The abiotic oxidation of pyrite requires the supply of oxygen and water only. In abiotic systems, pyrite oxidation may proceed via several paths, with multiple steps in each of the paths. Defect sites (S-deficient, Fe3+ bearing sites) on the pyrite surface have been shown reported to be the initial reaction sites on pristine pyrite surfaces. In neutral to slightly acidic solutions (3.5<pH<7), ferric iron hydroxide patches will form on the surface. These patches have been shown to be the predominant sites for electron exchange. Efforts were undertaken to suppress the electron transfer at these sites to inhibit pyrite oxidation. It has been shown that pyrite oxidation can be controlled by exposing the pyrite to phosphate under relatively high pH values (pH above 5.0). However, phosphate ceases to function as an inhibitor under lower pHs. The use of two-tail phospholipids instead of phosphate to inhibit the pyrite oxidation proved to be very effective under abiotic conditions. The purpose of the present study is to determine if the use of two-tail phospholipids can be extended to systems that have bacteria present. Batch experiments were conducted in which pyrite slurries were treated with two-tailed lipid either before or after exposure to bacteria. Iron release into the solution was used as a reaction progress variable and Atomic Force Microscopy was used to study the distribution of lipids and bacteria on the pyrite surface. AFM images showed that the formation of 7nm lipid bilayers contributes to the majority of lipid structures on pyrite surface. The bilayers render the pyrite surface hydrophobic and inhibit the reaction of water with the surface, which is known to be a critical reactant. AFM images also showed that phospholipids are capable of displacing a large fraction of bacteria attached to pyrite, reducing the oxidation rate of the mineral. However, addition of heterotrophic bacterial (Acidiphilum acidophilum) to the system resulted in the increase in pyrite oxidation rate again. Cross-linking of the 23:2 dyne phospholipids by exposing the lipid to UV light greatly enhanced the stability of the lipid in the presence of the heterotrophic bacteria. UV pretreated lipid layers reduced pyrite oxidation in the presence of heterotrophic bacteria for up to 30 days. / Chemistry

There is a need for the development of new technologies for the early detection of disease. Diverse initiatives are underway in academia and the pharmaceutical and biotechnology industries to develop highly-sensitive, high-throughput methods to detect disease-relevant biomarkers at the single-cell level. Biomarkers can define the progress of a disease or efficacy of disease treatment, and can include nucleic acids (RNA, DNA), proteins, small molecules, or even specific cells. While discovery research in this area is accelerating, there are a number of current experimental limitations. Most existing methodologies require a relatively large sample size. Also, amplification-based detection technologies are destructive to sample, and errors in amplification can occur, leading to an incorrect diagnosis. Nanomaterial-based devices (nanodevices) offer the promise of label-free, amplification-free detection strategies. Such nanodevices could allow analysis of minute biological samples without the requirement for amplification or incorporation of reporter groups. Loss of sample, due to handling and processing would be minimized and the sample could be recovered for further analysis. Atomic force microscopy (AFM) allows topographic imaging and compressibility/elasticity measurement of biomolecules on solid supports. AFM can enable assays of ligand binding with single molecule detection capability. Certain nucleic acid types, in particular double-stranded (ds) RNA, can act as a biomarker for specific cancers (e.g. leukemia) and viral infection. dsRNA also is of interest since it is a conserved structural feature of precursors to gene-regulatory RNAs, including micro (mi) RNAs and short interfering (si) RNAs. This project demonstrates a single-step, label-free, amplification-free approach for detecting the interaction of biomolecules that bind and/or process dsRNA, using a nanomanipulated, self-assembled monolayer (SAM) of a ds[RNA-DNA] chimera as imprinting matrix, a reference nuclease as imprinting agent, and AFM for imprint-readout. The action of the dsRNA-specific enzyme, ribonuclease III (RNase III), as well as the binding of an inactive, dsRNA-binding RNase III mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA] chimera, as measured by atomic force microscopy, provides a distinct digital output for each type of input. These findings provide the basis for developing imprinting-based nano-biosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale. RNA-DNA heteroduplexes are biomarkers for specific inflammatory conditions of genetic origin, and also are the product of capture of an RNA (e.g., miRNA) by a complementary DNA sequence. The approach used here to detect RNA-DNA hybrids is based on the ability of alkylthiol-modified ssDNA molecules to form monolayers and nanomatrices on gold surfaces (as described above) with density-dependent thickness, which increases upon formation of RNA-DNA hybrids following addition of a complementary oligoribonucleotide. Changes in hybrid matrix thickness can be measured by AFM, using a reference monolayer. RNA-DNA hybrid formation as well as subsequent processing by RNase H can be observed as a height increase or decrease, respectively, of the monolayer. When Mg2+ is omitted to prevent RNA cleavage, but not protein binding, a significant height increase is observed. The height increase is not observed with the corresponding ssDNA or ssRNA nanomatrices, and only occurs with nanomatrices having a hybrid density above a defined threshold. The data indicate formation of a stable multimeric RNase H assembly on the hybrid nanomatrix which provides a robust signal that is nondestructive to the RNA. The implications of these findings are discussed with respect to development of novel detection methodologies for RNA, dsRNA, and RNA-DNA hybrids. / Chemistry

