Understanding interactions of plasticisers with a phospholipid monolayer.

The use of DEHP (diethylhexyl phthalate) is now banned for most applications in Europe; the exception is for blood bags, where its toxicity is overshadowed by its ability to extend the storage life of red blood cells. Another plasticiser, BTHC (butanoyl trihexyl citrate), is used in paediatric blood bags but does not stabilise blood cells as effectively. Interactions between plasticisers and lipids are investigated with a phospholipid, DMPC, to understand the increased stability of blood cells in the presence of DEHP as well as bioaccumulation and identify differences with BTHC. Mixed monolayers of DMPC and DEHP or BTHC were studied on Langmuir troughs where surface pressure/area isotherms can be measured. Neutron reflection measurements were made to determine the composition and structure of these mixed layers. A large amount of plasticiser can be incorporated into a DMPC monolayer but once an upper limit is reached, plasticiser is selectively removed from the interface at high surface pressures. The upper limit is found to occur between 40-60 mol% for DEHP and 20-40 mol% for BTHC. The areas per molecule are also different with DEHP being in the range of 50-100 Å2 and BTHC being 65-120 Å2. Results indicate that BTHC does not fit as well as DEHP in DMPC monolayers which could help explain the differences observed with regards to the stability of blood cells.

Adsorption Behavior of the Coenzyme NADH at the Carbon/Electrolyte Interface Determined by Neutron Reflectometry.

The adsorption behavior of ß-nicotinamide adenine dinucleotide (NADH) at the carbon/electrolyte interface has been studied using a combination of neutron reflectometry (NR) and solution depletion isotherms. Coupling the NR technique with an electrochemical cell allowed in situ observation of the reversible adsorption and desorption of the molecule at the electrode surface over a range of applied potentials. The overall surface coverage was low (30-50%), suggesting adsorption only at specific defect sites on the surface. Isotherms conducted over a range of temperatures were used to extract thermodynamic parameters, which implied strong physisorption via electrostatic interactions. In addition, changes in the outermost layer of the carbon electrode were observed as the applied potential was varied, which were confirmed with ex situ X-ray reflectivity measurements (XRR). X-ray photoelectron spectroscopy (XPS) measurements of the carbon surface demonstrated the majority of carbon atoms were in an sp2 state.

Effect of Anionic Lipids on Mammalian Plasma Cell Membrane Properties.

The effect of lipid composition on models of the inner leaflet of mammalian cell membranes has been investigated. Grazing incidence X-ray diffraction and X-ray and neutron reflectivity have been used to characterize lipid packing and solvation, while electrochemical and infrared spectroscopic methods have been employed to probe phase behavior in an applied electric field. Introducing a small quantity of the anionic lipid dimyristoylphosphatidylserine (DMPS) into bilayers of zwitterionic dimyristoylphosphatidylethanolamine (DMPE) results in a significant change in the bilayer response to an applied field: the tilt of the hydrocarbon chains increases before returning to the original tilt angle on detachment of the bilayer. Equimolar mixtures, with slightly closer chain packing, exhibit a similar but weaker response. The latter also tend to incorporate more solvent during this electrochemical phase transition, at levels similar to those of pure DMPS. Reflectivity measurements reveal greater solvation of lipid layers for DMPS > 30 mol %, matching the greater propensity for DMPS-rich bilayers to incorporate water. Taken together, the data indicate that the range of 10-35 mol % DMPS provides optimum bilayer properties (in flexibility and function as a barrier), which may explain why the DMPS content of cell membranes tends to be found within this range.

Fully Aqueous Self-Assembly of a Gold-Nanoparticle-Based Pathogen Sensor.

Surface plasmon resonance (SPR) is a very sensitive measure of biomolecular interactions but is generally too expensive for routine analysis of clinical samples. Here we demonstrate the simplified formation of virus-detecting gold nanoparticle (AuNP) assemblies on glass using only aqueous buffers at room temperature. The AuNP assembled on silanized glass and displayed a distinctive absorbance peak due to the localized SPR (LSPR) response of the AuNPs. Next, assembly of a protein engineering scaffold was followed using LSPR and a sensitive neutron reflectometry approach, which measured the formation and structure of the biological layer on the spherical AuNP. Finally, the assembly and function of an artificial flu sensor layer consisting of an in vitro-selected single-chain antibody (scFv)-membrane protein fusion was followed using the LSPR response of AuNPs within glass capillaries. In vitro selection avoids the need for separate animal-derived antibodies and allows for the rapid production of low-cost sensor proteins. This work demonstrates a simple approach to forming oriented arrays of protein sensors on nanostructured surfaces that uses (i) an easily assembled AuNP silane layer, (ii) self-assembly of an oriented protein layer on AuNPs, and (iii) simple highly specific artificial receptor proteins.

