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Significance Modulation of cellular electrophysiology helps develop an understanding of cellular development and function in healthy and diseased states. We modulate the electrophysiology of neuronal cells in two-dimensional (2D) and 3D assemblies with subcellular precision via photothermal stimulation using a multiscale fuzzy graphene nanostructure. Nanowire (NW)-templated 3D fuzzy graphene (NT-3DFG) nanostructures enable remote, nongenetic photothermal stimulation with laser energies as low as subhundred nanojoules without generating cellular stress. NT-3DFG serves as a powerful toolset for studies of cell signaling within and between in vitro 3D models (human-based organoids and spheroids) and can enable therapeutic interventions., The ability to modulate cellular electrophysiology is fundamental to the investigation of development, function, and disease. Currently, there is a need for remote, nongenetic, light-induced control of cellular activity in two-dimensional (2D) and three-dimensional (3D) platforms. Here, we report a breakthrough hybrid nanomaterial for remote, nongenetic, photothermal stimulation of 2D and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photothermal stimulation at subcellular precision without the need for genetic modification, with laser energies lower than a hundred nanojoules, one to two orders of magnitude lower than Au-, C-, and Si-based nanomaterials. Photothermal stimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves as a powerful toolset for studies of cell signaling within and between tissues and can enable therapeutic interventions.

Microelectrode arrays (MEAs) have enabled investigation of cellular networks at sub-millisecond temporal resolution. However, current MEAs are limited by the large electrode footprint since reducing the electrode's geometric area to sub-cellular dimensions leads to a significant increase in impedance thus affecting its recording capabilities. We report a breakthrough ultra-microelectrodes platform by leveraging the outstanding surface-to-volume ratio of nanowire-templated out-of-plane synthesized three-dimensional fuzzy graphene (NT-3DFG). The enormous surface area of NT-3DFG leads to 140-fold reduction in electrode impedance compared to bare Au microelectrodes, thus enabling scaling down the geometric size by 625-fold to ca. 2 μm × 2 μm. The out-of-plane morphology of NT-3DFG leads to a tight seal with the cell membrane thus enabling recording of electrical signals with high signal-to-noise ratio (SNR) of > 6. This work highlights the possibility to push the limits of the conventional MEA technology to enable electrophysiological investigation at sub-cellular level without the need of any surface coatings. This presented approach would greatly impact our basic understanding of signal transduction within a single cell as well as complex cellular assemblies.

Fuzzy graphene and cellular optoporation enable intracellular recordings of cardiac action potentials on microelectrode arrays., Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.

Objective. Three-dimensional (3D) neuronal spheroid culture serves as a powerful model system for the investigation of neurological disorders and drug discovery. The success of such a model system requires techniques that enable high-resolution functional readout across the entire spheroid. Conventional microelectrode arrays and implantable neural probes cannot monitor the electrophysiology (ephys) activity across the entire native 3D geometry of the cellular construct. Approach. Here, we demonstrate a 3D self-rolled biosensor array (3D-SR-BA) integrated with a 3D cortical spheroid culture for simultaneous in vitro ephys recording, functional Ca2+ imaging, while monitoring the effect of drugs. We have also developed a signal processing pipeline to detect neural firings with high spatiotemporal resolution from the ephys recordings based on established spike sorting methods. Main results. The 3D-SR-BAs cortical spheroid interface provides a stable, high sensitivity recording of neural action potentials (µV peak-to-peak amplitude). The 3D-SR-BA is demonstrated as a potential drug screening platform through the investigation of the neural response to the excitatory neurotransmitter glutamate. Upon addition of glutamate, the neural firing rates increased notably corresponding well with the functional Ca2+ imaging. Significance. Our entire system, including the 3D-SR-BA integrated with neuronal spheroid culture, enables simultaneous ephys recording and functional Ca2+ imaging with high spatiotemporal resolution in conjunction with chemical stimulation. We demonstrate a powerful toolset for future studies of tissue development, disease progression, and drug testing and screening, especially when combined with native spheroid cultures directly extracted from humans.

Dopamine (DA) is a monoamine neurotransmitter involved in the modulation of various physiological brain functions, including learning, motivation, reward, and motor functions. The development of a high sensitivity real-time sensor for multi-site detection of DA with high spatial resolution has critical implications for both neuroscience and clinical communities to improve understanding and treatments of neurological and neuropsychiatric disorders. Here, we present high-surface area out-of-plane grown three-dimensional (3D) fuzzy graphene (3DFG) microelectrode arrays (MEAs) for highly selective, sensitive, and stable DA electrochemical sensing. 3DFG microelectrodes present a remarkable sensitivity to DA (2.87 ± 0.25 nA/nM, withLOD of 990±15 pM), the highest reported for nanocarbon MEAs using Fast Scan Cyclic Voltammetry (FSCV). The high surface area of 3DFG allows for miniaturization of electrode down to 2 x 2 μm^2, without compromising the electrochemical performance. Moreover, 3DFG MEAs are electrochemically stable under 7.2 million scans of continuous FSCV cycling, present exceptional selectivity over the most common interferents in vitro with minimum fouling by electrochemical byproducts, and can discriminate DA and serotonin (5-HT) in response to the injection of their 50:50 mixture. These results highlight the potential of 3DFG MEAs as a promising platform for FSCV based multi-site detection of DA with high sensitivity, selectivity, and spatial resolution.

