Nanocrown electrodes for parallel and robust intracellular recording of cardiomyocytes.

Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.

Graphene Electric Field Sensor Enables Single Shot Label-Free Imaging of Bioelectric Potentials.

The measurement of electrical activity across systems of excitable cells underlies current progress in neuroscience, cardiac pharmacology, and neurotechnology. However, bioelectricity spans orders of magnitude in intensity, space, and time, posing substantial technological challenges. The development of methods permitting network-scale recordings with high spatial resolution remains key to studies of electrogenic cells, emergent networks, and bioelectric computation. Here, we demonstrate single-shot and label-free imaging of extracellular potentials with high resolution across a wide field-of-view. The critically coupled waveguide-amplified graphene electric field (CAGE) sensor leverages the field-sensitive optical transitions in graphene to convert electric potentials into the optical regime. As a proof-of-concept, we use the CAGE sensor to detect native electrical activity from cardiac action potentials with tens-of-microns resolution, simultaneously map the propagation of these potentials at tissue-scale, and monitor their modification by pharmacological agents. This platform is robust, scalable, and compatible with existing microscopy techniques for multimodal correlative imaging.

Label-free optical detection of bioelectric potentials using electrochromic thin films.

Understanding how a network of interconnected neurons receives, stores, and processes information in the human brain is one of the outstanding scientific challenges of our time. The ability to reliably detect neuroelectric activities is essential to addressing this challenge. Optical recording using voltage-sensitive fluorescent probes has provided unprecedented flexibility for choosing regions of interest in recording neuronal activities. However, when recording at a high frame rate such as 500 to 1,000 Hz, fluorescence-based voltage sensors often suffer from photobleaching and phototoxicity, which limit the recording duration. Here, we report an approach called electrochromic optical recording (ECORE) that achieves label-free optical recording of spontaneous neuroelectrical activities. ECORE utilizes the electrochromism of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) thin films, whose optical absorption can be modulated by an applied voltage. Being based on optical reflection instead of fluorescence, ECORE offers the flexibility of an optical probe without suffering from photobleaching or phototoxicity. Using ECORE, we optically recorded spontaneous action potentials in cardiomyocytes, cultured hippocampal and dorsal root ganglion neurons, and brain slices. With minimal perturbation to cells, ECORE allows long-term optical recording over multiple days.

Membrane curvature underlies actin reorganization in response to nanoscale surface topography.

Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at ∼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior.

A nanostructure platform for live-cell manipulation of membrane curvature.

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.

Soft conductive micropillar electrode arrays for biologically relevant electrophysiological recording.

Multielectrode arrays (MEAs) are essential tools in neural and cardiac research as they provide a means for noninvasive, multiplexed recording of extracellular field potentials with high temporal resolution. To date, the mechanical properties of the electrode material, e.g., its Young's modulus, have not been taken into consideration in most MEA designs leaving hard materials as the default choice due to their established fabrication processes. However, the cell-electrode interface is known to significantly affect some aspects of the cell's behavior. In this paper, we describe the fabrication of a soft 3D micropillar electrode array. Using this array, we proceed to successfully record action potentials from monolayer cell cultures. Specifically, our conductive hydrogel micropillar electrode showed improved signal amplitude and signal-to-noise ratio, compared with conventional hard iridium oxide micropillar electrodes of the same diameter. Taken together, our fabricated soft micropillar electrode array will provide a tissue-like Young's modulus and thus a relevant mechanical microenvironment to fundamental cardiac and neural studies.

An integrated self-healable electronic skin system fabricated via dynamic reconstruction of a nanostructured conducting network.

