Nous étudions le transport de protéines natives, partiellement ou complètement dépliées à travers des nanopores protéiques ou solides à l’échelle de la molécule unique en utilisant une détection électrique. Le système modèle que nous avons choisi est la protéine MalE sauvage ou mutante, en particulier la protéine MalE 219 qui se déplie à de plus faibles concentrations d’agent dénaturant que la protéine sauvage. Nous montrons que la translocation de protéines partiellement dépliées à travers un canal protéique, l’hémolysine du staphylocoque doré, dépend des conformations individuelles que nous pouvons distinguer. Les molécules dépliées passent rapidement dans les nanopores. Nous mesurons directement leur fraction en fonction de la concentration en agent dénaturant. La technique est très sensible aux mutations affectant le repliement.. Nous avons également étudié le transport de protéines à travers des nanopores solides dans différents cas. Nous comparons d’abord le transport de protéines natives et de protéines complètement dépliées à travers un nanopore de grand diamètre puis nous étudions la translocation de protéines dépliées à travers un nanopore étroit de diamètre inférieur à la taille de la protéine. Nous observons différents régimes de translocation quand nous varions le champ électrique appliqué que nous interprétons à l’aide d’un modèle théorique simple. / We study the transport of native, partially or completely unfolded proteins through protein or solid-state nanopores at the single molecule level using an electrical detection. The model system that we use is the wild-type MalE or mutant protein, in particular MalE219, which unfolds at lower concentration of denaturing agent than the wild type. We show that the translocation of partially unfolded proteins through the Hemolysin protein channel, a toxin from Staphylococcus aureus, depends on of individual conformations that we can distinguish. The unfolded proteins pass rapidly through the nanopores. We directly measure their proportion as a function of the concentration of denaturing agent. The technique is very sensitive to the mutations affecting the folding properties. We also study the transport of proteins through solid-state nanopores in different situations. We first compare the transport of native and fully unfolded proteins through a nanopore of large diameter. We then study the tranlocation of unfolded proteins through a narrow pore, whose diameter is smaller than the protein size. We observe different regimes of translocation by varying the applied electric field, which we interpret using a simple theoretical model.

No description available.

Single-molecule biosensors offer distinct advantages over their ensemble-averaged counterparts by being able to extract information related to rare targets and specific molecular configurations within a sample. In particular, solid-state nanopores embody a promising single-molecule technique that is based on detecting target molecules by the amount of ionic current they block as they pass through a nanoscale aperture across a thin membrane. In this thesis, I present extensions of the basic nanopore system aimed at addressing some of its main limitations at present, namely: 1) the low rates at which nanopores capture molecules from a bulk volume, which restricts their ability to work with dilute (≲ nM) samples, and 2) the difficulty in using nanopores to distinguish small or closely related molecules by their direct current blockage signatures alone. I begin by describing the design and construction of a nanopore-based instrument that integrates an optical detection channel in parallel with ionic current sensing. A particular emphasis was placed on minimizing the electrical noise contributions of the added optical equipment on the original ionic current channel. Measuring the optical signals of translocating molecules together with their current blockages can improve the discrimination of two fluorescently labelled targets (or two configurations of a single target) that normally produce similar ionic current signatures. I next investigate the combination of nanopore sensing with target pre-concentration, specifically, by embedding a nanopore membrane within a fluidic cell that features an insulator-based dielectrophoretic (iDEP) trap. Applying large (≳ 100 V) AC voltages across the iDEP channels of the cells resulted in the accumulation of polarizable targets (dsDNA, polystyrene beads) at the locations of the membranes, thus pointing toward a convenient method for the detection of ultra-dilute target samples in future nanopore devices. Finally, I introduce improved protocols for the synthesis and nanopore signal analysis of dsDNA-based molecular carriers. In a molecular carrier scheme, in order to enhance the target specificity of the system, target molecules are not sensed directly by a nanopore but instead interact specifically with secondary molecules (“carriers”) to recognizably alter the carrier translocation signals. Here, I present proof-of-principle analyses of DNA carrier experiments that highlight the multiplexing capabilities of our carrier design, which are based on separating targets by their interactions with carriers of different lengths. Developments of the nanopore sensing platform such as those presented in this work, which leverage the intrinsic versatility of solid-state nanopores to be integrated within complex devices and to detect a wide range of target molecules, will play an important role in continuing to increase the precision of single-molecule measurements into the future and to expand their breadth of potential applications.

