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Building 3D ordered nanostructures by copolymer deposition on a substrate implies a full control beyond the thin film regime. We have used here block copolymers (BCPs) forming bulk lamellar phases to form thick, i.e. much thicker than the lamellar period, structured films on a substrate. Films are formed by a simple method of multiple successive coatings. The film structure is controlled using the combined action of surface templating and annealing time. Sections of the thick layers were characterized by scanning electron microscopy (SEM) after etching of one of the BCP moieties. We show that perfect hexagonally perforated films (HPL) with lamellae parallel to the substrate are formed for a wide thickness range up to 300 nm. Grazing incidence small angle X-ray scattering (GISAXS) confirms such an organization by revealing that perforations sit on a hexagonal lattice. A lamellar organization perpendicular to the substrate is shown to take over for thicker films. A scenario consistent with our observations is proposed, where the sequence of phases results from the balance between surface and stretching energy effects.
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In nature, biomolecules are often organized as functional thin layers in interfacial architectures, the most prominent examples being biological membranes. Biomolecular layers play also important roles in context with biotechnological surfaces, for instance, when they are the result of adsorption processes. For the understanding of many biological or biotechnologically relevant phenomena, detailed structural insight into the involved biomolecular layers is required. Here, we use standing-wave X-ray fluorescence (SWXF) to localize chemical elements in solid-supported lipid and protein layers with near-Ångstrom precision. The technique complements traditional specular reflectometry experiments that merely yield the layers' global density profiles. While earlier work mostly focused on relatively heavy elements, typically metal ions, we show that it is also possible to determine the position of the comparatively light elements S and P, which are found in the most abundant classes of biomolecules and are therefore particularly important. With that, we overcome the need of artificial heavy atom labels, the main obstacle to a broader application of high-resolution SWXF in the fields of biology and soft matter. This work may thus constitute the basis for the label-free, element-specific structural investigation of complex biomolecular layers and biological surfaces.
Assuntos
Bicamadas Lipídicas/química , Proteolipídeos/química , Espectrometria por Raios X/métodos , Antraquinonas/química , Glicosídeos/química , Humanos , Fosfatidilcolinas/química , Albumina Sérica Humana/química , Soluções , Espectrometria por Raios X/instrumentaçãoRESUMO
Synchrotron radiation reflectometry was used to access the transverse structure of model membranes under the action of the human sialidase NEU2, down to the Ångström length scale. Model membranes were designed to mimic the lipid composition of so-called Glycosphingolipids Enriched Microdomains (GEMs), which are membrane platforms specifically enriched in cholesterol and sphingolipids, and where also typical signalling molecules are hosted. Gangliosides, glycosphingolipids containing one or more sialic acid residues, are asymmetrically embedded in GEMs, in the outer membrane leaflet where gangliosides are claimed to interact directly with growth-factor receptors, modulating their activation and then the downstream intracellular signalling pathways. Thus, membrane dynamics and signalling could be strongly influenced by the activity of enzymes regulating the membrane ganglioside composition, including sialidases. Our results, concerning the structure of single membranes undergoing in-situ enzymatic digestion, show that the outcome of the sialidase action is not limited to the emergence of lower-sialylated ganglioside species. In fact, membrane reshaping occurs, involving a novel arrangement of the headgroups on its surface. Thus, sialidase activity reveals to be a potential tool to control dynamically the structural properties of the membrane external leaflet of living cells, influencing both the morphology of the close environment and the extent of interaction among active molecules belonging to signalling platforms.
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Gangliosídeos/metabolismo , Bicamadas Lipídicas/química , Neuraminidase/metabolismo , Síncrotrons , Digestão , Humanos , Microdomínios da Membrana/química , Transdução de SinaisRESUMO
The effect of ac electric fields on the elasticity of supported lipid bilayers is investigated at the microscopic level using grazing incidence synchrotron x-ray scattering. A strong decrease in the membrane tension up to 1 mN/m and a dramatic increase of its effective rigidity up to 300 k_{B}T are observed for local electric potentials seen by the membrane â²1 V. The experimental results are analyzed using detailed electrokinetic modeling and nonlinear Poisson-Boltzmann theory. Based on a modeling of the electromagnetic stress, which provides an accurate description of the bilayer separation versus pressure curves, we show that the decrease in tension results from the amplification of charge fluctuations on the membrane surface whereas the increase in bending rigidity results from the direct interaction between charges in the electric double layer. These effects eventually lead to a destabilization of the bilayer and vesicle formation. Similar effects are expected at the tens of nanometers length scale in cell membranes with lower tension, and could explain a number of electrically driven processes.
