Supported Bilayer Lipid Membranes Research Articles

Surface-enhanced Raman scattering of self-assembled thiol monolayers and supported lipid membranes on thin anodic porous alumina.

Thin anodic porous alumina (tAPA) was fabricated from a 500 nm thick aluminum (Al) layer coated on silicon wafers, through single-step anodization performed in a Teflon electrochemical cell in 0.4 M aqueous phosphoric acid at 110 V. Post-fabrication etching in the same acid allowed obtaining tAPA surfaces with ≈160 nm pore diameter and ≈80 nm corresponding wall thickness to be prepared. The tAPA surfaces were made SERS-active by coating with a thin (≈25 nm) gold (Au) layer. The as obtained tAPA–Au substrates were incubated first with different thiols, namely mercaptobenzoic acid (MbA) and aminothiol (AT), and then with phospholipid vesicles of different composition to form a supported lipid bilayer (SLB). At each step, the SERS substrate functionality was assessed, demonstrating acceptable enhancement (≥100×). The chemisorption of thiols during the first step and the formation of SLB from the vesicles during the second step, were independently monitored by using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The SLB membranes represent a simplified model system of the living cells membranes, which makes the successful observation of SERS on these films promising in view of the use of tAPA–Au substrates as a platform for the development of surface-enhanced Raman spectroscopy (SERS) biosensors on living cells. In the future, these tAPA–Au-SLB substrates will be investigated also for drug delivery of bioactive agents from the APA pores.

Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane. The technique was applied to two well-characterized aptamers, both of which undergo conformational changes upon binding either a protein or a small molecule ligand. In both cases, SHG was able to resolve conformational changes in these oligonucleotides sensitively and specifically, in solution and in real time, using nanogram amounts of material. In addition, we developed a competition assay for the oligonucleotides between the specific ligands and known, nonspecific binders, and we demonstrated that intercalators and minor groove binders affect the conformation of the DNA and RNA oligonucleotides in different ways upon binding and subsequently block specific ligand binding in all cases. Our work demonstrates the broad potential of SHG for studying oligonucleotides and their conformational changes upon interaction with ligands. As SHG offers a powerful, high-throughput screening approach, our results here also open an important new avenue for identifying novel chemical probes or sequence-targeted drugs that disrupt or modulate DNA or RNA structure and function.

Model membrane-mediated cell alignment through surface hydrophobicity

ABSTRACTWe show a realization of well-controlled alignments of human lung fibroblast cell parallel to the guided direction achieved by selectively patterned model membranes. The supported lipid bilayer membranes (SLBMs)-mediated cell alignment and selective growth in particular un- derlying surface properties were achieved through peeling-off of the polydimethylsiloxane (PDMS) stamp. We used the PDMS not only as a microfluidic channel to reconstruct model membrane on a hydrophilic surface, but also as a self-inking stamp for the substrate-surface modification by transfer of outmost low-molecular weight PDMS oligomers (S-PDMS). We found that surface hydrophobicity of solid supports is controlled by thermally-assisted stamping methods and this is crucial for the cell adhesion in lipid-free regions.

Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function.

Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties.

Study of the ion-channel behavior on glassy carbon electrode supported bilayer lipid membranes stimulated by perchlorate anion.

In this paper, a kind of didodecyldimethylammonium bromide (DDAB) layer membranes was supported on a glassy carbon electrode (GCE). We studied the ion channel behavior of the supported bilayer lipid membrane by scanning electrochemical microscopy (SCEM) in tris(2,2'-bipyridine) ruthenium(II) solution. Perchlorate anion was used as a presence of stimulus and ruthenium(II) complex cations as the probing ions for the measurement of SECM, the lipid membrane channel was opened and exhibited the behavior of distinct SECM positive feedback curve. The channel was in a closed state in the absence of perchlorate anions while reflected the behavior of SECM negative feedback curve. The rates of electron transfer reaction in the lipid membranes surface were detected and it was dependant on the potential of SECM.

