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Multi-Modal 16,384-Electrode CMOS MEA with 16 Independent Multi-Well Assays for Physiological Studies of Different Cellular Models

Beatrice Miccoli1, 2, Carolina Mora Lopez1, Ho Sung Chun1, Shiwei Wang1, Jan Putzeys1, Carl Van Den Bulcke1, 2, Andrea Firrincieli1, Nick Van Helleputte1, Veerle Reumers1 and Dries Braeken1*
  • 1 Interuniversity Microelectronics Centre (IMEC), Belgium
  • 2 KU Leuven, Belgium

Passive and active multi-electrodes arrays (MEAs) stand out as powerful platforms for characterizing and unveiling complex electrophysiological dynamics of cells and tissues (Huys et al., 2012). Among them, active complementary metal oxide semiconductors (CMOS) based MEAs allow for a higher spatial resolution due to the possibility of reducing the electrodes size down to the single-cell scale, noise reduction due to reduced external interconnections, and higher automatization and portability, which strongly widen the applicability range (Huys et al., 2012). Mainly focused on electrogenic cells, these technologies impact very different fields ranging from high-throughput cardiotoxicity assays, fundamental for drug development or personalized-medicine applications, to advanced neuroscience studies (Chi et al., 2015). Despite the high versatility of CMOS MEAs, most of them are single-modal systems, therefore limiting the actual range of physiological parameters, that can be monitored on the same chip (Chi et al., 2015). Nevertheless, cellular physiological processes are extremely complex and multi-faceted therefore requiring either the simultaneous monitoring of multiple parameters or multiple types of electrophysiological measurements, on the same chip (Chi et al., 2015). Moreover, being able to access on the same chip both measurements at the cellular and network level is another key requirement, fundamental e.g., to understand how specific cellular processes influence the overall network dynamics in neuronal populations (Müller et al., 2015). This is also translated in the possibility of concurrently performing extra-cellular and intra-cellular action potential recordings in situ, on the same platform. This not only eliminates the need for external patch-clamp instrumentation, hence allowing long-term intracellular measurements along with extracellular recording but, also, it results in higher portability and versatility (Braeken et al., 2012). Therefore, we developed a multimodal 16,384 titanium nitride (TiN) electrode CMOS MEA chip with 1,024 parallel recording channels for physiological studies of different types of cells. The MEA allows both intracellular and extracellular recording, current and voltage stimulation, as well as impedance measurements, i.e. impedance spectroscopy (10 Hz to 1 MHz) or fixed frequency impedance monitoring (1 and 10 kHz), which significantly broaden the applicability range, also to non-electrogenic cells (Lopez et al., 2018). The 16,384 TiN electrodes are grouped on the chip surface in 16 independent wells, each one including 256 pixels, in a 16 x 16 matrix configuration. Each pixel then contains 4 electrodes thus leading to 1,024 electrodes per well that can be connected to 1,024 channels. The 6 different functionalities can be independently and simultaneously assessed on the 16 different wells leading to a potential 16 multi-well assays. It is important to underline that, while the electrodes pitch is fixed at 15 µm, 4 different electrodes sizes are implemented ranging from 2.5 x 3.5 µm^2 up to 10.5 x 11.0 µm^2. The presence of electrodes of different sizes and a large range of amplifier gain settings are crucial not only to widen the applicability of the chip for cells of different types and size, but, also, to accurately assess different physiological phenomena, at slightly different dimensional scales. The detailed circuit architecture of the presented chip was reported in (Lopez et al., 2018). Here, we present several assays demonstrating the multi-modality of the platform. At first, rat cardiomyocytes extracellular and intracellular action potentials were recorded, both independently and simultaneously, by exploiting the internal stimulation and recording circuitry of the chip. The amplifier gain for each channel can be independently adjusted to accommodate signals ranging from hundreds of µV (extra-cellular) to tens of mV (intra-cellular). Specifically, a peak-to-peak voltage amplitude of 1.43 mV ± 0.007 mV was measured on a 3 DIV rat cardiomyocytes culture showing a beating rate of 65.78 ± 0.027 bpm. The intracellular measurements revealed, instead, a spike peak-to-peak amplitude of 27.77 mV. Further, we used the fixed frequency impedance measurement mode (at 1 kHz) to assess cardiomyocyte contractility. Moreover, large ‘pacing’ electrodes were implemented to apply defined beating frequency to the cell culture. The 16 different wells feature independent on-chip reference electrodes thus 16 different measurement conditions can be applied on a single silicon die. All above-described measurements can be performed simultaneously on the array and therefore multi-parametric information from the same cell culture experiment can be obtained. By taking advantage of the diverse possibilities both in terms of electrode fabrication and of multi-modal operation, complex physiological phenomena as well as multi-parametric experiments can be characterized and deeply investigated, not only with a tunable resolution down to subcellular processes but, also, with high throughput thanks to the 16 independently controlled wells.

References

Braeken, D., Jans, D., Huys, R., Stassen, A., Collaert, N., Hoffman, L., et al. (2012). Open-cell recording of action potentials using active electrode arrays. Lab Chip 12, 4397. doi:10.1039/c2lc40656j.
Chi, T., Park, J. S., Butts, J. C., Hookway, T. A., Su, A., Zhu, C., et al. (2015). A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening. IEEE Trans. Biomed. Circuits Syst. 9, 801–814. doi:10.1109/TBCAS.2015.2504984.
Huys, R., Braeken, D., Jans, D., Stassen, A., Collaert, N., Wouters, J., et al. (2012). Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip. Lab Chip 12, 1274. doi:10.1039/c2lc21037a.
Lopez, C. M., Chun, H. S., Berti, L., Wang, S., Bulcke, C. Van Den, Weijers, J., et al. (2018). A 16384-Electrode 1024-Channel Multimodal CMOS MEA for High-Throughput Intracellular Action Potential Measurements and Impedance Spectroscopy in Drug- Screening Applications. in 2018 International Solid-State Circuits Conference, 4–6.
Müller, J., Ballini, M., Livi, P., Chen, Y., Radivojevic, M., Shadmani, A., et al. (2015). High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab Chip 15, 2767–2780. doi:10.1039/C5LC00133A.

Keywords: multi-modal CMOS MEA, in vitro electrophysiology, CMOS multi-well assay, Intracellular action potential, impedance spectroscopy

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Oral Presentation

Topic: Microelectrode Array Technology

Citation: Miccoli B, Mora Lopez C, Chun H, Wang S, Putzeys J, Van Den Bulcke C, Firrincieli A, Van Helleputte N, Reumers V and Braeken D (2019). Multi-Modal 16,384-Electrode CMOS MEA with 16 Independent Multi-Well Assays for Physiological Studies of Different Cellular Models. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00033

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Received: 16 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Dr. Dries Braeken, Interuniversity Microelectronics Centre (IMEC), Leuven, Belgium, Dries.Braeken@imec.be