Globular protein assembly and network formation at fluid interfaces: effect of oil.

The formation of viscoelastic networks at fluid interfaces by globular proteins is essential in many industries, scientific disciplines, and biological processes. However, the effect of the oil phase on the structural transitions of proteins, network formation, and layer strength at fluid interfaces has received little attention. Herein, we present a comprehensive study on the effect of oil polarity on globular protein networks. The formation dynamics and mechanical properties of the interfacial networks of three different globular proteins (lysozyme, ß-lactoglobulin, and bovine serum albumin) were studied with interfacial shear and dilatational rheometry. Furthermore, the degree of protein unfolding at the interfaces was evaluated by subsequent injection of disulfide bonds reducing dithiothreitol. Finally, we measured the interfacial layer thickness and protein immersion into the oil phase with neutron reflectometry. We found that oil polarity significantly affects the network formation, the degree of interfacial protein unfolding, interfacial protein location, and the resulting network strength. These results allow predicting emulsion stabilization of proteins, tailoring interfacial layers with desired mechanical properties, and retaining the protein structure and functionality upon adsorption.

Designing Cellulose Nanofibrils for Stabilization of Fluid Interfaces.

Particles of biological origin are of increasing interest for the Pickering stabilization of biocompatible and environmentally friendly foams and emulsions. Cellulose nanofibrils (CNFs) are readily employed in that respect; however, the underlying mechanisms of interfacial stabilization remain widely unknown. For instance, it has not been resolved why CNFs are unable to stabilize foams while efficiently stabilizing emulsions. Here, we produce CNFs with varying contour lengths and charge densities to investigate their behavior at the air-water phase boundary. CNFs adsorbing at the air-water interface reduce surface tension and form interfacial layers with high viscoelasticity, which are attributed to the thermodynamic and kinetic stability of CNF-stabilized colloids, respectively. CNF adsorption is accelerated and higher surface pressures are attained at lower charge densities, indicating that CNF surface charges limit both adsorption and surface coverage. CNFs form monolayers with ∼40% coverage and are primarily wetted by the aqueous phase indicating a contact angle <90°, as demonstrated by neutron reflectometry. The low contact angle at the air-water interface is energetically unfavorable for adsorbed CNFs, which is proposed as a potential explanation why CNFs show poor foaming capacity.

Transient measurement and structure analysis of protein-polysaccharide multilayers at fluid interfaces.

The formation of electrostatic protein-polysaccharide multilayers has attracted attention for the design of fluid interfaces with enhanced stability and functionality. However, current techniques are often limited to measuring final multilayer properties. We present an interfacial shear rheology setup with simultaneous subphase exchange, allowing the transient measurement of biopolymer multilayers by their viscoelasticity. The successive and simultaneous adsorption of ß-lactoglobulin (ß-lg) and low-methoxyl pectin were investigated at the n-dodecane/water interface at pH 4. The successive injection of pectin increased the viscoelasticity of an adsorbed ß-lg layer by electrostatic complexation. On the other hand, simultaneous adsorption impeded adsorption kinetics and interfacial layer strength due to complexation in the bulk phase prior to adsorption. Neutron reflectometry at the air-water interface confirmed the formation of an initial ß-lg layer and electrostatic complexation of a secondary pectin layer, which desorbed upon pH-induced charge inversion. The layer formed by simultaneous adsorption mainly consisted of ß-lg. We conclude that protein-polysaccharide complexes show limited surface activity and result in a lower effective protein concentration available for adsorption.

Adsorption and Interfacial Layer Structure of Unmodified Nanocrystalline Cellulose at Air/Water Interfaces.

Nanocrystalline cellulose (NCC) is a promising biological nanoparticle for the stabilization of fluid interfaces.  However, the adsorption and interfacial layer structure of NCC are poorly understood as it is currently unknown how to form  NCC interfacial layers. Herein, we present parameters for the adsorption of unmodified NCC at the air-water (A/W) interface. Initial NCC adsorption is limited by diffusion, followed by monolayer saturation and decrease in surface tension at the time scale of hours. These results confirm the current hypothesis of a Pickering stabilization. NCC interfacial performance can be modulated by salt-induced charge screening, enhancing adsorption kinetics, surface load, and interfacial viscoelasticity. Adsorbed NCC layers were visualized by atomic force microscopy at planar Langmuir films and curved air bubbles, whereat NCC coverage was higher at curved interfaces. Structural analysis by neutron reflectometry revealed that NCC forms a discontinuous monolayer with crystallites oriented in the interfacial plane at a contact angle < 90°, favoring NCC desorption upon area compression. This provides the fundamental framework on the formation and structure of NCC layers at the A/W interface, paving the way for exploiting NCC interfacial stabilization for tailored colloidal materials.

