Structural Determinants of Chirally Selective Transport of Amino Acids through the α-Hemolysin Protein Nanopores of Free-Standing Planar Lipid Membranes.

Despite the importance of the enantioselective transport of amino acids through transmembrane protein nanopores from fundamental and practical perspectives, little has been explored to date. Here, we study the transport of amino acids through α-hemolysin (αHL) protein pores incorporated into a free-standing lipid membrane. By measuring the transport of 13 different amino acids through the αHL pores, we discover that the molecular size of the amino acids and their capability to form hydrogen bonds with the pore surface determine the chiral selectivity. Molecular dynamics simulations corroborate our findings by revealing the enantioselective molecular-level interactions between the amino acid enantiomers and the αHL pore. Our work is the first to present the determinants for chiral selectivity using αHL protein as a molecular filter.

Rheology and dynamics of a solvent segregation driven gel (SeedGel).

Bicontinuous structures promise applications in a broad range of research fields, such as energy storage, membrane science, and biomaterials. Kinetically arrested spinodal decomposition is found responsible for stabilizing such structures in different types of materials. A recently developed solvent segregation driven gel (SeedGel) is demonstrated to realize bicontinuous channels thermoreversibly with tunable domain sizes by trapping nanoparticles in a particle domain. As the mechanical properties of SeedGel are very important for its future applications, a model system is characterized by temperature-dependent rheology. The storage modulus shows excellent thermo-reproducibility and interesting temperature dependence with the maximum storage modulus observed at an intermediate temperature range (around 28 °C). SANS measurements are conducted at different temperatures to identify the macroscopic solvent phase separation during the gelation transition, and solvent exchange between solvent and particle domains that is responsible for this behavior. The long-time dynamics of the gel is further studied by X-ray Photon Correlation Spectroscopy (XPCS). The results indicate that particles in the particle domain are in a glassy state and their long-time dynamics are strongly correlated with the temperature dependence of the storage modulus.

Removal Analysis of Residual Photoresist Particles Based on Surface Topography Affected by Exposure Times of Ultraviolet and Developer Solution.

Particle removal from the surface of a substrate has been an issue in numerous fields for a long time. In semiconductor processes, for instance, the formation of clean surfaces by removing photoresist (PR) must be followed in order to create neat patterns. Although PR removal has been intensively investigated recently, little is known about how ultraviolet (UV) and developer solutions alter the PR resin (and in what manner) near the surface. While varying the exposure times of UV and developer solution, we investigated the topographic changes on the surfaces of PR resin films and particles. The measured surface properties were then correlated with the detachment force determined using films, and eventually with the residual PR particle removal percentages obtained in a microchannel. Using a positive PR and a base developer solution, we demonstrated that UV causes the surface of PR resin to become hydrophilic and wavy, whereas the developer solution produces a surface with a larger degree of roughness by swelling and partially dissolving the resin. Ultimately, the increased roughness decreased the effective contact area between PR resins, hence decreasing the detachment force and increasing the particle removal percentages. We anticipate that our findings will help understand residual particle issues, particularly on the removal mechanism of PR resins based on surface topography.

Vapor-phase synthesis of a reagent-free self-healing polymer film with rapid recovery of toughness at room temperature and under ambient conditions.

A rapidly self-healable polymer is highly desirable but challenging to achieve. Herein, we developed an elastomeric film with instant self-healing ability within 10 s at room temperature. For this purpose, a series of copolymers of poly(glycidyl methacrylate-co-2-hydroxyethyl acrylate) (poly(GMA-co-HEA), or pGH) were synthesized in the vapor phase via an initiated chemical vapor deposition (iCVD) process. The elastomer includes a large amount of hydroxyl groups in the 2-hydroxyethyl acrylate (HEA) moiety capable of forming rapid, reversible hydrogen bonding at room temperature, while glycidyl methacrylate (GMA) with a rigid methacrylic backbone chain in the copolymer provides mechanical robustness to the elastic copolymer. With the optimized copolymer composition, pGH indeed showed instant recovery of the toughness within a minute; a completely divided specimen could be welded within a minute at room temperature and under ambient conditions simply by placing the pieces in close contact, which showed the outstanding recovery performance of elastic modulus (93.2%) and toughness (15.6 MJ m-3). The rapid toughness recovery without supplementing any external energy or reagents (e.g. light, temperature, or catalyst) at room temperature and under ambient conditions will be useful in future wearable electronics and soft robotics applications.

The role of excess attractive particles in the elasticity of high internal phase Pickering emulsions.

