Droplet Polymer Bilayers for Bioelectronic Membrane Interfacing.

Model membranes interfaced with bioelectronics allow for the exploration of fundamental cell processes and the design of biomimetic sensors. Organic conducting polymers are an attractive surface on which to study the electrical properties of membranes because of their low impedance, high biocompatibility, and hygroscopic nature. However, establishing supported lipid bilayers (SLBs) on conducting polymers has lagged significantly behind other substrate materials, namely, for challenges in membrane electrical sealing and stability. Unlike SLBs that are highly dependent on surface interactions, droplet interface bilayers (DIBs) and droplet hydrogel bilayers (DHBs) leverage the energetically favorable organization of phospholipids at atomically smooth liquid interfaces to build high-integrity membranes. For the first time, we report the formation of droplet polymer bilayers (DPBs) between a lipid-coated aqueous droplet and the high-performing conducting polymer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). The resulting bilayers can be produced from a range of lipid compositions and demonstrate strong electrical sealing that outcompetes SLBs. DPBs are subsequently translated to patterned and planar microelectrode arrays to ease barriers to implementation and improve the reliability of membrane formation. This platform enables more reproducible and robust membranes on conducting polymers to further the mission of merging bioelectronics and synthetic, natural, or hybrid bilayer membranes.

Simultaneous Electrophysiology and Imaging Reveal Changes in Lipid Membrane Thickness and Tension upon Uptake of Amphiphilic Gold Nanoparticles.

Amphiphilic gold core nanoparticles (AmNPs) striped with hydrophilic 11-mercapto-1-undecanesulfonate (MUS) and hydrophobic 1-octanethiol (OT) ligands are promising candidates for drug carriers that passively and nondisruptively enter cells. Yet, how they interact with cellular membranes is still only partially understood. Herein, we use electrophysiology and imaging to carefully assess changes in droplet interface bilayer lipid membranes (DIBs) incurred by striped AmNPs added via microinjection. We find that AmNPs spontaneously reduce the steady-state specific capacitance and contact angle of phosphatidylcholine DIBs by amounts dependent on the final NP concentration. These reductions, which are greater for NPs with a higher % OT ligands and membranes containing unsaturated lipids but negligible for MUS-only-coated NPs, reveal that AmNPs passively embed in the interior of the bilayer where they increase membrane thickness and lateral tension through disruption of lipid packing. These results demonstrate the enhanced evaluation of nano-bio interactions possible via electrophysiology and imaging of DIBs.

Insight into the Mechanisms Driving the Self-Assembly of Functional Interfaces: Moving from Lipids to Charged Amphiphilic Oligomers.

Polymer-stabilized liquid/liquid interfaces are an important and growing class of bioinspired materials that combine the structural and functional capabilities of advanced synthetic materials with naturally evolved biophysical systems. These platforms have the potential to serve as selective membranes for chemical separations and molecular sequencers and to even mimic neuromorphic computing elements. Despite the diversity in function, basic insight into the assembly of well-defined amphiphilic polymers to form functional structures remains elusive, which hinders the continued development of these technologies. In this work, we provide new mechanistic insight into the assembly of an amphiphilic polymer-stabilized oil/aqueous interface, in which the headgroups consist of positively charged methylimidazolium ionic liquids, and the tails are short, monodisperse oligodimethylsiloxanes covalently attached to the headgroups. We demonstrate using vibrational sum frequency generation spectroscopy and pendant drop tensiometery that the composition of the bulk aqueous phase, particularly the ionic strength, dictates the kinetics and structures of the amphiphiles in the organic phase as they decorate the interface. These results show that H-bonding and electrostatic interactions taking place in the aqueous phase bias the grafted oligomer conformations that are adopted in the neighboring oil phase. The kinetics of self-assembly were ionic strength dependent and found to be surprisingly slow, being composed of distinct regimes where molecules adsorb and reorient on relatively fast time scales, but where conformational sampling and frustrated packing takes place over longer time scales. These results set the stage for understanding related chemical phenomena of bioinspired materials in diverse technological and fundamental scientific fields and provide a solid physical foundation on which to design new functional interfaces.

Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes.

In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interface bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ∼38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near transition temperatures by preventing unwanted defects and macroscopic demixing associated with high-enthalpy transitions commonly found in simpler mixtures.

