Doctor of Philosophy / Department of Chemical Engineering / Peter H. Pfromm / Characterization of the interactions of hydrogen with catalytic metal surfaces and the mass transfer processes involved in heterogeneous catalysis are important for catalyst development. Although a range of technologies for studying catalytic surfaces exists, much of it relies on high-vacuum conditions that preclude in-situ research. In contrast, atomic force microscopy (AFM) provides an opportunity for direct observation of surfaces under or near actual reaction conditions. Tapping-mode AFM was explored here because it expands AFM beyond the usual topographic information toward speciation and other more subtle surface information. This work describes using phase-angle data from tapping-mode AFM to follow the interactions of hydrogen with palladium. Both gas-solid and liquid-solid interfaces were studied. Real-time AFM phase-angle data allowed for the observation of multiphase mass transfer to and from the surface of palladium at atmospheric pressure and room temperature without the need for complex sample preparation. The AFM observations were quantitatively benchmarked against and confirm mass transfer predictions based on bulk hydrogen diffusion estimates. Additionally, they support recent studies that demonstrate the existence of multiple hydrogen states during interactions with palladium surfaces.

Tissue cells exhibit varying responses according to the stiffness of their extracellular matrix (ECM). The mechanism of this stiffness sensing is not fully understood; however, it is known that cells probe stiffness by applying intracellular force to the ECM via integrin-mediated focal adhesions. The bonds between integrins and ECM have been described as “catch bonds�, and it is unclear how ECM viscoelasticity affects these bonds. We have observed the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the dissociation force of individual integrin-ligand bonds of 3T3 fibroblasts on collagen-coated polyacrylamide gels using atomic force microscopy. Results show that integrins exhibit higher rates of activation on stiff substrates. Furthermore, increased matrix stiffness results in the occurrence of larger, multi-bond dissociation events, which suggests that substrate stiffness may affect the cellular response by promoting integrin clustering as well as by modulating the maximum possible force between individual integrins and the ECM.

The necessity to explore nanoscopic systems is ever increasing in the world of science and technology. This evolving need to study such physically small systems demands new experimental techniques and methodologies. Atomic force microscopy (AFM) is a versatile technique that can overcome many nanoscopic size limitations. AFM has been utilized in the world of nanotechnology to study physiochemical properties of particles, materials, and biomolecules through characterization of morphology, electrical and mechanical properties, binding interactions, and surface tension, among others. The work discussed herein is largely a report of several novel AFM methodologies that were developed to allow new characterization techniques of individual submicrometer particles and single biomolecular interactions. The effects of atmospheric aerosols on the radiative budget of the earth and climate are largely unknown. For this reason, characterizing the physiochemical properties of aerosols is vital. Since the particles that have relatively long lifetimes in the atmosphere are smaller than one micrometer in size, high resolution microscopy techniques are required to study them. AFM is a suitable technique for single particle studies because it has nanometer spatial resolution, can perform experiments under ambient pressure and variable relative humidity and temperature. These advantages were utilized here and AFM was used to study morphology, organic volume fraction, water uptake, and surface tension of nascent sea spray aerosol (SSA) particles as well as laboratory generated aerosols composed of relevant chemical model systems. The morphology of SSA was found, often times, to be composed of core-shell structure. With complementary microscopy techniques, the composition of the core and the shell was found to be inorganic and organic in nature, respectively. Novel methodology to measure water uptake and surface tension of single substrate deposited particles with AFM was established using chemical model systems. Furthermore, these methodologies were employed on nascent chemically complex SSA particles collected from a biologically active oceanic waveflume experiment. Finally, phase imaging was used to measure organic volume fraction on a single particle basis and was correlated with biological activity. Overall, this suite of single (submicrometer) particle AFM analysis techniques have been established, allowing future systematic studies of increasing complexity aimed at bridging the gap between the simplicity of laboratory generated particles and the complexity of nature. Another nanotechnology topic of interest is studying single biomolecular interactions. Virtually every biological process involves some amount of minute forces that are required for the biomolecular system to function properly. For example, there are picoNewton forces associated with enzymatic motions that are important for enzyme catalysis. The AFM studies reported here use a model enzyme/drug system to measure the forces associated with single molecule adhesion events. Escherichia Dihydrofolate Reductase (DHFR) is a target of cancer therapeutic studies because it can be inhibited by drugs like methotrexate (MTX) that are structurally similar to the natural folate binder but have much higher binding affinity. One of the obstacles of single molecular recognition force spectroscopy (MRFS) studies is the contribution of non-specific forces that create a source of uncertainty. In this study, DHFR and MTX are bound to the surface and the AFM tip, respectively, using several different linking molecules. These linking molecules included polyethylene glycol (PEG) and double stranded DNA (dsDNA) and the distribution of forces was compared to scenarios were a linker was not employed. We discovered that dsDNA and PEG both allow identification and removal of non-specific interaction forces from specific forces of interest, which increases the accuracy of the measurement compared to directly bound constructs. Traditionally, the linker of choice in the MRFS community is PEG. Here, we introduce dsDNA as a viable linker that offers more rigidity than PEG, which may be desirable in future molecular constructs. The majority of the work and data presented in this dissertation supports the establishment of new AFM methodologies that can be used to better explore single biomolecular interactions and individual submicrometer particles on the nanoscale.

