Bilayer Formation Research Articles

Curved Lipid Interfaces Studied with Grazing Incident SANS

Curved cell membranes are important for the function of the cell, both for compartmentalization in organelles within the cell, as well as for cellular mitosis. It has also been shown that the curvature of a lipid membrane can affect the concentration of membrane bound proteins. In this project we have used semiconductor nanowires in order to study the effect of curvature on phospholipid bilayers and membrane proteins. We have previously demonstrated the formation of a supported phospholipid bilayer on nano wires via vesicle fusion, which can be used as a proxy to curved membranes. Based on fluorescence recovery after photobleaching, FRAP, measurement, the phospholipid bilayers were found to follow the contours of the nano wires as continuous and fluid, locally curved bilayers. However these data do not reveal the coverage, composition and the dimensions of the bilayer. In this project we aim to revel this using grazing incident small angle neutron scattering (GISANS). For this purpose we have performed GISANS experiments at the KSW-1 at FRM II (Germany), VSANS at NIST (USA) and SANS2D at ISIS (UK) with phospholipid covered nanowires. The nanowires were covered with a phospholipid bilayer obtained from deposition from vesicular dispersion of mixture of 20mol% DOPE in DOPC. In these experiments we have successfully shown the formation of a lipid bilayer and the subsequent binding of membrane protein. These results shows that nanowire supported lipid bilayer is an excellent way to study biding membrane protein to curved lipid interfaces.

A platform for light-controlled formation of free-stranding lipid membranes

The engineering of artificial cells is one of the most significant scientific challenges. Thus, controlled fabrication and in situ monitoring of biomimetic nanoscale objects are among the central issues in current science and technology. Studies of transmembrane channels and cell mechanics often require the formation of lipid bilayers (LBs), their modification and their transfer to a particular place. We present here a novel approach for remotely controlled manipulation of LBs. Layer-by-layer deposition of polyethyleneimine and poly(sodium 4-styrenesulfonate) on a nanostructured TiO 2 photoanode was performed to obtain a surface with the desired net charge and to enhance photocatalytic performance. The LB was deposited on top of a multi-layer positive polymer cushion by the dispersion of negative vesicles. The separation distance between the electrostatically linked polyelectrolyte cushion and the LB can be adjusted by changing the environmental pH, as zwitter-ionic lipid molecules undergo pH-triggered charge-shifting. Protons were generated remotely by photoanodic water decomposition on the TiO 2 surface under 365 nm illumination. The resulting pH gradient was characterized by scanning vibrating electrode and scanning ion-selective electrode techniques. The light-induced reversible detachment of the LB from the polymer-cushioned photoactive substrate was found to correlate with suggested impedance models.

Uncovering Biophysical Properties and Interactions of Bacteria Membrane using An Outer Membrane Supported Bilayer

Antibiotic resistance is a growing global health concern that has been increasing in prevalence in the past few decades. In Gram-negative bacteria, changes in membrane properties such as surface charge, length of membrane components such as lipopolysaccharide (LPS), and in physical properties of the membrane such as domain clustering, can alter responses to antibiotics and mediate resistance. To study the effect of these properties, model membranes have been used to mimic bacterial membranes and to study changes in membrane properties using quantitative surface techniques. However, most model membrane platforms do not capture the complexity of bacterial membranes, which also contain proteins, LPS, and exhibit asymmetry and diverse lipid compositions. We developed a substrate-supported membrane platform using outer membrane vesicles (OMVs) to characterize biophysical membrane properties and investigate the interactions of bacterial membrane and antibacterial compounds. OMVs are naturally shed from the OM by many Gram-negative bacteria from the outer membrane (OM) and recapitulate OM composition and physical properties. Using five clinical bacterial isolates, we demonstrated the formation of fluid bilayers using a membrane fluorescent dye, octadecyl rhodamine B, and measured diffusion coefficients using fluorescence recovery after photobleaching (FRAP). Using total internal reflection fluorescence microscopy (TIRFM), we also confirmed the presence of outer membrane material such as lipid A of LPS and the proper orientation and asymmetry of the bilayer using genetic modifications involving cleavable fluorescent proteins. To study interactions between bacterial membrane and antibacterial compounds, we use optical techniques to observe changes in membrane properties and quantify membrane interactions using polymyxin B. Findings from this work demonstrate OMV bilayers as a platform for studying membrane interactions while retaining membrane components and asymmetry found in native bacterial membranes.

