Graphene nanostructures for input-output bioelectronics

Abstract

The ability to manipulate the electrophysiology of electrically active cells and tissues has enabled a deeper understanding of healthy and diseased tissue states. This has primarily been achieved via input/output (I/O) bioelectronics that interface engineered materials with biological entities. Stable long-term application of conventional I/O bioelectronics advances as materials and processing techniques develop. Recent advancements have facilitated the development of graphene-based I/O bioelectronics with a wide variety of functional characteristics. Engineering the structural, physical, and chemical properties of graphene nanostructures and integration with modern microelectronics have enabled breakthrough high-density electrophysiological investigations. Here, we review recent advancements in 2D and 3D graphene-based I/O bioelectronics and highlight electrophysiological studies facilitated by these emerging platforms. Challenges and present potential breakthroughs that can be addressed via graphene bioelectronics are discussed. We emphasize the need for a multidisciplinary approach across materials science, micro-fabrication, and bioengineering to develop the next generation of I/O bioelectronics.

FIG. 1.

I/O bioelectronic interfaces. (a) Schematic of the whole-cell patch-clamp technique for recording cellular electrophysiology. (b) Electrical equivalent circuit of the (I) cell–MEA and (II) cell–FET interfaces. R_J, R_NJ, R_seal, and R_e represent junctional, non-junctional, seal, and electrode resistances, respectively. C_J, C_NJ, C_coupling, and C_e represent junctional, non-junctional, coupling, and electrode capacitances, respectively. V_J, V_SD, V_G, V_rec, and I_SD represent junctional voltage across the cleft, source–drain voltage, gate voltage, recorded voltage, and source–drain current, respectively. RE represents reference electrode.

FIG. 2.

Evolution of microelectrodes for bioelectronics. (a) Electron micrograph of (I) an uncoated, sharpened tungsten wire; and (II) optical images of coated electrodes immersed in water to show the coating. Reproduced with permission from Hebul, Science 125, 3247 (1957). Copyright 2002 Science. (b) Scanning electron micrograph of Utah electrode array with 100 microelectrodes. Reproduced with permission from Normann et al., Vision Res. 39(15), 2577–2587 (1999). Copyright 1999 Elsevier Science Ltd. (c) Optical micrograph of Michigan probe with eight-channel recording gold sites. Reproduced with permission from Abidian et al., Adv. Funct. Mater. 19(4), 573–585 (2009). Copyright 2009 Wiley-VCH. (d) Photograph of a flexible 360-channel high density active electrode array. Reproduced with permission from Viventi et al., Nat. Neurosci. 14, 1599–1605 (2011). Copyright 2011 Nature Publishing Group. (e) Photograph of four threads on a NET-e device panel. Reproduced with permission from Wei et al., Adv. Sci. 5(6), 1700625 (2018). Copyright 2018 Author(s), licensed under a Creative Commons Attribution 4.0 License. (f) Optical image of mesh electronics emerging from the tip (upper right) of a 95 μm diameter needle into 1× phosphate-buffered saline (PBS) solution. Reproduced with permission from Liu et al., Nat. Nanotechnol. 10, 629–636 (2015). Copyright 2015 Springer Nature. (g) 3D confocal microscopy image of 3D-SR-BA with microelectrodes. Color bar represents the depth in micrometers. Reproduced with permission from Kalmykov et al., Sci. Adv. 5(8), eaax0729 (2020). Copyright 2019 Author(s), licensed under a Creative Commons Attribution-NonCommercial License 4.0.

FIG. 3.

Graphene nanostructures. (a) (I) A typical low-magnification transmission electron microscopy (TEM) image of synthesized graphene sheets. Reproduced with permission from Dato et al., Chem. Commun. 40, 6095–6097 (2009). Copyright 2009 The Royal Society of Chemistry. (II) Scanning electron micrograph of the microporous graphene foam (GF) structure showing a continuous network of 3D interconnected graphene sheets that comprise the walls of the foam-like structure. Reproduced with permission from Yavari et al., Sci. Rep. 1, 166 (2011). Copyright 2011 Author(s), licensed under a Creative Comments CC-BY-NC-ND License. (III) Scanning electron microscopy (SEM) image of the 3D porous LIG film patterned on polyimide (PI) substrates. Reproduced with permission from Lin et al., Nat. Commun. 5, 5714 (2014). Copyright 2014 Nature Publishing Group. (b) Truly 3D topology of graphene. (I–II) Representative SEM images of NT-3DFG synthesized for 30 min at 700 and 1100 °C, respectively. The insets represented by red-dashed boxes show out-of-plane graphene on SiNWs. (III) Number of flake edges along a 1 μm length of the nanowire of radius, r, for all synthesis conditions. Results are presented as mean ± SD (n = 3). Reproduced with permission from San Roman et al., ACS Catal. 10(3), 1993–2008 (2020). Copyright 2020 American Chemical Society.

