Continuous progress in the nanotechnology field has allowed for the emergence of powerful, nanopore-based detection technology. Solid-state nanopores were developed for next-generation sequencing and single-molecule detection. They are advantageous over their biological counterpart because they offer robustness, stability, tunable pore size and the ability to be integrated within a microfluidic device. With all of these attractive attributes, solid-state nanopores are a top contender for point-of-care diagnostic technologies. However, hindering their performance is an inability to distinguish between small molecules, pore-clogging, and the detection rate's dependence on sample concentration. The concentration-dependent detection rate becomes particularly evident at low sample concentrations (<1 nM), sometimes taking hours for the nanopore to sense a single molecule because of diffusion. The inability to distinguish between small molecules can be addressed using DNA nanostructures; however, pore-clogging and variable detection rates hinder its potential in a clinical setting. This thesis proposes a microfluidic device design and methodology that seeks to mitigate pore-clogging and improve the detection rate for dilute samples. DNA coated microbeads will create a bead column within the microfluidic device and confine the target molecules to an extremely small (20 nL) volume. The sample can be washed, ridding the contaminants, and eluted on-chip, so the sample is purified and concentrated, affording a more reliable sensing performance. First, a magnetic microbead DNA assay was optimized off-chip, and the capture and release efficiencies were monitored using a Biotek™ Epoch™ 2 spectrophotometer (Chapter 2). Next, a novel microfluidic device design was optimized and validated to ensure precise sample manipulation (Chapter 3). Finally, the microbead assay was incorporated into the microfluidic device for sample concentration (Chapter 4). Fluorescence microscopy results suggest successful DNA elution from the microbeads within the microfluidic device, allowing for a 28.5 X concentration increase. This platform shows promise for sample preconcentration by reducing the starting DNA sample volume of 25 µL to 20 nL, which could improve the speed of solid-state nanopore sensing.

The Nanopore Detector using á-hemolysin channel transcribes kinetics of a single molecule along the nanometer-scale pore. The transcribed data is represented by electrical measurements. We present accurate and computationally inexpensive tools to analyze single molecule kinetics. The HMM-EM level projection method de-noises data, retaining the transitions with very high precision. This approach doesn't require input number of levels. Another advantage is the minimal tuning required. The levels are then identified using Finite State Automata (FSAs). Spike Detector algorithm analyzes spikes characterizing behavior of molecule in pore. No commercial tools available are capable of analyzing spikes in presence of noise. The formulation of HMM-EM, FSAs and Spike Detector together provides a robust method for analysis of channel current data. Application of these methods is described for Vercoutere channel blockade dataset which contains signals of radiated and non-radiated molecules. The tools developed were used successfully to differentiate between these two molecules.

