The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.

This work explores the stochastic dynamics and important diagnostics of a mechanical resonator (nanobeam) used in cavity optomechanical sensors for atomic force microscopy. Atomic force microscopy (AFM) is a tool to image surface topology down to the level of individual atoms. Conventional AFM has been an essential tool for micro and nanoscale studies in physics, chemistry, and biology. Cavity optomechanical sensors for AFM extend the utility of conventional AFM into a new regime of high sensitivity k is approximately 1 N/m and high frequency f0 is approximately 10 MHz. Cavity optomechanical sensors for AFM are unique because they use near field optics to transduce the position of a nanobeam. The nanobeam is not able to be transduced by more conventional AFM techniques, such as laser interferometry, because the nanobeam is smaller than the spot size of the laser. This work determines the noise spectrum G of a nanobeam in water and in air. Also important diagnostics of the nanobeam are determined in air and in water. These important diagnostics include the quality factor Q and natural frequency in fluid omega_f. It is found that the nanobeam is overdamped in water. However, the nanobeam is underdamped in air and has quality factor of Q is approximately 4. The noise spectrum is determined from deterministic numerical calculations and the Fluctuation-Dissipation Theorem. This is possible because the same molecular processes, Brownian motion, cause both the fluctuations of the nanobeam and the dissipation of the nanobeam. / Master of Science

In this dissertation, experiments are described which elucidate how the ordering of symmetric diblock copolymers affects the dynamics within various geometries. In all studies presented herein, experimental techniques are used to probe the dynamics of symmetric diblock copolymer systems as they progress toward equilibrium and to study the role that nanoscale ordering plays in these processes. In the majority of work presented herein, experiments were performed on symmetric diblock copolymer thin films. This work focuses on the effect of various sample preparation techniques on the equilibration kinetics of lamellar forming films. Films are prepared with varying thicknesses in the homogeneous, disordered state and annealed to form islands and holes as the surface decomposes to form commensurate thicknesses. Both nucleated and spinodal growth patterns were observed for this surface decomposition dependent on the initial thickness and intermediate morphologies formed upon ordering. We also prepare equilibrium commensurate films and induce a step change in surface interactions, switching from asymmetric to symmetric wetting boundaries. Upon equilibration, a perforated lamella forms at the free surface to mediate the order-order transition, inducing hole growth with a ramified shape. In the final project, the effect that lamellar order has on dynamics is studied within unstable polymer melt bridges. Liquid bridges are what is formed when a droplet is stretched between two surfaces, like spit between two fingers. Disordered diblock bridges are shown to evolve similar to their homopolymer counterparts. However, ordered diblock copolymer exhibits an enhanced stability with an inhibition of flow proposed to be induced by the isotropic orientational order within the bridge. As well, shear thinning is observed that is believed to be caused by an alignment of ordered domains along the bridge axis due to shear strain rates, providing pathways for flow of diblock copolymer out of the unstable bridge. / Thesis / Doctor of Philosophy (PhD)

Cold spray (CS) deposition of metals is a process involving deposition of materials in the solid or semi-solid state. It also has lower operating temperatures, and oxidation is greatly reduced in the process. The process is beneficial for refractory metals, such as tantalum, which are tough and difficult to machine. The interface between the CS powder and the substrate is the most important region for the study of mechanical properties as it is where the bonding process occurs first; studying mechanical properties at the nanoscale will give us a better idea of the mechanical properties of the coated surface. The present work investigates multiple-sprayed conventional and low-hydrogen treated tantalum powders on stainless steel substrates and also single-sprayed nitrogen-treated tantalum powders on aluminum substrate using Atomic force microscopy (AFM). It also discusses the effects of topography on the local changes in modulus. AFM is an instrument that measures the site-specific property of the sample. In this work, the local Young's modulus is studied using force-distance curves. Calibration of the AFM cantilever and the photodetector used to measure the cantilever, is a vital step before the actual process. The conventional method of calibration can cause damage to the tip as it arbitrarily penetrates into the sample creating a cantilever deflection vs. tip penetration curve, giving the sensitivity of the photodetector. AFM is highly dependent on topographical features as the cantilever tip-sample interaction can vary, causing variations in the property mapped. This work, however uses a non-contact method of calibration which saves the cantilever tip from potential damages, saving the results from the detrimental effects of tip topography. The work also discusses the effects of local sample deformation and volume of tip-surface contact on local changes in Young's modulus at the interface of coating and substrate. This work uses Electron micro-probe analysis (EPMA) to show the presence of oxides at the interface. The presence of oxides changes the bond energy as compared to a pure tantalum bond, ultimately affecting the local modulus mapped using AFM. The effect of oxides on the local modulus at the coating-substrate interface is theoretically discussed.

