X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model.

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

Structure of graphene oxide-phospholipid monolayers: A grazing incidence X-ray diffraction and neutron and X-ray reflectivity study.

HYPOTHESIS: Graphene oxide-based nanotechnology has aroused a great interest due to its applications in the biomedical and optoelectronic fields. The wide use of these materials makes it necessary to study its potential toxicity associated with the inhalation of Graphene Oxide (GO) nanoparticles and its interaction with the lung surfactant. Langmuir monolayers have proven to be an excellent tool for studying the properties of the lung surfactant and the effect of intercalation of nanoparticles on its structure and properties. Therefore, to know the origin of the phospholipids/GO interaction and the structure of the lipid layer with GO, in this work we study the effect of the insertion of GO sheets on a Langmuir film of 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC). EXPERIMENTS: Surface pressure-area isotherms, Neutron (NR) and X-ray Reflectivity (XRR) and Grazing Incidence X-ray Diffraction (GIXD) measurements of hydrogenated and deuterated DPPC monolayers with and without GO have been carried out. FINDINGS: The results outline a strong interaction between the GO and the zwitterionic form of DPPC and prove that GO is in three regions of the DPPC monolayer, the aliphatic chains of DPPC, the head groups and water in the subphase. Comparison between results obtained with hydrogenated and deuterated DPPC allows concluding that both, electrostatic attractions, and dispersion forces are responsible of the interaction GO/DPPC. Results also demonstrated that the insertion of GO into the DPPC aliphatic chains does not induce significant changes on unit cell of DPPC.

Modulating Polymer Ultrathin Film Crystalline Fraction and Orientation with Nanoscale Curvature.

We investigated the effect of nanoscale curvature on the structure of thermally equilibrated poly-3-hexylthiophene (P3HT) ultrathin films. The curvature-induced effects were investigated with synchrotron grazing incidence X-ray diffraction (GIXRD) and atomic force microscopy (AFM). Our results demonstrate that nanoscale curvature reduces the polymer crystalline fraction and the crystal length. The first effect is strongest for the lowest curvature and results in a decrease in the out-of-plane thickness of the polymer crystals. On the other hand, the crystal in-plane length decreases with the increase in substrate curvature. Finally, the semi-quantitative analysis of crystal anisotropy shows a marked dependence on the substrate curvature characterized by a minimum at curvatures between 0.00851 nm-1 and 0.0140 nm-1. The results are discussed in terms of a curvature-dependent polymer fraction, which fills the interstices between neighboring particles and cannot crystallize due to extreme space confinement. This fraction, whose thickness is highest at the lowest curvatures, inhibits the crystal nucleation and the out-of-plane crystal growth. Moreover, because of the adhesion to the curved portion of the substrates, crystals adopt a random orientation. By increasing the substrate curvature, the amorphous fraction is reduced, leading to polymer films with higher crystallinity. Finally, when the thickness of the film exceeds the particle diameter, the curvature no longer affects the crystal orientation, which, similarly to the flat case, is predominantly edge on.

Closing the loop: autonomous experiments enabled by machine-learning-based online data analysis in synchrotron beamline environments.

Recently, there has been significant interest in applying machine-learning (ML) techniques to the automated analysis of X-ray scattering experiments, due to the increasing speed and size at which datasets are generated. ML-based analysis presents an important opportunity to establish a closed-loop feedback system, enabling monitoring and real-time decision-making based on online data analysis. In this study, the incorporation of a combined one-dimensional convolutional neural network (CNN) and multilayer perceptron that is trained to extract physical thin-film parameters (thickness, density, roughness) and capable of taking into account prior knowledge is described. ML-based online analysis results are processed in a closed-loop workflow for X-ray reflectometry (XRR), using the growth of organic thin films as an example. Our focus lies on the beamline integration of ML-based online data analysis and closed-loop feedback. Our data demonstrate the accuracy and robustness of ML methods for analyzing XRR curves and Bragg reflections and its autonomous control over a vacuum deposition setup.

X-ray scattering study of GaN nanowires grown on Ti/Al2O3 by molecular beam epitaxy.

