Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes.
We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional "Two Electrode Voltage Clamp" (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or "macropatch" techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between -100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration-inhibition curve displaying an inhibition constant IC(50) of about 50 nM, comparable to previously published values obtained with the TEVC technique.
Dahan
E
E
Ecole Polytechnique Fédérale de Lausanne, Institute of Microelectronics and Microsystems, Lausanne, Switzerland. elodie.dahan@epfl.ch
Bize
V
V
Lehnert
T
T
Horisberger
J-D
JD
Gijs
M A M
MA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004566:Electrodes; D053503:Epithelial Sodium Channels / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008564:Membrane Potentials; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation*; D012828:Silicones / Q000737:chemistry; D014981:Xenopus
D004129:Dimethylpolysiloxanes; D053503:Epithelial Sodium Channels; D012828:Silicones; C013407:benzamil; C013830:baysilon; D000584:Amiloride