(496d) Development of a Platform for the Monitoring of Extracellular Ionic Activities

In the past few years, there has been great interest in using micro- and nanostructures for the analysis of complex biological systems such as living cells. In this study we present the development of a generic platform for cell culture able to monitor extracellular ionic activities (K+, NH4+, Ca2+,?) for real-time monitoring of cell-based responses, such as necrosis and apoptosis, differentiation, etc. Here we focused on the development of potassium- and ammonium-selective microelectrodes. Potassium, which is present at high levels in intact viable cells, leaks out of dying cells and elevated extracellular potassium can be used as a marker of cell death. Ammonium is taken up by differentiated hepatocytes and a decrease in extracellular ammonium can be used as a marker of stem cell differentiation into hepatocytes. For this purpose, we developed a platform for cell culture that is equipped with an array of 16 silicon nitride micropipettes (with a diameter of either 2 or 6 um) housing ion-selective microelectrodes. This array consist of two rows of 8 micropipettes separated by 150 um located at the bottom of a 200 um wide and 350 um deep microchannel where the cells are cultured. The micropipettes are individually connected with a network of 50 um wide microchannels etched in a Pyrex substrate aligned and bonded at the bottom side of the silicon substrate. Extended characterization of the ion-selective microelectrodes arrays in different standard and physiological solutions was carried out. Near Nernstian slopes were obtained for potassium-selective microelectrodes (58.6 ± 0.8 mV/pK, n=15) and for ammonium-selective microelectrodes (59.4 ± 3.9 mV/pNH4, n=13), both after more than one week of use. The calibration curves were highly reproducible and showed very little drift (in average 4 mV/h, n=10). Long-term behavior as well as response after immersion in physiological solutions will also be presented.