Characterizing the electrical activity of cardiomyocytes and neurons is crucial in understanding the complex processes in the heart and brain tissues, both in healthy and diseased states. Micro- and nanotechnologies have significantly improved the electrophysiological investigation of cellular networks. Carbon-based nanomaterials or nanocarbons, such as carbon nanotubes (CNTs), nanodiamonds (NDs) and graphene are promising building blocks for bioelectronics platforms owing to their outstanding chemical and physical properties. In this review, we discuss the various bioelectronics applications of nanocarbons and their derivatives. Furthermore, we touch upon the challenges that remain in the field and describe the emergence of carbon-based hybrid-nanomaterials that will potentially address those limitations, thus improving the capabilities to investigate the electrophysiology of excitable cells, both as a network and at the single cell level.

The development of a high sensitivity real-time sensor for multi-site detection of dopamine (DA) with high spatial and temporal resolution is of fundamental importance to study the complex spatial and temporal pattern of DA dynamics in the brain, thus improving the understanding and treatments of neurological and neuropsychiatric disorders. In response to this need, here we present high surface area out-of-plane grown three-dimensional (3D) fuzzy graphene (3DFG) microelectrode arrays (MEAs) for highly selective, sensitive, and stable DA electrochemical sensing. 3DFG microelectrodes present a remarkable sensitivity to DA (2.12 ± 0.05 nA/nM, with LOD of 364.44 ± 8.65 pM), the highest reported for nanocarbon MEAs using Fast Scan Cyclic Voltammetry (FSCV). The high surface area of 3DFG allows for miniaturization of electrode down to 2 × 2 μm2, without compromising the electrochemical performance. Moreover, 3DFG MEAs are electrochemically stable under 7.2 million scans of continuous FSCV cycling, present exceptional selectivity over the most common interferents in vitro with minimum fouling by electrochemical byproducts and can discriminate DA and serotonin (5-HT) in response to the injection of their 50:50 mixture. These results highlight the potential of 3DFG MEAs as a promising platform for FSCV based multi-site detection of DA with high sensitivity, selectivity, and spatial resolution.

In recent years graphene has drawn considerable research interest for biomedical applications. However, applications of graphene in biological systems also raise concerns about its possible toxicity. Here, by using live cell imaging techniques, we investigate the effect of pristine graphene on the viability as well as stress of both nonneuronal and neuronal cells under physiological conditions. We find that graphene promotes cell adhesion and proliferation. Furthermore, we find that graphene has no detectable adverse effect on mitochondrial membrane potential and morphology, or autophagy levels in the cell, indicating that graphene does not induce cell stress. Our results highlight the potential of graphene to be used in biomedical applications by providing long-term and stable nonneural and neural interfaces.

An organ-on-electronic chip (organ-on-e-chip)—a three-dimensional (3D) biosensor array to decipher tissue electrical activity., Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.

The ability to modulate cellular electrophysiology is a fundamental aspect for investigation of tissue development, maturation and function. Currently, there is a need for remote, non-genetic, light-induced control of cellular activity in native-like state, three-dimensional (3D) tissues such as spheroids and organoids. Here, we report a breakthrough hybrid-nanomaterial for remote, non-genetic photostimulation of both two-dimensional (2D) and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photostimulation at subcellular precision with laser energies lower than hundred nanojoules, 1-2 orders of magnitude lower than Au-, C- and Si-based photostimulation. Photostimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves a novel powerful toolset for studies of cell signaling within and between tissues and can enable new therapeutic interventions.

Characterizing the electrical activity between neuronal cells is crucial in understanding the complex processes in the brain, both in healthy and diseased tissue. Neural interface technology that enables recording of the neuronal electrical activity as well as stimulation of the neurons has attracted great attention for both experimental and clinical applications. In this article, we discuss the fundamentals of the bioelectrical signals recording, and the advancements in the field of nano-bioelectronics, that is, the different kinds of materials and designs used to improve the cellular-device interface to enable recording and stimulation of the neuronal cells. Furthermore, we discuss the development of synthetic biomaterials that enable fusion of electronics and bioactive scaffolds which are essential to regenerative engineering. We also discuss the technical and scientific challenges associated with these technologies, and the future prospects and opportunities.

