Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.

Every biosensor, bioengineered scaffold or biomedical implant depends crucially on an ability to control protein adsorption at the material surface. Yet the adsorption of proteins to solid surfaces in aqueous media is a complex and poorly understood phenomenon. To gain further insights we study protein adsorption using the quartz crystal microbalance for 10 model globular proteins interacting with positive, negative, neutral, hydrophobic and mixed alkanethiol monolayers as well as silica, polystyrene and Teflon, equating to approximately 200 protein–surface combinations. The charge state of the materials in liquid was measured with atomic force microscopy using a colloidal probe and numerically solving the full non-linear Poisson–Boltzmann equation. This approach has allowed us to address some of the important questions surrounding the basic principles that govern protein adsorption including the relative importance of net charge and hydrophobicity and why some materials are protein resistant. With our set of mixed monolayer surfaces, we can modulate charge over a wide range whilst eliminating hydrophobic interactions and vice versa – thus permitting determination of the functional dependence of adsorption on these parameters. This has led us to develop two empirical predictive models with up to 90% accuracy that together encompass most materials relevant to biotechnological and biomedical applications.

Healthcare has advanced significantly, bringing with it longer life expectancies and a growing population of elders who suffer from dementia, specifically Alzheimer’s disease (AD). The amyloid beta (Aβ) peptide has been implicated in the cause of AD, where the peptides undergo a conformational change and form neurotoxic amyloid oligomers which cause neuronal cell death. While AD has no cure, preventative measures are being designed to either slow down or stop the progression of this neurodegenerative disease. One of these measures involves dietary supplements with polyunsaturated fatty acids such as docosahexaenoic acid (DHA). This omega-3 fatty acid is a key component of brain development and has been suggested to reduce the progression of cognitive decline. However, different studies have yielded different results as to whether DHA has positive, negative, or no effects on Aβ fibril formation. We believe that these discrepancies can be explained with varying concentrations of DHA. Here, we test the inhibitory effect of different concentrations of DHA on amyloid fibril formation using atomic force microscopy. Our results show that DHA has a strong inhibitory effect on Aβ_1–42 fibril formation at lower concentrations (50% reduction in fibril length) than higher concentrations above its critical micelle concentration (70% increase in fibril length and three times the length of those at lower concentrations). We provide evidence that various concentrations of DHA can play a role in the inhibitory effects of amyloid fibril formation in vitro and help explain the discrepancies observed in previous studies.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.