Probing the Separation Distance between Biological Nanoparticles and Cell Membrane Mimics Using Neutron Reflectometry with Sub-Nanometer Accuracy.

Nanoparticle interactions with cellular membranes are controlled by molecular recognition reactions and regulate a multitude of biological processes, including virus infections, biological nanoparticle-mediated cellular communication, and drug delivery applications. Aided by the design of various supported cell membrane mimics, multiple methods have been employed to investigate these types of interactions, revealing information on nanoparticle coverage, interaction kinetics, as well as binding strength; however, precise quantification of the separation distance across which these delicate interactions occur remains elusive. Here, we demonstrate that carefully designed neutron reflectometry (NR) experiments followed by an attentive selection and application of suitable theoretical models offer a means to quantify the distance separating biological nanoparticles from a supported lipid bilayer (SLB) with sub-nanometer precision. The distance between the nanoparticles and SLBs was tuned by exploiting either direct adsorption or specific binding using DNA tethers with different conformations, revealing separation distances of around 1, 3, and 7 nm with nanometric accuracy. We also show that NR provides precise information on nanoparticle coverage, size distribution, material composition, and potential structural changes in the underlying planar SLB induced upon nanoparticle binding. The precision with which these parameters could be quantified should pave an attractive path for investigations of the interactions between nanoparticles and interfaces at length scales and resolutions that were previously inaccessible. This thus makes it possible to, for example, gain an in-depth understanding of the molecular recognition reactions of inorganic and biological nanoparticles with cellular membranes.

Systematic in situ hydration neutron reflectometry study on Nafion thin films.

Reported herein is a neutron reflectometry (NR) study on hydrated Nafion thin films (∼30 nm) on a silicon substrate with native oxide. The Nafion morphology is investigated systematically across the whole relative humidity range using both H2O and D2O vapours to enable a comparative study. By utilising this systematic approach two key results have been obtained. The first is that by leveraging the strong positive scattering signal from the D2O vapour, a complete and systematic water adsorption isotherm (Type II) for a Nafion thin film is produced. Utilising the slight negative scattering signal of the H2O enabled the quantification of the hydration dependent evolution of the formation of Nafion/water lamellae near the substrate surface. The number of lamellae layers increases continuously with hydration, and does not form abruptly. We also report the effects of swelling on the thin films across the relative humidity ranges. The work reported should prove useful in quantifying other hydration dependent properties of Nafion thin films such as conductivity and understanding Nafion/semiconductor based devices, as well as showcasing a NR methodology for other hydrophilic polymers.

Spontaneous Formation of Cushioned Model Membranes Promoted by an Intrinsically Disordered Protein.

In this article, it is shown that by exposing commonly used lipids for biomembrane mimicking studies, to a solution containing the histidine-rich intrinsically disordered protein histatin 5, a protein cushion spontaneously forms underneath the bilayer. The underlying mechanism is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Hence, we anticipate that this novel "green" approach of forming cushioned bilayers can be an important tool to mimic the cell membrane without the disturbance of the solid substrate, thereby achieving a further understanding of protein-cell interactions.

Structural Changes in Adsorbed Cytochrome c upon Applied Potential Characterized by Neutron Reflectometry.

The structural behavior of an electron-transfer protein, cytochrome c, at the 316L stainless steel electrode/aqueous interface was investigated over a range of applied potentials using neutron reflectometry supported by solution depletion isotherms, X-ray reflectometry, and quartz crystal microbalance measurements. A custom-made electrochemical cell allowed in situ observation of the adsorbed protein across a range of applied potentials; models fitted to the NR data showed a compact inner protein layer at the metal/electrolyte interface and a further thicker but highly diffuse layer that could be removed by rinsing. The overall amount adsorbed was found to be strongly dependent on the applied potential and buffer pH. Subtle but significant changes in the structure of the adsorbed protein layer were seen as the potential was swept between ±0.40 V, reflecting changing attractive/repulsive interactions between the protein's charged side groups and the surface. At greater applied potentials, irreversible changes in the stainless steel film structure were also observed and attributed to deuterium absorption into the metal.

