Three-dimensional Microelectrode Arrays Research Articles

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.

Liquid-metal-based three-dimensional microelectrode arrays integrated with implantable ultrathin retinal prosthesis for vision restoration

Electronic retinal prostheses for stimulating retinal neurons are promising for vision restoration. However, the rigid electrodes of conventional retinal implants can inflict damage on the soft retina tissue. They also have limited selectivity due to their poor proximity to target cells in the degenerative retina. Here we present a soft artificial retina (thickness, 10 μm) where flexible ultrathin photosensitive transistors are integrated with three-dimensional stimulation electrodes of eutectic gallium–indium alloy. Platinum nanoclusters locally coated only on the tip of these three-dimensional liquid-metal electrodes show advantages in reducing the impedance of the stimulation electrodes. These microelectrodes can enhance the proximity to the target retinal ganglion cells and provide effective charge injections (72.84 mC cm−2) to elicit neural responses in the retina. Their low Young’s modulus (234 kPa), owing to their liquid form, can minimize damage to the retina. Furthermore, we used an unsupervised machine learning approach to effectively identify the evoked spikes to grade neural activities within the retinal ganglion cells. Results from in vivo experiments on a retinal degeneration mouse model reveal that the spatiotemporal distribution of neural responses on their retina can be mapped under selective localized illumination areas of light, suggesting the restoration of their vision.

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.

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.

Determination of Stiffness and the Elastic Modulus of 3D-Printed Micropillars with Atomic Force Microscopy-Force Spectroscopy.

Nowadays, many applications in diverse fields are taking advantage of micropillars such as optics, tribology, biology, and biomedical engineering. Among them, one of the most attractive is three-dimensional microelectrode arrays for in vivo and in vitro studies, such as cellular recording, biosensors, and drug delivery. Depending on the application, the micropillar's optimal mechanical response ranges from soft to stiff. For long-term implantable devices, a mechanical mismatch between the micropillars and the biological tissue must be avoided. For drug delivery patches, micropillars must penetrate the skin without breaking or bending. The accurate mechanical characterization of the micropillar is pivotal in the fabrication and optimization of such devices, as it determines whether the device will fail or not. In this work, we demonstrate an experimental method based only on atomic force microscopy-force spectroscopy that allows us to measure the stiffness of a micropillar and the elastic modulus of its constituent material. We test our method with four different types of 3D inkjet-printed micropillars: silver micropillars sintered at 100 and 150 °C and polyacrylate microstructures with and without a metallic coating. The estimated elastic moduli are found to be comparable with the corresponding bulk values. Furthermore, our findings show that neither the sintering temperature nor the presence of a thin metal coating plays a major role in defining the mechanical properties of the micropillar.

Fabrication and Characterization of 3D Printed, 3D Microelectrode Arrays for Interfacing with a Peripheral Nerve-on-a-Chip.

We present a nontraditional fabrication technique for the realization of three-dimensional (3D) microelectrode arrays (MEAs) capable of interfacing with 3D cellular networks in vitro. The technology uses cost-effective makerspace microfabrication techniques to fabricate the 3D MEAs with 3D printed base structures with the metallization of the microtowers and conductive traces being performed by stencil mask evaporation techniques. A biocompatible lamination layer insulates the traces for realization of 3D microtower MEAs (250 μm base diameter, 400 μm height). The process has additionally been extended to realize smaller electrodes (30 μm × 30 μm) at a height of 400 μm atop the 3D microtower using laser micromachining of an additional silicon dioxide (SiO2) insulation layer. A 3D microengineered, nerve-on-a-chip in vitro model for recording and stimulating electrical activity of dorsal root ganglion (DRG) cells has further been integrated with the 3D MEA. We have characterized the 3D electrodes for electrical, chemical, electrochemical, biological, and chip hydration stability performance metrics. A decrease in impedance from 1.8 kΩ to 670 Ω for the microtower electrodes and 55 to 39 kΩ for the 30 μm × 30 μm microelectrodes can be observed for an electrophysiologically relevant frequency of 1 kHz upon platinum electroless plating. Biocompatibility assays on the components of the system resulted in a large range (∼3%-70% live cells), depending on the components. Fourier-transform infrared (FTIR) spectra of the resin material start to reveal possible compositional clues for the resin, and the hydration stability is demonstrated in in-vitro-like conditions for 30 days. The fabricated 3D MEAs are rapidly produced with minimal usage of a cleanroom and are fully functional for electrical interrogation of the 3D organ-on-a-chip models for high-throughput of pharmaceutical screening and toxicity testing of compounds in vitro.