Paper-Supported Lipid Bilayers that can be Stored Before use

Supported lipid bilayers provide a biological membrane-like platform that can be engineered into devices for controlled small scale reactions, biosensing, or filtration purposes. Traditional lipid bilayer manufacturing techniques have time critical steps which limit their portability. Most lipid bilayer formation techniques begin with hydrophilic treatment of a substrate, e.g., plasma etching, followed immediately by the deposition of lipids upon the substrate. The hydrophilic treatments are reversible over time, so the time between treatment and lipid bilayer formation is critical, and likely the limiting step in manufacturing and distributing membranes. We have developed a lipid bilayer formation technique where we deposit stacks of lipids prior to plasma etching, which allows for a bilayer to be spread in aqueous solvent even several days later. This enables samples to be prepared and shipped, requiring only plasma etching and hydration at the point of use. Additionally, we have adapted this approach to a flexible substrate (PDMS cellulose) suitable for roll-to-roll deposition. We have compared lamellarity (by fluorescence quenching) and diffusion (by gradient relaxation) and found the flexible substrate's lipid membrane to be comparable to glass, and that the post-deposition plasma etching does not significantly alter the membrane's properties. Preliminary results suggest that the plasma etching of the lipids is mild enough that peroxidation is minimal. Together the post-deposition plasma etching and cellulose-PDMS substrates could lead to a bio-membrane products that are relatively cheap to produce and easy to distribute.

Removal of emerging contaminants from water by zeolite-rich composites: A first approach aiming at diclofenac and ketoprofen

Abstract In this study, composites of the natural zeolites and cationic surfactants cetylpyridinium chloride and Arquad® 2HT-75 were used for removal of two emerging contaminants – diclofenac sodium and ketoprofen. Modifying a clinoptilolite- and a phillipsite-rich tuff, with surfactants with one or two hydrophobic tails, resulted in composites in monolayer and bilayer forms. The intention was to better evaluate interactions of composites with selected molecules. Starting materials and composites were characterized by ATR–FTIR and STA coupled with EGA. The adsorption capacities of the prepared sorbents were estimated by determination of adsorption isotherms and kinetic runs. Maximum adsorption capacity, obtained from the Langmuir model, showed that the best results were for the bilayer form of the composites up to 35 mg/g. Between the two surfactants, composites with cetylpyridinium chloride gave better results. Zeta potential measurements showed that the surfactants turned out to be unstable on the zeolite surface, the only exception being bilayers prepared using the two-tailed surfactant Arquad® 2HT-75. These results suggested possible applications of these composites for water treatment purposes.

Continuous Curvature Change into Controllable and Responsive Onion-like Vesicles by Rigid Sphere-Rod Amphiphiles.

We observe the formation of highly controllable and responsive onion-like vesicles by using rigid sphere-rod amphiphilic hybrid macromolecules, composed of charged, hydrophilic Keggin-type clusters (spheres) and hydrophobic rod-like oligofluorenes (OFs). Unlike the commonly used approach, which mainly relies on chain bending of flexible molecules to satisfy different curvatures in onion-like vesicles, the rigid hybrids form flexible interdigitations by tuning the angles between OFs, leading to the formation of bilayers with different sizes. The self-assembled vesicles possess complete onion-like structures from most inner to outer layers, and their size (layer number) can be accurately manipulated by different solution conditions including solvent polarity, ionic strength, temperature, and hybrid concentration, with fixed interbilayer distance under all conditions. Moreover, the vesicle size (layer number) shows excellent reversibility to the change of temperature. The charged feature of spheres, rod length, and overall hybrid architecture shows significant effects on the formation of such onion-like vesicles.

Supported Lipid Bilayer Formation: Beyond Vesicle Fusion.

Supported lipid bilayers (SLBs) are cell-membrane-mimicking platforms that can be formed on solid surfaces and integrated with a wide range of surface-sensitive measurement techniques. SLBs are useful for unravelling details of fundamental membrane biology and biophysics as well as for various medical, biotechnology, and environmental science applications. Thus, there is high interest in developing simple and robust methods to fabricate SLBs. Currently, vesicle fusion is a popular method to form SLBs and involves the adsorption and spontaneous rupture of lipid vesicles on a solid surface. However, successful vesicle fusion depends on high-quality vesicle preparation, and it typically works with a narrow range of material supports and lipid compositions. In this Feature Article, we summarize current progress in developing two new SLB fabrication techniques termed the solvent-assisted lipid bilayer (SALB) and bicelle methods, which have compelling advantages such as simple sample preparation and compatibility with a wide range of material supports and lipid compositions. The molecular self-assembly principles underpinning the two strategies and important experimental parameters are critically discussed, and recent application examples are presented. Looking forward, we envision that these emerging SLB fabrication strategies can be widely adopted by specialists and nonspecialists alike, paving the way to enriching our understanding of lipid membrane properties and realizing new application possibilities.

