Graphene – Coated Microelectrode Arrays
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1
Universidade Estadual de Campinas, Faculty of Electrical and Computer Engineering, Brazil
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2
Centro de Tecnologia da Informação Renato Archer (CTI), Brazil
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3
George Mason University, Electrical and Computer Engineering Department, United States
Introduction
This work aims to present our version of graphene-based Microelectrode Arrays (MEAs) for use in recording and stimulation of neuronal cell cultures. On glass substrate, our devices are composed of SU-8 insulation and titanium nitride (TiN) electrodes coated with graphene, which combines several interesting features such as low noise, excellent electrical conductivity, good charge injection capacity, biocompatibility, and transparency [1].
We characterized the fabricated arrays using Raman spectroscopy, impedance spectroscopy and cyclic voltammetry. Biocompatibility tests were performed using dorsal root ganglion (DRG) neuronal cultures from Wistar rats. We show here results from tests in and it is biocompatible and adequate to measure extracellular potentials.
Materials and Methods:
MEA design is compatible with the commercially available Multichannel System Arrays (see Figure 1) and consist of 60 TiN/graphene microelectrodes (30 µm in diameter with inter-electrode distance of 200 µm), with an insulating layer to protect the tracks made of 2 µm thick SU – 8 through optical lithography process.
We have developed a process flow to enable the use of graphene on TiN as microelectrode material using silicon-based lithography adapted to glass. Graphene used here was grown by Chemical Vapor Deposition (CVD) on Copper foil and it was transferred to the active MEA surface over a patterned region by optical lithography. Its removal from undesired regions (outside the electrodes) is performed by oxygen plasma etching.
Raman spectroscopy (Renishaw spectrometer with excitation wavelength laser of 488 nm) from graphene shows a satisfactory graphene transference process with an acceptable defect density onto TiN microelectrodes. For electrochemical characterization both cyclic voltammetry and impedance spectroscopy were performed using phosphate – buffered saline (PBS) 10 mM, with platinum wire as counter electrode and Ag/AgCl electrode as reference electrode. Finally, biocompatibility test was performed using DRG culture from Wistar rats kept on the MEA surface for over one week, with no appreciable difference in growth between graphene-based MEAs and TiN controls.
Results:
Experimental analysis shows that our MEAs yield performance comparable to commercially available MEAs. Characterization of the electrodes was performed through cyclic voltammetry and impedance spectroscopy, with better results than those previously reported in the literature. Figure 2 shows cyclic voltammetry curves obtained for manufactured MEA. The most important result obtained from this measurement is Charge Injection Capacity (CIC), which is the maximum injected charge without exceeding the reduction water potential at -0,6 V [1]. The value obtained for our device is 0.5 mC.cm-², for a potential window of -1 V to 1V, while commercial MEAs with TiN electrodes with same dimensio) exhibit CIC of 0.16 mC.cm-² [2]. With respect to Impedance Spectroscopy, the crucial information is the impedance modulus at 1 kHz. If this value is within the range of 1 kΩ to 1 MΩ, at 1 kHz, decaying as the frequency increases, then the microelectrode is considered functional. When compared to literature, our MEA showed good results. Impedance average at 1 kHz for eight microelectrodes of our MEA is 50 kΩ, which is compatible with the levels presented by standard commercial MEAs from MultiChannel Systems, which can exhibit values between 30 – 400 kΩ [2]. Additionally, Raman spectra (Renishaw spectrometer with excitation wavelength laser of 488 nm) from our microelectrodes reveals approximately the same position of Raman typical peaks for graphene: G (~ 1580 cm-¹) and one Lorentzian profile of 2D band (~ 2750 cm-¹). Considering there are no other prominent peaks (D, at 1350 cm-¹, and G’, at 1620 cm-¹) it is possible to affirm that there is low density of defects and impurities in the graphene used here. Additionally, 2D is sharp and symmetrical with full width at half maximum (FWHM) close to 40 cm-¹ with the intensity ratio of G and 2D peaks approximately equal to 0.3, which confirms the expectation of monolayer [1]. Therefore, this measurement shows a satisfactory graphene transference process with an acceptable defect density.
Finally, biocompatibility test was performed, and it is an extremely important test since it allows evaluating the suitability of the device to be used for biological measurements. After 216 hours in culture, there was adhesion of a large number of neurons and growth of non - neuronal cells (glia) on MEA surface. Therefore, our device is biocompatible and adequate to measure cellular potentials.
Conclusion:
Since graphene is a promising conductive material it was chosen as coating for our microelectrodes. It has been shown that our devices exhibited reasonable response both compared to TiN microelectrodes and commercial MEAs. We conclude that our MEAs have low impedance, good charge injection capacity and are biocompatible and suitable for measuring cellular potentials and for stimulation. We intend to use these graphene electrodes to design novel sensing and biosensing arrays for multiple molecule detection in neuronal cultures with dopaminergic neurons and glial cells.
Acknowledgements
This work was performed in part at the Center for Semiconductor Components and Nanotechnologies, a member of SisNano (CNPq process 402299/2013-2). Thus, the authors would like to acknowledge the financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazilian foundation and from INCT/ CNPq – NAMITEC. We are also grateful to the Instituto de Física ‘Gleb Wataghin’ (IFGW), in State University of Campinas, for the manufacture of glass rings.
References
[1] B. Koerbitzer, P. Krauss, C, Nick, S. Yadav, J. J. Schneider, and C. Thielemann, Graphene electrodes for stimulation of neuronal cells, 2D Mater., vol. 3, no. 2, pp. 24004 (2016).
[2] M. Systems, “Microelectrode Array (MEA) Manual,” 131 (2017).
Keywords:
MEA,
Graphene,
Titanium nitride,
cell culture,
characterization
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Microelectrode Array Technology
Citation:
Gomes
VP,
Panepucci
RR,
Swart
JW and
Peixoto
N
(2019). Graphene – Coated Microelectrode Arrays.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00113
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Ms. Vanessa P Gomes, Universidade Estadual de Campinas, Faculty of Electrical and Computer Engineering, Campinas, São Paulo, 13083852, Brazil, pgomes.vanessa@gmail.com