Microelectrode Array Chip Research Articles

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.

A Silicon-Based Field-Effect Biosensor for Drug-Induced Cardiac Extracellular Calcium Ion Change Detection.

Calcium ions participate in the regulation of almost all biological functions of the body, especially in cardiac excitation-contraction coupling, acting as vital signaling through ion channels. Various cardiovascular drugs exert their effects via affecting the ion channels on the cell membrane. The current strategies for calcium ion monitoring are mainly based on fluorescent probes, which are commonly used for intracellular calcium ion detection (calcium imaging) and cannot achieve long-term monitoring. In this work, an all-solid-state silicone-rubber ion-sensitive membrane was fabricated on light-addressable potentiometric sensors to establish a program-controlled field-effect-based ion-sensitive light-addressable potentiometric sensor (LAPS) platform for extracellular calcium ion detection. L-type calcium channels blocker verapamil and calcium channel agonist BayK8644 were chosen to explore the effect of ion channel drugs on extracellular calcium ion concentration in HL-1 cell lines. Simultaneously, microelectrode array (MEA) chips were employed to probe the HL-1 extracellular field potential (EFP) signals. The Ca2+ concentration and EFP parameters were studied to comprehensively evaluate the efficacy of cardiovascular drugs. This platform provides more dimensional information on cardiovascular drug efficacy that can be utilized for accurate drug screening.

High-definition electroporation: Precise and efficient transfection on a microelectrode array.

Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.

A Taste Bud Organoid-Based Microelectrode Array Biosensor for Taste Sensing

The biological taste system has the unique ability to detect taste substances. Biomaterials originating from a biological taste system have been recognized as ideal candidates to serve as sensitive elements in the development of taste-based biosensors. In this study, we developed a taste bud organoid-based biosensor for the research of taste sensation. Taste bud organoids prepared from newborn mice were cultured and loaded onto the surface of a 64-channel microelectrode array (MEA) chip to explore the electrophysiological changes upon taste; an MEA chip was used to simultaneously record multiple-neuron firing activities from taste bud organoids under different taste stimuli, which helped to reveal the role of taste buds in taste sensing. The obtained results show that taste cells separated from the taste epithelium grew well into spherical structures under 3D culture conditions. These structures were composed of multiple cells with obvious budding structures. Moreover, the multicellular spheres were seeded on a 64-channel microelectrode array and processed with different taste stimuli. It was indicated that the MEA chip could efficiently monitor the electrophysiological signals from taste bud organoids in response to various taste stimuli. This biosensor provides a new method for the study of taste sensations and taste bud functions.

An In Vitro HL-1 Cardiomyocyte-Based Olfactory Biosensor for Olfr558-Inhibited Efficiency Detection

Some short-chain fatty acids with a pungent or unpleasant odor are important components of human body odor. These malodors severely threaten human health. The antagonists of malodors would help to improve odor perception by affecting the interaction between odors and their receptors. However, the traditional odor detection and analysis methods, such as MOS, electrochemical, conductive polymer gas sensors, or chromatography-mass spectrometry are not suitable for screening the antagonists since they are unable to detect the ligand efficacy after odor-receptor binding. In this study, RT-PCR results showed that HL-1 cardiomyocytes endogenously express the olfactory receptor 558 (Olfr558) which can be activated by several malodorous short-chain fatty acids. Therefore, an in vitro HL-1 cardiomyocyte-based olfactory biosensor (HCBO-biosensor) was developed by combining cardiomyocytes and microelectrode array (MEA) chips for screening the potential antagonists of the Olfr558. Firstly, it showed that the biosensor specifically responded to ligands of Olfr558 through odor stimulation experiments. Then, an odor response model of HL-1 cardiomyocytes was constructed by a ligand of Olfr558 (isovaleric acid). The response feature of the in vitro HCBO-biosensor to individual odors and mixtures with a potential antagonist (citral or β-damascenone) were extracted and compared. Finally, the Olfr558-inhibited efficiency was indirectly detected by comparing the half-maximal inhibitory concentration of isovaleric acid. The results showed that β-damascenone greatly inhibited Olfr558 while citral showed no significant inhibitory effect. In conclusion, we built a novel screening method for the antagonists of Olfr558 based on HL-1 cardiomyocytes and the MEA chip which will assist odor-related companies to develop novel antagonists of Olfr558.

