Methods for the Formation of Single Bimolecular Lipid Membranes in Aqueous Solution

Müeller, Paul; Rudin, Donald O.; Tien, H. Ti; Wescott, William C.

doi:10.1021/j100796a529

Cited by 665 publications

(202 citation statements)

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“…Bilayers were formed by using the paintbrush method of Mueller et al (42) using soybean phosphatidylcholine (95% lecithin; Avanti Polar Lipids) dissolved to 50 mg/ml in decane (99%; Alfa Aesar) supported on a circular hole of radius 0.5 mm punched into a square of PTFE sheet (0.025 mm thick; Goodfellow Cambridge) fixed as a diaphragm between a two-chamber PTFE cell with wax (Fig. 6).…”

Section: Methodsmentioning

confidence: 99%

“…The cell was similar to that developed previously by Bard and coworkers (31) for pure SECM experiments. This method of bilayer formation is a well established and popular protocol (42)(43)(44) and was thus a good choice for initial studies that needed to compare measurements on similar systems to those in the literature. The membrane formation was monitored until a clear black bilayer formed, with excess lipid and solvent restricted to the periphery of the bilayer structure that was not investigated.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative visualization of passive transport across bilayer lipid membranes

Grime

Edwards

Rudd

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest that the applicability of Overton's rule requires re-evaluation.Overton's rule ͉ permeation ͉ ultramicroelectrode ͉ laser confocal scanning microscopy ͉ finite element method

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative visualization of passive transport across bilayer lipid membranes

Grime

Edwards

Rudd

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…electrophysiology). Free-standing lipid bilayers are traditionally formed over small apertures in Teflon thin sheets by "painting" a droplet of lipid dissolved in organic solvent over the orifice 7 .…”

Section: Introductionmentioning

confidence: 99%

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

et al. 2016

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ABSTRACT. We report a simple, cost-effective and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive (NOA81).Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable DPhPC and DOPC/DPPC membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-Hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane, allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

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“…These requirements led us to incorporate a passivated yet hydrophilic well, with different chemical functionality to the hydrophobic aperture, to maintain the required water reservoir. The reservoir is small (subpicoliter) and was disrupted by typical BLM formation approaches, such as direct addition of lipid dissolved in organic solvent (32,33) or spreading from an air/water interface (29,34). For the trapped aqueous reservoir to consistently survive, addition of PEG (1.5%) to the trapped aqueous solution and a more gentle lipid addition as small vesicles in the upper aqueous layer were required.…”

Section: Discussionmentioning

confidence: 99%

A membrane interferometer

Ganesan

Boxer

2009

Proc. Natl. Acad. Sci. U.S.A.

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Freestanding phospholipid bilayers have been assembled spanning shallow, micrometer-sized wells etched into a Si wafer substrate so that the bilayers are near (within hundreds of nanometers) but not in contact with the wafer surface. The proximity of the bilayers to the highly reflective Si/SiO2 interface allows them to be probed by using fluorescence-interference techniques. These interferometry measurements show that the bilayers are curved and that the curvature can be varied by changes in osmotic pressure. Furthermore, the ionophore gramicidin can be incorporated into the bilayers, making them selectively permeable to monovalent cations. This freestanding architecture may overcome surface-interaction problems that occur when cell membrane proteins are introduced into solid supported bilayers, while also allowing for high-precision measurements of changes in fluorophore position by interferometry.bilayer lipid membrane ͉ black lipid membrane ͉ fluorescence interference contrast B ilayer or ''black'' lipid membranes (BLMs) are a widely used model system for the study of cell-membrane proteins (1). BLMs are prepared on an aperture, usually hundreds of micrometers in diameter, made in a hydrophobic substrate material. Advances in BLM platform architectures, particularly chipbased architectures, have been directed toward a range of different membrane geometries (2-5), improved reproducibility (6, 7) and stability (8, 9), and applying imaging techniques such as fluorescence microscopy (10-14). In parallel, efforts are ongoing to increase the utility of solid supported lipid bilayers (15, 16) for examining cell membrane proteins, including tethering (17), cushioning (18), and loosely associated second-story bilayers (19-21).We are particularly interested in using fluorescence to study membrane protein conformational dynamics in a native environment, but measuring small changes requires the determination of fluorophore position with a precision better than the optical diffraction limit. The interference pattern above a mirror can be used to determine the absolute distance of fluorescent objects from the mirror with high precision (22). The mirror reflects excitation light, and the reflected light interferes with the incident light so that the intensity of excitation (and similarly, the intensity of emitted light) varies with distance from the mirror. To that end, we have developed a platform consisting of a freestanding lipid bilayer, analogous to a BLM, suspended above a reflective Si mirror (Fig. 1A).In fluorescence interference contrast (FLIC) microscopy (22-25), this interferometry concept was adapted to determine distances of interest and distribution of fluorophores in SiO 2 -supported lipid bilayers and associated objects, often with subnanometer precision. The bilayers are assembled on a SiO 2 /Si wafer (because Si reflects visible light), with SiO 2 layers of varying thickness (sometimes called a FLIC-chip), to step the fluorophore through the interference pattern and generate multiple fluorescence intensit...

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Methods for the Formation of Single Bimolecular Lipid Membranes in Aqueous Solution

Cited by 665 publications

References 0 publications

Quantitative visualization of passive transport across bilayer lipid membranes

Quantitative visualization of passive transport across bilayer lipid membranes

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

A membrane interferometer

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