Continuous recording of intracellular activities in single cells is required for deciphering rare, dynamic and heterogeneous cell responses, which are missed by population or brief single-cell recording. Even if the field of intracellular recording is constantly proceeding, several technical challenges are still remained to conquer this important approach. Here, we demonstrate long-term intracellular recording by combining a vertical nanowire multi electrode array (VNMEA) with optogenetic stimulation to minimally disrupt cell survival and functions during intracellular access and measurement. We synthesized small-diameter and high-aspect-ratio silicon nanowires to spontaneously penetrate into single cells, and used light to modulate the cell's responsiveness. The light-induced intra-and extracellular activities of individual optogenetically-modified cells were measured simultaneously, and each cell showed distinctly different measurement characteristics according to the cell-electrode configuration. Intracellular recordings were achieved continuously and reliably without signal interference and attenuation over 24 hours. The integration of two controllable techniques, vertically grown nanowire electrodes and optogenetics, expands the strategies for discovering the mechanisms for crucial physiological and dynamic processes in various types of cells.Critical cellular dynamics, including transcriptional change, protein synthesis, receptor replacement, and synaptic plasticity in neural cells, take place over time periods ranging from several hours to days 1 . A common approach for discerning these cellular mechanisms in the single cell level is to measure its intracellular electrical activity using sharp microelectrodes or patch-clamping. However, as intracellular recording using these conventional electrodes is achieved by tearing the cell membrane, disruption of cell integrity limits the recording duration to several hours 2 , providing only brief 'snapshots' of cellular dynamics during limited experimental sessions 3 . Thus, such intermittent recordings have limitations in tracking single-unit activity over timescales relevant for most developmental and learning processes, or for pharmaceutical drug screening over long periods.Three-dimensional micro/nanostructure electrodes have shown superior feasibility for the electrophysiological study of single cells by accessing the cell interior and recent technologies allow simultaneous observations of the intracellular activity of individual cells in neuronal populations with high temporal/spatial resolution 4-17 . However, despite their advantages of high-sensitivity and minimal invasiveness, the reported time durations of intracellular recording using micro/nanostructure-based electrodes have not exceeded 80 min 4 . These intracellular recordings have been demonstrated by employing one of two agents: (i) external poration force achieved by a burst of electrical (electroporation) or optical (optoporation) 4 pulses applied through electrodes to increase the permeability ...

Electrical stimulation through direct electrical activation has been widely used to recover the function of neurons, primarily through the extracellular application of thin film electrodes. However, studies using extracellular methods show limited ability to reveal correlations between the cells and the electrical stimulation due to interference from external sources such as membrane capacitance and culture medium. Here, we demonstrate long-term intracellular electrical stimulation of undamaged pheochromocytoma (PC-12) cells by utilizing a vertical nanowire electrode array (VNEA). The VNEA was prepared by synthesizing silicon nanowires on a Si substrate through a vapor-liquid-solid (VLS) mechanism and then fabricating them into electrodes with semiconductor nanodevice processing. PC-12 cells were cultured on the VNEA for 4 days with intracellular electrical stimulation and then a 2-day stabilization period. Periodic scanning via two-photon microscopy confirmed that the electrodes pierced the cells without inducing damage. Electrical stimulation through the VNEA enhances cellular differentiation and neurite outgrowth by about 50% relative to extracellular stimulation under the same conditions. VNEA-mediated stimulation also revealed that cellular differentiation and growth in the cultures were dependent on the potential used to stimulate them. Intracellular stimulation using nanowires could pave the way for controlled cellular differentiation and outgrowth studies in living cells.

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A spatially and temporally confined single neuron activation method exploiting the strong interfaces between a neuron and a nanowire electrode.