Supra- and Sub-Threshold Intracellular-Like Recording of 2D and 3D Neuronal Networks Using Nanopillar Electrode Arrays

Nanoscale bioelectronic sensors which harness the cell/nanomaterial interface have recently emerged as a promising <i>in vitro</i> approach to correlate single cell behavior to network activity. Probing and manipulating cells within semi-native environments requires nanomaterials that do not cause immediate stress or ultimate degeneration. Typical gold standard electrophysiological techniques, such as patch clamp or calcium imaging, have enabled neuroscience breakthroughs, but today present limitations related to scalability, temporal resolution, and invasiveness.¹ One solution offered by previous researchers has been multiplexed, intracellular-like electrophysiology using nanostructured electrode arrays (NEA). These arrays of nano-scale vertical conductive or non-conductive structures leverage the malleable nature of a cell’s plasma membrane to create a region of high-seal resistance, allowing for tight electrical coupling between the electrode and cell.^2–4 Upon a brief electrical pulse, the membrane is temporarily permeabilized, allowing the electrode to gain intracellular access. While the nature of these intracellular signals is poorly understood, an ensemble of integrins and curvature-sensitive proteins allow the plasma membrane to adhere to the underlying nanostructures through curved adhesions.⁵ With these devices, signals obtained from cellular networks can be used to evaluate novel drugs,⁶ monitor maturation over time,² identify cellular subtypes,⁷ and even transfect single cells.⁸ A critical gap in NEA technology is the optimization of various nanostructures to target different kinds of cell sizes, morphologies, and structures. Here, we optimize the electrode size, chemical functionalization and device packaging to achieve intimate contacts with neurites and somata in diverse neuronal cultures. Using these optimized NEAs, we show that intracellular-like signals can be recorded from primary neurons, iPSC-derived neuronal networks, and brain organoids. We also use pharmacology to confirm the validity of these recordings. Furthermore, we present a novel spike detection algorithm for characterizing diverse spiking datasets, which include extracellular field potentials, extracellular action potentials, intracellular postsynaptic potentials, and intracellular action potentials. We believe that our findings will lead to biological discoveries relating subthreshold synaptic activity to neuronal network behavior, elucidate the role of excitatory-to-inhibitory imbalances in neurological disorders, and pave the way for analyzing diverse spiking datasets within neural signal processing.<br/><br/><br/>1. Fendyur, A. & Spira, M. E. Toward on-chip, in-cell recordings from cultured cardiomyocytes by arrays of gold mushroom-shaped microelectrodes. <i>Front Neuroeng </i><b>5</b>, 21 (2012).<br/>2. Jahed, Z. <i>et al.</i> Nanocrown electrodes for parallel and robust intracellular recording of cardiomyocytes. <i>Nat Commun </i><b>13</b>, 2253 (2022).<br/>3. Abbott, J. <i>et al.</i> A nanoelectrode array for obtaining intracellular recordings from thousands of connected neurons. <i>Nat Biomed Eng </i><b>4</b>, 232–241 (2020).<br/>4. Shokoohimehr, P. <i>et al.</i> High-Aspect-Ratio Nanoelectrodes Enable Long-Term Recordings of Neuronal Signals with Subthreshold Resolution. <i>Small </i><b>18</b>, 2200053 (2022).<br/>5. Lou, H.-Y., Zhao, W., Zeng, Y. & Cui, B. The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling. <i>Acc Chem Res </i><b>51</b>, 1046–1053 (2018).<br/>6. Yang, Y. <i>et al.</i> Cardiotoxicity drug screening based on whole-panel intracellular recording. <i>Biosensors and Bioelectronics </i><b>216</b>, 114617 (2022).<br/>7. Lin, Z. C. <i>et al.</i> Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease. <i>Microsyst Nanoeng </i><b>3</b>, 1–7 (2017).<br/>8. Liu, X., Zu, Y. & Wang, S. Cell Size-Specific Transfection by Micropillar Array Electroporation. <i>Methods Mol Biol </i><b>2050</b>, 3–12 (2020).