3D Microelectrode Arrays Research Articles

Microelectrothermoforming (μETF): one-step versatile 3D shaping of flexible microelectronics for enhanced neural interfaces

Increasing the proximity of microelectrode arrays (MEA) to targeted neural tissues can establish efficient neural interfaces for both recording and stimulation applications. This has been achieved by constructing protruding three-dimensional (3D) structures on top of conventional planar microelectrodes via additional micromachining steps. However, this approach adds fabrication complexities and limits the 3D structures to certain shapes. We propose a one-step fabrication of MEAs with versatile microscopic 3D structures via “microelectrothermoforming (μETF)” of thermoplastics, by utilizing 3D-printed molds to locally deform planar MEAs into protruding and recessing shapes. Electromechanical optimization enabled a 3D MEA with 80 μm protrusions and/or recession for 100 μm diameter. Its simple and versatile shaping capabilities are demonstrated by diverse 3D structures on a single MEA. The benefits of 3D MEA are evaluated in retinal stimulation through numerical simulations and ex vivo experiments, confirming a threshold lowered by 1.7 times and spatial resolution enhanced by 2.2 times.

Highly Stretchable 3D Microelectrode Array for Noninvasive Functional Evaluation of Cardiac Spheroids and Midbrain Organoids.

Organoids are 3D biological models that recapitulate the complex structures and functions of human organs. Despite the rapid growth in the generation of organoids, in vitro assay tools are still limited to 2D forms. Thus, a comprehensive and continuous functional evaluation of the electrogenic organoids remains a challenge. Here, a highly stretchable 3D multielectrode array (sMEA) with protruding microelectrodes is presented for functional evaluation of electrogenic organoids. The optimized serpentine structures with bridge structures cover the surface of the organoids conformally even in immersion. The protruding microelectrodes form a stable contact with the organoids and allow electrophysiological recordings with high signal-to-noise ratio (SNR). sMEAs are fabricated in wafer-scale for repeatable, scalable, and mass production and packed into an easy-to-use, user-friendly, and robust microwell for fast dissemination of technology. The versatility of sMEA is validated by measuring electrophysiological signals from cardiac spheroids and midbrain organoids with a wide range of sizes from 500 to 1500µm. Also, electrophysiological signals recorded with high SNR enable functional evaluation of the effects of drugs. The proposed sMEA with high SNR and user-friendly interface could be the key player in high-throughput drug screening, 3D spatiotemporal mapping of electrogenic organoids, and standardization of protocols for quality assessment.

The e-Flower: A hydrogel-actuated 3D MEA for brain spheroid electrophysiology.

Traditional microelectrode arrays (MEAs) are limited to measuring electrophysiological activity in two dimensions, failing to capture the complexity of three-dimensional (3D) tissues such as neural organoids and spheroids. Here, we introduce a flower-shaped MEA (e-Flower) that can envelop submillimeter brain spheroids following actuation by the sole addition of the cell culture medium. Inspired by soft microgrippers, its actuation mechanism leverages the swelling properties of a polyacrylic acid hydrogel grafted to a polyimide substrate hosting the electrical interconnects. Compatible with standard electrophysiology recording systems, the e-Flower does not require additional equipment or solvents and is ready to use with preformed 3D tissues. We designed an e-Flower achieving a curvature as low as 300 micrometers within minutes, a value tunable by the choice of reswelling media and hydrogel cross-linker concentration. Furthermore, we demonstrate the ability of the e-Flower to detect spontaneous neural activity across the spheroid surface, demonstrating its potential for comprehensive neural signal recording.

Organotypic culture of post-mortem adult human brain explants exhibits synaptic plasticity

BackgroundSynaptic plasticity is an essential process encoding fine-tuned brain functions, but models to study this process in adult human systems are lacking. ObjectiveWe aim to test whether ex vivo organotypic culture of post-mortem adult brain explants (OPABs) retain synaptic plasticity. MethodsOPABs were seeded on 3D microelectrode arrays to measure local field potential (LFP). Paired stimulation of distant electrodes was performed over three days to investigate our capacity to modulate specific neuronal connections. ResultsLong-term potentiation (LTP) or depression (LTD) did not occur within a single day. In contrast, after two and three days of training, OPABs showed a significant modulation of the paired electrodes’ response compared to the non-paired electrodes from the same array. This response was alleviated upon treatment with dopamine. ConclusionOur work highlights that adult human brain explants retain synaptic plasticity, offering novel approaches to neural circuitry in animal-free models.