A high internal phase emulsion (HIPE), which has a volume fraction of dispersed phase of over 74%, shows a solid-like property because of concentrated polyhedral droplets. Although many studies have proposed theoretical and empirical models to explain the rheological properties of HIPEs, most of them are only limited to the emulsions stabilized by surfactants. In the case of high internal phase Pickering emulsions (HIPPEs), much greater values of elastic modulus have been reported, compared to those of surfactant-stabilized HIPEs, but so far, there have been no clear explanations for this. In this study, we investigate how colloidal particles attribute to the significantly high elasticity of HIPPEs, specifically considering two different contributions, namely, interfacial rheological properties and bulk rheological properties. Our results reveal that the flocculated structures of colloidal particles that possess a significant elasticity can be interconnected between dispersed droplets. Furthermore, this elastic structure is a crucial factor in the high elasticity of HIPPEs, which is also supported by a simple theoretical model.

Ultra-Stable Freestanding Lipid Membrane Array: Direct Visualization of Dynamic Membrane Remodeling with Cholesterol Transport and Enzymatic Reactions.

Cell membranes actively change their local compositions, serving essential biological processes such as cellular signaling and endocytosis. Although membrane dynamics is vital in the cellular functions, the complexity of natural membranes has made its fundamental understanding and systematic assessment difficult. Here, a powerful artificial membrane system is developed for real-time visualization of the spatiotemporal dynamics of membrane remodeling. Through well-defined air/oil/water interfaces on grid holes, tens of planar lipid bilayer membranes are easily created, and their reproducibility, controllability, and generality are highlighted. The freestanding membranes are large but also highly stable, facilitating direct long-term monitoring of dynamic membrane reconstitution caused by external stimuli. As an example to demonstrate the superiority of this membrane system, the effect of cholesterol trafficking, which significantly affects biophysical properties of cell membranes, is investigated at different membrane compositions. Cholesterol transport into and out of the membranes at different rates causes anomalous lipid arrangements through cholesterol-mediated phase transitions and decomposition, which have never been witnessed before. Furthermore, enzyme-induced membrane dynamics is successfully shown in this platform; sphingomyelinases locally generate asymmetry between two membrane leaflets. This technique is broadly applicable for exploring the membrane heterogeneity under various membrane-based reactions, providing valuable insight into the membrane dynamics.

Direct Measurement of Contact Angle Change in Capillary Rise.

Capillary rise is important in many aspects of physical phenomena from transport in porous media to biotechnology. It is typically described by the Lucas-Washburn-Rideal equation (LWRE), but discrepancy between some experiments and the model still remains elusive. In this paper, we show that the discrepancy is simply from the contact angle change during the capillary rise with no help of any specific models, such as dynamic contact angle (DCA) models. To demonstrate this, we directly measure the contact angle change in the capillary rise for glycerol and carboxymethyl cellulose solutions as examples of Newtonian and non-Newtonian liquids. Unlike previous studies that used DCA models to explain the discrepancy, when the contact angle change is directly applied to the LWRE for all four tested fluids, the model agrees well with experimental data. The estimated contact angle from the capillary rise as a function of time is in good agreement with the directly measured contact angle within a narrow margin of error. To pinpoint the conditions for the discrepancy, we propose a new time scale when contact angle dynamics dominates. The contact angle dynamics that can be obtained from the macroscopic capillary rise may provide useful information for capillary flow in a more complicated geometry such as porous media.

Stratification of polymer-colloid mixtures via fast nonequilibrium evaporation.

In drying liquid films of polymer-colloid mixtures, stratification in which polymers are placed on top of larger colloids is studied. It is often presumed that the formation of segregated polymer-colloid layers is solely due to the proportion in size at fast evaporation as in binary colloid mixtures. By comparing experiments with a theoretical model, we found that the transition in viscosity near the drying interface was another important parameter for controlling the formation of stratified layers in polymer-colloid mixtures. At high evaporation rates, increased polymer concentrations near the surface lead to a phase transition from a semidilute to concentrated regime, in which colloidal particles are kinetically arrested. Stratification only occurs if the formation of a stratified layer precedes the evolution to the concentrated regime near the drying interfaces. Otherwise, the colloids will be trapped by the polymers in the concentrated regime before forming a segregated layer. Also, no stratification is observed if the initial polymer concentration is too low to form a sufficiently high polymer concentration gradient within a short period of time. Our findings are relevant for developing solution-cast polymer composites for painting, antifouling and antireflective coatings.

Deformation of soft particles with controlled elasticity by liquid-liquid interfacial tension.

Herein we report the deformation of PDMS-based particles at a liquid-liquid interface at varying degrees of softness. Direct visualization of the particle adsorption to the interface reveals at least five different modes of deformation from the complete spreading of a polymer resin droplet to a non-deforming, rigid particle.

Static and Dynamic Permeability Assay for Hydrophilic Small Molecules Using a Planar Droplet Interface Bilayer.

