Nanoelectronics-biology frontier: From nanoscopic probes for action potential recording in live cells to three-dimensional cyborg tissues

Abstract

Semiconductor nanowires configured as the active channels of field-effect transistors (FETs) have been used as detectors for high-resolution electrical recording from single live cells, cell networks, tissues and organs. Extracellular measurements with substrate supported silicon nanowire (SiNW) FETs, which have projected active areas orders of magnitude smaller than conventional microfabricated multielectrode arrays (MEAs) and planar FETs, recorded action potential and field potential signals with high signal-to-noise ratio and temporal resolution from cultured neurons, cultured cardiomyocytes, acute brain slices and whole animal hearts. Measurements made with modulation-doped nanoscale active channel SiNW FETs demonstrate that signals recorded from cardiomyocytes are highly localized and have improved time resolution compared to larger planar detectors. In addition, several novel three-dimensional (3D) transistor probes, which were realized using advanced nanowire synthesis methods, have been implemented for intracellular recording. These novel probes include (i) flexible 3D kinked nanowire FETs, (ii) branched intracellular nanotube SiNW FETs, and (iii) active silicon nanotube FETs. Following phospholipid modification of the probes to mimic the cell membrane, the kinked nanowire, branched intracellular nanotube and active silicon nanotube FET probes recorded full-amplitude intracellular action potentials from spontaneously firing cardiomyocytes. Moreover, these probes demonstrated the capability of reversible, stable, and long-term intracellular recording, thus indicating the minimal invasiveness of the new nanoscale structures and suggesting biomimetic internalization via the phospholipid modification. Simultaneous, multi-site intracellular recording from both single cells and cell networks were also readily achieved by interfacing independently addressable nanoprobe devices with cells. Finally, electronic and biological systems have been seamlessly merged in 3D for the first time using macroporous nanoelectronic scaffolds that are analogous to synthetic tissue scaffold and the extracellular matrix in tissue. Free-standing 3D nanoelectronic scaffolds were cultured with neurons, cardiomyocytes and smooth muscle cells to yield electronically-innervated synthetic or 'cyborg' tissues. Measurements demonstrate that innervated tissues exhibit similar cell viability as with conventional tissue scaffolds, and importantly, demonstrate that the real-time response to drugs and pH changes can be mapped in 3D through the tissues. These results open up a new field of research, wherein nanoelectronics are merged with biological systems in 3D thereby providing broad opportunities, ranging from a nanoelectronic/tissue platform for real-time pharmacological screening in 3D to implantable 'cyborg' tissues enabling closed-loop monitoring and treatment of diseases. Furthermore, the capability of high density scale-up of the above extra- and intracellular nanoscopic probes for action potential recording provide important tools for large-scale high spatio-temporal resolution electrical neural activity mapping in both 2D and 3D, which promises to have a profound impact on many research areas, including the mapping of activity within the brain.

Keywords: bioelectronics; brain activity mapping; cellular and subcellular resolution; field-effect transistor; flexible electronics; graphene; macroporous 3D electronics; nanodevice; nanowire; synthetic tissue.

Figure 1

Schematic illustration of the advantages for using SiNW based devices to interface with biological systems. (a) SiNW based devices are used for intra- and extracellular action potential recording. The small size of the functional element increases the spatial precision and resolution, also enables subcellular interfacing; (b) The bottom-up pathway used for making the nanoscale electronic devices allows us to realize multi-function on a single chip, make flexible electronics, also three-dimensional and free-standing devices to interface from inside the tissue.

Figure 2

SiNW based FET biosensors. (a) Scanning electron microscope image of a SiNW FET. Scale bar is 100 nm; (b) Conductance versus water-gate voltage trace for three representative SiNW FET devices. Inset: Scheme representing experimental setup, (red) nanowire, (yellow/navy) passivated contact electrodes, (blue) electrolyte solution, (green) Ag/AgCl reference/gate electrode.