Inkjet‐Printed 3D Electrode Arrays for Recording Signals from Cortical Organoids

AbstractMonitoring electrical activity across multiple planes in 3D cell cultures and organoids is imperative to comprehensively understand their functional connections and behavior. However, traditional planar microelectrode arrays (MEAs) are intended for surface recordings and are inadequate in addressing this aspect. The limitations, such as longer production times and limited adaptability imposed by standard clean‐room techniques, constrain the design possibilities for 3D electrode arrays and potentially hinder effective cell‐electrode coupling. To tackle this challenge, a novel approach is presented that leverages rapid prototyping processes and additive manufacturing in combination with wet etching and electrodeposition to enhance electrode fabrication and performance. The laser‐patterned MEAs on glass, polyimide (PI) foil, or polyethylene terephthalate (PET) foil substrates incorporate high‐aspect ratio (up to 44:1) ink‐jet printed 3D electrode structures with heights up to 1 mm at a pitch of 200 µm, enabling precise recording within cell tissues. The specific shapes of the electrode tips and customizable 3D structures provide great flexibility in electrode placement. The versatility of the 3D MEAs is demonstrated by recording the electrophysiological activity of cortical organoids in situ, paving the way for investigating neural activity under regular or various pathologically altered conditions in vitro in a high throughput manner.

Highly stretchable and customizable microneedle electrode arrays for intramuscular electromyography.

Stretchable three-dimensional (3D) penetrating microelectrode arrays have potential utility in various fields, including neuroscience, tissue engineering, and wearable bioelectronics. These 3D microelectrode arrays can penetrate and conform to dynamically deforming tissues, thereby facilitating targeted sensing and stimulation of interior regions in a minimally invasive manner. However, fabricating custom stretchable 3D microelectrode arrays presents material integration and patterning challenges. In this study, we present the design, fabrication, and applications of stretchable microneedle electrode arrays (SMNEAs) for sensing local intramuscular electromyography signals ex vivo. We use a unique hybrid fabrication scheme based on laser micromachining, microfabrication, and transfer printing to enable scalable fabrication of individually addressable SMNEA with high device stretchability (60 to 90%). The electrode geometries and recording regions, impedance, array layout, and length distribution are highly customizable. We demonstrate the use of SMNEAs as bioelectronic interfaces in recording intramuscular electromyography from various muscle groups in the buccal mass of Aplysia.

Enhancing the Deposition Rate and Uniformity in 3D Gold Microelectrode Arrays via Ultrasonic-Enhanced Template-Assisted Electrodeposition.

In the pursuit of refining the fabrication of three-dimensional (3D) microelectrode arrays (MEAs), this study investigates the application of ultrasonic vibrations in template-assisted electrodeposition. This was driven by the need to overcome limitations in the deposition rate and the height uniformity of microstructures developed using conventional electrodeposition methods, particularly in the field of in vitro electrophysiological investigations. This study employs a template-assisted electrodeposition approach coupled with ultrasonic vibrations to enhance the deposition process. The method involves utilizing a polymeric hard mask to define the shape of electrodeposited microstructures (i.e., micro-pillars). The results show that the integration of ultrasonic vibrations significantly increases the deposition rate by up to 5 times and substantially improves the uniformity in 3D MEAs. The key conclusion drawn is that ultrasonic-enhanced template-assisted electrodeposition emerges as a powerful technique and enables the development of 3D MEAs at a higher rate and with a superior uniformity. This advancement holds promising implications for the precision of selective electrodeposition applications and signifies a significant stride in developing micro- and nanofabrication methodologies for biomedical applications.

Highly Customizable 3D Microelectrode Arrays for In Vitro and In Vivo Neuronal Tissue Recordings.

Planar microelectrode arrays (MEAs) for - in vitro or in vivo - neuronal signal recordings lack the spatial resolution and sufficient signal-to-noise ratio (SNR) required for a detailed understanding of neural network function and synaptic plasticity. To overcome these limitations, a highly customizable three-dimensional (3D) printing process is used in combination with thin film technology and a self-aligned template-assisted electrochemical deposition process to fabricate 3D-printed-based MEAs on stiff or flexible substrates. Devices with design flexibility and physical robustness are shown for recording neural activity in different in vitro and in vivo applications, achieving high-aspect ratio 3D microelectrodes of up to 33:1. Here, MEAs successfully record neural activity in 3D neuronal cultures, retinal explants, and the cortex of living mice, thereby demonstrating the versatility of the 3D MEA while maintaining high-quality neural recordings. Customizable 3D MEAs provide unique opportunities to study neural activity under regular or various pathological conditions, both in vitro and in vivo, and contribute to the development of drug screening and neuromodulation systems that can accurately monitor the activity of large neural networks over time.

