Novel Multiparametric Bioelectronic Measurement System for Monitoring Virus-Induced Alterations in Functional Neuronal Networks.

Development and optimisation of bioelectronic monitoring techniques like microelectrode array-based field potential measurement and impedance spectroscopy for the functional, label-free and non-invasive monitoring of in vitro neuronal networks is widely investigated in the field of biosensors. Thus, these techniques were individually used to demonstrate the capabilities of, e.g., detecting compound-induced toxicity in neuronal culture models. In contrast, extended application for investigating the effects of central nervous system infecting viruses are rarely described. In this context, we wanted to analyse the effect of herpesviruses on functional neuronal networks. Therefore, we developed a unique hybrid bioelectronic monitoring platform that allows for performing field potential monitoring and impedance spectroscopy on the same microelectrode. In the first step, a neuronal culture model based on primary hippocampal cells from neonatal rats was established with reproducible and stable synchronised electrophysiological network activity after 21 days of cultivation on microelectrode arrays. For a proof of concept, the pseudorabies model virus PrV Kaplan-ΔgG-GFP was applied and the effect on the neuronal networks was monitored by impedance spectroscopy and field potential measurement for 72 h in a multiparametric mode. Analysis of several bioelectronic parameters revealed a virus concentration-dependent degeneration of the neuronal network within 24-48 h, with a significant early change in electrophysiological activity, subsequently leading to a loss of activity and network synchronicity. In conclusion, we successfully developed a microelectrode array-based hybrid bioelectronic measurement platform for quantitative monitoring of pathologic effects of a herpesvirus on electrophysiological active neuronal networks.

Novel high-dense microelectrode array based multimodal bioelectronic monitoring system for cardiac arrhythmia re-entry analysis.

In recent decades, significant progress has been made in the treatment of heart diseases, particularly in the field of personalized medicine. Despite the development of genetic tests, phenotyping and risk stratification are performed based on clinical findings and invasive in vivo techniques, such as stimulation conduction mapping techniques and programmed ventricular pacing. Consequently, label-free non-invasive in vitro functional analysis systems are urgently needed for more accurate and effective in vitro risk stratification, model-based therapy planning, and clinical safety profile evaluation of drugs. To overcome these limitations, a novel multilayer high-density microelectrode array (HD-MEA), with an optimized configuration of 512 sensing and 4 pacing electrodes on a sensor area of 100 mm2, was developed for the bioelectronic detection of re-entry arrhythmia patterns. Together with a co-developed front-end, we monitored label-free and in parallel cardiac electrophysiology based on field potential monitoring and mechanical contraction using impedance spectroscopy at the same microelectrode. In proof of principle experiments, human induced pluripotent stem cell (hiPS)-derived cardiomyocytes were cultured on HD-MEAs and used to demonstrate the sensitive quantification of contraction strength modulation by cardioactive drugs such as blebbistatin (IC50 = 4.2 µM), omecamtiv and levosimendan. Strikingly, arrhythmia-typical rotor patterns (re-entry) can be induced by optimized electrical stimulation sequences and detected with high spatial resolution. Therefore, we provide a novel cardiac re-entry analysis system as a promising reference point for diagnostic approaches based on in vitro assays using patient-specific hiPS-derived cardiomyocytes.

Analysis of Nipah Virus Replication and Host Proteome Response Patterns in Differentiated Porcine Airway Epithelial Cells Cultured at the Air-Liquid Interface.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respiratory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking in the determination of complex host response patterns in differentiated respiratory tract epithelia for the understanding of NiV replication and spread in swine. Here we characterized infection and spread of NiV in differentiated primary porcine bronchial epithelial cells (PBEC) cultivated at the air-liquid interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the apical or basal sides. Deep time course proteomics revealed pronounced upregulation of genes related to type I/II IFN, immunoproteasomal subunits, transporter associated with antigen processing (TAP)-mediated peptide transport, and major histocompatibility complex (MHC) I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II IFN host response with conversion from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.

Advanced 96-microtiter plate based bioelectrochemical platform reveals molecular short cut of electron flow in cytochrome P450 enzyme.