Protein-DNA interactions are central to many biological processes and form the bedrock of gene transcription, DNA replication, and DNA repair processes. Many proteins recognize specific sequences in DNA- a restriction enzyme must only cut at the correct sequence and a transcription factor should bind at its consensus sequence. Some proteins are designed to bind to specific structural or chemical features in DNA, such as DNA repair proteins and some DNA modifying enzymes. Target-specific DNA binding proteins initially bind to non-specific DNA and then search for their target sites through different types of diffusion mechanisms. Atomic force microscopy (AFM) is a single-molecule technique that is specifically well-suited to resolve the distinct states of target-specific as well as nonspecific protein-DNA interactions that are vital for a deeper insight into the target site search mechanisms of these enzymes. In this thesis, protein systems involved in epigenetic regulation, base excision repair (BER), and transcription are investigated by single-molecule AFM analyses complemented by biochemical and biophysical experiments. The first chapter of this thesis narrates the establishment of a novel, user-unbiased MatLab-based tool for automated DNA bend angle measurements on AFM data. This tool has then been employed to study the initial lesion detection step of several DNA glycosylases. These results promoted a model describing the altered plasticities of DNA at the target lesions of DNA glycosylases as the fundamental mechanism for their enhanced efficiency of lesion detection. In the second chapter of this thesis, the novel automated tool has been further extended to provide protein binding positions on the DNA along with corresponding DNA bend angles and applied to the study of DNMT3A DNA methyltransferase. These AFM studies revealed preferential co-methylation at specific, defined distances between two CpG sites by the enzyme and when combined with biochemical analyses and structural modelling supported novel modes of CpG co-methylation by DNMT3A. In the third chapter of this thesis, the role of 8-oxo-guanine glycosylase (hOGG1) in Myc-mediated transcription initiation has been investigated. AFM analyses revealed that in the presence of oxidative damage in DNA, Myc is recruited to its target site (E-box) by hOGG1 through direct protein-protein interactions, specifically under oxidizing conditions. Intriguingly, oxidation of hOGG1 was further observed to result in dimerization of hOGG1, which may also play a role in the mechanism of transcription regulation by hOGG1 under oxidative stress. / Protein-DNA-Wechselwirkungen sind für viele biologische Prozesse von zentraler Bedeutung und bilden die Grundlage der Gentranskription, der DNA-Replikation und der DNA-Reparaturprozesse. Viele Proteine erkennen bestimmte Bassen-Sequenzen in der DNA - ein Restriktionsenzym darf nur an der richtigen Sequenz schneiden, und ein Transkriptionsfaktor sollte an seine Konsenssequenz binden. Einige Proteine sind darauf ausgelegt, an bestimmte strukturelle oder chemische Merkmale der DNA zu binden, wie z. B. DNA-Reparaturproteine und verschiedene DNA-modifizierende Enzyme. Zielspezifische DNA-bindende Proteine binden zunächst an unspezifische DNA und suchen dann durch verschiedene Arten von Diffusionsmechanismen nach ihren Zielstellen in der DNA. AFM ist eine Einzelmolekültechnik, die besonders gut geeignet ist, um die verschiedenen Zustände sowohl der spezifisch gebundenen als auch unspezifischen Protein-DNA-Wechselwirkungen aufzulösen, die für einen tieferen Einblick in die Mechanismen der Zielstellensuche unerlässlich sind. In dieser Arbeit werden Proteinsysteme, die an der epigenetischen Regulation, der Basenexzisionsreparatur (BER) und der Transkription beteiligt sind, durch Einzelmolekül- AFM-Analysen untersucht, und diese Studien werden durch biochemische und biophysikalische Experimente komplementiert. ...

No description available.