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Membrana Celular/química , Bicamadas Lipídicas , Membrana Celular/fisiologia , Elasticidade , Lipídeos de Membrana , Radiografia , Raios XRESUMO
Multiple water-in-oil-in-water (W/O/W) emulsions are promising materials in designing carriers of hydrophilic molecules or drug delivery systems, provided stability issues are solved and biocompatible chemicals can be used. In this work, we designed a biocompatible amphiphilic copolymer, poly(dimethylsiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA), that can stabilize emulsions made with various biocompatible oils. The hydrophilic/hydrophobic properties of the copolymer can be adjusted using both pH and ionic strength stimuli. Consequently, the making of O/W (oil in water), W/O (water in oil), and W/O/W emulsions can be achieved by sweeping the pH and ionic strength. Of importance, W/O/W emulsions are formulated over a large pH and ionic strength domain in a one-step emulsification process via transitional phase inversion and are stable for several months. Cryo-TEM and interfacial tension studies show that the formation of these W/O/W emulsions is likely to be correlated to the interfacial film curvature and microemulsion morphology.
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We study the stability of a model Pickering emulsion system using fluorinated oil and functionalized silica nanoparticles. A special counter-flow microfluidic set-up was used to prepare monodisperse oil droplets in water. The wettability of the monodisperse silica nanoparticles (NPs) could be tuned by surface grafting and the surface coverage of the droplets was controlled using the microfluidic setup. For surface coverage as low as 23%, we observed a regime of Pickering emulsion stability where the surface coverage of emulsion droplets of constant size increases with time, coexisting with an excess of oil phase. Our results demonstrate that the previously observed limited coalescence regime where surface coverage tends to control the average size of the final droplets must be put in a broader perspective.
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Ion-surface interactions are of high practical importance in a wide range of technological, environmental and biological problems. In particular, they ultimately control the electric double layer structure, hence the interaction between particles in aqueous solutions. Despite numerous achievements, progress in their understanding is still limited by the lack of experimental determination of the surface composition with appropriate resolution. Tackling this challenge, we have developed a method based on X-ray standing waves coupled to nano-confinement which allows the determination of ion concentrations at a solid-solution interface with a sub-nm resolution. We have investigated mixtures of KCl/CsCl and KCl/KI in 0.1 mM to 10 mM concentrations on silica surfaces and obtained quantitative information on the partition of ions between bulk and Stern layer as well as their distribution in the Stern layer. Regarding partition of potassium ions, our results are in agreement with a recent AFM study. We show that in a mixture of KCl and KI, chloride ions exhibit a higher surface propensity than iodide ions, having a higher concentration within the Stern layer and being on average closer to the surface by ≈1-2 Å, in contrast to the solution water interface. Confronting such data with molecular simulations will lead to a precise understanding of ionic distributions at aqueous interfaces.
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Understanding interactions between membranes requires measurements on well-controlled systems close to natural conditions, in which fluctuations play an important role. We have determined, by grazing incidence X-ray scattering, the interaction potential between two lipid bilayers, one adsorbed on a solid surface and the other floating close by. We find that interactions in this highly hydrated model system are two orders of magnitude softer than in previously reported work on multilayer stacks. This is attributed to the weak electrostatic repulsion due to the small fraction of ionized lipids in supported bilayers with a lower number of defects. Our data are consistent with the Poisson-Boltzmann theory, in the regime where repulsion is dominated by the entropy of counter ions. We also have unique access to very weak entropic repulsion potentials, which allowed us to discriminate between the various models proposed in the literature. We further demonstrate that the interaction potential between supported bilayers can be tuned at will by applying osmotic pressure, providing a way to manipulate these model membranes, thus considerably enlarging the range of biological or physical problems that can be addressed.
