title: Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate.
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. Ca(2+) 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 Ca(2+) 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 (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), 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.
authors.affiliation: NTT Basic Research Laboratories, NTT Corporation, 3-1 Morinosato Wakamiya, Atsugi, Kanagawa 243-0198, Japan. sumitomo.koji@lab.ntt.co.jp
authors.forename: Koji
authors.initials: K
authors.lastname: Sumitomo
authors.forename: Arianna
authors.initials: A
authors.lastname: McAllister
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tamba
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kashimura
authors.forename: Aya
authors.initials: A
authors.lastname: Tanaka
authors.forename: Youichi
authors.initials: Y
authors.lastname: Shinozaki
authors.forename: Keiichi
authors.initials: K
authors.lastname: Torimitsu
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D015640:Ion Channel Gating; D017136:Ion Transport; D008051:Lipid Bilayers / Q000737:chemistry*; D046228:Microarray Analysis / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry
chemical_list: D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin; D002118:Calcium; D012825:Silicon