GaN nanowires (NWs) grown by molecular beam epitaxy on Ti films sputtered on Al2O3 are studied by X-ray diffraction (XRD) and grazing-incidence small-angle X-ray scattering (GISAXS). XRD, performed both in symmetric Bragg reflection mode and at grazing incidence, reveals Ti, TiN, Ti3O, Ti3Al and Ga2O3 crystallites with in-plane and out-of-plane lattice parameters intermediate between those of Al2O3 and GaN. These topotaxial crystallites in the Ti film, formed as a result of interfacial reactions and N exposure, possess little misorientation with respect to Al2O3. As a result, GaN NWs grow on the top TiN layer, possessing a high degree of epitaxial orientation with respect to the substrate. The measured GISAXS intensity distributions are modelled by the Monte Carlo method, taking into account the orientational distributions of NWs, the variety of their cross-sectional shapes and sizes, and the roughness of their side facets. The cross-sectional size distributions of the NWs and the relative fractions of the {1100} and {1120} side facets are determined.

Self-Organization of Lipid-Porphyrin Conjugates at the Air/Water interface.

Lipid-porphyrin conjugates are versatile compounds which can self-assemble into liposome-like structures with multifunctional properties. Most of the conjugates that have been described so far, consisted in grafting pyropheophorbide-a (Pyro-a) or other porphyrin derivatives through the esterification of the hydroxyl group in the sn-2 position of a lysophosphatidylcholine. However, despite the versatility of these conjugates, less is known about the impact of the lipid backbone structure on their 2D phase behavior at the air/water interface and more precisely on their fine structures normal to the interface as well as on their in-plane organization. Herein, we synthesized a new lipid-porphyrin conjugate (PyroLSM) based on the amide coupling of Pyro-a to a lysosphingomyelin backbone (LSM) and we compared its interfacial behavior to that of Pyro-a and Pyro-a conjugated lysophosphatidylcholine (PyroLPC) using Langmuir balance combined to a variety of other physical techniques. Our results provided evidence on the significant impact of the lipid backbone on the lateral packing of the conjugates as well as on the shape and size of the formed domains. Compared to Pyro-a and PyroLPC monolayers, PyroLSM exhibited the highest lateral packing which highlights the role of the lipid backbone in controlling their 2D organization which in turn may impact the photophysical properties of their assemblies.

Graphene at Liquid Copper Catalysts: Atomic-Scale Agreement of Experimental and First-Principles Adsorption Height.

Liquid metal catalysts have recently attracted attention for synthesizing high-quality 2D materials facilitated via the catalysts' perfectly smooth surface. However, the microscopic catalytic processes occurring at the surface are still largely unclear because liquid metals escape the accessibility of traditional experimental and computational surface science approaches. Hence, numerous controversies are found regarding different applications, with graphene (Gr) growth on liquid copper (Cu) as a prominent prototype. In this work, novel in situ and in silico techniques are employed to achieve an atomic-level characterization of the graphene adsorption height above liquid Cu, reaching quantitative agreement within 0.1 Å between experiment and theory. The results are obtained via in situ synchrotron X-ray reflectivity (XRR) measurements over wide-range q-vectors and large-scale molecular dynamics simulations based on efficient machine-learning (ML) potentials trained to first-principles density functional theory (DFT) data. The computational insight is demonstrated to be robust against inherent DFT errors and reveals the nature of graphene binding to be highly comparable at liquid Cu and solid Cu(111). Transporting the predictive first-principles quality via ML potentials to the scales required for liquid metal catalysis thus provides a powerful approach to reach microscopic understanding, analogous to the established computational approaches for catalysis at solid surfaces.

Cross-sectional shape evolution of GaN nanowires during molecular beam epitaxy growth on Si(111).

We study the cross-sectional shape of GaN nanowires (NWs) by transmission electron microscopy. The shape is examined at different heights of long NWs, as well as at the same height for NWs of different lengths. Two distinct trends in the evolution of the cross-sectional shape along the NW length are observed. At the top, merging NWs develop common {11̄00} side facets. At the bottom, the NWs acquire roundish shapes. This observation is explained by the entirely different NW environments at the top and the bottom of the NWs. At the top, NWs are exposed to the Ga and N atomic fluxes giving rise to axial growth, resulting in the equilibrium growth shape with zero growth rate at the {11̄00} facets. At the bottom, NWs are shadowed from the impinging fluxes and are only annealed, allowing them to eventually approach the equilibrium crystal shape. The study of identical samples by grazing incidence small-angle X-ray scattering independently confirms these trends in the shape evolution of the sidewall facets.

The Effects of Cholesterol Oxidation on Erythrocyte Plasma Membranes: A Monolayer Study.