FIG. 4.

Graphene biointerfaces. (a) The effect of graphene nanostructures on the viability of cardiomyocytes. (I) Quantification of %Viability of hESC-CMs cultured on glass (orange) and graphene (green) substrates. N.S. denotes no statistically significant difference. Results are presented as mean ± SD (n = 3). Reproduced with permission from Rastogi et al., Cel. Mol. Bioeng. 11, 407–418 (2018). Copyright 2018 Biomedical Engineering Society. (II) %Viability of hESC-CMs cultured on Si/SiO₂ control and 3DFG substrates. N.S. denotes no statistically significant difference. Results are presented as mean ± SD (n = 4). Reproduced with permission from Dipalo et al., Sci. Adv. 7(15), eabd5175 (2021). Copyright 2021 Author(s), licensed under Creative Commons Attribution-NonCommercial License 4.0. (b) TMRE assay performed on hESC-CMs at 6 days in vitro (DIV), cultured on (I) Si/SiO₂ control and (II) 3DFG substrates. Red (TMRE) and blue (Hoechst) denote mitochondria and cell nuclei, respectively. (III) Relative fluorescence readout of the TMRE-labeled hESC-CMs cultured on Si/SiO₂ control and 3DFG substrates. Results are presented as mean ± SD (n = 4). N.S. denotes no statistically significant difference. Reproduced with permission from Dipalo et al., Sci. Adv. 7(15), eabd5175 (2021). Copyright 2021 Author(s), licensed under Creative Commons Attribution-NonCommercial License 4.0. (c) Coupling between graphene nanostructures and electrogenic cells. SEM images (tilt 52°, backscattered electrons) of (I) 2D graphene-cell cross section, arrow identifies membrane invagination; (II) 3DFG-cell cross section; (III) NT-3DFG mesh-cell cross section, arrow identifies membrane wrapping at single wire. (IV) NT-3DFG vertically standing wires-cell cross section, arrow identifies a wire spontaneous penetration. Reproduced with permission from Mation et al., Adv. Mater. Interfaces 7(18), 2000699 (2020). Copyright 2020 Wiley-VCH GmbH.

FIG. 5.

Graphene nanostructures for electrophysiology recordings. (a) Graphene FET (gFET) for electrical recording. (I) Optical microscope image of polydimethylsiloxane (PDMS)/cells interfaced with large flake gFET. Graphene flake outline is marked by white-dashed line; the measured device is marked by red arrow. (II) Recorded averaged peak (red) and raw data (gray traces) for the gFET and cell in (I). Reproduced with permission from Cohen-Karni et al., Nano Lett. 10(3), 1098–1102 (2010). Copyright 2010 American Chemical Society. (b) Graphene transistor arrays for recording action potentials from electrogenic cells. (I) Combination of an optical microscopy image of a transistor array and a fluorescence image of the calcein-stained cell layer on the same array. (II) Exemplary single spikes. The current response has been converted to an extracellular voltage signal. The upper spike resembles a capacitive coupling followed by the opening of voltage-gated sodium channels, whereas in the bottom one, the ion channels dominate over the capacitive coupling. Reproduced with permission from Hess et al., Adv. Mater. 23(43), 5045–5049 (2011). Copyright 2011 Wiley-VCH Verlag GmbH. (c) Graphene MEAs for electrical and optical measurements of human stem cell-derived cardiomyocytes. (I) Differential interference contrast (DIC) image of graphene MEAs fabricated on glass coverslip. (II) Representative recorded field potential traces using graphene MEAs. Reproduced with permission from Rastogi et al., Cel. Mol. Bioeng. 11, 407–418 (2018). Copyright 2018 Biomedical Engineering Society. (d) 3DFG ultra-MEAs for subcellular electrical recordings. (I) SEM image of 10 μm NT-3DFG-MEAs. (II) Representative recorded field potential traces using 10, 5, and 2 μm NT-3DFG ultra-microelectrodes. Reproduced with permission from Rastogi et al., Nano Res. 13, 1444–1452 (2020). Copyright 2020 Springer. (e) Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene MEAs. (I) SEM image of 5 μm 3DFG electrodes. (I) Representative extracellular field potential recording of hiPSC-CMs using 3DFG-MEA with 50 μm electrodes (n = 80 electrodes). (III) Representative intracellular action potential recording on 3DFG-MEA with 50-μm electrodes after optoporation (n = 70 electrodes). Reproduced with permission from Dipalo et al., Sci. Adv. 7(15), eabd5175 (2021). Copyright 2021 Author(s), licensed under Creative Commons Attribution-NonCommercial License 4.0.

FIG. 6.

