Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Abstract

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

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

“…and capacitance were calculated considering that the membrane conductance is negligible compared to the conductance of the bulk solution 24,25 . The background capacitance is measured before the formation of the bilayer membrane, and subtracted from the measured total capacitance obtained from the bilayer membrane 26 .…”

Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip

“…KcsA was expressed, purified, and reconstituted into proteoliposomes as previously described. 15,33 The channel/lipid molar ratio in the proteoliposomes was 1/6000. It is known that the lipid composition modulates KcsA activity, 33 therefore the protein was reconstituted in liposomes and characterized in bilayers of a 1:1 lipid mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoylsn-glycero-3-phospho-(1 0 -rac-glycerol) (POPG).…”

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

“…The use of a nonvolatile hydrocarbon solvent is a common approach not only to improve BLM stability but also to promote channel integration (7)(8)(9)(10)(11)(12)(13)(14)(18)(19)(20)(21)(22)(24)(25)(26)(27)(28)(29). Several researchers reported that the presence of nonvolatile organic solvent in BLMs appeared to promote channel reconstitution (28,29).…”