SOL-GEL ROUTES TO MESOPOROUS TUNGSTEN OXIDES WITH MIXED ELECTRON/PROTON CONDUCTIVITY

The present thesis is focused on the development of novel, straightforward sol-gel techniques for the synthesis of highly mesoporous, mixed-conducting tungsten oxide monoliths and powders. Such materials are extremely interesting in view of potential applications for a variety of emerging electrochemical technologies, including electrode design in Polymer-Electrolyte-Membrane Fuel Cells. Both hydrolytic and non-hydrolytic methods are set up. The hydrolytic route is based on a proper steam-treatment as an effective way to control the supply of water molecules to the gelling phase and thus also the oxide formation rate, which is crucial in determining mesoporous features. The non-hydrolytic route is based on a metal halide/alcohol system and affords a variety of mesoporous frameworks. An extended investigation is carried out in order to establish a correlation between alcohol molecular structure and physical properties of final oxide materials. All samples are systematically characterized as to mesoporous properties, chemical composition and electrical properties. Mesoporosity is mainly investigated by means of nitrogen adsorption/desorption analysis, which allows determination of surface area and pore volume/size as well as surface fractal dimension. In particular, the fractal dimension is shown to be a fundamental parameter in controlling and tayloring the mesoporous properties. Additional structural information is obtained from Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and X-Ray Diffraction (XRD). Chemical composition (non-stoichiometry) plays a key role in electron conduction and is studied by X-Ray Photoelectron Spectroscopy (XPS). Finally, electrical properties are subjected to a detailed quantitative inspection by means of Electrical Impedance Spectroscopy (EIS). Electron Conductivity is discussed in terms of hopping-transport models. Proton conductivity takes place in humid conditions according to the Grotthuss mechanism and can be extracted from EIS data by fitting with a proper equivalent circuit. Fractal dimension has a deep influence on proton dynamics and two well-distinct transport regimes are observed for rough and smooth oxide matrices. Based on preparation and processing conditions, the following important values can be achieved: surface area up to 184 m2/g, pore volume up to 0.56 cm3/g, fairly monodisperse pore diameter in the range 3 ÷ 20 nm, electron conductivity up to 20 S/cm and proton conductivity up to 47 mS/cm

Nanopores with Fluid Walls for Characterizing Proteins and Peptides.

Nanopore-based, resistive-pulse sensing is a simple single-molecule technique, is label free, and employs basic electronic recording equipment. This technique shows promise for rapid, multi-parameter characterization of single proteins; however, it is limited by transit times of proteins through a nanopore that are too fast to be resolved, non-specific interactions of proteins with the nanopore walls, and poor specificity of nanopores for particular proteins. This dissertation introduces the concept of nanopores with fluid walls and their applications in sensing and characterization of proteins, disease-relevant aggregates of amyloid-beta peptides, and activity of membrane-active enzymes. Inspired by lipid-coated nanostructures found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to a nanopore such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision, well-defined control of protein transit times, simultaneous multi-parameter characterization of proteins, and an ability to monitor phospholipase D. Using these bilayer-coated nanopores with lipids presenting a ligand, proteins binding to the ligand were captured, concentrated on the surface, and selectively transported to the nanopore, thereby, conferring specificity to a nanopore. These assays enabled the first combined determination of a protein’s volume, shape, charge, and affinity for the ligand using a single molecule technique. For non-spherical proteins, the dipole moment and rotational diffusion coefficient could be determined from a single protein. Additionally, the fluid, biomimetic surface of a bilayer-coated nanopore was non-fouling and enabled characterization of Alzheimer’s disease-related amyloid-beta aggregates. The presented method and analysis fulfills a previously unmet need in the amyloid research field: a method capable of determining the size distributions and concentrations of amyloid-beta aggregates in solution. The experiments presented here demonstrate that the concept of a nanopore with fluid walls enables new nanopore-based assays. In particular, it demonstrates the benefits of this concept for simultaneous, multi-parameter characterization of proteins with a single-molecule method; this technique may, therefore, be well-suited for identification of proteins directly in complex biological fluids. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single-molecule assays and protein characterization.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96049/1/ecyus_1.pd

Resistive Pulse Sensing of Protein Unfolding and Transport in Solid-State Nanopores