The aim of DNA sequencing is to obtain the order of DNA composition comprising the base pairs A (adenine) T (thymine), and C (cytosine) G (guanine). The fast development of DNA sequencing technology allows us to better understand the relationships among diseases, inheritance, and individuality. Solid state nanopores have been recommended as the next generation platform for DNA sequencing due to its low-cost and high-throughput. In particular, nanopores fabricated from graphene sheets are extremely thin and structurally robust and have been extensively used in DNA detection in recent years. In DNA sequencing, the translocation of a DNA molecule through a nanopore is known to be a very complicated issue and is affected by many factors, such as ion concentration, thickness of the nanopore, and the nanopore diameter. The technique of molecular dynamic simulations has been a complementary tool to study DNA translocation through nanopores.       In this thesis, I summarize my work of computational studies of DNA sequencing using graphene nanopores. These studies include: DNA translocation through single-layer graphene nanopores of different diameters under conditions of various ion concentrations and applied voltages; DNA translocation through multilayer graphene nanopores varied from a single to a few layers; pulling out single strand DNA molecules from small graphene nanopores of different geometries. The major contributions of this work include: 1. Effects of bias voltage on DNA translocation time were investigated leading to the insight that lower applied voltages can extend the time of DNA translocation through monolayer graphene nanopores. The effect of salt concentration on the corresponding ionic current was studied. At a low ionic concentration (&lt; 0.3M), the current increases as DNA translocates through a nanopore. However, at a high ionic concentration (&gt;0.5M), the current decreases as DNA translocates through the nanopore. A theoretical model was proposed to explore the relationship between the current and the occupied nanopore area. We demonstrated that the DNA translocation time can be prolonged by narrowing the diameter of a nanopore properly and the reduction of the blockade current depends on the ratio of the unoccupied nanopore area to the total nanopore area. 2.  DNA translocation through multilayer graphene nanopores was studied by molecular dynamics simulations with the aim to achieve single-base resolution. We show that the DNA translocation time can be extended by increasing the graphene layers up to a moderate number (7) and that the current in DNA translocation undergoes a stepwise change upon DNA going through an multi-layer graphene (MLG) nanopore. A model was built to account for the relationship between the current change and the unoccupied volume of the MLG nanopore. We demonstrate that the blockade current is closely related to the unoccupied volume. The dynamics of DNA translocation depends specifically on the interaction of nucleotides with the graphene sheet. Thus, our study indicates that the resolution of DNA detection can be improved by increasing the number of graphene layers in a certain range and by modifying the surface of graphene nanopores. 3. The effect of graphene nanopore geometry on DNA sequencing has been assessed by steered molecular dynamics simulations. DNA fragments including A, T, C, G and 5-methylcytosine (MC) were pulled through graphene nanopores of different geometries with diameters down to ~1nm by steered molecular dynamics simulations. We demonstrated that the bases (A, T, C, G, and MC) can be indentified in single-base resolution by the characteristic force peak values in a circular graphene nanopore but not in graphene nanopores of other geometries. Symmetric nanopores are thus better suited to DNA sequence detection via force curves than asymmetric nanopores. This implies that the graphene nanopore surface should be modified as symmetric as possible to sequence DNA by an atomic force microscope or optical tweezers. This helps us to understand low-cost and time-efficient DNA sequencing in narrow nanopores. 4. The translocation time for different nucleotides to pass through graphene nanopores with certain diameters was investigated. It was found that the translocation times are different for different bases under a low electric field. The results indicate that DNA can be sequenced by the translocation time to pass through a graphene nanopore. 5. Inspired by the structure of K+ channel proteins, a series of oxygen doped graphene nanopores of different size were designed to discriminate the transport of K+ and Na+ ions. The results indicate that the ion selectivity of such biomimetic graphene nanopores can be simply controlled by the size of the nanopore.  Compared to K+, the smaller radius of Na+ leads to a much higher free energy barrier in the nanopore of a certain size. / <p>QC 20141212</p>

abstract: In the last few years, significant advances in nanofabrication have allowed tailoring of structures and materials at a molecular level enabling nanofabrication with precise control of dimensions and organization at molecular length scales, a development leading to significant advances in nanoscale systems. Although, the direction of progress seems to follow the path of microelectronics, the fundamental physics in a nanoscale system changes more rapidly compared to microelectronics, as the size scale is decreased. The changes in length, area, and volume ratios due to reduction in size alter the relative influence of various physical effects determining the overall operation of a system in unexpected ways. One such category of nanofluidic structures demonstrating unique ionic and molecular transport characteristics are nanopores. Nanopores derive their unique transport characteristics from the electrostatic interaction of nanopore surface charge with aqueous ionic solutions. In this doctoral research cylindrical nanopores, in single and array configuration, were fabricated in silicon-on-insulator (SOI) using a combination of electron beam lithography (EBL) and reactive ion etching (RIE). The fabrication method presented is compatible with standard semiconductor foundries and allows fabrication of nanopores with desired geometries and precise dimensional control, providing near ideal and isolated physical modeling systems to study ion transport at the nanometer level. Ion transport through nanopores was characterized by measuring ionic conductances of arrays of nanopores of various diameters for a wide range of concentration of aqueous hydrochloric acid (HCl) ionic solutions. Measured ionic conductances demonstrated two distinct regimes based on surface charge interactions at low ionic concentrations and nanopore geometry at high ionic concentrations. Field effect modulation of ion transport through nanopore arrays, in a fashion similar to semiconductor transistors, was also studied. Using ionic conductance measurements, it was shown that the concentration of ions in the nanopore volume was significantly changed when a gate voltage on nanopore arrays was applied, hence controlling their transport. Based on the ion transport results, single nanopores were used to demonstrate their application as nanoscale particle counters by using polystyrene nanobeads, monodispersed in aqueous HCl solutions of different molarities. Effects of field effect modulation on particle transition events were also demonstrated. / Dissertation/Thesis / Ph.D. Electrical Engineering 2011

