Hybrid Bilayer Membrane Research Articles

Dielectrically Addressable Microspheres Engineered Using Self-Assembled Monolayers

We have used self-assembled monolayer techniques to produce a new class of microspheres with specifically engineered dielectric properties to enable their dielectrophoretic manipulation and identification in microsystems. Dielectrophoresis is an electrokinetic phenomenon that exploits frequency-dependent polarizability differences between a particle and its suspending medium to drive the movement of the particle toward or away from the high-field regions of an inhomogeneous electric field. While dielectrophoretic methods have been used extensively for cell manipulation, separation, and identification, we wished to extend the applicability of dielectrophoresis to molecular analysis by developing a panel of dielectric microspheres or "handles". Dielectric shell theory was used to model the dielectrophoretic response for a biomimetic particle composed of a thin insulating shell over a conductive interior. We specifically sought to modulate the specific capacitance, and thereby the dielectric properties, of the particle by controlling the thickness of the insulating layer. Such a structure was fabricated by covering a gold-coated polystyrene core particle with self-assembled monolayers of alkanethiol and phospholipid. To test the prediction that the carbon chain length of these layers should dictate the dielectric properties of the particles, we constructed a panel of six microsphere types with shell compositions ranging from a C(9) alkanethiol monolayer to a C(32) hybrid bilayer membrane. These microsphere populations were distinguishable and manipulatable by dielectrophoresis in a characteristic, frequency-dependent manner as predicted by theory. Experimentally derived specific membrane capacitance values were inversely related to the insulating shell thickness and agreed with published capacitance values for planar layers of similar thicknesses. These proof of principle studies are the first to demonstrate that the dielectric properties of particles can be specifically engineered to allow their dielectrophoretic manipulation and are a first step toward the development of bead-based dielectrophoretic microsystems for multiplexed molecular separation and analysis.

The role of surface free energy on the formation of hybrid bilayer membranes.

The interaction of small phospholipid vesicles with well-characterized surfaces has been studied to assess the effect of the surface free energy of the underlying monolayer on the formation of phospholipid/alkanethiol hybrid bilayer membranes (HBMs). The surface free energy was changed in a systematic manner using single-component alkanethiol monolayers and monolayers of binary mixtures of thiols. The binary surfaces were prepared on gold by self-assembly from binary solutions of the thiols HS-(CH(2))(n)()-X (n = 11, X = CH(3) or OH) in THF. Surface plasmon resonance (SPR), electrical capacitance, and atomic force microscopy (AFM) measurements were used to characterize the interaction of palmitoyl,oleoyl-phosphatidylcholine (POPC) vesicles with the surfaces. For all surfaces examined, it appears that the polar part of surface energy influences the nature of the POPC assembly that associates with the surface. Comparison of optical, capacitance, and AFM data suggests that vesicles can remain intact or partially intact even at surfaces with a contact angle with water of close to 100 degrees. In addition, comparison of the alkanethiols of different chain lengths and the fluorinated compound HS-(CH(2))(2)-(CF(2))(8)-CF(3) that characterize with a low value of the polar part of the surface energy suggests that the quality of the underlying monolayer in terms of number of defects has a significant influence on the packing density of the resulting HBM layer.

Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5

A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained “inside out” and “right side out” membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the β-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.

Formation of a Supported Hybrid Bilayer Membrane on Gold: A Sterically Enhanced Hydrophobic Effect

Adsorption of a monolayer of didecanoyl-l-α-phosphatidylcholine (DDPC) from dispersions of small unilamellar vesicles onto hydrophobic surfaces was investigated by means of cyclic voltammetry and impedance spectroscopy. The hydrophobic surfaces were self-assembled monolayers of 2-mercapto-3-n-octylthiophene (MOT) on gold. One characteristic of the MOT monolayer is its permeability to organic molecules in aqueous solution, thus providing a more energetically favorable hydrophobic surface for the addition of phospholipid vesicles. The kinetics of the lipid monolayer formation were followed by measuring the time-dependent interfacial capacitance. Unusual values of thickness and capacitance of the MOT/DDPC bilayers were observed. An interdigitating conformation of the bilayer structure was proposed to interpret the experimental results. The horseradish peroxidase reconstituted into the bilayer demonstrated the expected protein activity, showing practical use in research and in biosensor application.