Air-stable droplet interface bilayers on oil-infused surfaces.

Droplet interface bilayers are versatile model membranes useful for synthetic biology and biosensing; however, to date they have always been confined to fluid reservoirs. Here, we demonstrate that when two or more water droplets collide on an oil-infused substrate, they exhibit noncoalescence due to the formation of a thin oil film that gets squeezed between the droplets from the bottom up. We show that when phospholipids are included in the water droplets, a stable droplet interface bilayer forms between the noncoalescing water droplets. As with traditional oil-submerged droplet interface bilayers, we were able to characterize ion channel transport by incorporating peptides into each droplet. Our findings reveal that droplet interface bilayers can function in ambient environments, which could potentially enable biosensing of airborne matter.

Lipid-Detergent Phase Transitions During Detergent-Mediated Liposome Solubilization.

We investigate the phase transition stages for detergent-mediated liposome solubilization of bio-mimetic membranes with the motivation of integrating membrane-bound Photosystem I into bio-hybrid opto-electronic devices. To this end, the interaction of two non-ionic detergents n-dodecyl-ß-D-maltoside (DDM) and Triton X-100 (TX-100) with two types of phospholipids, namely DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), are examined. Specifically, solubilization processes for large unilamellar liposomes are studied with the aid of turbidity measurements, dynamic light scattering, and cryo-transmission electron microscopy imaging. Our results indicate that the solubilization process is well depicted by a three-stage model, wherein the lamellar-to-micellar transitions for DPhPC liposomes are dictated by the critical detergent/phospholipid ratios. The solubilization of DPhPC by DDM is devoid of formation of a "gel-like" phase. Furthermore, our results indicate that DDM is a stable candidate for DPhPC solubilization and proteoliposome formation. Finally, although the solubilization of DPPG with DDM indicated the familiar three-stage process, the same process with TX-100 indicate structural deformation of vesicles into complex network of kinetically trapped micro- and nanostructured arrangements of lipid bilayers.

Reversible, voltage-activated formation of biomimetic membranes between triblock copolymer-coated aqueous droplets in good solvents.

Biomimetic membranes assembled from block copolymers attract considerable interest because they exhibit greater stability and longetivity compared to lipid bilayers, and some enable the reconstitution of functional transmembrane biomolecules. Yet to-date, block copolymer membranes have not been achieved using the droplet interface bilayer (DIB) method, which uniquely allows assembling single- and multi-membrane networks between water droplets in oil. Herein, we investigate the formation of poly(ethylene oxide)-b-poly(dimethyl siloxane)-b-poly(ethylene oxide) triblock copolymer-stabilized interfaces (CSIs) between polymer-coated aqueous droplets in solutions comprising combinations of decane, hexadecane and AR20 silicone oil. We demonstrate that triblock-coated droplets do not spontaneously adhere in these oils because all are thermodynamically good solvents for the hydrophobic PDMS middle block. However, thinned planar membranes are reversibly formed at the interface between droplets upon the application of a sufficient transmembrane voltage, which removes excess solvent from between droplets through electrocompression. At applied voltages above the threshold required to initiate membrane thinning, electrowetting causes the area of the CSI between droplets to increase while thickness remains constant; the CSI electrowetting response is similar to that encountered with lipid-based DIBs. In combination, these results reveal that stable membranes can be assembled in a manner that is completely reversible when an external pressure is used to overcome a barrier to adhesion caused by solvent-chain interactions, and they demonstrate new capability for connecting and disconnecting aqueous droplets via polymer-stabilized membranes.

Heating-enabled formation of droplet interface bilayers using Escherichia coli total lipid extract.

Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method to assemble DIBs using total lipid extract from Escherichia coli (eTLE); it is found that incubating oil-submerged aqueous droplets containing eTLE liposomes at a temperature above the gel-fluid phase transition temperature (Tg) promotes monolayer self-assembly that does not occur below Tg. Once monolayers are properly assembled via heating, droplets can be directly connected or cooled below Tg and then connected to initiate bilayer formation. This outcome contrasts immediate droplet coalescence observed upon contact between nonheated eTLE-infused droplets. Specific capacitance measurements confirm that the interface between droplets containing eTLE lipids is a lipid bilayer with thickness of 29.6 Å at 25 °C in hexadecane. We observe that bilayers formed from eTLE or DPhPC survive cooling and heating between 25 and 50 °C and demonstrate gigaohm (GΩ) membrane resistances at all temperatures tested. Additionally, we study the insertion of alamethicin peptides into both eTLE and DPhPC membranes to understand how lipid composition, temperature, and membrane phase influence ion channel formation. Like in DPhPC bilayers, alamethicin peptides in eTLE exhibit discrete, voltage-dependent gating characterized by multiple open channel conductance levels, though at significantly lower applied voltages. Cyclic voltammetry measurements of macroscopic channel currents confirm that the voltage-dependent conductance of alamethicin channels in eTLE bilayers occurs at lower voltages than in DPhPC bilayers at equivalent peptide concentrations. This result suggests that eTLE membranes, via composition, fluidity, or the presence of subdomains, offer an environment that enhances alamethicin insertion. For both membrane compositions, increasing temperature reduces the lifetimes of single channel gating events and increases the voltage required to cause an exponential increase in channel current. However, the fact that alamethicin insertion in eTLE exhibits significantly greater sensitivity to temperature changes through its Tg suggests that membrane phase plays an important role in channel formation. These effects are much less severe in DPhPC, where heating from 25 to 50 °C does not induce a phase change. The described technique for heating-assisted monolayer formation permits the use of other high transition temperature lipids in aqueous droplets for DIB formation, thereby increasing the types of lipids that can be considered for assembling model membranes.

Adsorption Kinetics Dictate Monolayer Self-Assembly for Both Lipid-In and Lipid-Out Approaches to Droplet Interface Bilayer Formation.

The droplet interface bilayer (DIB)--a method to assemble planar lipid bilayer membranes between lipid-coated aqueous droplets--has gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous droplet-oil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: "lipid-in", in which phospholipids in the form of liposomes are placed in water, and "lipid-out", in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods and exhibit different success rates for bilayer formation. In this study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. We also clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid placement and type are characterized.

Control of membrane permeability in air-stable droplet interface bilayers.

Air-stable droplet interface bilayers (airDIBs) on oil-infused surfaces are versatile model membranes for synthetic biology applications, including biosensing of airborne species. However, airDIBs are subject to evaporation, which can, over time, destabilize them and reduce their useful lifetime compared to traditional DIBs that are fully submerged in oil. Here, we show that the lifetimes of airDIBs can be extended by as much as an order of magnitude by maintaining the temperature just above the dew point. We find that raising the temperature from near the dew point (which was 7 °C at 38.5% relative humidity and 22 °C air temperature) to 20 °C results in the loss of hydrated water molecules from the polar headgroups of the lipid bilayer membrane due to evaporation, resulting in a phase transition with increased disorder. This dehydration transition primarily affects the bilayer electrical resistance by increasing the permeability through an increasingly disordered polar headgroup region of the bilayer. Temperature and relative humidity are conveniently tunable parameters for controlling the stability and composition of airDIB membranes while still allowing for operation in ambient environments.

Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer.

Thickness and tension are important physical parameters of model cell membranes. However, traditional methods to measure these quantities require multiple experiments using separate equipment. This work introduces a new multi-step procedure for directly accessing in situ multiple physical properties of droplet interface bilayers (DIB), including specific capacitance (related to thickness), lipid monolayer tension in the Plateau-Gibbs border, and bilayer tension. The procedure employs a combination of mechanical manipulation of bilayer area followed by electrowetting of the capacitive interface to examine the sensitivities of bilayer capacitance to area and contact angle to voltage, respectively. These data allow for determining the specific capacitance of the membrane and surface tension of the lipid monolayer, which are then used to compute bilayer thickness and tension, respectively. The use of DIBs affords accurate optical imaging of the connected droplets in addition to electrical measurements of bilayer capacitance, and it allows for reversibly varying bilayer area. After validating the accuracy of the technique with diphytanoyl phosphatidylcholine (DPhPC) DIBs in hexadecane, the method is applied herein to quantify separately the effects on membrane thickness and tension caused by varying the solvent in which the DIB is formed and introducing cholesterol into the bilayer. Because the technique relies only on capacitance measurements and optical images to determine both thickness and tension, this approach is specifically well-suited for studying the effects of peptides, biomolecules, natural and synthetic nanoparticles, and other species that accumulate within membranes without altering bilayer conductance.