A three-dimensional micro-electrode array for in-vitro neuronal interfacing

Objective. In this paper, we report on the development of an easy-to-fabricate three-dimensional Micro-Electrode Array (3D-MEA) specifically designed for brain-on-a-dish applications. Approach. The proposed device consists of pillar-shaped gold microelectrodes realized by electroplating directly on top of a standard MEA, making this approach highly versatile and convenient for batch fabrication. Moreover, with this simple technique, it is possible to obtain electrodes with a height of more than 100 µm onto different kind of substrates, ranging from glass to flexible plastic ones. Main results. This novel 3D-MEA structure has been validated with acute brain slices, successfully recording both epileptiform-like discharges (upon the administration of 4-AP), and electrically-evoked neuronal activity. The preliminary validation showed a substantial improvement in the signals amplitude with respect to both commercial and custom planar electrodes thanks to a better coupling offered by the peculiar shape of the three-dimensional electrodes. Significance. Beside the versatility of the fabrication approach, which allows to obtain 3D MEA devices onto both rigid and flexible substrates, the reported validation showed how the pillar approach can outperform standard planar MEA recordings in terms of signal amplitude. Moreover, thanks to the possibility of obtaining multi-level 3D structures within the same device, the proposed fabrication technique offers an interesting and flexible approach for the development of a new family of electrophysiological tools for 3D in vitro electrophysiology, in particular for acute brain slices and 3D neuronal cultures for brain-on-a-dish applications.

A Three-Dimensional Microelectrode Array to Generate Virtual Electrodes for Epiretinal Prosthesis Based on a Modeling Study.

Despite many advances in the development of retinal prostheses, clinical reports show that current retinal prosthesis subjects can only perceive prosthetic vision with poor visual acuity. A possible approach for improving visual acuity is to produce virtual electrodes (VEs) through electric field modulation. Generating controllable and localized VEs is a crucial factor in effectively improving the perceptive resolution of the retinal prostheses. In this paper, we aimed to design a microelectrode array (MEA) that can produce converged and controllable VEs by current steering stimulation strategies. Through computational modeling, we designed a three-dimensional concentric ring-disc MEA and evaluated its performance with different stimulation strategies. Our simulation results showed that electrode-retina distance (ERD) and inter-electrode distance (IED) can dramatically affect the distribution of electric field. Also the converged VEs could be produced when the parameters of the three-dimensional MEA were appropriately set. VE sites can be controlled by manipulating the proportion of current on each adjacent electrode in a current steering group (CSG). In addition, spatial localization of electrical stimulation can be greatly improved under quasi-monopolar (QMP) stimulation. This study may provide support for future application of VEs in epiretinal prosthesis for potentially increasing the visual acuity of prosthetic vision.

A 3D flexible microelectrode array for subretinal stimulation

Objective. Various retinal prostheses have been developed to restore the vision for blind patients, and some of them are already in clinical use. In this paper, we present a three-dimensional (3D) microelectrode array for a subretinal device that can effectively stimulate retinal cells. Approach. To investigate the effect of electrode designs on the electric field distribution, we simulated various electrode shapes and sizes using finite element analysis. Based on the simulation results, the 3D microelectrode array was fabricated and evaluated in in vitro condition. Main results. Through the simulation, we verified that an electrode design of square frustum was effective to stimulate with high contrast. Also, the 3D flexible and transparent microelectrode array based on silicon and polydimethylsiloxane was fabricated using micro-electro-mechanical system technologies. In in vitro experiments, the subretinally positioned 3D microelectrodes properly evoked spikes in retinal ganglion cells. The mean threshold current was 7.4 µA and the threshold charge density was 33.64 µC·cm−2 per phase. Significance. The results demonstrate the feasibility of the fabricated 3D microelectrodes as the subretinal prosthesis. The developed microelectrode array would be integrated with the stimulation circuitry and implanted in animals for further in vivo experiments.

3D engineered neuronal networks coupled to 3D-MEAs: a new experimental model for in-vitro electrophysiology

3D engineered neuronal networks coupled to 3D-MEAs: a new experimental model for in-vitro electrophysiology

A three-dimensional microelectrode array composed of vertically aligned ultra-dense carbon nanotube networks

Electrodes based on carbon nanotubes are a promising approach to manufacture highly sensitive sensors with a low limit of signal detection and a high signal-to-noise ratio. This is achieved by dramatically increasing the electrochemical active surface area without increasing the overall geometrical dimensions. Typically, carbon nanotube electrodes are nearly planar and composed of randomly distributed carbon nanotube networks having a limited surface gain for a specific geometrical surface area. To overcome this limitation, we have introduced vertically aligned carbon nanotube (VACNT) networks as electrodes, which are arranged in a microelectrode pattern of 60 single electrodes. Each microelectrode features a very high aspect ratio of more than 300 and thus a dramatically increased surface area. These microelectrodes composed of VACNT networks display dramatically decreased impedance over the entire frequency range compared to planar microelectrodes caused by the enormous capacity increase. This is experimentally verified by electrochemical impedance spectroscopy and cyclic voltammetry.

Enzyme immobilization on microelectrode arrays of CNT/Nafion nanocomposites fabricated using hydrogel microstencils

Enzyme-modified, three-dimensional (3D) microelectrode arrays for microfluidic biofuel cell applications were fabricated using carbon nanotube (CNT) reinforced Nafion® nanocomposites and hydrogel microstenciling. CNT/Nafion nanocomposites were prepared by dispersing oxidized, multi-walled CNTs in Nafion solution. A hydrogel microstencil was fabricated using photolithography, soft lithography, and capillary-force lithography. After structuring the nanocomposites with the microstencil, a plasma-etching process was applied to the surface of the nanocomposite in order to obtain a micro-porous structure. Enzymes could be successfully immobilized on the micro-porous structures of nanocomposite surfaces using direct covalent binding.

Cell electroporation with a three-dimensional microelectrode array on a printed circuit board

Electroporation is a commonly used approach to rapidly introduce exogenous molecules into cells without permanent damage. Compared to classical electroporation protocols, microchip-based electroporation approaches have the advantages of high transfection efficiency and low consumption, but they also commonly rely on costly and tedious microfabrication technology. Hence, it is desirable to develop a novel, more affordable, and effective approach to facilitate cell electroporation. In this study, we utilized a standard printed circuit board (PCB) technology to fabricate a chip with an interdigitated array of electrodes for electroporation of suspended cells. The electrodes (thickness~35μm) fabricated by PCB technology are much thicker than the two-dimensional (2D) planar electrodes (thickness<1μm) fabricated by conventional microfabrication techniques and possess a smooth corner edge. Numerical simulations showed that the three-dimensional (3D) electrodes fabricated by PCB technology can provide a more uniformly distributed electric field compared to 2D planar electrodes, which is beneficial for reducing the electrolysis of water and improving cell transfection efficiency. The chip constructed here is composed of 18 individually addressable wells for high throughput cell electroporation. HeLa, MCF7, COS7, Jurkat, and 3T3-L1 cells were efficiently transfected with the pEGFP-N1 plasmid using individually optimal electroporation parameters. This work provides a novel method for convenient and rapid cell transfection and thus holds promise for use as a low-cost disposable device in biomedical research.

