Tzahi Cohen-Karni

Three-dimensional, Flexible Nanoscale Field Effect Transistors as Localized Bioprobes

Nanoprobes of Cell Potential Direct electrical measurements of cell potentials usually face design compromises. Microelectrodes probe within the cytosol of cells but have a minimum size (hundreds of nanometers in width) for obtaining useful signals. Nanoscale field effect transistors (FETs) can have an active probe size of only tens of nanometers but generally allow only the outer cell potential to be measured. Tian et al. (p. 830) fabricated nanowires in which kinks could be introduced to create a sharp probe tip pointing away from the fabrication substrate. Coating the tip with a phospholipid bilayer allowed the probe to be inserted through the membranes of beating cardiac cells, where it could be used to follow temporal changes in cell potential. Kinked nanowire transistors can measure intracellular electric potentials. Nanoelectronic devices offer substantial potential for interrogating biological systems, although nearly all work has focused on planar device designs. We have overcome this limitation through synthetic integration of a nanoscale field-effect transistor (nanoFET) device at the tip of an acute-angle kinked silicon nanowire, where nanoscale connections are made by the arms of the kinked nanostructure, and remote multilayer interconnects allow three-dimensional (3D) probe presentation. The acute-angle probe geometry was designed and synthesized by controlling cis versus trans crystal conformations between adjacent kinks, and the nanoFET was localized through modulation doping. 3D nanoFET probes exhibited conductance and sensitivity in aqueous solution, independent of large mechanical deflections, and demonstrated high pH sensitivity. Additionally, 3D nanoprobes modified with phospholipid bilayers can enter single cells to allow robust recording of intracellular potentials.

Graphene and Nanowire Transistors for Cellular Interfaces and Electrical Recording

Nanowire field-effect transistors (NW-FETs) have been shown to be powerful building blocks for nanoscale bioelectronic interfaces with cells and tissue due to their excellent sensitivity and their capability to form strongly coupled interfaces with cell membranes. Graphene has also been shown to be an attractive building block for nanoscale electronic devices, although little is known about its interfaces with cells and tissue. Here we report the first studies of graphene field effect transistors (Gra-FETs) as well as combined Gra- and NW-FETs interfaced to electrogenic cells. Gra-FET conductance signals recorded from spontaneously beating embryonic chicken cardiomyocytes yield well-defined extracellular signals with signal-to-noise ratio routinely >4. The conductance signal amplitude was tuned by varying the Gra-FET working region through changes in water gate potential, V(wg). Signals recorded from cardiomyocytes for different V(wg) result in constant calibrated extracellular voltage, indicating a robust graphene/cell interface. Significantly, variations in V(wg) across the Dirac point demonstrate the expected signal polarity flip, thus allowing, for the first time, both n- and p-type recording to be achieved from the same Gra-FET simply by offsetting V(wg). In addition, comparisons of peak-to-peak recorded signal widths made as a function of Gra-FET device sizes and versus NW-FETs allowed an assessment of relative resolution in extracellular recording. Specifically, peak-to-peak widths increased with the area of Gra-FET devices, indicating an averaged signal from different points across the outer membrane of the beating cells. One-dimensional silicon NW- FETs incorporated side by side with the two-dimensional Gra-FET devices further highlighted limits in both temporal resolution and multiplexed measurements from the same cell for the different types of devices. The distinct and complementary capabilities of Gra- and NW-FETs could open up unique opportunities in the field of bioelectronics in the future.

Intracellular recordings of action potentials by an extracellular nanoscale field-effect transistor

The ability to make electrical measurements inside cells has led to many important advances in electrophysiology1-6. The patch clamp technique, in which a glass micropipette filled with electrolyte is inserted into a cell, offers both high signal-to-noise ratio and temporal resolution1,2. Ideally the micropipette should be as small as possible to increase the spatial resolution and reduce the invasiveness of the measurement, but the overall performance of the technique depends on the impedance of the interface between the micropipette and the cell interior1,2, which limits how small the micropipette can be. Techniques that involve inserting metal or carbon microelectrodes into cells are subject to similar constraints4,7-9. Field-effect transistors (FETs) can also record electric potentials inside cells10, and since their performance does not depend on impedance11,12, they can be made much smaller than micropipettes and microelectrodes. Moreover, FET arrays are better suited for multiplexed measurements. Previously we have demonstrated FET-based intracellular recording with kinked nanowire structures10, but the kink configuration and device design places limits on the probe size and the potential for multiplexing. Here we report a new approach where a SiO2 nanotube is synthetically integrated on top of a nanoscale FET. After penetrating the cell membrane, the SiO2 nanotube brings the cell cytosol into contact with the FET and enables the recording of intracellular transmembrane potential. Simulations show that the bandwidth of this branched intracellular nanotube FET (BIT-FET) is high enough for it to record fast action potentials even when the nanotube diameter is decreased to 3 nm, a length scale which is well below that accessible with other methods1,2,4. Studies of cardiomyocyte cells demonstrate that when brought close, the nanotubes of phospholipid-modified BIT-FETs spontaneously penetrate the cell membrane to yield stable, full-amplitude intracellular action potential recording, showing that a stable tight seal forms between the nanotube and cell membrane. We also show that multiple BIT-FETs can record multiplexed intracellular signals from both single cells and networks of cells.

