Determining membrane capacitance by dynamic control of droplet interface bilayer area

By making dynamic changes to the area of a droplet interface bilayer (DIB), we are able to measure the specific capacitance of lipid bilayers with improved accuracy and precision over existing methods. The dependence of membrane specific capacitance on the chain-length of the alkane oil present in the bilayer is similar to that observed in black lipid membranes. In contrast to conventional artificial bilayers, DIBs are not confined by an aperture, which enables us to determine that the dependence of whole bilayer capacitance on applied potential is predominantly a result of a spontaneous increase in bilayer area. This area change arises from the creation of new bilayer at the three phase interface and is driven by changes in surface tension with applied potential that can be described by the Young-Lippmann equation. By accounting for this area change, we are able to determine the proportion of the capacitance dependence that arises from a change in specific capacitance with applied potential. This method provides a new tool with which to investigate the vertical compression of the bilayer and understand the changes in bilayer thickness with applied potential. We find that, for 1,2-diphytanoyl-sn-glycero-3-phosphocholine membranes in hexadecane, specific bilayer capacitance varies by 0.6-1.5% over an applied potential of ±100 mV.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Triggered release of molecules across droplet interface bilayer lipid membranes using photopolymerizable lipids

A combination of nonpolymerizable phospholipids (DPPC or DPhPC) and a smaller amount of cross-linking photopolymerizable phospholipids (23:2 DiynePC) is incorporated in an unsupported artificial lipid bilayer formed using the droplet interface bilayer (DIB) approach. The DIB is formed by contacting lipid monolayer-coated aqueous droplets against each other in a dodecane-lipid medium. Cross-linking of the photopolymerizable lipids incorporated in the DIB was obtained by exposure to UV-C radiation (254 nm), resulting in pore formation. The effect of cross-linking on the DIB properties was characterized optically by measuring the diffusion of selectively encapsulated dye molecules (calcein) from one droplet of the DIB to the other droplet. Changes in DIB conductivity due to UV-C exposure were investigated using current-voltage (I-V) measurements. The leakage of dye molecules across the DIB and the increase in DIB conductivity after UV-C exposure indicates the formation of membrane pores. The results indicate that the DIB approach offers a simple and flexible platform for studying phototriggered drug delivery systems in vitro.     

A self-consistent field study of a hydrocarbon droplet at the air-water interface

A molecularly detailed self-consistent field (SCF) approach is applied to describe a sessile hydrocarbon droplet placed at the air-water interface. Predictions of the contact angle for macroscopic droplets follow from using Neumann's equation, wherein the macroscopic interfacial tensions are computed from one-gradient calculations for flat interfaces. A two-gradient cylindrical coordinate system with mirror-like boundary conditions is used to analyse the three dimensional shape of the nano-scale oil droplet at the air-water interface. These small droplets have a finite value of the Laplace pressure and concomitant line tension. It has been calculated that the oil-water and oil-vapour interfacial tensions are curvature dependent and increase slightly with increasing interfacial curvature. In contrast, the line tension tends to decrease with curvature. In all cases there is only a weak influence of the line tension on the droplet shape. We therefore argue that the nano-scale droplets, which are described in the SCF approach, are representative for macroscopic droplets and that the method can be used to efficiently generate accurate information on the spreading of oil droplets at the air-water interface in molecularly more complex situations. As an example, non-ionic surfactants have been included in the system to illustrate how a molecularly more complex situation will change the wetting properties of the sessile drop. This short forecast is aimed to outline and to stress the potential of the method.     

Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. Such DIB networks might be used as model systems for the study of membrane-based biological phenomena. In this study, we develop and experimentally validate an electrical modeling approach for DIB networks by applying it to describe the current flow through a simple network containing protein pores and blocking molecules. We demonstrate the use of SPICE (Simulation Program with Integrated Circuit Emphasis) for simulating the electrical behavior of DIB networks. The modular and scalable nature of DIB networks should enable a straightforward extension of the analysis presented in this paper to large, complex networks.     

Single channel recordings of alpha-hemolysin reconstituted in S-layer-supported lipid bilayers.