Mechanism and Kinetics of Lipid Bilayer Formation in Solid-State Nanopores.

Solid-state nanopores provide a highly versatile platform for rapid electrical detection and analysis of single molecules. Lipid bilayer coating of the nanopores can reduce nonspecific analyte adsorption to the nanopore sidewalls and increase the sensing selectivity by providing possibilities for tethering specific ligands in a cell-membrane mimicking environment. However, the mechanism and kinetics of lipid bilayer formation from vesicles remain unclear in the presence of nanopores. In this work, we used a silicon-based, truncated pyramidal nanopore array as the support for lipid bilayer formation. Lipid bilayer formation in the nanopores was monitored in real time by the change in ionic current through the nanopores. Statistical analysis revealed that a lipid bilayer is formed from the instantaneous rupture of individual vesicle upon adsorption in the nanopores, differing from the generally agreed mechanism that lipid bilayer forms at a high vesicle surface coverage on a planar support. The dependence of the lipid bilayer formation process on the applied bias, vesicle size, and concentration was systematically studied. In addition, the nonfouling properties of the lipid bilayer coated nanopores were demonstrated during long single-stranded DNA translocation through the nanopore array. The findings indicate that the lipid bilayer formation process can be modulated by introducing nanocavities intentionally on the planar surface to create active sites or changing the vesicle size and concentration.

Characterization and Modeling of Anodic Oxide Films on a Ti Alloy in Fluoride-Containing Electrolyte

The anodic oxidation of a titanium alloy (β-21S) in ethylene glycol—water—fluoride solution is studied by chronoamperometry, photocurrent and electrochemical impedance measurements in a wide range of applied potentials. The composition of the surface oxides is estimated by X-ray photoelectron spectroscopy. Data on pure Ti are presented for comparative purposes. All the experimental data are successfully described by a kinetic model previously proposed for anodic oxidation of Ti with bilayer formation. A three-step regression procedure is used to estimate the values of kinetic and transport parameters of electrochemically detectable reaction steps. The effect of alloying elements (Mo and Nb) is discussed in terms of the formation of non-stoichiometric TiO2 containing significant amounts of Mo(V), Mo(VI) and Nb(V) substitutional ions.

Bilayer Graphene: From Stacking Order to Growth Mechanisms

Due to the unique bandgap tunability of bilayer graphene, the preparation of large‐sized bilayer graphene has attracted a wide range of attention. Herein, the preparation of bilayer graphene, from stacking order to growth mechanism, is reviewed, and the chemical vapor deposition (CVD) of AB‐stacked bilayer graphene on copper substrate is emphasized. Various methods and growth strategies to synthesize bilayer graphene and the corresponding growth mechanisms are discussed. Mechanisms of layer‐by‐layer growth, the hydrogen passivation of graphene edges for the formation of bilayers, and carbon atoms penetrating through a copper wall for bilayer growth are included and highlighted for a better understanding of controlling bilayer graphene uniformity and forming its stacking order. Finally, the remaining challenges and the potential development of CVD‐controlled growth of bilayer graphene are outlined.

Silicon-graphene composite synthesis: Microstructural, spectroscopic and electrical conductivity characterizations

In this research work, we explored an idea to successfully prepare Si-graphene (0.5–1.5 wt%) composites by an uniquely in-house designed planetary ball milling process. XRD of Si-graphene composites show major peak of Si (1 1 1) and C(0 0 2). With increasing addition of graphene, intensity of C(0 0 2) peak is found to be increased in the composites. Raman spectra show peaks of Si along with different peaks of carbon such as D, G and 2D peaks. Micro Raman and TEM characterizations indicate that graphene in the Si-graphene (1–1.5 wt%) composite is developed in bi-layer form. HRTEM of typical Si-graphene (1 wt%) composite confirms the formation of composite between graphene and silicon. XPS spectra of de-convoluted high resolution C 1s and Si 2p further confirm the formation of Si-graphene composites. XPS spectra of composites show that more amount of Si is converted into composite with graphene along with also presence of only free Si. When graphene (0.5–1.5 wt%) is added to Si, it is observed that the specific surface area is improved significantly from 87 to 304 m2 g−1. In this work, it is found that the typical Si-graphene (1 wt%) composite exhibits 11% more electrical conductivity than pure Si.

Biological Functions of Plasmalogens.

