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Ion-selective microelectrode arrays for cell culture monitoring
Auteur(s)
Generelli, Silvia
Editeur(s)
De Rooij, Nicolas-F.
Date de parution
2008
Résumé
The design, microfabrication and characterization of a platform comprising an array of ion-selective microelectrodes (µISE) aimed at <i>in vitro</i> cellular physiology and toxicology is described. This study focusses on K<sup>+</sup> and Ca<sup>2+</sup> monitoring in cell culture environments. A potential promising application of such a platform is based on recent findings in molecular biology, revealing connections between certain diseases, as for example some types of cancer or parkinsonism, and a malfunction in cellular ion fluxes. The silicon microfabrication of the platform allowed the realization of ion-selective microelectrode arrays comprising 16 electrodes with diameters ranging from 1.5 µm to 6 µm, and an interelectrode gap of 150 µm or 300 µm. The µISE, inspired by the classical glass capillary microelectrodes, are based on a micropipette-like channel which, in two of the three realized geometries, protrude for 5 µm from the surface and are located in a 350 µm deep cavity. In the third geometry, the micropipettes are non-protruding, completely embedded in the silicon substrate. This planar platform allowed the implementation of an array of K<sup>+</sup>-selective electrodes combined with planar platinum electrodes. The metallic electrodes placed near the ion-selective electrodes are designed for the application of electrical pulses for localised electroporation of cells. Preliminary tests showed the feasibility of localised electroporation, and demonstrated that the application of several electroporation pulses does not affect the functionality of K<sup>+</sup> selective microelectrodes, however further investigation is necessary to optimize the electroporation protocol. The characterization of the electrodes in physiological concentration ranges was performed using electrodes of 1.5 µm diameter in contact with simple buffer calibration solutions. The observed detection limits, 10<sup>-5</sup> M for the K<sup>+</sup> -selective electrodes and 10<sup>-9</sup> M for the Ca<sup<sup>2+</sup> -selective electrodes, demonstrated that the µISE are adapted for the use in cell culture concentration ranges. The functional lifetime of the sensors, when conditioned in 10<sup>-3</sup> M KCl, or 10<sup>-4</sup> M CaCl<sub>2</sub> respectively, varied from 30 to 45 days for K<sup>+</sup> -selective electrodes, but is limited to 1 to 3 days for Ca<sup>2+</sup>-selective electrodes. This difference in the functional lifetimes of K<sup>+</sup>- and Ca<sup>2+</sup> -selective electrodes is due to the use of calcium-selective sensors in their non-equilibrium state. After several days of use the calcium detection limit rises to the micromolar range, which is adapted for extracellular calcium concentrations, but not for intracellular monitoring. In this case, the lifetime of the sensors is comparable to those of K<sup>+</sup>-selective microelectrodes. Tests performed in presence of culture medium showed a drastic diminution of the electrodes lifetime down to several days. On the other hand, a short-time contact of the sensors with the cells or cellular debris does not affect their functional characteristics. A short-time <i>in vitro</i> test was thus used to verify the possibility of identification of cell necrosis. Cell death, induced by a hypoosmotic shock, was successfully followed in real time by monitoring the intracellular potassium release in the culture medium. The µISE also allowed to quantify the necrotic cells, opening interesting possibilities in toxicological screening. Further development of this technique might lead to monitoring of early stage apoptosis. The observation of apoptosis before the process becoming irreversible, causing the death of the cell, could bring new information on the physiology of the cell cultures.
Notes
Thèse de doctorat : Université de Neuchâtel, 2008 ; Th. 2042
Identifiants
Type de publication
doctoral thesis
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