Parallel Recordings of Transmembrane hERG Channel Currents Based on Solvent-Free Lipid Bilayer Microarray.

The reconstitution of ion-channel proteins in artificially formed bilayer lipid membranes (BLMs) forms a well-defined system for the functional analysis of ion channels and screening of the effects of drugs that act on these proteins. To improve the efficiency of the BLM reconstitution system, we report on a microarray of stable solvent-free BLMs formed in microfabricated silicon (Si) chips, where micro-apertures with well-defined nano- and micro-tapered edges were fabricated. Sixteen micro-wells were manufactured in a chamber made of Teflon®, and the Si chips were individually embedded in the respective wells as a recording site. Typically, 11 to 16 BLMs were simultaneously formed with an average BLM number of 13.1, which corresponded to a formation probability of 82%. Parallel recordings of ion-channel activities from multiple BLMs were successfully demonstrated using the human ether-a-go-go-related gene (hERG) potassium channel, of which the relation to arrhythmic side effects following drug treatment is well recognized.

Optical transport of sub-micron lipid vesicles along a nanofiber.

Enhanced manipulation and analysis of bio-particles using light confined in nano-scale dielectric structures has proceeded apace in the last several years. Small mode volumes, along with the lack of a need for bulky optical elements give advantages in sensitivity and scalability relative to conventional optical manipulation. However, manipulation of lipid vesicles (liposomes) remains difficult, particularly in the sub-micron diameter regime. Here we demonstrate the optical trapping and transport of sub-micron diameter liposomes along an optical nanofiber using the nanofiber mode's evanescent field. We find that nanofiber diameters below a nominal diffraction limit give optimal results. Our results pave the way for integrated optical transport and analysis of liposome-like bio-particles, as well as their coupling to nano-optical resonators.

Modulation of Photoinduced Transmembrane Currents in a Fullerene-Doped Freestanding Lipid Bilayer by a Lateral Bias.

We report on a novel lipid bilayer system, in which a lateral bias can be applied in addition to a conventional transmembrane voltage. Freestanding bilayer lipid membranes (BLMs) doped with [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) were formed in a microaperture, around which metal electrodes were deposited. Using this system, it was possible to modulate and amplify photoinduced transmembrane currents by applying a lateral bias along the BLM. The results indicate that the microfabricated Si chip with embedded electrodes is a promising platform for the formation of transistor-like devices based on PCBM-doped BLMs and have potential for use in a wide variety of nanohybrid devices.

Formation and Characterization of Air-Stable Lipid Bilayer Membranes Incorporated with Phthalocyanine Molecules.

Bilayer lipid membranes (BLMs) are used as basic frameworks for biosensors and biohybrid devices due to their unique properties, which include ultrathin thickness, ultrahigh resistivity, and self-assembling ability. However, BLMs can only form and maintain their structure in aqueous environments, which pose significant limitations to their use. In this work, we report on the formation of highly uniform hybrid BLMs at a water/air interface through self-assembly by simply doping the BLMs with a functional organic molecule, copper(II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc). By transferring the membrane onto substrates, we were able to produce stable hybrid BLMs under anhydrous conditions. Atomic force microscopy and X-ray diffraction measurements confirmed that the hybrid membranes were composed of single, highly uniform BLMs or stacks of BLMs. Fluorescence resonance energy transfer measurements indicated that the CuPc molecules were located between the hydrophobic tails of lipid molecules, forming a sandwich structure in the hybrid membranes. The hybrid BLMs fabricated by this method substantially expand the range of applications of BLMs to solid-state devices.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels.

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.