Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip

We developed a highly reproducible method for planar lipid bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar lipid bilayers are formed at apertures, 100 microm in diameter, by flowing lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the lipid solution at the aperture determines the state of the lipid bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of lipid solution is distributed at the aperture. Then, the layer of lipid solution was thinned by applying an external pressure and finally became a bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for bilayer formation was 90%. Using this technique, four lipid bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.     

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.     

Determination of the lipid bilayer breakdown voltage by means of linear rising signal

Electroporation is characterized by formation of structural changes within the cell membrane, which are caused by the presence of electrical field. It is believed that "pores" are mostly formed in lipid bilayer structure; if so, planar lipid bilayer represents a suitable model for experimental and theoretical studies of cell membrane electroporation. The breakdown voltage of the lipid bilayer is usually determined by repeatedly applying a rectangular voltage pulse. The amplitude of the voltage pulse is incremented in small steps until the breakdown of the bilayer is obtained. Using such a protocol each bilayer is exposed to a voltage pulse many times and the number of applied voltage pulses is not known in advance. Such a pre-treatment of the lipid bilayer affects its stability and consequently the breakdown voltage of the lipid bilayer. The aim of this study is to examine an alternative approach for determination of the lipid bilayer breakdown voltage by linear rising voltage signal. Different slopes of linear rising signal have been used in our experiments (POPC lipids; folding method for forming in the salt solution of 100 mM KCl). The breakdown voltage depends on the slope of the linear rising signal. Results show that gently sloping voltage signal electroporates the lipid bilayer at a lower voltage then steep voltage signal. Linear rising signal with gentle slope can be considered as having longer pre-treatment of the lipid bilayer; thus, the corresponding breakdown voltage is lower. With decreasing the slope of linear rising signal, minimal breakdown voltage for specific lipid bilayer can be determined. Based on our results, we suggest determination of lipid bilayer breakdown voltage by linear rising signal. Better reproducibility and lower scattering are obtained due to the fact that each bilayer is exposed to electroporation treatment only once. Moreover, minimal breakdown voltage for specific lipid bilayer can be determined.     

Large scale biomimetic membrane arrays

To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical–electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO₂ laser micro-structured 8?×?8 aperture partition arrays with average aperture diameters of 301?±?5 μm. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24?×?24 and hexagonal 24?×?27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays.

Structure of a gel phase lipid bilayer prepared by the Langmuir-Blodgett/Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy

The structure of a planar supported lipid bilayer (PSLB) prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method was investigated by sum-frequency vibrational spectroscopy (SFVS). By using asymmetric lipid bilayers composed of selectively deuterated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids, the orientation of the fatty acid chains and phosphocholine headgroups has been determined independently for both leaflets of the bilayer. The alkyl chains of the lipids were found to be orientated approximately 13 degrees +/- 4 degrees from the surface normal for both leaflets. The lipid chains in both leaflets also contain some gauche content, which is consistent with previous NMR and FTIR studies of similar lipid systems. More importantly, the relative number of gauche defects does not seem to be influenced by the deposition method, LB versus LS. The headgroup orientation for the lipid film in contact with the silica support was determined to be 69 degrees +/- 3 degrees , whereas that in contact with the aqueous phase was 66 degrees +/- 4 degrees from the surface normal. The SFVS results indicate that the structure of the DSPC lipid film in contact with the solid support and the film adjacent to the aqueous phase are nearly identical in structure. These results suggesting the LB/LS deposition method do indeed produce symmetric lipid bilayers. These studies further add to the growing information on the efficacy of PSLBs as suitable models for biological membrane studies.     

Substrate-supported lipid nanotube arrays

This Communication describes the self-assembly of phospholipids into lipid nanotubes inside nanoporous anodic aluminum oxide substrate. Orientations of the lipid molecules in such lipid nanoscale structures were verified by high-resolution spin labeling EPR at 95 GHz. The static order parameter of lipids in such nanotube arrays was determined from low-temperature EPR spectra and was found to be exceptionally high, Sstatic approximately 0.9. We propose that substrate-supported lipid nanotube arrays have potential for building robust biochips and biosensors in which rigid nanoporous substrates protect the bilayer surface from contamination. The total bilayer surface in the lipid nanotube arrays is much greater than that in the planar substrate-supported membranes. The lipid nanotube arrays seem to be suitable for developing patterned lipid deposition and could be potentially used for patterning of membrane-associated molecules.     

