Abstract
Planar lipid bilayers constitute a versatile method for measuring the activity of protein channels and pores on a single molecule level. Ongoing efforts attempt to tailor this method for detecting biomedically relevant target analytes or for high-throughput screening of drugs. To improve the mechanical stability of bilayer recordings, we use a thin-film epoxy resist ADEX as septum in free-standing vertical bilayers. Defined apertures with diameters between 30 µm and 100 µm were micro-fabricated by photolithography. The performance of these septa was tested by functional reconstitution of the K+ channel KcvNTS in lipid bilayers spanned over apertures in ADEX or Teflon films; the latter is conventionally used in bilayer recordings and serves as reference. We observe that the functional properties of the K+ channel are identical in both materials while ADEX provides no advantage in terms of capacitance and signal-to-noise ratio. In contrast to Teflon, however, ADEX enables long-term experimental recordings while the stability of the lipid bilayer is not compromised by pipetting solutions in and out of the recording chamber. Combined with the fact that the ADEX films can be cleaned with acetone, our results suggest that ADEX carries great potential for multiplexing bilayer chambers in robust and reusable sensing devices.Graphical
Highlights
Channel proteins catalyze the diffusion of ions across lipid membranes
Many attempts were made over the past decades: In a recent study, we reported a new technique which allows efficient synthesis and incorporation of ion channel proteins into planar lipid bilayers (Winterstein et al 2018)
Dry ADEX film sheets are suitable for fabricating complex apertures by photolithographic methods
Summary
Channel proteins catalyze the diffusion of ions across lipid membranes. This can occur in a highly regulated and selective manner through canonical ion channels (Hille 2001), or less selectively via highly conductive beta-barreled pores (Delcour 2002). Pore-forming proteins, so-called protein nanopores, have received great attention from protein engineers (Ayub and Bayley 2016). These proteins can be genetically modified to sense various physical stimuli (e.g. voltage, mechanical stress) and chemical signals and molecules (e.g. ligands, pH, DNA etc.) in a highly sensitive and selective fashion. The most prominent example of such a biotechnological application of conducting pores is the MinION device from Oxford Nanopore Technologies, which serves as a portable long-read DNA sequencing device (Fraiture et al 2018)
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