Dynamic morphologies of microscale droplet interface bilayers.

Droplet interface bilayers (DIBs) are a powerful platform for studying the dynamics of synthetic cellular membranes; however, very little has been done to exploit the unique dynamical features of DIBs. Here, we generate microscale droplet interface bilayers (µDIBs) by bringing together femtoliter-volume water droplets in a microfluidic oil channel, and characterize morphological changes of the µDIBs as the droplets shrink due to evaporation. By varying the initial conditions of the system, we identify three distinct classes of dynamic morphology. (1) Buckling and fission: when forming µDIBs using the lipid-out method (lipids in oil phase), lipids in the shrinking monolayers continually pair together and slide into the bilayer to conserve their mass. As the bilayer continues to grow, it becomes confined, buckles, and eventually fissions one or more vesicles. (2) Uniform shrinking: when using the lipid-in method (lipids in water phase) to form µDIBs, lipids uniformly transfer from the monolayers and bilayer into vesicles contained inside the water droplets. (3) Stretching and unzipping: finally, when the droplets are pinned to the wall(s) of the microfluidic channel, the droplets become stretched during evaporation, culminating in the unzipping of the bilayer and droplet separation. These findings offer a better understanding of the dynamics of coupled lipid interfaces.

Evaporation-induced buckling and fission of microscale droplet interface bilayers.

Droplet interface bilayers (DIBs) are a robust platform for studying synthetic cellular membranes; however, to date no DIBs have been produced at cellular length scales. Here, we create microscale droplet interface bilayers (µDIBs) at the interface between aqueous femtoliter-volume droplets within an oil-filled microfluidic channel. The uniquely large area-to-volume ratio of the droplets results in strong evaporation effects, causing the system to transition through three distinct regimes. First, the two adjacent droplets shrink into the shape of a single spherical droplet, where an augmented lipid bilayer partitions two hemispherical volumes. In the second regime, the combined effects of the shrinking monolayers and growing bilayer force the confined bilayer to buckle to conserve its mass. Finally, at a critical bending moment, the buckling bilayer fissions a vesicle to regulate its shape and mass. The µDIBs produced here enable evaporation-induced bilayer dynamics reminiscent of endo- and exocytosis in cells.

Entrapment and Voltage-Driven Reorganization of Hydrophobic Nanoparticles in Planar Phospholipid Bilayers.

Engineered nanoparticles (NPs) possess diverse physical and chemical properties, which make them attractive agents for targeted cellular interactions within the human body. Once affiliated with the plasma membrane, NPs can become embedded within its hydrophobic core, which can limit the intended therapeutic functionality and affect the associated toxicity. As such, understanding the physical effects of embedded NPs on a plasma membrane is critical to understanding their design and clinical use. Here, we demonstrate that functionalized, hydrophobic gold NPs dissolved in oil can be directly trapped within the hydrophobic interior of a phospholipid membrane assembled using the droplet interface bilayer technique. This approach to model membrane formation preserves lateral lipid diffusion found in cell membranes and permits simultaneous imaging and electrophysiology to study the effects of embedded NPs on the electromechanical properties of the bilayer. We show that trapped NPs enhance ion conductance and lateral membrane tension in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers while lowering the adhesive energy of the joined droplets. Embedded NPs also cause changes in bilayer capacitance and area in response to applied voltage, which are nonmonotonic for DOPC bilayers. This electrophysical characterization can reveal NP entrapment without relying on changes in membrane thickness. By evaluating the energetic components of membrane tension under an applied potential, we demonstrate that these nonmonotonic, voltage-dependent responses are caused by reversible clustering of NPs within the unsaturated DOPC membrane core; aggregates form spontaneously at low voltages and are dispersed by higher transmembrane potentials of magnitude similar to those found in the cellular environment. These findings allow for a better understanding of lipid-dependent NP interactions, while providing a platform to study relationships between other hydrophobic nanomaterials and organic membranes.