Previous studies demonstrated that lipid membranes attached to a proteinaceous crystalline surface-layer (S-layer) revealed a prolonged lifetime and showed a reduced tendency to rupture in the presence of membrane active molecules. In addition, comparative studies on folded and S-layer-supported lipid membranes (SsLM) revealed an uniform capacitance of 0.64 +/- 0.04 microF/cm(2) for both composite membranes. In the present study, the feasibility to reconstitute the channel-forming protein alpha-hemolysin (alpha HL) into SsLM at single channel resolution was investigated. Single alpha HL channels could be recorded and the intrinsic properties like unitary conductance, current-voltage characteristics, and closure was found to be similar at both membranes. Thus, the tightly attached S-layer allowed complete reconstitution of alpha HL channels in SsLM.     

Electrowetting of aligned carbon nanotube films

Electrowetting is one approach to reducing the interfacial tension between a solid and a liquid. In this method, an electrical potential is applied across the solid/liquid interface which modifies the wetting properties of the liquid on the solid without changing the composition of the solid and liquid phases. Electrowetting of aligned carbon nanotube (CNT) films is investigated by the sessile drop method by dispensing deionized (DI) water or 0.03 M NaCl droplets (contacted by Au wire) onto aligned CNT films assembled on a copper substrate. The results demonstrate that electrowetting can greatly reduce the hydrophobicity of the aligned CNTs; the contact angle saturation for DI water and 0.03 M NaCl droplets occurs at 98 and 50 degrees , respectively. The combined effects of the geometrical roughness and the electrical potential on the contact angle are briefly discussed and modeled. Such a strategy may be invoked to controllably reduce the interfacial tension between carbon nanotubes (CNTs) and polymer precursors when infiltrating the monomers into the prealigned nanotube films.     

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.     

Distribution of local anesthetics in lipid membranes

Absorption of local anesthetics into lipid membranes and adsorption onto their surfaces were studied as a function of the pH of aqueous bulk solutions by measuring lipid vesicle electrophoretic mobility, the partition of the anesthetics between the aqueous and membrane phases by the use of fluorescence and radioactive tracer methods, and the effect of the anesthetics on interfacial tension of lipid monolayers formed at the oil/aqueous interface.

At a pH much lower than the pK_a value of the local anesthetic, the charged form of the local anesthetic was only adsorbed onto the membrane surface, as determined from vesicle electrophoretic mobility, radioisotope tracer and the monolayer surface tension studies. Surface partition coefficients of the charged form of the local anesthetics on phosphatidylcholine and phosphatidylserine membranes were obtained from the data of electrophoretic mobilities for lipid vesicles. The surface partition coefficients of various local anesthetics paralleled those of the bulk partition coefficients.

As the pH of the solutions increased, the adsorbed amount of the charged form of the anesthetic at the membrane interface decreased, while the absorption of the uncharged form of the local anesthetic into the membrane increased. The total amount of local anesthetic adsorbed per unit area of the membrane generally increased as the pH of the solution increased. This was also observed from the measurements of the fluorescence of local anesthetics adsorbed into the membranes. At lower pH than that corresponding to the pK_a value of the local anesthetic, the amount of anesthetic adsorbed depended greatly upon the membrane surface charge. At a higher pH than its pK_a, it did not depend appreciably on the surface charge density of the membrane but did depend on the bulk partition coefficients between the aqueous and oil phases.     

A Comparison of Membrane Emulsification Obtained Using SPG (Shirasu Porous Glass) and PTFE [Poly(Tetrafluoroethylene)] Membranes

SPG (Shirasu porous glass) membrane emulsification used to prepare uniform polymeric microspheres is briefly reviewed, and the performance of a hydrophilically treated PTFE [poly(tetrafluoroethylerie)] membrane is described and compared with that of the SPG membrane. A mixture of styrene. divinyl benzene and hexadecane (HD) was extruded through the membranes and dispersed in an aqueous phase containing polyvinylalcohol (PVA) and sodium lauryl sulfate (SLS) as mixed stabilizers. A hvdrophilically treated PTFE membrane was used with a stainless steel mesh support so that the membrane would not expand to affect the pore size during the emulsification. The nominal pore size of the PTFE membrane was replaced with the calculated one using a theoretical expression derived from the force balance between the external pressure and the interfacial tension between oil and water phases. The emulsion droplets prepared with the PTFE membrane revealed a broader size distribution than those obtained with the SPG membrane, and the rate of emulsificaton was nearly same for both membranes. Droplet size control was readily possible. The performance was significantly affected by the adsorption behavior of the stabilizers on the membrane surfaces. The contact angle profile of oil droplets on the PTFE membrane implied that the hydrophilically treated PTFE membrane is still hydrophobic compared to the SPG membrane. This tendency was reflected by the dependence of the average droplet diameter (and coefficient of variation, CV) on the concentration and composition of mixed stabilizers.     

