Integration of Impedimetric Sensors for In Situ Electrochemical Impedance Spectroscopy in Free-Flow Electrophoresis Applications in Lab-on-Chip Systems.

Miniaturization and integration of chemical reactions into fluidic systems in combination with product purification or buffer exchange can reduce the amount of solvents and reactants required while increasing synthesis efficiency. A critical step is the regulation of flow rates to realize optimal synthesis conditions and high purification rates, so real-time, label-free monitoring is required in methods such as free-flow electrophoresis. Optical detection methods are widely used, but they often have complex excitation and detection setups that are disadvantageous for point-of-care applications. The method we have chosen is electrochemical impedance spectroscopy for detecting charged compounds in aqueous buffers with low ionic strength. Propranolol was selected for proof of concept and was separated from the organic solvent and the precursor oxirane by free-flow electrophoresis. For this purpose, electrode structures were fabricated in microfluidic channels by photolithographic lift-off technique and optimized in terms of positioning, electrode size and distance for sensitive detection, and quantification of propranolol in the nanomolar range. It is also noteworthy that the organic solvent dimethyl sulfoxide (DMSO) could be detected and quantified by an increased impedance magnitude. Subsequently, the optimized interdigital electrode structures were integrated into the outlet channels of the electrophoretic separation chamber to monitor the various outgoing fluidic streams and provide in-line control of the fluidic flows for the purification step. In conclusion, we can provide a microfluidic chip to monitor the separation efficiency of a substance mixture during free-flow electrophoresis without the need of complex analytical techniques using electrochemical impedance spectroscopy.

Multielectrode biosensor chip for spatial resolution screening of 3D cell models based on microcavity arrays.

Three-dimensional cell models represent the native in vivo situation more closely than two-dimensional cultures and are therefore preferred today for in vitro studies. In this context, there is a great demand for fast, non-invasive, real-time, and label-free methods that are capable for detailed analyses of three-dimensional cultures. To characterize heterogeneous cultures or to detect localized drug effects, a measurement method such as impedance spectroscopy in combination with microcavity arrays (MCAs) is desirable, which additionally offers spatial resolution. To overcome these limitations of the previously described MCA based on opaque silicon substrates and a square shape with four measurement electrodes imposed by the crystal structure, we used the selective laser etching (SLE) method to fabricate microcavities in fused silica and borosilicate glass without geometric constraints. We successfully developed MCAs with variable base including up to eight measurement electrodes in one cavity, which allows the increase in the number of electrode combinations to improve spatial resolution. In addition, we integrated a central cone electrode at the cavity bottom to extend the spatial resolution on the z-axis. To demonstrate the capability of the MCAs, we used MDA-HB-231 spheroids with an enclosed glass sphere to show that the heterogeneity of the model is evident in the relative impedance spectra. Analyses on various cell spheroids highlight the broad applicability of glass MCAs. In conclusion, our SLE-fabricated MCA clearly improve bioelectronic analyses of cellular changes in heterogeneous 3D models. Thus, bioelectronic analysis of electrophysiologically active cells and tumor biopsy samples could significantly benefit from our development.

Novel PMMA based 96-well microelectrode arrays for bioelectronic high throughput monitoring of cells in a live mode.

Microelectrode arrays (MEA) are widely used for bioelectronic monitoring of alterations in cells and tissues. MEAs are based on a substrate that is structured with electrodes and conducting paths. While cheap substrates like printed circuit board materials offers easy production and flexible contacting, there are limitations regarding microstructure resolution, optical transparency and biocompatibility. In contrast, glass substrates are favored due to its biocompatibility, chemical resistance and optical transparency. Drawbacks are high substrate costs and limited flexibility for routing of conducting paths. To overcome these limitations, we wanted to use optical transparent polymer-based substrates. Therefore, we identified the polymer poly-methyl-methacrylate (PMMA) as a promising substrate material, due to its good optical and mechanical properties as well as biocompatibility. To achieve sufficient chemical resistance for high resolution photolithographic structuring a novel process had to be developed involving a protection coating. After optimization of the structuring process, we achieved a comparable resolution and thus, microelectrodes with diameter of less than 100 µm. Moreover, the use of PMMA allowed the simple integration of more than 400 vias directly into the substrate for contacting of the microelectrode array from the bottom without the need of complex and error prone redirecting adapters with hundreds of additional bonding sides. In order to show that the PMMA based MEA is comparable to glass based MEA in terms of signal quality and sensitivity as well as optical and surface properties, we cultivated different cell models on the MEAs and validated our 96-well PMMA MEAs by different bioelectronic monitoring techniques.

Reactive Sputtered Silicon Nitride as an Alternative Passivation Layer for Microelectrode Arrays in Sensitive Bioimpedimetric Cell Monitoring.