Because numerous drugs are administered through an oral route and primarily absorbed at the intestine, the prediction of drug permeability across an intestinal epithelial cell membrane has been a crucial issue in drug discovery. Thus, various in vitro permeability assays have been developed such as the Caco-2 assay, the parallel artificial membrane permeability assay (PAMPA), the phospholipid vesicle-based permeation assays (PVPA) and Permeapad. However, because of the time-consuming and quite expensive process for culturing cells in the Caco-2 assay and the unknown microscopic membrane structures of the other assays, a simpler yet more accurate and versatile technique is still required. Accordingly, we developed a new platform to measure the permeability of small molecules across a planar freestanding lipid bilayer with a well-defined area and structure. The lipid bilayer was constructed within a conventional UV spectrometer cell, and the transport of drug molecules across the bilayer was recorded by UV absorbance over time. We then computed the permeability from the time-dependent diffusion equation. We tested this assay for five exemplary hydrophilic drugs and compared their values with previously reported ones. We found that our assay has a much higher permeability compared to the other techniques, and this higher permeability is related to the thickness of the lipid bilayer. Also we were able to measure the dynamic permeability upon the addition of a membrane-disrupting surfactant demonstrating that our assay has the capability to detect real-time changes in permeability across the lipid bilayer.

Blending Mechanism of PS- b-PEO and PS Homopolymer at the Air/Water Interface and Their Morphological Control.

We report a blending mechanism of polystyrene- b-poly(ethylene oxide) (PS- b-PEO) and PS homopolymer (homoPS) at the air/water interface. Our blending mechanism is completely different from the well-known "wet-dry brush theory" for bulk blends; regardless of the size of homoPS, the domain size increased and the morphology changed without macrophase separation, whereas the homoPS of small molecular weight (MW) leads to a transition after blending into the block copolymer domains, and the large MW homoPS is phase-separated in bulk. The difference in blending mechanism at the interface is attributed to adsorption kinetics at a water/spreading solvent interface. Upon spreading, PS- b-PEO is rapidly adsorbed to the water/spreading solvent interface and forms domain first, and then homoPS accumulates on them as the solvent completely evaporates. On the basis of our proposed mechanism, we demonstrate that rapid PS- b-PEO adsorption is crucial to determine the final morphology of the blends. We additionally found that spreading preformed self-assemblies of the blends slowed down the adsorption, causing them to behave similar to bulk blends, following the "wet-dry brush theory". This new mechanism provides useful information for various block copolymer-homopolymer blending systems with large fluid/fluid interfaces such as emulsions and foams.

Controllable one-step double emulsion formation via phase inversion.

Double emulsions, the simplest form of multiple emulsion, have been intensively utilized in various industries as well as in fundamental research. A variety of strategies to effectively form double emulsions have been developed, but no simple yet controlled and scalable technique has been achieved yet. Herein, we examine the mechanism of the entire process of double emulsion formation by phase inversion, and we propose a universal one-step strategy for the formation of an oil/water/oil double emulsion using oil soluble polymers and hydrophobic silica nanoparticles. We demonstrate that this new approach enables control of both the fraction and the number of inner small droplets; even high internal phase double emulsions could be achieved.

Nonlinear chiral rheology of phospholipid monolayers.

Microbutton rheometry reveals that the chiral morphology of dipalmitoylphosphatidylcholine (DPPC) monolayers imparts a chiral nonlinear rheological response. The nonlinear elastic modulus and yield stress of DPPC monolayers are greater when sheared clockwise (C), against the natural winding direction of DPPC domains, than counter-clockwise (CC). Under strong CC shear strains, domains deform plastically; by contrast, domains appear to fracture under strong C shearing. After CC shearing, extended LC domains develop regular patterns of new invaginations as they recoil, which we hypothesize reflect the nucleation and growth of new defect lines across which the tilt direction undergoes a step change in orientation. The regular spacing of these twist-gradient defects is likely set by a competition between the molecular chirality and the correlation length of the DPPC lattice. The macroscopic mechanical consequences of DPPC's underlying molecular chirality are remarkable, given the single-component, non-cross-linked nature of the monolayers they form.

Effect of cholesterol nanodomains on monolayer morphology and dynamics.