Figure 3

Extracellular action potential recording from cultured neurons. (a) Left: optical image of a cortical neuron aligned across a SiNW FET; scale bar is 10 μm. Right: High-resolution image of the region where axon (red arrow) crosses a SiNW (yellow arrow). (b) Red trace: intracellular potential of an cortex neuron (after 6 days in culture) recorded by glass micropipette during stimulation; Black trace: time-correlated signal from axon measured using a p-type SiNW FET; (c) Optical image of a cortical neuron interfaced to three of the four functional NWFETs in an array; scale bar is 20 μm; (d) Trace of intracellular current stimulation and resulting electrical responses from the four SiNW FETs in c. Reprinted with permission from Ref. [27]. Copyright 2006 American Association for the Advancement of Science.

Figure 4

Building interface between SiNW FETs and spontaneously firing cardiomyocytes. (a) Schematic illustration of manipulating the PDMS/cell substrate to make the interface with an NWFET device. (b) Distinct patch of beating cells (red dashed oval) over a SiNW device (yellow arrow). scale bar is 20 μm; (c) Two representative traces recorded with same SiNW FET from a spontaneously firing cardiomyocyte with different PDMS/cell displacement value; (d) High resolution comparison of single peaks recorded with increasing displacement values (from purple to red); (e) Data recorded in distinct experiment at displacement value close to cell failure. Reprinted with permission from Ref. [51]. Copyright 2009 National Academy of Sciences.

Figure 5

Extracellular action potential recording from nanoFETs with different projection area. (a) Schematic illustrating the chip design incorporating graphene and SiNW FET devices; (b) Optical microscope image of PDMS/cells interfaced with large flake graphene FET. Graphene flake outline is marked by white dashed line; measured device is marked by red arrow. Scale bar is 30 μm; (c) Optical microscope image of PDMS/cells interfaced with smaller flake graphene FET and SiNW FET. Graphene flake outline is marked by white dashed line, measured graphene device is marked by red arrow, and measured SiNW device is marked by blue arrow. Scale bar is 13.6 μm; (d) Recorded averaged peak (red) and raw data (gray traces) for the Gra1-FET and cell in b; (e) Thirteen raw signal peaks (gray traces) from the Gra2-FET (upper data) and SiNW FET (lower data) devices marked by red and blue arrows, respectively in c. The average of the peaks was plotted in red and blue, respectively. Reprinted with permission from Ref. [37]. Copyright 2010 American Chemical Society.

Figure 6

Extracellular action potential recording with short channel NWFETs. (a) Illustration of Au-nanocluster-catalyzed nanowire growth with well-controlled axial dopant profile introduced during VSS growth; (b) Short-channel n⁺⁺/i/n⁺⁺ SiNWs with channel lengths of 150 nm, 80 nm, and 50 nm respectively. Scale bars are 150 nm. The Au-nanoclusters were ~80 nm in diameter, and nanowires were selectively etched to reveal the active channel; (c) Summary of the peak-to-peak widths for the 150, 80, and 50 nm channel length devices. In addition, a previously published 2.3 μm channel length SiNW device (black) is shown for comparison [37]. Inset, an example of single peaks from each of the short-channel devices; (d) Optical image of cardiomyocytes interfaced with three 130 nm channel length devices (labeled d1, d2, and d3). White dashed lines illustrate the nanowire position. Scale bar is 15 μm; (e) Representative recorded signals from the three devices in d; (f) Histogram of the time lag between devices d1 and d2 (red; separation distance 1.9 μm) and between devices d1 and d3 (blue; separation distance 73 μm). Reprinted with permission from Ref. [59]. Copyright 2012 American Chemical Society.

Figure 7

Extracellular field and action potential recording from brain slices with NWFETs. (a) Measurement schematics. Top: overview of a NWFET array interfacing with slice oriented with pyramidal cell layer over the devices. Bottom Left: zoom-in of device region illustrating interconnected neurons and NWFETs. Bottom Right: photograph of the assembled sample chamber. 1, 2, and 3 indicate the mitral cells in the olfactory bulb, the lateral olfactory tract, and the pyramidal cells, respectively. 4 and 5 mark the stimulation electrode and the patch clamp pipette, respectively; (b) Optical image of an acute slice over a 4 × 4 NWFET array. Crosses along the LOT fiber region of the slice mark the stimulation spots a–h. Scale bar is 100 μm; (c) Laminar organization and input circuitry of the piriform cortex (Layer I–III); (d) a zoom-in signal recorded from two SiNW FET devices. The Open Triangle and Dashed Oval mark the stimulation and presynaptic features, resp. The Plus and Asterisk mark the postsynaptic features; (e) Maps of the relative signal intensity or activity for devices 1–8. Reprinted with permission from Ref. [69]. Copyright 2010 National Academy of Sciences.

