Simulation and fabrication of in vitro microfluidic microelectrode array chip for patterned culture and electrophysiological detection of neurons

To enable the detection and modulation of modularized neural networks in vitro,this study proposes a microfluidic microelectrode array chip for the cultivation,compartmentalization,and control of neural cells.The chip was designed based on the specific structure of neurons and the requirements for detection and modulation.Finite-element analysis of the chip’s flow field was conducted using the COMSOL Multiphysics software,and the simulation results show that the liquid within the chip can flow smoothly,ensuring stable flow fields that facilitate the uniform growth of neurons within the microfluidic channels.By employing MEMS technology in combination with nanomaterial modification techniques,the microfluidic microelectrode array chip was fabricated successfully.Primary hippocampal neurons were cultured on the chip,forming a well-defined neural network.Spontaneous electrical activity of the detected neurons was recorded,exhibiting a 23.7%increase in amplitude compared to neuronal discharges detected on an open-field microelectrode array.This study provides a platform for the precise detection and modulation of patterned neuronal growth in vitro,potentially serving as a novel tool in neuroscience research.

Microelectrode array for bioelectrical signal stimulation and recording

A microelectrode array（MEA） is presented, which is composed of 60 independent electrodes with 59 working ones and one reference one, and they are divided into 30 pairs. Except for the reference electrode, each pair consists of one stimulating electrode and one recording electrode. Supported by the peripheral circuits, four electrode states to study the bioelectrical signal of biological tissue or slice cultured in-vitro on the surface of the electrodes can be realized through each pair of electrodes. The four electrode states are stimulation, recording, stimulation and recording simultaneously, and isolation. The state of each pair of working electrodes can be arbitrarily controlled according to actual needs. The MEAs are fabricated in printed circuit board （PCB） technology. The total area of the PCB-based MEA is 49 mm × 49 mm. The impedance measurement of MEA is carried out in 0.9% sodium chloride solution at room temperature by means of 2-point measurements with an Agilent LCR meter, and the test signal for the impedance measurement is sinusoidal （AC voltage 50 mV, sweeping frequency 20 Hz to 10 kHz）. The electrode impedance is between 200 and 3 kΩ while the frequency is between 500 and 1 000 Hz. The electrode impedance magnitude is inversely proportional to the frequency. Experiments of toad sciatic nerve in-vitro stimulation and recording and signal regeneration between isolated toad sciatic nerves are carried out on the PCB-based MEA. The results show that the MEA can be used for bioelectrical signal stimulation, recording, stimulation and recording simultaneously, and isolation of biological tissues or slices in-vitro.

Microelectrode array recordings of excitability of low Mg^(2+)-induced acute hippocampal slices

Neuronal connections can be detected by neuronal network discharges in hippocampal neurons cultured on multi-electrodes.However,the multi-electrode-array（MEA）has not been widely used in hippocampal slice culture studies focused on epilepsy.The present study induced spontaneous synchronous epileptiform activity using low Mg2＋artificial cerebrospinal fluid on acute hippocampal slices to record hippocampal discharges with MEA.Results showed that burst duration and average number of spikes in a burst were significantly greater in the CA3 compared with dentate gyrus and CA1 areas.In Schaffer cut-off group,CA1 area discharges disappeared,but synchronous discharges remained in the CA3 area.Moreover,synchronous discharge frequency in the Schaffer cut-off group was similar to control.However,burst duration and average number of spikes in a burst were significantly decreased compared with control（P 〈 0.05）.Results demonstrated that highest neuronal excitability occurred in the CA3 area,and synchronous discharges induced by low Mg2＋originated from the CA3 region.

THE STUDY ON NOVEL MICROELECTRODE ARRAY CHIPS FOR THE DETECTION OF HEAVY METALS IN WATER POLLUTION

Qualitative and quantitative analysis of trace heavy metals in aqueous environment are rapidly assuming significance along with the rapid development of industry.In this paper,gold microelectrode array(MEA)plated with mercury film was used for simultaneous voltammetric detection of zinc,cadmium,lead and copper ions in water.The electrochemical behavior and the actual surface area of the MEA were investigated by cyclic voltammetry in K_(3)[Fe(CN)_(6)].Electrochemical impedance spectrum(EIS)was utilized to examine the deposition of mercury on the electrode surface.Based on anodic stripping voltammetry,mercury film
Au MEA was applied to the detection of heavy metals in artificial analyte,where good calibrate linearity was obtained for cadmium,lead and copper ions,but with zinc exhibiting poor linearity.

Highly-compliant,conformal and stretchable microelectrode arrays

Most biological tissues are supple and elastic, while current electronic devices fabricated by semiconductors and metals are usually stiff and brittle. As a result, implanted electronic devices can irritate and damage surrounding tissues, causing immune reaction and scarring. In this work, we develop stretchable microelectrode arrays, with the development of a novel soft lithography technology, which are designed and fabricated with a polymer/stretchable metal/polymer sandwich structure. With the great deformability of stretch, compression, bend and twisting, while preserving electrical property, this technology overcomes the fundamental mismatch of mechanical properties between biological tissues and electronic devices, and provides highly-compliant, confonnal and stretchable bio-electronic interfaces. Here we also describe the following three applications of the stretchable electrode arrays： a. monitoring intracranial electroencephalography （EEG）; b. stimulating peripheral nerves to drive muscles; c. monitoring epicardial electrocardiography （ECG）. Stretchable microelectrode arrays create a promising field in biomedical applications for its better modulus match with biological tissues and robust mechanical and electrical properties. They allow for construction of electronic integrated circuits spread over on complex and dynamic curved surfaces, providing a much friendlier bio-electronic interface for diagnosis, treatment and in- telligent bio-control.

An implantable microelectrode array for simultaneous L-glutamate and electrophysiological recordings in vivo

L-glutamate,the most common excitatory neurotransmitter in the mammalian central nervous system(CNS),is associated with a wide range of neurological diseases.Because neurons in CNS communicate with each other both electrically and chemically,dualmode(electric and chemical)analytical techniques with high spatiotemporal resolution are required to better understand glutamate function in vivo.In the present study,a silicon-based implantable microelectrode array(MEA)composed of both platinum electrochemical and electrophysiological microelectrodes was fabricated using micro-electromechanical system.In the MEA probe,the electrophysiological electrodes have a low impedance of 0.018 MΩat 1 kHz,and the electrochemical electrodes show a sensitivity of 56 pAμM^(−1) to glutamate and have a detection limit of 0.5μM.The MEA probe was used to monitor extracellular glutamate levels,spikes and local field potentials(LFPs)in the striatum of anaesthetised rats.To explore the potential of the MEA probe,the rats were administered to KCl via intraperitoneal injection.K+significantly increases extracellular glutamate levels,LFP low-beta range(12–18 Hz)power and spike firing rates with a similar temporal profile,indicating that the MEA probe is capable of detecting dual-mode neuronal signals.It was concluded that the MEA probe can help reveal mechanisms of neural physiology and pathology in vivo.

Microelectrode Array-evaluation of Neurotoxic Effects of Magnesium as an Implantable Biomaterial

Magnesium （Mg）-based biomaterials have shown great potential in clinical applications. However, the cytotoxic effects of excessive Mg2. and the corrosion products from Mg-based biomaterials, particularly their effects on neurons, have been little studied. Although viability tests are most commonly used, a functional evaluation is critically needed. Here, both methyl thiazolyl tetrazolium （MTT） and lactate de- hydrogenase （LDH） assays were used to test the effect of Mg2. and Mg-extract solution on neuronal viability. Microelectrode arrays （MEAs）, which provide long-term, real-time recording of extracellular electro- physiological signals of in vitro neuronal networks, were used to test for toxic effects. The minimum effective concentrations （ECmin） of Mg2. from the MTr and LDH assays were 3 mmol/L and 100 mmol/L respec- tively, while the ECmin obtained from the MEA assay was 0.1 mmol/L MEA data revealed significant loss of neuronal network activity when the culture was exposed to 25% Mg-extract solution, a concentra- tion that did not affect neuronal viability. For evaluating the biocompatibility of Mg-based biomaterials with neurons, MEA electrophysiological testing is a more precise method than basic cell-viability testing.

