This diploma thesis deals with taxonomy independent methods for classification of metagenomic signals, aquired by a MinION sequencer. It describes the formation and character of metagenomic data and already existing methods of metagenomic data classification and their development. This thesis also evaluates an impact of the third generation sequencing techniques in the world of metagenomics and further specialises on the function of the Oxford Nanopore MinION sequencing device. Lastly, a custom method for metagenomic data classification, based on data obtained from a MinION sequencer, is proposed and compared with an already existing method of classification.

Microorganisms exist ubiquitously on Earth, yet their functions and ecological roles remain elusive. Investigating these microbes is accomplished by using culture-dependent and culture-independent methodologies. This study employs both methodologies to characterize: 1) the genomic potential of the novel deep-subsurface bacterial isolate Thermanaerosceptrum fracticalcis strain DRI-13T by combining next-generation and nanopore sequencing technologies and 2) the microbiome of the artificial marine environment for the Hawaiian Bobtail Squid in aquaculture using next-generation sequencing of 16S rRNA gene. Microbial ecology of the deep-subsurface remains understudied in terms of microbial diversity and function. The genomic information of DRI-13T revealed a potential for syntrophic relationships, diverse metabolic potential including prophages/antiviral defenses, and novel methylation motifs. Artificial marine environments housing marine the Hawaiian Bobtail Squid (Euprymna scolopes) contain microorganisms that can directly influence animal and aquaculture health. No studies presently show if bacterial communities of the tank environment correlate with the health and productivity of E. scolopes. This study sought to address this by sampling from a year of unproductive aquaculture yield and comparing the bacterial communities from productive cohorts. Bacterial communities from unproductive samples show less bacterial diversity and abundance coupled with shifts in bacterial composition. Nitrate and pH levels between the tanks were found to be strong influences on determining the bacterial populations of productive and unproductive cohorts.

Nanofabrication techniques produce materials with enhanced physicochemical properties through a combination of nanoscale roughness and the use of chemically diverse polymers which enable advanced applications in separation science (air/water purification), tissue engineering, and biosensing. Since the late 1990’s, electrospinning has been extensively studied and utilized to produce nano- to microfiber meshes with 3D porosity on the gram scale. By combining a high surface area to volume ratio and tunable surface chemistry, electrospinning is a facile platform for generating non-woven polymeric fibers for many biomedical and industrial applications. This thesis describes three applications of electrospun nano- and microfiber meshes spun from both commercially available and novel polymer systems for: 1) oil and water separation after an accidental oil spill; 2) ultraviolet light controlled protein and cell patterning throughout 3-dimensional nanofiber meshes; and 3) novel diagnostic platform by combining electrospun nanofiber meshes with solid state nanopores for enhanced single molecule nucleic acid and protein detection. Each application embodies the philosophy that electrospun materials have the potential to solve a wide variety of problems by simply tuning the physicochemical properties and mesh morphologies towards the design requirements for a specific problem. For example, to solve the problem of recovering crude oil after an oil spill while generating a minimal waste burden, a hydrophobic and biodegradable microfiber mesh was designed to repeatedly separate oil and water and naturally biodegrade after use. In order to solve the problem of spatiotemporal placement of cells within a 3-dimensional tissue engineering construct, an ultraviolet light activated mesh was designed to transition from hydrophobic (water impermeable) to hydrophilic (water permeable) upon exposure to ultraviolet light facilitating protein and cell patterning. Finally to address two problems with single molecule solid state nanopore biosensors, namely rapid nucleic acid translocation rates and limited protein identification capabilities, a new biosensor platform was developed based on two novel polymeric systems which were synthesized and electrospun into high surface area nanofiber mesh coatings. / 2018-02-17T00:00:00Z

abstract: My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale. My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule. My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

No description available.

Drying and evaporation from nanoscale conduits are two ubiquitous phenomena found in nature. As these two nanoscale liquid-vapor phase change phenomena are significantly “accelerated” compared with the corresponding ones at micro- and macro-scales, various industrial applications, including oil recovery, electronic cooling, membrane desalination, and energy harvesting, have been developed. Despite their important implications, the fundamental mechanisms for these two accelerated phase-change processes have not been completely understood. For drying, it is widely accepted that liquid corner flow and film flow could significantly enhance mass transport in microscale conduits other than the sole contribution by vapor diffusion. However, it is unclear if the same principles apply to smaller scales and if the vapor diffusivity will change at the nanoscale. For evaporation, the evaporation kinetics at the nanoscale interface, rather than liquid/vapor transport toward/from the interface, determine the ultimate transport limit, which can be significantly higher than the classical prediction derived under quasi-equilibrium evaporation conditions. Still, the contributions to such enhanced kinetically limited evaporation remain unclear. This thesis aims to answer these unsolved questions by conducting systematic experimental studies on drying and evaporation from single nanochannels and nanopores. We used state-of-art fabrication to create close-end 2D nanochannels with heights from 29 to 122 nm and measure water drying in such channels using an optical microscope. Combining with the channel confinement study and relative humidity study, we decoupled the individual contributions from vapor and liquid transport to the drying and extracted the water vapor diffusivity in nanochannels. We also developed a hybrid nanochannel-nanopore design to achieve and measure kinetically limited evaporation flux from silicon nitride nanopores and graphene nanopores with pore diameters ranging from 24 to 347 nm. Our results show that the evaporation flux increases with the decreasing diameter for both types of nanopores. Furthermore, graphene nanopores overall exhibit higher evaporation fluxes than silicon nitride nanopores with similar diameters. We attribute the diameter-dependent evaporation flux to the diameter-dependent hydronium ion concentration in silicon nitride nanopores and the edge-facilitated evaporation in graphene nanopores, respectively. We expect this work to advance our understanding of nanoscale fast drying and evaporation and provide design guidance for novel nanoporous membrane evaporators.

