Optimising Recording & Stimulation Performance of Neuronal Microelectrode Arrays through Macroporous Conducting Polymer Modification

Abstract

Microelectrodes are commonly used to interface with electrically active cells, such as neurons (typically as an array of microelectrodes) allowing for their monitoring (recording) and manipulation (stimulation). Recent trends towards smaller electrodes for improved spatial resolution have revealed limitations of conventional planar microelectrode materials, such as gold and platinum, due to the subsequent reduction in electrochemical surface area. Strategies utilised to increase electrochemical surface area whilst maintaining the desired geometric area involve modification with rough, conductive electrode coatings, such as conducting polymers (CPs). This research aims to investigate a macroporous templated CP based on poly(3,4-ethylenedioxythiophene)/polystyrene sulphonate (PEDOT/PSS) hypothesised to further increase electrochemical surface area of conventional CP coatings. The enlarged electrochemical surface area offered is hypothesised to improve (i) recording, through reduced impedance and thermal noise and (ii) stimulation, through increased cathodic charge storage capacity (CSCc) and charge injection limits (CIL). Alongside microelectrode modification, we hypothesise that the refinement of recording instrumentation (amplifier and digitiser) required to acquire signals from MEA devices will compliment reduced thermal noise of modified microelectrodes, yielding a low noise recording and stimulation set-up. This research aims to produce an amplifier with intrinsic noise levels 45% (or less) that of microelectrode thermal noise. Conventional and macroporous PEDOT/PSS coated microelectrodes were deposited onto custom made gold MEAs at four increasing charge densities (32, 127, 318 and 637 mC cm−2) using both potentiostatic and galvanostatic electrochemical deposition. Scanning electron microscopy confirmed the presence of macropores within the CP structure. Electrochemical characterisation for properties central to neuronal recording and stimulation, comprised (i) cyclic voltammetry (CV) to yield CSCc, (ii) voltage transient measurements (VTM) to yield CIL and (iii) electrochemical impedance spectroscopy (EIS) to yield impedance spectra and thermal noise estimates. All CP coatings drastically improved electrochemical properties of gold and this effect was more apparent as deposition charge density increased. Comparisons between conventional PEDOT/ PSS and macroporous PEDOT/PSS coatings revealed (i) no significant difference in CSCc for all deposition charge densities, (ii) a significant increase in CIL at the highest deposition charge density of 637 mC cm−2 from 6.4 ± 0.3 mC cm−2 to 8.8 ± 0.3 mC cm−2 (p < 0.05), (iii) a significant reduction in impedance magnitude (at 100 Hz) at the highest deposition charge density of 637 mC cm−2 from 59 ± 5 kΩ to 41 ± 1 kΩ (p < 0.05) and (iv) a significant reduction in predicted thermal noise amplitude at the highest deposition charge density of 637 mC cm−2 from 12.6 ± 0.3 μVp−p to 10.5 ± 0.6 μVp−p (p < 0.05). No differences in electrochemistry were observed between potentiostatic and galvanostatic deposition methods. Stability studies, performed through longterm biphasic current injection, revealed conventional PEDOT/PSS coatings were less prone to delamination. However, macroporous PEDOT/PSS coatings exhibited an improvement in impedance throughout the time-course of the experiment, followed by abrupt delamination. Neurite outgrowth and viability assays, indicated the biocompatibility of both conventional and macroporous PEDOT/PSS coatings alongside primary hippocampal neurons. An amplification system was designed and coupled with an analog to digital converter from National Instruments to produce a low noise neuronal data acquisition system for the MEA devices. The amplifier was (i) operated between 0 and 5 V utilising a DC battery power supply, (ii) had a bandwidth of 10 kHz, (iii) removed DC voltage created at the electrode/electrolyte interface with a high-pass cut-off frequency of 0.7 Hz and (iv) had a gain of 2000. An offset voltage of 2.5 V was employed to allow for amplification of both negative and positive signals, due to the biphasic nature of neuronal action potentials. Strategies to reduce environment electromagnetic interference at the amplifier front end were employed and involved a customised neural interface board connected between the MEA and amplifier. A final intrinsic noise amplitude of 4.6 μVp−p was achieved and met the threshold target of < 45% of the microelectrode thermal noise for unmodified gold as well as for the lower thermal noise of conventional PEDOT/PSS and macroporous PEDOT/PSS coatings. In-vitro characterisation involved noise floor measurements pre- and post-culture to investigate microelectrode biofouling susceptibility and measurement of spontaneous neuronal activity alongside primary hippocampal cells. Conventional and macroporous PEDOT/PSS coatings both outperformed gold with preculture noise amplitudes at 18 ± 1 and 19 ± 2 μVp−p, compared to 36 ± 3 μVp−p for gold. All microelectrode materials were susceptible to biofouling, however, both CP coatings still exhibited a smaller noise floor than gold. Conventional PEDOT/PSS coatings displayed the highest number of active recordings and recording performance, as measured via a signal to noise ratio (SNR). The maximum SNR values achieved for gold, conventional PEDOT/PSS and macroporous PEDOT/PSS were 2.7, 12.6 and 17.6, respectively. Results for in-vitro stimulation efficacy were not obtained due to limitations of the MEA devices used. The large inter-electrode distance on the MEA devices resulted in the use of the same microelectrode for both stimulation and recording. Noise introduced from the voltage source and post-stimulus artifacts made recording direct neuronal responses difficult. Furthermore, the young age of cultures (< DIV 14) may have contributed to insufficient activity post-stimulus. In conclusion, the work presented here reveals macroporous CP coatings, at deposition charge densities of above 318 mC cm−2, provide subtle advantages in electrochemical parameters central to neuronal recording and stimulation as compared to conventional PEDOT/PSS coatings. Macroporous PEDOT/PSS coatings displayed potential for long-term stimulation applications as observed through high CIL and improvements in impedance during long-term biphasic stimulation. However, strategies to improve substrate adhesion are required for their successful implementation. Recording instrumentation was successfully designed and constructed, facilitating low-noise recording of neuronal activity from primary hippocampal neurons. No additional benefit was introduced from macroporous PEDOT/PSS coatings when compared to conventional PEDOT/ PSS coatings during in-vitro spontaneous recordings. Unfortunately, no data was obtained following in-vitro stimulation at both DIV 7 and DIV 14. Future in-vitro stimulation experiments could be refined through the use of paired stimulating and recording microelectrodes and by exploring cultures aged > DIV 14.