Wearing a single DNA molecule with an AFM tip

While the fundamental limit on the resolution achieved in an atomic force microscope (AFM) is clearly related to the tip radius, the fact that the tip can creep and/or wear during an experiment is often ignored. This is mainly due to the difficulty in characterizing the tip, and in particular a lack of reliable methods that can achieve this in situ. Here, we provide an in situ method to characterize the tip radius and monitor tip creep and/or wear and biomolecular sample wear in ambient dynamic AFM. This is achieved by monitoring the dynamics of the cantilever and the critical free amplitude to observe a switch from the attractive to the repulsive regime. The method is exemplified on the mechanically heterogeneous sample of single DNA molecules bound to mica mineral surfaces. Simultaneous monitoring of apparent height and width of single DNA molecules while detecting variations in the tip radius R as small as one nanometer are demonstrated. The yield stress can be readily exceeded for sharp tips (R10nm). The ability to know the AFM tip radius in situ and in real-time opens up the future for quantitative nanoscale materials properties determination at the highest possible spatial resolution.Comment: 26 pages, 6 figure

Cantilever dynamics in amplitude modulation AFM: continuous and discontinuous transitions

Transitions between the attractive and the repulsive force regimes for amplitude modulation atomic force microscopy (AFM) can be either discontinuous, with a corresponding jump in amplitude and phase, or continuous and smooth. During the transitions, peak repulsive and average forces can be up to an order of magnitude higher when these are discrete. Under certain circumstances, for example, when the tip radius is relatively large (e.g. R > 20–30 nm) and for high cantilever free amplitudes (e.g. A0 > 40–50 nm), the L state can be reached with relatively low set-points only (e.g. Asp/A0 < 0.30). We find that these cases do not generally lead to higher resolution but increase the background noise instead. This is despite the fact that the imaging can be non-contact under these conditions. The appearance of background noise is linked to increasing cantilever mean deflection and tip–surface proximity with increasing free amplitude in the L state. Cantilever dynamics in amplitude modulation AFM: Continuous and discontinuous transitions (PDF Download Available). Available from: https://www.researchgate.net/publication/231025693_Cantilever_dynamics_in_amplitude_modulation_AFM_Continuous_and_discontinuous_transitions [accessed Mar 27, 2017].Peer ReviewedPostprint (author's final draft

Real Space Imaging of Nanoparticle Assembly at Liquid-Liquid Interfaces with Nanoscale Resolution.

Bottom up self-assembly of functional materials at liquid-liquid interfaces has recently emerged as method to design and produce novel two-dimensional (2D) nanostructured membranes and devices with tailored properties. Liquid-liquid interfaces can be seen as a "factory floor" for nanoparticle (NP) self-assembly, because NPs are driven there by a reduction of interfacial energy. Such 2D assembly can be characterized by reciprocal space techniques, namely X-ray and neutron scattering or reflectivity. These techniques have drawbacks, however, as the structural information is averaged over the finite size of the radiation beam and nonperiodic isolated assemblies in 3D or defects may not be easily detected. Real-space in situ imaging methods are more appropriate in this context, but they often suffer from limited resolution and underperform or fail when applied to challenging liquid-liquid interfaces. Here, we study the surfactant-induced assembly of SiO2 nanoparticle monolayers at a water-oil interface using in situ atomic force microscopy (AFM) achieving nanoscale resolved imaging capabilities. Hitherto, AFM imaging has been restricted to solid-liquid interfaces because applications to liquid interfaces have been hindered by their softness and intrinsic dynamics, requiring accurate sample preparation methods and nonconventional AFM operational schemes. Comparing both AFM and grazing incidence X-ray small angle scattering data, we unambiguously demonstrate correlation between real and reciprocal space structure determination showing that the average interfacial NP density is found to vary with surfactant concentration. Additionally, the interaction between the tip and the interface can be exploited to locally determine the acting interfacial interactions. This work opens up the way to studying complex nanostructure formation and phase behavior in a range of liquid-liquid and complex liquid interfaces

Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels

Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care

Smoking in asthma is associated with elevated levels of corticosteroid resistant sputum cytokines—an exploratory study

&lt;p&gt;Background: Current cigarette smoking is associated with reduced acute responses to corticosteroids and worse clinical outcomes in stable chronic asthma. The mechanism by which current smoking promotes this altered behavior is currently unclear. Whilst cytokines can induce corticosteroid insensitivity in-vitro, how current and former smoking affects airway cytokine concentrations and their responses to oral corticosteroids in stable chronic asthma is unclear.&lt;/p&gt; &lt;p&gt;Objectives: To examine blood and sputum cytokine concentrations in never, ex and current smokers with asthma before and after oral corticosteroids.&lt;/p&gt; &lt;p&gt;Methods: Exploratory study utilizing two weeks of oral dexamethasone (equivalent to 40 mg/day prednisolone) in 22 current, 21 never and 10 ex-smokers with asthma. Induced sputum supernatant and plasma was obtained before and after oral dexamethasone. 25 cytokines were measured by multiplex microbead system (Invitrogen, UK) on a Luminex platform.&lt;/p&gt; &lt;p&gt;Results: Smokers with asthma had elevated sputum cytokine interleukin (IL) -6, -7, and -12 concentrations compared to never smokers with asthma. Few sputum cytokine concentrations changed in response to dexamethasone IL-17 and IFNα increased in smokers, CCL4 increased in never smokers and CCL5 and CXCL10 reduced in ex-smokers with asthma. Ex-smokers with asthma appeared to have evidence of an ongoing corticosteroid resistant elevation of cytokines despite smoking cessation. Several plasma cytokines were lower in smokers wi&lt;/p&gt; &lt;p&gt;Conclusion: Cigarette smoking in asthma is associated with a corticosteroid insensitive increase in multiple airway cytokines. Distinct airway cytokine profiles are present in current smokers and never smokers with asthma and could provide an explanatory mechanism for the altered clinical behavior observed in smokers with asthma.&lt;/p&gt

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP > Heparin or HepS > DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies