Ultraflat Gold QCM Electrodes Fabricated with Pressure-Forming Template Stripping for Protein Studies at the Nanoscale.

Single-molecule imaging of proteins using atomic force microscopy (AFM) is crucially dependent on protein attachment to ultraflat substrates. The template-stripping (TS) technique, which can be used to create large areas of atomically flat gold, has been used to great effect for this purpose. However, this approach requires an epoxy, which can swell in solution, causing surface roughening and substantially increasing the thickness of any sample, preventing its use on acoustic resonators in liquid. Diffusion bonding techniques should circumvent this problem but cannot be used on samples containing patterned features with mismatched heights because of cracking and poor transfer. Here, we describe a new technique called pressure-forming TS (PTS), which permits an ultraflat (0.35 ± 0.05 nm root-mean-square roughness) layer of gold to be transferred to the surface of a patterned substrate at low temperature and pressure. We demonstrate this technique by modifying a quartz crystal microbalance (QCM) sensor to contain an ultraflat gold surface. Standard QCM chips have substantial roughness, preventing AFM imaging of proteins on the surface after measurement. With our approach, there is no need to run samples in parallel: the modified QCM chip is flat enough to permit high-contrast AFM imaging after adsorption studies have been conducted. The PTS-QCM chips are then used to demonstrate adsorption of bovine serum albumin in comparison to rough QCM chips. The ability to attach thin layers of ultraflat metals to surfaces of heterogeneous nature without epoxy will have many applications in diverse fields where there is a requirement to observe nanoscale phenomena with multiple techniques, including surface and interfacial science, optics, and biosensing.

Melatonin directly interacts with cholesterol and alleviates cholesterol effects in dipalmitoylphosphatidylcholine monolayers.

Melatonin is a pineal hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimer's disease, and certain types of cancers. Cholesterol is a major membrane constituent with both a structural and functional influence. It is also known that melatonin readily partitions into cellular membranes. We investigated the effects of melatonin and cholesterol on the structure and physical properties of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer as a simple membrane model using the Langmuir-Blodgett (L-B) monolayer technique and molecular dynamics (MD) simulations. We report that melatonin increases the area per lipid and elastic compressibility of the DPPC monolayer in a concentration dependent manner, while cholesterol has the opposite effect. When both melatonin and cholesterol were present in the monolayer, the compression isotherms showed normalization of the area per molecule towards that of the pure DPPC monolayer, thus indicating that melatonin counteracts and alleviates cholesterol's effects. Atomistic MD simulations of melatonin enriched DPPC systems correlate with our experimental findings and illustrate the structural effects of both cholesterol and melatonin. Our results suggest that melatonin is able to lessen the influence of cholesterol through two different mechanisms. Firstly, we have shown that melatonin has a fluidizing effect on monolayers comprising only lipid molecules. Secondly, we also observe that melatonin interacts directly with cholesterol. Our findings suggest a direct nonspecific interaction of melatonin may be a mechanism involved in reducing cholesterol associated membrane effects, thus suggesting the existence of a new mechanism of melatonin's action. This may have important biological relevance in addition to the well-known anti-oxidative and receptor binding effects.

Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy.

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.

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

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.

A simple bioconjugate attachment protocol for use in single molecule force spectroscopy experiments based on mixed self-assembled monolayers.

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.

Understanding how charge and hydrophobicity influence globular protein adsorption to alkanethiol and material surfaces.

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.

Core-Shell Electrospun Polycrystalline ZnO Nanofibers for Ultra-Sensitive NO2 Gas Sensing.

This Research Article discusses the growth of polycrystalline, self-supporting ZnO nanofibers, which can detect nitrogen dioxide (NO2) gas down to 1 part per billion (ppb), one of the smallest detection limits reported for NO2 using ZnO. A new and innovative method has been developed for growing polycrystalline ZnO nanofibers. These nanofibers have been created using core-shell electrospinning of inorganic metal precursor zinc neodecanoate, where growth occurs at the core of the nanofibers. This process produces contamination-free, self-supporting, polycrystalline ZnO nanofibers of an average diameter and grain size 50 and 8 nm, respectively, which are ideal for gas sensing applications. This process opens up an exciting opportunity for creating nanofibers from a variety of metal oxides, facilitating many new applications especially in the areas of sensors and wearable technologies.

All Subdomains of the Talin Rod Are Mechanically Vulnerable and May Contribute To Cellular Mechanosensing.

