High-Density Multilayer Graphene Microelectrode Arrays for Optogenetic Electrophysiology in Human Embryonic Kidney Cells

Optogenetic electrophysiology offers high precision cellular analysis by electrophysiological recording under optogenetic control [1] , [2] . Such studies often use microelectrode arrays (MEA) to obtain massively parallel recording from densely packed cells. Among the MEA materials, graphene has been suggested to be well suited for optogenetic electrophysiology [1] - [3] , enabling transparent, flexible, and low-noise MEAs for in vivo recording of the local field potential (LFP) [1] . To date, most graphene microelectrodes were 25-300 μm in size to achieve high signal-to-noise ratios, and placed in a 150-900 μm pitch for single-unit recording [1] , [3] . Such device dimension however has limited spatial resolution compared to closely-packed silicon MEAs [4] , and cannot offer spatial oversampling of the cell activity. Here we present a 28-μm pitched multilayer graphene MEA with 13-μm sized electrodes, the smallest in literature, for high-density optogenetic electrophysiology in human embryonic kidney (HEK) cells. Our MEA was made of CVD-grown multilayer graphene for its low sheet resistance, which was one-time transferred onto a Si/SiO 2 substrate, instead of layer-by-layer transfer steps (see [2] ) that may increase the electrode impedance by contaminants. Our electrodes had 2 MΩ impedance at 1 kHz (2.38 Ω•cm 2 ) in electrochemical impedance spectroscopy (EIS), 6 times smaller than those made by layer-by-layer transfer steps if they were made in the same size [2] . Our MEA was able to record optogenetically evoked extracellular signals in HEK cells co-expressed with opsins ( ChR2 ) and Ca 2+ reporters ( jRCAMP1a ) [5] . The signal amplitude increased with the intensity (not the duration) of the optogenetic stimulus, and qualitatively matched the position of optogenetically responsive cells (confirmed by jRCAMP1a imaging). Our work suggests the possible use of multiplayer graphene MEA for optogenetic electrophysiology in HEK cells.