Modifying the Contact Angle of Anisotropic Cellulose Nanocrystals: Effect on Interfacial Rheology and Structure.

Cellulose nanocrystals (CNCs) are an emerging natural material with the ability to stabilize fluid/fluid interfaces. Native CNC is hydrophilic and does not change the interfacial tension of the stabilized emulsion or foam system. In this study, rodlike cellulose particles were isolated from hemp and chemically modified to alter their hydrophobicity, i.e., their surface activity, which was demonstrated by surface tension measurements of the particles at the air/water interface. The buildup and mechanical strength of the interfacial structure were investigated using interfacial shear and dilatational rheometry. In contrast to most particle or protein-based interfacial adsorption layers, we observe in shear flow a Maxwellian behavior instead of a glasslike frequency response. The slow and reversible buildup of the layer and its unique frequency dependence indicate a weakly aggregated system, which depends on the hydrophobicity and, thus, on the contact angle of the CNC particles at the air/water interface. Exposed to dilatational flow, the weakly aggregated particles cluster and form compact structures. The interfacial structure generated by the different flow fields is characterized by the contact angle, immersion depth, and layer roughness obtained by neutron reflectometry with contrast variation while the size and local structural arrangement of the CNC particles were investigated by AFM imaging.

Complexation of phospholipids and cholesterol by triterpenic saponins in bulk and in monolayers.

The interactions between three triterpene saponins: α-hederin, hederacoside C and ammonium glycyrrhizate with model lipids: cholesterol and dipalmitoylphosphatidylcholine (DPPC) are described. The oleanolic acid-type saponins (α-hederin and hederacoside C) were shown to form 1:1 complexes with lipids in bulk, characterized by stability constants in the range (4.0±0.2)·10(3)-(5.0±0.4)·10(4) M(-1). The complexes with cholesterol are generally stronger than those with DPPC. On the contrary, ammonium glycyrrhizate does not form complexes with any of the lipids in solution. The saponin-lipid interactions were also studied in a confined environment of Langmuir monolayers of DPPC and DPPC/cholesterol with the saponins present in the subphase. A combined monolayer relaxation, surface dilational rheology, fluorescence microscopy and neutron reflectivity (NR) study showed that all three saponins are able to penetrate pure DPPC and mixed DPPC/cholesterol monolayers. Overall, the effect of the saponins on the model lipid monolayers does not fully correlate with the lipid-saponin complex formation in the homogeneous solution. The best correlation was found for α-hederin, for which even the preference for cholesterol over DPPC observed in bulk is well reflected in the monolayer studies and the literature data on its membranolytic activity. Similarly, the lack of interaction of ammonium glycyrrhizate with both lipids is evident equally in bulk and monolayer experiments, as well as in its weak membranolytic activity. The combined bulk and monolayer results are discussed in view of the role of confinement in modulating the saponin-lipid interactions and possible mechanism of membranolytic activity of saponins.

Self-Diffusion in Amorphous Silicon.

The present Letter reports on self-diffusion in amorphous silicon. Experiments were done on ^{29}Si/^{nat}Si heterostructures using neutron reflectometry and secondary ion mass spectrometry. The diffusivities follow the Arrhenius law in the temperature range between 550 and 700 °C with an activation energy of (4.4±0.3) eV. In comparison with single crystalline silicon the diffusivities are tremendously higher by 5 orders of magnitude at about 700 °C, which can be interpreted as the consequence of a high diffusion entropy.

On the Interaction between Digitonin and Cholesterol in Langmuir Monolayers.

In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a combination of tensiometric (surface pressure and dilatational surface elasticity), spectroscopic (infrared reflection absorption spectroscopy, IRRAS), microscopic (fluorescence microscopy), and scattering techniques (neutron reflectivity, NR, and grazing incidence X-ray diffraction, GIXD). The monolayers of individual lipids and their 10:9 (mol/mol) mixture were exposed to an aqueous solution of digitonin (10(-4) M) by subphase exchange using a setup developed recently in our laboratory. The results confirm that digitonin can adsorb onto both bare and lipid-covered water-air interfaces. In the case of DPPC, a relatively weak interaction can be observed, but the presence of cholesterol drastically enhances the effect of digitonin. The latter is shown to dissociate the weak cholesterol-DPPC complexes and to bind cholesterol in an additional layer attached to the original lipid monolayer.

Semifluorinated Alkanes at the Air-Water Interface: Tailoring Structure and Rheology at the Molecular Scale.