Flexible three-dimensional microelectrode array for neural applications

A neural electrode array design is proposed with 3mm long sharpened pillars made from an aluminum-based substrate. The array is composed by 25 electrically insulated pillars in a 5×5 matrix, in which each aluminum pillar was precisely machined via dicing saw technique. The result is an aluminum structure with high-aspect-ratio pillars (19:1), each with a tip radius of 10μm. A thin-film of platinum was deposited via sputtering technique to perform the ionic signal transduction. Each pillar was encapsulated with an epoxy resin insulating the entire pillar excluding the tip. This process resulted in mechanically robust electrodes each capable of withstanding loads up to 200mN before bending. The array implantation tests were conducted on agar gel at speeds of 50mm/min, 120mm/min and 180mm/min which resulted in average implantation forces of 119mN, 145mN and 150mN, respectively. Insertion and withdrawal tests were also performed in porcine cadaver brain showing a necessary force of 66mN for successful explantation. A three point flexural test demonstrated a displacement of 0.8mm before array's breakage. The electrode's impedance was characterized showing a near resistive impedance of 385Ω in the frequency range from 2kHz to 125kHz. The resultant array, as well as the fabrication technique, is an innovative alternative to silicon-based electrode solutions, avoiding some fabrication methods and limitations related to silicon and increasing the mechanical flexibility of the array.

A methodology for fast assessments to the electrical activity of barrel fields in vivo: from population inputs to single unit outputs

Here we propose a methodology to analyze volumetric electrical activity of neuronal masses in the somatosensory barrel field of Wistar rats. The key elements of the proposed methodology are a three-dimensional microelectrode array, which was customized by our group to observe extracellular recordings from an extended area of the barrel field, and a novel method for the current source density analysis. By means of this methodology, we were able to localize single barrels from their event-related responses to single whisker deflection. It was also possible to assess the spatiotemporal dynamics of neuronal aggregates in several barrels at the same time with the resolution of single neurons. We used simulations to study the robustness of our methodology to unavoidable physiological noise and electrode configuration. We compared the accuracy to reconstruct neocortical current sources with that obtained with a previous method. This constitutes a type of electrophysiological microscopy with high spatial and temporal resolution, which could change the way we analyze the activity of cortical neurons in the future.

Preparation and Characterization of a Micro-Supercapacitor with Three-Dimensional Microelectrode Arrays

With the development of the MEMS technology and the more widely-used micro-systems, micro-power devices are developed for portable consumer electronics.[The MEMS supercapacitor is one of the most important parts for micro-powers. In this paper, a micro-supercapacitor was designed and fabricated with three-dimensional microelectrode arrays configuration. It has been tested by scanning electron microscopy, Cyclic Voltammeter, Galvanostatical charge/discharge curve and electrochemical impedance spectroscopy. These posts were 50 μm in diameter and 100 μm tall; with foot print area of 2×2mm2. The capacitance is 89.52mF/cm2. The biggest feature in this paper is that the two plates of the supercapacitor are both three-dimensional microelectrode arrays which are shown to offer significant advantages.

A study on three-dimensional electrode arrays fabricated by PolyMUMPs® for AC electro-osmotic pumping

This paper presents four three-dimensional (3D) micro-electrode arrays for pumping liquid by utilizing AC electro-osmosis (ACEO) mechanism. Fabricated by using PolyMUMPs® process, these electrode arrays are made of polysilicon and subject to a surface oxide layer, which does not exist on conventional gold electrodes. An ACEO finite element model (FEM) accounting for the surface oxide on polysilicon electrodes is integrated in this work. The relationship of the velocity of driven liquid vs. the frequency of the electrical driving signal is obtained for the four electrode arrays through simulation and testing. Impacts of the electrode configuration and surface oxide are analyzed by comparing simulation and experimental results.

Micromachined three-dimensional electrode arrays for transcutaneous nerve tracking