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Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Químicos , Pressão Osmótica , Eletricidade Estática , Entropia , Espalhamento de RadiaçãoRESUMO
Spontaneous ordering of nanoparticles (NPs) occurring as a consequence of solvent evaporation can yield highly ordered and extended NP superlattices bearing both fundamental scientific interest and potential for technological application. A versatile experimental chamber has been developed allowing (i) controlled in situ deposition of NP solutions on solid substrates, (ii) rate-controlled evaporation of the bulk solvent, and (iii) adsorption/desorption of nano-thick solvent films onto preformed NP assemblies. Within this hermetically sealed chamber all the stages of self-assembly, including macroscopic solution evaporation, NP thin-film formation and its subsequent structural transformation induced by nano-thick solvent films, can be characterized in situ by X-ray scattering techniques. Here, technical design and calibration details are provided, as well as three experimental examples highlighting the chamber's performances and potential. Examples include the controlled adsorption of thin toluene films on flat silicon wafers, the observation of transient accumulation of gold NPs near the toluene-vapour interface, and preliminary data on the structural effects of fast macroscopic solvent evaporation followed by nanoscale solvent adsorption/desorption from a vapour phase. By combining bulk evaporation rate control, fine tuning of the thickness of adsorbed solvent films and in situ X-ray characterization capabilities, this cell enables explorations of both near-to-equilibrium and far-from-equilibrium routes to NP self-assembly.
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We report Angstrom-resolved x-ray reflectivity analysis of externally polarized liquid-Hg surface in contact with molar LiCl, LiBr, and MgSO4 aqueous electrolytes. Interpretation of reflectivity curves demonstrates a dependence of Hg-surface layering on both applied potential and ion nature. It further highlights how interfacial polarization degree impacts electron density profiles at a molecular scale. These profiles indicate accumulation of anions and cations at the Hg surface. Upon decrease of the potential from the point of zero charge, anions are gradually expelled from the Hg surface. The study challenges traditional thermodynamic approaches for deriving countercharge composition at the Hg-electrolyte-solution interface from macroscopic Hg-surface tension data. It further dismisses the long-standing approximation that assimilates the Hg surface to a smooth, perfect chemically inert conductor with a uniformly smeared-out surface charge density.
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Progress in the determination of structure and fluctuation spectrum of a floating bilayer system, as well as potential applications for biological studies, is reviewed. The system described here was first introduced by Charitat et al. (Eur Phys J B 8:583-593, 1999) and consists of a planar bilayer floating at 2-3 nm away from an adsorbed one on a solid surface in contact with bulk water. This model has been widely used for surface scattering studies using both neutrons and synchrotron radiation and its use in studies of relevance for physics and biology research areas will be described, together with the progress towards the production of complex biomimetic samples for use with scattering techniques.
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Bicamadas Lipídicas/química , Modelos Moleculares , Difração de Nêutrons/métodos , Síncrotrons , Difração de Raios X/métodosRESUMO
Amphiphilic cyclodextrins (CDs) are good candidates to functionalize natural membranes as well as synthetic vesicles. In this paper, we provide a full description of the interfacial behavior of pure 6I,6IV-(ß-cholesteryl)succinylamido-6I,6IV-(6-deoxy-per-(2,3,6-O-methyl))cycloheptaose (TBdSC) and how it inserts in dipalmitoyl-l-α-phosphatidylcholine (DPPC) monolayers as a membrane model. Langmuir isotherms of pure TBdSC suggest a reorganization upon compression, which could be clarified using X-ray reflectivity. The CD head can adjust its conformation to the available area per molecule. A compatible model involving a rotation around a horizontal axis defined by the two selectively substituted glucose units is proposed. The in-plane structure is characterized at all scales by Brewster angle microscopy (BAM) on the water surface and atomic force microscopy (AFM) on monolayers deposited on solid substrates. The same tools are used for its mixtures with DPPC. We show in particular that TBdSC seems to be soluble in the liquid-expanded DPPC. However, phase segregation occurs at higher pressure, allowing for sequentially liquid-condensed DPPC and high-pressure conformation of TBdSC. This gives rise to a remarkable contrast inversion in both imaging methods.
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Colesterol/química , Ciclodextrinas/química , Membranas Artificiais , Solubilidade , Microscopia de Força AtômicaRESUMO
Amphiphilic peptides that induce catalysis are interesting alternatives to natural enzymes thanks to robustness of their synthesis and the ability to induce certain types of conformations by specific motifs of amino acid sequences. Various studies aimed at mimicking the activity of serine proteases by designed peptides. Here we demonstrate that the order by which the catalytic triad residues are positioned along amphiphilic ß-strands influences both assembly structures and catalytic activity. A set of three ß-sheet amphiphilic peptides, decorated with different orders of the catalytic triad amino acids, Glu, His and Ser along the strands were evaluated for their catalytic hydrolysis efficiency of p-nitrophenyl acetate (pNPA) substrate. Among the three peptides, Ac-Cys-Phe-Glu-Phe-Ser-Phe-His-Phe-Pro-NH2 (ESH) achieved the greatest catalytic efficiency with a value of 0.19 M-1 s-1, at peptide concentration of 250 µM. This study sheds light on an overlooked factor in designing catalytic amphiphilic assemblies whereby charged residues that make up the active sites, are in fact engaged in intermolecular stabilizing interactions that in turn may hamper their catalytic action.