As knowledge of the human genome has accelerated, various diseases and individuals’ responses to drugs have been pinpointed to specific DNA variations in one’s genome. Among many different types of variants, the most common and simplest is the single nucleotide polymorphism (SNP) in which a single base substitution occurs. Although there have been considerable improvements in technologies that can reveal a single base difference in a DNA strand, simple and affordable methods that have high detection sensitivity and require small sample volume are expected to facilitate widespread adoption of routine SNP analysis in clinical settings. One such method that meets these requirements is to use nanopore as a single molecule detector, an emerging analytic system that detects changes in current related to molecules occupying a nanometer aperture. This dissertation thus chronicles our endeavors in developing a nanopore-based SNP assay using polymer tagged dideoxynucleotides (ddNTPs). The fundamental principles of this method rely on single base extension (SBE) of a primer by DNA polymerase using polymer tagged ddNTP analogs for allele discrimination and simple electronic readout of an alpha hemolysin (αHL) nanopore current for allele detection at the single molecule level. Using four uniquely tagged ddNTPs, a characteristic current level that is specific to each base is produced, thus identifying the SNP alleles present and the genotype at the site. To demonstrate the feasibility of this approach, four polymer attached ddNTPs, each with a different tag that generates a characteristic current blockade level in the αHL nanopore, were designed and synthesized. To search for a DNA polymerase that can accept these tagged ddNTP analogs as substrates, several candidate DNA polymerases were surveyed and their relative efficiencies for incorporation of the analogs were compared (Chapter 2). To generate a steady and stable blockade event for accurate SNP analysis, two different means of positioning a tag molecule in the αHL nanopore after the SBE reaction have been explored: covalent conjugation of DNA primer to the pore and immobilization of biotinylated primer within the pore by streptavidin. To find a suitable position for primer attachment on the pore, three αHL mutants, each with a different single conjugation point, were constructed. Using these mutants, different DNA-pore conjugates were produced and purified via various chromatography systems (Chapter 3). In the nanopore system, charged molecules such as DNA are electrophoretically driven through the pore under an applied voltage, thereby modulating the ionic current through the nanopore. This current reveals useful information about the structure and dynamic motion of the molecule at the single molecule level. Before performing SNP analysis, we first studied single molecule behaviors of oligonucleotides of different lengths and structures in the αHL pore and their ensuing current signatures in the system (Chapter 4). Finally, harnessed with tools and insights from the nanopore single molecule studies, actual SNP assays were performed in our nanopore system using the polymer tagged ddNTPs and SBE. Chapter 5 discusses the integrated approach where SBE is achieved on a primer-conjugated αHL nanopore and Chapter 6 presents the results using a biotin-streptavidin complex for immobilization of tag molecules in the pore. Overall, this thesis validates adaptation of the nanopore detection system for SNP analysis using the polymer tagged ddNTPs.

The high surface to volume ratio which is a property of nanoscale devices means the interfacial effects from these devices on the mass transport of analyte can be significant. Quartz nanopipette effect on the mass transport behavior of inorganic monovalent salts such as potassium chloride is shown to differ from those of conical nanopore. Quartz nanopipettes demonstrate a more significant interfacial impact on the mass transport behavior of inorganic salts. This is evidenced by significant impacts on ionic transport even at high electrolyte concentration where nanopore interfacial effects do not significantly impact the ion transport. Nanopipettes have been use to precipitate salts such as lithium chloride in bulk concentrations three orders of magnitude below the saturation concentration. These novel interfacial interactions have opened new avenues for crystallization of more complex organic biomolecules using inorganic systems as model systems on which to base the approach for these more complex systems.