Potassium, Calcium, and Magnesium Bridging of AOT to Mica at Constant Ionic Strength.

The bridging effect of a series of common cations between the anionic mica surface and the AOT anion has been studied in a condition of constant ionic strength and surfactant concentration. It was found that sodium ions did not show any bridging effect in this system; however, calcium, magnesium, and potassium all caused adsorption of the organic to the mica surface. The concentrations at which bridging occurred was probed, revealing that only a very low bridging cation concentration was required for binding. The bridged layer stability was also investigated, and the interaction was shown to be a weak one, with the bound layer in equilibrium with the species in the bulk and easily removed. Even maintaining ionic strength and bridging ion concentration was not sufficient to retain the layer when the free organic in solution was removed.

An X-ray and Neutron Reflectometry Study of Iron Corrosion in Seawater.

The corrosive breakdown of thin iron films supported on silicon substrates under a number of conditions is presented-in particular to understand better how iron, and hence ferritic steel, behaves in a salty water environment. A combination of X-ray and neutron reflectometry was used to monitor the structures of both metal and oxide surface layers and also organic corrosion inhibitors adsorbed at the iron/aqueous interface. A range of behavior in seawater was observed, including complete dissolution and void formation under the metal surface. Importantly, two simple treatments-UV/ozone or soaking in ultrapure water-were found to significantly protect the iron surface for considerable lengths of time, although evidence of pitting corrosion began after around 10 days. The underlying causes of the efficacies of these treatments were further investigated using X-ray photoelectron spectroscopy. In addition, three potential corrosion inhibitors were investigated: (i) dodecyltrimethylammonium bromide (DTAB) demonstrated no ability to protect the surface; (ii) sodium dodecyl sulfate (SDS) appeared to accelerate corrosion; and (iii) bis(2-ethylhexyl)phosphate showed an impressive level of protection (the neutron reflectometry results indicated a thick diffuse layer of surfactant of 23% surface coverage). These findings have been interpreted in terms of preferential inhibitor adsorption at cathodic and anodic surface sites (depending on the nature of the inhibitor).

Neutron Reflectometry of an Anionic Surfactant at the Solid-Liquid Interface under Shear.

Neutron reflectometry with in situ rheology is used to measure the shear response of an adsorbed anionic surfactant (sodium bis(2-ethylhexyl) sulfosuccinate, AOT) at the alumina-water interface. A low surfactant concentration is measured where a single bilayer adsorbs at the interface as well as a higher concentration where a multilamellar structure forms. The low concentration structure does not change with the imposed shear (oscillatory or steady). However, the lamellar phase shows a loss of structure under both steady and oscillatory shear. There are differences between the steady and oscillatory cases, which are discussed, with both showing a strong dependence on the strain amplitude.

Adsorption of Methyl Ester Sulfonate at the Air-Water Interface: Can Limitations in the Application of the Gibbs Equation be Overcome by Computer Purification?

We describe a new laboratory synthesis of the α-methyl ester sulfonates based on direct sulfonation of the methyl ester by SO3 introduced from the vapor phase. This was used to synthesize a chain deuterated sample of αC14MES, which was then used to measure the surface excess of αC14MES directly at the air/water interface over a wide range of concentration using neutron reflection. The adsorption isotherm could be fitted to an empirical equation close to a Langmuir isotherm and gave a limiting surface excess of (3.4 ± 0.1) × 10-6 mol m-2 in the absence of added electrolyte. The neutron-measured surface excesses were combined with the integrated Gibbs equation to fit the variation in surface tension with concentration (σ-ln C curve). The fit was exact provided that we used a prefactor consistent with the counterion at the surface being an impurity divalent ion, as has previously been found for sodium diethylhexylsulfosuccinate (aerosol OT or AOT) and various perfluorooctanoates. The critical micelle concentration (CMC) was determined from this fit to be 2.4 ± 0.3 mM in the absence of electrolyte. In the presence of 100 mM NaCl, this contamination was suppressed and the σ-ln C curve could be fitted using the integrated Gibbs equation with the expected prefactor of 1. The new data were used to reinterpret measurements by Danov et al. on an unpurified sample of αC14MES for which computer refinement was used to try to eliminate the effects of the impurities.