Homogeneous hybrid droplet interface bilayers assembled from binary mixtures of DPhPC phospholipids and PB-b-PEO diblock copolymers.

Hybrid membranes built from phospholipids and amphiphilic block copolymers seek to capitalize on the benefits of both constituents for constructing biomimetic interfaces with improved performance. However, hybrid membranes have not been formed or studied using the droplet interface bilayer (DIB) method, an approach that offers advantages for revealing nanoscale changes in membrane structure and mechanics and offers a path toward assembling higher-order tissues. We report on hybrid droplet interface bilayers (hDIBs) formed in hexadecane from binary mixtures of synthetic diphytanoyl phosphatidylcholine (DPhPC) lipids and low molecular weight 1,2 polybutadiene-b-polyethylene oxide (PBPEO) amphiphilic block copolymers and use electrophysiology measurements and imaging to assess the effects of PBPEO in the membrane. This work reveals that hDIBs containing up to 15 mol% PBPEO plus DPhPC are homogeneously mixtures of lipids and polymers, remain highly resistive to ion transport, and are stable-including under applied voltage. Moreover, they exhibit hydrophobic thicknesses similar to DPhPC-only bilayers, but also have significantly lower values of membrane tension. These characteristics coincide with reduced energy of adhesion between droplets and the formation of alamethicin ion channels at significantly lower threshold voltages, demonstrating that even moderate amounts of amphiphilic block copolymers in a lipid bilayer provide a route for tuning the physical properties of a biomimetic membrane.

Modeling the saturation of detergent association in mixed liposome systems.

Cells tune the lipid types present in their membranes to adjust for thermal and chemical stability, as well as to promote association and dissociation of small molecules and bound proteins. Understanding the influence of lipid type on molecule association would open doors for targeted cell therapies, in particular when molecular association is observed in the presence of competing membranes. For this reason, we modeled and experimentally observed the association of a small molecule with two membrane types present by measuring the association of the detergent Triton X-100 with two types of liposomes, egg phosphatidylcholine (ePC) liposomes and egg phosphatidic acid (ePA) liposomes, at varying ratios. We called this mixed liposomes, as each liposome population was formed from a different lipid type. Absorbance spectrometry was used to observe the stages of detergent association with mixed liposomes and to determine the detergent concentration at which the liposomes were fully saturated. A saturation model was also derived that predicts the detergent associated with each liposome type when the lipid bilayers are fully saturated with detergent. The techinical input parameters for the model are the detergent to lipid ratio and the relative absorbance intensity for each of the pure liposome species at saturation. With that, the association of detergent with any mixture of those liposome types at saturation can be determined.

Nanoparticle-Induced Disorder at Complex Liquid-Liquid Interfaces: Effects of Curvature and Compositional Synergy on Functional Surfaces.

The self-assembly of surfactant monolayers at interfaces plays a sweeping role in tasks ranging from household cleaning to the regulation of the respiratory system. The synergy between different nanoscale species at an interface can yield assemblies with exceptional properties, which enhance or modulate their function. However, understanding the mechanisms underlying coassembly, as well as the effects of intermolecular interactions at an interface, remains an emerging and challenging field of study. Herein, we study the interactions of gold nanoparticles striped with hydrophobic and hydrophilic ligands with phospholipids at a liquid-liquid interface and the resulting surface-bound complexes. We show that these nanoparticles, which are themselves minimally surface active, have a direct concentration-dependent effect on the rapid reduction of tension for assembling phospholipids at the interface, implying molecular coassembly. Through the use of sum frequency generation vibrational spectroscopy, we reveal that nanoparticles impart structural disorder to the lipid molecular layers, which is related to the increased volumes that amphiphiles can sample at the curved surface of a particle. The results strongly suggest that hydrophobic and electrostatic attractions imparted by nanoparticle functionalization drive lipid-nanoparticle complex assembly at the interface, which synergistically aids lipid adsorption even when lipids and nanoparticles approach the interface from opposite phases. The use of tensiometric and spectroscopic analyses reveals a physical picture of the system at the nanoscale, allowing for a quantitative analysis of the intermolecular behavior that can be extended to other systems.

Physical encapsulation of droplet interface bilayers for durable, portable biomolecular networks.