The heart, as a vital organ, must pump continuously to deliver oxygenated blood to the tissues of the body. The physical stress of pumping is supported by the extracellular matrix (ECM), a dynamic protein scaffold inside and around the heart. While a regulated ECM is required to maintain heart function, aberrant or excessive ECM remodelling, called fibrosis, is associated with disease states and is a hallmark of cardiovascular disease. One major trigger of cardiovascular disease is obesity, and fibrotic remodelling is known to occur in this context. In order to study the impact of increased body size on heart function and the molecular and biophysical characteristics of the ECM, a larval overgrowth model for obesity in the genetic model Drosophila melanogaster has been developed and characterised. This model produces giant larvae twice as heavy as their wildtype counterparts, and allows a unique opportunity to study changes in the cardiac ECM in a simple genetic model. Results demonstrate a remarkable ability of the ECM to accommodate this increase in size. The muscles of the heart are particularly robust, and there are no obvious observable defects to the matrix. Preliminary results suggest Collagen fibres are thicker and more disperse. When observing heart functionality, the cross-sectional area of the heart lumen is increased significantly in giant larvae, both at diastole and systole. However, giant larvae display defects in contraction of the heart tube, characterised by an inability to contract fully at systole. This results in a less than proportional increase in stroke volume, and an increase in heart rate. Heart function of giant larvae is clearly affected by the increase in body size. To quantify the impact to the biophysical structure of the ECM, an atomic force microscopy protocol is being developed. / Thesis / Master of Science (MSc) / A known side effect of cardiovascular disease is fibrosis of the heart, a form of pathological extracellular matrix (ECM) remodelling. Fibrosis causes the stiffening of heart muscle, leading to impaired cardiac function. One of the main risk factors for the development of cardiovascular disease is obesity, and fibrosis is known to occur in this context. I have characterised changes in the morphology and physiology of the heart in a Drosophila model for obesity. The resulting cardiac hypertrophy reveals significant plasticity in the heart ECM, while heart contraction and output is compromised.

Living systems possess seemingly bottomless complexity. Attempts to parse the details of one cellular process from all other concurrent processes are challenging, if not daunting undertakings. The apparent depth of this problem, as it pertains to biomineralization, is related to the small number of existing studies focused on the development of a mechanism-based understanding of intracellular mineralization processes. Molecular biologists and geneticists have only begun to turn their attention towards identification and characterization of molecules involved in regulating and controlling biomineral formation. With this new knowledge, a number of new and exciting research opportunities are currently awaiting development upon a barren landscape. Silica biomineralization is one of these emerging frontiers. As new information about the chemical and structural nature of the macromolecules involved in biosilicification is revealed, the means these species employ to control the temporal and spatial onset of silica deposition in vivo become available for exploration. The first chapter of this dissertation outlines those aspects of silicate metabolism that are directly relevant to the controlled biomineralization of silica in eukaryotic organisms and identifies pervasive and unanswered questions surrounding biosilica formation. Particular attention is paid to the diatoms, which are the most abundant, and extensively investigated silica-mineralizing organisms in modern seas. The extent, and mechanism through which specific organic moieties work individually or in concert to direct mineral formation at biological interfaces is a central concern of modern biomineralization research. Chapter two addresses this forefront issue for silica mineralizing systems, and reports the results of an experimental investigation designed to measure the effects of individual surface-bound organic functional groups on the rate of surface-directed silica nucleation. Chapter three discusses an additional aspect of this research aimed at investigating the reactivity of nanoparticulate biogenic silica produced by marine phytoplankton and terrestrial plants in natural environments. Density Functional Theory and ab initio molecular orbital calculations are employed to explore potential mechanisms underlying the catalytic activity of divalent metal cations during the hydrolysis of Si – O bonded networks. / Ph. D.

