Microelectrode Arrays Research Articles

Probing the efficiency and mechanism of hydrogen permeation inhibition in pipeline steel by organic inhibitors

A hydrogen economy will require the use of steel pipelines to transport and store hydrogen. Methods and materials are urgently needed to prevent the embrittlement of steel pipelines in hydrogen service. In this work, we show that hydrogen permeation into pipeline steel can be mitigated by organic inhibitors, as demonstrated by a typical organic inhibitor benzotriazole (BTA) under simulated X65 steel pipeline cathodic protection conditions. Electrochemical and surface analytical techniques were used to probe the efficiency and mechanism of hydrogen permeation inhibition. In particular, a novel multi-electrode array method was designed to probe the inhibitor film by measuring local impedance and hydrogen charging currents. The ability and efficiency of BTA to inhibit hydrogen permeation were found to be closely associated with the formation and coverage of inhibitor films on steel surfaces. A model is proposed to illustrate the mechanism of hydrogen permeation inhibition by organic inhibitors.

A 4096 channel event-based multielectrode array with asynchronous outputs compatible with neuromorphic processors

Bio-signal sensing is pivotal in medical bioelectronics. Traditional methods focus on high sampling rates, leading to large amounts of irrelevant data and high energy consumption. We introduce a self-clocked microelectrode array (MEA) that digitizes bio-signals at the pixel level by encoding changes as asynchronous digital address-events only when they exceed a threshold, significantly reducing off-chip data transmission. This novel MEA comprises a 64 × 64 electrode array, an asynchronous 2D-arbiter, and an Address-Event Representation (AER) communication block. Each pixel operates autonomously, monitoring voltage fluctuations from cellular activity and producing digital pulses for significant changes. Positive and negative signal changes are encoded as “up” and “down” events and are routed off-chip via the asynchronous arbiter. We present results from chip characterization and experimental measurements using electrogenic cells. Additionally, we interface the MEA to a mixed-signal neuromorphic processor, demonstrating a prototype for end-to-end event-based bio-signal sensing and processing.

Understanding responses to multi-electrode epiretinal stimulation using a biophysical model.

Neural interfaces are designed to evoke specific patterns of electrical activity in populations of neurons by stimulating with many electrodes. However, currents passed simultaneously through multiple electrodes often combine nonlinearly to drive neural responses, making evoked responses difficult to predict and control. This response nonlinearity could arise from the interaction of many excitable sites in each cell, any of which can produce a spike. However, this multi-site activation hypothesis is difficult to verify experimentally. We developed a biophysical model to study retinal ganglion cell (RGC) responses to multi-electrode stimulation and validated it using data collected from ex vivo preparations of the macaque retina using a microelectrode array (512 electrodes; 30µm pitch; 10µm diameter). First, the model was validated by reproducing essential empirical findings from single-electrode stimulation and recording, including spike waveforms over the electrode array and sigmoidal responses to injected current. Then, stimulation with two electrodes was modeled to test how the positioning of the electrodes relative to the cell affected the degree of response nonlinearity. Currents passed through pairs of electrodes positioned near the cell body or far from the axon (>40 µm) exhibited linear summation. Currents passed through pairs of electrodes close to the axon summed linearly when their locations along the axon were similar, and nonlinearly otherwise. Over a range of electrode placements, several distinct, localized spike initiation sites were observed, and the number of these sites covaried with the degree of response nonlinearity. Similar trends were observed for three-electrode stimuli. All of these trends were consistent with experimental observations. These findings support the multi-site activation hypothesis for nonlinear activation of neurons, providing a biophysical interpretation of previous experimental results and potentially enabling more efficient use of multi-electrode stimuli in future neural implants.

Flexible Microelectrode Arrays Based on Vacuum Filling for Electrophysiology Sensing of Cardiomyocytes

Flexible Microelectrode Arrays Based on Vacuum Filling for Electrophysiology Sensing of Cardiomyocytes

Fibrin-Polycaprolactone Scaffolds for the Differentiation of Human Neural Progenitor Cells into Dopaminergic Neurons.