Nanofluidic devices have been of great interest due to their applications in variety of fields, including energy conversion and storage, water desalination, biological and chemical separations, and lab-on-a-chip devices. Although these applications cross the boundaries of many different disciplines, they all share the demand for understanding transport in nanoscale conduits. In this thesis, different elusive aspects of molecule and particle transport through nanofluidic conduits are investigated, including liquid and ion transport in nanochannels, diffusion- and reaction-governed enzyme transport in nanofluidic channels, and finally translocation of nanobeads through nanopores. Liquid or solvent transport through nanoconfinements is an essential yet barely characterized component of any nanofluidic systems. In the first chapter, water transport through single hydrophilic nanochannels with heights down to 7 nm is experimentally investigated using a new measurement technique. This technique has been developed based on the capillary flow and a novel hybrid nanochannel design and is capable of characterizing flow in both single nanoconduits as well as nanoporous media. The presence of a 0.7 nm thick hydration layer on hydrophilic surfaces and its effect on increasing the hydraulic resistance of the nanochannels is verified. Next, ion transport in a new class of nanofluidic rectifiers is theoretically and experimentally investigated. These so called nanofluidic diodes are nanochannels with asymmetric geometries which preferentially allow ion transport in one direction. A nondimensional number as a function of electrolyte concentration, nanochannel dimensions, and surface charge is derived that summarizes the rectification behavior of this system. In the fourth chapter, diffusion- and reaction-governed enzyme transport in nanofluidic channels is studied and the theoretical background necessary for understanding enzymatic activity in nanofluidic channels is presented. A simple analytical expression that describes different reaction kinetics is derived and confirmed against available experimental data of reaction of Trypsin with Poly-L-lysine. Finally, in the last chapter translocation of nanobeads through synthetic nanopores is experimentally investigated using resistive pulse sensing. The emphasis is placed on elucidating the effect of nanobead size on the translocation current and time. The key goals pursued in this study are multiplex detection of different nanobead sizes in a mixture of nanobeads as well as determining the concentration of each component. This problem other than its fundamental significance paves the way for developing new biosensing mechanisms for detection of biomolecules. This thesis further explores the molecule and particle transport in nanoscale conduits and serves for better characterization and development of nanofluidic devices for various applications.

This thesis investigates DNA dynamics and translocation through nanopores using Brownian dynamics (BD) simulations, offering insights into sequencing technologies, DNA marker detection, and accurate barcoding utilizing solid-state nanopore platforms. First, we in silico study the intricate process of capture and translocation in a single nanopore. Our simulation reveals a high probability of hairpin loop formation during the capture process. However, attaching a charged tag to one end of DNA improves multi-scan rates and enhances unidirectional translocations. We use modulating voltage biases to multi-scan a lambda-phage dsDNA with oligonucleotide flap markers (tags) through a single and double nanopore system. Our study shows that the bulkier tags introduce velocity variations along the chain length that lead to potential inaccuracies in genetic distance (barcode) estimations. We introduce an interpolation scheme that incorporates both the tag velocities and the average velocity of the chain to improve barcode precision. Subsequently, we include bead and side-chain tags to explain asymmetric dwell time distributions as observed in double nanopore experiments. Our findings indicate that local charge interactions between tags and the nanopore's electric field introduce dwell time asymmetries that can be used for discriminating tags based on their net charges. Finally, we obtain the current blockades of the molecular motifs attached to a dsDNA using electrokinetic Brownian dynamics (EKBD) simulation. Our simulation demonstrates that divalent salt reduces the translocation speed, facilitating precise measurement of the motif's dwell time. Finally, we formulate a volumetric ansatz to construct current blockade diagrams from the ordinary BD simulation in a computationally efficient way and show that using simple scale factors, these volumetric blockades can be mapped accurately to the ionic current blockades obtained from more expensive EKBD simulation. Our studies present comprehensive explorations of DNA translocation and barcoding methods in solid-state nanopores, demonstrating their utility in nanopore sequencing and nanobiotechnology

Room-temperature ionic liquids (RTILs) and their derivatives are promising electrolytes for electrochemical devices including supercapacitors. Understanding the behavior of RTILs in these devices is critical for improving their performance. The energy density of supercapacitors can be improved greatly by using RTILs as electrolytes and nanoporous carbon as electrodes, but the mechanism of the charge storage using these materials is not well understood. In this dissertation, the diffusion and charging dynamics of RTILs in nanopores are studied. The results show that ion packing typically plays the most important role in ion diffusion. The study also demonstrates that the cyclic charging and discharging of a pore can exhibit a number of interesting features (e.g., sloshing of ionic charge along the pores during cyclic scans), which help explain experimental observations such as the negligible contribution of co-ions to charge storage at high scan rates. Solid electrolytes with both high ionic conductivities and excellent mechanical strength are needed in many electrochemical devices. The invention of ion gels featuring aligned polyanions immersed inside RTILs has shown promise in meeting this demand, but the mechanism behind their superior mechanical strength remains elusive. Using molecular simulations, it is discovered that the high elastic moduli of model PBDT ion gels originate from the RTIL-mediated interactions between the polyanions. This insight is useful for future design of ion gels to improve their transport and mechanical properties. / Ph. D.

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.