Interfacing living cells and spherically supported bilayer lipid membranes.

Spherically supported bilayer lipid membranes (SS-BLMs) exhibiting co-existing membrane microdomains were created on spherical silica substrates. These 5 μm SiO2-core SS-BLMs are shown to interact dynamically when interfaced with living cells in culture, while keeping the membrane structure and lipid domains on the SS-BLM surface intact. Interactions between the SS-BLMs and cellular components are examined via correlating fluorescently labeled co-existing microdomains on the SS-BLMs, their chemical composition and biophysical properties with the consequent organization of cell membrane lipids, proteins, and other cellular components. This approach is demonstrated in a proof-of-concept experiment involving the dynamic organization of cellular cytoskeleton, monitored as a function of the lipid domains of the SS-BLMs. The compositional versatility of SS-BLMs provides a means to address the relationship between the phenomenon of lipid phase separation and the other contributors to cell membrane lateral heterogeneity.

Immunosensor for trace penicillin G detection in milk based on supported bilayer lipid membrane modified with gold nanoparticles

In this work, we developed an immunosensor for electrochemical detection of penicillin G at trace level. The biosensor was fabricated by immobilizing anti-penicillin G in a supported bilayer lipid membrane (s-BLM) modified with gold nanoparticles, and the modified electrodes were characterized by the scanning electron microscope (SEM), cyclic voltammetry and electrochemical impedance spectroscopy. The biosensor was able to detect penicillin G with a linear correlation ranging from 3.34×10−3ng/L to 3.34×103ng/L and a detection limit of 2.7×10−4ng/L, much lower than the maximum residue limit (MRL) of penicillin G in milk (4ppb, equal to 4×103ng/L) set out by the European Union. The mean coefficient variation (CV) of the intra-assays and the inter-assays were 5.4% and 7.7%, respectively. In addition, the concentration of penicillin G in milk samples determined by this biosensor was in good agreement with that determined by high performance liquid chromatography (HPLC) assay.

Dynamic behavior of DNA cages anchored on spherically supported lipid bilayers.

We report the anchoring of 3D-DNA-cholesterol labeled cages on spherically supported lipid bilayer membranes (SSLBM) formed on silica beads, and their addressability through strand displacement reactions, controlled membrane orientation and templated dimerization. The bilayer-anchored cages can load three different DNA-fluorophores by hybridization to their "top" face (furthest from bilayer) and unload each of them selectively upon addition of a specific input displacement strand. We introduce a method to control strand displacement from their less accessible "bottom" face (closest to the bilayer), by adding cholesterol-substituted displacing strands that insert into the bilayer themselves in order to access the toehold region. The orientation of DNA cages within the bilayer is tunable by positioning multiple cholesterol anchoring units on the opposing two faces of the cage, thereby controlling their accessibility to proteins and enzymes. A population of two distinct DNA cages anchored to the SSLBMs exhibited significant membrane fluidity and have been directed into dimer assemblies on bilayer via input of a complementary linking strand. Displacement experiments performed on these anchored dimers indicate that removal of only one prism's anchoring cholesterol strand was not sufficient to release the dimers from the bilayer; however, removal of both cholesterol anchors from the dimerized prisms via two displacement strands cleanly released the dimers from the bilayer. This methodology allows for the anchoring of DNA cages on supported lipid bilayers, the control of their orientation and accessibility within the bilayer, and the programmable dimerization and selective removal of any of their components. The facile coupling of DNA to other functional materials makes this an attractive method for developing stimuli-responsive protein or nanoparticle arrays, drug releasing biomedical device surfaces and self-healing materials for light harvesting applications, using a highly modular, DNA-economic scaffold.