A biomimetic sensor using neurotransmitter detection to decode odor perception by an olfactory network

Mammalian olfactory perception is an important physiological function for tasks such as finding food, identifying species, and avoiding enemies. Previous studies have demonstrated the molecular basis of receptors and the anatomical structure of the olfactory bulb (OB), which is the initial processing center for odor perception. However, there remains some controversy about the coding and transmission mode of olfactory information in the OB through neuronal interaction mediated by different neurotransmitters. In this paper, a biomimetic sensor based on OB neuronal network was developed to detect trace amounts of typical neurotransmitters and decode odor perception in vitro. Primary OB neurons were seeded on a microelectrode array (MEA) chip to capture multichannel extracellular electrical activities. The firing features of OB neurons were statistically analyzed after stimulation with graded concentrations of the glutamate and gamma-aminobutyric acid (GABA). Cross-correlation analysis between channels was used to evaluate the connection status in the neuronal networks. The concentration-dependent responses to these two neurotransmitters were assessed over a range of values, and the lower limits of detection for glutamate and GABA were 100 nM and 50 nM, respectively. Stimulation with excessive concentrations produced response tolerance and neurotoxicity, and transmission between cells in the neural network was modulated by different neurotransmitters. This device could serve as a novel biosensor for detecting trace amounts of common neurotransmitters and for screening the effects of drugs on the physiology of olfaction. The response patterns of this biomimetic sensor are conducive to revealing the coding mechanism of information processing in the olfactory system.

Biohybrid Tongue for Evaluation of Taste Interaction between Sweetness and Sourness

The past decade has witnessed tremendous progress achieved in taste research, while few studies focus on interactions among taste compounds. Indeed, sweeteners and acidulants are commonly used food additives, and sweet-sour mixtures always provide improved tastes. For example, sensory studies have shown that sourness suppresses sweetness. However, the degree of sweetness suppression by sourness is difficult to evaluate quantitatively and objectively. Therefore, we propose a biohybrid tongue that is constructed by integrating mammalian gustatory epithelium with a microelectrode array chip. The taste quality and intensity information is coded in time-frequency patterns of local field potential. Different response patterns evoked by sweet and sour stimuli are observed, and the response is dose-dependent. Then, interaction effects of sourness against sweetness are quantified. The results indicate that suppression of sweetness by sourness occurs by increasing sourness concentrations. In summary, this study provides a powerful new tool for quantitative evaluation of sweet, sour, and their binary taste interactions that mimic the mammalian taste system.

Capacitive Aptasensor Coupled with Microfluidic Enrichment for Real-Time Detection of Trace SARS-CoV-2 Nucleocapsid Protein.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for almost 2 years. Stemming its spread has posed severe challenges for clinical virus detection. A long turnaround time, complicated operation, and low accuracy have become bottlenecks in developing detection techniques. Adopting a direct antigen detection strategy, we developed a fast-responding and quantitative capacitive aptasensor for ultratrace nucleocapsid protein detection based on a low-cost microelectrode array (MEA) chip. Employing the solid–liquid interface capacitance with a sensitivity of picofarad level, the tiny change on the MEA surface can be definitively detected. As a result, the limit of detection reaches an ultralow level of femtogram per milliliter in different matrices. Integrated with efficient microfluidic enrichment, the response time of this sensor from the sample to the result is shortened to 15 s, completely meeting the real-time detection demand. Moreover, the wide linear range of the sensor is from 10–5 to 10–2 ng/mL, and a high selectivity of 6369:1 is achieved. After application and evaluation in different environmental and body fluid matrices, this sensor and the detection method have proved to be a label-free, real-time, easy-to-operate, and specific strategy for SARS-CoV-2 screening and diagnosis.