Electrical Recording from Hearts with Flexible Nanowire Device Arrays

We show that nanowire field-effect transistor (NWFET) arrays fabricated on both planar and flexible polymeric substrates can be reproducibly interfaced with spontaneously beating embryonic chicken hearts in both planar and bent conformations. Simultaneous recordings from glass microelectrode and NWFET devices show that NWFET conductance variations are synchronized with the beating heart. The conductance change associated with beating can be tuned substantially by device sensitivity, although the voltage-calibrated signals, 4-6 mV, are relatively constant and typically larger than signals recorded by microelectrode arrays. Multiplexed recording from NWFET arrays yielded signal propagation times across the myocardium with high spatial resolution. The transparent and flexible NWFET chips also enable simultaneous electrical recording and optical registration of devices to heart surfaces in three-dimensional conformations not possible with planar microdevices. The capability of simultaneous optical imaging and electrical recording also could be used to register devices to a specific region of the myocardium at the cellular level, and more generally, NWFET arrays fabricated on increasingly flexible plastic and/or biopolymer substrates have the potential to become unique tools for electrical recording from other tissue/organ samples or as powerful implants.

Flexible electrical recording from cells using nanowire transistor arrays

Semiconductor nanowires (NWs) have unique electronic properties and sizes comparable with biological structures involved in cellular communication, thus making them promising nanostructures for establishing active interfaces with biological systems. We report a flexible approach to interface NW field-effect transistors (NWFETs) with cells and demonstrate this for silicon NWFET arrays coupled to embryonic chicken cardiomyocytes. Cardiomyocyte cells were cultured on thin, optically transparent polydimethylsiloxane (PDMS) sheets and then brought into contact with Si-NWFET arrays fabricated on standard substrates. NWFET conductance signals recorded from cardiomyocytes exhibited excellent signal-to-noise ratios with values routinely >5 and signal amplitudes that were tuned by varying device sensitivity through changes in water gate–voltage potential, Vg. Signals recorded from cardiomyocytes for Vg from −0.5 to +0.1 V exhibited amplitude variations from 31 to 7 nS whereas the calibrated voltage remained constant, indicating a robust NWFET/cell interface. In addition, signals recorded as a function of increasing/decreasing displacement of the PDMS/cell support to the device chip showed a reversible >2× increase in signal amplitude (calibrated voltage) from 31 nS (1.0 mV) to 72 nS (2.3 mV). Studies with the displacement close to but below the point of cell disruption yielded calibrated signal amplitudes as large as 10.5 ± 0.2 mV. Last, multiplexed recording of signals from NWFET arrays interfaced to cardiomyocyte monolayers enabled temporal shifts and signal propagation to be determined with good spatial and temporal resolution. Our modular approach simplifies the process of interfacing cardiomyocytes and other cells to high-performance Si-NWFETs, thus increasing the experimental versatility of NWFET arrays and enabling device registration at the subcellular level.