Creating Free-Standing Lipid Bilayers on Fused Silica Substrates with Nanograting Structure

Supported lipid bilayer membranes have been widely used to study protein-lipid membrane interactions. However, the membrane physical properties are often influenced by the solid support, which may affect the interested interactions. Some previous studies have created free-standing bilayers by depositing unilamellar vesicles on the pores with sizes smaller than the vesicles or by using shear-driven bilayers to span over nanopores at high pH. However, these methods may not form continuous bilayers in a large scale or may not apply to various types of lipids. Here, we developed a theory, based on extended-Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and Helfrich bending theory, to estimate the appropriate conditions for creating free-standing lipid bilayers on solid supports with nanograting structure. We calculated how the buffer conditions, lipid types, and the solid support geometry can influence the affinity between the lipid membrane and the solid support, and the bending energy of the lipid membrane. When the bending energy dominates the entire energy of the system, we predicted that the membrane would form free standing bilayers above the trenched area of the nanograting solid support. On the other hand, if the support-membrane affinity dominates, the lipid bilayer can easily bend and follow the contour of the solid support. In order to tune the relative size of the support-membrane affinity, we prepared buffers with a series of ionic strength (from 5 mM to 100 mM NaCl) and fused silica substrates with periodic 100 nm and 200 nm nanograting structure. The characterization of membrane states by fluorescence recovery after photobleaching measurement shows that our experiment results well match the theoretical predictions and that the developed theory could be used to predict the suitable conditions for the free-standing bilayers to form on solid supports with various kinds of geometry.

Spatial Organization of Oncogenic Ras on Supported Membranes

Ras is a membrane protein which acts as a molecular switch in the activation of many signal transduction pathways involved in cellular processes such as proliferation and apoptosis. Ras has been a subject of great interest due to the causal role found between its hyperactivity and cancer, and studies have shown that approximately 30% of all human cancers have oncogenic Ras mutations. Although Ras has been studied for more than thirty years, the spatial organization of oncogenic Ras on membranes is still not well characterized. It has been suggested that Ras may organize into clusters in cell membranes but the molecular scale structure of these organizations remain unknown. Molecular dimerization of Ras has also been suggested and we quantitatively characterize this in an array of Ras mutants in supported lipid bilayer membranes using fluorescence correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The implications of Ras clustering on downstream signaling will be discussed.

Pore Formation and Dynamics of Cytolysin-A on Supported Lipid Bilayer Membranes

Ayush Agrawal1, Rajat Desikan1, Pradeep S.3, K. G. Ayappa1, J. K. Basu2, Sandhya Visweswariah41 Department of Chemical Engineering, Indian Institute of Science2 Centre for Condensed Matter Theory, Department of Physics, Indian Institute of Science3 Bioengineering program, Indian Institute of Science4 Department of Molecular Reproduction, Development and Genetics, Indian Institute of ScienceObserving the structure and dynamics of large protein assemblies on membranes has implications on understanding the general principles behind protein aggregation and engineering drug therapies. Dynamic and structural data on mesoscopic length and time scales are scarce for these systems. Cytolysin-A (ClyA) is a pore forming toxin that is known to cause unregulated pores on various cell membranes. In order to study pore formation of ClyA on model membranes, supported lipid bilayers (SLBs) of DMPC were incubated with varying concentrations of ClyA. Pore densities were quantified using atomic force microscopy (AFM) and led to the observation that a threshold toxin concentration is necessary for pore formation. The influence of the bilayer preparation protocol was observed by contrasting the pore densities on SLBs prepared using Langmuir-Blodgett (LB) deposition as well as vesicle deposition. Pore formation at varying deposition pressures on different substrates (mica, silicon) were investigated using LB prepared bilayers. Interestingly, at a high toxin concentration of 8.82 nM (large pore density regime), membrane undulations were prominently observed in the AFM images. Understanding the lipid dynamics around protein assemblies has implications on protein signalling, stability and function. Fluorescence imaging using FRAP and FCS on SLBs incubated with ClyA, and complementary molecular dynamics simulations were carried out to investigate the inherent heterogeneity in lipid dynamics in the vicinity of the ClyA pore.

