Development Of A Microelectrode Array With Semitransparent Carbon Nanotube Electrodes for In-Vitro Applications And Optogenetics
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1
Natural and Medical Sciences Institute at the University Tübingen, Microsystems and Nanotechnology, Germany
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2
Natural and Medical Sciences Institute at the University Tübingen, Electrophysiology, Germany
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3
University of Tübingen, Plasmonic Nanostructures, Germany
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4
Natural and Medical Sciences Institute at the University Tübingen, Technical Engineering & Biophysics, Germany
Motivation
Commonly microelectrode materials are selected in consideration of their electrical interface properties, chemical stability and biocompatibility. With these points in mind, there is a small set of standard microelectrode materials available, namely gold, platinum and titanium nitride. Although there are some carbon materials, e.g. PEDOT:PSS, graphite and diamond, none of these offer a reasonable optical transparency.
However, in several applications electrode transparency is an integral necessity. For example culture chambers (wells), microfluidic channels and fluid in-/outlets are built on top of MEA substrates and thus, optical imaging from the top with high resolution is impractical. Here, transparent electrodes allow the observation of cell cultures and tissue from the bottom of the well [1].
Furthermore, in the field of optogenetics transparent electrodes enable light stimulation of the cells in the very contact with the electrode through the substrate, as demonstrated by the CLEAR device [2]. There, an all transparent microelectrode array was fabricated out of graphene and successfully utilized.
Here, we present a transparent microelectrode array for in-vitro applications, which is based on a thin layer of carbon nanotubes as electrode interface.
Materials and Methods
A MEA substrate was fabricated utilizing indium-tin-oxide conducting paths and silicon oxide insulator with established cleanroom processes. After opening the electrodes and connection pads by reactive ion etching an ultra-thin catalyst layer of nickel (around 1 nm to 5 nm) is deposited by PVD. After lift-off, thermal CNT deposition is performed in a mixed ammonia-acetylene atmosphere [3]. Substrate transparency after different process steps is measured using an UV-vis spectrometer on reference substrates and impedance measurements in PBS are performed using a MCS MEA2100 system and a platinum reference electrode.
For cell culture experiments, ventricular myocytes were isolated from 14 days old embryonic chick hearts as previously described [4]. Field action potentials (fAP) of spontaneously beating cells were recorded after five days in culture at 37°C, 5% CO2.
Results and Discussion
Following the described process, MEAs with transparent microelectrodes were fabricated. It was observed, that the thickness of the nickel catalyst layer and the CNT height are the critical factors which determine the morphology and thus the optical transparency of the electrode layer. Optimal parameters were found to be 5 mins of pretreatment with 200 sccm ammonia at 3 mbar at 550°C followed by CNT synthesis for 5 mins in 200 sccm ammonia / 200 sccm acetylene at 5 mbar at 550°C. The total transparency of the final CNT electrodes was measured to be around 40% in the visible range, see Figure 1. The impedance of these MEAs with electrode diameters of 30 µm was measured to be in the range of 50 kO to 100 kO at 1 kHz in PBS buffer. SEM images showed a layer of CNTs with heights of around 200 nm.
Cultivation of ventricular myocytes on the fabricated MEAs was successful. fAPs could be measured with a good signal-to-noise ratio. The basic noise level of the electrodes was approximately ± 10 µV while the biological signals reached peak-to-peak amplitudes of up to 2,5 mV.
The advantage of these MEAs in comparison to the standard MEAs is the transparency of the electrodes which allows visualizing the plated cells directly on the respective electrodes, see Figure 2.
Conclusions and Outlook
We developed a process to fabricate transparent microelectrodes based on carbon nanotubes. These MEAs were successfully used to measure field action potentials from ventricular myocytes and allowed optical cell visualization from the bottom through the electrode.
We will further improve the transparency and electrical properties of these MEAs and investigate other carbon materials, e.g. graphene, for their feasibility as transparent electrode material.
Experiments to test the applicability of the fabricated MEAs for optogenetic measurements and for calcium imaging are planned.
Figure 1: Measured transparency on a reference substrate after different process steps.
Figure 2: SEM image of a CNT microelectrode (left) and optical image of cardio cells through the electrodes and conducting lines (right). An electrode and its conducting line is dotted in red for visualization. Signals in the right image are 1.5 s long and peak-to-peak is cropped to 200 µV to show repolarization peak.
References:
[1] K.Y. Kwon et al., IEEE Trans Biomed Circuits Syst. 7, 593–600 (2013)
[2] D.-W. Park et al., Nature Communications 5, 5258 (2014)
[3] K. Schneider et al., 12th Conference on Nanotechnology (IEEE-NANO), 1–5 (2012)
[4] P. Connolly et al., Biosensors and Bioelectronics 5(3), 223–234 (1990)
Acknowledgements
This work is supported by the Federal Ministry of Education and Research (BMBF), PTKA-PFT, grant 02P14Z200 (M-ERA NET project C4HEALTH).
Keywords:
Carbon nanotubes,
optogenetics,
Transparent electrodes,
C4HEALTH
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,
Besca
K,
Kshirsagar
P,
Buckenmaier
S,
Schneider
K,
Fleischer
M,
Kern
D,
Kraushaar
U,
Stett
A and
Burkhardt
C
(2016). Development Of A Microelectrode Array With Semitransparent Carbon Nanotube Electrodes for In-Vitro Applications And Optogenetics.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00085
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Claus Burkhardt, Natural and Medical Sciences Institute at the University Tübingen, Microsystems and Nanotechnology, Reutlingen, Germany, Claus.Burkhardt@nmi.de