Central to tapping mode atomic force microscopy is an oscillating cantilever whose tip interacts with a sample surface. The tip-surface interactions are strongly nonlinear, rapidly changing, and hysteretic. We explore numerically a lumped-mass model that includes attractive, adhesive, and repulsive contributions as well as the interaction of the capillary fluid layers that cover both tip and sample in the ambient conditions common in experiment. To accomplish this, we have developed and used numerical techniques specifically tailored for discontinuous, nonlinear, and hysteretic dynamical systems. In particular, we use forward-time simulation with event handling and the numerical pseudo-arclength continuation of periodic solutions. We first use these numerical approaches to explore the nonlinear dynamics of the cantilever. We find the coexistence of three steady state oscillating solutions: (i) periodic with low-amplitude, (ii) periodic with high-amplitude, and (iii) high-periodic or irregular behavior. Furthermore, the branches of periodic solutions are found to end precisely where the cantilever comes into grazing contact with event surfaces in state space corresponding to the onset of capillary interactions and the onset of repulsive forces associated with surface contact. Also, the branches of periodic solutions are found to be separated by windows of irregular dynamics. These windows coexist with the periodic branches of solutions and exist beyond the termination of the periodic solution. We also explore the power dissipated through the interaction of the capillary fluid layers. The source of this dissipation is the hysteresis in the conservative capillary force interaction. We relate the power dissipation with the fraction of oscillations that break the fluid meniscus. Using forward-time simulation with event handling, this is done exactly and we explore the dissipated power over a range of experimentally relevant conditions. It is found that the dissipated power as a function of the equilibrium cantilever-surface separation has a characteristic shape that we directly relate to the cantilever dynamics. We also find that despite the highly irregular cantilever dynamics, the fraction of oscillations breaking the meniscus behaves in a fairly simple manner. We have also performed a large number of forward-time simulations over a wide range of initial conditions to approximate the basins of attraction of steady oscillating solutions. Overall, the simulations show a complex pattern of high and low amplitude periodic solutions over the range of initial conditions explored. We find that for large equilibrium separations, the basin of attraction is dominated by the low-amplitude periodic solution and for the small equilibrium separations by the high-amplitude periodic solution. / Ph. D.

The foundational studies of this dissertation concern the characterization of segmented polyurethanes and polyureas synthesized without the use of chain extenders'molecules that are typically used to promote a microphase separated morphology that gives these materials their useful characteristics. Polyurethanes in which a single asymmetric diisocyanate comprising the whole of the hard segment were found to display poor microphase separation. Conversely, polyurethanes in which a single symmetric diisocyanate composed the hard segment were found to display good microphase separation. The more efficient packing of the symmetric hard segments also led to an increase in hard segment connectivity and hence higher values of storage moduli in these systems. When hydroxyl-terminated diisocyanates were replaced with amine-terminated diisocyanates, polyureas were formed. Here too, diisocyanate symmetry was found to play a key role with symmetric diisocyanates leading to better microphase separation. In addition, the polyurea materials displayed broader service temperature windows than their polyurethane counterparts as the relatively stronger bidentate hydrogen bonding replaced monodentate hydrogen bonding in these materials. A thread-like, microphase separated morphology was visually confirmed using atomic force microscopy. Other techniques such as ambient temperature tensile testing, and wide and small angle x-ray scattering were employed to confirm the presence of the microphase separated structure. The investigation into the effects of diisocyanate chemistry and its symmetry was broadened to incorporate non-chain extended polyurethane materials with different soft segment molecular weights, as well as polyurethanes that did contain chain extenders. Once again the effect of using symmetric versus asymmetric diisocyanates was evident in the structure–property behavior of these systems, with symmetric diisocyanates forming materials that displayed better microphase separation and more connectivity of their hard domains. Lastly, in a departure from the segmented copolymer area, a study was conducted into the influence of casting variables on the solid-state structure of synthetic polypeptide films based on glutamate chemistry. The effect of solvent evaporation was determined to play a key role in the morphology of these polypeptide films. Measured small angle light scattering patterns were compared to computer calculated patterns to reveal information about the structure, shape, and length scale of the polypeptide structure. / Ph. D.