An Anionic Surfactant on an Anionic Substrate: Monovalent Cation Binding.

Neutron reflectometry has been used to study the adsorption of the anionic surfactant bis(2-ethylhexyl) sulfosuccinate cesium salt on the anionic surface of mica. Evidence of significant adsorption is reported. The adsorption is reversible and changes little with pH. This unexpected adsorption behavior of an anionic molecule on an anionic surface is discussed in terms of recent models for surfactant adsorption such as cation bridging, where adsorption has been reported with the divalent ion calcium but not previously observed with monovalent ions.

Polarized Neutron Reflectometry of Nickel Corrosion Inhibitors.

Polarized neutron reflectometry has been used to investigate the detailed adsorption behavior and corrosion inhibition mechanism of two surfactants on a nickel surface under acidic conditions. Both the corrosion of the nickel surface and the structure of the adsorbed surfactant layer could be monitored in situ by the use of different solvent contrasts. Layer thicknesses and roughnesses were evaluated over a range of pH values, showing distinctly the superior corrosion inhibition of one negatively charged surfactant (sodium dodecyl sulfate) compared to a positively charged example (dodecyl trimethylammonium bromide) due to its stronger binding interaction with the surface. It was found that adequate corrosion inhibition occurs at significantly less than full surface coverage.

Neutron reflection study of the adsorption of the phosphate surfactant NaDEHP onto alumina from water.

The adsorption of a phosphorus analogue of the surfactant AOT, sodium bis(2-ethylhexyl) phosphate (NaDEHP), at the water/alumina interface is described. The material is found to adsorb as an essentially water-free bilayer from neutron reflection measurements. This is similar to the behavior of AOT under comparable conditions, although AOT forms a thicker, more hydrated layer. The NaDEHP shows rather little variation with added salt, but a small thickening of the layer on increasing the pH, in contrast to the behavior of AOT.

Experimental and simulation study of self-assembly and adsorption of glycerol monooleate in n-dodecane with varying water content onto iron oxide.

The self-assembly and surface adsorption of glycerol monooleate (GMO) in n-dodecane are studied using a combination of experimental and molecular dynamics simulation techniques. The self-assembly of GMO to form reverse micelles, with and without added water, is studied using small-angle neutron scattering and simulations. A large-scale simulation is also used to investigate the self-assembly kinetics. GMO adsorption onto iron oxide is studied using depletion isotherms, neutron reflectometry, and simulations. The adsorbed amounts of GMO, and any added water, are determined experimentally, and the structures of the adsorbed films are investigated using reflectometry. Detailed fitting and analysis of the reflectometry measurements are presented, taking into account various factors such as surface roughness, and the presence of impurities. The reflectometry measurements are complemented by molecular dynamics simulations, and good consistency between both approaches is demonstrated by direct comparison of measured and simulated reflectivity and scattering length density profiles. The results of this analysis are that in dry systems, GMO adsorbs as self-assembled reverse micelles with some molecules adsorbing directly to the surface through the polar head groups, while in wet systems, the GMO is adsorbed onto a thin layer of water. Only at high surface coverage is some water trapped inside a reverse-micelle structure; at lower surface coverages, the GMO molecules associate primarily with the water layer, rather than self-assemble.

Hexadecylamine adsorption at the iron oxide-oil interface.

The adsorption behavior of a model additive, hexadecylamine, onto an iron surface from hexadecane oil has been characterized using polarized neutron reflectometry, sum-frequency generation spectroscopy, solution depletion isotherm, and X-ray photoelectron spectroscopy (XPS). The amine showed a strong affinity for the metal surface, forming a dense monolayer at relatively low concentrations; a layer thickness of 16 (±3) Å at low concentrations, increasing to 20 (±3) Å at greater amine concentrations, was determined from the neutron data. These thicknesses suggest that the molecules in the layer are tilted. Adsorption was also indicated by sum-frequency generation spectroscopy and XPS, the latter indicating that the most dominant amine-surface interaction was via electron donation from the nitrogen lone pair to the positively charged iron ions. Sum-frequency generation spectroscopy was used to determine the alkyl chain conformation order and orientation on the surface.