Figure 8

Action potential recording from whole hearts with NWFETs fabricated on flexible plastic substrate. (a) Photograph of a flexible SiNW FETs chip interfaced with a chicken heart in concave configuration. Yellow arrow marks the location of the heart; (b) Top-down photograph of same system, which enables overall registration between heart and lithographically defined markers on the substrate; (c) Photograph of a flexible SiNW FETs chip interfaced with a chicken heart in convex configuration; (d) A representative recorded conductance data from a SiNW FET interfacing with chicken heart in bent configuration. Reprinted with permission from Ref. [36]. Copyright 2009 American Chemical Society.

Figure 9

Intracellular action potential recording with kinked nanowire FET devices. (a) SEM image of a doubly kinked nanowire with a cis configuration. scale bar: 200 nm; (b) Top: schematic of kinked nanowire probe with encoded active region (pink) by dopant level modulation. The blue regions designate the source/drain (S/D). Low: schematic of kinked nanowire probe with p-n junction; (c) Superposition of tmSGM images on AFM topographic images of a representative kinked p n nanowire device under V_tip of +5 V. Scale bar is 0.5 μm. The blue/red arrows indicate the p-type and n-type depletion/accumulation regions (left panel), respectively. Inset: line profile of the tmSGM signal along the white dashed lines; (d) A 3D, free-standing kinked nanowire FET probe bent up by stress release of the metal interconnects. The yellow arrow and pink star mark the nanoscale FET and SU-8, respectively. Scale bars, 5 μm; (e) Schematic of intracellular recording from spontaneously beating embryonic chicken cardiomyocytes cultured on PDMS substrate using kinked nanowire nanoprobes; (f) Transition from extracellular to intracellular recordings during cellular entrance recorded by a kinked nanowire FET probe from beating cardiomyocytes. Green and pink stars denote the peak positions of intracellular and extracellular signal components, respectively; (g) Steady-state intracellular recording; (h) Zoom-in signals of an intracellular action potential peak. Blue and orange stars designate features that are possibly associated with inward sodium and outward potassium currents, respectively. The letters ‘a’ to ‘e’ denote five characteristic phases of a cardiac intracellular potential, as defined in text. The red-dashed line is the baseline corresponding to intracellular resting state. Reprinted with permission from Ref. [28][32]. Copyright 2010 American Association for the Advancement of Science, 2012 American Chemical Society.

Figure 10

Intracellular action potential recording with the BIT-FET. (a) Schematics illustrating the working principle of intracellular electrical recording with the BIT-FET; (b) SEM image of a BIT-FET device. Scale bar is 200 nm; (c) Calculated bandwidth of the BIT-FET device versus the inner diameter of the nanotube (ALD SiO2 thickness was the same as the nanotube inner diameter, and the nanotube length was fixed at 1.5 μm); (d) A representative trace corresponding to the second entry of the nanotube from a BIT-FET around the same position on the cardiomyocyte cell. Reprinted with permission from Ref. [29]. Copyright 2012 Nature Publishing Group.

Figure 11

Intracellular action potential recording with the ANTT. (a) Schematic illustration the working principle of intracellular electrical recording with the ANTT; (b) SEM image of an ANTT probe. Scale bar, 10 μm. Inset, zoom of the probe tip from the dashed red box. Scale bar, 100 nm; (c) A representative intracellular action potential peak recorded with an ANTT. The five characteristic phases of the action potential peak are denoted by 1–5; (d) Schematic of chip-based vertical ANTT probe arrays fabricated from epitaxial Ge/Si nanowires for enhanced integration. Reprinted with permission from Ref. [33]. Copyright 2012 American Chemical Society.

