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In Vitro And In Vivo Probes With Mushroom-shaped Microelectrodes - Tools For In-cell Electrophysiology

Peter D. Jones1, Clemens Barthold2, Meike Beer1, Claus Burkhardt2, Katja Gutöhrlein3, Gerhard Heusel2, Udo Kraushaar4, Pranoti Kshirsagar2, Manuel Martina2, Sebastian Roehler2, Ramona Samba1, Birgit Schroeppel2, Angelika Stumpf2, Simon Werner1 and Martin Stelzle1*
  • 1 Natural and Medical Sciences Institute at the University Tübingen, BioMEMS and Sensors, Germany
  • 2 Natural and Medical Sciences Institute at the University Tübingen, Microsystems / Nanotechnology, Germany
  • 3 Natural and Medical Sciences Institute at the University Tübingen, Microsystems / Nanotechnology, Germany
  • 4 Natural and Medical Sciences Institute at the University Tübingen, Electrophysiology, Germany

Motivation An ambitious goal for neuroscience and cardiac research is to apply microelectrode array (MEA) technology to simultaneously record from large cell populations with signal quality matching intracellular patch clamp recordings (Spira & Hai, 2013). Conventional MEAs record extracellular field action potentials, but cannot measure details such as subthreshold synaptic potentials. MEA-based cardiac electrophysiology struggles to resolve details of cardiac action potentials. Patch clamp techniques can resolve such details, but cannot record and stimulate for long times, and even skilled operators achieve low throughput with neural and cardiac cells. Inspired by the shape of dendritic spines, MEAs with mushroom-shaped microelectrodes have enabled ‘in-cell’ recordings of monophasic action potentials and subthreshold activity, based on enhanced coupling between cells and extracellular electrodes (Hai et al., 2009; Spira & Hai, 2013). Here, we present reliable methods to produce MEAs with micrometer-scale, three-dimensional, mushroom-shaped electrodes for in vitro and in vivo investigations towards high density in cell recordings. Materials and Methods Mushroom-shaped electrodes were integrated on in vitro and in vivo MEAs (Figure 1) by electroplating gold through micrometer-sized holes in the insulator and a sacrificial layer. In vitro MEAs on glass used silicon nitride (SiNx) as insulator and photoresist (AZ ECI 3027) as sacrificial layer. In vivo devices were fabricated on polyimide (PI) on glass handling substrates with PI as insulator and SiNx as sacrificial layer. Holes for mushrooms were produced by photolithography and plasma etching. Electrical leads were gold with titanium for adhesion on both sides. Electroplating was controlled to yield mushroom cap diameters below 2.5 µm. After electroplating, sacrificial layers were removed: photoresist by KOH solution and SiNx by CF4 plasma. Gold mushrooms were optionally coated with poly(3,4-ethylenedioxythiophene) (PEDOT) by electropolymerization (Gerwig et al., 2012). Before cell plating, in vitro MEAs were coated with polyethylene imine and either laminin or fibronectin. Cardiomyocytes derived from human induced pluripotent stem (hiPS) cells (Cellartis ChiPSC22) were cultivated for 6 days before recordings. Recordings were performed at 37 °C (MEA2100 amplifier, sampling 20 kHz, bandwidth 0.1–3000 Hz). Implantation of flexible in vivo probes was tested in agarose brain models similar to previously reported methods (Kozai and Kipke, 2009). Results Gold mushroom electrodes with cap diameters below 2.5 µm were reliably integrated on in vitro MEAs and 8 µm thick flexible in vivo probes (Figure 2). Gold mushroom surface areas of ~20 µm2 were confirmed by cyclic voltammetry in 0.5 M sulfuric acid (Trasatti & Petrii, 1992). These electrodes have an estimated impedance at 1 kHz (Z1 kHz) of 16 MΩ based on a specific capacitance of 0.5 pF/µm2 (Mirsky et al., 1997), although the system capacitance prevents measurement of this impedance. Conformal electrodeposition of PEDOT (Figure 2H) reduced impedance at 1 kHz to 700 kΩ while maintaining small electrode dimensions. Recordings from cardiomyocytes with gold mushroom electrodes (Figure 3) spontaneously showed monophasic action potentials with amplitudes of up to 1.3 mV, in contrast to similar recordings obtained only after electrical stimulation (Fendyur & Spira, 2012; Xie et al., 2012). We interpret these monophasic signals as indicating enhanced coupling between cells and electrodes. The recorded waveforms were attenuated from the intracellular potential, and further investigation is required to understand the mechanisms required to record monophasic signals; these points are discussed below. Flexible probes were implanted into agarose brain models by adhesive-free shuttles (Figure 4). Impedance measurements of PEDOT-coated mushrooms showed no damage due to implantation. Discussion Our