A membrane interferometer

Abstract

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...

Lipid Bilayers on a Picoliter Microdroplet Array for Rapid Fluorescence Detection of Membrane Transport

“…Although liposomes are generally used as the lipid bilayer chambers, it is difficult to control the chamber size of these systems, owing to which they are unsuitable for high‐throughput quantitative analyses. Two systems have been proposed for use as volume‐controllable chambers: micromachined chambers sealed with lipid bilayers and droplet chambers in which monodisperse microdroplets come into contact with other droplets in a lipid‐dispersed organic solvent . Micromachined chambers provide controlled volume reduction, resulting in a variable S/V ratio; however, it is difficult to achieve reproducible sealing of the chamber .…”

“…Two systems have been proposed for use as volume‐controllable chambers: micromachined chambers sealed with lipid bilayers and droplet chambers in which monodisperse microdroplets come into contact with other droplets in a lipid‐dispersed organic solvent . Micromachined chambers provide controlled volume reduction, resulting in a variable S/V ratio; however, it is difficult to achieve reproducible sealing of the chamber . On the other hand, droplet chambers provide high reproducibility of the sealing; nevertheless, the S/V ratio is relatively low (S/V < 4 μm 2 /pL) owing to the large droplet volume (several nanoliters) .…”

Enhanced stability of freestanding lipid bilayer and its stability criteria

“…Engineering artificial model cell membranes, such as lipid vesicles12, supported lipid bilayers2345, and freestanding lipid bilayers6789101112131415161718192021222324, is an important issue in physical science, life science and biomedical engineering2526. Such model membranes can provide a useful in vitro platform for studying a variety of biological problems related to cell membranes2728.…”

“…Accordingly, a lot of new fabrication methods for stable freestanding lipid bilayers have been introduced678910111213141516171819202122. Recently, an excellent technique, termed as droplet interface bilayers (DIBs) was developed181920212223 where two lipid-coated water droplets immersed in solvent bring together to form a stable freestanding lipid bilayer with large area.…”

Surface nanostructures for fluorescence probing of supported lipid bilayers on reflective substrates

“…Medium of immersion is water, with n w = 1.333. polymer brushes with nanometer precision. [33][34][35] Here, we have used structures on the substrate that confer a known distance between the fluorophores and the reflecting surface to increase the fluorescence signal sufficiently to enable imaging of supported lipid bilayer (see Fig. 1B).…”