ZnO nanowire microelectrode arrays for integration with neuronal networks
Authors:   , Maddah Mohsen 
Published: December 2021

<p>Microelectrode arrays (MEAs) have been shown as a successful approach for neuroscientists to monitor the signal communication within the neuronal networks for understanding the functionality of the nervous system. However, using conventional planar MEAs is shown to be incapable of precise signal recording from neuronal networks at single-cell resolution due to low signal-to-ratio (SNR). This thesis looks at developing an electronic platform that comprises of zinc oxide nanowires (ZnO-NWs) on MEAs as a future device to record action potential (AP) signals with high SNR from human neuronal networks at single-cell resolution. Specifically, I studied the controlled growth of ZnO nanowires with various morphologies at exact locations across the substrate. I then investigated the biocompatibility of ZnO nanowires with different morphology and geometry for interaction with human NTera2.D1 (hNT) neurons. Finally, I examined the electrical characteristics of MEAs that were integrated with ZnO nanowires and metal encapsulated ZnO nanowires in comparison to the planar MEAs.  The hydrothermal growth of ZnO nanowires is thoroughly investigated as a technique to allow synthesis of the nanowires at a low temperature (95°C) with a low cost and high scalability that can also be applied on flexible substrates. The morphology of the ZnO nanowires was varied (diameters of 20–300 nm, lengths of 0.15–6.2 µm, aspect ratios of 6–95 and densities of 10–285 NWs/µm²) by controlling the critical growth parameters such as the precursor concentration (2.5–150 mM), growth time (1–20 h) and additive polyethylenimine (PEI) concentration (0–8 mM). The diameter and length of the ZnO nanowires were increased by increasing the precursor concentration and growth time. Using the standard precursor concentration of 25 mM, growth times of up to 4 h were found effective for the active growth of the nanowires due to the consumption of the precursor ions and precipitation of ZnO. The addition of 6 mM PEI to the growth solution was shown to mediate the growth solution, allowing the extension of the nanowire growth to 20 h or longer. The PEI molecules were also attached to the lateral faces of the nanowires that confined their lateral growth and promoted their axial growth (enhanced aspect ratio from 12 ± 3 to 67 ± 21).  Standard photolithography techniques were also introduced to selectively grow ZnO nanowires on exact locations across the substrates. The role of the ZnO seed layer geometry, seed layer area and gap, on the growth of ZnO nanowires was also investigated. Despite using the constant growth parameters (25 mM of precursor concentration with 4 h of growth time) changing the seed line widths (4 µm–1 mm) and the gap between the seed lines (2 µm–800 µm) resulted in the morphology of the nanowires to vary across the same substrate (diameters of 50–240 nm, lengths of 1.2–4.6 µm, aspect ratios of 9–34 and densities of 28–120 NWs/µm²). The seed area ratio of 50% was determined as a threshold to influence the nanowire morphology, where decreasing the seed area ratio below 50% (by increasing the adjacent gap or decreasing the seed layer area) increased the growth rate of the nanowires.  The biocompatibility of ZnO nanowires with human hNT neurons was investigated in this work for the first time. The adhesion and growth of hNT neurons on the arrays of ZnO nanowire florets were determined to be influenced by both geometry and morphology of the nanowires. The growth of the hNT neurons was promoted by 30% compared to the control Si/SiO₂ substrate surface when ZnO nanowires with lengths shorter than 500 nm and densities higher than 350 NWs/µm² were grown. The hNT neurons on all nanowires were also demonstrated to be functionally viable as they responded to the glutamate stimulation.  ZnO nanowires were shown to improve the electrical properties of the MEAs by reducing the electrochemical impedance due to the increased 3D surface area. The ZnO nanowires that were grown with 50 mM of precursor concentration for 4 h of growth time lowered the impedance from 835 ± 40 kΩ of planar Cr/Au MEAs to 540 ± 20 kΩ at a frequency of 1 kHz. In contrast, the ZnO nanowires that were grown with PEI for 35 h showed that despite the increased surface area by a factor of 45× the impedance was found to be quite high, 2.25 ± 0.2 MΩ at 1 kHz of frequency. The adsorption of PEI molecules to the lateral surfaces of the nanowires was thought to behave as a passivation layer that could have restricted the charge transfer characteristics of the ZnO-NW MEAs.  Encapsulation of the pristine ZnO nanowires that were grown with standard precursor concentration of 25 mM for 4 h of growth time with different metallic layers (Cr/Au, Ti and Pt) further improved the electrical characteristics of the MEAs. The ZnO nanowires that were encapsulated with a 10 nm thin layer of Ti and Pt achieved the lowest electrochemical impedance of 400 ± 25 kΩ at 1 kHz in this work. The robustness of the Ti encapsulated ZnO nanowires were also improved in comparison to the PEI ZnO nanowires. The improved electrochemical characteristics and mechanical stability of the MEAs integrated with metal encapsulated ZnO nanowires have shown a great promise for improving the SNR of recording signals from neuronal cells for long term measurements.  This work concludes that both pristine ZnO nanowire MEAs and metal encapsulated ZnO nanowire MEAs will be capable of recording AP signals from human neuronal networks at single-cell resolution. However, further optimisation and extensions of the work are required to record AP signals from human neuronal cells.</p>