Micro and nanoscale systems are rapidly evolving to improve the resolution of experimental measurements. Experiments involving such small scale devices are difficult and expensive, and the available analytical theory to describe their dynamics is idealized. The dynamics of microscopic cantilevers in fluid are complicated and include significant contributions from many sources in an actual experiment. Some examples are: complex cantilever geometries, near-wall effects, thermal and external actuation techniques, and a variety of measurement techniques. Numerical simulations are a powerful approach to describe the dynamics of micro and nanoscale systems for the precise conditions of experiment. This thesis provides a numerical approach capable of addressing these inherent challenges and quantifies the dynamics of microscopic cantilevers in fluid for experimentally relevant conditions. A thermodynamic approach based upon the fluctuation-dissipation theorem allows for the calculation of stochastic dynamics from deterministic dynamics. Using numerical simulations, the thermal motion can be described for the precise conditions of experiment. It is found that the measured dynamics of cantilevers differs depending on the quantity being measured. In particular, the dynamics of displacement and angle of the cantilever tip distribute energy differently to the higher flexural modes. The externally driven dynamics of microscale cantilevers in fluid are also considered. The driven dynamics are calculated using numerical simulations of the cantilever response to a force impulse. It is found that the driven dynamics depend upon the type of actuation in addition to the quantity measured. A comparison of the driven dynamics to the corresponding stochastic dynamics yields insight into the nature of the Brownian force acting on the cantilever. Another experimentally relevant condition is the use of cantilevers with V-shaped planforms in fluid. The resulting flow field is three-dimensional and complex in contrast to what is found for a long and slender rectangular cantilever. Despite the flow complexity, the stochastic and driven dynamics of the fundamental mode can be predicted using a two-dimensional model with an appropriately chosen length scale. An experimentally motivated magnetomotive actuation technique is investigated. Results show that this approach generates power spectra nearly equivalent to the noise spectra. Furthermore, the case of a V-shaped cantilever in fluid and oscillating in proximity of a solid boundary is investigated. In the presence of a solid surface the fluid damping and added mass of fluid on the cantilever are larger than for a cantilever far from boundaries. This results in a lower frequency and quality factor for the fundamental resonance. This can impede experimental efforts because broad peaks lack distinct features that can be used to identify experimental signals. An option to overcome the large viscous damping is to take advantage of higher modes of cantilever oscillation. The higher frequency oscillations of the higher modes generate a smaller viscous boundary layer and have a reduced added mass. As a result, the quality factor increases with increasing mode number. The frequency dependence of the fluid dynamics around a fluctuating microscale cantilever is also studied. The mass of fluid entrained by the cantilever and the viscous damping quantify the interaction of a cantilever with the surrounding fluid and are computed. Analytical expressions for these parameters are derived for moderate mode number. The techniques and findings of this thesis have broad applicability to a wide range of micro and nanotechnologies that rely upon understanding the dynamics of small scale structures in fluid. / Ph. D.

Poly(N-isopropyl-acrylamide) (PNIPAM) is a thermoresponsive polymer with a wide range of biological applications, including drug delivery, biosensing, and tissue engineering. The tunability of the structural and mechanical properties of PNIPAM makes it particularly at- tractive in emulating cell environments and dynamic cytoskeletal deformations. This thesis discusses PNIPAM's properties and applications in different forms i.e., solution, brushes, hydrogels, and surface patterned hydrogels, with specific focus on lithographically patterned substrates coated with PNIPAM films. The scaffolds are investigated for structural and me- chanical responses to thermally driven changes in the PNIPAM hydration states using atomic force microscopy (AFM). AFM measurements on our lithographically patterned substrates show that the substrate pattern and coating method enable the fabrication of scaffolds with different topographic and mechanical properties across a wide thermal range. Importantly, these scaffolds exhibit variations in both lateral topography and Young's modulus, rendering them well suited for investigations of differential mechanical stresses experienced by cells and cell membranes. / Master of Science / Poly(N-isopropyl-acrylamide) (PNIPAM) is a polymer which can change its water absorption depending on the temperature of its aqueous environment. It transitions from a swollen state at room temperature to a collapsed state at around 32 °C. These thermally tunable properties make PNIPAM an attractive candidate in a variery of applications, including biomedical and biophysical applications. In this thesis, PNIPAM is coated on lithographically patterned substrates to emulate the cellular cytoskeleton. Atomic force microscopy (AFM) measurements are performed to measure the topography and mechanical properties of the fabricated scaffolds. The results show that the coating method and the features of the used substrate allow the fabrication of different surface topographies with biologically relevant mechanics.