Laser-patterned epoxy-based 3D microelectrode arrays for extracellular recording.

Microelectrode arrays are commonly used to study the electrophysiological behavior of cells. Recently, there has been a growing interest in fabricating three-dimensional microelectrode arrays. Here, we present a novel process for the fast fabrication of epoxy-based 3D microelectrode array platforms with the assistance of laser-patterning technology. To this end, we photopatterned 3D pillars as scaffolds using epoxy-based dry films. Electrodes and conductor traces were fabricated by laser patterning of sputtered platinum films on top of the 3D structures, followed by deposition of parylene-C for insulation. Microelectrodes at the tip of the 3D structures were exposed using a vertical laser ablation process. The final electrodes demonstrated a low impedance of ∼10 kΩ at 1 kHz in electrochemical impedance spectroscopy measurements under physiological conditions. We investigated the maximum compression force of the 3D structures, which could withstand approximately 0.6 N per pillar. The 3D microelectrode arrays were used to record extracellular signals from HL-1 cells in culture as a proof of principle. Our results show regular firing of action potentials recorded at the tip of the 3D structures, demonstrating the possibility of recording cell signals in non-planar environments.

Long-term in vitro culture of 3D brain tissue model based on chitosan thermogel

Methods for studying brain function and disease heavily rely on in vivo animal models, ex-vivo tissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects of in vivo human brain are needed as additional preclinical tools. In this context, 3D hydrogel-based in vitro models of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-β-glycerophosphate (CHITO-β-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-β-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-β-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

Development of multi-depth probing 3D microelectrode array to record electrophysiological activity within neural cultures

Microelectrode arrays (MEAs) play a crucial role in investigating the electrophysiological activities of neuronal populations. Although two-dimensional neuronal cell cultures have predominated in neurophysiology in monitoring in-vitro the electrophysiological activity, recent research shifted toward culture using three-dimensional (3D) neuronal network structures for developing more sophisticated and realistic neuronal models. Nevertheless, many challenges remain in the electrophysiological analysis of 3D neuron cultures, among them the development of robust platforms for investigating the electrophysiological signal at multiple depths of the 3D neurons’ networks. While various 3D MEAs have been developed to probe specific depths within the layered nervous system, the fabrication of microelectrodes with different heights, capable of probing neural activity from the surface as well as from the different layers within the neural construct, remains challenging. This study presents a novel 3D MEA with microelectrodes of different heights, realized through a multi-stage mold-assisted electrodeposition process. Our pioneering platform allows meticulous control over the height of individual microelectrodes as well as the array topology, paving the way for the fabrication of 3D MEAs consisting of electrodes with multiple heights that could be tailored for specific applications and experiments. The device performance was characterized by measuring electrochemical impedance, and noise, and capturing spontaneous electrophysiological activity from neurospheroids derived from human induced pluripotent stem cells. These evaluations unequivocally validated the significant potential of our innovative multi-height 3D MEA as an avant-garde platform for in vitro 3D neuronal studies.

Exploring retinal ganglion cells encoding to multi-modal stimulation using 3D microelectrodes arrays.

Microelectrode arrays (MEA) are extensively utilized in encoding studies of retinal ganglion cells (RGCs) due to their capacity for simultaneous recording of neural activity across multiple channels. However, conventional planar MEAs face limitations in studying RGCs due to poor coupling between electrodes and RGCs, resulting in low signal-to-noise ratio (SNR) and limited recording sensitivity. To overcome these challenges, we employed photolithography, electroplating, and other processes to fabricate a 3D MEA based on the planar MEA platform. The 3D MEA exhibited several improvements compared to planar MEA, including lower impedance (8.73 ± 1.66kΩ) and phase delay (-15.11° ± 1.27°), as well as higher charge storage capacity (CSC = 10.16 ± 0.81mC/cm2), cathodic charge storage capacity (CSCc = 7.10 ± 0.55mC/cm2), and SNR (SNR = 8.91 ± 0.57). Leveraging the advanced 3D MEA, we investigated the encoding characteristics of RGCs under multi-modal stimulation. Optical, electrical, and chemical stimulation were applied as sensory inputs, and distinct response patterns and response times of RGCs were detected, as well as variations in rate encoding and temporal encoding. Specifically, electrical stimulation elicited more effective RGC firing, while optical stimulation enhanced RGC synchrony. These findings hold promise for advancing the field of neural encoding.