Interfacial complexation of a neutral amphiphilic 'tardigrade' co-polymer with a cationic surfactant: Transition from synergy to competition.

HYPOTHESIS: Neutral amphiphilic PEG-g-PVAc co-polymer (a "tardigrade" polymer consisting of a hydrophilic polyethylene glycol, PEG, backbone with hydrophobic polyvinyl acetate, PVAc, grafts) can form complexes at the air-water interface with cationic dodecyltrimethylammonium bromide (DTAB) via self-assembly. Compared to anionic SDS, cationic DTAB headgroups are expected to interact strongly with the negatively charged OH- groups from the partial dissociation of the PVAc grafts. We anticipate a transition from synergistic to competitive behaviour, which is expected to be dependent on the surfactant structural characteristics and concentration. EXPERIMENTS: DTAB/PEG-g-PVAc mixtures were investigated using a combination of dynamic and equilibrium surface tension measurements, neutron reflectivity (NR) at the air-water interface, and foaming tests. We varied the concentrations of both the DTAB (0.05 to 5 critical micelle concentration, cmc) and that of PEG-g-PVAc (0.2 and 2 critical aggregation concentration, cac). FINDINGS: Our results show that the interfacial interactions between DTAB and PEG-g-PVAc were both synergistic and antagonistic, depending sensitively on the surfactant concentration. At DTAB concentrations below its cmc, a pronounced cooperative adsorption behaviour was likely driven by the hydrophobic interactions between the DTAB tail and the PVAc grafts and the attraction between the DTAB headgroups and the partially dissociated -O- groups in the partially hydrolysed PVAc grafts, forming a mixed layer. This synergistic adsorption behaviour transitioned to a competitive adsorption behaviour at DTAB concentrations above its cmc, leading to polymer-surfactant partition, forming a "hanging" polymer layer underlying a surfactant monolayer at the interface. We postulate that DTAB/PEG-g-PVAc complexation in the bulk contributed to partial depletion of the mixture from the interface. We therefore consider this polymer/surfactant system to be a moderately interacting system at the air-water interface. No discernible differences in the foaming behaviour were observed between the DTAB/PEG-g-PVAc systems and the pure surfactant. Our results suggest that surfactant headgroup characteristics (particularly charges) were crucial in determining the structure and composition of polymer-surfactant complexes at the air-water interface, as well as the foamability and foam stability, whilst the coexistence of the synergistic and competitive adsorption behaviour is attributed to the unique architecture of the tardigrade polymer with amphiphilicity and partial charge, facilitating different surfactant-polymer interactions at different DTAB concentrations.

MUC5B mucin films under mechanical confinement: A combined neutron reflectometry and atomic force microscopy study.

HYPOTHESIS: Among other functions, mucins hydrate and protect biological interfaces from mechanical challenges. Mucins also attract interest as biocompatible coatings with excellent lubrication performance. Therefore, it is of high interest to understand the structural response of mucin films to mechanical challenges. We hypothesized that this could be done with Neutron Reflectometry using a novel sample environment where mechanical confinement is achieved by inflating a membrane against the films. EXPERIMENTS: Oral MUC5B mucin films were investigated by Force Microscopy/Spectroscopy and Neutron Reflectometry both at solid-liquid interfaces and under mechanical confinement. FINDINGS: NR indicated that MUC5B films were almost completely compressed and dehydrated when confined at 1 bar. This was supported by Force Microscopy/Spectroscopy investigations. Force Spectroscopy also indicated that MUC5B films could withstand mechanical confinement by means of steric interactions for pressures lower than âˆ¼ 0.5 bar i.e., mucins could protect interfaces from mechanical challenges of this magnitude while keeping them hydrated. To investigate mucin films under these pressures by means of the employed sample environment for NR, further technological developments are needed. The most critical would be identifying or developing more flexible membranes that would still meet certain requirements like chemical homogeneity and very low roughness.