Surfactants, polymers, and their mixtures are widely used in commercial formulations of paints, water-based adhesives, detergents, food, and other products. This thesis describes measurements of the forces acting on colloidal particles in surfactant and polymer solutions. The change in force on addition of surfactants and polymers is usually caused by adsorption to an interface. In this thesis, I also describe the effect of surface charge density, surface crystallinity, surface heterogeneity, and preadsorbed polymer on surfactant adsorption. A new method for the stabilization of colloidal particles is introduced via the synthesis and adsorption of unnatural proteins. Unnatural proteins can be synthesized using the natural "machinery" of a bacterial cell with almost any primary sequence, and provide an environmentally friendly route to colloidal stabilization. As a model system, we study the stabilization of alumina, because alumina has a high Hamaker constant and is therefore difficult to stabilize. An unnatural protein with the sequence, thioredoxin-Pro39Glu10 is used. The Glu10 is anionic (pH > 3) and is designed to adsorb to positively charged alumina (pH<9). The thioredoxin-Pro39 is hydrophilic so it should remain in solution, thereby providing a steric barrier to the approach of two particles in a range of salt and pH conditions. Ellipsometry experiments show that thioredoxin-Pro39Glu10 adsorbs to alumina. Force measurements with the Atomic Force Microscopy (AFM) colloid probe technique show that adsorption of the unnatural protein leads to repulsive forces that decay exponentially with the separation between the surfaces, and are independent of salt concentration. The loss of a salt-dependent force shows that adsorption of the unnatural protein has effectively neutralized the charge on the alumina. Thus, I have shown that an unnatural protein can be used to control the stability of a colloidal system. In general, the same hydrophilic block can probably be added to a variety of anchoring blocks to stabilize different colloidal particles. Electrostatic forces are frequently responsible for the stabilization of colloidal particles. The decay length of these forces is dictated by the electrolyte concentration. The relationship between the decay length and the concentration is well understood for fully dissociated 1:1 electrolytes. Here, I examine the decay-length in solutions where the ions associate strongly. The forces are measured between silica surfaces in aqueous carboxylic acid and surfactant solutions. The decay lengths of the electrostatic double-layer force in both these solutions are well described by the usual expression for decay length when the concentration of ions is obtained from an activity measurement. The effect of the surface properties of the solid substrate on surfactant adsorption is also described in this thesis. The adsorption characteristics of a charged surfactant onto fixed charged surfaces as a function of surface charge density is reported. This is the first time that a method has been introduced for making a series of known fixed charged surfaces. Investigating surfactant adsorption to these surfaces has improved our understanding of the role of charge density in surfactant adsorption and desorption. The desired surface charge density is achieved by the use of gold-thiol self-assembled monolayers (SAMs) of different Ï -groups ("OH and "N+(CH3)3). The mole fraction of "N+(CH3)3 on the mixed SAM dictates the surface charge density. The charge on "N+(CH3)3 is fixed and does not self-regulate. The adsorption of sodium dodecyl sulfate (SDS) to the interface between these model surfaces and aqueous solutions of SDS is investigated. Atomic Force Microscopy (AFM) of the adsorbed surfactant reveals no surface micelles above the critical micelle concentration, cmc, over a wide variety of "N+(CH3)3 densities. This shows that the lateral mobility of ions other than surfactant at the interface is important for the formation of surface micelles of ionic surfactants. Adsorption isotherms of SDS (with no added salt) measured by Surface Plasmon Resonance (SPR) show a plateau region in which the surface excess of SDS is equal to the known fixed surface charge. This demonstrates that the adsorption is electrostatically driven. There is no critical surface charge density at which adsorption rises rapidly. Thus there appears to be no 'hemimicelle concentration'. My work suggests that the formation of hemimicelles depends on the lateral mobility of the surface ions. Desorption experiments starting above the cmc show rapid desorption of SDS into water until the surface excess is equal to the surface charge density. The rapid desorption is followed by a much slower desorption. The elucidation of this fast-slow desorption pattern based on charge density is made possible by the preparation of a set of constant charge surfaces. / Ph. D.

Incorporation of silver nanoparticles (AgNPs) into an increasing number of consumer products has led to concern over the potential ecological impacts of their unintended release to the environment. Dissolution is an important environmental transformation that affects the form and concentration of AgNPs in natural waters; however, studies on AgNP dissolution kinetics are complicated by nanoparticle aggregation. Herein, nanosphere lithography (NSL) was used to fabricate uniform arrays of AgNPs immobilized on glass substrates. Nanoparticle immobilization enabled controlled evaluation of AgNP dissolution in an air-saturated phosphate buffer (pH 7, 25 °C) under variable NaCl concentrations in the absence of aggregation. Atomic force microscopy (AFM) was used to monitor changes in particle morphology and dissolution. Over the first day of exposure to ≥10 mM NaCl, the in-plane AgNP shape changed from triangular to circular, the sidewalls steepened, and the height increased by 6-12 nm. Subsequently, particle height and in-plane radius decreased at a constant rate over a 2-week period. Dissolution rates varied linearly from 0.4 to 2.2 nm/d over the 10-550 mM NaCl concentration range tested. NaCl-catalyzed dissolution of AgNPs may play an important role in AgNP fate in saline waters and biological media. This study demonstrates the utility of NSL and AFM for the direct investigation of un-aggregated AgNP dissolution. / Master of Science