Neutron Reflectometry Experiments Research Articles

On the lipid flip-flop and phase transition coupling.

We measured the passive lipid flip-flop of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in solid supported lipid bilayers across their main gel to fluid (Lβ → Lα) phase transition. By performing time and temperature resolved neutron reflectometry experiments, we demonstrated that asymmetric systems prepared in the gel phase are stable for at least 24 hours. Lipid flip-flop was found to be intrinsically linked to the amount of lipid molecules in the fluid phase. Moreover, the increase of this amount during the broad phase transition was found to be the main key factor for the timing of the flip-flop process. By measuring different temperature scan rate, we could demonstrate that, in the case of supported bilayers and for the temperature investigated, the lipid flip flop is characterised by an activation energy of 50 kJ mol-1 and a timescale on the order of few hours. Our results demonstrate the origin on the discrepancies between passive flip-flop in bulk systems and at interfaces.

Investigating partitioning of free versus macrocycle bound guest into a model POPC lipid bilayer.

We report on the permeation of free and macrocycle-bound avobenzone across a POPC lipid bilayer through combined neutron reflectometry experiments and molecular dynamics simulations. Results indicate that the p-phosphonated calix[8]arene macrocycle limits the avobenzone penetration into the upper leaflet of the membrane. Hence, it could serve as a useful vehicle for safer formulations.

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers.

Understanding the molecular mechanisms governing nanoparticle-membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse-grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di-stearoyl-phosphatidylcholine (DSPC) and anionic di-stearoyl-phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC-DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP-membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse-grained MD simulations as a lipid-crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.

Molecular Mechanisms of the Interaction between Arf1 and ASAP1 pH Domain at the Membrane Interface

Pleckstrin Homology (PH) domains have dual functions: localizing proteins to specific membrane regions and modulating membrane protein function through interdomain interactions. Here, we focused on the PH domain of ASAP1, which is necessary for phosphatidylinositol 4, 5- bisphosphate (PIP2)- dependent conversion of Arf1, a member of the RAS superfamily, from its GTP to GDP form. The interaction of the PH domain with the lipid surface was studied by 15N, 13C methyl NMR and SPR, using PIP2 and phosphatidylserine (PS) - containing nanodiscs as mimics of the membrane surface. Comparison of the chemical shift perturbations within PH upon interaction with nanodiscs- containing a single PIP2, multiple PIP2 lipids or a single PIP2 and increasing PS mole fraction showed that (i) the two loops between β sheets 1, 2 and 3, 4 delineate two, equivalent and independent PIP2 lipid binding sites and (ii) the PH domain is attracted to the membrane surface predominantly by non- specific electrostatic interactions with the background anionic lipids, before docking to the target lipid PIP2. Constraints obtained from neutron reflectometry and Paramagnetic Relaxation NMR experiments suggest that the PH domain is oriented with its long axis parallel to the membrane normal. Interestingly, deletion of 14 amino acids immediately N term of the first beta strand of the PH domain strongly reduces ASAP1 catalytic efficiency even if the deletion has no effect on ASAP1 equilibrium binding to PIP2. Taken together, this suggests that simultaneous binding to two PIP2 strictly orients the PH domain at the membrane interface, promoting functionally critical contacts between Arf1 and the N-term of ASAP1.

Neutron reflectometry to measure in situ the rate determining step of lithium ion transport through thin silicon layers and interfaces.

Li ion transport through thin (14-22 nm) amorphous silicon layers adjacent to lithium metal oxide layers (lithium niobate) was studied by in situ neutron reflectometry experiments and the control mechanism was determined. It was found that the interface between amorphous silicon and the oxide material does not hinder Li transport. It is restricted by Li diffusion in the silicon material. This finding based on in situ experiments confirms results obtained ex situ and destructively by secondary ion mass spectrometry (SIMS) depth profiling investigations. The Li permeabilities obtained from the present experiments are in agreement with those obtained from ex situ SIMS measurements showing similar activation enthalpies.

Lipid composition in fungal membrane models: effect of lipid fluidity.

The creation of effective fungal membrane models for neutron and X-ray reflectometry experiments is a key step in the development of new antifungal pharmaceuticals and agrochemicals to allow in vitro investigation of their mode of interaction with target cells. The structure of the obtained models depends on the properties of the lipids used and the final composition of the leaflets, and can be subject to the spontaneous translocation of phospholipids across the bilayer. The effect of phospholipid acyl-chain unsaturation and the presence of steroids in the membrane on the bilayer asymmetry were examined by means of neutron reflectometry. The measurements showed that membrane stability was higher if a zwitterionic, saturated acyl-chain phospholipid is present as the inner leaflet. Furthermore, membrane asymmetry was higher in the case of fully saturated lipid systems. As a result, membrane models consisting of fully saturated acyl chains within the inner leaflet are recommended as the starting point for subsequent studies of antifungal interactions owing to the simplicity of the models and their relative stability, thus allowing better control over the exact lipid composition facing the tested antifungal.