processes reliably integrate mushroom electrodes with diameters below 2.5 µm on in vitro MEAs and flexible in vivo probes. Such electrodes should encourage engulfment by cells, thereby increasing seal resistance of cells on the electrodes; their shape and dimensions should enable suitable engulfment by mammalian neurons. For comparison, diameters of 3.5 µm would be unsuitable (Ojovan et al., 2015) and mushrooms are preferred over cylindrical electrodes (Santoro et al., 2014). A critical factor for in-cell recording is to reduce junctional membrane impedance. This has been achieved with Aplysia neurons by biochemical functionalization (Hai et al., 2010) or electroporation (Hai & Spira, 2012), and with rodent cardiomyocytes by electroporation on mushroom electrodes (Fendyur & Spira, 2012) or nanowire electrodes (Xie et al., 2012). Consistent with work pioneered by the lab of Micha Spira, we recorded large signals from cardiomyocytes despite using small, high impedance electrodes. Surprisingly, we observed monophasic action potentials without special consideration to reduce membrane impedance. No electroporation was needed to record monophasic signals, in contrast to other reports (Fendyur & Spira, 2012; Xie et al., 2012). The mechanisms of engulfment and reduction of junctional membrane impedance which lead to these results must be further investigated. The recorded monophasic action potentials (Figure 3) were attenuated and distorted versus the intracellular potential. Attenuation depends on electrical coupling and seal resistance (Ojovan et al., 2015). Additionally, capacitance of insulated conducting leads was 10–50 pF, which is large when compared to the gold mushroom electrodes (estimated Z1 kHz = 16 MΩ, capacitance of 8 pF). The insulation may act as a low pass filter, shunting high frequency signals to the electrolyte to produce the observed waveform. PEDOT coatings should minimize this problem but the interaction between cells and PEDOT mushrooms has not yet been investigated. Investigation of mushroom electrodes on in vivo probes is still required. Engulfment could help to achieve stable single unit recordings. The use of flexible substrates will be critical to minimize motion between electrodes and cells. Micromotions of tens of micrometers, many times larger than our electrodes themselves, have been measured in rat cortex (Gilletti & Muthuswamy, 2006). PEDOT coatings with biochemical functionalization can encourage recognition by specific cells (Zhu et al., 2014). Similar functionalization could encourage interactions such as engulfment by specific cell types and recruitment of ion channels to reduce membrane impedance. Conclusion We have developed reliable methods to produce both in vitro and in vivo MEAs with integrated mushroom-shaped microelectrodes as tools to study in-cell recording of electrical activity. Recordings of monophasic action potentials from cardiomyocytes support the possibility of MEA-based in-cell recording. These tools can be applied to reveal and improve the mechanisms by which cells recognize and interact with 3D mushroom microelectrodes, moving us closer to the goal of stable, patch-clamp-quality signals from large cell populations with MEA technology. Acknowledgements This work was funded by the European Commission (FP7 Information and Communication Technologies, Future Emerging Technology programme, BRAINLEAP grant n. 306502). Helpful discussions with Stefano Ferraina, Luc Gentet, Michele Giugliano, and Micha Spira are acknowledged. We thank Sandra Buckenmaier for preparing cell cultures. References Fendyur, A., and Spira, M. E. (2012). Toward on-chip, in-cell recordings from cultured cardiomyocytes by arrays of gold mushroom-shaped microelectrodes. Front. Neuroeng. 5, 21. doi:10.3389/fneng.2012.00021. Gerwig, R., Fuchsberger, K., Schroeppel, B., Link, G. S., Heusel, G., Kraushaar, U., et al. (2012). PEDOT-CNT Composite Microelectrodes for Recording and Electrostimulation Applications: Fabrication, Morphology, and Electrical Properties. Front. Neuroeng. 5, 8. doi:10.3389/fneng.2012.00008. Gilletti, A., and Muthuswamy, J. (2006). Brain micromotion around implants in the rodent somatosensory cortex. J. Neural Eng. 3, 189–95. doi:10.1088/1741-2560/3/3/001. Hai, A., Dormann, A., Shappir, J., Yitzchaik, S., Bartic, C., Borghs, G., et al. (2009). Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices. J. R. Soc. Interface 6, 1153–65. doi:10.1098/rsif.2009.0087. Hai, A., Shappir, J., and Spira, M. E. (2010). Long-term, multisite, parallel, in-cell recording and stimulation by an array of extracellular microelectrodes. J. Neurophysiol. 