Plasmalogens (Pls) are one kind of phospholipids enriched in the brain and other organs. These lipids were thought to be involved in the membrane bilayer formation and anti-oxidant function. However, extensive studies revealed that Pls exhibit various beneficial biological activities including prevention of neuroinflammation, improvement of cognitive function, and inhibition of neuronal cell death. The biological activities of Pls were associated with the changes in cellular signaling and gene expression. Membrane-bound GPCRs were identified as possible receptors of Pls, suggesting that Pls might function as ligands or hormones. Aging, stress, and inflammatory stimuli reduced the Pls contents in cells, and addition of Pls inhibited inflammatory processes, which could suggest that reduction of Pls might be one of the risk factors for the diseases associated with inflammation. Oral ingestion of Pls showed promising health benefits among Alzheimer's disease (AD) patients, suggesting that Pls might have therapeutic potential in other neurodegenerative diseases.

The Biosurfactant β-Aescin: A Review on the Physico-Chemical Properties and Its Interaction with Lipid Model Membranes and Langmuir Monolayers.

This review discusses recent progress in physicochemical understanding of the action of the saponin -aescin (also called -escin), the biologically active component in the seeds of the horse chestnut tree Aesculus hippocastanum. -Aescin is used in pharmacological and cosmetic applications showing strong surface activity. In this review, we outline the most important findings describing the behavior of -aescin in solution (e.g., critical micelle concentration () and micelle shape) and special physicochemical properties of adsorbed -aescin monolayers at the air–water and oil–water interface. Such monolayers were found to posses very special viscoelastic properties. The presentation of the experimental findings is complemented by discussing recent molecular dynamics simulations. These simulations do not only quantify the predominant interactions in adsorbed monolayers but also highlight the different behavior of neutral and ionized -aescin molecules. The review concludes on the interaction of -aescin with phospholipid model membranes in the form of bilayers and Langmuir monolayers. The interaction of -aescin with lipid bilayers was found to strongly depend on its . At concentrations below the , membrane parameters are modified whereas above the , complete solubilization of the bilayers occurs, depending on lipid phase state and concentration. In the presence of gel-phase phospholipids, discoidal bicelles form; these are tunable in size by composition. The phase behavior of -aescin with lipid membranes can also be modified by addition of other molecules such as cholesterol or drug molecules. The lipid phase state also determines the penetration rate of -aescin molecules into lipid monolayers. The strongest interaction was always found in the presence of gel-phase phospholipid molecules.

Supported Lipid Membranes at the Au‐Buffer Interface by Solvent Exchange: The Effect of Initial Solvent Concentration

The recently introduced solvent‐assisted lipid bilayer (SALB) formation method has proved a fast, simple, and versatile alternative to vesicle fusion in forming membrane‐mimicking platforms on solid supports such as SiO2, TiO2, graphene, and Au. This confers SALB with a broad applicability for developing biomaterial coatings for implants, biomimetic cell culture platforms, and cancer diagnostics. Although significant understanding on the interplay of the parameters controlling SALB (type of solvent, lipid concentration, and flow rate) has been achieved to date, there still exists several unexplored specific questions to be tackled. Herein, the question of how the initial solvent concentration affects the formation and organization of (zwitterionic) lipid membranes formed by SALB on Au surfaces is addressed. Using quartz crystal microbalance with dissipation monitoring, differences in frequency and dissipation responses are observed upon dissolving the lipid compound dimyristoyl phosphatidylcholine (DMPC) in either pure isopropanol or mixtures of different volume ratios of isopropanol and aqueous buffer. At high and medium initial solvent concentrations, thin and rigid supported lipid bilayers (SLBs) are typically formed at the Au–liquid interface, irrespective of the lipid structures formed in bulk, whereas at low solvent concentrations, irreversible vesicle adsorption takes place hindering the homogeneous SLB formation.

Probing the influence of tether density on tethered bilayer lipid membrane (tBLM)-peptide interactions

Tethered bilayer lipid membranes (tBLMs) represent a promising model membrane system that can host transmembrane proteins to serve as biosensors with exceptional detection performance. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we systematically investigated the influence of tether density on tBLM-peptide interactions and characterized the membrane binding dynamics of membrane-active peptides on tBLMs using AH peptide as a model. To achieve both physical stability and nanoscale separation from the support substrate, the tBLMs were fabricated on self-assembled monolayers (SAMs) obtained using a mixture of tether and spacer molecules with controlled tether-to-spacer ratio from 1:99 (T1) to 100:0 (T100). The solvent-assisted lipid bilayer (SALB) formation method was then employed to form the tBLMs, before the introduction of AH peptide. The QCM-D measurement responses indicated that the interactions between AH peptide and tBLMs involved peptide adsorption on the membrane layer followed by peptide translocation across the membrane. With increasing tether density, the abundance of hydrophobic groups within the tether chain led to stronger interactions and greater amount of translocated peptide. Depending on the tether density, this could result in significant structural transformation within the tBLM. Taken together, our work highlights the prospect of modulating membrane-peptide interactions by means of controlling the tBLM architecture, which will facilitate the creation of model membrane systems with highly tailored functionalities.

