Patterning Single Cell-Electrode Pairs for Electrochemical Measurement of Quantal Exocytosis on Microchips

Abstract

We are developing transparent multi- electrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Design goals are that one and only one cell be positioned directly over each electrode and working electrodes have μm-scale dimensions in order to resolve pA-level currents. Patterning of cell-adhesion molecules in register with electrodes using conventional photolithographic approaches is problematic because organic solvents can disable sensitive biomolecule films. We report the parylene “dry liftoff” approach pioneered by Ilic and Craighead (Biomed Microdev 2: 317, 2000) can be used to pattern single cell-electrode pairs on the chip. A 1 μm-thick parylene C film is deposited on the multi-electrode array and S1813 photoresist is spin coated onto the device and patterned. The unprotected parylene over the electrodes is then removed using Reactive Ion Etch. Poly-l-lysine (PLL) is then added to promote cell attachment. Chromaffin cells are loaded on the chip in standard culture media and left in an incubator overnight. Finally, the parylene film is peeled off to remove excess cells and PLL, leaving tightly adhered chromaffin cells at the desired locations. Importantly, we find that promoting cell attachment with PLL films does not passivate the electrochemical electrodes. Experiments are in process to explore an alternative approach whereby PLL is patterned using the dry liftoff approach but cells are added after peel off of the parylene. With this approach, cell attachment to inactive areas of the chip is blocked by using “cytophobic” materials such as Teflon AF. This alternative approach may allow efficient targeting of cells at lower cell densities as cells migrate from cytophobic areas to the electrode binding sites (Supported by NIH BRP grant RO1 NS048826).