(Invited) Toward Rapid Profiling of Proteases Activities for Cancer Diagnosis Based on Multiplex Microelectrode Array Sensors

We report the development of an electrochemical biosensor platform based on multiplex micro-/nano- electrode arrays toward cancer diagnosis based on rapid profiling of protease activities. Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of a nanoelectrode array platform based on embedded vertically aligned carbon nanofibers for electrochemical detection of protease activities and further development into an individually addressed 3x3 gold thin-film microelectrode array for rapid profiling of multiple protease activities.Peptides with specific sequences of 4 to 8 amino acids are designed and synthesized as the substrates for selective proteolysis by the cognate proteases, which are attached to the electrode surface as the specific probes. The far end is covalently attached with a ferrocene (Fc) group as the redox moiety for electrochemical measurements. The quantity of the intact peptides on the electrode surface can be sensitively detected with an AC voltammetry method and presents as the peak current at the specific potential for Fc oxidation into ferrocenium (Fc+). As the peptide is cleaved by the cognate protease, the peak current decreases exponentially in the continuously repeated AC voltammetry measurements. The recorded kinetic proteolytic curve can be quantitatively described by a surface-based heterogeneous Michaelis-Menten model. The inverse of exponential decay time constant, 1/t, is found to represent the protease activity which equals to [E](kcat /KM), where [E] is the protease concentration and kcat /KM is the specificity constant defined by the kinetic proteolytic reaction constant kcat and the Michaelis equilibrium constant KM. We have systematically investigated the factors that affect the value of kcat /KM, including the peptide sequence, peptide length, temperature and buffer composition, and optimized the conditions for highly sensitive detection of protease activity of cathepsin B, a potential cancer biomarker.Toward rapid profiling of multiple proteases, we have developed a multiplex electrochemical sensor chip based on a 3x3 gold microelectrode array. The nine individual gold microelectrodes are partially buried underneath the surrounding SiO2 thin film, which show highly consistent cyclic voltammetric signals in gold surface cleaning experiments and detecting benchmark redox species in solution. Upon selectively functionalizing the individual gold microelectrodes with the specific ferrocene-labeled peptide probes, simultaneous detection of the proteolytic curves of the target proteases can be obtained over 9 channels by monitoring the decay of the AC voltammetry signal of the ferrocene-labeled peptide molecules. So far, the algorithm for fitting the kinetic proteolytic curves to accurately derive the activity of cathepsin B has been established. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides has been demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases.Interestingly, the above-discussed electrochemical method detects the activity of the proteases, which reflects the true biological function and is fundamentally different from the concentration derived from commonly used affinity biosensors or assays such as enzyme-linked immunosorbent assay (ELISA). The direct comparison has shown that ELISA measurements will give the same results no matter the original proenzyme form (zymogen) in the sample is activated or not, while the electrochemically measured activity shows dramatic increase after being chemically activated. In addition, in the current practice, each protease is measured separately in its optimal buffer with the pH value varying from 5 to 9. It is not possible to measure them in a common buffer simultaneously. We have demonstrated that the electrochemical method can be applied for protease activity profiling in a buffer at pH = 7.4 that is compatible to the physiology conditions. This enables measuring the activity of multiple proteases in human serum, which is a critical step toward protease activity for cancer diagnosis.The figure shows the optical images of (a) the whole 3x3 Au microelectrode array chip and (b) the active electrode area, and the schematic illustration of (c) the mechanism of proteolysis of the peptide probes, (d) the change of the AC voltammetry signal before and after proteolysis and (e) the kinetic proteolytic curves, i.e. the exponential peak current decay over time, at different protease concentration [E]. Figure 1

Customised micro-electrode array (MEA) test setup featuring a silicone-casted overlay with two chambers for separated cell seedings