Parkinson's disease (PD), a progressive central nervous system disorder marked by involuntary movements, poses a significant challenge in neurodegenerative research due to the gradual degeneration of dopaminergic (DA) neurons. Early diagnosis and understanding of PD's pathogenesis could slow disease progression and improve patient management. In vitro modeling with DA neurons derived from human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) offers a promising approach. These neurons can be cultured on electrospun (ES) nanofibrous polycaprolactone (PCL) scaffolds, but PCL's hydrophobic nature limits cell adhesion. We investigated the ability of ES PCL scaffolds coated with hydrophilic extracellular matrix-based biomaterials, including cell basement membrane proteins, Matrigel, and Fibrin, to enhance NPC differentiation into DA neurons. We hypothesized that fibrin-coated scaffolds would maximize differentiation based on fibrin's known benefits in neuronal tissue engineering. The scaffolds both coated and uncoated were characterized using scanning electron microscopy (SEM), transmission electron microscopy, Fourier transform infrared spectroscopy-attenuated total reflectance, and dynamic mechanical analysis to assess their properties. NPCs were seeded on the coated scaffolds, differentiated, and matured into DA neurons. Immunocytochemistry targeting tyrosine hydroxylase (TH) and SEM confirmed DA neuronal differentiation and morphological changes. Electrophysiology via microelectrode array recorded their neuronal firing. Results showed enhanced neurite extension, increased TH expression, and active electrical activity in cells on fibrin-coated scaffolds. Diluted fibrin coatings particularly promoted more pronounced neuronal differentiation and maturation. This study introduces a novel tissue-on-a-chip platform for neurodegenerative disease research using DA neurons.

Effects of chronic insecticide exposure on neuronal network development in vitro in rat cortical cultures

Developmental exposure to carbamates, organophosphates, and pyrethroids has been associated with impaired neurodevelopmental outcomes. Sex-specific differences following chronic insecticide exposure are rather common in vivo. Therefore, we assessed the chronic effects of in vitro exposure to different carbamates (carbaryl, methomyl and aldicarb), organophosphates [chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), and 3,5,6,trichloropyridinol (TCP)], and pyrethroids [permethrin, alpha-cypermethrin and 3-phenoxy benzoic acid (3-PBA)] on neuronal network development in sex-separated rat primary cortical cultures using micro-electrode array (MEA) recordings. Our results indicate that exposure for 1 week to carbaryl inhibited neurodevelopment in male cultures, while a hyperexcitation was observed in female cultures. Methomyl and aldicarb evoked a hyperexcitation after 2 weeks of exposure, which was more pronounced in female cultures. In contrast to acute MEA results, exposure to ≥ 10 µM CPF caused hyperexcitation in both sexes after 10 days. Interestingly, exposure to 10 µM CPO induced a clear hyperexcitation after 10 days of exposure in male but not female cultures. Exposure to 100 µM CPO strongly inhibited neuronal development. Exposure to the type I pyrethroid permethrin resulted in a hyperexcitation at 10 µM and a decrease in neuronal development at 100 µM. In comparison, exposure to ≥ 10 µM of the type II pyrethroid alpha-cypermethrin decreased neuronal development. In female but not in male cultures, exposure to 1 and 10 µM permethrin changed (network) burst patterns, with female cultures having shorter (network) bursts with fewer spikes per (network) burst. Together, these results show that MEA recordings are suitable for measuring sex-specific developmental neurotoxicity in vitro. Additionally, pyrethroid exposure induced effects on neuronal network development at human-relevant concentrations. Finally, chronic exposure has different effects on neuronal functioning compared to acute exposure, highlighting the value of both exposure paradigms.