Cholesterol plays a key role in the molecular and mesoscopic organisation of lipid membranes and it is expected that changes in its molecular structure (e.g., through environmental factors such as oxidative stress) may affect adversely membrane properties and function. In this study, we present evidence that oxidation of cholesterol has significant effects on the mechanical properties, molecular and mesoscopic organisation and lipid-sterol interactions in condensed monolayers composed of the main species found in the inner leaflet of the erythrocyte membrane. Using a combination of experimental methods (static area compressibility, surface dilatational rheology, fluorescence microscopy, and surface sensitive X-ray techniques) and atomistic molecular dynamics simulations, we show that oxidation of cholesterol to 7-ketocholesterol leads to stiffening of the monolayer (under both static and dynamic conditions), significant changes in the monolayer microdomain organisation, disruption in the van der Waals, electrostatic and hydrophobic interactions between the sterol and the other lipid species, and the lipid membrane hydration. Surface sensitive X-ray techniques reveal that, whilst the molecular packing mode is not significantly affected by cholesterol oxidation in these condensed phases, there are subtle changes in membrane thickness and a significant decrease in the coherence length in monolayers containing 7-ketocholesterol.

X-ray reflectivity from curved surfaces as illustrated by a graphene layer on molten copper.

The X-ray reflectivity technique can provide out-of-plane electron-density profiles of surfaces, interfaces, and thin films, with atomic resolution accuracy. While current methodologies require high surface flatness, this becomes challenging for naturally curved surfaces, particularly for liquid metals, due to the very high surface tension. Here, the development of X-ray reflectivity measurements with beam sizes of a few tens of micrometres on highly curved liquid surfaces using a synchrotron diffractometer equipped with a double crystal beam deflector is presented. The proposed and developed method, which uses a standard reflectivity θ-2θ scan, is successfully applied to study in situ the bare surface of molten copper and molten copper covered by a graphene layer grown in situ by chemical vapor deposition. It was found that the roughness of the bare liquid surface of copper at 1400 K is 1.25 ± 0.10 Å, while the graphene layer is separated from the liquid surface by a distance of 1.55 ± 0.08 Šand has a roughness of 1.26 ± 0.09 Å.

Influence of the porphyrin structure and linker length on the interfacial behavior of phospholipid-porphyrin conjugates.

HYPOTHESIS: Phospholipid-porphyrin (Pl-Por) conjugates consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone. Owing to their structural similarities with phospholipids, Pl-Por conjugates can self-assemble into liposome-like assemblies. However, there is a significant lack of information concerning the impact of the porphyrin type and the length of the alkyl chain bearing the porphyrin on the interfacial behavior of the Pl-Por conjugates. We hypothesized that changing the chain length and the porphyrin type could impact their two-dimensional phase behavior and modulate the alignment between the two chains. EXPERIMENTS: 6 Pl-Por conjugates with different alkyl chain lengths in the sn2 position of C16 lysophosphatidylcholine and coupled to either pheophorbide-a or pyropheophorbide-a were synthesized. Their interfacial behavior at the air/water interface was assessed using Langmuir balance combined to a variety of other physical techniques including Brewster angle microscopy, atomic force microscopy and X-ray reflectometry. FINDINGS: Our results showed that all 6 Pl-Por form stable monolayers with the porphyrin moiety at the air/water interface. We also showed that changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains. The chain length dictated the structure of the formed domains with no evidence of the alignment between the two chains.

De Novo Synthesis of Free-Standing Flexible 2D Intercalated Nanofilm Uniform over Tens of cm2.

Of a variety of intercalated materials, 2D intercalated systems have attracted much attention both as materials per se, and as a platform to study atoms and molecules confined among nanometric layers. High-precision fabrication of such structures has, however, been a difficult task using the conventional top-down and bottom-up approaches. The de novo synthesis of a 3-nm-thick nanofilm intercalating a hydrogen-bonded network between two layers of fullerene molecules is reported here. The two-layered film can be further laminated into a multiply film either in situ or by sequential lamination. The 3 nm film forms uniformly over an area of several tens of cm2 at an air/water interface and can be transferred to either flat or perforated substrates. A free-standing film in air prepared by transfer to a gold comb electrode shows proton conductivity up to 1.4 × 10-4 S cm-1 . Electron-dose-dependent reversible bending of a free-standing 6-nm-thick nanofilm hung in a vacuum is observed under electron beam irradiation.

Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Real-Time Multiscale Monitoring and Tailoring of Graphene Growth on Liquid Copper.