104, 559–68. doi:10.1152/jn.00265.2010. Hai, A., and Spira, M. E. (2012). On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes. Lab Chip 12, 2865. doi:10.1039/c2lc40091j. Kozai, T. D. Y., and Kipke, D. R. (2009). Insertion shuttle with carboxyl terminated self-assembled monolayer coatings for implanting flexible polymer neural probes in the brain. J. Neurosci. Methods 184, 199–205. doi:10.1016/j.jneumeth.2009.08.002. Mirsky, V. M., Riepl, M., and Wolfbeis, O. S. (1997). Capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol films on gold electrodes. Biosens. Bioelectron. 12, 977–989. doi:10.1016/S0956-5663(97)00053-5. Ojovan, S. M., Rabieh, N., Shmoel, N., Erez, H., Maydan, E., Cohen, A., et al. (2015). A feasibility study of multi-site, intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes. Sci. Rep. 5, 14100. doi:10.1038/srep14100. Santoro, F., Dasgupta, S., Schnitker, J., Auth, T., Neumann, E., Panaitov, G., et al. (2014). Interfacing Electrogenic Cells with 3D Nanoelectrodes: Position, Shape, and Size Matter. ACS Nano 8, 6713–6723. doi:10.1021/nn500393p. Spira, M. E., and Hai, A. (2013). Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 8, 83–94. doi:10.1038/nnano.2012.265. Trasatti, S., and Petrii, O. A. (1992). Real surface area measurements in electrochemistry. J. Electroanal. Chem. 327, 353–376. doi:10.1016/0022-0728(92)80162-W. Xie, C., Lin, Z., Hanson, L., Cui, Y., and Cui, B. (2012). Intracellular recording of action potentials by nanopillar electroporation. Nat. Nanotechnol. 7, 185–190. doi:10.1038/nnano.2012.8. Zhu, B., Luo, S.-C., Zhao, H., Lin, H.-A., Sekine, J., Nakao, A., et al. (2014). Large enhancement in neurite outgrowth on a cell membrane-mimicking conducting polymer. Nat. Commun. 5, 4523. doi:10.1038/ncomms5523. Figure Legend Figure 1: Fabrication process for mushroom MEAs. Holes in the sacrificial layer and insulator were produced by photolithography and plasma etching to define the mushroom stalks. Electroplating through and beyond the holes produced mushroom-shaped gold electrodes, which were free-standing after removal of the sacrificial layer. In vitro MEAs were produced on glass substrates with silicon nitride insulators and photoresist as the sacrificial layer. In vivo MEAs were produced on polyimide substrates (supported by glass, not illustrated). The insulator was also polyimide, and the sacrificial layer was silicon nitride. Figure 2: Mushroom microelectrode arrays. A–D: Photos of MEAs and microscopic images of mushroom electrodes on (A, B) in vitro and (C, D) in vivo MEAs. The texture in (D) is the surface supporting the transparent device. The offset of the electrodes from the center was intentional. E–H: Scanning electron images of (E) an intact mushroom electrode and cross-sections of (F) an in vitro mushroom electrode, (G) an in vivo mushroom electrode, and (H) a mushroom electrode coated with PEDOT. Cross-sections were prepared by focused ion beam milling. Mushroom cap diameters were 2.0–2.2 µm (F–G). The gold mushroom in H had a diameter of 2.8 µm before conformal deposition of approximately 150 nm PEDOT. The scale bar in E applies to E–H. Figure 3: Cardiac action potentials. In comparison to intracellular action potentials recorded by patch clamp (A) and extracellular field potentials recorded by planar microelectrodes (B), the signal recorded with mushroom microelectrodes appears similar to a filtered and attenuated intracellular action potential. Similar signals were recorded on several electrodes; selected traces from the same experiment are shown in D. Figure 4: Implantation of flexible probes in agarose. Removable shuttles (A) were produced with hydrophilic self-assembled monolayer surfaces. Flexible probes were easily aligned on the shuttles (B) with a drop of ethanol. After evaporation, the adhesion with the shuttle was sufficient to lift the weight of the probe. The shuttles were used to implant probes into agarose gel (C). Wicking of water between the shuttle and probe allowed removal of the shuttle (D).

Figure 1
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Keywords: PEDOT, cardiomyocytes, microfabrication, in vivo probes, in vitro MEA, mushroom electrodes

Conference: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.

Presentation Type: oral

Topic: MEA Meeting 2016

Citation: Jones PD, Barthold C, Beer M, Burkhardt C, Gutöhrlein K, Heusel G, Kraushaar U, Kshirsagar P, Martina M, Roehler S, Samba R, Schroeppel B, Stumpf A, Werner S and Stelzle M (2016). In Vitro And In Vivo Probes With Mushroom-shaped Microelectrodes - Tools For In-cell Electrophysiology. Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00076

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Received: 22 Jun 2016; Published Online: 24 Jun 2016.

* Correspondence: Dr. Martin Stelzle, Natural and Medical Sciences Institute at the University Tübingen, BioMEMS and Sensors, Reutlingen, Germany, martin.stelzle@nmi.de