Evaluation of in-cell like recordings using different types of 3D electrodes
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1
Natural and Medical Sciences Institute, Germany
Motivation
The main goal of this work was to explore in-cell like recordings obtained upon electroporation using different types of three-dimensional electrodes. The development of three dimensional (3D) micro structured electrodes has been shown to improve the electrical contact between cell and electrode [1] leading to signal shapes which resemble intracellular action potentials. 3D nano - and micro electrodes have been interfaced to cardiomyocytes to obtain recordings resembling intracellular action potentials by applying a relatively strong electrical stimulus [2-4]. This phenomenon is believed to be mostly attributed to electroporation of the cell membrane which transiently increases the leakage membrane conductance. In this work we developed different 3D electrodes shapes (mushroom-shaped, hollow pillar, pillar) processed either individually or in groups (3, 11, 25) per electrode. We investigated the efficacy for obtaining in-cell like signals on cardiomyocytes cell cultures.
Material and methods For the electrodes fabrication, Silicon nitride is applied as an insulator, covered with photoresist by means of spin coating. Upon UV exposure through a mask and subsequent plasma etching, the exposed areas reaches the gold underneath, thereby defining the electrode pads. The three dimensional structures are electroplated using a potentiostat using different deposition volume and current. Subsequently the electrodes are coated with poly(3,4-ethylenedioxythiophene) (PEDOT) by electropolymerization. For electrochemical characterization, cyclic voltammetry and impedance spectroscopy measurements are performed. Cardiomyocytes of chicken embryo ventricles were cultivated on the MEAs over an active area of 2mm2. After 5 days of incubation electrical stimulation and recording was performed. Extracellular recordings as well as electrical stimulation were performed using a 60-channel amplifier (MEA-2100-System, Multichannel System, Reutlingen, Germany) at 37°C at a sampling rate of 10 kHz. Data was hardware filtered using a bandpass of 0.1 Hz – 3.5 kHz.
Stimulation and data analysis
Stimulation electrodes were selected after identification of reliable spontaneous signals on these electrodes. Electrical stimuli ranged between 100-1600 mV at a frequency between 100-250 Hz and the stimulus burst duration was between 120-300 ms. Within this parameter range we investigated how long in-cell signals persisted, the threshold voltage and the repeatability over minutes and hours. The persistence of the in-cell state was associated with the time constant of the decreasing signal amplitude. For comparison we repeated the experiments on planar MEA, and MEAs with 3D tips (60-3DMEA200/12iR-Ti, Multi Channel Systems MCS).
Results
We successfully processed solid and hollow pillars as well as mushroom-shaped electrodes with impedances ranging between 100-600 kΩ. On these electrodes we recorded extracellular action potential from cardiomyocytes. The electroporation protocols successfully evoked in-cell like signals on all 3D electrodes. On average in-cell like recordings lasted 65 seconds (n = 44 cells). Electroporation could be repeated onto the same cell over several hours and occasionally days. The shape of the in-cell like signal was similar to an intracellular action potential obtained in a control experiment using a patch-electrode.
Conclusion
We showed that reliable in-cell like recordings can be obtained with different 3D electrode shapes. Ongoing experiments investigate to what degree the shape of the 3D electrode and their number relates to the observed signal shape.
Figure Legend
(A) Electrode comprising three pillar-shaped electrodes (B) Electrode comprising one single gold mushroom-shaped electrode (C) Spontaneous extracellular recordings on a 3D electrode prior to electroporation (D) In-cell like recording upon application of voltage pulses.
Acknowledgements
We thank Pranoti Kshirsagar for assistance with the electron microscope. This project was funded in part by the German Ministry of Education and Research (BMBF, 031L0059A)
References
[1] Micha E Spira and Aviad Hai. "Multi-electrode array technologies for neuroscience and cardiology". Nature nanotechnology 8.2 (2013), p. 83.
[2] Ziliang Carter Lin et al. "Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease". Microsystems & Nanoengineering 3 (2017), p. 16080.
[3] Dries Braeken et al. “Open-cell recording of action potentials using active electrode arrays”. Lab on a chip 12.21 (2012), pp. 4397-4402.
[4] Aviad Hai and Micha E Spira. "On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes". Lab on a Chip 12.16 (2012), pp. 2865-2873.
Keywords:
3d-microstructured electrodes,
Electroporation,
In-cell recordings,
Cardiomyocytes cultures,
mushroom-shaped electrodes,
MEA - Multi-electrodes arrays
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:
Del Torre
L,
Jones
PD,
Buckenmaier
S,
Kraushaar
U and
Zeck
G
(2019). Evaluation of in-cell like recordings using different types of 3D electrodes.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00062
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Received:
16 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mr. Luca Del Torre, Natural and Medical Sciences Institute, Reutlingen, Germany, luca.deltorre9@gmail.com