PLE presents an alternative or complementary probe-based tool to DPN, PPL, and NFL. Unlike most scanning probe techniques, where patterning by deposition is usually employed, PLE is unique because it is capable of deposition and removal in one or multiple steps. Therefore, PLE allows rectification of patterning errors, and it can be employed for both additive and subtractive patterning through molecular deposition and chemical and electrochemical etching, respectively. PLE is a technique that exploits the intrinsic porosity of hydrogels like agarose and polyacrylamide. The probes are made by polymerizing a liquid mixture of agarose or acrylamide monomers in a conical or pyramidal master. The polymeric probe is hydrated in deionized water or ink of interest after polymerization. For deposition, PLE has shown promising results in the selective deposition of fluorescent inks on bare or functionalized glass substrates. Erasing via PLE has been done in two ways: the first method involves selectively erasing the fluorescent molecules using a probe loaded with deionized water by bringing the probe in contact with the area of interest. Thus, solvation and transportation of the molecules into the polymeric probes render selective removal of materials (fluorescent inks) from a substrate. On the other hand, erasing or removal of metals deposited on a substrate was demonstrated using redox reactions. Here, the probe is loaded with an etchant, which is selectively delivered onto the substrate by bringing the probe close to or in contact with the surface. Thus, the etchant molecules passively diffuse from the probe to the substrate through a meniscus formed at the probe-substrate interface. Removal of molecules occurs after the redox reaction between the ink, and the substrate is completed. Many in-length microscale complex patterns can be easily made by translocating the probe over the substrate while the probe’s tip is in contact with the surface. Since the probes used in PLE are made of polymers, the probe-substrate contacting area can be easily modulated, and damage to the substrate by the probe is minimum. Moreover, it has been shown that the probes can be used multiple times, a hurdle frequently faced by probes made of hard materials such as silicon-based probes. We explored the capabilities of a polymeric probe made of PAAM to selectively deliver and remove (erase) material deposited on a surface. PLE, pioneered by our group, takes advantage of the hydrophilic and porous nature of polyacrylamide. In addition, the conformability of PAAM hydrogels was exploited to make patterns of various sizes and to the pattern on non-planar surfaces. The main advantage of PLE is removing materials from various substrates. Additionally, selective delivery of material to planar and non-planar substrates was demonstrated. Whereas DPN and sister techniques require multiple steps for patterning through the etching process, PLE can perform etching in one step. Therefore, using PLE, microscale patterning on surfaces can save considerable time, labor, and cost. Further, chemical and supplies waste are minima in PLE. Notably, the deposition and etching at the microscale level can be simultaneously achieved in one single step, providing an extremely high throughput patterning rate (on the order of 1000 mm2/s). The PLE patterning rate is two to three orders larger than DPN-based patterning. However, PLE inherently deposits and removes materials with features much larger (microscale) than that can be achieved with DPN (sub-nanoscale). Therefore, PLE is an alternative to DPN, PPL, and related probe-based deposition and erasing techniques, and in some cases, PLE provides enhanced capabilities than its contemporary techniques. In this dissertation, I intend to demonstrate the potential of PLE for fabricating working devices at a lower cost as an alternative to contemporary fabrication. Chapter 2 involves the fabrication of micro-electrodes on rigid and flexible substrates by selectively removing copper and ITO from a glass and a PET substrate. As proof of concept, substrates coated with the PLE patterned surfaces were used to fabricate a photodetector, and LEDs were assembled on the electrodes made on ITO-PET substrates. Chapter 3 describes a series of experiments involving the evaluation of ink withholding capacity, large area patterning, and the effect of modification of substrate surface energy on PLE patterning. These experiments an increased understanding of processes involved in PLE editing and microscale patterning. A potential pitfall of PLE-based etching was also observed in these experiments, where a thin layer of material was left behind after subtractive editing with a PLE probe. EDS analysis indicated that the material was composed of iron, chlorine, and copper ─ components of the etchant solution and the copper film. The ring structure was attributed to the coffee-ring effect pinning the water meniscus to the substrate. By understanding the potential causes of the formation of the coffee-ring possible solutions to this problem were formulated. Chapter 4 describes the physical and mechanical properties of the hydrogel PAAM probes at the nanoscale. ESEM and AFM were employed to investigate the structural and mechanical properties of the probes after impregnation with metal etchants of various concentrations. The effect of local RH on PLE patterns was also investigated. More importantly, these experiments show critical structural differences of PAAM hydrogels composed of various monomer and crosslinker concentrations. ESEM showed the significant influence exerted by RH on meniscus size and its interaction with the substrate. The behavior of the water meniscus observed in ESEM shows that large RH promotes water spreading on the substrate generating larger patterning features. Chapter 5 describes the capability of PLE to selectively deliver metallic inks on a non-linear curved substrate to fabricate a microscale battery. PLE was used to deposit silver nitrate onto a non-planar flexible substrate which was used to grow a thin electrically conductive copper film via copper electroless deposition. Electrodeposition of zinc on the copper substrate was accomplished. By coupling a zinc electrode to a manganese oxide-graphite composite cathode, we demonstrated a working Zn-MnO2 aqueous microscale battery.