Abstract Micro-electrode array (MEA) systems are noninvasive platforms for the investigation of electrophysiological properties of cell layers, such as spontaneously active cardiomyocytes. An MEA chip is composed of two-dimensional grids of dot-like electrodes embedded into glass. Here we present a test setup featuring a customised two-chamber silicone overlay. The overlay is designed to be placed on an MEA with two separate electrode fields and enables the seeding of two distinct cell sub-types on the MEA for synchronised drug testing applications while giving the possibility of analysing intersubtype-specific cellular interactions. The overlay has a full size of 10 x 10 x 5 mm (width x length x height), each chamber has a size of 2.5 x 6 x 5 mm (V = 75 mm³). The chambers are separated by a wall with a thickness of 0.3 mm. The overlay was manufactured via silicone-casting, utilising a 3D printed model. The model is 3D printed via high accurate digital light processing (DLP). In addition, a DLP 3D printed cover optimises the attachment of the overlay on an MEA. A proofof- principle of the utilisation of the overlay is demonstrated.

Single-cell individualized electroporation with real-time impedance monitoring using a microelectrode array chip

The ability to precisely deliver molecules into single cells while maintaining good cell viability is of great importance to applications in therapeutics, diagnostics, and drug delivery as it is an advancement toward the promise of personalized medicine. This paper reports a single-cell individualized electroporation method with real-time impedance monitoring to improve cell perforation efficiency and cell viability using a microelectrode array chip. The microchip contains a plurality of sextupole-electrode units patterned in an array, which are used to perform in situ electroporation and real-time impedance monitoring on single cells. The dynamic recovery processes of single cells under electroporation are tracked in real time via impedance measurement, which provide detailed transient cell states and facilitate understanding the whole recovery process at the level of single cells. We define single-cell impedance indicators to characterize cell perforation efficiency and cell viability, which are used to optimize electroporation. By applying the proposed electroporation method to different cell lines, including human cancer cell lines and normal human cell lines individually, optimum stimuli are determined for these cells, by which high transfection levels of enhanced green fluorescent protein (EGFP) plasmid into cells are achieved. The results validate the effectiveness of the proposed single-cell individualized electroporation/transfection method and demonstrate promising potential in applications of cell reprogramming, induced pluripotent stem cells, adoptive cell therapy, and intracellular drug delivery technology.

A biohybrid nose for evaluation of odor masking in the peripheral olfactory system

Olfaction is a synthetic sense in which odor mixtures elicit emergent perceptions at the expense of perceiving the individual components. The most common result of mixing two odors is masking one component by another. However, there is lack of analytical techniques for measuring the sense of smell, which is mediated by cross-odorant interactions. Here, we propose a biohybrid nose for objective and quantitative evaluation of malodor masking efficiency of perfumed products. This biohybrid nose is constructed by integrating mammalian olfactory epithelium with microelectrode array chip to read out the olfactory information as electrical signal from multiple tissue sites. The intrinsic odor response of olfactory epithelium is found to be represented by widespread spatiotemporal oscillatory activity. The masking efficiency of fragrance is quantified by calculating the relative difference between the malodor and the binary mixture (malodor + fragrance) response patterns. Results indicate that masking efficiency of fragrance is concentration-dependent, whereas completely masking may occurs when fragrance is employed at a concentration 2–3 orders of magnitude higher than malodor. This study demonstrates for the first time that capitalizing on the biological sense of smell to create biohybrid system provides an effective technique to resolve more complex biosensing-related issues such as odor interactions in mixtures.

Label-Free Detection of Zeptomol miRNA via Peptide Nucleic Acid Hybridization Using Novel Cyclic Voltammetry Method.

To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π–π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity.

A microelectrode array chip for osteogenic differentiation of mesenchymal stem cells under electrical stimulation.

