Batch Fabrication of Three-Dimensional Microelectrode Arrays For The In-Vitro Investigation Of Tissue Slices And 3D Cell Cultures
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1
NMI Natural and Medical Sciences Institute at the University Tubingen, Microsystems / Nanotechnology, Germany
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2
NMI Naturwissenschaftliches und Medizinisches Institut, Microsystems / Nanotechnology, Germany
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3
NMI Natural and Medical Sciences Institute at the University Tubingen, Technical Engineering & Biophysics, Germany
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4
NMI Naturwissenschaftliches und Medizinisches Institut, Neurochip Research, Germany
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5
NMI at the University Tuebingen, Neurochip Research, Germany
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6
NMI Natural and Medical Sciences Institute at the University Tubingen, Microsystems / Nanotechnology, Germany
Motivation
Three dimensional microelectrode arrays pose a valuable tool for the in-vitro investigation of acute tissue slices [1,2]. The reason for this is a dead-cell layer, which occurs due to the preparation of tissue slices and dampens the electrical signal when using planar electrodes. 3D MEAs can overcome the dead-cell layer by penetrating the tissue and moving the active electrode array in direct proximity to the living cells.
Furthermore, three dimensional cell cultures become an increasingly hot topic and are subject of more and more investigations [3,4]. Here, a 3D MEA can be applied to measure signals, preferable in different defined heights from the MEA surface. A microelectrode array combining planar and 3D electrodes, with heights ranging from some ten micrometers to a hundred micrometer, can be a valuable tool to progress in this field of research.
Here, we present a batch fabrication process, which allows to combine planar electrodes and 3D electrodes with tunable geometry.
Material and Methods
A process scheme of the fabrication is depicted in Figure 1. First, a silicon-on-glass wafer (consisting of a 50 µm thick silicon layer bonded to a thick glass wafer) is coated with a 50 nm thick silicon nitride layer. The silicon nitride layer is structured by optical lithography into square masks, where the electrode tips will be located. The mask size correlates to the later tip height. After anisotropic etching in potassium hydroxide, the majority of the silicon is removed, yielding a transparent substrate with 3D silicon structures. From here, structuring into a MEA is done by standard processes. During the final step, namely the deposition of the electrode material, height and size of the active electrode area is determined.
Results and Discussion
Following the developed process, an electrode array with 50 µm high tips is fabricated. Figure 2 shows SEM images of the finished 3D electrodes. The active electrode area, consisting of a fractal titanium nitride layer is located at the upper 20 µm, respectively 15 µm. The aspect ratio (tip height / base diameter) of the structures is measured to 2 and given by the silicon crystal orientation and mask shape. The apex angle is measured to 28°, with a tip radius of 1 µm. This geometry should be well suited for tissue penetration.
The developed process possess several advantages over already reported ways to fabricate 3D MEAs, e.g. the isotropic wet etching of glass or the dry etching of SU-8 resist for structure fabrication. Structures fabricated using a silicon-on-glass wafer are completely chemically inert in organic solvents (which are used in lithography processes), mechanically stable during fabrication as well as in application and provide an aspect ratio which cannot be achieved by isotropic wet etching.
Conclusions and Outlook
We developed a stable and batch process compatible way to fabricate MEAs with 3D electrodes. Although the electrode aspect ratio is fixed, tip height, tip distance and the size of the active electrode area can be tuned to one’s needs. The fabrication of these MEAs utilizes parallel processing and allows to produce these substrate in high numbers to affordable prices. Electrophysiological measurements with these MEAs are currently conducted.
In the future, tip geometry and aspect ratio can be further tuned by adapting the mask shape. With proper mask geometry and orientation, triangular tips with apex angles below 25° (known from AFM probes) can be fabricated. It might be possible to fabricate tips with heights of 100 µm and more by using a silicon-on-glass wafer with a thicker silicon layer.
Figure Legend
Figure 1: Process scheme for the fabrication of an electrode array, combining planar electrodes and 3D electrodes of different heights.
Figure 2: SEM images of 50 µm high tips with different sized active electrode areas (red).
References:
[1] M. O. Heuschkel et al., Journal of Neuroscience Methods 114, 135-148 (2002)
[2] S. Röhler et al., Microelectronic Engineering 98, 453-457 (2012)
[3] Y. Torisawa et al., Biomaterials 26, 2165-2172 (2015)
[4] M. Jang et al., Biomicrofluidics 9, 034113 (2015)
Acknowledgements
Funding of this work by the German ministry of education and research (BMBF) within the project DREPHOS (031L0059A) is kindly acknowledged.
Keywords:
3D cell culture,
acute tissue slices,
MEA fabrication,
3D MEA
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Martina
M,
Klaus
S,
Stett
A,
Herrmann
T,
Zeck
G and
Burkhardt
C
(2016). Batch Fabrication of Three-Dimensional Microelectrode Arrays For The In-Vitro Investigation Of Tissue Slices And 3D Cell Cultures.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00133
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Claus Burkhardt, NMI Natural and Medical Sciences Institute at the University Tubingen, Microsystems / Nanotechnology, Reutlingen, Germany, Claus.Burkhardt@nmi.de