Ion Beam Assisted E-Beam Deposited TiN Microelectrodes—Applied to Neuronal Cell Culture Medium Evaluation

Abstract

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in in vitro electrophysiological studies. Here, we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover, the produced IBAD TiN microelectrodes were characterized by impedance, charge transfer capacity (CTC) and noise measurements for electrical properties, AFM and SEM for topological imaging, and EDS for material composition. The impedance (at 1 kHz) of brand new 30 μm IBAD TiN microelectrodes was found to be double but still below 100 kΩ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary, the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm2) also outperformed the sputtered counterparts (2.0 mC/cm2). To verify the suitability of IBAD TiN microelectrodes for cell measurements, human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However, BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1–2 weeks. In addition, BPH was found to corrode the Si3N4 insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements.

Accelerated neuronal and synaptic maturation by BrainPhys medium increases Aβ secretion and alters Aβ peptide ratios from iPSC-derived cortical neurons

“…Next, we used a multi-electrode array (MEA) system to analyze the effects of BrainPhys culturing on spontaneous activity. Although the neurons cultured in BrainPhys medium detached from the electrodes in MEA plates during long-term culturing, as was previously observed 39 , switching to BrainPhys for six to ten days was enough to increase both the number of spikes and the number of population bursts in mature neurons differentiated according to Shi et al 12 . Taken together, our findings are in line with earlier findings 15 and support the observation of increased neuronal networks and synaptic activity.…”

Microelectrode Array With Transparent ALD TiN Electrodes

“…Openings for the electrodes and the contact pads were dry etched with SF 6 + O 2 RIE process (Williams et al, 2003). Two MEAs of each ALD TiN thicknesses were fabricated, and for another of each thickness an additional 400 nm of TiN coating was deposited on the contact pads and large ground electrodes with IBAD technique (Ryynänen et al, 2018b) to enhance the mechanical durability.…”

“…Such small electrodes have rather high impedance and noise levels due to small contact area with the cells and culturing medium. In order to decrease noise and impedance, the active surface area of the electrode can be increased by using columnar or porous electrode materials like titanium nitride (TiN) (Egert et al, 1998; Ryynänen et al, 2018b) or platinum black (PtB) (Thomas et al, 1972; Pine, 1980; Oka et al, 1999). However, in addition to electrical measurements, it is often necessary to image and observe the cells growing or fixed on the MEA.…”

“…However, usually this means that the film thickness is at maximum only a few tens of nanometers and at that low thicknesses it is not at all self-evident whether the film is fully continuous and homogenous. In sputtering (Egert et al, 1998) and ion beam assisted e-beam deposition (IBAD) (Ryynänen et al, 2018b) which so far have been used for depositing TiN electrodes, the thin film formation is somewhat flocculated. On the contrary, in atomic layer deposition (ALD), where the precursors are dosed one-by-one and self-terminating surface reactions take place, films are grown on atomic or at least molecular layer-by-layer manner, the film can be expected to be uniform already at sub nanometer thicknesses (Sintonen et al, 2014).…”

Transparent Microelectrode Arrays Fabricated by Ion Beam Assisted Deposition for Neuronal Cell In Vitro Recordings

“…Sputter deposition is the standard method for depositing both ITO [21,22] and TiN [13] thin films. However, we have successfully shown that ion beam assisted electron beam deposition (IBAD) can also be used as a deposition method in fabrication of opaque TiN electrodes [23]. In this paper, IBAD TiN has been applied not only as an opaque layer, but also as a very thin transparent top layer for the ITO electrodes.…”