Since the invention of the atomic force microscope (AFM), this technology has had profound implications in the study of material science and molecular biology. The ability to visualize and perform quantitative analysis of the nanoscale properties of surfaces has provided great insights into these nanoscale landscapes. The present dissertation manuscript exploits this technology for the measurement of charge transport through molecular films and the development of combinatorial force microscopy. Firstly, this work probed, for the first time, charge transport through molecular films derived from diazonium salts grafted to carbon electrodes using conductive atomic force microscopy. We found the charge transport properties of a molecular junction are dependent upon the chemical structure of the molecular film. We also investigated the effect of molecular film compression and deformation has on charge transport. In this, we observed increases in current densities associated with increases in applied load to the molecular film. Furthering these initial findings, PPF/NAB/Cu molecular junctions were fabricated having junction sizes ranging between micro-scale and nanoscale. The charge transport experiments reveal an agreement of electron transport properties between the metal deposited PPF/NAB/Cu junction and a PPF/NAB/Cu AFM tip junction at an applied load of approximately 60nN. This form of molecular layer charge transport control may potentially open new horizons for integration of molecular films into the microelectronics industry. This dissertation manuscript also describes the development of the quantitative interrogation opposing chemical libraries involved in combinatorial inverted atomic force microscopy. Tipless cantilevers were patterned with chemically modified nanorods. These modified nanorods were then used as chemical identifiers during a combinatorial force microscopy experiment and for the first time 16 interactions were monitored within one experiment in a continuous medium. Thus, providing excellent for the validation that combi-AFM is a truly quantitative high-throughput technology.

A number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin. Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence. Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner. We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity. Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death. These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.

Advances in several research areas increase the need for more sophisticated fabrication techniques and better performing materials. Tackling this problem from a bottom-up perspective is currently an active field of research. The bottom-up fabrication procedure offers sub-nanometer accurate manipulation. At this time, candidates to achieve nanomanipulation include chemical (self-assembly), biotechnology methods (DNA-based), or using controllable physical forces (e.g. electrokinetic forces, mechanical forces). In this thesis, new methods and techniques for mechanical nanomanipulation using probe force interaction are developed. The considered probes are commonly used in Atomic Force Microscopes (AFMs) for high resolution imaging. AFM-based mechanical nanomanipulation will enable arranging nanoscale entities such as nanotubes and molecules in a precise and controlled manner to assemble and produce novel devices and systems at the nanoscale. The novelty of this research stems from the development of new modeling of the physics and mechanics of the tip interaction with nanoscale entities, coupled with the development of new smart cantilevers with multiple degrees of freedom. The gained knowledge from the conducted simulations and analysis is expected to enable true precision and repeatability of nanomanipulation tasks which is not feasible with existing methods and technologies.

