Bilayer lipid membranes from falling droplets

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid–liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid–alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.     

Miniaturized planar lipid bilayer: increased stability, low electric noise and fast fluid perfusion

A microfluidic device was designed allowing the formation of a planar lipid bilayer across a micron-sized aperture in a glass slide sandwiched between two polydimethylsiloxane channel systems. By flushing giant unilamellar vesicles through a 500-μm-wide channel above the hole, we were able to form a planar lipid bilayer across the hole, resulting in a giga-seal. We demonstrate incorporation of biological nanopores into the bilayer. This miniaturized system offers noise recordings comparable to open headstage noise (under 1 pA RMS at 10 kHz), fast precision perfusion on each side of the membrane and the use of nanoliter analyte volumes. This technique shows a promising potential for automation and parallelization of electrophysiological setups.     

Planar lipid bilayer reconstitution with a micro-fluidic system

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.     

Controlled delivery of proteins into bilayer lipid membranes on chip

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.     

Lipid bilayer deposition and patterning via air bubble collapse

We report a new method for forming patterned lipid bilayers on solid substrates. In bubble collapse deposition (BCD), an air bubble is first "inked" with a monolayer of phospholipid molecules and then touched to the surface of a thermally oxidized silicon wafer and the air is slowly withdrawn. As the bubble shrinks, the lipid monolayer pressure increases. Once the monolayer exceeds the collapse pressure, it folds back on itself, depositing a stable lipid bilayer on the surface. These bilayer disks have lateral diffusion coefficients consistent with high quality supported bilayers. By sequentially depositing bilayers in overlapping areas, fluid connections between bilayers of different compositions are formed. Performing vesicle rupture on the open substrate surrounding this bilayer patch results in a fluid but spatially isolated bilayer. Very little intermixing was observed between the vesicle rupture and bubble-deposited bilayers.     

Detection of membrane biointeractions based on fluorescence superquenching

Assays for biointeractions of molecules with supported lipid bilayers using fluorescence superquenching are described. A conjugated cationic polymer was adsorbed on to silica microspheres, which were then coated with an anionic lipid bilayer. The lipid bilayer attenuated superquenching by acting as a barrier between the conjugated polymer and its quencher. Biointeractions of the lipid bilayer with a membrane lytic peptide, melittin, were detected and quantitated by superquenching of the conjugated polyelectrolyte in flow cytometric and microfluidic bioassays. A higher sensitivity for detecting melittin lysis of the lipid bilayer at lower concentrations and shorter times for melittin action was found using flow cytometry in this study in comparison to other existing methods. This study combined the sensitivity of superquenching and flow cytometry to detect biointeractions with a lipid bilayer, which serves as a platform for developing functional assays for sensor applications, lipid enzymology, and investigations of molecular interactions. In addition, this study demonstrated proof-of-concept for using superquenching detected as a result of lipid bilayer disruption in a microfluidic format.     

Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10⁻⁹ cm²/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks

Droplet interface bilayer (DIB) networks have vast potential in the field of membrane biophysics, synthetic biology, and functional bio-electronics. However a technological bottleneck exists in network fabrication: existing methods are limited in terms of their automation, throughput, versatility, and ability to form well-defined 3-D networks. We have developed a series of novel and low-cost methodologies which address these limitations. The first involves building DIB networks around the contours of a microfluidic chip. The second uses flow rate and droplet size control to influence droplet packing geometries within a microfluidic chamber. The latter method enables the controlled formation of various 3-D network arrays consisting of thousands of interconnected symmetric and asymmetric lipid bilayers for the first time. Both approaches allow individual droplet position and composition to be controlled, paving the way for complex on-chip functional network synthesis.     

Vesicle adsorption on mesoporous silica and titania

Lipid bilayer formation via vesicle fusion on mesoporous silica and mesoporous titania was investigated using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescent recovery after photobleaching (FRAP). Results showed that lipid bilayers were formed on mesoporous silica and that intact vesicle adsorption was obtained on mesoporous titania. From the FRAP results, it could be concluded that the lipid bilayer was fluid; however, it had a smaller diffusivity constant compared to bilayers supported on a nonporous silica.     