Voltage dependence of bilayer membrane capacitance

The change in capacitance of cholesterol-hexadecyltrimethylammonium bilayer membranes upon application of relatively high dc and ac potentials was measured as a function of frequency. Capacitance increases proportional to the square of voltage were observed (0.5–1% for 100 mV dc). The amplitude of the ac component of the capacitance variation decreased with frequency until a break frequency was reached, above which the capacitance response was constant at a value about two orders of magnitude lower than the dc response. By comparison with the elastic response of the membrane and border, it was concluded the capacitance response at higher frequency was due to bilayer thickness decrease with voltage (electrostriction) while the lower frequency response was associated with a bilayer area increase. Agreement was obtained between the measured electrostriction and that calculated from membrane elasticity. Area increases with voltage calculated assuming border shrinkage as a result of an increase in contact angle were in order of magnitude agreement with the observed dc response. The effects of lenses and bowed membranes are discussed.     

Functional bionetworks from nanoliter water droplets

We form networks from aqueous droplets by submerging them in an oil/lipid mixture. When the droplets are joined together, the lipid monolayers surrounding them combine at the interface to form a robust lipid bilayer. Various protein channels and pores can incorporate into the droplet-interface bilayer (DIB), and the application of a potential with electrodes embedded within the droplets allows ionic currents to be driven across the interface and measured. By joining droplets in linear or branched geometries, functional bionetworks can be created. Although the interfaces between neighboring droplets comprise only single lipid bilayers, the structures of the networks are long-lived and robust. Indeed, a single droplet can be "surgically" excised from a network and replaced with a new droplet without rupturing adjacent DIBs. Networks of droplets can be powered with internal "biobatteries" that use ion gradients or the light-driven proton pump bacteriorhodopsin. Besides their interest as coupled protocells, the droplets can be used as devices for ultrastable bilayer recording with greatly reduced electrolyte volume, which will permit their use in rapid screening applications.     

Giant lipid vesicles impaled with glass microelectrodes: GigaOhm seal by membrane spreading

Giant unilamellar lipid vesicles could be perfect systems to study ion channels in the environment of lipid membranes with defined chemical and physical properties. Prerequisite for electrical measurements is an intravesicular electrical contact. We describe the impalement of giant lipid vesicles by glass micropipet electrodes with a tight seal. To avoid displacement or burst during impalement, the vesicles are immobilized in relaxed conditions by microscopic picket fences of polyimide. The outer surface of the pipets is selectively coated with silanes or polylysine. Structurally, the impalement is verified by ejecting a fluorescent solution out of the pipet. For electrical characterization, current pulses are applied to the pipet and voltage transients are recorded. The data are evaluated in terms of the capacitance and effective resistance of the membrane. Directly after impalement, we observe a seal resistance up to 1.2 GOmega that continuously decays within a period of up to 20 min until it suddenly disappears without burst of the vesicle. During impalement, a spreading of the vesicle membrane along the outer surface of the pipets is observed using a fluorescent membrane-bound dye. We assign the tight pipet-vesicle contact to spreading of the lipid bilayer by a rolling mechanism and the loss of resistance to micro- and macropores that are induced by the resulting membrane tension. Limitation of spreading is attempted with barriers on the pipet.     

Optical measurement of the deformation of giant lipid vesicles driven by a micropipet electrode

We investigate the deformation of giant lipid vesicles driven by a micropipet electrode by use of differential confocal microscopy. This optical technique provides nanometer depth resolution without mechanical contact and hence prevents large tension or perforation of the soft membrane. For dipalmitoyl phosphatidylcholine (DPPC) membranes in the gel phase, we observed deformations of several hundreds of nanometers when the driving voltage was about 0.1 V. The voltage and frequency responses of the vesicle deformation can be explained by the balance between the electroosmotic force inside the micropipet and the membrane tension. We also used DPPC:cholesterol vesicles to check the validity of this model. In the fluid phase, however, the deformation is independent of the modulation signal because micrometer-scale thermal fluctuations dominate the membrane motion.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Alkylated glass partition allows formation of solvent-free lipid bilayer by Montal-Mueller technique

