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Nanoporous microbead supported bilayers : stability, physical characterizationand incorporation of functional transmembrane proteins. Davis, Ryan W. The introduction of functional transmembrane proteins into supported bilayer -based biomimetic systems presents a ificant challenge for biophysics.

Among the various methods for producing supported bilayersliposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined.

Supported bilayers on porous silica microspheres show a ificant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin bR and the human type 3 serotonin receptor 5HT3R are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency.

In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are ificantly enhanced through detergent-mediated protein reconstitution. The of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform. Lipid bilayers supported by substrates with nanometer-scale surface corrugations holds interest in understanding both nanoparticle-membrane interactions and the challenges of constructing models of cell membranes on surfaces with desirable properties, e.

Here, we successfully form a two-phase gel-fluid lipid bilayer supported by nanoporous silica xerogel. Surface topology, diffusion, and lipid density in comparison to mica- supported lipid bilayers were characterized by AFM, FRAP, FCS, and quantitative fluorescence microscopy, respectively.

We found that the two-phase lipid bilayer follows the xerogel surface contours. The corrugation imparted on the lipid bilayer in a lipid density that is twice that on a flat mica surface.

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Furthermore, the gel-phase domains on xerogel compared to mica were larger and less numerous. Possible mechanism of adhesion in a mica supported phospholipid bilayer. Phospholipid bilayers supported on hydrophilic solids like silica and mica play a substantial role in fundamental studies and technological applications of phospholipid membranes. In both cases the molecular mechanism of adhesion between the bilayer and the support is of primary interest.

Since the possibilities of experimental methods in this specific area are rather limited, the methods of computer simulation acquire great importance. In this paper we use the grand canonical Monte Carlo technique and an atomistic force field to simulate the behavior of a mica supported phospholipid bilayer in pure water as a function of the distance between the bilayer and the support. The simulation reveals a possible adhesion mechanism, where the adhesion is due to individual lipid molecules that protrude from the bilayer and form widely spaced links with the support. Simultaneously, the bilayer remains separated from the bilayer by a thin water interlayer which maintains the bilayer fluidity.

Biological organisms are potentially the most sensitive and selective biological detection systems known, yet we are currently severely limited in our ability to exploit biological interactions in sensory devices, due in part to the limited stability of biological systems and derived materials. This proposal addresses an important aspect of integrating biological sensory materials in a solid state device. If successful, such technology could enable entirely new classes of robust biosensors that could be miniaturized and deployed in the field.

The critical aims of the proposed work were 1 the calibration of a more versatile approach to measuring pH, 2 the use of this method to monitor pH changes caused by the light-induced pumping of protons across vesicles with bacteriorhodopsin integrated into the membranes either polymer or lipid ; 3 the preparation of bilayer assemblies on platinum surfaces; 4 the enhanced detection of lightinduced pH changes driven by bR-loaded supported bilayers.

I have developed a methodology that may enable that at interfaces and developed a methodology to characterize the functionality of bilayer membranes with reconstituted membrane proteins. The integrity of the supported bilayer films however must be optimized prior to the full realization of the work originally envisioned in the original proposal.

Nevertheless, the work performed on this project and the encouraging it has produced has demonstrated that these goals are challenging yet within reach. Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids.

We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterizedusing single molecule tracking and moment scaling spectrum MSS analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer.

For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in ificant scrambling of protein orientation and typically immobilized membrane proteins in SLBs.

The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions. Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy. Fluorescence correlation spectroscopy FCS is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycerophosphocholine DPPC lipid domains in 1,2-dioleoyl-sn-glycerophosphocholine DOPC.

Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes.

We attribute the latter to domain heterogeneities that could be induced by the probe. Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.

Directory of Open Access Journals Sweden. Full Text Available 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 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 bilayerwhich 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.

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In situ atomic force microscope imaging of supported lipid bilayers. For one-component DPPC bilayers an enhanced enzymatic activity is observed towards preexisting defects in the bilayer. Phase separation is observed in two-co Full Text Available To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy.

The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE- supported bilayers.

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Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method. Functional liposomes and supported lipid bilayers : towards the complexity of biological archetypes.

This perspective paper provides some illustrative examples on the interplay between information gathered on planar supported lipid bilayers SLB and unilamellar lipid vesicles ULV to get an integrated description of phenomena occurring at the nanoscale that involve locally bilayered structures.

Similarities and differences are underlined and critically compared in terms of biomimetic fidelity and instrumental accessibility to structural and dynamical parameters, focusing on some recent reports that either explicitly address this comparison or introducing some studies that separately investigate the same process in SLB and lipid vesicles. Despite the structural similarity on the nanoscale, the different topology implies radically different characterization techniques that have evolved in sectorial and separated approaches.

The quest for increasing levels of compositional complexity for bilayered systems should not result in a loss of structural and dynamical control: this is the central challenge of future research in this area, where the integrated approach highlighted in this contribution would enable improved levels of understanding. The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane.

In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used ly to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion.

In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis.