Nanopores are apertures of nanometric dimensions in an insulating matrix. They are routinely used to sense and measure properties of single molecules such as DNA. This sensing technique relies on the process of translocation, whereby a molecule in aqueous solution moves through the pore under an applied electric field. The presence of the molecule modulates the ionic current through the pore, from which information can be obtained regarding the molecule's properties. Whereas the electrical properties of the nanopore are relatively well known, much less work has been done regarding their fluidic properties. In this thesis I investigate the effects of fluid flow within the nanopore system. In particular, the charged nature of the DNA and pore walls results in electrically-driven flows called electroosmosis. Using a setup which combines the nanopore with an optical trap to measure forces with piconewton sensitivity, we elucidate the electroosmotic coupling between multiple DNA molecules inside the confined environment of the pore. Outside the pore, these flows produce a nanofluidic jet, since the pore behaves like a small electroosmotic pump. We show that this jet is well-described by the low Reynolds number limit of the classical Landau-Squire solution of the Navier-Stokes equations. The properties of this jet vary in a complex way with changing conditions: the jet reverses direction as a function of salt concentration, and exhibits asymmetry with respect to voltage reversal. Using a combination of simulations and analytic modelling, we are able to account for all of these effects. The result of this work is a more complete understanding of the fluidic properties of the nanopore. These effects govern the translocation process, and thus have consequences for better control of single molecule sensing. Additionally, the phenomena we have uncovered could potentially be harnessed in novel microfluidic applications, whose technological implications range from lab-on-a-chip devices to personalised medicine.

No description available.