Fully Integrated 3D Microelectrode Arrays with Polydopamine-Mediated Silicon Dioxide Insulation for Electrophysiological Interrogation of a Novel 3D Human, Neural Microphysiological Construct.

Advances within in vitro biological system complexity have enabled new possibilities for the "Organs-on-a-Chip" field. Microphysiological systems (MPS) as such incorporate sophisticated biological constructs with custom biological sensors. For microelectromechanical systems (MEMS) sensors, the dielectric layer is critical for device performance, where silicon dioxide (SiO2) represents an excellent candidate due to its biocompatibility and wide utility in MEMS devices. Yet, high temperatures traditionally preclude SiO2 from incorporation in polymer-based BioMEMS. Electron-beam deposition of SiO2 may provide a low-temperature, dielectric serving as a nanoporous MPS growth substrate. Herein, we enable improved adherence of nanoporous SiO2 to polycarbonate (PC) and 316L stainless steel (SS) via polydopamine (PDA)-mediated chemistry. The resulting stability of the combinatorial PDA-SiO2 film was interrogated, along with the nature of the intrafilm interactions. A custom polymer-metal three-dimensional (3D) microelectrode array (3D MEA) is then reported utilizing PDA-SiO2 insulation, for definition of novel dorsal root ganglion (DRG)/nociceptor and dorsal horn (DH) 3D neural constructs in excess of 6 months for the first time. Spontaneous/evoked compound action potentials (CAPs) are successfully reported. Finally, inhibitory drugs treatments showcase pharmacological responsiveness of the reported multipart biological activity. These results represent the initiation of a novel 3D MEA-integrated, 3D neural MPS for the long-term electrophysiological study.

Fabrication of a transparent array of penetrating 3D microelectrodes with two different heights for both neural stimulation and recording

Microelectrode array (MEA) is one of the representative platforms for recording and stimulating neurons to explore their populational electrophysiological activities. Due to the layered structures of the nervous system, various three-dimensional (3D) MEAs have been used to reach specific target depths of neuronal tissues. But it is still difficult to monolithically fabricate various heights of microelectrodes, particularly with optical transparency. Here, we present a 3D MEA with different heights fabricated by multi-step silicon deep reactive ion etching. The microelectrodes were embedded in reflowed glass for high transparency which enables both neural tissue observation and optical stimulation. The heights of microelectrodes were well controlled by our fabrication processes and electrical connections were made with through glass vias (TGVs) to the bottom side for individual addressing. To confine electric stimulation in a small area, we used microelectrodes neighboring a stimulation electrode as local returns. The feasibility of the fabricated MEA was demonstrated by stimulating the mouse retina and successfully recording spikes of ganglion cells located near the stimulation site. Consistent with our COMSOL simulation, the ex vivo experimental results showed the stimulation current confinement was effective. Our study provides a new tool for 3D nervous systems and its fabrication method.

Direct laser writing of 3D electrodes on flexible substrates

This report describes a 3D microelectrode array integrated on a thin-film flexible cable for neural recording in small animals. The fabrication process combines traditional silicon thin-film processing techniques and direct laser writing of 3D structures at micron resolution via two-photon lithography. Direct laser-writing of 3D-printed electrodes has been described before, but this report is the first to provide a method for producing high-aspect-ratio structures. One prototype, a 16-channel array with 300 µm pitch, demonstrates successful electrophysiological signal capture from bird and mouse brains. Additional devices include 90 µm pitch arrays, biomimetic mosquito needles that penetrate through the dura of birds, and porous electrodes with enhanced surface area. The rapid 3D printing and wafer-scale methods described here will enable efficient device fabrication and new studies examining the relationship between electrode geometry and electrode performance. Applications include small animal models, nerve interfaces, retinal implants, and other devices requiring compact, high-density 3D electrodes.

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

Organoid intelligence (OI): the new frontier in biocomputing and intelligence-in-a-dish