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.     

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   

Probing organization and communication at layered interfaces

We have investigated the local organization intrinsic to a variety of interfacial structures, by both electrochemical and spectroscopic means. Our focus has been on the design and construction of biomimetic interfaces, where a lipid bilayer or a hybrid bilayer membrane can be bound to an interface. The goal of this work is ultimately to create an interface on a transducer surface that can support an enzyme in its active form. To this point, we have examined the extent of organization that is achievable in monolayers that will be used to bind bilayer structures to a transducer surface. Our electrochemical data point to the important role of the substrate surface in determining adlayer organization. We have also investigated the fluidity and structural heterogeneity of lipid bilayers using time-resolved and steady state fluorescence spectroscopy. Our data point to the highly interactive nature of lipid bilayer constituents, where perturbations introduced to one region have significant consequences on other regions of the bilayer. Such information is directly relevant to the existence and properties of lipid raft structures in both model and biological bilayers.     

Glutamate receptor incorporated in a mixed hybrid bilayer lipid membrane array, as a sensing element of a biosensor working under flowing conditions

The realization of a reliable receptor biosensor requires stable, long-lasting, reconstituted biomembranes able to supply a suitable biomimetic environment where the receptor can properly work after incorporation. To this end, we developed a new method for preparing stable biological membranes that couple the biomimetic properties of BLMs (bilayer lipid membranes) with the high stability of HBMs (hybrid bilayer membranes); this gives rise to an innovative assembly, named MHBLM (mixed hybrid bilayer lipid membrane). The present work deals with the characterization of biosensors achieved by embedding an ionotropic glutamate receptor (GluR) on MHBLM. Thanks to signal (transmembrane current) amplification, which is typical of natural receptors, the biosensor here produced detects glutamate at a level of nmol L(-1). The transmembrane current changes linearly vs glutamate up to 100 nmol L(-1), while the limit of detection is 1 nmol L(-1). In addition, the biosensor response can be modulated both by receptor agonists (glycine) and antagonists (Mg(2+)) as well, and by exploiting the biosensor response, the distribution of different kinds of ionotropic GluR present in the purified sample, and embedded in MHBLM, was also evaluated. Finally, one of the most important aspects of this investigation is represented by the high stability of the biomimetic system, which allows the use of biosensor under flowing conditions, where the solutions flow on both biomembrane faces.     

Kinetics of channel formation in bilayer lipid membranes (BLMs) and tethered BLMs: monazomycin and melittin

The kinetics of channel formation by the polyene-like antibiotic monazomycin, both in a bilayer lipid membrane (BLM) and in a tethered BLM (tBLM), and by the peptide melittin in a tBLM, is investigated. Stepping the applied potential from a value at which channels are not formed to one at which they are formed yields current vs time curves that are sigmoidal on a BLM, while they show a maximum on a tBLM; in the latter case, sigmoidal curves are obtained by plotting the charge against time. These curves are interpreted on the basis of a general kinetic model, which accounts for the potential-dependent penetration of adsorbed monomeric molecules into the lipid bilayer, followed by their aggregation with channel formation by a mechanism of nucleation and growth. In the case of monazomycin, which is present in the solution in the form of relatively hydrophilic clusters and is adsorbed as such on top of the lipid bilayer, penetration into the bilayer following a potential jump is assumed to be preceded by a potential-independent disaggregation of the adsorbed clusters into adsorbed monomers.     

非支撑磷脂双层膜制备及温度对其力学性质的影响

结合聚苯乙烯球刻蚀和微机电系统技术加工氮化硅纳米多孔膜, 并在其上用囊泡法制备非支撑磷脂双层膜, 通过温控原子力显微术(AFM)的成像模式和力曲线模式对非支撑磷脂双层膜的形貌和力学性质进行研究. 实验结果表明, 该方法制备的非支撑磷脂双层膜具有流动性, 能进行自我修复, 该特点有利于提供足够的非支撑磷脂双层膜区域用于其性质研究; 非支撑磷脂双层膜的膜破力和粘滞力均随着温度的升高而减小, 即膜的机械稳定性随着温度的升高而降低. 非支撑磷脂双层膜膜破力小于支撑磷脂双层膜的膜破力, 并且非支撑磷脂双层膜粘滞力随温度的变化趋势与支撑磷脂双层膜的变化趋势相反.     

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.     