La technique de détection à l'aide de nanopores au niveau de la molécule unique est l'une des plus puissantes pour l'analyse de diverses molécules, dont les polymères biologiques et synthétiques, les protéines et les peptides, les molécules de sucre ou les nanoparticules métalliques. Ces pores peuvent également servir de plate-forme pour l'étude de phénomènes physiques et biologiques fondamentaux. Dans le cadre de l'analyse de molécules, ce travail, expérimenté en utilisant la technique de la peinture de bicouche lipidique, porte principalement sur la détection des polymères et leur utilité pour sonder les processus fondamentaux des de l'α-hémolysine et de l'aérolysine.Le premier chapitre de résultats décrit l’analyse des flux à travers l'hémolysine et l'aérolysine à l’aide des polyéthylèneglycols (PEG) et des α-cyclodextrines, ainsi que les effets des sels de KCl et de LiCl sur l'interaction des PEG avec ces pores. L'une des principales conclusions est qu'il existe un flux électoosmotique plus fort dans l'aérolysine, responsable du transport des molécules neutres, les α-cyclodextrines. La seconde constatation concerne la dynamique des PEG avec les nanopores qui semblait être fortement dépendante du sel, montrant des différences drastiques de fréquence et de durée d’interaction en fonction de la tension pour les deux sels, bien que la détection de la masse de PEG dans les deux conditions indique que la nature de l'interaction avec le pore est similaire dans les deux types de sels.Le but des travaux présentés dans le deuxième chapitre de résultats était de détecter les polymères de précision et à trouver les meilleures conditions pouvant conduire à leur séquençage avec des nanopores. Des homopolymères et copolymères de poly(phosphodiester)s ont été sondés en utilisant l'hémolysine, l'aérolysine et MspA. Le premier type de polymères étudiés contenant une amorce 3-polythymidine et une suite de comonomères de type (0) a montré une forte interaction avec les pores qui a été interprétée comme la promotion de la liaison avec les pores, due à l'amorce d’ADN simple brin, combinée à une grande flexibilité du premier type de polymères. Les polymères qui contenaient des chaînes latérales alcyne et triazole se sont révélés avoir des interactions plus complexes, mais ont interagi pendant des durées plus courtes avec les pores indiquant qu'ils étaient plus rigides. Le second type de polymères semble s’agréger en solution du fait de l’interaction entre les chaînes latérales, ce qui prouve l’importance de la caractérisation de ces molécules en solution par diffusion de rayons, dans le cadre de la détection et finalement de leur séquençage.L'étude du troisième chapitre de résultats, a porté sur la dynamique de petits oligonucléotides avec le pore d’aerolysine. Les polyadénines (A3, A4, A5) ont montré une dynamique complexe d’interaction avec le pore, qui a été étudiée par l'analyse et la quantification des différentes propriétés des événements. L'ensemble du processus s'est avéré être régi par deux sites de liaison et des barrières énergétiques à l'intérieur du pore que les molécules doivent surmonter. Ces résultats ont été combinés à un modèle cinétique qui a permis une description complète de la liaison et de la translocation (ou son non succès) des polyadénines.Le dernier chapitre des résultats décrit l’interaction de plus grandes polyadénines (A6-A7-A8-A9-A10) avec l’aérolysine. L’analyse de l'amplitude des courants des blocs induits par l'adénine à l'intérieur de ce pore montre une interaction dépendante de l'orientation des molécules avec le pore. Cette interaction dépendante de l'orientation a commencé à apparaître pour la molécule A7 et est devenue l'effet dominant pour A9 et A10. En raison de la flexibilité de l'ANDsb, cet effet n'est pas observé pour les molécules de plus petite taille (A6 et inférieures) en raison de leur possibilité de réorientation à l'intérieur du pore. / The technique of detection with nanopores at the single molecule level, is one of the most powerful method for the analysis of various molecules, of which biological and synthetic polymers, proteins and peptides, sugar molecules or metal nanoparticles. These pores can also serve as a platform for the study of fundamental physical and biological phenomenons. In the context of molecule analysis, this work, which is experimented using the technique of planar lipid bilayer painting, focuses mainly on the detection of polymers and their utility to portray fundamental processes of the α-hemolysin and aerolysin biological nanopores.The first results chapter described the probing of flows through α-hemolysin and aerolysin using polyethylene glycols (PEGs) and α-cyclodextrines, and the effects of KCl and LiCl salts on the interaction of PEGs with these pores. One main finding was that there exists a stronger electoosmotic flow in aerolysin, responsible for the transport of the neutral molecules α-cyclodextrines. The second finding was that the dynamics of PEGs with the nanopores are strongly dependent on the salt, showing drastic differences of frequency and dwell times vs. voltage for the two salts, although, the results of detection of mass of PEGs pointed to the fact that the nature of the interaction with the pore is similar in both salts.The aim of the work presented in the second results chapter, was to detect precision polymers, and find the best conditions, which can lead to their sequencing with nanopores. The homo- an copolymers of poly(phosphodiester)s were probed using α-hemolysin, aerolysin and MspA. The first type of polymers investigated which contained a 3-polythymidine primer and a sequence of comonomers of type (0) showed a strong interaction with the pores that was interpreted as the promotion of ssDNA-primer to the binding with the pore, combined to a high flexibility of the first type of polymers. The polymers which contained alkyne and triazole side chains, were found to have more complex interactions, but interacted for shorter durations with the pore indicating them to be stiffer. The second type of polymers seemed to be clustering in solution due the interaction between side chains, which proved the importance of performing characterization of these molecules in solution using wave scattering in the context of detection and ultimately sequencing.The study of the third result chapter, focused on the dynamics of small oligonucleotides with the aerolysin pore. The interaction of polyadenines (A3, A4, A5) showed complex dynamics and kinetics with pore, which was investigated via analysis of the events pattern. The whole process was found to be governed by two binding sites and energy barriers inside the pore that the molecules have to overcome. These results were combined to a developed kinetic model which allowed a complete description of the binding and translocation (or failure of it) of these polyadenines.The last results chapter described the interaction of bigger polyadenines (A6-A7-A8-A9-A10) with the aerolysin nanopore. The analysis of amplitude of currents of the adenine-induced blocks inside this pore showed an orientation dependent interaction of the molecules with the pore. This orientation dependent interaction started to be apparent for the A7 molecule and became the dominant effect for A9 and A10. Due to the flexibility of ssDNA, this effect is not observed for smaller sized molecules (A6 and below) because of their possibility of reorientation while inside the pore.