Recent advances in human stem cell-derived brain organoids promise to replicate critical molecular and cellular aspects of learning and memory and possibly aspects of cognition in vitro. Coining the term “organoid intelligence” (OI) to encompass these developments, we present a collaborative program to implement the vision of a multidisciplinary field of OI. This aims to establish OI as a form of genuine biological computing that harnesses brain organoids using scientific and bioengineering advances in an ethically responsible manner. Standardized, 3D, myelinated brain organoids can now be produced with high cell density and enriched levels of glial cells and gene expression critical for learning. Integrated microfluidic perfusion systems can support scalable and durable culturing, and spatiotemporal chemical signaling. Novel 3D microelectrode arrays permit high-resolution spatiotemporal electrophysiological signaling and recording to explore the capacity of brain organoids to recapitulate the molecular mechanisms of learning and memory formation and, ultimately, their computational potential. Technologies that could enable novel biocomputing models via stimulus-response training and organoid-computer interfaces are in development. We envisage complex, networked interfaces whereby brain organoids are connected with real-world sensors and output devices, and ultimately with each other and with sensory organ organoids (e.g. retinal organoids), and are trained using biofeedback, big-data warehousing, and machine learning methods. In parallel, we emphasize an embedded ethics approach to analyze the ethical aspects raised by OI research in an iterative, collaborative manner involving all relevant stakeholders. The many possible applications of this research urge the strategic development of OI as a scientific discipline. We anticipate OI-based biocomputing systems to allow faster decision-making, continuous learning during tasks, and greater energy and data efficiency. Furthermore, the development of “intelligence-in-a-dish” could help elucidate the pathophysiology of devastating developmental and degenerative diseases (such as dementia), potentially aiding the identification of novel therapeutic approaches to address major global unmet needs.

3D microelectrode arrays, pushing the bounds of sensitivity toward a generic platform for point-of-care diagnostics

The increased sensitivity of microelectrode arrays (MEAs) over macroelectrodes for biosensing is well established, and results from reducing the diffusion gradient of the target species to and from the electrode surfaces. The current study describes the fabrication and characterisation of a polymer-based MEA, which exploits the advantages of three dimensionality (3D). Firstly, the unique 3D formfactor promotes release of the gold tips from an inert layer in a controlled fashion, to form a highly reproducible array of microelectrodes in a single step. The 3D topography of the fabricated MEAs significantly enhances the diffusion profile of the target species to the electrode which results in higher sensitivity. Furthermore, the “sharpness” of the 3D structure induces differential current distribution that is focused at the apices of the individual electrodes, reducing the active area, and thereby overcoming the requirement for the electrodes to be sub-micron in size before true MEA behaviour can be achieved. The electrochemical characteristics of the 3D MEAs shows ideal micro-electrode behaviour, with a level of sensitivity of three orders of magnitude greater than that of enzyme-linked immunosorbent assays (ELISA), as the optical based gold standard. The application of the 3D MEAs uses the combination of enzyme-label and substrate approach employed in ELISAs as a generic basis for biosensing and can hence be applied to the plethora of targets that utilise the ELISA approach. As an example, the 3D MEAs are applied to the detection of RNA and demonstrate a level of detection down to single digit picomolar concentrations.

Inkjet-printed 3D micro-ring-electrode arrays for amperometric nanoparticle detection.

Chip-based impact electrochemistry can provide means to measure nanoparticles in solution by sensing their stochastic collisions on appropriately-polarized microelectrodes. However, a planar microelectrode array design still restricts the particle detection to the chip surface and does not allow detection in 3D environments. In this work, we report a fast fabrication process for 3D microelectrode arrays by combining ink-jet printing with laser-patterning. To this end, we printed 3D pillars from polyacrylate ink as a scaffold. Then, the metal structures are manufactured via sputtering and laser-ablation. Finally, the chip is passivated with a parylene-C layer and the electrode tips are created via laser-ablation in a vertical alignment. As a proof of principle, we employ our 3D micro-ring-electrode arrays for single impact recordings from silver nanoparticles.

A Novel 3D Helical Microelectrode Array for In Vitro Extracellular Action Potential Recording.

Recent advances in cell and tissue engineering have enabled long-term three-dimensional (3D) in vitro cultures of human-derived neuronal tissues. Analogous two-dimensional (2D) tissue cultures have been used for decades in combination with substrate integrated microelectrode arrays (MEA) for pharmacological and toxicological assessments. While the phenotypic and cytoarchitectural arguments for 3D culture are clear, 3D MEA technologies are presently inadequate. This is mostly due to the technical challenge of creating vertical electrical conduction paths (or ‘traces’) using standardized biocompatible materials and fabrication techniques. Here, we have circumvented that challenge by designing and fabricating a novel helical 3D MEA comprised of polyimide, amorphous silicon carbide (a-SiC), gold/titanium, and sputtered iridium oxide films (SIROF). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) testing confirmed fully-fabricated MEAs should be capable of recording extracellular action potentials (EAPs) with high signal-to-noise ratios (SNR). We then seeded induced pluripotent stems cell (iPSC) sensory neurons (SNs) in a 3D collagen-based hydrogel integrated with the helical MEAs and recorded EAPs for up to 28 days in vitro from across the MEA volume. Importantly, this highly adaptable design does not intrinsically limit cell/tissue type, channel count, height, or total volume.