Molecular Addressability of Lipid Membrane Embedded Calixarenes towards Cytochrome C

By means of the atomic force microscopy techniques we studied the surface topography of the supported bilayer lipid membranes (sBLM) composed of 1,2-sn-glycerodimyristoylfosfatidylcholine (DMPC) with incorporated calixarenes tOct [6]CH2COOH (CX) specific to cytochrome c (cyt c). It is supposed that cyt c interacts with CX through amino groups of lysine residues at its surface. Therefore we also applied single molecule force spectroscopy (SMFS) to analyze the mechanisms of interaction of cyt c with the CX. In later case cyt c or individual NH2 group have been connected to the AFM tip through special linker. The topography of bare sBLM in a gel state (T=19oC) revealed relatively smooth surface (RRMS=0.18 nm) and thickness ~5.1 nm which agrees well with previous studies. Incorporation of CX into DMPC bilayer resulted in increase of the surface roughness (RRMS= 0.39 nm) and in increase of thickness in average by 0.5 nm. The incubation of the layer with30 nM of cyt c resulted in a surface smoothing (RRMS=0.32 nm) and in a further increase of the thickness between 0.7 to 1.2 nm. The SMFS experiments with cyt c modified AFM tips approved its specific binding to CX and allowed us to determine the binding parameters koff (1.14 ± 0.59 s-1) and xβ (3.98 ± 0.63A). SMFS experiments with an amino-ended linker also resulted in highly specific interactions with comparable values for koff (2.74 ± 0.66 s-1) and xβ (5.91 ± 2.55 A). This suggests that both electrostatic and amino group specific interactions between cyt c and CX cavity exist.

Probing Simultaneously Membrane Dynamics and Protein Activity in Suspended Bilayers in a Microfluidic Format

Membrane dynamics affect the structure and function of ion channels, a point that deserves more attention while studying membrane proteins. One important factor in the local lipidic environment of the ion channels, is the membrane fluidity which is directly connected to the free diffusion and packing of the phospholipids. Typically, FRAP is utilized to investigate this parameter in simplified cell membrane models, and preferably supported bilayer lipid membranes (BLMs), which are more stable than their free-standing counterparts. However, supported membranes are less suitable for the incorporation of ion channels and electrophysiological monitoring of their activity than suspended ones.

Extreme pH-induced lateral reorganization of supported lipid bilayer by fluorescence microscope

The extreme pH-induced lateral reorganization of supported lipid bilayer membranes are studied by fluorescence microscopy. The results show that the fluid dioleoyl-phosphatidylcholine bilayers in extreme acidic or basic solution presents a similar phenomenon to endocytosis and exocytosis, such as rupture, detachment, budding, formation of microtubules etc. In the view of the interaction of polar molecules with H+/H3O+ or OH ions, we conclude that the zwitterionic phospholipid headgroup as core adsorbs the H+/H3O+ or OH ions in electrolyte solution. The asymmetric charge adsorption quantity of the lipid headgroups leads to the effective area discrepancy between the outer and inner leaflets of lipid bilayers. The asymmetric membrane curvatures induce a variety of structures and dynamic responses. The present study helps explain lipid membranes reorganization under extreme pH conditions and provides some guidelines for deformation process of lipid membranes.

On-chip electrophoresis in supported lipid bilayer membranes achieved using low potentials.

A micro supported lipid bilayer (SLB) electrophoresis method was developed, which functions at low potentials and appreciable operating times. To this end, (hydroxymethyl)-ferrocene (FcCH2OH) was employed to provide an electrochemical reaction at the anode and cathode at low applied potential to avoid electrolysis of water. The addition of FcCH2OH did not alter the SLB characteristics or affect biomolecule function, and pH and temperature variations and bubble formation were eliminated. Applying potentials of 0.25–1.2 V during flow gave homogeneous electrical fields and a fast, reversible, and strong build-up of a charged dye-modified lipid in the direction of the oppositely charged electrode. Moreover, streptavidin mobility could be modulated. This method paves the way for further development of analytical devices.

