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Abstract: For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca2+ ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca2+ ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1–100fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm2, and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices. [Copyright &y& Elsevier]