Single‐Particle Electrochemical Cycling Single‐Crystal and Polycrystalline NMC Particles

AbstractLi(Ni,Mn,Co)O2 (NMC) is one of the most widely used cathode materials for lithium‐ion batteries. Most commercial cathodes utilize polycrystalline particle morphologies, which have a characteristic “meatball” shape. Recently, there has been interest in replacing polycrystalline particles with single‐crystal particles, which are believed to have improved cycle life due to the absence of intergranular cracking. However, the effects of single‐crystal particles on rate capability show conflicting results. In this work, single‐particle electrochemistry using a microelectrode array to test the kinetics of 40 polycrystalline (3–12 µm) and 13 single‐crystal (2–4 µm) NMC cathode particles is conducted. The results show that single‐crystal particles have much higher reaction overpotentials than polycrystalline ones. Moreover, larger single‐crystal particles show higher overpotentials than smaller ones, while the polycrystalline particles display no size effects despite the greater variability. These findings are attributed to intergranular cracking and the penetration of the liquid electrolyte, which enables much faster reaction kinetics. In characterizing the electrochemistry of individual particles, the results confirm that these single‐crystal particles would have much poorer rate kinetics and more challenges with fast charge and discharge compared to polycrystalline particles of the same composition.

Micro-electrode array recording of extracellular electrical potentials of liquid static surface fermented Hericium erinaceus

Hericium erinaceus is a basidiomycetes fungus with previously uncharacterised extracellular electrophysiology. Here, we present results of recordings of the electrical potentials of fungal biofilms of this species using microelectrode arrays (MEAs). In particular, we focused on modelling the temporal and spatial progression of the low frequency (≤ 1 Hz) potentials. Culture media control studies showed that the electrical potential activity results from the growth and subsequent spiking behaviours of the mycelium extracellular matrices. An antifungal assay using nystatin suspension, 10,000 unit/mL in DPBS, provided evidence for the biological origin of electrical potentials due to targeting of the selective permeability of the cell membrane and subsequent cessation of electrical activity. Conversely, injection of L-glutamic acid increased the combined multi-channel mean firing rate from 0.04 Hz to 0.1 Hz. Analysis of bursting and spatial propagation of the extracellular signals are also presented.

An Accurate and Rapidly Calibrating Speech Neuroprosthesis

BackgroundBrain–computer interfaces can enable communication for people with paralysis by transforming cortical activity associated with attempted speech into text on a computer screen. Communication with brain–computer interfaces has been restricted by extensive training requirements and limited accuracy.MethodsA 45-year-old man with amyotrophic lateral sclerosis (ALS) with tetraparesis and severe dysarthria underwent surgical implantation of four microelectrode arrays into his left ventral precentral gyrus 5 years after the onset of the illness; these arrays recorded neural activity from 256 intracortical electrodes. We report the results of decoding his cortical neural activity as he attempted to speak in both prompted and unstructured conversational contexts. Decoded words were displayed on a screen and then vocalized with the use of text-to-speech software designed to sound like his pre-ALS voice.ResultsOn the first day of use (25 days after surgery), the neuroprosthesis achieved 99.6% accuracy with a 50-word vocabulary. Calibration of the neuroprosthesis required 30 minutes of cortical recordings while the participant attempted to speak, followed by subsequent processing. On the second day, after 1.4 additional hours of system training, the neuroprosthesis achieved 90.2% accuracy using a 125,000-word vocabulary. With further training data, the neuroprosthesis sustained 97.5% accuracy over a period of 8.4 months after surgical implantation, and the participant used it to communicate in self-paced conversations at a rate of approximately 32 words per minute for more than 248 cumulative hours.ConclusionsIn a person with ALS and severe dysarthria, an intracortical speech neuroprosthesis reached a level of performance suitable to restore conversational communication after brief training. (Funded by the Office of the Assistant Secretary of Defense for Health Affairs and others; BrainGate2 ClinicalTrials.gov number, NCT00912041.)

Temporal dynamics of neocortical development in organotypic mouse brain cultures: a comprehensive analysis.

Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the broad application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Using a holistic approach, we studied organotypic whole hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a 5-wk period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed multielectrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by 2 wk in culture, followed by the transition of spontaneous activity toward more complex patterns including high-frequency oscillations. However, culturing weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders.NEW & NOTEWORTHY The development of the mouse neocortex in vitro mimics the in vivo development. Mouse brain cultures can serve as a model system for cortical development for the first 2 wk in vitro and as a model system for the adult cortex from 2 to 4 wk in vitro. Mouse organotypic brain slice cultures develop high-frequency network oscillations at γ frequency after 2 wk in vitro. Mouse brain cultures exhibit increased heterogeneity and variability after 4 wk in culture.

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Biofilm Spatial Monitoring By Multiple Electrochemical Techniques Integrated with Optical Microscopy and a Microfluidic Device

Biofilm represents the inherent structure of bacterial populations, consisting of bacteria encapsulated within extracellular polymeric substances they secrete. The natural formation of a biofilm acts as a physical barrier, offering protection against environmental changes and increasing bacterial antibiotic resistance, often up to 1,000 times. To enhance our understanding of these intricate biosystems, electrochemical analytical micro-systems are crucial to mimic the biofilm’s behavior in nature and to monitor responses to diverse treatments. Current electrochemical micro-systems, however, face a fundamental limitation, employing a single electrode to characterize biofilm behavior [1], despite biofilms being inherently three-dimensional and non-uniformly distributed across the electrode. Here, we introduce a novel electrochemical micro-systems that is based on an array of microelectrodes and microfluidics to provide spatial monitoring of the biofilm. Fabricated through conventional photolithography and lift-off techniques, the micro-system comprises five individual gold microelectrode arrays covered with a polydimethylsiloxane microfluidic channel (Fig. 1A) and linked to a reference Ag/AgCl electrode (Fig. 1B). In a proof-of-concept study focusing on monitoring the biofilm formation and functionality, we cultivated Pseudomonas aeruginosa, a pathogenic bacterium secreting electroactive molecules known as phenazines when reaching high cell density. Subsequently, we monitored biofilm formation over 30 hours by repetitively measuring the electrochemical signal using differential pulse voltammetry (DPV). The analysis of anodic (Fig. 1C) and cathodic (Fig. 1D) currents unveiled an increasing oxidation peak at -0.28V vs Ag/AgCl, varying among the five electrodes, attributed to phenazine formation (Fig. 1E). Furthermore, both anodic and cathodic currents decreased at the negative applied potential (-0.8 V vs Ag/AgCl), correlating with bacterial growth and phenazines production (Fig. 1F). Electrochemical impedance spectroscopy (EIS) at different DC potentials (-400, +50, and +400 mV vs Ag/AgCl; Fig. 1G, +400mV) was performed, and the impedance generated was correlated with 10 different equivalent electrical circuits (optimal circuit with the best fitting score is shown in Fig. 1H) [2]. Hourly monitoring of biofilm growth was conducted using microscopy imaging at multiple locations along the microfluidic channel (Fig. 1I, image after 10hr). The experiment's conclusion involved validating biofilm formation through fluorescent staining of the biofilm's sugars (Figs. 1J – 1L, phase image, fluorescent image, and overlay image). By integrating the developed electrochemical micro-system with microscopy imaging, real-time and direct monitoring of biofilm formation becomes achievable. Furthermore, this integrated system can be employed across electrochemical and microscopic platforms to explore biofilm properties in response to diverse conditions. Figure 1. (A) Microfluidic electrochemical array illustration (B) photograph of the experimental setup (C) 30-hours anodic and cathodic (D) voltammograms recorded in the presence of Pseudomonas aeruginosa (PA14). (E) transient oxidation peak current at -0.28V (F) the anodic and cathodic currents at -0.8V (G) transient impedance spectrographs recorded from PA14 (H) raw data and fitted electric circuit. Microscopic imaging of biofilm growth at (I) 10 hr, end of experiment (J) phase image, (K) biofilm polysaccharides fluorescent staining and (L) overlayed image.[1] Estrada-Leypon, O. et. al, Bioelectrochemistry 2015, 105, 56–64.[2] Ben-Yoav, H. et.al, Electrochim. Acta 2011, 56 (23), 7780–7786. Figure 1