At low mole fractions, cholesterol segregates into 10- to 100-nm-diameter nanodomains dispersed throughout primarily dipalmitoylphosphatidylcholine (DPPC) domains in mixed DPPC:cholesterol monolayers. The nanodomains consist of 6:1 DPPC:cholesterol "complexes" that decorate and lengthen DPPC domain boundaries, consistent with a reduced line tension, λ. The surface viscosity of the monolayer, ηs, decreases exponentially with the area fraction of the nanodomains at fixed surface pressure over the 0.1- to 10-Hz range of frequencies common to respiration. At fixed cholesterol fraction, the surface viscosity increases exponentially with surface pressure in similar ways for all cholesterol fractions. This increase can be explained with a free-area model that relates ηs to the pure DPPC monolayer compressibility and collapse pressure. The elastic modulus, G', initially decreases with cholesterol fraction, consistent with the decrease in λ expected from the line-active nanodomains, in analogy to 3D emulsions. However, increasing cholesterol further causes a sharp increase in G' between 4 and 5 mol% cholesterol owing to an evolution in the domain morphology, so that the monolayer is elastic rather than viscous over 0.1-10 Hz. Understanding the effects of small mole fractions of cholesterol should help resolve the controversial role cholesterol plays in human lung surfactants and may give clues as to how cholesterol influences raft formation in cell membranes.

Optical vortex arrays from smectic liquid crystals.

We demonstrate large-area, closely-packed optical vortex arrays using self-assembled defects in smectic liquid crystals. Self-assembled smectic liquid crystals in a three-dimensional torus structure are called focal conic domains. Each FCD, having a micro-scale feature size, produces an optical vortex with consistent topological charge of 2. The spiral profile in the interferometry confirms the formation of an optical vortex, which is predicted by Jones matrix calculations.

Fluorescence recovery after merging a surfactant-covered droplet: a novel technique to measure the diffusion of phospholipid monolayers at fluid/fluid interfaces.

We present a novel technique to measure diffusion coefficients of insoluble surfactant monolayers. We merge a surfactant-coated droplet with a fluorescently labeled planar monolayer. During the merging process, a monolayer on a droplet displaces the existing planar monolayer, leaving a dark area when viewed under a fluorescence microscope. We measure fractional intensities as the dyes recover, which allows diffusion coefficients to be computed. We validate this technique with the two most common phospholipid monolayers (DPPC and DOPC) and study the diffusion of their mixtures. The proposed technique has several advantages over the FRAP technique and is potentially capable of measuring the diffusion of any soluble/insoluble surfactant monolayers.

Surface charge regulation of carboxyl terminated polystyrene latex particles and their interactions at the oil/water interface.

We study electrostatic interactions of polystyrene particles at an oil/water interface controlled by a chemical reaction of carboxylate surface functional groups. By replacing the carboxyl functional groups with hydrocarbon chains using the well-known EDC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide) coupling reaction, the surface charge density decreases while the hydrophobicity of the colloid surface increases. Direct visualization of the particle-laden interface reveals that, depending on the extent of hydrocarbon coupling, the strength of the electrostatic repulsion can be regulated: the repulsive interaction increases with the reaction, removing aggregates, but rapidly decreases if the reaction proceeds too much, forming a large aggregation. This simple reaction, thus, dramatically changes the structures of the colloidal monolayers at the oil/water interface. We conclude that such structural change is the result of change of the repulsive interactions from the oil phase, although interactions in the water phase are also changed slightly.

Influence of molecular coherence on surface viscosity.

Adding small fractions of cholesterol decreases the interfacial viscosity of dipalmitoylphosphatidylcholine (DPPC) monolayers by an order of magnitude per wt %. Grazing incidence X-ray diffraction shows that cholesterol at these small fractions does not mix ideally with DPPC but rather induces nanophase separated structures of an ordered, primarily DPPC phase bordered by a line-active, disordered, mixed DPPC-cholesterol phase. We propose that the free area in the classic Cohen and Turnbull model of viscosity is inversely proportional to the number of molecules in the coherence area, or product of the two coherence lengths. Cholesterol significantly reduces the coherence area of the crystals as well as the interfacial viscosity. Using this free area collapses the surface viscosity data for all surface pressures and cholesterol fractions to a universal logarithmic relation. The extent of molecular coherence appears to be a fundamental factor in determining surface viscosity in ordered monolayers.

Surface charge effects on optical trapping of nanometer-sized lipid vesicles.

Optical trapping of nanometer-sized lipid vesicles has been challenging due to the low refractive index contrast of the thin lipid bilayer to the aqueous medium. Using an "optical bottle", a recently developed technique to measure interactions of nanoparticles trapped by an infrared laser, we report, for the first time, quantitative measurements of the trapping energy of charged lipid vesicles. We found that the trapping energy increases with the relative amount of anionic lipids (DOPG) to neutral lipids (DOPC) in vesicles. Moreover, as monovalent salt is added into the exterior solution of vesicles, the trapping energy rapidly approaches zero, and this decrease in trapping energy strongly depends on the amount of anionic lipids in vesicles. A simple model with our experimental observations explains that the trapping energy of charged lipid vesicles is highly correlated with the surface charge density and electric double layer. In addition, we demonstrated selective trapping of a binary mixture of vesicles in different mole fractions of charged lipids, a strategy that has potential implications on charge selective vesicle sorting for engineering applications.

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.