Nous avons étudié le couplage dépliement-transport de la Maltose Binding Protein (MBP ou MalE), une protéine périplasmique d'E. Coli et d'un mutant instable, le MalE219, en fonction de la concentration d'un agent dénaturant, le chlorure de guanidium (GdnHCl) à l'échelle de la molécule unique. La technique utilisée est basée sur la détection électrique du transport de macromolécules à travers un nanopore protéique (l'Aérolysine d'Aeromonas Hydrophila) inséré dans une bicouche lipidique plane. Les résultats obtenus ont été comparés à ceux obtenus lors d'une précédente étude réalisée à travers un autre nanopore protéique, l'alpha-hémolysine du Staphylocoque doré, de géométrie et de charge nette différente. Nous avons montré l'existence de temps courts et longs de blocage du courant associés à des protéines dépliées ou partiellement repliées. La fréquence des blocages du courant permet d'obtenir la fraction de protéine dépliée passant à travers le pore en fonction de la concentration en GdnHCl. Les courbes de dénaturation obtenues avec les deux pores montrent un comportement sigmoïdale très similaire. Le type de pore n'influence donc pas la dénaturation des protéines, mais uniquement leur dynamique de transport. En revanche, la courbe de dénaturation du mutant instable présente un déplacement vers les concentrations plus faibles en GdnHCl. Il a été montré également que la présence du maltose comme ligand sur le MalE219 stabilise nettement sa structure. Pour La MBP, les temps de blocages longs diminuent avec l'augmentation de la concentration de GdnHCl montrant une dynamique de transition vitreuse . Cette technique est appropriée à l'étude du dépliement et des changements de conformation de protéines, mais ne permet pas d'obtenir des informations structurales sur les états intermédiaires de repliement. Ainsi, la spectroscopie RMN a été utilisée pour tenter de caractériser ces états intermédiaires de repliement, notamment par la méthode d'échange proton-deutérium. Elle consiste à suivre les cinétiques d'échange des résidus de la protéine sur des spectres 2D 1H-15N HSQC à différentes concentrations de GdnHCl.Ainsi 180 résidus sur les 370 que compte la MBP ont été suivis lors de la dénaturation en présence de GdnHCl. Les deux hélices en C-terminal sont très accessibles au solvant et se dénaturent facilement. La MBP est composée de deux domaines globulaires, le domaine N-ter et le domaine C-ter. Les éléments de structures secondaires situés dans la zone intermédiaire entre les deux domaines (principalement des brins β) sont particulièrement affectés par l'agent dénaturant. D'autres structures secondaires dans les domaines globulaires sont très protégées et plutôt stables. Il est donc proposé que les protéines partiellement dépliées s'insèrent dans le pore par l'extrémité C-terminal et que des parties de structuration tertiaire restent stable entraînant le blocage du pore. / We study the unfolding-transport mechanism of the Maltose Binding Protein (MBP or MalE), a periplasm protein of E. Coli and a destabilised variant, the MalE219, as the function of the concentration of denaturing agent, Guanidine Hydrochloride(GdnHCl) at the single molecule level. The technique is based on the electrical detection of the macromolecule transport through a nanometer-scale channel, Aerolysin channel, inserted into a planar lipid bilayer. Results obtained were compared to previous data with another channel, the alpha-Hemolysin. Both channels have different geometry and net charge.We show that we can distinguish unfolded states from partially folded ones with aerolysin pore.Unfolded proteins induce short current blockades, their duration is constant as a function of the concentration of denaturing agent. Partially folded proteins exhibit long blockades whose life times decrease as the concentration of GdnHCl increase, this indicates a possible glassy dynamics.The frequency of the short current blockades increases as the concentration of denaturing agent increases, following a sigmoidal denaturation curve.The unfolding curve of native MBP with Aerolysin pore is similar to the one previously measured with Hemolysin channel. The denaturation curve of the destabilized variant obtained with Aerolysin is shifted towards lower value of GdnHCl concentration in agreement with bulk measurements. We show also that the addition of maltose stabilizes the structure of MalE219. This nanopore recording technique is also suitable for the study of unfolding and conformation changes of proteins.In order to obtain structural informations that nanopore recording cannot provides, the structure of MBP along its denaturation curve was studied by NMR spectroscopy. The Hydrogen-exchange method known to be sensitive to folding intermediates was specially used. It consists in tracking hydrogen-deuterium exchange rates for amino on the 2D 1H-15N HSQC spectra.Thus, 180 residus of 370 for MBP was followed during denaturation in the presence of GdnHCl. The two last helices in C-terminal of MBP are accessible to the solvent and are denaturated easily. MBP is a two domains protein, N-ter domain and C-ter domain. It was found out that the C- and D-domain of MBP (mainly alpha-helices) could be relatively stable in presence of denaturing agent and that beta strands which make the link between the two domains would be affected by the denaturing agent. It was proposed that partially unfolded proteins enter the pore by the C-terminal end and that stable tertiary structure still present block the pore.

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.