Role of lipid charge in organization of water/lipid bilayer interface: insights via computer simulations

Anionic unsaturated lipid bilayers represent suitable model systems that mimic real cell membranes: they are fluid and possess a negative surface charge. Understanding of detailed molecular organization of water-lipid interfaces in such systems may provide an important insight into the mechanisms of proteins' binding to membranes. Molecular dynamics (MD) of full-atom hydrated lipid bilayers is one of the most powerful tools to address this problem in silico. Unfortunately, wide application of computational methods for such systems is limited by serious technical problems. They are mainly related to correct treatment of long-range electrostatic effects. In this study a physically reliable model of an anionic unsaturated bilayer of 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) was elaborated and subjected to long-term MD simulations. Electrostatic interactions were treated with two different algorithms: spherical cutoff function and particle-mesh Ewald summation (PME). To understand the role of lipid charge in the system behavior, similar calculations were also carried out for zwitterionic bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). It was shown that, for the charged DOPS bilayer, the PME protocol performs much better than the cutoff scheme. In the last case a number of artifacts in the structural organization of the bilayer were observed. All of them were attributed to inadequate treatment of electrostatic interactions of lipid headgroups with counterions. Electrostatic properties, along with structural and dynamic parameters, of both lipid bilayers were investigated. Comparative analysis of the MD data reveals that the water-lipid interface of the DOPC bilayer is looser than that for DOPS. This makes possible deeper penetration of water molecules inside the zwitterionic (DOPC) bilayer, where they strongly interact with carbonyls of lipids. This can lead to thickening of the membrane interface in zwitterionic as compared to negatively charged bilayers.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Absorption and fluorescence of PRODAN in phospholipid bilayers: a combined quantum mechanics and classical molecular dynamics study

Absorption and fluorescence spectra of PRODAN (6-propionyl-2-dimethylaminonaphthalene) were studied by means of the time-dependent density functional theory and the algebraic diagrammatic construction method. The influence of environment, a phosphatidylcholine lipid bilayer and water, was taken into account employing a combination of quantum chemical calculations with empirical force-field molecular dynamics simulations. Additionally, experimental absorption and emission spectra of PRODAN were measured in cyclohexane, water, and lipid vesicles. Both planar and twisted configurations of the first excited state of PRODAN were taken into account. The twisted structure is stabilized in both water and a lipid bilayer, and should be considered as an emitting state in polar environments. Orientation of the excited dye in the lipid bilayer significantly depends on configuration. In the bilayer, the fluorescence spectrum can be regarded as a combination of emission from both planar and twisted structures.     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

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.     

Transmembrane Signaling with Lipid‐Bilayer Assemblies as a Platform for Channel‐Based Biosensing

Artificial and natural lipid membranes that elicit transmembrane signaling is are useful as a platform for channel‐based biosensing. In this account we summarize our research on the design of transmembrane signaling associated with lipid bilayer membranes containing nanopore‐forming compounds. Channel‐forming compounds, such as receptor ion‐channels, channel‐forming peptides and synthetic channels, are embedded in planar and spherical bilayer lipid membranes to develop highly sensitive and selective biosensing methods for a variety of analytes. The membrane‐bound receptor approach is useful for introducing receptor sites on both planar and spherical bilayer lipid membranes. Natural receptors in biomembranes are also used for designing of biosensing methods.     

Lipids out of equilibrium: energetics of desorption and pore mediated flip-flop

The potential of mean force (PMF) of a phospholipid in a bilayer is a key thermodynamic property that describes the energetic cost of localized lipid defects. We have calculated the PMF by umbrella sampling using molecular dynamics simulations. The profile has a deep minimum at the equilibrium position in the bilayer and steeply rises for displacements both deeper into the bilayer and moving away from the bilayer. As the lipid loses contact with the bilayer, the profile abruptly flattens without a significant barrier. The calculated free energy difference of 80 kJ/mol between the minimum of the PMF and the value in water agrees well with the free energy difference calculated from the experimentally measured critical micelle concentration. Significant water/lipid defects form when a lipid is forced into the bilayer interior, in the form of a small water pore that spans the membrane. The energy required to form such a water pore is also found to be 80 kJ/mol. On the basis of this energy, we estimate the lipid flip-flop rate and permeability rate of sodium ions. The resulting rates are in good agreement with experimental measurements, suggesting lipid flip-flop and basal permeability of ions are pore mediated.