A perforated CMOS microchip for immobilization and activity monitoring of electrogenic cells

Abstract

CMOS-based microelectrode systems offer decisive advantages over conventional micro-electrode arrays, which include the possibility to perform on-chip signal conditioning or to efficiently use larger numbers of electrodes to obtain statistically relevant data, e.g., in pharmacological drug screening. A larger number of electrodes can only be realized with the help of on-chip multiplexing and readout schemes, which require integrated electronics. Another fundamental issue in performing high-fidelity recordings from electrogenic cells is a good electrical coupling between the cells and the microelectrodes, in particular, since the recorded extracellular signals are in the range of only 10–1000 µV. In this paper we present the first CMOS microelectrode system with integrated micromechanical cell-placement features fabricated in a commercial CMOS process with subsequent post-CMOS bulk micromachining. This new microdevice aims at enabling the precise placement of single cells in the center of the electrodes to ensure an efficient use of the available electrodes, even for low-density cell cultures. Small through-chip holes have been generated at the metal-electrode sites by using a combination of bulk micromachining and reactive-ion etching. These holes act as orifices so that cell immobilization can be achieved by means of pneumatic anchoring. The chip additionally hosts integrated circuitry, i.e., multiplexers to select the respective readout electrodes, an amplifier with selectable gain (2×, 10×, 100×), and a high-pass filter (100 Hz cut-off). In this paper we show that electrical signals from most of the electrodes can be recorded, even in low-density cultures of neonatal rat cardiomyocytes, by using perforated metal electrodes and by applying a small underpressure from the backside of the chip. The measurements evidenced that, in most cases, about 90% of the electrodes were covered with single cells, approximately 4% were covered with more than one cell due to clustering and approximately 6% were not covered with any cell, mostly as a consequence of orifice clogging. After 4 days of culturing, the cells were still in place on the electrodes so that the cell electrical activity could be measured using the on-chip circuitry. Measured signal amplitudes were in the range of 500–700 µV, while the input-referred noise of the readout was below 15 µVrms (100 Hz–4 kHz bandwidth). We report on the development and fabrication of this new cell-biological tool and present first results collected during the characterization and evaluation of the chip. The recordings of electrical potentials of neonatal rat cardiomyocytes after several days in vitro, which, on the one hand, were conventionally cultured (no pneumatic anchoring) and, on the other hand, were anchored and immobilized, will be detailed.