INTRODUCTION: Cell–cell communication plays a pivotal role in biological systems’ coordination and function. Electrical properties have been linked to specification and differentiation of stem cells into targeted progeny, such as neurons and cardiomyocytes. Currently, there is a critical need in developing new ways to complement fluorescent indicators, such as Ca(2+)-sensitive dyes, for direct electrophysiological measurements of cells and tissue. Here, we report a unique transparent and biocompatible graphene-based electrical platform that enables electrical and optical investigation of human embryonic stem cell-derived cardiomyocytes’ (hESC-CMs) intracellular processes and intercellular communication. METHODS: Graphene, a honeycomb sp(2) hybridized two-dimensional carbon lattice, was synthesized using low pressure chemical vapor deposition system, and was tested for biocompatibility. Au and graphene microelectrode arrays (MEAs) were fabricated using well-established microfabrication methods. Au and graphene MEAs were interfaced with hESC-CMs to perform both optical and electrical recordings. RESULTS: Optical imaging and Raman spectroscopy confirmed the presence of monolayer graphene. Viability assay showed biocompatibility of graphene. Electrochemical characterization proved graphene’s functional activity. Nitric acid treatment further enhanced the electrochemical properties of graphene. Graphene electrodes’ transparency enabled both optical and electrical recordings from hESC-CMs. Graphene MEA detected changes in beating frequency and field potential duration upon β-adrenergic receptor agonist treatment. CONCLUSION: The transparent graphene platform enables the investigation of both intracellular and intercellular communication processes and will create new avenues for bidirectional communication (sensing and stimulation) with electrically active tissues and will set the ground for investigations reported diseases such as Alzheimer, Parkinson’s disease and arrhythmias. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-018-0525-z) contains supplementary material, which is available to authorized users.

For more than a century, blood agar plates have been the only test for beta-hemolysis. Although blood agar cultures are highly predictive for bacterial pathogens, they are too slow to yield actionable information. Here, we show that beta-hemolytic pathogens are able to lyse and release fluorophores encapsulated in sterically stabilized liposomes whereas alpha and gamma-hemolytic bacteria have no effect. By analyzing fluorescence kinetics, beta-hemolytic colonies cultured on agar could be distinguished in real time with 100% accuracy within 6 h. Additionally, end point analysis based on fluorescence intensity and machine-extracted textural features could discriminate between beta-hemolytic and cocultured control colonies with 99% accuracy. In broth cultures, beta-hemolytic bacteria were detectable in under an hour while control bacteria remained negative even the next day. This strategy, called beta-hemolysis triggered-release assay (BETA) has the potential to enable the same-day detection of beta-hemolysis with single-cell sensitivity and high accuracy.

Graphene, a honeycomb sp2 hybridized carbon lattice, is a promising building block for hybrid-nanomaterials due to its electrical, mechanical, and optical properties. Graphene can be readily obtained through mechanical exfoliation, solution-based deposition of reduced graphene oxide (rGO), and chemical vapor deposition (CVD). The resulting graphene films’ topology is two-dimensional (2D) surface. Recently, synthesis of three-dimensional (3D) graphitic networks supported or templated by nanoparticles, foams, and hydrogels was reported. However, the resulting graphene films lay flat on the surface, exposing 2D surface topology. Out-of-plane grown carbon nanostructures, such as vertically aligned graphene sheets (VAGS) and vertical carbon nanowalls (CNWs), are still tethered to 2D surface. 3D morphology of out-of-plane growth of graphene hybrid-nanomaterials which leverages graphene's outstanding surface-to-volume ratio has not been achieved to date. Here we demonstrate highly controlled synthesis of 3D out-...

In recent years, there has been a growing interest in using graphene as a synthesis platform for polymers, zero-dimensional (0D) materials, one-dimensional materials (1D), and two-dimensional (2D) materials. Here, we report the investigation of the growth of germanium nanowires (GeNWs) and germanium nanocrawlers (GeNCs) on single-layer graphene surfaces. GeNWs and GeNCs are synthesized on graphene films by gold nanoparticles catalyzed vapor-liquid-solid growth mechanism. The addition of hydrogen chloride gas (HCl) at the nucleation step increased the propensity toward GeNCs growth on the surface. As the time lag before HCl introduction during the nucleation step increased, a significant change in the number of out-of-plane GeNWs versus in-plane GeNCs was observed. The nucleation temperature and time played a key role in the formation of GeNCs as well. The fraction of GeNCs (χNCs) decreased from 0.95 ± 0.01 to 0.66 ± 0.07 when the temperature was kept at 305 °C for 15 s versus maintained at 305 °C throughout the process, respectively. GeNCs exhibit ⟨112⟩ as the preferred growth direction whereas GeNWs exhibit both ⟨112⟩ and ⟨111⟩ as the preferred growth directions. Finally, our growth model suggests a possible mechanism for the preference of an in-plane GeNC growth on graphene versus GeNW on SiO2. These findings open up unique opportunities for fundamental studies of crystal growth on graphene, as well as enable exploration of new electronic interfaces between group IV materials and graphene, potentially toward designing new geometries for hybrid materials sensors.