These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few. Lipid bilayers suspended on microfabricated supports. Ogier, Simon D. The plasma membrane, that exists as part of many animal and plant cells, is a regulator for the transport of ions and small molecules across cell boundaries.

Two main components involved are the phospholipid bilayer and the transport proteins. This paper details the construction of a micromachined support for bilayers MSB as a first step towards the development of highly selective and highly sensitive ion-channel based biosensors. Electrodes attached to the structure allow the resistance of the membranes to be measured using d. The MSB is made in two halves, using SU8 ultra-thick resist, which are subsequently bonded together to make the final structure.

A layer of gold, surrounding the aperture, enables self-assembled monolayers of alkanethiols to be used to make the polymeric structure biocompatible. The ion-channel gramicidin has successfully been incorporated into the bilayer and its activity monitored. It is proposed that this type of device could be used not only for studying membrane transport phenomena but also as part of an ion-channel based biosensor.

SFG studies on interactions between antimicrobial peptides and supported lipid bilayers. The mode of action of antimicrobial peptides AMPs in disrupting cell membrane bilayers is of fundamental importance in understanding the efficiency of different AMPs, which is crucial to de antibiotics with improved properties. Recent developments in the field of sum frequency generation SFG vibrational spectroscopy have made it a powerful and unique biophysical technique in investigating the interactions between AMPs and a single substrate supported planar lipid bilayer.

We will review some of the recent progress in applying SFG to study membrane lipid bilayers and discuss how SFG can provide novel information such as real-time bilayer structure change and AMP orientation during AMP-lipid bilayer interactions in a very biologically relevant manner. This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy AFM images of phase-separated Langmuir-Blodgett bilayers with imaging load.

The mechanisms of formation and conditions of the existence of the ripple phase are fundamental thermodynamic questions with practical implications for medicine and pharmaceuticals. This ripple phase is detected by FRAPP using diffusion coefficient measurements as a function of temperature: a diffusivity plateau is observed. It occurs in the same temperature range where ripple phase existence has been observed using other methods. When AFM experiments are performed in the appropriate temperature range the ripple phase is confirmed.

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Full Text Available Complex multi-lamellar structures play a critical role in biological systems, where they are present as lamellar bodies, and as part of biological assemblies that control energy transduction processes. Multi-lamellar lipid layers not only provide interesting systems for fundamental research on membrane structure and bilayer -associated polypeptides, but can also serve as components in bioinspired materials or devices. Although the ability to pattern stacked lipid bilayers at the micron scale is of importance for these purposes, limited work has been done in developing such patterning techniques.

Here, we present a simple and direct approach to pattern stacked supported lipid bilayers SLBs using polymer stencil lift-off and the electrostatic interactions between cationic and anionic lipids. Both homogeneous and phase-segregated stacked SLB patterns were produced, demonstrating that the stacked lipid bilayers retain lateral diffusivity. We demonstrate patterned SLB stacks of up to four bilayerswhere fluorescence resonance energy transfer FRET and quenching was used to probe the interactions between lipid bilayers.

Furthermore, the study of lipid phase behaviour showed that gel phase domains align between adjacent layers. The proposed stacked SLB pattern platform provides a robust model for studying lipid behaviour with a controlled of bilayersand an attractive means towards building functional bioinspired materials or devices. Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur.

In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycerophosphocholine and 1,2-dimyristoyl-sn-glycero[phospho-rac- 1-glycerol ] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Formation of supported lipid bilayers of charged E. Full Text Available We describe a simple way of fusing E.

Supported lipid bilayers on metal surfaces are interesting for several reasons: transducing a biological al to an electric readout, using surface analytical tools such as Surface Plasmon Resonance SPR, Infrared Reflection Absorption Spectroscopy, Neutron Reflectivity or Electrochemistry.

The most widely used method to prepare supported lipid membranes is fusion of preexisting liposomes. It is quite efficient on hydrophilic surfaces such as glass, mica or SiO2, but vesicle fusion on metals and metal oxide surfaces as gold, titanium oxide or indium tin oxide, remains a challenge, particularly for vesicles containing charged lipids, as is the case of bacterial lipids.

We describe a simple method based on modifying the gold surface with a charged mercaptopropionic acid self-assembled monolayer and liposomes partially solubilized with detergent. Some advantages of this protocol are that the stability of the self-assembled monolayer allows for repeated use of the substrate after detergent removal of the bilayer and that the amount of detergent required for optimal fusion can be determined ly using the lipid-detergent solubility curve.

Supported lipid bilayers with controlled curvature via colloidal lithography. Supported lipid bilayers SLBs at surfaces provide a route to quantitatively study molecular interactions with and at lipid membranes via different surface-based analytical techniques.

Here, a method to fabricate SLBs with controlled curvatures, in the nanometer regime over large areas, is prese Development and characterization of sugar palm starch and poly lactic acid bilayer films. The development and characterization of environmentally friendly bilayer films from sugar palm starch SPS and poly lactic acid PLA were conducted in this study.

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