Solid-state nanopore sensors have attracted considerable attraction as a tool for solution-based single-molecule studies and have been successfully utilized for characterization of biomolecules such as nucleic acids, proteins, glycans, viruses, etc. Among these, characterization of proteins has been more challenging due to their charge heterogeneity and the complex energy landscape associated with different protein conformations. Presented in this thesis is the fabrication of solid-state nanopores and their application for characterizing proteins and understanding their transport through nanopores. Fabrication of nanometer-sized pores in SixNy membranes was achieved using the conventional controlled dielectric breakdown method as well as a simple modified version of it that resulted in ultra-stable nanopores devoid of legacy issues associated with the conventional nanopores. The noise characteristics of the fabricated nanopores were studied as a function of solution pH, electrolyte type and concentration, applied voltage and pore diameter. SixNy-based solid-state nanopores were used for studying the voltage and pH-induced conformational changes of the human serum transferrin protein and for distinguishing between its two forms – apo (iron-free) and holo (iron-rich). Finally, the transport of protein through nanopores was studied, first in symmetric salt conditions and then in asymmetric salt conditions. Investigating protein transport phenomena in different electrolyte types and concentrations as well as different electrolyte concentration gradients provided valuable insights into the electrokinetic phenomena such as electrophoresis and electroosmosis that govern analyte capture and transport through solid-state nanopores

Computational modeling of biological nanopores

Throughout our history, we, humans, have sought to better control and understand our environment. To this end, we have extended our natural senses with a host of sensors-tools that enable us to detect both the very large, such as the merging of two black holes at a distance of 1.3 billion light-years from Earth, and the very small, such as the identification of individual viral particles from a complex mixture. This dissertation is devoted to studying the physical mechanisms that govern a tiny, yet highly versatile sensor: the biological nanopore. Biological nanopores are protein molecules that form nanometer-sized apertures in lipid membranes. When an individual molecule passes through this aperture (i.e., "translocates"), the temporary disturbance of the ionic current caused by its passage reveals valuable information on its identity and properties. Despite this seemingly straightforward sensing principle, the complexity of the interactions between the nanopore and the translocating molecule implies that it is often very challenging to unambiguously link the changes in the ionic current with the precise physical phenomena that cause them. It is here that the computational methods employed in this dissertation have the potential to shine, as they are capable of modeling nearly all aspects of the sensing process with near atomistic precision. Beyond familiarizing the reader with the concepts and state-of-the-art of the nanopore field, the primary goals of this dissertation are fourfold: (1) Develop methodologies for accurate modeling of biological nanopores; (2) Investigate the equilibrium electrostatics of biological nanopores; (3) Elucidate the trapping behavior of a protein inside a biological nanopore; and (4) Mapping the transport properties of a biological nanopore. In the first results chapter of this thesis (Chapter 3), we used 3D equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at https://github.com/willemsk/phdthesis-tex

Protein separation in ion-exchange membrane partitioned free -flow isoelectric focusing (IEM-FFIEF) system

Ph.DDOCTOR OF PHILOSOPH

Direct Current Electrokinetic Particle Transport in Micro/Nano-Fluidics

Electrokinetics has been widely used to propel and manipulate particles in micro/nano-fluidics. The first part of this dissertation focuses on numerical and experimental studies of direct current (DC) electrokinetic particle transport in microfluidics, with emphasis on dielectrophoretic (DEP) effect. Especially, the electrokinetic transports of spherical particles in a converging-diverging microchannel and an L-shaped microchannel, and cylindrical algal cells in a straight microchannel have been numerically and experimentally studied. The numerical predictions are in quantitative agreement with our own and other researchers\u27 experimental results. It has been demonstrated that the DC DEP effect, neglected in existing numerical models, plays an important role in the electrokinetic particle transport and must be taken into account in the numerical modeling. The induced DEP effect could be utilized in microfluidic devices to separate, focus and trap particles in a continuous flow, and align non-spherical particles with their longest axis parallel to the applied electric field. The DEP particle-particle interaction always tends to chain and align particles parallel to the applied electric field, independent of the initial particle orientation except an unstable orientation perpendicular to the electric field imposed. The second part of this dissertation for the first time develops a continuum-based numerical model, which is capable of dynamically tracking the particle translocation through a nanopore with a full consideration of the electrical double layers (EDLs) formed adjacent to the charged particles and nanopores. The predictions on the ionic current change due to the presence of particles inside the nanopore are in qualitative agreement with molecular dynamics simulations and existing experimental results. It has been found that the initial orientation of the particle plays an important role in the particle translocation and also the ionic current through the nanopore. Furthermore, field effect control of DNA translocation through a nanopore using a gate electrode coated on the outer surface of the nanopore has been numerically demonstrated. This technique offers a more flexible and electrically compatible approach to regulate the DNA translocation through a nanopore for DNA sequencing