This study uses in situ atomic force microscopy (AFM) to directly observe the atomic scale effects of Sr on the monomolecular layer growth of abiotic calcite. These insights are coupled with quantitative measurements of the kinetics and thermodynamics of growth to determine the direction-specific effects of Sr on the positive and negative surface coordination environments that characterize calcite step edges. Low concentrations of strontium enhance calcite growth rate through changes in kinetics. A new conceptual model is introduced to explain this behavior. Higher concentrations of strontium inhibit and ultimately stop calcite growth by a step blocking mechanism. The critical supersaturation required to initiate growth (sigma*) increases with increasing levels of strontium. At higher supersaturations, strontium causes growth rates to increase to levels greater than those for the pure system. The step blocking model proposed by Cabrera and Vermilyea in 1958 does not predict the experimental data reported in this study because the dependence of sigma* upon strontium concentration is not the same for all supersaturations. Strontium inhibits calcite growth by different mechanisms for positive and negative step directions. Preliminary evidence indicates that strontium is preferentially incorporated into the positive step directions suggesting that impurity concentrations are not homogeneous throughout the crystal structure. Despite geochemical similarities, this study demonstrates that strontium and magnesium have different surface interaction mechanisms. The findings of this study demonstrate the importance of understanding microscopic processes and the significance of interpreting biominerals trace element signatures in the context of direction-specific interactions. / Master of Science

Through enhancing aerosol particle measurement accuracy by determining particle shape factors using Atomic Force Microscopy (AFM) combined with machine learning techniques, this study aims to provide a methodology that will improve the precision of aerosol measurements and contribute to the development of more effective filtration technologies. Accurate shape factor measurement is crucial for devices such as the Scanning Mobility Particle Sizer (SMPS), which often assume spherical particles of uniform density. By identifying and analyzing particles in AFM scans using machine learning techniques, this research provides a better understanding of shape factors, improving the quality of aerosol measurements. These advancements contribute to a deeper understanding of aerosol properties and their impact on filtration systems, aiding in the development of more effective filtration technologies and improving our capability to measure and control particulate matter in various environmental and industrial applications.

"The adhesion of bacteria to surfaces has been analyzed in terms of surface charge, surface energy, and the characteristics of polymers on bacteria, to understand the factors that control bacterial adhesion. Pseudomonas aeruginosa has received a great deal of interest because it is responsible for a variety of chronic bacterial infections such as airway infections in cystic fibrosis patients and ulcerative bacterial keratitis in soft contact lens users. Over the past few years, force measurement techniques such as atomic force microscopy (AFM) have made it possible to examine interactions between colloidal particles and surfaces. In the present study, the AFM was used to study the interactions between each of two Pseudomonas aeruginosa strains with proteins. Topographical images and force cycles of bacterial cells and proteins were analyzed. Bovine serum albumin (BSA) and concanavalin A (Con A) were the model proteins chosen to represent protein molecules that might affect bacterial adhesion. In addition, the role of LPS structure in bacterial adhesion was investigated. The magnitude of adhesive forces for two P. aeruginosa stains was not statistically significant when they interact with silicon. Although it is not clear if the pull-off distances are accurate representatives of the absolute length of bacterial surface molecules, the trend indicates that the surface molecules of strain AK1401 are shorter than those of strain PAO1. The semi-rough strain AK1401 was more hydrophobic than the smooth strain PAO1, according to the water contact angle measurements. However, surface free energy components and zeta potential values were not significantly different for both strains. Zeta potential of bacterial cells decreased when they were suspended in HEPES/DTT buffer instead of ultrapure water. The AFM results demonstrate the importance of nano-scale interactions between proteins and bacterial cells. Our results show that the lipid A and core oligosaccharides are the most important molecules influencing the interactions of P. aeruginosa with protein molecules. The interactions of P. aeruginosa with model proteins in our study were weak. Therefore, the role of protein molecules may be inadequate for the purpose of enhancing subsurface delivery for bioremediation. Our results suggest that the semi-rough mutant, AK1401, can adhere to the protein receptors of the epithelial cells or protein coated implants stronger than the smooth strain, PAO1, and therefore can cause serious infections."