3D lithography of shaped lipid bilayer apertures
Electrical measurements of ion channel activity can be performed by patch clamping of cell membranes or by suspending lipid bilayer model membranes, with incorporated channels, in an aperture positioned inbetween two aqueous compartments. The latter method is in principle more suitable for miniaturization, parallelization and automation of ion channel measurements, but it critically depends on the stability of the suspended bilayer. Conventional apertures in thin Teflon sheets have a diameter of ~150 µm and are produced by mechanical punching or electric sparks. These methods do not give reproducible aperture geometries and consequently only a number of apertures are suitable for suspended bilayer formation, and even these tend to be relatively fragile, limiting measurement throughput.
In this project we fabricate apertures in photoresist sheets by 3D lithography, which enables not only precise control of the aperture diameter but also of the shape of the aperture side walls. Ideally the sheets should be relatively thick to reduce the capacitance of the septum, but it is hypothesized that a thinner aperture edge improves bilayer stability. We have shown that bilayers formed at the thin tip of our tapered apertures display drastically increased lifetimes, typically >20 hours, and mechanical stability, being able to withstand extensive perturbation of the aqueous compartments as required for ion channel assays. Single-channel electrical recordings of peptides and proteoliposome-delivered channels demonstrate channel measurements with low noise, enabling observation of the ~10 pA channel current steps.
These shaped apertures with micrometer edge thickness should substantially enhance the throughput of channel characterisation by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms, which are developed in a related project.
- Birkbeck College, University of London
- ECS studentship contribution
- Mr Sumit Kalsi
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