Insulator materials

The basic purpose of the insulator layer in an MEA is to isolate the cells and the cell culturing medium from the tracks connecting the electrodes to the contact pads.

This is very important because otherwise the tracks would also act as electrodes and it would be impossible to know whether the measured signal came from a cell on

top of an electrode or from cells located on top of the track. The thickness and dielectric constant of the insulator affect the capability of the layer to reduce the parasitic capacitance between the tracks and the cell culturing medium (Heuschkel et al. 2006) and thus ultimately the noise level of the MEA. As the insulator layer is typically the top layer of the MEA, another function which the insulator layer could have is promoting cell adhesion on the MEA surface (Blau 2013). Surprisingly, not much effort has been put into studying that function, at least not specifically from a purely MEA perspective. On the contrary, cell biologists usually apply their own cell-favoring or repelling coatings on the insulator layer. Polyethyleneimine (PEI), laminin or gelatin applied as a molecular layer are typical examples of such coatings.

PEI, for example, changes the charge on the glass surface from negative to positive (MCS 2011).

As the fabrication of normal MEAs strongly relies on traditional IC processes, it is obvious that two common silicon compound insulator materials, Si3N4 (sometimes just referred to as SiN or SiNx) (Berdondini et al. 2006; Buehler et al. 2016; Gabay et al. 2007; Kim and Nam 2015; Nisch et al. 1994) and SiO2 (Jing et al. 2009; Kim et al.

2013; Zhou et al. 2015) or some sandwich structure incorporating them both (Buitenweg et al. 1998; Eick et al. 2009; Xiang et al. 2007; Yeung et al. 2007) are also favored on MEAs. The most common of these is Si3N4. Despite its easy processing and decent insulation properties, Si3N4 has three drawbacks. Firstly, it is a relatively hard material compared to the naturalin vivo environment or the polystyrene favored in cell-culturing dishes. Therefore, Si3N4 is not an optimal surface for the cells. This causes problems in cell adhesion and forces the biologists to use additional coatings, as has already been mentioned. Secondly, another annoying characteristic of Si3N4, (albeit often glossed over by the manufacturers) is its poor tolerance of common cell-culturing mediums. For example, in a comprehensive study by Herrera Morales (2015), the Si3N4 degraded at about 1 μm/year in PBS at 37 °C, and in Publication III the 500 nm of Si3N4 500 had already disappeared in a couple of weeks in a cell culturing medium. This severely limits the MEA’s life time and usability for long term cell experiments. As a solution for that problem, Nam et. al (2006) suggested inserting a replaceable PDMS insulator sheet on the degraded Si3N4 layer. The third drawback is that, in practice the thickness of Si3N4 is limited to about 0.5-1 μm. This is not always considered sufficient for the reduction of parasitic capacitance (Heuschkel et al. 2006). With its somewhat lower relative permittivity and more cell-friendly hydrophilic nature, SiO2 should have benefits over Si3N4, but being

permeable to sodium ions, it is not a good choice as a sole insulator material. The SiO2-Si3N4-SiO2 sandwich structure combines the benefits of both materials. In addition, Schmitt et al. (2000) propose that the counteracting intrinsic stresses in the sandwiched layers improve the corrosion resistance of such structures compared with just one layer. Furthermore, in a layered structure it is also highly unlikely that there are equally positioned pinholes in all the layers throughout the structure.

However, the need for more deposition steps and the longer etching process of the sandwich structure have persuaded most designers to opt for just Si3N4 as the insulator material.

The most common alternatives to Si3N4 and SiO2 are SU-8 (Gawad et al. 2009;

Heuschkel 2001; Ren et al. 2015) and polyimide (Du et al. 2015; Novak and Wheeler 1986; Oka et al. 1999; Stett et al. 2005). There are also other options, such as parylene-C (Charkhkar et al. 2016; Tonomura et al. 2010), PDMS (Blau et al. 2009;

Gross 1979), silicone-based positive photoresist (Jimbo et al. 2003), spin-on-glass (SOG) (Morin et al. 2006), nanocrystalline diamond (Maybeck et al. 2014), and an acrylic resin used by Alpha MED on its MEAs. Excluding SOG and diamond, they are often justified by being more natural or more polystyrene-like surfaces than Si3N4, and as they are available in much higher thicknesses than Si3N4, they have the possibility of offering better electrical insulation properties. However, at least sometimes the real reason for choosing one of the above materials may well have been, once again, simply practical, process-related issues. For example, a photoresist SU-8, as well as some polyimide variants, can easily be patterned by photolithography, which saves at least one etching step in the MEA fabrication process. It is also possible to use laser pulses to make openings for electrodes on a PDMS surface without the need for any lithography or etching (Gross 1979).

However, polymers also have problems of their own. Depending on the material there might be issues related to their thermal and chemical compatibility, as has already been mentioned in the substrate section of this chapter. One more issue is the adhesion of PDMS structures on non-glass surfaces. Especially for SU-8 and acrylic resin, this is a well-known problem (Morin et al. 2006; Ren et al. 2015). Thus, particularly if reversible but still non-leaking bonding is needed, those materials are not suitable candidates for the insulator material.

In addition to the materials already mentioned above, there are several other insulator materials commonly used in IC processes, which may occasionally have also been applied on MEAs, such as Tetraethyl Orthosilicate, TEOS (Gaio et al.

2016), and Al2O3, TiO2, HfO2 and SiC. But as with any material, they all have issues of their own (Herrera Morales 2015) or perhaps it is just that nobody has put in enough effort on trying to sell the idea of some new material to the biologists. This may explain why those materials have not gained any notable popularity among the MEA community. As already mentioned, the major part of cell culturing is traditionally done on polystyrene dishes. Surprisingly, for a long time the only study about MEAs with polystyrene as an insulator layer was a short experiment by the author (Ryynänen et al. 2010), and it is only recently that Hammack et al. (2018) have published another report on the topic. The major fabrication challenge with polystyrene is how to make openings for the electrodes without damaging the polystyrene layer, as it has limited compatibility with the bakes and solvents included in the normal photolithography process.