We report the development of metal transfer micromolded (MTM) three-dimensional microelectrode arrays (3D MEAs) for a transcutaneous nerve tracking application. The measurements of electrode–skin–electrode impedance (ESEI), electromyography (EMG) and nerve conduction utilizing these minimally invasive 3D MEAs are demonstrated in this paper. The 3D MEAs used in these measurements consist of a metalized micro-tower array that can penetrate the outer layers of the skin in a painless fashion and are fabricated using MTM technology. Two techniques, an inclined UV lithography approach and a double-side exposure of thick negative tone resist, have been developed to fabricate the 3D MEA master structure. The MEAs themselves are fabricated from the master structure utilizing micromolding techniques. Metal patterns are transferred during the micromolding process, thereby ensuring reduced process steps compared to traditional silicon-based approaches. These 3D MEAs have been packaged utilizing biocompatible Kapton® substrates. ESEI measurements have been carried out on test human subjects with standard commercial wet electrodes as a reference. The 3D MEAs demonstrate an order of magnitude lower ESEI (normalized to area) compared to wet electrodes for an area that is 12.56 times smaller. This compares well with other demonstrated approaches in literature. For a nerve tracking demonstration, we have chosen EMG and nerve conduction measurements on test human subjects. The 3D MEAs show 100% improvement in signal power and SNR/√area as compared to standard electrodes. They also demonstrate larger amplitude signals and faster rise times during nerve conduction measurements. We believe that this microfabrication and packaging approach scales well to large-area, high-density arrays required for applications like nerve tracking. This development will increase the stimulation and recording fidelity of skin surface electrodes, while increasing their spatial resolution by an order of magnitude or more. Although biopotential electrode systems are not without their challenges, the non-invasive access to neural information, along with the potential for automation with associated electronic and software development, is precisely what makes this technology an excellent candidate for the next generation in diagnostic, therapeutic, and prosthetic devices.

A three-dimensional flexible microprobe array for neural recording assembled through electrostatic actuation

We designed, fabricated and tested a novel three-dimensional flexible microprobe to record neural signals of a lateral giant nerve fiber of the escape circuit of an American crayfish. An electrostatic actuation folded planar probes into three-dimensional neural probes with arbitrary orientations for neuroscientific applications. A batch assembly based on electrostatic forces simplified the fabrication and was non-toxic. A novel fabrication for these three-dimensional flexible probes used SU-8 and Parylene technology. The mechanical strength of the neural probe was great enough to penetrate into a bio-gel. A flexible probe both decreased the micromotion and alleviated tissue encapsulation of the implant caused by chronic inflammation of tissue when an animal breathes or moves. The cortex consisted of six horizontal layers, and the neurons of the cortex were arranged in vertical structures; the three-dimensional microelectrode arrays were suitable to investigate the cooperative activity for neurons in horizontal separate layers and in vertical cortical columns. With this flexible probe we recorded neural signals of a lateral giant cell from an American crayfish. The response amplitude of action potentials was about 343 µV during 1 ms period; the average recorded data had a ratio of signal to noise as great as 30.22 ± 3.58 dB. The improved performance of this electrode made feasible the separation of neural signals according to their distinct shapes. The cytotoxicity indicated a satisfactory biocompatibility and non-toxicity of the flexible device fabricated in this work.

Characterization of the in vitro propagation of epileptiform electrophysiological activity in organotypic hippocampal slice cultures coupled to 3D microelectrode arrays

Dynamic aspects of the propagation of epileptiform activity have so far received little attention. With the aim of providing new insights about the spatial features of the propagation of epileptic seizures in the nervous system, we studied in vitro the initiation and propagation of traveling epileptiform waves of electrophysiological activity in the hippocampus by means of substrate three-dimensional microelectrode arrays (MEAs) for extracellular measurements. Pharmacologically disinhibited hippocampal slices spontaneously generate epileptiform bursts mostly originating in CA3 and propagating to CA1. Our study specifically addressed the activity-dependent changes of the propagation of traveling electrophysiological waves in organotypic hippocampal slices during epileptiform discharge and in particular our question is: what happens to the epileptic signals during their propagation through the slice? Multichannel data analysis enabled us to quantify an activity-dependent increase in the propagation velocity of spontaneous bursts. Moreover, through the evaluation of the coherence of the signals, it was possible to point out that only the lower-frequency components (<95Hz) of the electrical activity are completely coherent with respect to the activity originating in the CA3, while components at higher frequencies lose the coherence, possibly suggesting that the cellular mechanism mediating propagation of electrophysiological activity becomes ineffective for those firing rates exceeding an upper bound or that some noise of neuronal origin was added to the signal during propagation.