Cytokines are small-molecule signaling proteins involved in cell-cell regulation. The detection of low-abundance cytokines is challenging since the currently available techniques are limited by sensitivity and are time-consuming. Nanopore sensing is an emerging technique in nanotechnology that is catalyzing key breakthroughs in many areas, including the analysis and study of proteins at the single-molecule level. Solid-state nanopore sensing has the advantage of analyzing small copy numbers of biomolecules, such as DNA, with high throughput. However, protein detection using nanopores is still in in infancy because the mechanisms of native protein translocation inside the solid-state nanopore are highly complicated. The goal of this project is to develop a novel solid-state nanopore device for identification and quantification of cancer cytokines directly from cell culture. Vascular endothelial growth factor (VEGF) is chosen as a model cytokine due to its high abundance in cancerous tissue, and its well-characterized molecular structure. Firstly, we used a nanopore sensor to monitor individual VEGF proteins in solution while simultaneously obtaining tertiary and quaternary structural information. Next, we used the translocation signature to identify VEGF secreted directly from the culture media of the breast cancer cell line. A series of DNA and RNA aptamers was screened to selectively bind to secreted VEGF, enhancing the detection rate and creating a unique translocation signature for easy protein discrimination. Finally, we integrated the nanopore with a hard microfluidic device designed to facilitate the on-chip sample preparation prior to nanopore sensing. This nanopore-microfluidic device may allow scientists and clinicians to directly detect biomarkers secreted from a small population of cultured cells, which would revolutionize cancer diagnostics and prognostics. / 2020-10-22T00:00:00Z

Solid-state nanopores are an emerging class of single-molecule detectors that provide information about molecular identity via the analysis of transient fluctuations in the ionic current flowing across a nanoscale pore in a thin membrane. The transport of biomolecules across a pore is a key step in nanopore-based sensing of DNA, RNA and proteins. The dynamics of biomolecular transport are complex and depend on the strength of many interactions, which can be tuned with temperature. However, temperature is rarely controlled during solid-state nanopore experiments because of the added electrical noise from the temperature control and measurement systems, greatly reducing the signal-to-noise ratio when detecting individual molecules. So far, the use of electric-based heating and cooling strategies has limited the recording bandwidth to the kHz range, restricting the studies to long polymers translocating via the pore relatively slowly. Yet, many molecules translocate through the pore orders of magnitude faster. This research presents the development and testing of an instrument to allow low-noise electrical recording of nanopore signals at MHz bandwidth as a function of temperature. Initial experiments using this custom-built instrument for the study of linear DNA polymers confirm previously observed translocation behaviours, while providing a higher temporal resolution. Overall results show that high-speed nanopore experiments are possible while controlling the temperature up to 70 °C, opening up exciting opportunities to study the unfolding of proteins toward single-molecule protein sequencing and the passage of DNA nanostructures for different bioassays. Future work will focus on realizing microfluidic flow cells and nanopore performance at higher temperature for longer recording times.

In this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables, the size of liposomes is found to be one of the dominating parameters in liposome based drug delivery, and the correlation between liposome size and delivery efficiency is discussed. To help improving the size evaluation ability, a few mainstream methods for liposome size detection and measurements are reviewed. As a reliable and accessible alternative method for liposomes detection, the resistive pulse method is introduced and the measurement on liposomes size change upon pH gradient was performed using this method. With our current liposome composition, we found the size increases as environmental pH increases. Further investigation is performed with vesicular pH=6, 7, and 8, respectively. Lastly, the stability of the small unilamellar vesicles (SUV) was studied via resistive pulse method, by monitoring the size change of 50nm liposomes as function of time. A significant size change in freshly prepared 50nm liposomes is recorded. This information will provide invaluable knowledge for targeting tumor with tight tissues, where small size liposomes are needed.