Electrical stimulation (ES) as an easy and effective inducing method has been widely used in induction differentiation of stem cells, e.g. osteogenic differentiation of mesenchymal stem cells (MSCs) for bone healing and bone tissue therapies. However, the micro-effect of an inhomogeneous electric field has rarely been investigated for ES in induction differentiation, and conventionally used ex situ assays may preclude accurate assessment due to variation from cell inoculation and treatments. Here, a novel electrical stimulation method with a microelectrode array chip is proposed for osteogenic differentiation of MSCs. The electric field applied onto the MSCs by the microelectrode array is designed similarly with a natural aggregation distribution of differentiated MSCs. The proposed ES method accelerates osteoblast proliferation and differentiation in the electrode array region and generates a larger amount of mineralized deposits, which are assayed via in situ alizarin red staining and morphology observation as well as immunocytochemistry. In addition, this method allows a direct in situ assessment to compare the osteogenic differentiation of MSCs with and without ES on a single chip to avoid culture environment difference. The method provides a fundamental platform for investigating induced differentiation of stem cells and allows integration with multifunctional cell assays to achieve in situ tracking for the differentiation process of stem cells.

In vitro pathological model of Alzheimer's disease based on neuronal network chip and its real-time dynamic analysis

Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-β oligomers (AβOs) and the intracellular accumulation of neurofibrillary tangles formed by hyperphosphorylated tau protein. In this paper, an in vitro pathological model of AD based on neuronal network chip and its real-time dynamic analysis were presented. The hippocampal neuronal network was cultured on the microelectrode array (MEA) chip and induced by AβOs as an AD model in vitro to simultaneously record two firing patterns from the interneurons and pyramidal neurons. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. This biosensor enabled the detection of the AβOs toxicity responses, and the identification of connectivity and interactions between neuronal networks, which can be a novel technique in the research of AD pathological model in vitro.

Abstract 3978: Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform

Abstract Exosomes carry signals between cells and may influence cancer development, metastasis, and drug resistance. Unfortunately, current tools for exosome isolation and analysis have been limited to examining a specific class of markers, such as DNA, RNA, and proteins. We have developed a microelectrode array chip that uses a novel Alternating Current Electrokinetic method to perform rapid, affinity-free isolation of exosomes. The chip runs on our proprietary ExoVeritaTM Flex platform. Isolated exosomes can then be analyzed in situ or can be lysed to obtain DNA and RNA for use in downstream assays, such as sequencing. In this study, we used the ExoVerita Flex to isolate exosomes directly from plasma specimens obtained from cancer subjects with lung, colorectal, or pancreatic cancers as well as healthy donors. Isolated exosomes were analyzed in situ for exosome-bound proteins and nucleic acids, and then purified following isolation to examine their nucleic acid expression profiles. Using the ExoVerita Flex with plasma specimens and antibodies targeting the exosome-bound proteins CEA, PD-L1, GPC-1 and CD63, we observed differences in protein expression levels among the different cancer types (N = 35) and healthy donors (N = 20), such as increased PD-L1 expression in non-small cell lung cancer subject samples and elevated levels of CEA in colorectal cancer subject samples (N = 10). Furthermore, we co-stained samples with antibodies and DNA- or RNA-selective dyes to simultaneously measure exosome-bound proteins and circulating nucleic acid levels in the same sample at the same time. RNA sequencing analysis was performed by extracting RNA from the isolated exosomes using a proprietary buffer, and then performing RT-ddPCR and miRNA-Sequencing. RT-ddPCR analysis of the PGK-1 housekeeping gene RNA transcript confirmed the presence of viable RNA following extraction. miRNA profiles from isolated exosomes were generated using a targeted RNA sequencing assay and used for repeatability and differential expression studies. The ExoVerita Flex platform offers a novel, unbiased method for isolation and characterization of exosome-bound proteins and circulating nucleic acids while being compatible with standard downstream analysis. The ability to perform two-dimensional protein & nucleic acid analysis in the same sample run, at the same time, is both unique and unprecedented and will enable promising future clinical studies in cancer looking at diverse classes of biomarkers directly from blood. Citation Format: Jean M. Lewis, David Searson, Alfred Kinana, Heath Balcer, Debrah Thompson, David Bodkin, Juan P. Hinestrosa, Rajaram Krishnan. Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3978.