Semifluorinated alkanes form monolayers with interesting properties at the air-water interface due to their pronounced amphi-solvophobic nature and the stiffness of the fluorocarbons. In the present work, using a combination of structural and dynamic probes, we investigated how small molecular changes can be used to control the properties of such an interface, in particular its organization, rheology, and reversibility during compression-expansion cycles. Starting from a reference system perfluor(dodecyl)dodecane, we first retained the linear structure but changed the linkage groups between the alkyl chains and the fluorocarbons, by introducing either a phenyl group or two oxygens. Next, the molecular structure was changed from linear to branched, with four side chains (two fluorocarbons and two hydrocarbons) connected to extended aromatic cores. Neutron reflectivity at the air-water interface and scanning force microscopy on deposited films show how the changes in the molecular structure affect molecular arrangement relative to the interface. Rheological and compression-expansion measurements demonstrate the significant consequences of these changes in molecular structure and interactions on the interfacial properties. Remarkably, even with these simple molecules, a wide range of surface rheological behaviors can be engineered, from viscous over viscoelastic to brittle solids, for very similar values of the surface pressure.

Blocking Gastric Lipase Adsorption and Displacement Processes with Viscoelastic Biopolymer Adsorption Layers.

Delayed fat digestion might help to fight obesity. Fat digestion begins in the stomach by adsorption of gastric lipases to oil/water interfaces. In this study we show how biopolymer covered interfaces can act as a physical barrier for recombinant dog gastric lipase (rDGL) adsorption and thus gastric lipolysis. We used ß-lactoglobulin (ß-lg) and thermosensitive methylated nanocrystalline cellulose (metNCC) as model biopolymers to investigate the role of interfacial fluid dynamics and morphology for interfacial displacement processes by rDGL and polysorbate 20 (P20) under gastric conditions. Moreover, the influence of the combination of the flexible ß-lg and the elastic metNCC was studied. The interfaces were investigated combining interfacial techniques, such as pendant drop, interfacial shear and dilatational rheology, and neutron reflectometry. Displacement of biopolymer layers depended mainly on the fluid dynamics and thickness of the layers, both of which were drastically increased by the thermal induced gelation of metNCC at body temperature. Soft, thin ß-lg interfaces were almost fully displaced from the interface, whereas the composite ß-lg-metNCC layer thermogelled to a thick interfacial layer incorporating ß-lg as filler material and therefore resisted higher shear forces than a pure metNCC layer. Hence, with metNCC alone lipolysis by rDGL was inhibited, whereas the layer performance could be increased by the combination with ß-lg.

Unusual penetration of phospholipid mono- and bilayers by Quillaja bark saponin biosurfactant.

The interactions between a model phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a biosurfactant Quillaja Bark Saponin (QBS) obtained from the bark of Quillaja saponaria Molina were studied using simple models of biological membranes. QBS is known to interact strongly with the latter, exerting a number of haemolytic, cytotoxic and anti-microbial actions. The interaction of QBS dissolved in the subphase with DPPC monolayers and silicon-supported bilayers was studied above the cmc (10(-3)M). Surface pressure relaxation and surface dilatational rheology combined with quartz crystal microbalance (QCM) and neutron reflectivity (NR) were employed for this purpose. The DPPC-penetrating abilities of QBS are compared with those of typical synthetic surfactants (SDS, CTAB and Triton X-100). We show that the penetration studies using high surface activity (bio)surfactants should be performed by a subphase exchange, not by spreading onto the surfactant solution. In contrast to the synthetic surfactants of similar surface activity, QBS does not collapse DPPC mono- and bilayers, but penetrates them, improving their surface dilatational elastic properties even in the highly compressed solid state. The dilatational viscoelasticity modulus increases from 204 mN/m for pure DPPC up to 310 mN/m for the QBS-penetrated layers, while it drops to near zero values in the case of the synthetic surfactants. The estimated maximum insertion pressure of QBS into DPPC monolayers exceeds the maximum surface pressure achievable in our setup, in agreement with the surface rheological response of the penetrated layers.

Response of Plasma-Polymerized Hexamethyldisiloxane Films to Aqueous Environments.

Thin plasma polymer films were deposited in hexamethyldisiloxane (HMDSO) and HMDSO/O2 low-pressure discharges and their chemical structures analyzed using infrared (IR) spectroscopy and neutron reflectometry (NR). The (plasma-polymerized) ppHMDSO film exhibits hydrophobic, poly(dimethylsiloxane)-like properties, while the retention of carbon groups is reduced by O2 addition, yielding a more inorganic, hydrophilic ppSiOx film. Both films show a minor (vertical) density gradient perpendicular to the substrate, where the exposed film surface seems to be more oxidized, indicating oxidative aging reactions upon contact with air. The hydration and water uptake abilities of the films in aqueous environments were investigated in humid environments using ellipsometry, NR in D2O, and multiple transmission-reflection IR measurements after equilibration of the films in water.