Small-Angle Neutron Scattering by Highly Oriented Hybrid Bilayer Membranes Confined in Anisotropic Porous Alumina

Small-angle neutron scattering (SANS) is used to characterize a phospholipid/alkoxysilane hybrid bilayer membrane (HBM), a model of biological membrane, supported in anisotropic porous alumina (Al2O3). The bilayer is obtained by fusion of phospholipid vesicles with a hydrophobic alkoxysilane monolayer chemically bound to the microporous alumina support. We first characterized the bare alumina material, then the alkoxysilane (OTS) layer bound to alumina, and finally the hybrid bilayer. By orienting the anisotropic support, we show that the intensity can be considerably increased, enabling the scattering to be measured in a wide q range (6 × 10-4 − 0.5 A-1) corresponding to 9−10 decades in intensity and down to 10-4 cm-1. This enables us to cover the structure factor of the oxide at large scale, the wide Porod regime, and the membrane form factor. Analysis of the scattering curves indicates that both the OTS layer and the HBM produce very smooth, uniform, and continuous layers at the alumina/solvent interfa...

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association ( k a) and dissociation ( k d) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl- l-α-phosphatidyl- dl-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl- l-α-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.

Investigation of Hybrid Bilayer Membranes with Neutron Reflectometry: Probing the Interactions of Melittin

Recent improvements in neutron reflectometry methodology have afforded enhanced sensitivity for the study of biomimetic membranes. The technique has been used to probe the interactions of the peptide toxin, melittin, with supported bilayers of phospholipid and octadecanethiol or thiahexa(ethylene oxide) alkane on gold. Improvements in instrumentation and experimental design permit neutron reflectivity measurements out to a wavevector transfer of 0.7 A-1 and down to reflectivities approaching 10-8, allowing unprecedented resolution of structural details in the bilayer. The data indicate that melittin strongly perturbs the phospholipid headgroup region and also affects the alkane chain region of the bilayer. There is no evidence for hydration of the ethylene oxide spacer region between the gold and alkane regions of the thiahexa(ethylene oxide) alkane/phospholipid bilayer, but a distinct shift of up to 3 A in the apparent location of the interface between the alkane and phospholipid regions is observed. Thi...

First-Principles Determination of Hybrid Bilayer Membrane Structure by Phase-Sensitive Neutron Reflectometry

The application of a new, phase-sensitive neutron reflectometry method to reveal the compositional depth profiles of biomimetic membranes is reported. Determination of the complex reflection amplitude allows the related scattering length density (SLD) profile to be obtained by a first-principles inversion without the need for fitting or adjustable parameters. The SLD profile so obtained is unique for most membranes and can therefore be directly compared with the SLD profile corresponding to the chemical compositional profile of the film, as predicted, for example, by a molecular dynamics simulation. Knowledge of the real part of the reflection amplitude, in addition to enabling the inversion, makes it possible to assign a spatial resolution to the profile for a given range of wavevector transfer over which the reflectivity data are collected. Furthermore, the imaginary part of the reflection amplitude can be used as a sensitive diagnostic tool for recognizing the existence of certain in-plane inhomogeneities in the sample. Measurements demonstrating the practical realization of this phase-sensitive technique were performed on a hybrid bilayer membrane (self-assembled monolayer of thiahexa (ethylene oxide) alkane on gold and a phospholipid layer) in intimate contact with an aqueous reservoir. Analysis of the experimental results shows that accurate compositional depth profiles can now be obtained with a spatial resolution in the subnanometer range, primarily limited by the background originating from the reservoir and the roughness of the film's supporting substrate.

Cytochrome c at Model Membrane Surfaces: Exploration via Second Harmonic Generation-Circular Dichroism and Surface-Enhanced Resonance Raman Spectroscopy

The novel nonlinear optical method of second harmonic generation-circular dichroism (SHG-CD) has been used to follow the adsorption and redox properties of a peripheral membrane protein, horse heart cytochrome c, adsorbed at several model membrane surfaces. The SHG-CD response is shown to be affected by the oxidation state of the heme within surface-adsorbed cytochrome c, as is consistent with the sensitivity of SHG to the chirality of the heme. SHG-CD measurements show that adsorbed cytochrome c is reducible by ascorbate at alkanethiol surfaces, but not at phospholipid/alkanethiol hybrid bilayer membranes (HBMs). The adsorption of cytochrome c at the model membrane surfaces was verified by surface plasmon resonance. Surface-enhanced resonance Raman measurements show that cytochrome c adsorbed on a hybrid bilayer membrane retains the Fe-heme conformation associated with solution-phase cytochrome c and is reducible by applying potential to the supporting electrode. The inability of ascorbic acid to reduce ...

