In situ generation of electrochemical gradients across pore-spanning membranes

Abstract

Silicon substrates with cavities in the micrometre range were micro-fabricated and appropriately functionalized to allow for the generation of pore-spanning membranes (PSMs) sealing the pore cavities. PSMs were either formed by applying lipids dissolved in organic solvent (painting technique) on a hydrophobically functionalized silicon surface followed by ‘solvent freeze-out’, or solvent-free PSMs were prepared by spreading of giant unilamellar vesicles on hydrophilically functionalized substrates. The geometry of the silicon cavities in conjunction with three dimensional confocal laser scanning microscopy images enabled us to simultaneously monitor the PSMs and a pH-sensitive dye entrapped into the picolitre-sized cavities. The excellent sealing properties of both PSM types allowed an in situ generation of proton gradients across these membranes. In the presence of nigericin, a proton/potassium-antiporter, a preformed potassium ion gradient was transformed into a stable proton gradient across the PSMs, which was visualized by the pH-sensitive dye pyranine entrapped in the silicon cavities in a time resolved manner by means of confocal laser scanning fluorescence microscopy.