Electronic skin devices capable of monitoring physiological signals and displaying feedback information through closed-loop communication between the user and electronics are being considered for next-generation wearables and the 'Internet of Things'. Such devices need to be ultrathin to achieve seamless and conformal contact with the human body, to accommodate strains from repeated movement and to be comfortable to wear. Recently, self-healing chemistry has driven important advances in deformable and reconfigurable electronics, particularly with self-healable electrodes as the key enabler. Unlike polymer substrates with self-healable dynamic nature, the disrupted conducting network is unable to recover its stretchability after damage. Here, we report the observation of self-reconstruction of conducting nanostructures when in contact with a dynamically crosslinked polymer network. This, combined with the self-bonding property of self-healing polymer, allowed subsequent heterogeneous multi-component device integration of interconnects, sensors and light-emitting devices into a single multi-functional system. This first autonomous self-healable and stretchable multi-component electronic skin paves the way for future robust electronics.

Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization.

The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.

Constructing Highly Uniform Onion-Ring-like Graphitic Carbon Nitride for Efficient Visible-Light-Driven Photocatalytic Hydrogen Evolution.

The introduction of microstructure to the metal-free graphitic carbon nitride (g-C3N4) photocatalyst holds promise in enhancing its catalytic performance. However, producing such microstructured g-C3N4 remains technically challenging due to a complicated synthetic process and high cost. In this study, we develop a facile and in-air chemical vapor deposition (CVD) method that produces onion-ring-like g-C3N4 microstructures in a simple, reliable, and economical manner. This method involves the use of randomly packed 350 nm SiO2 microspheres as a hard template and melamine as a CVD precursor for the deposition of a thin layer of g-C3N4 in the narrow space between the SiO2 microspheres. After dissolution of the microsphere template, the resultant g-C3N4 exhibits uniquely uniform onion-ring-like microstructures. Unlike previously reported g-C3N4 powder morphologies that show various degrees of agglomeration and irregularity, the onion-ring-like g-C3N4 is highly dispersed and uniform. The calculated band gap for onion-ring-like g-C3N4 is 2.58 eV, which is significantly narrower than that of bulk g-C3N4 at 2.70 eV. Experimental characterization and testing suggest that, in comparison with bulk g-C3N4, onion-ring-like g-C3N4 facilitates charge separation, extends the lifetime of photoinduced carriers, exhibits 5-fold higher photocatalytic hydrogen evolution, and shows great potential for photocatalytic applications.

Interfacing Cells with Vertical Nanoscale Devices: Applications and Characterization.

Measurements of the intracellular state of mammalian cells often require probes or molecules to breach the tightly regulated cell membrane. Mammalian cells have been shown to grow well on vertical nanoscale structures in vitro, going out of their way to reach and tightly wrap the structures. A great deal of research has taken advantage of this interaction to bring probes close to the interface or deliver molecules with increased efficiency or ease. In turn, techniques have been developed to characterize this interface. Here, we endeavor to survey this research with an emphasis on the interface as driven by cellular mechanisms.

Biocompatible and totally disintegrable semiconducting polymer for ultrathin and ultralightweight transient electronics.

Increasing performance demands and shorter use lifetimes of consumer electronics have resulted in the rapid growth of electronic waste. Currently, consumer electronics are typically made with nondecomposable, nonbiocompatible, and sometimes even toxic materials, leading to serious ecological challenges worldwide. Here, we report an example of totally disintegrable and biocompatible semiconducting polymers for thin-film transistors. The polymer consists of reversible imine bonds and building blocks that can be easily decomposed under mild acidic conditions. In addition, an ultrathin (800-nm) biodegradable cellulose substrate with high chemical and thermal stability is developed. Coupled with iron electrodes, we have successfully fabricated fully disintegrable and biocompatible polymer transistors. Furthermore, disintegrable and biocompatible pseudo-complementary metal-oxide-semiconductor (CMOS) flexible circuits are demonstrated. These flexible circuits are ultrathin (<1 µm) and ultralightweight (∼2 g/m2) with low operating voltage (4 V), yielding potential applications of these disintegrable semiconducting polymers in low-cost, biocompatible, and ultralightweight transient electronics.

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease.

The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.

Imaging electric field dynamics with graphene optoelectronics.