Nanoliposome-encapsulated caged-GABA for modulating neural electrophysiological activity with simultaneous detection by microelectrode arrays

Effective and precise neural modulation with real-time detection in the brain is of great importance and represents a significant challenge.Nanoliposome-encapsulated light-sensitive compounds have excellent characteristics such as high temporal and spatial resolution,delayed drug clearance,and restricted drug biodistribution for neural modulation.In this study,we developed a nanoliposome-based delivery system for ruthenium-based caged GABA compounds(Nanolipo-Ru)to modulate neural activity and allow for real-time monitoring using the microelectrode arrays(MEAs).The Nanolipo-Ru nanoparticles had an average size of 134.10±4.30 nm and exhibited excellent stability for seven weeks.For the in vivo experiment in the rat,release of GABA by Nanolipo-Ru under blue light illumination resulted in an average firing rate reduction in interneurons and pyramidal neurons in the same brain region of 79.4%and 81.6%,respectively.Simultaneously,the average power of local field potentials in the 0–15 Hz range degraded from 4.34 to 0.85 mW.In addition,the Nanolipo-Ru nanoparticles have the potential to provide more flexible timing of modulation than unencapsulated RuBi-GABA in the experiments.These results indicated that Nanolipo-Ru could be an effective platform for regulating neuronal electrophysiology.Furthermore,nanoliposomes with appropriate modifications would render promising utilities for targeting of specific types of neurons in the future.

Spatio-temporally resolved measurement of quantal exocytosis from single cells using microelectrode array modified with poly L-lysine and poly dopamine

The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array （MEA）, fabricated by MEMS technology, was applied successfully for real-time monitoring of quantal exocytosis from single pheochromocytoma （PC12） cell, The developed MEA was evaluated by dopamine （DA） using electrochemical methods and the results revealed that the sensitivity of DA was improved to 12659.24 p.A L mmol 1 cm 2. The modified MEA was used to detect in vitro vesicular exocytosis of DA from single PCI 2 cells stimulated by concentrated 100 mmol L-1 K＋ cell solution. A total of 592 spikes were measured and analyzed by three parameters and the statistical results revealed the population of each parameter was an approximate Gaussian distribution, and on average, 1.31 × 106 ± 9.25× 104 oxidizable molecules were released in each quantal exocytosis. In addition, results also indicate that a single PC12 cell probably releases the spikes with T ranging from 25.6 ms to 35.4 ms corresponding to/max ranging from 45.6 pA to 65.2 pA. The devices, including a homemade computer interface and the MEA modified with polymer film, provides a new means for further research on the neural, intercellular, communication mechanism.

Wireless closed-loop deep brain stimulation using microelectrode array probes

Deep brain stimulation(DBS),including optical stimulation and electrical stimulation,has been demonstrated considerable value in exploring pathological brain activity and developing treatments for neural disorders.Advances in DBS microsystems based on implantable microelectrode array(MEA)probes have opened up new opportunities for closed-loop DBS(CL-DBS)in situ.This technology can be used to detect damaged brain circuits and test the therapeutic potential for modulating the output of these circuits in a variety of diseases simultaneously.Despite the success and rapid utilization of MEA probe-based CL-DBS microsystems,key challenges,including excessive wired communication,need to be urgently resolved.In this review,we considered recent advances in MEA probe-based wireless CL-DBS microsystems and outlined the major issues and promising prospects in this field.This technology has the potential to offer novel therapeutic options for psychiatric disorders in the future.

Fabrication of a Silicon-Based Microprobe for Neural Interface Applications

A two-dimensional （2D） multi-channel silicon-based microelectrode array is developed for recording neural signals. Three photolithographic masks are utilized in the fabrication process. SEM images show that the microprobe is 1.2mm long, 100μm wide,and 30μm thick,with recording sites spaced 200μm apart for good signal isolation. For the individual recording sites, the characteristics of impedance versus frequency are shown by in vitro testing. The impedance declines from 14MΩ to 1.9kΩ as the frequency changes from 0 to 10MHz. A compatible PCB （print circuit board） aids in the less troublesome implantation and stabilization of the microprobe.

