title: Positioning of the sensor cell on the sensing area using cell trapping pattern in incubation type planar patch clamp biosensor.
abstract: Positioning the sensor cell on the micropore of the sensor chip and keeping it there during incubation are problematic tasks for incubation type planar patch clamp biosensors. To solve these problems, we formed on the Si sensor chip's surface a cell trapping pattern consisting of a lattice pattern with a round area 5 μm deep and with the micropore at the center of the round area. The surface of the sensor chip was coated with extra cellular matrix collagen IV, and HEK293 cells on which a chimera molecule of channel-rhodopsin-wide-receiver (ChR-WR) was expressed, were then seeded. We examined the effects of this cell trapping pattern on the biosensor's operation. In the case of a flat sensor chip without a cell trapping pattern, it took several days before the sensor cell covered the micropore and formed an almost confluent state. As a result, multi-cell layers easily formed and made channel current measurements impossible. On the other hand, the sensor chip with cell trapping pattern easily trapped cells in the round area, and formed the colony consisted of the cell monolayer covering the micropore. A laser (473 nm wavelength) induced channel current was observed from the whole cell arrangement formed using the nystatin perforation technique. The observed channel current characteristics matched measurements made by using a pipette patch clamp.
authors.affiliation: Nagoya University, Nagoya 464-8603, Japan.
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Wang
authors.forename: Noriko
authors.initials: N
authors.lastname: Takada
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Uno
authors.forename: Toru
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Hiromu
authors.initials: H
authors.lastname: Yawo
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Urisu
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D000075402:Channelrhodopsins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000075402:Channelrhodopsins; D049452:Green Fluorescent Proteins