Physically-encapsulated droplet interface bilayers are formed by confining aqueous droplets encased in lipid monolayers within connected compartments of a solid substrate. Each droplet resides within an individual compartment and is positioned on a fixed electrode built into the solid substrate. Full encapsulation of the network is achieved with a solid cap that inserts into the substrate to form a closed volume. Encapsulated networks provide increased portability over unencapsulated networks by limiting droplet movement and through the integration of fixed electrodes into the supporting fixture. The formation of encapsulated droplet interface bilayers constructed from diphytanoyl phosphocoline (DPhPC) phospholipids is confirmed with electrical impedance spectroscopy, and cyclic voltammetry is used to measure the effect of alamethicin channels incorporated into the resulting lipid bilayers. The durability of the networks is quantified using a mechanical shaker to oscillate the bilayer in a direction transverse to the plane of the membrane and the results show that single droplet interface bilayers can withstand 1-10g of acceleration prior to bilayer failure. Observed failure modes include both droplet separation and bilayer rupturing, where the geometry of the supporting substrate and the presence of integrated electrodes are key contributors. Physically-encapsulated DIBs can be shaken, moved, and inverted without bilayer failure, enabling the creation of a new class of lab-on-chip devices.

Regulated attachment method for reconstituting lipid bilayers of prescribed size within flexible substrates.

A new method called the regulated attachment method (RAM) for reproducibly forming lipid bilayers within flexible substrates has been developed that enables precise control over the size of the bilayer. This technique uses a deformable flexible substrate to open and close an aperture that subdivides aqueous volumes submersed in an organic solvent. Phospholipids incorporated as vesicles in the aqueous phase self-assemble at the oil/water interface to form lipid monolayers that encapsulate each aqueous volume. Controlled attachment of opposing lipid monolayers is achieved by regulating the dimensions of the aperture in the substrate that separates the adjacent aqueous volumes. In this manner, the size of a lipid bilayer formed within a flexible substrate is a function of the substrate and aperture dimensions, and not determined by the sizes or shapes of the aqueous volumes. Lipid bilayers formed within the prototype flexible substrate exhibit DC resistances consistently higher than 10 GOmega and can survive 20-30x changes in area without rupture. Furthermore, RAM permits lipid bilayers to be completely unzipped after thinning by applying sufficient force to fully close the dividing aperture and even allows the introduction of species, such as alamethicin channels, into preformed lipid bilayers via controlled injection through an intersecting channel within the substrate. Controlling the size of the interface through indirect interactions with the supporting substrate offers a new platform for assembling durable lipid bilayers. We envision that this technology can be scaled to higher dimensions consisting of multiple apertures required for creating aqueous networks partitioned by functional lipid bilayers and to smaller length scales to produce very small lipid bilayers capable of hosting single proteins.

Lipid shape determination of detergent solubilization in mixed-lipid liposomes.

The effects of lipid charge and head group size on liposome partitioning by detergents is an important consideration for applications such as liposomal drug delivery or proteoliposome formation. Yet, the solubilization of mixed-lipid liposomes, those containing multiple types of lipids, by detergents has received insufficient attention. This study examines the incorporation into and subsequent dissolution of mixed-lipid liposomes comprised of both egg phosphatidylcholine (ePC) and egg phosphatidic acid (ePA) by the detergent Triton-X100 (TX). Liposomes were prepared with mixtures of the two lipids, ePC and ePA, at molar ratios from 0 to 1, then step-wise solubilized with TX. Changes in turbidity, size distribution, and molar heat power at constant temperature throughout the solubilization process were assessed. The data suggest that the difference in lipid shapes (shape factors = 0.74 and 1.4 [1,2]) affects packing in membranes, and hence influences how much TX can be incorporated before disruption. As such, liposomes containing the observed ratios of ePA incorporated higher concentrations of TX before initiating dissolution into detergent and lipid mixed-micelles. The cause was concluded to be increased mismatching in the bilayer from the conical shape of ePA compared to the cylindrical shape of ePC. Additionally, the degree to which ePA is approximated as conical versus cylindrical was modulated with pH. It was confirmed that less conical ePA behaved more similarly to ePC than more conical ePA. The understanding gained here on lipid shape in liposome incorporation of TX enables research to use in vitro liposomes that more closely mimic native membranes.