Self-Assembly of Monolayer Vesicles via Backbone-Shiftable Synthesis of Janus Core–Shell Bottlebrush Polymer

We report the self-assembly of monolayer vesicles from Janus core–shell bottlebrush polymers. A route was developed to synthesize doubly grafted bottlebrush copolymers (DGBCPs) possessing A-b-B and B′-b-C side chains on a single repeating unit. Graft-through ring-opening metathesis polymerization of a norbornene moiety installed by single unit monomer insertion allowed us to place the backbone on any repeating unit of the core (B and B′) block. By decorating each core chain end with different chains via reversible addition–fragmentation chain transfer polymerization, we can obtain nanoobjects with an asymmetric B core and a phase-separated A/C shell. We demonstrate that polystyrene-branch-polystyrene′ and polylactide-b-polystyrene-branch-polystyrene′-b-poly(n-butyl acrylate) macromonomers can be successfully synthesized and polymerized to produce DGBCPs in high yields (81–94% conversion) with an absolute molar mass of 149–395 kg mol–1 and a dispersity of 1.18–1.38. In a solvent slightly more selective to A than C, self-assembly of monolayer vesicles with diameter of <100 nm was observed by transmission electron microscopy. Dissipative particle dynamics simulations suggest that increasing the backbone length and moving the backbone toward the B′/C interface increases the backbone bending energy and favors a lower curvature. The spontaneous curvature appears to prefer a particular layer radius, avoiding bilayer formation.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid.

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

Human Colon-on-a-Chip Enables Continuous InVitro Analysis of Colon Mucus Layer Accumulation and Physiology.

Background & AimsThe mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology.MethodsA human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures noninvasively.ResultsThe Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel–dependent increase in its hydration state, but no increase in de novo mucus secretion.ConclusionsThis study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer.

Direct observation of van der Waals stacking-dependent interlayer magnetism.

Controlling the crystal structure is a powerful approach for manipulating the fundamental properties of solids. In van der Waals materials, this control can be achieved by modifying the stacking order through rotation and translation between the layers. Here, we observed stacking-dependent interlayer magnetism in the two-dimensional (2D) magnetic semiconductor chromium tribromide (CrBr3), which was enabled by the successful growth of its monolayer and bilayer through molecular beam epitaxy. Using in situ spin-polarized scanning tunneling microscopy and spectroscopy, we directly correlate the atomic lattice structure with the observed magnetic order. Although the individual monolayer CrBr3 is ferromagnetic, the interlayer coupling in bilayer depends on the stacking order and can be either ferromagnetic or antiferromagnetic. Our observations pave the way for manipulating 2D magnetism with layer twist angle control.

Preparation of an integrated porous substrate of 11-mercaptoundecanoic acid and chitosan on gold for electrochemical study of pores and pore forming interactions in lipid bilayers

In this work formation of a planar phospholipid bilayer of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine on an integrated porous cushion and its interaction with the ionic liquid 1-ethyl-3-methylimidazolium tosylate was studied. The integrated porous cushion was prepared by grafting chitosan covalently on porous 11-mercaptoundecanoic acid self-assembled monolayer on gold. Pores on the self-assembled monolayer were created by partial electro-oxidative desorption of the thiol molecules from the gold surface. Chitosan was grafted on the porous self-assembled monolayer by EDC/NSH activation procedure. Lipid bilayer was formed through solvent exchange method and evidenced with QCM-D, fluorescence microscopy, and electrochemical impedance spectroscopy. A lowering of QCM frequency of 26.4±1.5 Hz was observed during the bilayer formation. Impedance measurement showed that the lipid bilayer deposited integrated membrane can attain a high electrical resistance of 27 MΩcm2. The said ionic liquid created pores in the bilayer through interaction that was evidenced by significant decrease in charge transfer resistance in the impedance spectra and corroborated with fluorescence imaging. The work has indicated that the ionic liquid 1-ethyl-3-methylimidazolium tosylate can be cytotoxic. The new strategy for the formation of polymer cushion on a porous tether has shown new possibility of studying membrane attack interaction and ion diffusion in lipid bilayers through simple impedance analysis technique.