Gene-Embedded Nanostructural Biotic-Abiotic Optoelectrode Arrays Applied for Synchronous Brain Optogenetics and Neural Signal Recording.

Optogenetics is a recently established neuromodulation technique in which photostimulation is used to manipulate neurons with high temporal and spatial precision. However, sequential genetic and optical insertion with double brain implantation tends to cause excessive tissue damage. In addition, the incorporation of light-sensitive genes requires the utilization of viral vectors, which remains a safety concern. Here, by combining device fabrication design, nanotechnology, and cell targeting technology, we developed a new gene-embedded optoelectrode array for neural implantation to enable spatiotemporal electroporation (EP) for gene delivery/transfection, photomodulation, and synchronous electrical monitoring of neural signals in the brain via one-time implantation. A biotic-abiotic neural interface (called PG) composed of reduced graphene oxide and conductive polyelectrolyte 3,4-ethylenedioxythiophene-modified amphiphilic chitosan was developed to form a nanostructural hydrogel with assembled nanodomains for encapsulating nonviral gene vectors (called PEI-NT-pDNA) formulated by neurotensin (NT) and polyethylenimine (PEI)-coupled plasmid DNA (pDNA). The PG can maintain high charge storage ability to respond to a minimal current of 125 μA for controllable gene delivery. The in vitro analysis of PG-PEI-NT-pDNA on the microelectrode array chip showed that the microelectrodes provided electrically inductive electropermeabilization, which permitted gene transfection into localized rat adrenal pheochromocytoma cells with a strong green fluorescent protein expression that was up to 8-fold higher than that in nontreated cells. Furthermore, the in vivo implantation enabled on-demand spatiotemporal gene transfection to neurons with 10-fold enhancement of targeting ability compared with astrocytes. Finally, using the real optogenetic opsin channelrhodopsin-2, the flexible neural probe incorporated with an optical waveguide fiber displayed photoevoked extracellular spikes in the thalamic ventrobasal region after focal EP for only 7 days, which provided a proof of concept for the use of photomodulation to facilitate neural therapies.

Voltammetric Application of Polypyrrole-Modified Microelectrode Array for the Characterization of DNA Methylation in Glutathione S-Transferase Pi 1

ABSTRACTDirect and efficient label-free voltammetric detection of glutathione S-transferase Pi 1 (GSTP1) hypermethylation is reported using a custom-developed 16-channel microelectrode array chip. The microelectrode array chip is used in a dipstick configuration allowing detection of DNA hybridization in a solution volume of only 0.35 mL. Platinum microelectrode disks (n = 16) 30 µm in diameter have been modified with a polypyrrole bilayer before any contact with the oligonucleotides. The attachment of 15-mer Probe DNA to the bilayer is random but controlled by the presence of aliphatic tether groups allowing it to form a bidentate complex with the probe DNA. The voltammetric detection procedure of methylated GSTP1-specific target DNA is combined with bisulfite treatment of target DNA. Changes at the interface of the modified microelectrodes in an array configuration are used to record simultaneously cyclic voltammetry on all of the devices. The detection of hybridization is evaluated statistically by a yes or no event by comparing the changes in recorded cyclic voltammograms before and after exposure to the target DNA. All cyclic voltammograms of the methylated target show a greater percentage change than those with the nonmethylated target exposure and show a greater change in cyclic voltammogram area after methylated target exposure. We observe an average percentage difference of 25.6 ± 4.9% with a variation of 19.1%. These results demonstrate that the fast sensing strategy possesses sensitivity and good specificity. Furthermore, this technology can potentially support rapid, accurate diagnosis and risk assessment of patients with prostate cancer.