Transmembrane protein ion channels can regulate intercellular transport in response to external stimulus, playing a vital role in diverse physiological functions. Replicating such stimulus-responsive behaviors in the artificial counterparts, e.g. solid-state nanopores, is of great interest in a variety of cross-disciplinary studies and applications, yet has remained challenging due to complicated structures of naturally occurring protein channels and anomalous transport phenomena of the nanoscale fluid. Current stimulus-responsive solid-state nanopores are achieved by employing functional materials and/or geometrical/surface charge asymmetry but suffer from low sensitivity, slow response, and limited reversibility. To tackle the existing challenges, this thesis investigates electromechanical coupled transport phenomena in a new type of stimulus-responsive nanopores, i.e., nanoparticle-blocked nanopores, and their potential applications in gating and sensing. The first part of this thesis describes a bio-inspired liposome-enabled nanopore gating strategy inspired by the ''ball-and-chain'' inactivation mechanism in voltage-gated protein ion channels. By manipulating the position of the liposome nanoparticles around the nanopore, we demonstrate an electromechanically gated nanopore with rapid, reversible, and complete gating response, which allows unprecedented spatial and temporal control of ion/chemical transport across the nanopore. In the second part of the thesis, we report an ultra-mechanosensitive ion transport across the single nanopore blocked by the rigid nanoparticles. The observed pressure-suppressed ion conduction partially mimics the behavior of stretch-inactivated ion channels and is rationalized with mechanical-induced particle motion. Finally, in the third part of the thesis, we further utilize the mechanosensitive ion conduction in nanoparticle-blocked nanopores to develop a nanopore-based platform for mechanical characterization of single nanoparticles. This new platform overcomes the limitations of current characterization techniques and provides an alternative nano-mechanical characterization approach in an efficient and cost-effective manner. We expect this work to provide a convenient platform to achieve natural stimulus-responsive functionalities as well as to develop emerging applications in drug delivery, biosensing, single-molecule manipulation, and ionic-based computation and storage. / 2024-01-25T00:00:00Z

Injecting CO2 into unconventional reservoirs to enhance oil recovery has been widely studied due to its potential to improve the profitability of these reservoirs. CO2 Huff-n-Puff is emerging as a promising method, but exploiting its full potential is challenging due to difficulties in optimizing its operations. The latter arises from the limited understanding of CO2 and oil transport in unconventional reservoirs. This dissertation used molecular dynamics simulations to study the storage and transport of oil and CO2 in unconventional reservoirs in single nanopores. The first study examined the modulation of oil flow in calcite pores by CO2. It is discovered that CO2 molecules adsorb strongly on calcite walls and can change decane permeability through 8 nm-wide pores by up to 30%. They impede decane flow at moderate adsorption density but enhance flow as adsorption approaches saturation. The second study investigated the CO2 transport in 4 nm-wide calcite pores during the soaking phase of Huff-n-Puff operations. CO2 entering the pore can become adsorbed on pore walls and diffuse on them or diffuse as free CO2 molecules. The accumulation of CO2 follows a diffusion behavior with an effective diffusivity ~50% smaller than bulk CO2. Two dimensionless groups are proposed to gauge the importance of surface adsorption and diffusion in CO2 storage and transport in nanopores. The third study examined the extraction of decane initially sealed in a 4 nm-wide calcite pore through exchange with CO2 and CH4 in a reservoir. The CO2-decane exchange is significantly driven by the evolution of adsorbed oil and gas initially, but a transition to dominance by free oil and gas occurs later; for CH4-decane exchange, the opposite occurs. The net gas accumulation and decane extraction follow the diffusive law, but their effective diffusivities do not always align well with the self-diffusion coefficients of CO2, CH4, and decane in the nanopore. The three studies identified the essential roles of gas/oil adsorption in their net transport in nanopores and, thus, unconventional reservoirs. Delineating these roles and formulating dimensionless groups to gauge their importance help develop better models for enhanced oil recovery from unconventional reservoirs by CO2 injection. / Doctor of Philosophy / Unconventional reservoirs are hydrocarbon-bearing formations with ultralow permeabilities, and they have emerged as a critical source of liquid petroleum production in the United States over the past decade. However, because oil is trapped in nanoscale pores in these reservoirs, the oil recovery rate is low. Therefore, many methods have been developed to enhance the oil recovery from unconventional reservoirs. One of the popular methods is to inject gas into reservoirs to enhance oil recovery. Improving this method's efficacy requires a fundamental understanding of the thermodynamic and transport phenomena underlying its operation is needed. This dissertation used molecular dynamics simulations to study the storage and transport of oil and CO2 in unconventional reservoirs at the single nanopore scale. Three series of studies have been performed to elucidate how CO2 modulates the flow of oil inside nanopores, how CO2 enters a nanopore filled with oil, and how oil is extracted from the nanopore by the ingression of CO2. These studies showed that when CO2 molecules adsorb strongly on a nanopore's walls, they can either enhance or impede the permeation of oil through the pore. The ingression of CO2 into an oil-filled nanopore and the concurrent oil extraction can be described by the same equation for the conduction of heat in one-dimensional objects. The CO2 ingression and oil extraction rates are heavily affected by the adsorption of CO2 and oil on the nanopore's walls. These results highlight the important effects of surface adsorption on the storage and transport of gas and oil in nanopores and, thus, unconventional oil reservoirs. Incorporating these effects into oil recovery models will improve their predictive power, and thus help model-guided optimization of oil recovery.