Electrophysiological investigations and mapping. (a) The effect of β-adrenergic receptor agonist on the extracellular electrophysiology using graphene MEAs. Averaged trace (70 peaks) before (red, −) and after (green, +) the application of β-adrenergic receptor agonist, isoproterenol. Reproduced with permission from Rastogi et al., Cel. Mol. Bioeng. 11, 407–418 (2018). Copyright 2018 Biomedical Engineering Society. (b) Intracellular electrophysiology investigations of the effect drugs on human-derived cardiomyocytes. (I) Representative cardiac action potentials before and after the administration of nifedipine at various concentrations. (II) Representative cardiac action potentials before and after the administration of 100 nM dofetilide (DOF). REF, reference signal in physiological conditions. Reproduced with permission from Dipalo et al., Sci. Adv. 7(15), eabd5175 (2021). Copyright 2021 Author(s), licensed under Creative Commons Attribution-NonCommercial License 4.0. (c) Mapping electrical signal propagation in 3D using the 3D-SR-BA. (I) A 3D confocal microscopy image of 3D cardiac spheroid labeled with Ca²⁺ indicator dye (Fluo-4, green fluorescence) encapsulated by the 3D-SR-BA. (II) Averaged field potential peak (red trace) and raw data (gray traces, n = 100 peaks recorded by channel 4). (III) 2D representation of the isochronal map of time latencies. White arrow represents average conduction velocity direction. Reproduced with permission from Kalmykov et al., Sci. Adv. 5(8), eaax0729 (2020). Copyright 2019 Author(s), licensed under a Creative Commons Attribution-NonCommercial License 4.0.

FIG. 7.

Graphene and carbon nanostructures for stimulation. (a) Electrical neural stimulation transparent graphene electrode arrays implanted in GCaMP6f mice. (I) Demonstration of micro-electrocorticography (micro-ECoG) implantation over sensorimotor cortex and electrical stimulation in GCaMP6f mice. (II) Visualization of the intensity of neural response to 100 μA electrical stimulation at times −130 to +670 ms of peak response with a graphene electrode array. Reproduced with permission from Park et al., ACS Nano 12(1), 148–157 (2018). Copyright 2017 American Chemical Society. (b) Flexible neural electrodes array based-on porous graphene for cortical stimulation. (I) Tilt SEM image of a 64-spot porous graphene array. (II) Stimulus evoking current (representing movement) response of the flex sensor in arbitrary units. Reproduced with permission from Lu et al., Sci. Rep. 6, 33526 (2016). Copyright 2016 Author(s), licensed under Creative Commons Attribution 4.0 License. (c) Simultaneous deep brain stimulation and fMRI with graphene fiber electrodes. (I) A representative SEM image of the axial external surface of a GF fiber. Inset, magnified image of the region in the dashed box. (II) B0 distortion maps observed in rats implanted with a GF (upper) and PtIr (lower) bipolar electrodes (electrodes are pointed by arrows). Reproduced with permission from Zhao et al., Nat. Commun. 11, 1788 (2020). Copyright 2020 Author(s), licensed under Creative Commons Attribution 4.0 License. (d) Micelle-enabled self-assembly of porous and monolithic carbon membranes for bioelectronic interfaces. (I) Cross-sectional view (upper panels) and associated top view (lower panels) of the hierarchical porous material. The hierarchical structures display two components: a bottom layer constructed from an ordered mesoporous structure and layers of porous vesicles assembled into multiple layers (as separated by the dashed lines). (II) Top: a retinal calcium image showing activated retinal ganglion cells (RGCs) upon the stimulation. Middle: representative calcium traces from individual RGCs (numbered in the upper image). Bottom: the input current density during the stimulation. Reproduced with permission from Fang et al., Nat. Nanotechnol. 16, 206–213 (2021). Copyright 2020 Author(s), licensed under Springer Nature Ltd. (e) Remote nongenetic optical modulation of neuronal activity using NT-3DFG. (I) Schematic illustrating an NT-3DFG interfaced with a neuron for photothermal stimulation. Purple spot indicates laser illumination area. (II) A 3D reconstruction of a fluorescence images of a representative DRG neuron labeled with plasma membrane stain (red, CellMask plasma membrane stain) and interfaced with NT-3DFG (white). (III) Representative recorded membrane potential of a repetitively stimulated DRG neuron (DRG neuron was patch-clamped in whole-cell configuration and current clamp mode). Electrical stimulation was performed by injecting a pulse of 100 nA for 1 ms. Photothermal stimulation was performed with 405-nm laser with a pulse of 2.28 mW power and 0.6-ms pulse duration (1.37 μJ). Reproduced with permission from Rastogi et al., Proc. Natl. Acad. Sci. U. S. A. 117(24), 13339–13349 (2020). Copyright 2020 Author(s), licensed under a creative Commons Attribution 4.0 License.