Advances in electrical impedance tomography-based brain imaging

Novel advances in the field of brain imaging have enabled the unprecedented clinical application of various imaging modalities to facilitate disease diagnosis and treatment. Electrical impedance tomography(EIT) is a functional imaging technique that measures the transfer impedances between electrodes on the body surface to estimate the spatial distribution of electrical properties of tissues. EIT offers many advantages over other neuroimaging technologies,which has led to its potential clinical use. This qualitative review provides an overview of the basic principles,algorithms, and system composition of EIT. Recent advances in the field of EIT are discussed in the context of epilepsy,stroke, brain injuries and edema, and other brain diseases. Further, we summarize factors limiting the development of brain EIT and highlight prospects for the field. In epilepsy imaging, there have been advances in EIT imaging depth,from cortical to subcortical regions. In stroke research, a bedside EIT stroke monitoring system has been developed for clinical practice, and data support the role of EIT in multi-modal imaging for diagnosing stroke. Additionally, EIT has been applied to monitor the changes in brain water content associated with cerebral edema, enabling the early identification of brain edema and the evaluation of mannitol dehydration. However, anatomically realistic geometry,inhomogeneity, cranium completeness, anisotropy and skull type, etc., must be considered to improve the accuracy of EIT modeling. Thus, the further establishment of EIT as a mature and routine diagnostic technique will necessitate the accumulation of more supporting evidence.

Role of induced pluripotent stem cells in diagnostic cardiology

Ethical concerns about stem cell-based research have delayed important advances in many areas of medicine,including cardiology.The introduction of induced pluripotent stem cells(iPSCs)has supplanted the need to use human stem cells for most purposes,thus eliminating all ethical controversies.Since then,many new avenues have been opened in cardiology research,not only in approaches to tissue replacement but also in the design and testing of antiarrhythmic drugs.This methodology has advanced to the point where induced human cardiomyocyte cell lines can now also be obtained from commercial sources or tissue banks.Initial studies with readily available iPSCs have generally confirmed that their behavioral characteristics accurately predict the behavior of beating cardiomyocytes in vivo.As a result,iPSCs can provide new ways to study arrhythmias and heart disease in general,accelerating the development of new,more effective antiarrhythmic drugs,clinical diagnoses,and personalized medical care.The focus on producing cardiomyocytes that can be used to replace damaged heart tissue has somewhat diverted interest in a host of other applications.This manuscript is intended to provide non-specialists with a brief introduction and overview of the research carried out in the field of heart rhythm disorders.

Automatic Monitoring Electronic Tongue with MEAs for Environmental Analysis

An automatic monitoring electronic tongue based on differential pulse stripping voltammetry (DPSV) was developed for heavy metals analysis.Simultaneous detections of trace Zn(Ⅱ),Cd(Ⅱ),Pb(Ⅱ),Cu(Ⅱ),Fe(Ⅲ) and Cr(Ⅲ) in water samples were performed with three electrochemical sensors.The sensor chip combined a silicon-based Hg-coated Au microelectrode array (MEA) as the working electrode on one side with an Ag/AgCl reference electrode and a Pt counter electrode on the other side.With a computer controlled multipotentiostat,pumps and valves,the electronic tongue realized in-situ real-time detection of the six metals mentioned above at parts-per-billion level without manual operation.

Quantitative evaluation of extrinsic factors influencing electrical excitability in neuronal networks： Voltage Threshold Measurement Method(VTMM)

The electrical excitability of neural networks is influenced by different environmental factors. Effective and simple methods are required to objectively and quantitatively evaluate the influence of such factors, including variations in temperature and pharmaceutical dosage. The aim of this paper was to introduce ‘the voltage threshold measurement method＇, which is a new method using microelectrode arrays that can quantitatively evaluate the influence of different factors on the electrical excitability of neural networks. We sought to verify the feasibility and efficacy of the method by studying the effects of acetylcholine, ethanol, and temperature on hippocampal neuronal networks and hippocampal brain slices. First, we determined the voltage of the stimulation pulse signal that elicited action potentials in the two types of neural networks under normal conditions. Second, we obtained the voltage thresholds for the two types of neural networks under different concentrations of acetylcholine, ethanol, and different temperatures. Finally, we obtained the relationship between voltage threshold and the three influential factors. Our results indicated that the normal voltage thresholds of the hippocampal neuronal network and hippocampal slice preparation were 56 and 31 m V, respectively. The voltage thresholds of the two types of neural networks were inversely proportional to acetylcholine concentration, and had an exponential dependency on ethanol concentration. The curves of the voltage threshold and the temperature of the medium for the two types of neural networks were U-shaped. The hippocampal neuronal network and hippocampal slice preparations lost their excitability when the temperature of the medium decreased below 34 and 33°C or increased above 42 and 43°C, respectively. These results demonstrate that the voltage threshold measurement method is effective and simple for examining the performance/excitability of neuronal networks.

Effect of visual stimulus locations on pattern-reversal visual evoked potential An epidural electrocorticogram study

To explore the effect of the location of a visual stimulus on neural responses in the primary visual cortex （V1）, a micro-electromechanical system-based microelectrode array with nine channels was implanted on the cerebral dura mater of V1 in adult cats. 2 Hz pattern reversal checkerboard stimul were used to stimulate the four visual quadrants （i.e., upper left, upper right, lower left, and lower right fields）. The results showed that there was a N75 component of the visual evoked potential around 50-80 ms after the onset of a checkerboard stimulus, and the onset of these N75 peaks varied with different stimulus locations. The checkerboard stimuli Jnduced shorter latencJes in the contralateral V1 than in the ipsilateral V1, while the checkerboard stimulus in the upper half visual field induced shorter latencies for N75. These results suggested that the pattern-reversal stimuli induced neural activities in V1 that can be recorded with multichannel microelectrodes, and more detailed temporal and spatial properties can be measured.

Microelectrode Recording of Tissue Neural Oscillations for a Bionic Olfactory Biosensor

In olfactory research, neural oscillations exhibit excellent temporal regularity, which are functional and necessary at thephysiological and cognitive levels. In this paper, we employed a bionic tissue biosensor which treats intact epithelium as sensing element to record the olfactory oscillations extracellularly. After being stimulated by odorant of butanedione, the olfactory receptor neurons generated different kinds of oscillations, which can be described as pulse firing oscillation, transient firing oscillation, superposed firing oscillation, and sustained firing oscillation, according to their temporal appearances respectively. With a time-frequency analysis of sonogram, the oscillations also demonstrated different frequency properties, such as δ, θ, α, β and γ oscillations. The results suggest that the bionic biosensor cooperated with sonogram analysis can well improve the in- vestigation of olfactory oscillations, and provide a novel model for artificial olfaetion sensor design.

Three-dimensional fuzzy graphene ultra-microelectrodes for subcellular electrical recordings

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

Dopamine detection using a patch-clamp system on a planar microeletrode array electrodeposited by polypyrrole/graphene nanocomposites

To achieve a dopamine （DA） response with high sensitivity and high signal-to-noise ratio （S/N） with a patch-clamp system, polypyrrole/graphene （PPy/GR） nanocomposites were steadily electrodeposited by an electrochemical method on a planar mi- croelectrode array （pMEA） fabricated by a standard micromachining process. The electrodeposition process was carried out by chronopotentimetry measurement scanning from 0.1 to 0.8 C/cm2 at the current of 2 mA; 0.5 C/cm2 was found to be optimal. The pMEA modified by PPy/GR at the 0.5 C/cm2 exhibits remarkable properties; for instance, the standard deviation （SD） de- creases from 8.4614×10-al to 5.62×10 11 A, reduced by 33.52%, and the sensitivity increases from 2566.88 to 76114.65 gAmMcm2 , 29.65 times higher than the bare Pt （platinum）. A good linear relationship between the current and DA concentra- tion in the range of 0.30 to 61.71 grn was obtained, with a correlation coefficient of 0.997. The sensor is meaningful for neuro- science research and the treatment of neurological diseases.

Spatiotemporal dynamics of high-K^+-induced epileptiform discharges in hippocampal slice and the effects of valproate

The epileptic seizure is a dynamic process involving a rapid transition from normal activity to a state of hypersynchronous neuronal discharges. Here we investigated the network properties of epileptiform discharges in hippocampal slices in the presence of high K ＋ concentration （8.5 mmol/L） in the bath, and the effects of the anti-epileptic drug valproate （VPA） on epileptiform discharges, using a microelectrode array. We demonstrated that epileptiform discharges were predominantly initiated from the stratum pyramidale layer of CA3a-b and propagated bi-directionally to CA1 and CA3c. Disconnection of CA3 from CA1 abolished the discharges in CA1 without disrupting the initiation of discharges in CA3. Further pharmacological experiments showed that VPA at a clinically relevant concentration （100 μmol/L） suppressed the propagation speed but not the rate or duration of high-K＋-induced discharges. Our findings suggest that pacemakers exist in the CA3a-b region for the generation of epileptiform discharges in the hippocampus. VPA reduces the conduction of such discharges in the network by reducing the propagation speed.