gain a better understanding of nanopore sensing as a single-molecule technique. The analysis of retro, inversed, and retro-inversed isomers of glucagon and α-helical Fmoc-D2A10K2 peptide showed that nanopore sensing utilizing a wild-type α-hemolysin pore can distinguish between all four isomers while circular dichroism can only distinguish between chiral isomers, but not between directional isomers. The investigation of a series of proteins of different chemical and physical properties revealed important information about nanopore analysis of proteins. Contrary to some reports in the literature, all proteins analysed here induced large blockade events. The frequency of total events and the proportion of large blockade events were significantly reduced in tris(hydroxymethyl)aminomethane or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffers and were only restored by the addition of ethylenediaminetetraacetic acid or the use of phosphate buffer, both of which can sequester metal ions. Furthermore, the results obtained with the proteins in the presence of ligands demonstrated that transient or partial unfolding of proteins can be detected by nanopore analysis confirming the usefulness of this technique for conformational studies or for protein/ligand interactions. Interestingly, while the blockade current histograms were different for each protein there was no obvious correlation between the properties of the proteins and the blockade current histograms. In an attempt to identify whether the large blockade events were translocation or intercalation, both an indirect and a direct approach were taken. The indirect approach which relies on the effect of voltage on the interaction of the molecule with the pore provided no conclusive answer to the question of protein translocation through the α-hemolysin pore. In contrast, the direct approach in which ribonuclease A is added to the cis side of the pore and then the trans side is tested for enzyme activity showed that ribonuclease A doesn't translocate through the α-hemolysin pore.

Metallic quantum clusters are stable structures that can exhibit many useful magnetic, chemical, and optical properties. Developing clusters for specific applications requires accurate methods for characterizing their physical and chemical properties. Most cluster characterization methods are ensemble-based measurements that can only measure the average values of the cluster properties. Single cluster measurements improve upon this by yielding information about the distribution of cluster parameters. This investigation describes the initial results on a new approach to detecting and characterizing individual gold nanoclusters (Au25(SG)18) in an aqueous solution with nanopore-based resistive pulse sensing. We also present a new application where the clusters are shown to increase the mean residence time of polyethylene glycol (PEG) molecules within an alpha hemolysin (αHL) nanopore. The effect appears over a range of PEG sizes and ionic strengths. This increases the resolution of the peaks in the single molecule mass spectrometry (SMMS) current blockade distribution and suggests a means for reducing the ionic strength of the nanopore solute in the SMMS protocol.

Nanopore sensing is a label-free method used to characterize water-soluble molecules. Recent work describes how Au25(SG)18 clusters improve the single molecule nanopore spectrometry (SMNS) technique when analyzing polyethylene glycol (PEG). This thesis will further study and optimize the enhancement effect resulting from a cluster’s presence. Additionally, a model describing the interaction between a cluster and PEG is developed to assist in understanding this mechanism of enhancement. This thesis will also discuss expanding the SMNS method to detect peptides, using Au25(SG)18 for enhancement, and adjusting solution conditions to improve the sensitivity of the SMNS system for peptide detection. Finally, a model describing the relationship between nanopore current blockades and molecular weight is developed to demonstrate the feasibility of using SMNS as a viable analytical technique for characterizing a wide variety of water-soluble molecules.

At its core, a nanopore detector has a nanometer-scale biological membrane across which a voltage is applied. The voltage draws a DNA molecule into an á-hemolysin channel in the membrane. Consequently, a distinctive channel current blockade signal is created as the molecule flexes and interacts with the channel. This flexing of the molecule is characterized by different blockade levels in the channel current signal. Previous experiments have shown that a nanopore detector is sufficiently sensitive such that nearly identical DNA molecules were classified successfully using machine learning techniques such as Hidden Markov Models and Support Vector Machines in a channel current based signal analysis platform [4-9]. In this paper, methods for improving feature extraction are presented to improve both classification and to provide biologists and chemists with a better understanding of the physical properties of a given molecule.

Aquatic ecotoxicology is the study of toxic chemicals and its effects on aquatic biological systems with the aim of minimising threats to human health and ensure self-sustained ecosystems. Freshwater bivalves are excellent sentinels for use in ecotoxilogical research due to their filter feeding properties, stationary lifestyle and inability to regulate body temperature. This project aimed to assess the feasibility and use of nanopore sequencing, a real-time single-molecule sequencing technology in comparative expression analysis by sequencing transcriptomic RNA from the freshwater mussel Anodonta anatina following exposure to copper. RNAs were extracted from 80 mg hepatopancreas tissue, followed by poly(A) RNA selection. Furthermore, the poly(A) RNA was used to construct a nanopore sequencing library. Sequencing a total amount of 560 ng poly(A) RNA over the course of two separate runs generated 239,448 reads, in which 75% of the reads were obtained during the first run (control) and 25% of the reads were obtained during the second run (case). The median read lengths ranged between 534-650 nucleotides, with a base call accuracy <90%. Due to the big differences in sequence data output between the two sequencing runs, the data was ineligible for comparative analysis. The findings conclude that nanopore sequencing is capable of generating longer read lengths when compared to other sequencing platforms. However, the technology is error-prone in terms of accurate base call identifications and relies on other platforms for error corrections. Future advances include de novo transcriptome assembly for efficient use of Anodonta anatina as a bioindicator in aquatic ecotoxicology.

This work presents membrane development applicable in nanofluidic devices. These membranes can also be termed suspended thin films, supported on two or more edges. I first discuss motivation and background for developing these structures. Then I derive the formative principles for nanofluidic systems. Following the derivation of the Navier-Stokes and Washburn equations, I discuss applying these theories to planar nanofluidic capillaries and finish the derivation by discussing the forces that drive liquid flow in nanochannels. I next discuss the membrane development process, starting with my work in static height traps, and develop the concept of analyzing nanoparticles using suspended membranes. After reviewing the lessons learned from the double-nanopore project I discuss developing an oxide layer tuned to the needs of a membrane and present the design of an adjustable membrane structure. Afterward, I discuss modeling and simulating the structure, and present a procedure for fabricating robust membranes. I then explain applying the membrane structure to form a nanofluidic pump and document the process for recording and analyzing the pumping characteristics for nanodevices. As part of the pump section I propose a theory and model for predicting the behavior of the pumps. I next present applying active membranes as nanoparticle traps. I document a quick-turn optical profilometry method for charicterizing the devices, then present experimental data involving trapping. Early results show that the device functions as a nanoparticle concentrator and may work well as a size-based trap for nanoparticles. I conclude by summarizing the main contributions made during my course of study and by providing supplemental material to guide future research.

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.

Il existe différentes techniques de manipulation et de détection de molécules uniques. Parmi elles, la mesure du courant ionique traversant un pore nanométrique unique est la plus ancienne. Le travail effectué dans cette thèse utilise cette technique et s'articule autour de deux objectifs. Tout d'abord, l'utilisation d'un nanopore unique obtenu par attaque de trace permet d'imiter les expériences déjà faites dans la littérature avec d'autres types de pores. Certains phénomènes, tels que la rectification par exemple, ont pu être observés, mais d'autres comme la translocation n'ont pu l'être. Ensuite, une étude du bruit de conductance électrique démontre l'existence d'un phénomène qui n'avait jusqu'alors pas été soulevé dans la littérature. En effet, ce bruit ne peut pas être imputé aux fluctuations des caractéristiques de la géométrie du pore ou de sa paroi, mais plutôt à des effets coopératifs sur la mobilité des ions dans un milieu confiné.

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.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.