Although the relevance of mechanotransduction in cell signaling is currently appreciated, the mechanisms that drive this process remain largely unknown. Mechanical unfolding of proteins may trigger distinct downstream signals in cells, providing a mechanism for cellular mechanotransduction. Force-induced unfolding of talin, a prominent focal adhesion protein, has been demonstrated previously for a small portion of its rod domain. Here, using single-molecule atomic force microscopy (smAFM), we show that the entire talin rod can be unfolded by mechanical extension, over a physiological range of forces between 10 and 40 pN. We also demonstrate, through a combination of smAFM and steered molecular dynamics, that the different bundles within the talin rod exhibit a distinct hierarchy of mechanical stability. These results provide a mechanism by which different force conditions within the cell control a graduated unfolding of the talin rod. Mechanical unfolding of the rod subdomains, and the subsequent effect on talin's binding interactions, would allow for a finely tuned cellular response to internally or externally applied forces.

Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment.

Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour.

Cu(2+) affects amyloid-ß (1-42) aggregation by increasing peptide-peptide binding forces.

The link between metals, Alzheimer's disease (AD) and its implicated protein, amyloid-ß (Aß), is complex and highly studied. AD is believed to occur as a result of the misfolding and aggregation of Aß. The dyshomeostasis of metal ions and their propensity to interact with Aß has also been implicated in AD. In this work, we use single molecule atomic force spectroscopy to measure the rupture force required to dissociate two Aß (1-42) peptides in the presence of copper ions, Cu(2+). In addition, we use atomic force microscopy to resolve the aggregation of Aß formed. Previous research has shown that metal ions decrease the lag time associated with Aß aggregation. We show that with the addition of copper ions the unbinding force increases notably. This suggests that the reduction of lag time associated with Aß aggregation occurs on a single molecule level as a result of an increase in binding forces during the very initial interactions between two Aß peptides. We attribute these results to copper ions acting as a bridge between the two peptide molecules, increasing the stability of the peptide-peptide complex.

A novel and cost-effective monitoring approach for outcomes in an Australian biodiversity conservation incentive program.

We report on the design and implementation of ecological monitoring for an Australian biodiversity conservation incentive scheme - the Environmental Stewardship Program. The Program uses competitive auctions to contract individual land managers for up to 15 years to conserve matters of National Environmental Significance (with an initial priority on nationally threatened ecological communities). The ecological monitoring was explicitly aligned with the Program's policy objective and desired outcomes and was applied to the Program's initial Project which targeted the critically endangered White Box-Yellow Box-Blakely's Red Gum Grassy Woodland and Derived Native Grassland ecological community in south eastern Australia. These woodlands have been reduced to <3% of their original extent and persist mostly as small remnants of variable condition on private farmland. We established monitoring sites on 153 farms located over 172,232 sq km. On each farm we established a monitoring site within the woodland patch funded for management and, wherever possible, a matched control site. The monitoring has entailed gathering data on vegetation condition, reptiles and birds. We also gathered data on the costs of experimental design, site establishment, field survey, and data analysis. The costs of monitoring are approximately 8.5% of the Program's investment in the first four years and hence are in broad accord with the general rule of thumb that 5-10% of a program's funding should be invested in monitoring. Once initial monitoring and site benchmarking are completed we propose to implement a novel rotating sampling approach that will maintain scientific integrity while achieving an annual cost-efficiency of up to 23%. We discuss useful lessons relevant to other monitoring programs where there is a need to provide managers with reliable early evidence of program effectiveness and to demonstrate opportunities for cost-efficiencies.

Effect of surfaces on amyloid fibril formation.

Using atomic force microscopy (AFM) we investigated the interaction of amyloid beta (Aß) peptide with chemically modified surfaces in order to better understand the mechanism of amyloid toxicity, which involves interaction of amyloid with cell membrane surfaces. We compared the structure and density of Aß fibrils on positively and negatively charged as well as hydrophobic chemically-modified surfaces at physiologically relevant conditions. We report that due to the complex distribution of charge and hydrophobicity amyloid oligomers bind to all types of surfaces investigated (CH3, COOH, and NH2) although the charge and hydrophobicity of surfaces affected the structure and size of amyloid deposits as well as surface coverage. Hydrophobic surfaces promote formation of spherical amorphous clusters, while charged surfaces promote protofibril formation. We used the nonlinear Poisson-Boltzmann equation (PBE) approach to analyze the electrostatic interactions of amyloid monomers and oligomers with modified surfaces to complement our AFM data.