微流控芯片电泳/电化学检测人单个肝癌细胞中的谷胱甘肽

采用微流控装置结合电化学检测研究了测定人单个血红细胞中谷胱甘肽(GSH)的方法。在该方法中,细胞的进样、定位、溶膜以及细胞中谷胱甘肽的转移和检测都在配有通道端安培检测器的双T形芯片中完成。单个细胞用液压导入到双T的交界面,在电泳缓冲液中毛地黄皂苷的作用下,细胞膜被穿孔。再施加直流电压,细胞被溶膜。释放出来的GSH被此直流电压电迁移至通道端并在Au/Hg电极上被检测。用校正曲线法可以定量测定单个细胞中的GSH。     

The design of molecular sensing interfaces with lipid-bilayer assemblies

We describe the current status of techniques for preparing planar lipid bilayers, the fusion of sensory elements and the design of bilayer interfaces for analytical applications. Advances in bilayer fabrication have allowed preparation of lipid bilayers with membrane resistance of >1 GΩ (“gigaseal”) in flow, microfluidic and array formats, which have enabled single-channel and multi-channel recording. Not only biological but also engineered channels have been adopted as sensory elements for lipid-bilayer-based biosensors. Synthetic and inorganic channels are also emerging for designing membranes for lipid-bilayer sensors. We discuss the potential of lipid bilayers containing biological, engineered or synthetic channels for the design of biosensors, including drug-screening biosensors.     

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.     

On-chip extrusion of lipid vesicles and tubes through microsized apertures

In this work we present the formation of micrometre-sized lipid vesicles and tubes with perfectly homogeneous diameter and extraordinary length. The method is a novel approach for unconventional fabrication of soft-matter microstructured devices based on the combination of top-down and bottom-up fabrication processes. Photolithography techniques are applied to fabricate microsized apertures that provide the requirements to form lipid structures with predictable size and to align and guide the vesicles and tubes in microstructured channels. The formation is facilitated by self-assembly of polar lipids to a lipid membrane that is afterwards forced to undergo a shape transformation by extrusion through a microsized aperture. Both the geometrical restriction by the small aperture and the pressure difference between the top and bottom sides of the aperture determine the form and length of the vesicles and tubes. A strong pressure difference favors the formation of lipid tubes, while a low pressure difference results in the formation of vesicle bunches with spherical and cylindrical shapes. Potential applications for the formed lipid structures could be as microreactors and transport channels as well as in the construction of flexible microfluidic networks.     

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.     

Long-term storable and shippable lipid bilayer membrane platform

The fragility and short lifetimes characteristic of conventionally formed lipid bilayer membranes has necessitated their preparation to be at the time and point of use. By using high freezing-point lipid-solvent mixtures, the process of lipid bilayer self-assembly may be reversibly arrested. In solid form, the bilayer precursor can be stored indefinitely and is sufficiently robust to withstand commercial shipping. Upon thawing, bilayer self-assembly resumes, resulting in a biologically functional membrane. Combination of this membrane precursor with an inexpensive chip results in a compact, practical, and disposable platform for ion channel measurements.     

Groove-spanning behavior of lipid membranes on microfabricated silicon substrates

We report on a spreading behavior of phospholipid membranes that arise from a lump of phospholipid (a lipid source) on topographically patterned substrates immersed in an aqueous solution. Microgrooves with well-defined shapes were prepared on Si111 surfaces by anisotropic etching in an alkaline solution. A spreading front that consists of membrane lobes and a single lipid bilayer was observed on the patterned silicon substrates by utilizing fluorescence interference contrast (FLIC) microscopy. FLIC images indicate that the membrane lobes span the microgrooves, while the underlying single lipid bilayer spread along the surface of the microgrooves. In fact, fluorescent polystyrene nanoparticles could be encapsulated in the microgrooves that were completely covered with the membrane lobes. The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid bilayers.     

Tethered bilayer lipid membranes self-assembled on mercury electrodes

In order to incorporate integral proteins in a functionally active state, metal-supported lipid bilayers must have a hydrophilic region interposed between the bilayer and the metal. This region is realized with a hydrophilic molecule terminating at one end with a sulfhydryl or disulfide group that anchors this "hydrophilic spacer" to the surface of a metal, such as gold or mercury. The other end of the hydrophilic spacer may be covalently linked to the polar head of a phospholipid molecule, giving rise to a supramolecule called "thiolipid" (TL). With respect to gold, mercury has the advantage of providing a defect-free and fluid surface to the self-assembling spacer. Hydrophilic spacers consisting of a polyethyleneoxy or a hexapeptide chain, as well as thiolipids derived from these spacers, were employed to fabricate mercury-supported lipid bilayers. The formation of a lipid bilayer on top of a self-assembled monolayer of a hydrophilic spacer, or of a single-lipid monolayer on top of a self-assembled monolayer of a thiolipid, was realized by simply immersing the coated mercury electrode into an aqueous solution across a lipid film previously spread on its surface at its spreading pressure. Particularly stable mercury-supported lipid bilayers were obtained by using thiolipids. The biomimetic properties of these lipid bilayers were tested by incorporating channel-forming polypeptides (gramicidin and melittin) and proteins (OmpF porin). The effect of the transmembrane potential on the function of these channels was estimated by using a simple electrostatic model of the mercury-solution interphase.