Microchips

A total number of 20 microchips were used for cell culture and electroporation experiments. Each chip contained two linear arrays of octagonal capacitive microelectrodes with a 15 nm SiO₂ film and whose structure and manufacturing process are described in detail elsewhere⁴¹ (see Results and Fig. 1). A plastic chamber was mounted on the chip for cell culture and stimuli delivered to the capacitive microelectrodes through a function generator (33250A 80 MHz, Agilent Technologies). Chips could be re-used multiple times (approximately 60, in average) before incurring in deterioration or rupture of capacitive microelectrodes. The integrity of the oxide film was monitored periodically throughout the experiments by DC I-V measurements and by assessing the response to pulsed waveforms with a capillary Ag/AgCl microelectrode immersed in the electrolyte and measuring the potential above the stimulation site.

Briefly n-type silicon wafers (diameter 100 mm) with a (100) surface and a resistance of 0.45 ± 0.75 Ω cm were used⁴¹. Local oxidation of silicon (LOCOS) was employed to insulate the stimulator areas with SiO₂. Highly conductive leads to the stimulation sites were created by bulk Si implantation with boron. A 660 nm passivation oxide was deposited by plasma enhanced chemical vapor deposition (PECVD) all over the chip and removed in the central area by etching. The 15 nm film of SiO₂ was grown by dry thermal oxidation (TCA/O₂ ambient) at 900 °C. The relative permittivity of the oxide estimated in electrolyte by impedance spectroscopy was about 4 (Birkenmeier & Fromherz, personal communication). The octagonal shape of the microelectrodes was chosen to reduce sharp edge effects on the electric field in the electrolyte.

Before each use, the microchips were gently cleaned with 5% (v/v) 70 °C Tickopur R33 detergent (Bandelin) for 1 min, sterilized for at least 30 min with 70% (v/v) ethanol, rinsed with sterile deionized water and air-dried under a sterile laminar hood. Thus, after addition of the cells suspension and seeding, they were kept in the cell incubator.