Even under the best management strategies contemporary crops face yield losses from diverse threats such as, pathogens, pests, and environmental stress. Adding to this management challenge is that under current global climate projections these impacts are predicted to become even greater. Natural genetic variation, long used by traditional plant breeders, holds great promise for adapting high performing agronomic lines to these stressors. Yet, efforts to bolster crop plant resilience using wild relatives have been hindered by time consuming efforts to develop genomic tools and/or identify the genetic basis for agronomic traits. Thus, increasing crop plant resilience requires developing and deploying approaches that leverage current high-throughput sequencing technologies to more rapidly and robustly develop genomic tools in these systems. Here we report the integration of bioinformatic and statistical tools to leverage high-throughput sequencing to 1) develop a machine learning approach to determine factors impacting transcriptome assembly and quantitatively evaluate transcriptome completeness, 2) dissect complex physiological pathway interactions in Solanum pimpinellifolium under combined stresses—using comparative transcriptomics, and 3) develop a genome assembly pipeline that can be deployed to rapidly assemble a more contiguous genome, unraveling previously hidden complexity, using Phytopthora capsici as a model. As a result, we have generated strategic guidelines for transcriptome assembly and developed an orthologue and reference free, machine learning based tool "WWMT" to quantitatively score transcriptome completeness from short read data. Secondly, we identified "hub genes" and describe genes involved with "cross-talk" between drought and herbivore stress response pathways. Finally, we demonstrate a protocol for combining long-read sequencing from the Oxford Nanopore Technologies MinION, and short-read data, to rapidly assembly a cost-effective, contiguous and relatively complete genome. Here we uncovered hidden variation in a well-known plant pathogen finding that the genome was 92% bigger than previous estimates with more than 39% of duplicated regions, supporting a hypothesized recent whole genome duplication in this clade. This community resource will support new functional and evolutionary studies in this economically important pathogen. / Doctor of Philosophy / Meeting the food production demands of a burgeoning population in a changing environment, means adapting crop plants to become more resilient to environmental stress. One of the greatest barriers to understanding and predicting crop responses to future environmental change is our poor understanding of the functional and genomic basis of stress resistance traits for contemporary crops. This impediment presents a barrier for rapid crop improvement technologies, such as, gene editing or genomic selection, that is only partially overcome by generating large amounts of sequencing data. Here we need tools that allow us to process and evaluate huge amounts of data generated from next generation sequencing studies to help identify genomic regions associated with agronomic traits. We also need technical approaches that allow us to disentangle the complex genetic interactions that drive plant stress responses. Here we present work that used statistical analysis and recent advances of artificial intelligence to develop a bioinformatic approach to evaluate genomic sequencing data prior to downstream analyses. Secondly, we used a reductionist approach to filter thousands of genes to key genes associated with combined stress responses (herbivory and drought), in the most widely used vegetable in the world, tomato. Finally, we developed a method for generating whole genome sequences that is low-cost and time sensitive and tested it using a well-known plant pathogen genome, wherein we unraveled significant hidden complexity. Overall this work provides community-wide genomic tools and information to promote crop resilience.