On the in-depth density distribution of layered assemblies of Au nanoparticles on planar interfaces

The assemblies of optically active gold nanoparticles on a functionalized silicon planar substrate were considered in terms of the interface depth density distribution obtained by atomic-force microscopy, X-ray diffraction/reflectometry and neutron reflectometry. Two layers of nanoparticles were sequentially deposited using an additional cross-linking agent. First layer was made of nanoparticles being synthesized in light water while the second one in light/heavy water mixture. The isotope difference in layers observed in the neutron reflectometry experiment was related to water molecules incorporation into the organic shell around nanoparticles. The results are discussed with respect to the possibilities in resolving the structure of complex multilayer assemblies.

On Enhancement of the Adsorption-Layer Effect at the Metallic Electrode−Liquid Electrolyte Interface in Specular Neutron Reflectometry Experiments

The possibilities for optimizing the substrate/electrode/electrolyte structure are considered in order to obtain the maximum change in the specular-reflection curves obtained in neutron reflectometry experiments at the electrochemical interfaces between a metallic electrode and liquid electrolyte containing Li+ ions during their operation. The characteristic relations between the scattering length densities of the components, for which the reflection curves most fully provide information about the structure of the solid electrolyte interphase layer formed on the electrode surface during the charge–discharge processes, are determined and analyzed.

Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform

We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Interactions between model cell membranes and the neuroactive drug propofol

Vibrational sum frequency spectroscopy (VSFS) complemented by surface pressure isotherm and neutron reflectometry (NR) experiments were employed to investigate the interactions between propofol, a small amphiphilic molecule that currently is the most common general anaesthetic drug, and phospholipid monolayers. A series of biologically relevant saturated phospholipids of varying chain length from C18 to C14 were spread on either pure water or propofol (2,6-bis(1-methylethyl)phenol) solution in a Langmuir trough, and the change in the molecular structure of the film, induced by the interaction with propofol, was studied with respect to the surface pressure. The results from the surface pressure isotherm experiments revealed that propofol, as long as it remains at the interface, enhances the fluidity of the phospholipid monolayer. The VSF spectra demonstrate that for each phospholipid the amount of propofol in the monolayer region decreases with increasing surface pressure. Such squeeze out is in contrast to the enhanced interactions that can be exhibited by more complex amphiphilic molecules such as peptides. At surface pressures of 22–25 mN m−1, which are relevant for biological cell membranes, most of the propofol has been expelled from the monolayer, especially in the case of the C16 and C18 phospholipids that adopt a liquid condensed phase packing of its alkyl tails. At lower surface pressures of 5 mN m−1, the effect of propofol on the structure of the alkyl tails is enhanced when the phospholipids are present in a liquid expanded phase. Specifically, for the C16 phospholipid, NR data reveal that propofol is located exclusively in the head group region, which is rationalized in the context of previous studies. The results imply a non-homogeneous distribution of propofol in the plane of real cell membranes, which is an inference that requires urgent testing and may help to explain why such low concentration of the drug are required to induce general anaesthesia.

Design and realization of a sputter deposition system for the in situ- and in operando-use in polarized neutron reflectometry experiments

We report on the realization of a sputter deposition system for the in situ- and in operando-use in polarized neutron reflectometry experiments. Starting with the scientific requirements, which define the general design considerations, the external limitations and boundaries imposed by the available space at a neutron beamline and by the neutron and vacuum compatibility of the used materials, are assessed. The relevant aspects are then accounted for in the realization of our highly mobile deposition system, which was designed with a focus on a quick and simple installation and removability at the beamline. Apart from the general design, the in-vacuum components, the auxiliary equipment and the remote control via a computer, as well as relevant safety aspects are presented in detail.