The synthesis of large, defect-free two-dimensional materials (2DMs) such as graphene is a major challenge toward industrial applications. Chemical vapor deposition (CVD) on liquid metal catalysts (LMCats) is a recently developed process for the fast synthesis of high-quality single crystals of 2DMs. However, up to now, the lack of in situ techniques enabling direct feedback on the growth has limited our understanding of the process dynamics and primarily led to empirical growth recipes. Thus, an in situ multiscale monitoring of the 2DMs structure, coupled with a real-time control of the growth parameters, is necessary for efficient synthesis. Here we report real-time monitoring of graphene growth on liquid copper (at 1370 K under atmospheric pressure CVD conditions) via four complementary in situ methods: synchrotron X-ray diffraction and reflectivity, Raman spectroscopy, and radiation-mode optical microscopy. This has allowed us to control graphene growth parameters such as shape, dispersion, and the hexagonal supra-organization with very high accuracy. Furthermore, the switch from continuous polycrystalline film to the growth of millimeter-sized defect-free single crystals could also be accomplished. The presented results have far-reaching consequences for studying and tailoring 2D material formation processes on LMCats under CVD growth conditions. Finally, the experimental observations are supported by multiscale modeling that has thrown light into the underlying mechanisms of graphene growth.

Small-angle X-ray scattering from GaN nanowires on Si(111): facet truncation rods, facet roughness and Porod's law.

Small-angle X-ray scattering from GaN nanowires grown on Si(111) is measured in the grazing-incidence geometry and modelled by means of a Monte Carlo simulation that takes into account the orientational distribution of the faceted nanowires and the roughness of their side facets. It is found that the scattering intensity at large wavevectors does not follow Porod's law I(q) ∝ q-4. The intensity depends on the orientation of the side facets with respect to the incident X-ray beam. It is maximum when the scattering vector is directed along a facet normal, reminiscent of surface truncation rod scattering. At large wavevectors q, the scattering intensity is reduced by surface roughness. A root-mean-square roughness of 0.9 nm, which is the height of just 3-4 atomic steps per micrometre-long facet, already gives rise to a strong intensity reduction.

Dendronized oligoethylene glycols with phosphonate tweezers for cell-repellent coating of oxide surfaces: coarse-scale and nanoscopic interfacial forces.

Dendronized oligoethylene glycols (dendron OEGs) with two phosphonate groups (phosphonate tweezers) have been drawing significant attention as a new class of coating materials for superparamagnetic iron oxide surfaces. However, despite dendron OEGs showing outstanding stability in physiological fluids in previous studies, little is understood about their structure and mechanical properties. Herein we report the surface and internal structures and mechanical properties of dendron OEGs, and quantitatively determine their ability to avoid non-specific adhesion of blood platelets. To gain insight into the interfacial force interactions, we measured the coarse-scale surface force acting on cell-sized particles and mapped the nanoscopic pinning centers by fast force mapping.

XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces.

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.

Quantification of the Structure of Colloidal Gas-Liquid Interfaces.

We have quantified the structure of the colloidal gas-liquid interface using synchrotron X-ray reflectivity measurements on a model colloid-polymer mixture. The interfacial width shows mean-field scaling with the colloid density difference, and the density profiles appear to be monotonic. Furthermore, our measurements allow us to distinguish between different theoretical polymer descriptions commonly used to model colloid-polymer mixtures. Our results highlight the importance of capturing the correct polymer physics in obtaining a quantitative theoretical description of the colloidal gas-liquid interface.

Ion-Mediated Cross-linking of Biopolymers Confined at Liquid/Liquid Interfaces Probed by In Situ High-Energy Grazing Incidence X-ray Photon Correlation Spectroscopy.

As manifested in biological cell membranes, the confinement of chemical reactions at the 2D interfaces significantly improves the reaction efficacy. The interface between two liquid phases is used in various key processes in industries, such as in food emulsification and floatation. However, monitoring the changes in the mechanics and dynamics of molecules confined at the liquid/liquid interfaces still remains a scientific challenge because it is nontrivial to access the interface buried under a liquid phase. Herein, we report the in situ monitoring of the cross-linking of polyalginate mediated by Ca2+ ions at the oil/water interface by grazing incidence X-ray photon correlation spectroscopy (GIXPCS). We first optimized the reaction conditions with the aid of interfacial shear rheology and then performed GIXPCS using a high-energy synchrotron X-ray beam (22 keV) that guarantees sufficiently high transmittance through the oil phase. The intensity autocorrelation functions implied that the formation of a percolated network of polyalginate is accompanied by increasing relaxation time. Moreover, the relaxation rate scales linearly with the momentum transfer parallel to the interface, suggesting that the process is driven by hyperdiffusive propagation but not by Brownian diffusion. Our data indicated that high-energy GIXPCS has potential for in situ monitoring of changes in the dynamics of polymers confined between two liquid phases.