Characterization Of Graphene-Coated Microelectrode Arrays For Recording And Stimulation Of Neuronal Cells
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1
University of Applied Sciences Aschaffenburg, BioMEMS Lab, Germany
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2
Technische Universität Darmstadt, Eduard-Zintl Institut für Anorganische und Physikalische Chemie, Germany
Motivation
In this study we investigate the benefits of graphene as microelectrode coating for recording and stimulation of neuronal cells. Due to its unique combination of characteristics graphene is a promising material for interfacing electrically excitable cells with electronic devices. Well-known properties include stability, high conductivity, biocompatibility as well as cell adhesion promotion [1]. For recording, graphene electrodes have already been successfully applied [2-4], but the question
whether graphene microelectrodes are also suitable for stimulation applications has not yet been addressed. To this end, we integrate graphene onto microelectrode arrays and evaluate the performance of the device regarding recording and stimulation of electrogenic cells. Characterization methods include Raman spectroscopy as well as electrochemical easurements. For biocompatibility studies, embryonic cortical rat neurons are cultured on the devices. Based on this, the usage of graphene microelectrodes for recording and safe extracellular stimulation is discussed.
Material and Methods
In order to modify the microelectrode array, several processing steps are required. First, graphene, grown by low-pressure chemical vapor deposition of methane on copper foil, is transferred onto a lithographically pre-patterned substrate using modified polymer-free chemical etching without common polymer coating. This guarantees high-quality few-layer graphene without polymer residues and a good electrical contact to the conductor paths. In a second step graphene microelectrodes are defined by a lift-off process and an insulating layer of SU-8 is deposited and structured in order to insulate the electrode connections. The resulting array consists of 60 electrodes with a diameter of 30 µm each and a distance between electrodes of 200 µm. As electrical conducting material gold and polycrystalline silicon (poly-Si) is used respectively.Raman spectroscopy (Horiba LabRAM HR8000, Lab Spec 5, Kyoto, Japan) confirms the successful transfer and lift-off process of graphene onto pre-patterned microelectrodes of different materials. For electrochemical characterization cyclic voltammetry, impedance spectroscopy and voltage transient measurements are performed using a potentiostat system (VersaSTAT 4, Princeton Applied Research, Princeton, USA). All measurements are conducted at room temperature in phosphate-buffered saline using an Ag/AgCl pellet (Multi Channel Systems, Reutlingen, Germany) as reference and platinum mesh as counter electrode.
Results
Graphene microelectrodes on gold are fabricated and electrochemically characterized. For comparison, the properties of commercially available titanium nitride microelectrodes (Multi Channel Systems MCS) and uncoated gold microelectrodes are also measured. In Figure 1a results of cyclic voltammetry measurements of these three microelectrode materials taken at a scan rate of 1 V/s are depicted. The gold electrode shows the highest hysteresis, but also distinct reaction peaks. For stimulation, these reactions are unwanted and a featureless voltammetry curve, as the other two materials exhibit, is preferable. Impedance spectrograms of the same microelectrodes are shown in Figure 1b. They reveal values of 61 kOhm for titanium nitride, 500 kOhm for plain gold and 950 kOhm for graphene on gold at 1 kHz. These values are all comparable to findings in the literature [2].Furthermore, cell culture tests display good biocompatibility of graphene electrodes for embryonic cortical rat neurons (Lonza Ltd, Basel, Switzerland). A graphene-coated gold microelectrode array with a cortical neuronal network is shown in Figure 2a. Figure 2b shows recorded spikes and proves recordings from the neuronal cells are possible.
Discussion
The comparatively lower impedance of titanium nitride is due to the microfold structure of the material which increases the electrochemical surface area. In contrast, the fabricated gold and graphene microelectrodes have a planar topography. Covering gold electrodes with graphene reduces significantly their electrochemical activity, but at the same time increases their impedance. This makes them more suitable for stimulation applications than for recording. However, despite high impedance, we have shown that recording is possible and spikes are clearly detectable.
Conclusion and Outlook
Graphene has the ability to improve the electrical interface between neuronal cells and electrodes used for recording and stimulation purposes. However, the properties of graphene on gold could still be improved. It has been shown for larger graphene electrodes that the underlying material strongly influences the electrochemical behavior of graphene [5]. Therefore, a suitable material needs to be identified. We believe that poly-Si is a promising candidate. Furthermore, stimulation electrodes have to meet special requirements regarding their electrochemical characteristics. Therefore, future experiments need to address long-term behavior such as the stability of the electrode material, adhesion to substrate and changes in electrochemical properties over time.
References:
[1] Park, Sung Young et al., Enhanced Differentiation of Human Neural Stem Cells into Neurons on Graphene, Advanced Materials, 23, 2011.
[2] Kuzum et al., Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging, Nature Communications, 5, 2014.
[3] Du et al., Graphene microelectrode arrays for neural activity detection, Journal Of Biological Physics, 2015.
[4] Park, Dong-Wook et al., Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications, Nature Communications, 5, 2014.
[5] Körbitzer et al., Graphene electrodes for stimulation of neuronal cells, 2D Materials, 3, 2016.
Figure Legend
Figure 1: Results of a) cyclic voltammetry measured with a scan rate of 1 V/s and b) impedance spectroscopy for gold, gold/graphene and titanium nitride microelectrodes.
Figure 2: a) Embryonic cortical rat neurons on gold/graphene microelectrode array, 10 DIV. b) Recording from neurons on a gold/graphene microelectrode array.
Acknowledgements
This work was funded by the BMBF Germany within the project Neurointerface (Förderkennzeichen 03FH061PX3).
Keywords:
Graphene,
Neuronal Cells,
impedance spectroscopy,
Electrochemical characterization,
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:
Körbitzer
B,
Krauß
P,
Schneider
J and
Thielemann
C
(2016). Characterization Of Graphene-Coated Microelectrode Arrays For Recording And Stimulation Of Neuronal Cells.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00013
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Berit Körbitzer, University of Applied Sciences Aschaffenburg, BioMEMS Lab, Aschaffenburg, Germany, berit.koerbitzer@h-ab.de