Integrated microfluidic system for non-invasive electrophysiological measurements on Xenopus oocytes
We developed a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. The Xenopus oocyte is a well-known expression system for various kinds of ion channels that are of potential interest for 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 for the classical patch-clamp or "macropatch" techniques, this method does not require removal of the fibrous 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. In a second part of this work, a rapid fluidic exchange system was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimeter-size cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The fabrication process for this disposable microchip, based on PDMS micromolding, is cost-efficient and simple. Moreover, an innovative integration of agar gel-based reference electrodes was developed. A conductive liquid junction is injected by capillary force filling of suitable microchannels, overcoming the problems encountered with the integration of Ag/AgCl thin film or wire reference electrodes. We tested this new microdevice by performing electrophysiological measurements on oocytes expressing the human Epithelial Sodium Channel (hENaC). Transmembrane currents could be recorded with a large signal-to-noise ratio. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of hENaC. Our results show that the response time of this ion channel to a specific reactant is about one order of magnitude faster than estimated with the traditional TEVC technique. We conclude that this new microdevice has high potential for improved electrophysiological investigations with oocytes in the field of pharmacology and toxicology.
Keywords: Microfluidics ; Lab-on-a-chip ; Poly-dimethylsiloxane (PDMS) ; SU-8 photoresist ; Integrated microvalves ; Ag/AgCl reference electrodes ; Xenopus oocytes ; Electrophysiology ; Voltage-clamp ; Microfluidique ; Laboratoire sur puce ; Poly-diméthylsiloxane (PDMS) ; résine photosensible SU-8 ; Microvannes intégrées ; Electrodes de référence en Ag /AgCl ; Ovocytes de Xenopus ; Electrophysiologie ; Voltage-ClampThèse École polytechnique fédérale de Lausanne EPFL, n° 4151 (2008)
Programme doctoral Microsystèmes et Microélectronique
Faculté des sciences et techniques de l'ingénieur
Institut de microtechnique
Laboratoire de microsystèmes 2
Record created on 2008-05-29, modified on 2016-08-08