Formation of bilayer lipid membrane (BLM) by Montal-Mueller technique across a small aperture in a partition film traditionally requires coating of the aperture with a hydrophobic substance, often just an organic solvent. However, we demonstrate here that the most effective coating is not strictly hydrophobic but rather provides water/oil repellent properties. BLM were formed from diphytanoylphosphatidylcholine (DPhPC) on small 0.1-0.8 mm apertures made in specially prepared alkylated glass coverslips. The coverslips were either fluorosiliconized by 3,3,3-Trifluoropropyl-trimethoxysilane, which reduces adsorption of DPhPC in addition to creation of hydrophobic surface, or silanized, which promote adsorption of DPhPC. At fluorosiliconized surfaces stable BLM were formed. Specific capacitance of these BLM was 0.86 microF/cm(2)+/-5%, while their lateral tension was estimated as 4.3+/-0.4 mN/m. BLM were stable for hours under moderate voltage applied. At silanized surfaces stable BLM were formed only in acidic medium (3 相似文献   

Stabilization of polymer blend structure by diblock copolymers

The stabilizing (emulsifying) effect of a symmetric diblock copolymer in the mixture of two immiscible homopolymers is considered. The equilibrium value of the interfacial area per copolymer chain is calculated via minimization of the free energy of the mixture for a fixed number of copolymer chains adsorbed to the interface. The size and concentration of droplets of the minor component are determined for the equilibrium state. The particles' radius is shown to be inversely proportional to the copolymer concentration, the coefficient of proportionality being dependent on the Flory-Huggins parameter and chain length. The penetration of homopolymer segments into the copolymer layer on the interface is taken into account and proved to be important for stabilization of the droplets by symmetric copolymers. The conditions of the validity of the presented approach are discussed in detail.     

Lipid barrier layers on surfaces of synthetic water permeable membranes

Barrier layers (area 0.79 cm²) from oxidized cholesterol or mixtures of oxidized cholesterol with phosphatidyl serine or phosphatidyl ethanol amine were formed on surfaces of different water-permeable synthetic membranes in 0.1 M NaCl as models of biological membranes. Ionic conductivity across the membranes decreased from 10⁻²-10⁻³ to 10⁻⁷-10⁻⁸ Ω⁻¹ cm⁻² when the barrier layers were formed on their surfaces. Average thicknesses of barrier layers 4.5–11 nm were estimated from electric capacitance. The layers were unstable with lifetimes ranging from several minutes to 50 hr according to the support membrane used. The interfacial tension between synthetic membrane surface and either water or lipid solution was calculated from contact angle measurement. The relation between barrier layer stability and hydrophobic and polar interaction of lipids with support surface was studied. The most stable barrier layers (lifetimes 30–50 hr) were formed on cellophane and gelatin membranes with surfaces hydrophobized by reaction with palmytoyl chloride.     

Influence of ethanol on lipid membranes: from lateral pressure profiles to dynamics and partitioning

We have combined experiments with atomic-scale molecular dynamics simulations to consider the influence of ethanol on a variety of lipid membrane properties. We first employed isothermal titration calorimetry together with the solvent-null method to study the partitioning of ethanol molecules into saturated and unsaturated membrane systems. The results show that ethanol partitioning is considerably more favorable in unsaturated bilayers, which are characterized by their more disordered nature compared to their saturated counterparts. Simulation studies at varying ethanol concentrations propose that the partitioning of ethanol depends on its concentration, implying that the partitioning is a nonideal process. To gain further insight into the permeation of alcohols and their influence on lipid dynamics, we also employed molecular dynamics simulations to quantify kinetic events associated with the permeation of alcohols across a membrane, and to characterize the rotational and lateral diffusion of lipids and alcohols in these systems. The simulation results are in agreement with available experimental data and further show that alcohols have a small but non-vanishing effect on the dynamics of lipids in a membrane. The influence of ethanol on the lateral pressure profile of a lipid bilayer is found to be prominent: ethanol reduces the tension at the membrane-water interface and reduces the peaks in the lateral pressure profile close to the membrane-water interface. The changes in the lateral pressure profile are several hundred atmospheres. This supports the hypothesis that anesthetics may act by changing the lateral pressure profile exerted on proteins embedded in membranes.