A novel bio-mimicking, planar nano-edge microelectrode enables enhanced long-term neural recording

Abstract

Our inability to accurately monitor individual neurons and their synaptic activity precludes fundamental understanding of brain function under normal and various pathological conditions. However, recent breakthroughs in micro- and nano-scale fabrication processes have advanced the development of neuro-electronic hybrid technology. Among such devices are three-dimensional and planar electrodes, offering the advantages of either high fidelity or longer-term recordings respectively. Here, we present the next generation of planar microelectrode arrays with "nano-edges" that enable long-term (≥1 month) and high fidelity recordings at a resolution 15 times higher than traditional planar electrodes. This novel technology enables better understanding of brain function and offers a tremendous opportunity towards the development of future bionic hybrids and drug discovery devices.

Figure 1. Biomimetic nano-edge microelectrode mimicking the morphological structure of a synaptic cleft.

(a) Schematic representation of two synaptically connected neurons (the box depicts a chemical synapse between the cells). The post-synaptic terminal is shown as engulfing the pre-synaptic terminal; thereby enhancing tight physical and dielectric coupling between the neurons. (b) Schematic layout developed further from Spira and Hai, illustrating an electrode-neuron interface with its analogue passive electrical circuit. Only the junctional membrane (part of the membrane in contact with the microelectrode) of the cell body is represented here (blue) – depicted to be in close contact with the electrode (yellow) and its nano-edges (orange). The non-junctional membrane (not shown in this diagram) refers to the part of the membrane not juxtaposed against the electrode. The electrode was fabricated on a silicon dioxide substrate (green) and connected to a recording system (MEA1060; Multichannel Systems, Reutlingen, Germany). The junctional membrane resistance (Rj) and conductance (Cj) are represented in parallel, similar to the electrode resistance (Re) and impedance (Ce). The sealing resistance (R_seal) was enhanced due to the nano-edges implemented on the electrodes. (c) Characterization of the nano-edge microelectrodes using atomic force microscopy. A three-dimensional representation of a 30 μm microelectrode with a 40° tilt is depicted. The nano-edge is discernable around the microelectrode perimeter (blue arrow), and can be seen continuing along the connecting electrode wire (bottom right). Having the nano-edge along the wire and not limited to the circular area has the advantage that it increases the sealing resistance even when a neuron is not placed exactly on top of the microelectrode. This configuration also increased the surface area of the microelectrode that was in contact with the neuronal cell membrane (when bigger than 30 μm in diameter). (d) Cross-section of the microelectrode height showing the shape of the nano-edge. The microelectrodes are 30 ± 1 μm in diameter, 200 ± 15 nm in height, and the nano-edges varied between 5 and 15 nm in height and 2 to 3 μm in width. Letters ‘A’ and ‘B’ refer to the location of the cross-section taken from (c).

Figure 2. Nano-edge microelectrodes permit unprecedented resolution and long-term neural recording at the single neuron level.

(a) Neurons were cultured on a custom designed MEA with multiple nano-edge microelectrodes grouped into clusters of 4 or 6 microelectrodes per set. The number of electrodes per set could be increased depending on the fabrication design and experimental needs. We continuously monitored neuronal activity - even if the cells had moved away from their initial culture site as described previously. This setup also allows us to characterize and differentiate activity patterns from various cell types over time. An example is provided in (d). (b) Recording of action potentials from a single neuron showing distinguishable patterned activity from selected Lymnaea neurons. (c) Single action potential with clearly defined depolarization followed by rebound hyperpolarization. Average of the recorded action potentials amplitude was 4.44 mV peak-to-peak (n = 13) with a maximum measured value of 10.6 mV. (d) Examples of distinctive spontaneous activity patterns associated to two different neurons (LPeD1 and RPeD1) resected from the mollusk Lymnaea. These specific activity changes recorded within identified neurons can now be studied over months and advanced drug-screening can be performed to better understand the effect of the extracellular milieu on the cells activity. (e) Comparison of maximum-recorded peak-to-peak action potential between the nano-edge microelectrodes compared with other types of extra-cellular electrode, showing that the nano-edge microelectrodes record higher action potentials than all other planar microelectrodes, including some three-dimensional ones (e.g. vertical nanowire, Mushroom shape electrode (gMμE). (f) Comparison of the most commonly used micro-/nano-electrodes used to record neural activity in-vitro. The maximum coupling coefficient and the longest reported recording time were used to evaluate electrodes capabilities. Our nano-edge microelectrodes (green circle) permit monitoring of action potentials with a coupling coefficient comparable to that of 3D electrodes (red square), and for a period of time equivalent to traditional planar microelectrodes (blue triangles).

Figure 3. Schematic representation of the simulated elements using COMSOL Multiphysics and their physical characteristics.

(a) Both glass substrate and extracellular fluid were modeled as infinite boundaries. The microelectrode height was set at 200 nm and its width to 30 μm as per our experimental needs. The neuron was positioned 50 nm above the microelectrodes to mimic the gap found at the neuron-electrode interfaces, and was modeled with diameters in the range of 5 to 80 μm, which is representative of most vertebrate and invertebrate cell diameters. Finally, the nano-edge was modeled at various heights from 0 (no nano-edge, similar to traditional planar electrodes) to 50 nm (same height as the cleft). (b) Table of physical values of electrical conductivity and relative permittivity used to run the computational simulation (Refs , , , are listed in brackets in the table). (c) Graphical representation of the sealing resistance disparity when computationally varying the nano-edge height and the cell’s diameter using a heat map, function of the cell’s diameter and the nano-edge height. Note the rapid increase in sealing resistance when the nano-edge is present and the cell’s diameter is equal or larger than the electrode (here 30 μm in diameter). (d) Variation of the sealing resistance for each cell diameter when the nano-edge increases in height. When the cell’s diameter reaches a diameter equal to or larger than the microelectrode and that an edge is present, the sealing resistance reached a plateau of 7.49 ± 0.34 MΩ. (e) Variation of the sealing resistance for each nano-edge height when the cell diameters increases. A dip between 10 and 25 μm can be attributed to current leakages happening when a cell’s diameter is smaller than the electrode. A similar plateau as for (d) can be seen.