Analysis of ion Channel Activities in Lipid Bilayers Suspended Over Microwells on SI Substrates

Abstract

One of the key techniques to fabricate a nanobio device that works with functioning membrane proteins is the creation of stable lipid bilayers, guaranteeing protein activity and the detection of their functions. In order to develop the measurement system of ion channel activities, here, suspended lipid bilayers were formed over the microwells fabricated on the Si substrate. Giant unilamellar vesicles (GUVs) with and without gramicidin A (gA) were ruptured over the microwells and the pH indicator pyranine was confined in the microwells by a lipid bilayer. Activity of gA ion channels contained in the suspended lipid bilayer was analyzed by the change of fluorescent intensity of pyranine. Increasing the pH outside of the microwells by exchanging solution led to an increase in the fluorescent emission of pyranine confined in the microwells. Sealing of the micro-wells by the lipid bilayer was good enough such that no leakage of pyranine was observed during functional analysis. A clear difference in the rate of fluorescent intensity changes is seen between lipid bilayers with and without gA ion channels, indicating that transport of protons through gA ion channels was observed. Proton leak through the lipid membrane was estimated to be 10 x 10−6 ∼ 10 x 10−5 cm/sec. This successful creation of stable lipid bilayers on Si chips with peptides as biological signal transducers is an important step towards the development of large and versatile sensor arrays which allow parallel single channel recordings.