Alternating Current Voltammetry Studies of the Effect of Melittin on Heterogeneous Electron Transfer across a Hybrid Bilayer Membrane Supported on a Gold Electrode

Hybrid bilayer membranes (HBM) that consist of two different layers (a self-assembled monolayer of alkanethiols on a gold electrode and a second layer of phospholipids) dramatically attenuate the heterogeneous electron transfer between soluble redox couples and the gold electrode. Alternating current voltammetry indicates the presence of a low density of defects in the HBM through which the redox couples can reach the electrode. Adsorption of a pore-forming cationic peptide, melittin, into the HBM increases the HBM capacitance, accelerates the faradic reaction for the negatively charged redox couple Fe(CN)63-, decelerates the faradic reaction for the positively charged redox couple Ru(NH3)63+, and has little effect on the faradic reaction of the neutral hydroxymethylferrocene. These results are discussed in terms of several possible mechanisms, including pore-forming processes and electrostatic effects.

Photoinduced electron transfer across a gold supported octadecanethiol/phosphatidylcholine hybrid bilayer membrane mediated by C 60 in different redox species solution

Electron transfer across the biomembrane is central to photosynthesis, to mitochondrial respiration and to the design of molecular systems for solar energy conversion. We report here that gold supported octadecanethiol/phosphatidylcholine hybrid bilayer membrane mediated by C 60 (abbreviated C 60 HBM) can act as both a photosensitizer for electron transfer from a donor molecule and a mediator for electron transport across a hybrid bilayer membrane. The steady-state photocurrent behaviors in different concentration of ascorbate, Co(bpy) 3 2+/3+ or Fe(CN) 6 4−/3− solution have been studied. The rate-limiting step of the whole photoinduced electron transfer depends on the applied potential and the redox concentration in solution. The C 60 HBM electrode was fabricated for the first time and there is no investigation on C 60 incorporated octadecanethiol/phosphatidylcholine hybrid bilayer membrane in literature. The sigmoidal-shaped steady-state photocurrent versus the applied potential curve is first observed and it is different from the linear-shaped i ph/ E curve of C 60 modified bilayer lipid membrane reported by Bensasson et al. [R.V. Bensasson, J.-L. Garaud, S. Leach, G. Miquel, P. Seta, Chem. Phys. Lett. 210 (1993) 141].

Ferrocene Mediated Transmembrane Electron Transfer on Gold Electrode Modified by Hybrid Bilayer Membrane Consisting of Octadecanethiol and Phospholipid

Supported hybrid bilayers consisting of octadecanethiol and phospholipid incorporated with ferrocene were fabricated through self-assembling methods. Its electrochemical behaviors in solutions containing different concentrations of Fe(CN)6 3-/4− were investigated by ac impedance and cyclic voltammetry. The transmembrane electron transfer across octadecanethiol self-assembled film and an adsorbed phospholipid layer mediated by ferrocene has been observed. It is determined that the apparent heterogeneous rate constant is 2.3 × 10−8 cm·s−1.

Assessing the Apparent Effective Thickness of the Supported Hybrid Bilayer Membranes Consisting of Octadecanethiol and Phospholipid by ac Impedance Spectroscopy

The electrochemical behavior of the gold electrode modified by hybrid bilayer membranes in different concentrations of Fe(CN)63−/Fe(CN)64− was investigated by electrochemical impedance spectroscopy and cyclic voltammetry technology. The electron transfer between gold and the redox species separated by the hybrid bilayers assembly was completed by an electron tunneling process. A detailed equivalent circuit for electron transport across the HBMs is proposed. It was found that the apparent effective thickness of the hybrid bilayer membranes was lower than that of the sum of the chain length of octadecanethiol and phospholipid so some possible collapsed sites might exist at the hybrid bilayer membranes.

Infrared Spectroscopic Characterization of Lipid−Alkylsiloxane Hybrid Bilayer Membranes at Oxide Substrates

Successive depositions of precompressed Langmuir monolayers have been shown to allow reproducible formation of air-stable, lipid−alkylsiloxane hybrid bimolecular architectures at oxidic supports. Specifically, prepolymerized Langmuir−Blodgett films of n-octadecylsiloxane (OTS) monolayers on oxided silicon substrates were used as the hydrophobic templates, upon which compressed monolayers of dipalmitoyl-sn1-glycerophosphatidylcholine (DPPC) and monosialogangliosides (Gm1) were deposited from a low-temperature air−water interface by the horizontal deposition method. Structural features of each leaflet of the resultant bimolecular architectures, namely DPPC/OTS/SiO2/Si and Gm1/OTS/SiO2/Si, were characterized using a combined application of infrared spectroscopy, null-ellipsometry, and surface wetting measurements. In both cases, the outer lipid leaflet (DPPC or Gm1) was found to be structurally decoupled with respect to the inner OTS layer. The inner silane layer was composed of essentially untilted (cant an...