Photoswitching the mechanical properties in Langmuir layers of semifluorinated alkyl-azobenzenes at the air-water interface.

Semifluorinated alkyl-azobenzene derivatives (SFAB) can form stable Langmuir layers at the air-water interface. These systems combine the amphiphobic character of semifluorinated alkyl units as structure-directing motifs with photochromic behavior based on the well-known reversible cis-trans isomerization upon irradiation with UV and visible light. Herein, we report our investigations of the structural and dynamic tunability of these SFAB layers at the air-water interface in response to an external light stimulus. The monolayer structures and properties of [4-(heptadecafluorooctyl)phenyl](4-octylphenyl)diazene (F8-azo-H8) and bis(4-octylphenyl)diazene (H8-azo-H8) were studied by neutron reflectivity, surface pressure-area isotherms with compression-expansion cycles, and interfacial rheology. We find that UV irradiation reversibly influences the packing behavior of the azobenzene molecules and interpret this as a transition from organized layer structures with the main axis of the molecule vertically oriented in the trans form to random packing of the cis isomer. Interestingly, this trans-cis isomerization leads to an increase in surface pressure, which is accompanied by a decrease in viscoelastic moduli. These results suggest ways of tailoring the properties of responsive fluid interfaces.

Foams stabilized by multilamellar polyglycerol ester self-assemblies.

The importance of surfactant self-assemblies in foam stabilization is well-known. The aim of the current study was to investigate the self-assemblies of the nonionic surfactant polyglycerol ester (PGE) in bulk solutions, at the interface and within foams, using a combined approach of small-angle neutron scattering, neutron reflectivity, and electron microscopy. PGE bulk solutions contain vesicles as well as open lamellar structures. Upon heating of the solutions the lamellar spacing increases, with significant differences in the presence of NaCl or CaCl(2) as compared to the standard solution. The adsorption of the multilamellar structures present in the bulk solutions lead to a multilayered film at the air-water interface. The ordering within this film was increased as a result of a 20% area compression mimicking a coalescence event. Finally, PGE foams were shown to be stabilized not only by strong interfacial films but also by agglomerated self-assemblies within the interstitial areas of the foams.

Tuning the structure and the magnetic properties of metallo-supramolecular polyelectrolyte-amphiphile complexes.

Self-assembly of Fe(2+) ions and the rigid ditopic ligand 1,4-bis(2,2':6',2''-terpyridin-4'-yl)benzene results in metallo-supramolecular coordination polyelectrolytes (MEPE). Sequential self-assembly of MEPE and dialkyl phosphoric acid esters of varying chain length via electrostatic interactions leads to the corresponding polyelectrolyte-amphiphile complexes (PAC), which have liquid-crystalline properties. The PACs have a stratified architecture where the MEPE is embedded in between the amphiphile layers. Upon heating above room temperature, the PACs show either a reversible or an irreversible spin-crossover (SCO) in a temperature range from 360 to 460 K depending on the architecture of the amphiphilic matrix. As the number of amphiphiles per metal ion is increased in the sequence 1:2, 1:4, and 1:6, the temperature of the SCO is shifted to higher values whereas the amphiphile chain length does not have a significant impact on the SCO temperature. In summary, we describe in this article how the structure and the magnetic response function of PACs can be tailored through the design of the ligand and the composition. To investigate the structure and the magnetic behavior, we use X-ray scattering, X-ray absorption spectroscopy, differential scanning calorimetry, faraday-balance, and superconducting quantum interference measurements in combination with molecular modeling.

Effect of the molecular structure on the hierarchical self-assembly of semifluorinated alkanes at the air/water interface.

Semifluorinated alkanes (C(n)F(2n+1)C(m)H(2m+1)), short FnHm display local phase separation of mutually incompatible hydrocarbon and fluorocarbon chain moieties, which has been utilized as a structure-forming motif in supramolecular architectures. The packing of semifluorinated alkanes, nominally based on dodecyl subunits, such as perfluoro(dodecyl)dodecane (F12H12) and perfluoro(dodecyl)eicosane (F12H20), as well as a core extended analogue, 1,4-dibromo-2-((perfluoroundecyl)methoxy)-5-(dodecyloxy)benzene) (F11H1-core-H12), was studied at the air/water interface. Langmuir monolayers were investigated by means of neutron reflectivity directly at the air/water interface and scanning force microscopy after transfer to silicon wafers. Narrowly disperse surface micelles formed in all three cases; however, they were found to bear different morphologies with respect to molecular orientation and assembly dimensionality, which gives rise to different hierarchical aggregate topologies. For F12H12, micelles of ca. 30 nm in diameter, composed of several circular or "spherical cap" substructures, were observed and a monolayer model with the fluorocarbon block oriented toward air is proposed. F12H20 molecules formed larger (ca. 50 nm diameter) hexagonally shaped surface micelles that were hexagonally, densely packed, besides more elongated but tightly interlocked wormlike structures. Conversely, F11H1-core-H12 films organized into linear rows of elongated surface micelles with comparable width, but an average length of ca. 400 nm, apparently formed by antiparallel molecular packing.

How the dynamics of subsurface hydration regulates protein-surface interactions.

The role of water structure near surfaces has been scrutinized extensively because it is accepted to control protein-surface interactions, however, often avoiding effects of hydration dynamics. Relating to this, we have recently discussed how the amount and state of water, accumulated within various hydrophobic-to-hydrophilic subsurface gradients of plasma polymer films, influence the magnitude of adsorbed bovine serum albumin, spurring the hypothesis of the presence of a subsurface dipolar field. This study now analyzes the kinetics of hydration by systematically introducing modified gradient architectures and relating different hydration times to the adsorption of a dipolar probing protein. We find that dry-stored subsurface gradients, owing nominally identical surface characteristics, exhibits comparable surface potential and protein adsorption values, while they behave in a different manner at transient hydration times of few hours, before reaching near-equilibrium state of the hydration. A characteristic hydration time is found where protein adsorption on gradient films is minimal, unveiling the transient nature of the effect. In general, protein adsorption is sensitive to the time allowed for hydration of the adsorbent surface, supporting our initial hypothesis inasmuch as the quantity as well as quality of water inside the subsurface matrix is crucial for controlling protein-surface interactions.

Liquid crystalline phase transition induces spin crossover in a polyelectrolyte amphiphile complex.

Self-assembly of Fe(2+) or Ni(2+) ions and the ditopic ligand 6,6',6''-bis(2-pyridyl)-2,2':4',4'':2'',2'''-quaterpyridine (btpy) through coordinative binding results in rodlike metallosupramolecular coordination polyelectrolytes (Fe-MEPE or Ni-MEPE). Sequential self-assembly with dihexadecyl phosphate (DHP) via electrostatic interactions between MEPE and DHP leads to the corresponding polyelectrolyte amphiphile complex (PAC) with liquid crystalline properties. The MEPE rods are embedded in between the interdigitated DHP layers. Upon heating above room temperature, the Fe-PAC shows an irreversible spin-crossover (SCO) from a diamagnetic low-spin (LS) to a paramagnetic high-spin (HS) state accompanied by a color change from dark blue to pale blue. The SCO is nearly complete (95%) and directly associated with the structure changes induced by the melting of the amphiphilic matrix. The original Fe-PAC architecture does not reassemble upon cooling and remains in a disordered frozen HS state. However, dissolving the heated PAC induces reassembly, and the original dark blue, diamagnetic, ordered material is completely recovered. In comparison to Fe-PAC, Ni-PAC shows the same lamellar structure and the same temperature depended structure changes but has a constant magnetic moment. In contrast to Fe-PAC, in neat Fe-MEPE the SCO depends on the history of the sample and in particular on the amount of included solvent as thermogravimetric analysis, differential scanning calorimetry (DSC), and magnetic measurements indicate. Solid MEPE does not have liquid crystalline properties, and, therefore, the induced structure changes upon heating are constrained by the solid-state architecture, and thus, the SCO in Fe-MEPE is incomplete.

Extending the Range of Controlling Protein Adsorption via Subsurface Architecture.

Recently, it has been shown that water, confined in a plasma polymer subsurface chemical gradient, nanometers below the surface, significantly reduced the amount of adsorbed protein bovine serum albumin (BSA). Relating to this effect, we proposed the hypothesis that oriented water molecules within the subsurface gradient generate a long-range dipolar field, which interacts with dipolar proteins such as BSA near the surface region. This study extends the above used in situ multistep plasma deposition process to introduce plasma oxidation modifications of the subsurface architecture with the aim to further control the effect on protein adsorption. Neutron reflectivity measurements reveal that the oxidation time increases the amount of matrix-confined water. There is, however, an optimal oxidation time to obtain minimal protein adsorption, which suggests that a minimal distance between confined water molecules plays an important role. Altogether we can extend the range of controlling the adsorbed protein mass by the introduction of this additional plasma oxidation step.