In bioelectrocatalysis, immobilised redox enzymes are activated in a bioelectronic interface without redox equivalents such as NADPH, thus enabling heterogeneous flow chemistry. The functional contact between enzyme and electrode requires a high degree of optimisation regarding choice of electrode material, electrode pre-treatment, enzyme immobilisation and reaction conditions. So far, however, there are no systems that can easily enable an optimisation procedure at a higher throughput. Here, we present an advanced platform with a vertical divided cell architecture in conjunction with a developed 96-multipotentiostat to be able to drive redox enzymes in 96 well microtiter plate based multielectrode arrays. This platform controls 96 independent three-electrode setups with arbitrary working electrode materials. We demonstrate its applicability in a mutation study of cytochrome P450 BM3 using indium tin oxide as electrode material and the 7-ethoxycoumarin product quantification assay. We show that the bioelectrocatalytic activity of P450 BM3 can be amplified when the cofactor FAD is erased from the enzyme by a single point mutation, so that FMN becomes the first electron entry point. Bioelectrocatalysis thus offers an approach to enzyme simplification as a remedy for the inherent instability of self-sufficient cytochrome P450 enzymes. In addition, we examined native and artificial enzyme activation with respect to ionic strength and buffer composition. The optimal conditions of the activation types differ substantially from each other and exhibit a new molecular facet in enzyme characteristics. In a proof-of-principle we demonstrate that the platform is also compatible with raw cell extracts, thus opening the door for random mutagenesis screenings.

FEM-based design of optical transparent indium tin oxide multielectrode arrays for multiparametric, high sensitive cell based assays.

Multielectrode array (MEA) technology is widely used for the bioelectronic monitoring of cellular alterations. In general, noble metal based MEAs are preferred e.g. for impedance spectroscopy because of their high conductivity and biocompatibility. Today's research focuses on combining different readout methods in a single measurement setup, such as sensitive electronic and optical readouts, where noble metal-based electrodes are excluded and transparent electrodes and optimized MEAs are required. In this context, we used optical transparent indium tin oxide (ITO) as electrode material. As a drawback, the decreased conductivity can lead to drastically decreased cell signals and it is hardly to predict which layout changes lead to a substantial signal increase. To overcome this limitation, we introduce an approach where equivalent circuit modelling (ECM) on reference multielectrode arrays is used to determine cell type specific electrical parameters, which then are used in finite element method (FEM) simulations to predict achievable cell signals and signal-noise-ratios (SNR) and thus use simulation to efficiently optimize multielectrode arrays. To evaluate our approach, MEAs with a wide range of electrode sizes were fabricated with ITO and gold. HEK-A cells were used to compare achievable cell signals for impedimetric monitoring. Our study revealed that especially for large ITO electrodes, the sensitivity drastically decreases. To overcome this drawback, we designed an optimized dual layer ITO MEA with gold support structures and more strikingly, successfully predict the cell signal increase by using our combined ECM and FEM simulation based approach.

Electrically Switchable Monostable Actuatoric Polymer-Based Nanovalve Arrays with a Long-Term Stability.

Here we present a novel electrically switchable nanovalve array based on an intrinsic conductive polymer that has the capabilities to change its volume depending on its redox state. The polymer is created by anodic deposition of a sodium dodecylbenzenesulfonate (DBS)-doped polypyrrole (PPy). Optimization of the DBS-doped PPy layers revealed an actuatoric performance of up to 10% out of plane volume change. More interestingly, the electrochemical characterization revealed an actuatoric monostable polymer that could be used to fabricate nanovalve arrays that have a native opened state when no potential is applied and that can be closed when a reductive potential is applied. As a proof of concept, Atto488-labeled biotin (Biotin-Atto488) was used as a model compound and defined nanovalve arrays with nanopores in the range of 10 nm in diameter (opened state) were fabricated. Afterward, we were able to successfully prove the functionality of our nanovalve array by monitoring the flow-through rates of the Biotin-Atto488. More strikingly, we could demonstrate for the first time the robust and long-term stability of our nanovalve array without any performance loss for at least 72 h and retention capabilities of up to 90%. Furthermore, the demonstrated long-term stability was achieved under biocompatible conditions without the need of toxic dopant supplementation of the flow-through solution. Thus, our novel functional long-term stable nanovalve array offers the capabilities for practical applications.