Evidence of Formation of Superdense Nonmagnetic Cobalt

Because of the presence of 3d transition metals in the Earth’s core, magnetism of these materials in their dense phases has been a topic of great interest. Theory predicts a dense face-centred-cubic phase of cobalt, which would be nonmagnetic. However, this dense nonmagnetic cobalt has not yet been observed. Recent investigations in thin film polycrystalline materials have shown the formation of compressive stress, which can increase the density of materials. We have discovered the existence of ultrathin superdense nonmagnetic cobalt layers in a polycrystalline cobalt thin film. The densities of these layers are about 1.2–1.4 times the normal density of Co. This has been revealed by X-ray reflectometry experiments, and corroborated by polarized neutron reflectometry (PNR) experiments. Transmission electron microscopy provides further evidence. The magnetic depth profile, obtained by PNR, shows that the superdense Co layers near the top of the film and at the film-substrate interface are nonmagnetic. The major part of the Co film has the usual density and magnetic moment. These results indicate the possibility of existence of nonmagnetic Co in the earth’s core under high pressure.

Neutron detectors for the ESS diffractometers

The ambitious instrument suite for the future European Spallation Source whose civil construction started recently in Lund, Sweden, demands a set of diverse and challenging requirements for the neutron detectors. For instance, the unprecedented high flux expected on the samples to be investigated in neutron diffraction or reflectometry experiments requires detectors that can handle high counting rates, while the investigation of sub-millimeter protein crystals will only be possible with large-area detectors that can achieve a position resolution as low as 200 μm. This has motivated an extensive research and development campaign to advance the state-of-the-art detector and to find new technologies that can reach maturity by the time the ESS will operate at full potential. This paper presents the key detector requirements for three of the Time-of-Flight (TOF) diffraction instrument concepts selected by the Scientific Advisory Committee to advance into the phase of preliminary engineering design. We discuss the detector technologies commonly employed at the existing similar instruments and their major challenges for ESS. The detector technologies selected by the instrument teams to collect the diffraction patterns are also presented. Analytical calculations, Monte-Carlo simulations, and real experimental data are used to develop a generic method to estimate the event rate in the diffraction detectors. We apply this method to make predictions for the future diffraction instruments, and thus provide additional information that can help the instrument teams with the optimisation of the detector designs.

Amyloid-β peptides in interaction with raft-mime model membranes: a neutron reflectivity insight.

The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment.

Lithium Transport through Thin Silicon Layers: On the Origin of Bragg Peaks in Neutron Reflectometry Experiments

Lithium transport through ultrathin silicon layers can be measured non-destructively by neutron reflectometry (NR) using a multilayer composed of silicon layers embedded between solid state Li reservoirs. An established model system is a multilayer with a repetition of five [Si / natLiNbO3 / Si / 6LiNbO3] units. Two types of Bragg peaks are detectable in reflectivity patterns. These Bragg peaks result from the interference of neutrons reflected at periodic interfaces. One type of Bragg peak originates from the periodicity of the LiNbO3/Si chemical contrast (first order peak), while the other Bragg peak results from a superstructure with double periodicity. This superstructure may arise from 6Li/7Li isotope contrast or alternatively from periodic thickness variations, as shown by simulations based on the Parratt algorithm. The intention of the present paper was to elucidate the origin of the second Bragg peak. Experiments done by Secondary Ion Mass Spectrometry (SIMS) isotope sensitive depth profiling showed in a direct way that annealing at 360 °C destroys indeed the 6Li/7Li contrast, whereas the LiNbO3/Si chemical contrast remains unchanged. This evidences that the experimentally observed decrease of the second Bragg peak in the reflectivity pattern during annealing is a measure for Li transport through the Si layer.

Volume Expansion during Lithiation of Amorphous Silicon Thin Film Electrodes Studied by In-Operando Neutron Reflectometry

Amorphous silicon is a promising high-capacity anode material for the next generation of lithium-ion batteries. However, the enormous volume expansion of the active material during lithiation up to 400% (V/V0) is held responsible for capacity fading during cycling. In this study we measured continuously the volume modifications taking place during galvanostatic lithiation of amorphous silicon thin film electrodes by in-operando neutron reflectometry experiments. The results indicate (after initial effects) a linear increase in volume as a function of lithiation time and lithium content independent of current density and initial film thickness. The experimental results are in agreement with recent atomistic calculations.