Synthetic Chemistry and Microelectrode Arrays for Point of Care Diagnostics

Molecular diagnostics typically require a molecular interaction that identifies a specific target, a device that detects, quantifies, and records that interaction, and an interface between those two elements. Each feature is critical for the success of the device. Yet while the first two are matters of intense interest an research, the surface itself is often ignored. This is problematic because it provides the platform for the whole "experiment". For a "point-of-care" diagnostic, the surface on the device must be stable both in terms of chemistry and time while still allowing the signal from the molecular interaction to be sensed and recorded.With this in mind, we have been focusing on the use of diblock copolymers as molecular surfaces for the construction of "point-of-care" diagnostics. We have developed a toolbox of chemical reactions that can be used site-selectively on such surfaces, and we have recently demonstrated that the approach taken is compatible with the use of DNA-aptamers to do multiplex sensing on a high density microelectrode array using a surface that is stable for a year. We have also illustrated how the quality of the signaling study conducted on an array is dependent on optimization of the chemical reaction.But exactly how good are the chemical reactions employed on the arrays to build a surface in a site-selective manner? One can tell with the use of fluorescence based studies that reactions happen where you want then to happen, but are there background reactions that can be problematic for a multiplex sensing experiment, do the reactions happen in high yield, and how much of an expensive, difficult to obtain biological reagent might be lost due to the confinement strategy? Are some reactions better than others in this regard? In short, if we are to optimize the surfaces constructed on a diagnostic device, then we need not only have tools in place for conducting synthetic chemistry on the device, but also the tools in place necessary to analyze those reactions. Understanding the chemistry that can and/or does occur at any electrode in a high density array requires more than looking at pretty pictures of fluorescent spots. In the talk to be given, we will utilize a combination of a "Kenner-safety-catch" linker strategy and molecular biology techniques to answer key questions about the synthetic chemistry used to construct the DNA-aptamer based multiplex sensor mentioned above.

Measurement of Neuropeptides and Amino Acids with Carbon-Fiber Multielectrode Array Enzyme-Modified Biosensors

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. These assays typically measure small molecule neurotransmitters such as dopamine and serotonin. The carbon fiber is relatively small, biocompatible, and makes minimally invasive measurements at high spatial and temporal resolution. Carbon Fiber Multielectrode arrays have been utilized to measure multiple neurotransmitters in several brain regions simultaneously with multi-waveform application on each electrode. We have extended this work to measure larger molecule neuropeptides such as Neuropeptide Y and Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and peptide, decreased surface fouling, and codetection with other catecholamines. As both oxytocin and Neuropeptide Y contain tyrosine, the MSW was also used to detect these neuropeptides. Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied neuropeptides on rat brain slices. These results may serve as novel assays for neuropeptide detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of neuropeptides such as Neuropeptide Y and oxytocin.Moreover, we have also developed enzyme modified microelectrodes for the measurement of glutamate (L-glutamic acid), which is an important excitatory amino acid and biomarker for epilepsy along with the inhibitory GABA. Since glutamate is not redox active at carbon electrodes, we modified CFMEs with glutamate oxidase enzyme to metabolize glutamate to hydrogen peroxide, which was then oxidized at carbon electrodes to produce readout cyclic voltammograms (CVs). The enzyme coating was optimized by varying the concentration of enzyme, chitosan binder, solvent, and deposition time. The coating was further analyzed electrochemically and imaged with scanning electron microscopy (SEM) for thickness and uniformity of surface coverages. Energy-Dispersive Spectroscopy (EDS/EDX) was utilized for chemical surface functionalization analysis. Glutamate oxidation was found to be adsorption controlled to CFMEs and characterized at various scan rates, concentrations, and stability times as well with an approximate 100 nM limit of detection. Glutamate was co-detected in complex mixtures with several monoamines such as dopamine, serotonin, norepinephrine, and others. Glutamate will furthermore be measured in several food samples and ex vivo in rat coronal brain slices and in vivo in anesthetized and freely behaving animals.

