Dye delivery from PEDOT electrodes in vivo - a new way to reconstruct recording sites
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1
Albert-Ludwigs Universität Freiburg, Department of Microsystems Engineering and Cluster of Excellence BrainLinks-BrainTools, Germany
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2
Albert-Ludwigs Universität Freiburg, Department of Microsystems Engineering and Cluster of Excellence BrainLinks-BrainTools, Germany
Motivation
Neuronal diseases, such as Epilepsy and Parkinson’s disease, are commonly investigated in animal models by recording neurons extracellularly with microelectrode arrays, for example in the hippocampus, over long periods. Subsequent histology is used to correlate the recorded signals to the recording area or neuron type and the status of the neuronal network in this region. At the experimental end point, the probes are removed and the tissue sectioned to reconstruct the recording locations, a step conventionally based on tissue damage and glial scaring. The necessity for precise reconstruction of the recording locations to identify recorded neurons precludes using electrocoagulation as a marking method. To find the exact location of each single electrode, which is particularly difficult for microelectrode arrays on silicon shafts, we developed a new concept to identify the tissue juxtaposed to the electrodes. Coating the electrodes with conducting polymer makes it possible to load charged neurotracers into the polymeric network. The dye can be actively released from the polymer even after long term recordings by applying an electrochemical trigger. The released lipophilic and positively charged dye molecules label the cells in direct vicinity of the electrodes, which makes microscopic detection in the histological slice possible.
Material and Methods
Microelectrode arrays on chips, based on iridiumoxide (IrOx), were used to test the functionality of the dye delivery system in vitro. The processing steps were as follows: The conducting polymer poly (3, 4-ethylenedioxythiophene/polystyrenesulfonate) (PEDOT/PSS) was grown in a three electrode setup with constant current of 500 µA/cm2 vs. Ag/AgCl on the IrOx electrodes until a charge of 300 mC/cm2 was reached. The neurotracer 3,3’-Dilinoleyloxacarbocyanine perchlorate (FastDiO) was incorporated into the PEDOT/PSS polymer by reducing the PEDOT backbone (-0.45 V to 0.2 V vs. Ag/AgCl; 5 sweeps, scan rate 0.1 V/s ). The positively charged dye is thereby attracted and electrostatically bound to the polymer material. Release of dye was first quantified without cells in organic solvent (0.1 M KClO4/DMSO) using spectro-fluorometry. For neuroblastoma cell culture experiments, chips were sterilized in 70 % ethanol before cells were cultured on the chip to analyze the cellular dye uptake. Release of dye was driven by applying a positive potential to the PEDOT-film. Fig. 1a shows a schematic of the dye exchange process within the PEDOT system. Uptake of dye by cells took place within 24 hours after triggering release.
For perfect labelling precision, all areas except the electrode must be free from dye contami-nation. In a preliminary study, a Parylene C polymer layer was used as sacrificial layer (5 µm) before dye loading. After the loading process the polymer was peeled off.
Results
The proof of concept measurements in vitro show the possibility to controllably exchange FastDiO via reduction and oxidation of the PEDOT film. Fig.1b shows passive leakage and active release from one chip with actively incorporated dye and one chip which was only immersed into a dye solution. Dye release highly depended on the active triggering process of PEDOT as well as on the method used to load the dye. Confirming the concept, the released dye locally stained the neuroblastoma cells (Fig. 1c). A 5 µm Parylene C layer protected the substrate surfaces underneath from dye contaminations and could be peeled off after the PEDOT/dye processing step.
Discussion
The new dye delivery concept allows precise labelling of cells in vitro. Coating the recording electrodes with a dye exchanging conducting polymer provides a convenient and precise method for neuronal tracing. The precision of the dye loading process can be increased by introducing a patterned sacrificial Parylene C layer that is peeled off after the dye loading step. This approach could avoid the necessity of washing steps to remove dye adhering unspecifically outside the electrode area and accidental washout from the electrodes. Thereby the amount of remaining dye molecules in the polymer and the precision of labelling of recorded neurons is expected to be enhanced.
Conclusion
We successfully demonstrated the applicability of a PEDOT-based dye delivery system to label neuronal cells in the vicinity of an electrode in a cell culture-based test system. This technology could provide a precise and convenient way for neuronal tracing after the experimental end point of an in vivo study using multisite recordings with silicon electrode arrays.
Figure caption:
Fig. 1 a) Dye incorporation and release were controlled via electrochemical reduction and oxidation of the PEDOT film. b) Spectro-fluorometric detection of passive leakage and active release of dye from PEDOT electrodes that were either only immersed to a FastDiO solution (black curve) or had dye actively incorporated into the PEDOT matrix. For both versions passive leakage and electrochemically controlled release were measured. c) Neuroblastoma culture on (1) bare IrOx electrode, (2) PEDOT+dye electrode, actively triggered, and (3) passive leakage control. Blue dots mark DAPI-stained cellular nuclei, green colour indicates uptake of dye by the cells. Significant uptake was only observed in (2), i.e. when dye was actively released from the PEDOT layer.
Acknowledgements
This work was supported by the DFG within the Cluster of Excellence BrainLinks-BrainTools (EXC 1086) and the BMBF (FKZ 01GQ0830).
Keywords:
Conductive polymers,
Micro-Electrode Arrays,
dye delivery system
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:
Heizmann
S,
Holzhammer
T,
Kilias
A,
Egert
U,
Ruther
P and
Asplund
M
(2016). Dye delivery from PEDOT electrodes in vivo - a new way to reconstruct recording sites.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00132
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Stefanie Heizmann, Albert-Ludwigs Universität Freiburg, Department of Microsystems Engineering and Cluster of Excellence BrainLinks-BrainTools, Freiburg, Germany, stefanie.heizmann@imtek.uni-freiburg.de