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Aminoácidos , Peptídeos , Sequência de Aminoácidos , Catálise , HidróliseRESUMO
The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale.
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Bacteriorodopsinas , Bicamadas Lipídicas , Nêutrons , Técnicas de Microbalança de Cristal de QuartzoRESUMO
Nanometer-sized gold nanoparticles have been prepared and surface-modified in order to stabilize alkane-in-water emulsions. A mixed hexane-undecanol ligand layer at the surface of the nanoparticles allowed us to tune their wettability and thus the adsorption at the oil-water interface. Oil droplets of the stable emulsions have been evidenced by confocal fluorescence microscopy, freeze-fracture transmission electron microscopy, and dynamic light scattering. Prepared emulsions were stable during performed cooling-heating cycles, in which the temperature stability of the emulsions has been studied by means of dynamic light scattering. The interfacial structure of the oil droplets was investigated by small-angle X-ray scattering. The obtained area per nanoparticle at the oil droplet interface was 30 nm(2). The investigation of the nanoparticle adsorption at the curved interface of the emulsion droplets is in agreement with our previous study at a planar oil-water interface, in which the nanoparticles started to interact with each other at about the same area per particle.
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We present and fully characterize a flow cell dedicated to imaging in liquid at the nanoscale. Its use as a routine sample environment for soft X-ray spectromicroscopy is demonstrated, in particular through the spectral analysis of inorganic particles in water. The care taken in delineating the fluidic pathways and the precision associated with pressure actuation ensure the efficiency of fluid renewal under the beam, which in turn guarantees a successful utilization of this microfluidic tool for in situ kinetic studies. The assembly of the described flow cell necessitates no sophisticated microfabrication and can be easily implemented in any laboratory. Furthermore, the design principles we relied on are transposable to all microscopies involving strongly absorbed radiation (e.g. X-ray, electron), as well as to all kinds of X-ray diffraction/scattering techniques.
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We investigate the interaction between highly charged lipid bilayers in the presence of monovalent counterions. Neutron and X-ray reflectivity experiments show that the water layer between like-charged bilayers is thinner than for zwitterionic lipids, demonstrating the existence of counterintuitive electrostatic attractive interaction between them. Such attraction can be explained by taking into account the correlations between counterions within the Strong Coupling limit, which falls beyond the classical Poisson-Boltzmann theory of electrostatics. Our results show the limit of the Strong Coupling continuous theory in a highly confined geometry and are in agreement with a decrease in the water dielectric constant due to a surface charge-induced orientation of water molecules.
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We describe how a versatile amphiphilic diblock copolymer can form oil-in-water (o/w) or water-in-oil (w/o) emulsions depending on pH and temperature. At high pH and temperature, this copolymer is mostly hydrophobic and forms w/o emulsions. Its spontaneous curvature is greatly increased upon pH and/or temperature lowering (due to protonation and/or hydration, respectively), which allows the formation of o/w emulsions. Conductivity measurements and confocal fluorescence micrographs evidence the two kinds of structures obtained over a wide range of pH and temperature. We also show how the emulsion type can be reversibly switched along a temperature scan under stirring. The lower stability of the w/o emulsions as compared to the o/w ones is attributed to a lack of electrostatic repulsion. The importance of the copolymer architecture and conformation with regards to droplet stability is discussed.
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Absolute ion concentration and its profile across polyelectrolyte multilayer films were studied. The films were prepared by alternating adsorption of polyanions and polycations from aqueous solution. Standing-wave X-ray fluorescence was used to map the ion profile. The well-studied multilayer system PSS/PAH was investigated, and bromide ions were used as probe entities. The results show that the sign of the charge of the outermost layer and the washing procedure after finishing the preparation have a decisive effect on the ion concentration and the ion profile. Multilayers with PSS as the outermost layer contain fewer bromide ions than the PAH-terminated multilayers. Exposure to water washes the ions out, but even after 6 h of washing, not all of the bromide ions had been removed.