Extracellular field potential recording of single cardiomyocytes in agarose microchambers using microelectrode array

To study cell communication, it is essential that single cell responses in cellular networks are analyzed. Cardiomyocyte networks are known to simultaneously change membrane potential with spontaneous excitation. To measure the extracellular potential change in single cardiomyocytes in networks, we developed a microelectrode array (MEA) chip consisting of 8-µm-diameter and 3-µm-thick platinum-black (Pt-B)-coated indium tin oxide (ITO) electrodes with agarose microchambers. Single cardiomyocytes were seeded individually on this MEA chip with a micropipette. The extracellular potential of a single cardiomyocyte could be measured with this MEA chip. The amplitude of the extracellular potential change in a single cardiomyocyte was very small (<100 µV) and the beating rate was not constant. We expected that these results would accelerate the measurement of membrane potential changes of single cardiomyocytes in cellular networks.

Functional epilepsy modelling – a novel platform for studying human stem cell -derived neuronal networks

Event Abstract Back to Event Functional epilepsy modelling – a novel platform for studying human stem cell -derived neuronal networks Anssi Pelkonen1*, Lassi Sukki2, Tomi Ryynänen3, Tanja Hyvärinen1, Jukka Lekkala3, Pasi Kallio2 and Susanna Narkilahti1 1 University of Tampere, BioMediTech and Faculty of Medicine and Life Sciences, Finland 2 Tampere University of Technology, Faculty of Biomedical Sciences and Engineering, Finland 3 Tampere University of Technology, Faculty of Biomedical Sciences and Engineering, Finland Motivation Epilepsy is characterized by spread of abnormal electrical activity within and between neuronal networks. New epilepsy treatments are tested in rodent models, but less than 10 % of putative drugs are accepted for use[1] and 30 % of epilepsy patients still do not respond to existing treatments[2]. Therefore, new models are needed for epilepsy modelling and drug testing. Human pluripotent stem cell (hPSC) –derived neural networks are the most promising base material for such models, but they alone cannot model the connections, feedback loops and spread of abnormal activity between neuronal networks. Material and Methods Our solution is an innovative new platform which combines human stem cell –derived neural networks, microfluidic devices and microelectrode array (MEA) technology. hPSC–derived cortical neurons are cultured in the platform where they form functional networks. The networks are cultured in a microfluidic cell culture device which isolates the stem cell derived networks and the axonal connections between them. The device is comprised of polydimethylsiloxane (PDMS), and shaped using a micro- and millimeter-scale mold which consists of SU-8 and stainless steel (3D printed using selective laser melting (SLM)). The device is connected to a custom made MEA chip for measuring the electrical activity within and between the networks. The MEA chip is compatible with a commercially available MEA system (MEA2100, Multi Channel Systems, Reutlingen, Germany). Results The produced neuronal networks are highly active and respond to glutamatergic and GABAergic factors. The PDMS device allows the formation of axonal connections between separated neural networks. The PDMS device and the MEA chip are a suitable environment for the neural networks, which remained viable and active up to 12 weeks. The MEA data shows that the network activity in the device develops into synchronous, network-to-network connected bursts in 3-5 weeks. Individual networks can be subjected to seizure-inducing drugs, and the effects of the drug treatments are clearly visible in the MEA-data. Conclusion The platform is suitable for studying network activity and connectivity of human stem cell –derived neural networks. Therefore, it has the potential to be used in disease modelling and drug testing for epilepsy and other central nervous system disorders. Acknowledgements Funding: Business Finland (formerly known as Finnish Funding Agency for Technology and Innovation (TEKES)); Academy of Finland (Decision No. 311017); Finnish Cultural Foundation (personal grant, TR); Juliana von Wendt fund (personal grant, AP). The work was supported by the Imaging Facility, Facility of Electrophysiological Measurements and iPS Cells Facility (Uni. Tampere). Thanks to Arla Tanner for technical assistance and Juha Heikkilä for assistance with measurement and analysis software.