Nanowire transistor arrays for mapping neural circuits in acute brain slices

Revealing the functional connectivity in natural neuronal networks is central to understanding circuits in the brain. Here, we show that silicon nanowire field-effect transistor (Si NWFET) arrays fabricated on transparent substrates can be reliably interfaced to acute brain slices. NWFET arrays were readily designed to record across a wide range of length scales, while the transparent device chips enabled imaging of individual cell bodies and identification of areas of healthy neurons at both upper and lower tissue surfaces. Simultaneous NWFET and patch clamp studies enabled unambiguous identification of action potential signals, with additional features detected at earlier times by the nanodevices. NWFET recording at different positions in the absence and presence of synaptic and ion-channel blockers enabled assignment of these features to presynaptic firing and postsynaptic depolarization from regions either close to somata or abundant in dendritic projections. In all cases, the NWFET signal amplitudes were from 0.3–3 mV. In contrast to conventional multielectrode array measurements, the small active surface of the NWFET devices, ∼0.06 μm2, provides highly localized multiplexed measurements of neuronal activities with demonstrated sub-millisecond temporal resolution and, significantly, better than 30 μm spatial resolution. In addition, multiplexed mapping with 2D NWFET arrays revealed spatially heterogeneous functional connectivity in the olfactory cortex with a resolution surpassing substantially previous electrical recording techniques. Our demonstration of simultaneous high temporal and spatial resolution recording, as well as mapping of functional connectivity, suggest that NWFETs can become a powerful platform for studying neural circuits in the brain.

Slow DNA Transport through Nanopores in Hafnium Oxide Membranes

We present a study of double- and single-stranded DNA transport through nanopores fabricated in ultrathin (2-7 nm thick) freestanding hafnium oxide (HfO2) membranes. The high chemical stability of ultrathin HfO2 enables long-lived experiments with <2 nm diameter pores that last several hours, in which we observe >50 000 DNA translocations with no detectable pore expansion. Mean DNA velocities are slower than velocities through comparable silicon nitride pores, providing evidence that HfO2 nanopores have favorable physicochemical interactions with nucleic acids that can be leveraged to slow down DNA in a nanopore.

Outside Looking In: Nanotube Transistor Intracellular Sensors

Nanowire-based field-effect transistors, including devices with planar and three-dimensional configurations, are being actively explored as detectors for extra- and intracellular recording due to their small size and high sensitivities. Here we report the synthesis, fabrication, and characterization of a new needle-shaped nanoprobe based on an active silicon nanotube transistor, ANTT, that enables high-resolution intracellular recording. In the ANTT probe, the source/drain contacts to the silicon nanotube are fabricated on one end, passivated from external solution, and then time-dependent changes in potential can be recorded from the opposite nanotube end via the solution filling the tube. Measurements of conductance versus water-gate potential in aqueous solution show that the ANTT probe is selectively gated by potential changes within the nanotube, thus demonstrating the basic operating principle of the ANTT device. Studies interfacing the ANTT probe with spontaneously beating cardiomyocytes yielded stable intracellular action potentials similar to those reported by other electrophysiological techniques. In addition, the straightforward fabrication of ANTT devices was exploited to prepare multiple ANTT structures at the end of single probes, which enabled multiplexed recording of intracellular action potentials from single cells and multiplexed arrays of single ANTT device probes. These studies open up unique opportunities for multisite recordings from individual cells through cellular networks.

Nanocomposite Gold-Silk Nanofibers

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNF(seed)). Following gold reduction, there was a 2-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFs(Au)). The Youngs modulus of the SNFs(Au) was almost 70% higher than that of SNFs. SNFs(Au) were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNF(Au) had a more than 2-fold larger cell area compared to the cells cultured on bare SNFs; SNF(Au) also increased cell size. This approach may be used to alter the cell-material interface in tissue engineering and other applications.

The Smartest Materials: The Future of Nanoelectronics in Medicine

Electronics have become central to many aspects of biomedicine, ranging from fundamental biophysical studies of excitable tissues to medical monitoring and electronic implants to restore limb movement. The development of new materials and approaches is needed to enable enhanced tissue integration, interrogation, and stimulation and other functionalities. Nanoscale materials offer many avenues for progress in this respect. New classes of molecular-scale bioelectronic interfaces can be constructed using either one-dimensional nanostructures, such as nanowires and nanotubes, or two-dimensional nanostructures, such as graphene. Nanodevices can create ultrasensitive sensors and can be designed with spatial resolution as fine as the subcellular regime. Structures on the nanoscale can enable the development of engineered tissues within which sensing elements are integrated as closely as the nervous system within native tissues. In addition, the close integration of nanomaterials with cells and tissues will also allow the development of in vitro platforms for basic research or diagnostics. Such lab-on-a-chip systems could, for example, enable testing of the effects of candidate therapeutic molecules on intercellular, single-cell, and even intracellular physiology. Finally, advances in nanoelectronics can lead to extremely sophisticated smart materials with multifunctional capabilities, enabling the spectrum of biomedical possibilities from diagnostic studies to the creation of cyborgs.