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A novel 3D-microstructured multielectrode array configuration for high-yield and high-fidelity electrophysiological recordings

José C. Mateus1, 2, 3, Mónica Cerquido4, Cátia Lopes1, 2, Diana Leitão5, Susana Cardoso5, João Ventura4 and Paulo Aguiar1, 2*
  • 1 INEB - Instituto Nacional de Engenharia Biomédica, University of Porto, Portugal
  • 2 i3S, Instituto de Investigação e Inovação em Saúde, Portugal
  • 3 Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Portugal
  • 4 Instituto de Física dos Materiais, Faculdade de Ciências, Universidade do Porto (IFIMUP), Portugal
  • 5 Instituto de Engenharia de Sistemas e Computadores Microsistemas e Nanotecnologias, Portugal

Motivation: Advances in microfabrication procedures have reduced the gap between intra- and extracellular recordings quality by greatly increasing the resistance seal and, consequently, the signal-to-noise ratio (SNR) of multielectrode array (MEA) recordings (1,2). Nonplanar microelectrodes that bio-mimic morphological structures dedicated to cell coupling, such as the synaptic cleft (3), or the shape and dimension (mushroom-like) of dendritic spines (4,5), have shown great promise in large invertebrate neurons for noninvasive, multisite, high-fidelity electrophysiological recordings. In particular, work pioneered by Spira and collaborators has enabled intracellular-like recordings using mushroom-shaped microelectrodes, though the translation of their recording capabilities to smaller mammalian neurons remains challenging (6,7). To parallelize and enhance signal detection, somata and neurites need to be in close vicinity with the multiple sensing microelectrodes. A growing body of knowledge demonstrates that topographical features can be used to manipulate neuron placement, growth and connectivity (8–10), thus 3D-microstructured MEAs can be designed to promote neuron-microelectrode contact. Here, we present a novel MEA configuration, where the sensing microelectrodes are structured with a 3×3 array of mushroom-shaped 3D microstructures. These islet-like agglomerates of 3D microstructures, replacing planar electrodes in normal MEA chips, have the potential of increasing the yield and SNR of conventional MEA technology. Methods: The fabrication process of the 3D-microstructured microelectrodes was performed on 49 mm x 49 mm glass substrate coated with Cr (5 nm)/Au (40 nm). A three-level photolithography process was then used to obtain the intended array. In the first level, one defines the electrical leads and reading pads by optical lithography and uses wet-etching to remove the Cr/Au thin layer of the unprotected parts. The second level is then used to passivate the electrical leads protecting them with 80 nm of Al2O3. The last level defines the array of 3×3 micro-holes with 2 µm of diameter separated by 10 µm. To create the mushroom shape, gold was electrodeposited inside the micro-holes under a constant potential of -1.0 V for 39 minutes using an Orosene bath at ambient temperature (approximately 23.5 oC). After this process, the photoresist was removed using acetone. Before cell plating, chips were rinsed in distilled water to remove residues from fabrication. Subsequently, chips were sterilized by a brief immersion in 70% ethanol, followed by UV-sterilization, and coated with poly(D-lysine) and laminin. Biocompatibility of the chips was confirmed with viable cultures of embryonic rat cortical neurons (1000 viable cells/mm2). The topography effect on the spatial organization of individual cells and the network itself was assessed via cell fixation at 3 days in vitro (DIV), followed by immunofluorescence staining and confocal imaging analysis. Extracellular recordings were performed inside a Faraday cage at room temperature (22ºC). All data was analog-filtered (0.3-20 kHz) and -amplified (×100). The amplifier was connected to the contact pads of the chips via pogo pins held in place by micromanipulators. Signals were digitized with 16-bit resolution at a sampling rate of 1 gigasamples/sec. Results: MEA chips composed by 3D-microstructured sensing microelectrodes were successfully developed and tested experimentally. The 3D microstructures had the intended mushroom shape with a stalk of 1.5 µm, a cap width of approximately 2.8 µm and a cap height of 0.5 µm. Beyond increasing the effective area per microelectrode, the islets of 3D microstructures per se influenced neuronal localization and increased the propensity for neuron-microelectrode contact. From our statistics, the probability for somata to localize in the islets increased near 2-fold when compared to the planar substrate (*p = 0.0195, two tailed unpaired t-test with Welch’s correction). Moreover, the presence of a microstructured substrate influenced neurite navigation, thus every 3D-microstructured area was covered with electrogenic cell compartments as soon as at 3 DIV. A plausible mechanism is that the 3D microstructures may function as anchors, where the neurons establish strong focal adhesion points. Although our configuration does not allow for high-controllability of neuron placement, the topography effect is enough to increase the yield of active electrodes in MEA experiments. Long-term recordings of rat cortical neurons revealed spontaneous activity in multiple 3D-microstructured microelectrodes. Most of the spike waveforms were negative biphasic spikes with amplitudes bigger than 100 µV. The 3D-microstructured microelectrodes seem to achieve enough physical coupling to detect subtler changes in the components of the signal in comparison to flat microelectrodes. Conclusion: We have developed, fabricated and tested gold 3D-microstructured MEA chips. They are composed of multiple islets of mushroom-shaped 3D microstructures that act as strong physical cues. This topographical feature causes topotaxis of cells, and increases the yield of MEA recordings. The electrophysiological capabilities of the chips were further confirmed by recordings of embryonic rat cortical neurons spontaneous activity. Our findings should help the MEA community moving closer to extracellular high-yield and high-fidelity recordings from mammalian neurons. Figure 1: a) Photo of the 3D-microstructured multielectrode chip. b) Microscopic image of the multiple 3D-microstructured microelectrodes. c) Confocal image of cortical neurons (3 days in vitro) on a single islet (3×3 array of 3D microstructures).

Figure 1
Image

Acknowledgements

The authors gratefully acknowledge Fundação para a Ciência e Tecnologia (FCT) of the Ministério da Educação e Ciência, Portugal, for funding (PTDC/CTM-NAN/3146/2014). JCM is a recipient of a FCT Ph.D. fellowship (PD/BD/135491/2018).

References

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Keywords: gold mushroom-shaped microelectrodes, microelectrode array, Topotaxis, neuron-electrode interface, Microfabrication techniques

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

Presentation Type: Poster Presentation

Topic: Microelectrode Array Technology

Citation: Mateus JC, Cerquido M, Lopes C, Leitão D, Cardoso S, Ventura J and Aguiar P (2019). A novel 3D-microstructured multielectrode array configuration for high-yield and high-fidelity electrophysiological recordings. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00028

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

* Correspondence: PhD. Paulo Aguiar, INEB - Instituto Nacional de Engenharia Biomédica, University of Porto, Porto, Portugal, pauloaguiar@ineb.up.pt