La compréhension des mécanismes d’absorption de l’eau, ainsi que l’arrangement moléculaire adopté par le réseau de molécules d’eau lors du confinement à l’échelle nanométrique, est crucial que ce soit pour l’optimisation de plusieurs applications, telles que la production d’énergie propre, la purification et le dessalement de l’eau, ou pour élucider certains processus complexes qui ont lieu dans les systèmes biologiques. Dans le cadre de cette thèse, l’hydratation contrôlée de trois systèmes poreux modèles suivie par spectroscopie infrarouge montre les effets de la nature des surfaces et des limitations stériques qui, en altérant les liaisons hydrogène établies entre les molécules d’eau, déclenche la formation de réseaux atypiques. La brillance de la source de rayonnement synchrotron infrarouge, exploitée par la ligne de lumière AILES au sein du synchrotron SOLEIL, a permis l’étude d’échantillons très absorbants en permettant de mettre en évidence la faible contribution de monocouches de molécules d’eau voire même de chaînes linéaires. L'effet d'une surface hydrophile sur la structure, la densité et la dynamique des molécules d'eau à l'interface a été étudié en mesurant l’absorbance d’une lamelle de Vycor poreux (pores d’environ 5 nm) à l’équilibre avec une pression de vapeur d'eau donnant lieu à la formation d’un réseau d’eau bi-dimensionnel. Les interactions fortes entre l’eau interfaciale et la surface hydrophile provoquent plusieurs transitions structurales du réseau d’eau expliquant ainsi les observations par d’autres techniques. Des similitudes et des différences surprenantes ont été observés entre l’hydratation d’une surface hydrophile et celle d’un système hydrophobe dans des conditions de confinement extrême : les nanotubes de carbone à paroi unique (SWCNTs) ayant un diamètre de quelques Angstrom. Nos mesures ont montré que l’établissement d’un réseau de molécules d’eau unique peut être à l’origine du déplacement rapide des molécules d’eau à l’intérieur des nanotubes de carbone. Enfin, l’étude des mécanismes d’adsorption de la membrane Nafion, membrane modèle dans les piles à combustible à membrane échangeuse de protons (PEMFCs), a permis de clarifier les processus en jeu lors de l’ionisation et la formation d’espèces protonique responsable de la remarquable conductivité ionique propre à cette famille de membranes.Cette méthodologie pourra être étendue à des systèmes plus complexes, tels que les réseaux de molécules d’eau dans les systèmes biologiques. / Understanding the water absorption mechanisms, and the molecular arrangement adopted by the water molecules upon confinement at the nanoscale is crucial both for technological applications, such as clean energy production, purification and desalination of water, and to unveil some of the complex processes occurring in biological systems.In this thesis, the controlled hydration of three porous model systems monitored by infrared spectroscopy shows the effects of surfaces forces and steric limitations that triggers the formation atypical networks by altering the hydrogen bonds established between water molecules. The brightness of the infrared synchrotron radiation source on the AILES beamline at synchrotron SOLEIL allowed to highlight the low contribution coming from water molecules arranged as monolayer or linear chains in highly infrared absorbent matrices. The effect of a hydrophilic surface on structure, density and dynamic of water molecules at interfaces was examined by measuring the absorbance of a porous Vycor slab (pores of about 5 nm) at equilibrium with the water vapor pressure needed to obtain a two-dimensional water network. The strong interfacial interactions between water and the hydrophilic surface cause several structural transitions on the water network elucidating the observations made by other techniques. Surprising similarities and differences are observed between the hydration of a hydrophilic surface and that of a hydrophobic system under extreme confinement: single-wall carbon nanotubes (SWCNTs) having a diameter of several Angstroms. Our measurements have shown that the arrangement of a peculiar water network may be the origin of the enhanced water flux in carbon nanotubes. Finally, the study of the adsorption mechanisms of the Nafion membrane, the benchmark electrolyte in proton exchange membrane fuel cells (PEMFCs), has shed new light on the processes involved in the ionization and the formation of protonic species, accountable for the remarkable ionic conductivity typical of these membranes.This approach may be also extended to more complex systems, such as water molecules networks in biological systems.

Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas. DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient. The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores.