000124906 001__ 124906
000124906 005__ 20190509132206.0
000124906 0247_ $$2doi$$a10.5075/epfl-thesis-4151
000124906 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis4151-7
000124906 02471 $$2nebis$$a5582831
000124906 037__ $$aTHESIS
000124906 041__ $$aeng
000124906 088__ $$a4151
000124906 245__ $$aIntegrated microfluidic system for non-invasive electrophysiological measurements on Xenopus oocytes
000124906 269__ $$a2008
000124906 260__ $$bEPFL$$c2008$$aLausanne
000124906 300__ $$a168
000124906 336__ $$aTheses
000124906 520__ $$aWe 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.
000124906 6531_ $$aMicrofluidics
000124906 6531_ $$aLab-on-a-chip
000124906 6531_ $$aPoly-dimethylsiloxane (PDMS)
000124906 6531_ $$aSU-8 photoresist
000124906 6531_ $$aIntegrated microvalves
000124906 6531_ $$aAg/AgCl reference electrodes
000124906 6531_ $$aXenopus oocytes
000124906 6531_ $$aElectrophysiology
000124906 6531_ $$aVoltage-clamp
000124906 6531_ $$aMicrofluidique
000124906 6531_ $$aLaboratoire sur puce
000124906 6531_ $$aPoly-diméthylsiloxane (PDMS)
000124906 6531_ $$arésine photosensible SU-8
000124906 6531_ $$aMicrovannes intégrées
000124906 6531_ $$aElectrodes de référence en Ag /AgCl
000124906 6531_ $$aOvocytes de Xenopus
000124906 6531_ $$aElectrophysiologie
000124906 6531_ $$aVoltage-Clamp
000124906 700__ $$aDahan, Elodie
000124906 720_2 $$aGijs, Martin$$edir.$$g113762$$0242801
000124906 720_2 $$aLehnert, Thomas$$edir.$$g113381$$0240962
000124906 8564_ $$uhttps://infoscience.epfl.ch/record/124906/files/EPFL_TH4151.pdf$$zTexte intégral / Full text$$s7793021$$yTexte intégral / Full text
000124906 909C0 $$xU10322$$0252094$$pLMIS2
000124906 909CO $$pthesis$$pthesis-bn2018$$pDOI$$ooai:infoscience.tind.io:124906$$qDOI2$$qGLOBAL_SET$$pSTI
000124906 918__ $$dEDMI$$cIMT$$aSTI
000124906 919__ $$aLMIS2
000124906 920__ $$b2008
000124906 970__ $$a4151/THESES
000124906 973__ $$sPUBLISHED$$aEPFL
000124906 980__ $$aTHESIS