Characterization and application of fusogenic liposomes

Conventional drug delivery strategies use the endocytic pathway to introduce biomolecules like proteins, DNA, or antibiotics into living cells. The main disadvantage of endocytic uptake is the quick intercellular degradation of the cargo. Compared to this, a more promising alternative for efficient molecular delivery is the induction of membrane fusion between liposomes and mammalian cells. Therefore special liposomes with extraordinary high fusion efficiency, so-called fusogenic liposomes (FLs), have been developed for such purposes. Due to the complete fusion of the liposomal membrane and the cellular plasma membrane, the cargo molecules can be effectively released into the cell cytoplasm, avoiding their degradation. In the last decade, applications relying on FLs became more and more relevant, however, the exact fusion mechanism is still to be elucidated. Therefore the aims of this work have been to investigate those liposomes and their fusogenicity with living mammalian cells dependent on lipid composition as well as environmental conditions to elucidate the most important factors inducing fusogenic structures within the liposomes. For structural characterization of the liposomes dynamic light and neutron scattering as well as solid state-NMR, freeze-fracture-STEM, Cryo-TEM, and differential scanning calorimetry were applied. Fusion efficiency was investigated by fluorescence microscopy and flow cytometry using Chinese hamster ovary (CHO) cells as an in vitro mammalian cell model. The first results showed that fusogenic liposomes (FLs) need cationic lipids with inverted conical molecular shapes and aromatic components at a distinct concentration as well as a neutral lipid for the best fusion induction. Neutral lipids with long and unsaturated chains and a small head group (e.g., PEs) do not change the liposomal fusion ability while those with saturated short chains and a big head group (e.g., PCs) do, and in most extreme cases revert the uptake mechanism to endocytosis. Additionally, a new application of fusogenic liposomes was established. For the first time, cationic liposomes with high fusion ability were successfully used as carrier particles for the delivery of the radionuclide 131I into mammalian breast cancer cells in vitro. The FLs reached the cancer cells with high efficiency and delivered their cargo into the cell cytoplasm. The control treatment of human red blood cells did not give positive results on fusion, and in this case, the delivery of the cargo was neglectable. These results considered FLs as an appropriate tool for applications in nuclear medicine. Further results showed that as the structural reorganization of the liposomal membrane supply the total required driving force to overcome the energy barrier of the different fusion intermediate steps, like in the case of FLs, changes of the fusion conditions such as temperature, osmolality or ionic concentration of the buffer did not influence the fusion success. In the case of the endocytic liposomes (ELs), buffer conditions played a crucial role in successful fusion, however, fusion efficiency remains infinitesimal under physiological conditions. To elucidate the correlation between efficient membrane fusion and liposomal characteristics, structural investigations of FLs with the best fusion efficiency were also carried out. Here, the simultaneous presence of lipid bilayers and small micelles of around 50 to 100 nm in diameter with high surface curvatures were found. Based on the obtained results, a theoretical mechanism of membrane fusion between FLs and cellular membranes could be proposed. The positively charged lipid is necessary for establishing contact between the two membranes. The micelles are formed by the neutral, phosphoethanolamine, lipids. The lipid bilayer enclosing inverted micelles has a high positive membrane curvature, which is especially favorable for the positively charged lipid molecules. Such curvature stress usually promotes the fusion-stalk formation and subsequent membrane fusion; therefore, the proposed fusion mechanism is called a modified stalk mechanism. Moreover, traces of other three-dimensional (3D) phases with high membrane curvature such us sponge-, inverted hexagonal-, and cubic phases could not be excluded. The present structures are probably metastable precursors, such as a rhombohedral phase, that reduce bilayer stability, which is leading to the pore formation occurring. In comparison to this, ELs formed only lamellar phases shown as non-fusogenic under physiological conditions. These results give rise to the hypothesis that the predominant presence of 3D-like and 3D phases with high membrane curvatures is the most important criterion for efficient membrane fusion induction