Figure 12

Multiplexed intracellular action potential recording. (a) Optical image of two BIT-FET devices (positions marked with dots) coupled to a single cardiomyocyte cell, with the cell boundary marked by the yellow dashed line. Scale bar is 10 μm; (b) Simultaneously recorded traces from the two devices in a, corresponding to the transition from extracellular to intracellular recording; (c) Design and SEM image of a probe with two independent ANTT devices sharing a common source contact. Horizontal scale bar, 5 μm; (d) Intracellular recording from a single cardiomyocyte using a probe with two independent ANTT devices. The interval between tick marks corresponds to 1 s; (e) Optical image of three BIT-FET devices coupled to a beating cardiomyocyte cell network. Scale bar is 30 μm; (f) Representative traces recorded simultaneously from the devices shown in e. Reprinted with permission from Ref. [29][33]. Copyright 2012 Nature Publishing Group, 2012 American Chemical Society.

Figure 13

Merging nanoelectronics with artificial tissues seamlessly for three-dimensional electrical interfacing. Reprinted with permission from Ref. [39]. Copyright 2012 Nature Publishing Group.

Figure 14

Nanowire nanoES, 3D nanoelectronics/tissue hybrids, and 3D action potential recording. (a) 3D reconstructed confocal fluorescence micrographs of reticular nanoES viewed along the y (I) and x (II) axes. Solid and dashed open magenta squares indicate two nanowire FET devices located on different planes along the x axis. The overall size of the structure, x-y-z=300-400-200 μm. Scale bars, 20 μm; (b) SEM image of a single kinked nanowire FET within a reticular scaffold, showing (1) the kinked nanowire, (2) metallic interconnects (dashed magenta lines) and (3) the SU-8 backbone. Scale bar, 2 μm; (c, d) 3D reconstructed confocal images of rat hippocampal neurons after a two-week culture on reticular nanoES/Matrigel. The white arrow highlights a neurite passing through a ring-like structure supporting a nanowire FET. Dimensions in c, x: 317 μm; y: 317 μm; z: 100 μm; in d, x: 127 μm; y: 127 μm; z: 68 μm; (e) Confocal fluorescence micrographs of a synthetic cardiac patch. (II and III), Zoomed-in view of the upper and lower dashed regions in I, Scale bar, 40 μm; (f) Epifluorescence micrograph of the surface of the cardiac patch. Green: α-actin; blue: cell nuclei. The dashed lines outline the position of the S/D electrodes. Scale bar, 40 μm; (g) Conductance versus time traces recorded from a single NW FET before (black) and after (blue) applying noradrenaline; (h) Multiplexed electrical recording of extracellular field potentials from four nanowire FETs at different depth in a mesh nanoES/cardiac hybrid. Data are conductance versus time traces of a single spike recorded at each nanowire FET. Reprinted with permission from Ref. [39]. Copyright 2012 Nature Publishing Group.

Figure 15

Synthetic vascular construct enabled 3D sensing. (a) Schematic of the smooth muscle nanoES. The upper panels are the side view, and the lower one is a zoom-in view. Grey: mesh nanoES; blue fibers: collagenous matrix secreted by HASMCs; yellow dots: nanowire FETs; pink: HASMCs; (b) (I) Photograph of a single HASMC sheet cultured with sodium L-ascorbate on a nanoES. (II) Zoomed-in view of the dashed area in I. Scale bar, 5 mm; (c) Photograph of the vascular construct after rolling into a tube and maturation in a culture chamber for three weeks. Scale bar, 5 mm; (d) Haematoxylin-Eosin- (I) and Masson-Trichrome- (II; collagen is blue) stained sections (~6 μm thick) cut perpendicular to the tube axis; lumen regions are labelled. The arrows mark the positions of SU-8 ribbons of the nanoES. Scale bars, 50 μm; (e) Changes in conductance over time for two nanowire FET devices located in the outermost (red) and innermost (blue) layers. The inset shows a schematic of the experimental set-up. Outer tubing delivered bathing solutions with varying pH (red dashed lines and arrows); inner tubing delivered solutions with fixed pH (blue dashed lines and arrows). Reprinted with permission from Ref. [39]. Copyright 2012 Nature Publishing Group.

Figure 16

Overview of nanoelectronics-biology interfacing enabled new fundamental studies and novel directions in biomedical research and applications. These new studies benefit from different features of the nanoelectronics.