Frauke Greve

Switch-Matrix-Based High-Density Microelectrode Array in CMOS Technology

We report on a CMOS-based microelectrode array (MEA) featuring 11, 011 metal electrodes and 126 channels, each of which comprises recording and stimulation electronics, for extracellular bidirectional communication with electrogenic cells, such as neurons or cardiomyocytes. The important features include: (i) high spatial resolution at (sub)cellular level with 3150 electrodes per mm2 (electrode diameter 7 ¿m, electrode pitch 18 ¿m); (ii) a reconflgurable routing of the recording sites to the 126 channels; and (iii) low noise levels.

An 11k-Electrode 126-Channel High-Density Microelectrode Array to Interact with Electrogenic Cells

A microelectrode array allows an arbitrary group of 126 electrodes to be selected from a total of 11,016 in order to do cell or neural recordings from areas of interest with 18 mum spatial resolution and 2.4 muv input-referred noise. Signals are amplified by 0 to 80dB, bandpass filtered (0.3 to 4kHz), and finally digitized (20kS/s, 8b). Example recordings from acute brain slices are shown

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

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.

CMOS Bidirectional Electrode Array for Electrogenic Cells

We report on a CMOS-based microelectrode-array chip (6.5 by 6.5 mm2) for bidirectional communication (stimulation and recording) with electrogenic cells such as cardiomyocytes or neurons targeted at investigating electrical signal propagation within cellular networks in vitro. The integration of on-chip circuitry, which includes analog signal amplification and filtering stages, analog-to-digital converters, a digital-to-analog converter, stimulation buffers, temperature sensors, and a digital interface for data transmission notably improves the overall system performance. Additionally, the interconnect challenge that limits the size of currently used microelectrode arrays is overcome. Measurements with cardiomyocytes and neuronal cells were successfully carried out, and the circuitry characterization evidenced a total equivalent input noise of 11.7 µ VRMS(0.1 Hz -100 kHz) at a gain of 1,000.

A hybrid microsystem for parallel perfusion experiments on living cells

A fully integrated microchip device for performing a complete and automated sample-perfusion experiment on living cells is presented. Cells were trapped and immobilized in a defined grid pattern inside a small 0.5 ?l volume incubation chamber by pneumatic anchoring on 1000 5-?m orifices. This new cell trapping technique assures a precise and repeatable cell quantity for each experiment and enables the formation of a homogeneous cell population in the incubation chamber. The microsystem includes a perforated silicon chip seamlessly integrated by a new embedding technique in a larger elastomer substrate, which features the microfluidic network. The latter forms the incubation chamber and allows for economic logarithmic dilution of the sample reagent over a range of three orders of magnitude with subsequent perfusion of the cell population. First, the logarithmic dilution stage was validated using quantitative fluorescent imaging of fluorescein solution. Then, the cell adhesion and culturing inside the incubation chamber was studied using primary normal human dermal fibroblasts (NHDFs). The cells adhered well on laminin-coated surfaces and proliferated to form a confluent cell layer after 6 days in vitro. Finally, the complete system was tested by a perfusion experiment with cultured NHDFs, which were exposed to a fluorescent cell tracker at dilutions of 100 ?m, 10 ?m, 1 ?m, 0.1 ?m and 0 ?m at a flow rate of 1.25 ?l min?1 for 20 min. Fluorescence imaging of the cell array after incubation and image analysis showed a logarithmic relationship between sample concentration and the fluorescence signal. This paper describes the fabrication of the components and the assembly of the microsystem, the design approach and the validation of the sample diluter, cell-adhesion and cell-culturing experiments over several days.

Perforated CMOS microchip platform for immobilization and activity monitoring of electrogenic cells

A unique combination of micromachining and CMOS microelectronics for physical cell immobilization and subsequent electrical activity recording is presented: Electrogenie cells are placed onto electrode arrays by pneumatic anchoring on perforated metal electrodes. After several days in culture, the electrical activity of the cells is measured after amplification by on-chip circuitry. This paper describes the fabrication of the bio-electronic chip, the electrical characterization of the circuitry, the culturing, and the recording of the electric signals of cardiomyocytes after several days in culture.

High-throughput cell-based screening system with on-chip dilution stage

A microchip-based approach for performing a complete and automated drug-screening assay on living cells is presented. Cells are trapped and immobilized in a small 0.5-mul-volume incubation chamber by means of orifice microstructures and are subsequently incubated with drug dilutions ranging over three orders of magnitude. The microsystem includes a perforated silicon chip embedded in a larger elastomer substrate, which features the microfluidic network and the incubation chamber. This article describes the modeling and the fabrication of the microchip components, immobilization of normal human dermal fibroblasts (NHDFs) and a screening experiment with cultured NHDFs, which have been exposed to a fluorescent cell tracker

Precise cell placement by pneumatic anchoring

This work presents a novel approach to trapping and placing a defined number of living cells on a microchip for biosensing and drug-screening applications. Cells are attracted from a liquid suspension by pneumatic anchoring through small orifices (3 /spl mu/m) on the chip surface, which allow for applying suction from the backside of the chip. In contrast to other cell handling techniques, pneumatic anchoring is a very mild, physical technique, which allows for permanent cell immobilization in a parallel fashion. In most cases, pressure needs only to be applied during cell placement, as they afterwards adhere to the protein-conditioned surface and remain at their position. The chip is fabricated using silicon-on-insulator (SOI) wafers by using combined front- and backside etching. Pneumatic particle handling has been studied using polymer beads and living cells suspended in physiological buffer solutions.