Alzheimer's Disease (AD) is a neurodegenerative disorder that is prevalent among the elderly population. Aß protein has been heavily implicated in the pathogenesis of AD. This protein in its fibrillar form is a major component in the senile plaques that form on neuronal cellular membranes during the course of AD. Despite substantial efforts the exact mechanism of Aß toxicity towards a cell membrane is not well-understood. The determination of this mechanism, however, is of utmost importance, since the membrane presents the first site of Aß interaction with neurons, which in turn maybe the origin of Aß neurotoxicity. The purpose of this study was to find a lipid composition that can be used as a model of neuronal membrane for subsequent studies of the role of topographical heterogeneity (domain formation) on Aß-membrane interaction as related to AD. The lipids used in the study were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), cholesterol (Chol), sphingomyelin (SM) and ganglioside GM1 (GM1). These lipids were combined in different proportions and deposited on a mica substrate to form supported monolayers. They were then imaged with an atomic force microscope (AFM) to determine if any of them exhibited domain formation. Three of the studied samples: POPC/POPG/SM 40:40:20 +5%Chol, POPC/SM/Chol 75:20:5 and POPC/SM/GM1/Chol 74:2:1:23 were found to possess interesting topography, rich in structural features: pores and domains. The average height difference between the domain features for each sample was found to be 0.58±015 nm, 0.61±0.12 nm and 0.27±0:07 nm.

<p>This research describes the development of a state-of-the-art atomic force microscope (AFM) for improved force spectroscopy. Although the AFM has been used extensively in this field of research, the performance of the instrument has been limited by inefficient operation techniques, incorrect experimental assumptions, and inadequate controller design. This research focuses on overcoming these deficiencies by providing precise control over the instrument for specialized research in a manner that is conducive to the natural science researcher.</p><p>To facilitate this research, a custom, multi-axis AFM system was constructed. The instrument was designed primarily for AFM-based force spectroscopy and as a result a substantial amount of research focused on the development of a wide variety of approach/retraction methods for the instrument. Defining research in this area included the development of methods to minimize potentially damaging compressive forces, form polymer bridges at different tip-sample gap widths, produce clean, deconvoluted force-extension curves, and limit single molecule force spectroscopy pulling geometry errors. In an effort to increase the efficiency of the instrument, the programs developed during this research were fully automated, allowing autonomous operation of the instrument for long periods of time. To compliment the data collection programs, both manual and automated analysis programs with force-volume imaging capabilities were also developed.</p><p>By studying the AFM from a dynamic systems, measurements, and controls approach, the resulting controllers were tailored to meet the process requirements of the intended applications. In doing so, the sensitivity of the instrument was improved for applications of interest. By incorporating control over the environment, contact force, loading rate, and pulling angle, the research has increased the accuracy of the AFM such that molecules and receptor-ligand binding events can be investigated with greater detail. Furthermore, the incorporation of a graphical user interface and automated data collection and analysis tools has made the AFM a more user-friendly, efficient instrument for the natural science researcher.</p> / Dissertation

No description available.

Atomic force microscopy is one of the powerful instruments used to explore the mechanical properties of nanoscale materials. It not only can produce high-resolution images and surface mechanical properties, but also can make use of its probe for surface etching. In this study, we first use atomic force microscopy to measure the Adhesion Map of insulin amyloid fibers, then conduct mechanical lithography on the surface with the probe. In the end, we discuss the effect on insulin amyloid fibrils due to exert different forces and different speeds with the probe. According to Nanoindentation theory and Hertzian model, we can derive the Young's modulus of insulin amyloid fibrils from force-indentation relations. Then we cut the Insulin amyloid fibers with probe. The results showed that when we applied 3.23 nN force by the probe, the insulin amyloid fibers began to break. When we applied 7.07 nN force, insulin amyloid fibers are cut off easily. Therefore, we can bite off insulin amyloid fibers of different lengths and sections, and arrange in the desired pattern by atomic force microscope.