The use of electric fields for signalling and control in liquids is widespread, spanning bioelectric activity in cells to electrical manipulation of microstructures in lab-on-a-chip devices. However, an appropriate tool to resolve the spatio-temporal distribution of electric fields over a large dynamic range has yet to be developed. Here we present a label-free method to image local electric fields in real time and under ambient conditions. Our technique combines the unique gate-variable optical transitions of graphene with a critically coupled planar waveguide platform that enables highly sensitive detection of local electric fields with a voltage sensitivity of a few microvolts, a spatial resolution of tens of micrometres and a frequency response over tens of kilohertz. Our imaging platform enables parallel detection of electric fields over a large field of view and can be tailored to broad applications spanning lab-on-a-chip device engineering to analysis of bioelectric phenomena.

A skin-inspired organic digital mechanoreceptor.

Human skin relies on cutaneous receptors that output digital signals for tactile sensing in which the intensity of stimulation is converted to a series of voltage pulses. We present a power-efficient skin-inspired mechanoreceptor with a flexible organic transistor circuit that transduces pressure into digital frequency signals directly. The output frequency ranges between 0 and 200 hertz, with a sublinear response to increasing force stimuli that mimics slow-adapting skin mechanoreceptors. The output of the sensors was further used to stimulate optogenetically engineered mouse somatosensory neurons of mouse cortex in vitro, achieving stimulated pulses in accordance with pressure levels. This work represents a step toward the design and use of large-area organic electronic skins with neural-integrated touch feedback for replacement limbs.

Impact of branching on the supramolecular assembly of thioethers on Au(111).

Alkanethiolate monolayers are one of the most comprehensively studied self-assembled systems due to their ease of preparation, their ability to be functionalized, and the opportunity to control their thickness perpendicular to the surface. However, these systems suffer from degradation due to oxidation and defects caused by surface etching and adsorbate rotational boundaries. Thioethers offer a potential alternative to thiols that overcome some of these issues and allow dimensional control of self-assembly parallel to the surface. Thioethers have found uses in surface modification of nanoparticles, and chiral thioethers tethered to catalytically active surfaces have been shown to enable enantioselective hydrogenation. However, the effect of structural, chemical, and chiral modifications of the alkyl chains of thioethers on their self-assembly has remained largely unstudied. To elucidate how molecular structure, particularly alkyl branching and chirality, affects molecular self-assembly, we compare four related thioethers, including two pairs of structural isomers. The self-assembly of structural isomers N-butyl methyl sulfide and tert-butyl methyl sulfide was studied with high resolution scanning tunneling microscopy (STM); our results indicate that both molecules form highly ordered arrays despite the bulky tert-butyl group. We also investigated the effect of intrinsic chirality in the alkyl tails on the adsorption and self-assembly of butyl sec-butyl sulfide (BSBS) with STM and density functional theory and contrast our results to its structural isomer, dibutyl sulfide. Calculations provide the relative stability of the four stereoisomers of BSBS and STM imaging reveals two prominent monomer forms. Interestingly, the racemic mixture of BSBS is the only thioether we have examined to date that does not form highly ordered arrays; we postulate that this is due to weak enantiospecific intermolecular interactions that lead to the formation of energetically similar but structurally different assemblies. Furthermore, we studied all of the molecules in their monomeric molecular rotor form, and the surface-adsorbed chirality of the three asymmetric thioethers is distinguishable in STM images.

Experimental demonstration of a single-molecule electric motor.

For molecules to be used as components in molecular machines, methods that couple individual molecules to external energy sources and that selectively excite motion in a given direction are required. Significant progress has been made in the construction of molecular motors powered by light and by chemical reactions, but electrically driven motors have not yet been built, despite several theoretical proposals for such motors. Here we report that a butyl methyl sulphide molecule adsorbed on a copper surface can be operated as a single-molecule electric motor. Electrons from a scanning tunnelling microscope are used to drive the directional motion of the molecule in a two-terminal setup. Moreover, the temperature and electron flux can be adjusted to allow each rotational event to be monitored at the molecular scale in real time. The direction and rate of the rotation are related to the chiralities of both the molecule and the tip of the microscope (which serves as the electrode), illustrating the importance of the symmetry of the metal contacts in atomic-scale electrical devices.