Reconstitution of ion channels in agarose-supported silicon orifices

Abstract

A silicon wafer with eight individually addressable microfabricated orifices was used for ion channel reconstitution and single-channel recording. A spin-on fluoropolymer created an insulating, hydrophobic interface that was more stable than silane. Total capacitance of the membranes was <10 pF, making it easy to evaluate bilayer formation by capacitance change. Orifices of 50–250 μm diameter were tested for ease and stability of bilayer formation; only those >100 μm resulted in ion channel function. Bilayers were formed over an agarose supporting layer by application of lipid in decane with a paintbrush; a second layer of agarose could then be added to stabilize the structure and prevent evaporation. Microfluidic wells were constructed on glass plates for ease of assembly and visualization of fluid flow, as well as high-resolution microscopy for studies using fluorescent lipids and channels. The microfluidics consisted of reversibly bonded silicone rubber (PDMS), so that the entire device could be washed and reused. Total electrical noise in the device was low enough to permit single-channel resolution. Successful channel insertions were observed with a self-assembling ionophore (alamethicin) as well as a complex, vesicle-associated mammalian channel (human glycine receptor, GlyR). A "hands-free" approach to bilayer formation was also tested, where lipid in solvent was applied to the wafer by spin-coating, dried, and then "sandwiched" between layers of agarose above and below the nitride. Electrical properties consistent with bilayers were observed and alamethicin recordings were obtained, however this method is not compatible with the fusion of vesicles containing mammalian channels.

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