Effect of Pyridoxine on the Transport of Iron Across Microfilter Supported Bilayer Lipid Membrane

Effect of Pyridoxine on the Transport of Iron Across Microfilter Supported Bilayer Lipid Membrane

Interactions of the baicalin and baicalein with bilayer lipid membranes investigated by cyclic voltammetry and UV–Vis spectroscopy

The baicalin and baicalein are the major flavonoids found in Radix Scutellariae, an essential herb in traditional Chinese medicine for thousands of years. The interactions of the baicalin and baicalein with lipid bilayer membranes were studied using cyclic voltammetry and UV–Vis spectroscopy. The thickness d of supported bilayer lipid membranes was calculated as d=4.59(±0.36) nm using AC impedance spectroscopy. The baicalein interacted with egg PC bilayer membranes in a dose-dependent manner. The responses of K3Fe(CN)6 on lipid bilayer membrane modified Pt electrode linearly increased in a concentration range of baicalein from 6.25μM to 25μM with a detection limit of 0.1μM and current-concentration sensitivity of 0.11(±0.01) μA/μM, and then reached a plateau from 25μM to 50μM. However the baicalin showed much weaker interactions with egg PC bilayer membranes. UV–Vis spectroscopy also confirmed that the baicalein could interact with egg PC membranes noticeably, but the interaction of baicalin with membranes was hard to be detected. The results provide useful information on understanding the mechanism of action of Radix Scutellariae in vivo.

Aptasensor for adenosine triphosphate based on electrode–supported lipid bilayer membrane

We have developed an aptasensor for adenosine triphosphate (ATP) based on an electrode-supported lipid bilayer membrane. The assay is based on a conformational change that is induced after binding the target which modulates the electron transfer rate in the conductive path. The method is highly sensitive, stable, and repeatable. The detection limit for ATP is 50 nM, and the dynamic range extends to 3.2 μM, which covers the concentration range of ATP in cell lysates (from 0.1 to 1 μM). The method also holds promise in that it may be transferred to submicro- or nano-scale electrodes so to enable intracellular monitoring of ATP.

Probing into the interaction of β-amyloid peptides with bilayer lipid membrane by electrochemical techniques

Alzheimer's disease (AD) is the most common degenerative disease in the elderly that is a severe threat to human health. In this paper, we report a simple but efficient electrochemical method to probe into the interaction between β-amyloid peptides and bilayer lipid membrane for revealing the toxic mechanism of AD. For this study, a supported bilayer lipid membrane is firstly modified on an electrode surface as the mimic of cell membrane, which can induce significant steric hindrance to prohibit the electrochemical probe [Fe(CN)6]3−/4− approaching the electrode. However, after incubation of the modified electrode with Aβ peptides, a noticeable electrochemical response of [Fe(CN)6]3−/4− can be obtained on the lipid membrane modified electrode, indicating the formation of ion channels in the lipid membrane. Further experimental results show that the peak current of [Fe(CN)6]3−/4− increases along with Aβ peptides concentration and incubation time. Meanwhile, the electrochemical response can be inhibited by (−)-epigallocatechin gallate, an inhibitor of the aggregation of Aβ peptides. Therefore, electrochemical technique might provide a convenient and powerful approach for in vitro studies of protein-misfolding diseases.

Diffusion of complex objects embedded in free and supported lipid bilayer membranes: role of shape anisotropy and leaflet structure

We present a versatile numerical scheme to predict diffusion coefficients for arbitrarily shaped objects embedded in lipid bilayer membranes. Diffusion coefficients for micron-scale diamond-shaped solid domains are calculated for direct comparison to recent experiments. In supported membranes, identical objects in the distal and proximal leaflets may diffuse differently from one another; quantitative predictions for this asymmetry are provided, both for experimental systems and coarse-grained molecular simulations. We show that though recent experiments comparing the diffusion of monomeric, dimeric and trimeric protein assemblies moving over the surface of supported bilayers are inconsistent with the simplest Saffman–Delbrück model, they may be explained by a hydrodynamic model appropriate for supported membranes.