Magnetically-Enhanced Electrochemical ELISA for the Detection of Sars-Cov-2 Nucleoprotein

Here we report on the development of a novel, magnetically-enhanced electrochemical (MagEC) bioassay targeting SARS-CoV-2 nucleoprotein. The sensing strategy is similar to microbead-based enzyme linked immunosorbent assays (ELISA) with optical detection. The target analyte is captured using a magnetic microbead functionalized with an antibody specific to SARS-CoV-2 nucleoprotein. Once SARS-CoV-2 nucleoprotein has been captured a detection construct, consisting of polystyrene microbeads functionalized with horseradish peroxidase (HRP) and antibodies specific to SARS-CoV-2 nucleoprotein are attached to form the full microbead construct.Once the full microbead constructs are assembled, they are injected into an electrochemical cell containing an assay solution where they are magnetically enriched at the electrode surface. This magnetic localization brings all of the reactants close to their point of detection at the electrode surface and also creates a positive feedback loop, drastically enhancing signal. The electrochemical assay was optimized using model constructs to emulate the fully assembled bead constructs and identify the optimal electrode configuration. Using the model constructs, we found a 60-fold decrease in limit of detection using the MagEC ELISA compared to optical detection with model bead constructs. We compared performance of a bare gold electrode to several geometries of gold microelectrode arrays. We obtained preliminary results for the detection of SARS-CoV-2 nucleoprotein. Additionally, the assay was expanded to incorporate a fluidic manifold to enhance signal, enhance throughput and enable operation in microgravity environments.

Fabrication of high-resolution, flexible, laser-induced graphene sensors via stencil masking

The advent of wearable sensing platforms capable of continuously monitoring physiological parameters indicative of health status have resulted in a paradigm shift for clinical medicine. The accessibility and adaptability of such portable, unobtrusive devices enables proactive, personalized care based on real-time physiological insights. While wearable sensing platforms exhibit powerful capabilities for continuously monitoring physiological parameters, device fabrication often requires specialized facilities and technical expertise, restricting deployment opportunities and innovation potential. The recent emergence of rapid prototyping approaches to sensor fabrication, such as laser-induced graphene (LIG), provides a pathway for circumventing these barriers through low-cost, scalable fabrication. However, inherent limitations in laser processing restrict the spatial resolution of LIG-based flexible electronic devices to the minimum laser spot size. For a CO2 laser–a commonly reported laser for device production–this corresponds to a feature size of ∼120 μm. Here, we demonstrate a facile, low-cost stencil-masking technique to reduce the minimum resolvable feature size of a LIG-based device from 120 ± 20 μm to 45 ± 3 μm when fabricated by CO2 laser. Characterization of device performance reveals this stencil-masked LIG (s-LIG) method yields a concomitant improvement in electrical properties, which we hypothesize is the result of changes in macrostructure of the patterned LIG. We showcase the performance of this fabrication method via production of common sensors including temperature and multi-electrode electrochemical sensors. We fabricate fine-line microarray electrodes not typically achievable via native CO2 laser processing, demonstrating the potential of the expanded design capabilities. Comparing microarray sensors made with and without the stencil to traditional macro LIG electrodes reveals the s-LIG sensors have significantly reduced capacitance for similar electroactive surface areas. Beyond improving sensor performance, the increased resolution enabled by this metal stencil technique expands capabilities for scalable fabrication of high-performance wearable sensors in low-resource settings without reliance on traditional fabrication pathways.

Chloride deregulation and GABA depolarization in MTOR related malformations of cortical development.

Focal Cortical Dysplasia, Hemimegalencephaly and Cortical Tuber are pediatric epileptogenic malformations of cortical development (MCDs) frequently pharmaco-resistant and mostly surgically treated by the resection of epileptic cortex. Availability of cortical resection samples allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes were identified. GABAa mediated paradoxical depolarization, related to altered chloride (Cl-) homeostasis, was shown to participate to ictogenesis in human pediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is still unclear. Here we studied the post translational interactions between the mTOR pathway and the regulation of cation-chloride cotransporters (CCC), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl- and ultimately GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo controls, diagnosed by histology plus genetic profiling) were operated for drug resistant epilepsy. Postoperative cortical tissues were recorded on multielectrode array (MEA) to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled Cl- conductance and equilibrium potential (EGABA) for GABA measurement. Of the 25 clinical cases, half harbored a somatic mutation in the mTOR pathway, while pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, that was reversed by WNK1 and SPAK antagonists (NEM and Staurosporine). mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR specific antagonist Rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in EGABA by rapamycin, measured after incorporation of MCD membranes to X. laevis oocytes, in line with a reestablishment of normal ECl-. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to chloride cotransporters deregulation, increased neuronal chloride levels and GABAa dysfunction in malformations of Cortical Development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.

Overexpression of Toxic Poly(Glycine-Alanine) Aggregates in Primary Neuronal Cultures Induces Time-Dependent Autophagic and Synaptic Alterations but Subtle Activity Impairments.

The pathogenic expansion of the intronic GGGGCC hexanucleotide located in the non-coding region of the C9orf72 gene represents the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation leads to the accumulation of toxic RNA foci and dipeptide repeats (DPRs), as well as reduced levels of the C9orf72 protein. Thus, both gain and loss of function are coexisting pathogenic aspects linked to C9orf72-ALS/FTD. Synaptic alterations have been largely described in C9orf72 models, but it is still not clear which aspect of the pathology mostly contributes to these impairments. To address this question, we investigated the dynamic changes occurring over time at the synapse upon accumulation of poly(GA), the most abundant DPR. Overexpression of this toxic form induced a drastic loss of synaptic proteins in primary neuron cultures, anticipating autophagic defects. Surprisingly, the dramatic impairment characterizing the synaptic proteome was not fully matched by changes in network properties. In fact, high-density multi-electrode array analysis highlighted only minor reductions in the spike number and firing rate of poly(GA) neurons. Our data show that the toxic gain of function linked to C9orf72 affects the synaptic proteome but exerts only minor effects on the network activity.

Motor somatotopy impacts imagery strategy success in human intracortical brain-computer interfaces.

The notion of a somatotopically organized motor cortex, with movements of different body parts being controlled by spatially distinct areas of cortex, is well known. However, recent studies have challenged this notion and suggested a more distributed representation of movement control. This shift in perspective has significant implications, particularly when considering the implantation location of electrode arrays for intracortical brain-computer interfaces (iBCIs). We sought to evaluate whether the location of neural recordings from the precentral gyrus, and thus the underlying somatotopy, has any impact on the imagery strategies that can enable successful iBCI control. Three individuals with a spinal cord injury were enrolled in an ongoing clinical trial of an iBCI. Participants had two intracortical microelectrode arrays implanted in the arm and/or hand areas of the precentral gyrus based on presurgical functional imaging. Neural data were recorded while participants attempted to perform movements of the hand, wrist, elbow, and shoulder. We found that electrode arrays that were located more medially recorded significantly more activity during attempted proximal arm movements (elbow, shoulder) than did lateral arrays, which captured more activity related to attempted distal arm movements (hand, wrist). We also evaluated the relative contribution from the two arrays implanted in each participant to decoding accuracy during calibration of an iBCI decoder for translation and grasping tasks. For both task types, imagery strategy (e.g., reaching vs. wrist movements) had a significant impact on the relative contributions of each array to decoding. Overall, we found some evidence of broad tuning to arm and hand movements; however, there was a clear bias in the amount of information accessible about each movement type in spatially distinct areas of cortex. These results demonstrate that classical concepts of somatotopy can have real consequences for iBCI use, and highlight the importance of considering somatotopy when planning iBCI implantation.

Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.