Control of morphology and function of low band gap polymer–bis-fullerene mixed heterojunctions in organic photovoltaics with selective solvent vapor annealing

Replacing PCBM with a bis-adduct fullerene (i.e. ICBA) has been reported to significantly improve the open circuit voltage (VOC) and power conversion efficiency (PCE) in P3HT bulk heterojunctions. However, for the most promising low band-gap polymer (LBP) systems, replacing PCBM with ICBA results in very poor short-circuit current (JSC) and PCE although the VOC is significantly improved. Therefore, in this work, we have completed small angle neutron scattering and neutron reflectometry experiments to study the impact of post-deposition solvent annealing (SA) with control of solvent quality on the morphology and performance of LBP–bis-fullerene BHJ photovoltaics. The results show that SA in a solvent that is selective for the LBP results in a depletion of bis-fullerene near the air surface, which limits device performance. SA in a solvent vapor which has similar solubility for polymer and bis-fullerene results in a higher degree of polymer ordering, bis-fullerene phase separation, and segregation of the bis-fullerene to the air surface, which facilitates charge transport and increases power conversion efficiency (PCE) by 100%. The highest degree of polymer ordering combined with significant bis-fullerene phase separation and segregation of bis-fullerene to the air surface is obtained by SA in a solvent vapor that is selective for the bis-fullerene. The resultant morphology increases PCE by 190%. These results indicate that solvent annealing with judicious solvent choice provides a unique tool to tune the morphology of LBP–bis-fullerene BHJ system, providing sufficient polymer ordering, formation of a bis-fullerene pure phase, and segregation of bis-fullerene to the air surface to optimize the morphology of the active layer. Moreover, this process is broadly applicable to improving current “disappointing” LBP–bis-fullerene systems to optimize their morphology and OPV performance post-deposition, including higher VOC and power conversion efficiency.

Quantitative Analysis of Interdigitation Kinetics between a Polymer Melt and a Polymer Brush

We present an investigation of the swelling dynamics of end-grafted hydrogenated polystyrene (h-PS) chains confined between a solid substrate and a deuterated polystyrene (d-PS) melt, based on neutron reflectometry experiments. The segment density profile of the grafted chains was measured for different annealing times. The kinetics of relaxation was quantified by measuring the amount of melt chains that have crossed the initial h-PS/d-PS sharp interface as a function of the annealing time. The dependence of this kinetics versus the molecular weight of the grafted chains, the melt chains, and the grafting density is investigated and compared to a proposed scaling model inspired from the previous work of Milner, McLeish, and O’Connor.1,2 Moreover, the relaxation kinetics is found to highly depend on the polydispersity of the polymer brushes, and we propose a way for taking that parameter into account.

Effect of Functionalized Gold Nanoparticles on Floating Lipid Bilayers

The development of novel nano-engineered materials poses important questions regarding the impact of these new materials on living systems. Possible adverse effects must be assessed in order to prevent risks for health and the environment. On the other hand, a thorough understanding of their interaction with biological systems might also result in the creation of novel biomedical applications. We present a study on the interaction of model lipid membranes with gold nanoparticles (AuNP) of different surface modifications. Neutron reflectometry experiments on zwitterionic lipid double bilayers were performed in the presence of AuNP functionalized with cationic and anionic head groups. Structural information was obtained that provided insight into the fate of the AuNPs with regard to the integrity of the model cell membranes. The AuNPs functionalized with cationic head groups penetrate into the hydrophobic moiety of the lipid bilayers and cause membrane disruption at an increased concentration. In contrast, the AuNPs functionalized with anionic head groups do not enter but seem to impede the destruction of the lipid bilayer at an alkaline pH. The information obtained might influence the strategy for a better nanoparticle risk assessment based on a surface charge evaluation and contribute to nano-safety considerations during their design.

In situ study of the impact of acidic and neutral deposition pH on alkane phosphate film formation and stability on TiO2

TiO2 has been used as a model system for the surface of medical implant devices based upon titanium alloys. Self Assembled Monolayers (SAM) can be applied via a specific phosphate interaction to modify the surface properties of the TiO2 substrate. Here it is demonstrated via in situ Quartz Crystal Microbalance and Neutron Reflectometry experiments that the deposition of dodecyl phosphate onto TiO2 proceeds more rapidly at pH 4.5 than at pH 7.0. Conversely, the film stability was enhanced for films deposited at pH 7.0, demonstrating that while the initial association with the surface is driven by electrostatics it does not determine the SAM density. While the adsorbed amount appeared to be relatively constant after a few minutes incubation time it was found that washing with buffer removed about 50% of the adsorbed material after these short incubation times. With incubation time of the order of hours the proportion of the film washed off the surface decreased demonstrating that the specific phosphate–TiO2 interaction was a slow process. The slower initial surface interaction at pH 7.0 therefore allowed greater re-arrangement of the dodecyl phosphate resulting in more complete and robust monolayers than at pH 4.5. This was demonstrated by washing the film with buffer of increasing pH of up to 9.5. For SAM longevity on titanium alloys it is clear that slow deposition at pH 7 produces more robust films than rapid deposition at lower pH values.