Supported Hybrid Bilayer Membranes as Rugged Cell Membrane Mimics

Supported planar lipid bilayers based on alkanethiol-tethering chemistry are becoming increasingly important biomimetic materials. Hybrid bilayers containing thiol-derivatized alkane moieties plus natural lipids provide a biomimetic matrix that permits the successful reconstitution of membrane protein activity. The hybrid membrane is self-assembled and sufficiently rugged to be of practical use in research and in commercial sensing applications. The coupling of the bilayer to a metal support allows a wide range of analytical techniques to be applied to this model membrane. This article reviews some of the fundamental studies of the formation, structure, and response of hybrid bilayer membranes.

Self assembly driven by hydrophobic interactions at alkanethiol monolayers: mechanism of formation of hybrid bilayer membranes

The mechanism for the formation of biomimetic model cell membranes consisting of bilayers composed of alkanethiols and phospholipids was probed with a kinetic study using surface plasmon resonance. The kinetics of formation of a monolayer of phospholipid from vesicles in solution onto a hydrophobic alkanethiol monolayer is described by a model that takes into account the lipid concentration, diffusion, and a surface reorganization rate constant. Monomer phospholipid apparently does not play a direct role in determining the kinetics of bilayer formation. Expressions for the limiting cases of this model describe the behavior of two distinct vesicle concentration conditions. At high concentrations of lipid vesicles the formation of the bilayer appears to be limited by the diffusion of vesicles to the surface; at lower concentrations of vesicles, the rate-limiting step is apparently the surface reorganization of lipid. This kinetic model can also be used to describe the formation of a biomimetic bilayer from an alkanethiol monolayer and cell membranes.

Hybrid Bilayer Membranes in Air and Water: Infrared Spectroscopy and Neutron Reflectivity Studies

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 Å −1, and provided a sensitivity to changes in total layer thickness on the order of 1–2 Å. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.

Assessing the Molecular Structure of Alkanethiol Monolayers in Hybrid Bilayer Membranes with Vibrational Spectroscopies

Hybrid bilayer membranes (HBMs), consisting of a lipid monolayer covering a self-assembled alkanethiol monolayer on a metal surface, are useful models for studying the structure and function of cell membranes. Surface-enhanced Raman spectroscopy (SERS) and reflection absorption infrared spectroscopy (RAIRS) are used to study HBMs with various alkanethiol and lipid components. Together, these two techniques clearly indicate that the lipid forms a well-ordered, non-interdigitated layer on the alkanethiol with the head groups of the lipids oriented away from the metal surface. Both techniques reveal that the formation of an HBM produces small changes in the alkanethiols, which are similar to those caused by a reduction in temperature, indicative of an increase in order. The small magnitude of the perturbations in the alkanethiol monolayer upon addition of the lipid layer will simplify the further study of HBMs.

Phospholipid/Alkanethiol Bilayers for Cell-Surface Receptor Studies by Surface Plasmon Resonance

Supported hybrid bilayer membranes (HBM) composed of a monolayer of phospholipid and a monolayer of alkanethiol associated with a thin gold film on glass are useful as model lipid bilayer membranes for studying membrane receptor-ligand and cell-cell binding events by surface plasmon resonance (SPR). Measurements of specific binding of proteins and lipid vesicles to well-defined HBMs have been performed under conditions of continuous flow using a commercial SPR instrument (BIAcore). HBMs are shown to be stable in flow and to block nonspecific adsorption of proteins to the alkanethiol/gold surface. The use of such supported lipid bilayers in flow provides a means of conducting equilibrium and kinetic studies of models of ligand-cell and cell-cell interactions with receptors or ligands in a membrane environment. Compared to the extended dextran polymer layer that is currently used for surface modification of BIAcore "sensor chips," the described HBMs provide a well-defined surface that will permit less ambiguous modeling of these important biological interactions.

Supported phospholipid/alkanethiol biomimetic membranes: insulating properties

A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes.