The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism. Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic Aβ oligomers at physiological concentrations, and monitored in real time the change of size and mole fraction of oligomers in the presence of dissociating agents or conditions. The higher-order dissociation process caused by DHBA isomers produced no transient oligomeric intermediates, a desirable feature for an anti-oligomer therapeutic. Urea and guanidine hydrochloride, in contrast, produced a linear dissociation with a progressive decrease of size and mole fraction of oligomers. FCS allows the facile distinction of small molecule Aβ oligomer dissociators that do not produce stable potentially toxic oligomeric Aβ intermediates.

<p>One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. </p><p>We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, &#945;-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.</p><p>Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.</p> / Dissertation

No description available.

Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors. Part II aims to encapsulate in model lipid bilayers, metallic nanoparticles, which are used as probes in surface enhanced Raman spectroscopy. Three strategies of encapsulating particles, and incorporating Raman-active dyes are demonstrated, each using a different dye: malachite green, rhodamine-PE, and Tryptophan. Dye incorporation is verified by SERS and the bilayer is visualized and measured by TEM, with support from DLS and UV-Vis spectroscopy. In both parts, lipid-coated sensors are successfully fabricated and characterized. These results represent important and novel solutions to the functionalization of plasmonic surfaces with biologically relevant cell membrane mimics.

Hydrophobic interactions guide important molecular self-assembly processes such as protein folding. On the macroscale, hydrophobic interactions consist of the aggregation of "oil-like" objects in water by minimizing the interfacial energy. However, the hydration mechanism of small hydrophobic molecules on the nanoscale (~1 nm) differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic cross-over length scale is critically important to hydrophobic interactions in polymers, proteins and other macromolecules. Accurate experimental determination of hydration mechanisms and their interaction strengths are needed to understand protein folding. This thesis reports the development of experimental and analytical techniques that allow for direct measurements of hydrophobic interactions in a single molecule. Using single molecule force spectroscopy, the mechanical unfolding of a single hydrophobic homopolymer was identified and modeled. Two experiments examined how hydrophobicity at the molecular scale differ from the macroscopic scale. The first experiment identifies macroscopic interfacial tension as a critical parameter governing the molecular hydrophobic hydration strength. This experiment shows that the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways, consistent with theoretical predictions. The second experiment probes the hydrophobic size effect by studying how the size of a non-polar side-chain affects the thermal signatures of hydration. Our experimental results reveal a cross-over length scale of approximately 1 nm that bridges the transition from entropically driven microscopic hydration mechanism to enthalpically driven macroscopic hydration mechanism. These results indicate that hydrophobic interactions at the molecular scale differ from macroscopic scale, pointing to potential ways to improve our understanding and predictions of molecular interactions. The system established in this thesis forms the foundation for further investigation of polymer hydrophobicity.

In this thesis, a direct method of Atomic Force Microscopy (AFM) technique has been developed to measure the adhesion forces between BSA and two different surfaces: mica (a hydrophilic surface); and polystyrene (a hydrophobic surface); in PBS solution. We have shown possible to measure interactions between proteins and substrate surface directly without any modification to the substrate and the AFM tip; this means protein molecules can keep the natural elastic property within the force measurements. The average measured value of adhesion forces between BSA and mica is 0.036 ± 0.002 nN, and between BSA and polystyrene is 0.066 ± 0.003 nN. The polystyrene surface is more adhesive to BSA than the mica surface. This is consistent with previous research, which assessed that hydrophobic surfaces enhance protein adhesion but hydrophilic surfaces do not.

Interactions between hydrophobic surfaces at short separation distances (at the nanometer scale) are very important in a number of industrial applications. For example, in the froth flotation mineral separation process it is the interaction between the hydrophobic particle and the bubble which is paramount in separating the valuable minerals from the gangue. A number of studies, most notably using the atomic force microscope (AFM) and the surface force apparatus (SFA) have found the existence of a long range hydrophobic attractive force between hydrophobic surfaces that cannot be explained by classical colloidal science theories. In many cases, this force is an artefact due to the accumulation of sub-microscopic bubbles, the so called nanobubbles, at the liquid-hydrophobic solid interface. Thus, what was thought to be a hydrophobic force was actually a capillary force resulting from the gaseous bridge formed from the coalescence of nanobubbles, that is, the nanobubble bridging capillary force (NBCF). It is the purpose of this thesis to provide further insight into the accumulation of soluble gases at the liquid-hydrophobic solid interface and the resulting NBCF. Specifically, this thesis studies these phenomena from a fundamental standpoint and additionally relates the findings to froth flotation mineral separation. A systematic method to measure the NBCF by controlling the size of the gaseous capillary bridge was devised in this thesis. Control of the capillary bridge was achieved by utilising the solvent-exchange method to accumulate nanobubbles at the surface, followed by surface scanning of the colloidal probe over the flat surface to harvest nanobubbles. Thus, the NBCF has been controlled to allow for greater success in modelling the interaction, understanding the geometric parameters of the bridge, observing changes in friction force due to nanobubbles and understanding the influence of ethanol on the force. An outcome of this thesis was the development of a capillary force model which describes the NBCF. The model considers a constant volume and constant contact angle assumption for a gaseous capillary bridge of toroidal geometry. The model was very successful in describing the NBCF at long separation distances (>20nm) for both the approach and retract interactions. The close fitting between the experimental data and the model allowed accurate determinations of the advancing and receding contact angles, bridge geometry and volume. The successful implementation of the capillary force model allowed a link between the bridge volume, and the resulting adhesion to the friction force between hydrophobic solid surfaces in water. Additionally, the model allowed the change from an attractive to a repulsive NBCF to be described by a change from a concave to convex bridge geometry. Thus, this thesis has added considerable knowledge to the fundamental aspects of nanobubbles and the NBCF. The final chapters of this thesis utilised the knowledge gained from the fundamental studies to understand the influence of nanobubbles on flotation. In the first study, the influence of NaCl concentration on the morphology of gaseous domains on a graphite surface is discussed in relation to the increased recovery of coal in saline water. In the second study, methanol treatment of a ZnS ore was found to increase the floatability due to slime removal and the artificial formation of nanobubbles.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: p. 198-203.

Les globules gras du lait sont entourés d’une membrane biologique extrêmement complexe en composition et en structure, appelée MFGM (milk fat globule membrane). L’investigation de cette membrane, in situ dans le lait, par microscopie confocale nous suggère que les lipides polaires à haute température de transition de phase (Tm) forment des domaines en phase gel ou liquide ordonné, dispersés dans une phase continue fluide. Sur la base de cette observation, ce projet vise à comprendre en quoi la composition en lipides polaires laitiers et leur état de phase peuvent moduler les propriétés élastiques de la MFGM, en vue d’une meilleure maîtrise de la stabilité des globules gras en industrie laitière.L’hétérogénéité mécanique générée par la coexistence de différents types de phase a ainsi été caractérisée par spectroscopie de force AFM en utilisant des bicouches de lipides modèles de la membrane réelle, à basse (T<Tm) et haute températures (T>Tm). Pour analyser finement les déterminants de l’élasticité de la membrane, et tenir compte de la courbure, une étude approfondie des effets de l’état de phase et de la composition hétérogène en lipides polaires a été entreprise par spectroscopie de force atomique, en complément d’une analyse structurale par microscopie électronique ou diffraction des rayons X. Nous y avons montré, en particulier, que la présence de molécules de longueur de chaîne acyles et d’insaturation variables rend les membranes de sphingomyéline de lait en phase gel moins rigides qu’attendu, bien que significativement plus rigide qu’une membrane fluide. Cette approc / The milk fat globules are enveloped by a biological membrane, called MFGM, of highly complex composition and structure. Investigation of this membrane, in situ in milk, using confocal microscopy suggested that polar lipids with high transition temperature (Tm) form domains in gel or liquid-ordered phase, dispersed in a continuous fluid phase. From this observation, the aim of this project was to understand how the composition and organization of dairy polar lipids can modulate the elastic properties of the MFGM, in order to better control stability of the fat globules in the dairy industry. The mechanical heterogeneity created by the coexistence of phases was then characterized by AFM force spectroscopy using lipid bilayers models at low (T<Tm) and high temperatures (T>Tm).In order to closely analyze the factors that direct membrane elasticity, force spectroscopy measurements were undertaken on curved liposome membranes, in combination with structural characterization by TEM and SAXS. We showed, in particular, that heterogeneity in acyl chain length and unsaturation made gel-phase milk sphingomyelin membranes less rigid than expected, although more rigid than a fluid phase membrane. This approach was finally applied to native milk fat globules, where mechanical heterogeneity was visible. However, elasticity values were somewhat different from those calculated on model systems, probably because of the presence of membrane proteins.

With increase in the number of three dimensional (3-D) nanometer-scale objects that are being either fabricated or studied, there is a need to accurately characterize their geometry. While the Atomic force microscope (AFM) is a versatile tool for performing nano-metrology, it suffers from issues of poor accessibility of 3-D features and inability to measure 3-D forces that limit its applicability in 3-D nano-metrology. This thesis investigates the design and development of a novel probing system based on AFM that improves accessibility and enables direct measurement of 3-D forces acting on the AFM tip. Two approaches are investigated to address the issue of poor accessibility. The first is to develop a novel system that enables in-situ replacement and reuse of specialized AFM tips that improve accessibility, and the second is to design a special AFM tip that can actively re-orient about two independent axes. In order to perform in-situ tip replacement, a liquid meniscus based micro-gripper is developed and integrated on to a conventional AFM probe. The stiffness of the gripper is analyzed and shown to be adequately high along all three axes for AFM imaging to be performed. Tip replacement and re-use are both experimentally demonstrated by employing a novel tip-exchange station. The replaced tips are employed to show artifact-free AFM imaging of a standard calibration grating in both tapping-mode and contact-mode. To actively re-orient a conventional tip, a novel magnetically-actuated micro-scale ball-and-socket joint is integrated onto an AFM probe. The quasi-static behavior of the joint is experimentally characterized, and the ability of the tip to independently re-orient about two axes is demonstrated. The achieved range is about +/- 90 degrees about both X- and Y-axes. In order to realize the potential of the proposed probes for 3-D nano-metrology, an AFM is developed in-house that possesses the capability to make direct measurement of 3-D forces. Optimization of the measurement system to achieve identical sensitivities and resolution along all three axes is studied. Subsequently, the necessary electronics for measurement, actuation and control are developed. All the subsystems are experimentally calibrated and integrated. The overall AFM is shown to have a resolution of about 0.2 nm when operated in tapping-mode. The developed AFM is employed to showcase the following applications: characterization of the coefficient of kinetic friction of Muscovite mica, force controlled nano-scribing on polymethyl methacrylate (PMMA) and tapping-mode imaging of a calibration grating with the developed re-orientable AFM probe. Finally, the unique ability of the re-orientable AFM probe to control its tip-orientation is employed to develop a nanometer-scale coordinate measurement machine (CMM). The developed nano-CMM is shown to access the vertical wall of a sample and obtain its topography.