Microelectrode arrays (MEAs) are widely used to study the behavior of cells noninvasively and in real time. While the design of MEAs focuses mainly on the electrode material or its application-dependent modification, the passivation layer, which is crucial to define the electrode area and to insulate the conducting paths, remains largely unnoticed. Because often most cells are in direct contact with the passivation layer rather than the electrode material, biocompatible photoresists such as SU-8 are almost exclusively used. However, SU-8 is not without limitations in terms of optical transmission, optimal cell support, or compatibility within polymer-based microfluidic lab on chip systems. Here, we established a silicon nitride (SiN) passivation by physical vapor deposition (PVD), which was optimized and evaluated for impedance spectroscopy-based monitoring of cells. Surface characteristics, biocompatibility, and electrical insulation capability were investigated and compared to SU8 in detail. To investigate the influence of the SiN passivation on the impedimetric analysis of cells, HEK-293 A and MCF-7 were chosen as adherent cell models and measured on microelectrodes of 50-200 µm in diameter. The results clearly revealed an overall suitability of SiN as alternative passivation. While for the smallest electrode size a cell line dependent comparable or slightly decreased cell signal could be observed in comparison with SU-8, a significant higher cell signal was observed for microelectrodes larger than 50 µm in diameter. Furthermore, a high suitability for the bonding of PEGDA and PDMS microfluidic structures on the SiN passivation layer without any leakage could be demonstrated.

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.

Multifunctional coatings combining bioactive peptides and affinity-based cytokine delivery for enhanced integration of degradable vascular grafts.

Insufficient endothelialization of cardiovascular devices is a high-risk factor for implant failure. Presentation of extracellular matrix (ECM)-derived coatings is a well-known strategy to improve implant integration. However, the complexity of the system is challenging and strategies for applying multifunctionality are required. Here, we engineered mussel-derived surface-binding peptides equipped with integrin (c[RGDfK]) and proteoglycan binding sites (FHRRIKA) for enhanced endothelialization. Surface-binding properties of the platform containing l-3,4-dihydroxyphenylalanine (DOPA) residues were confirmed for hydrophilized polycaprolactone-co-lactide scaffolds as well as for glass and polystyrene. Further, heparin and the heparin-binding angiogenic factors VEGF, FGF-2 and CXCL12 were immobilized onto the peptide in a modular assembly. Presentation of bioactive peptides greatly enhanced human umbilical vein endothelial cell (HUVEC) adhesion and survival under static and fluidic conditions. In subsequent investigations, peptide-heparin-complexes loaded with CXCL12 or VEGF had an additional increasing effect on cell viability, differentiation and migration. Finally, hemocompatibility of the coatings was ensured. This study demonstrates that coatings combining adhesion peptides, glycosaminoglycans and modulators are a versatile tool to convey ECM-inspired multifunctionality to biomaterials and efficiently promote their integration.

Electrochemical live monitoring of tumor cell migration out of micro-tumors on an innovative multiwell high-dense microelectrode array.

Understanding of cell migration and spreading out of tumor tissue is of great interest concerning the mechanism and causes of tumor malignancy and metastases. Although there are methods available for studying cell migration on monolayer cell cultures like transwell assays, novel techniques for monitoring cell spreading out of 3D organoids or tumor tissue samples are highly required. In this context, we developed an innovative high-dense microelectrode array for impedimetric monitoring of cell migration from 3D tumor cultures. For a proof of concept, a strongly migrating breast cancer cell line (MDA-MB-231) and two malignant melanoma cell lines (T30.6.9, T12.8.10ZII) were used for generating viable micro-tumor models. The migration propensity was determined by impedimetric monitoring over 144 hours, correlated by microscopy and validated by transwell assays. The impedimetric analysis of covered electrodes and the relative impedance maximum values revealed extended information regarding the contribution of proliferative effects. More strikingly, using reference populations of mitomycin C treated spheroids where proliferation was suppressed, distinction of proliferation and migration was possible. Therefore, our high-dense microelectrode array based impedimetric migration monitoring has the capability for an automated quantitative analysis system that can be easily scaled up as well as integrated in lab on chip devices.

Nonaqueous Micro Free-Flow Electrophoresis for Continuous Separation of Reaction Mixtures in Organic Media.

The continuous separation mechanism of micro free-flow electrophoresis (µFFE) is a straightforward, suitable tool for microscale purification of reaction mixtures. However, aqueous separation buffers and organic reaction solvents limit the applicability of this promising combination. Herein, we have explored nonaqueous micro free-flow electrophoresis for this purpose and present its suitability for a continuous workup of organic reactions performed in acetonitrile. After successful nonaqueous FFE separation of organic dyes, the approach was applied to continuously recover the photocatalyst [Ru(bpy)3]2+ from a homogeneous, acetonitrile-based reaction mixture. This approach opens up possibilities for further downstream processing of purified products and is also attractive for recycling of precious catalyst species.

Large-Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals.

Large-ring cyclodextrins (CD) are cyclic glucans composed of 9 or more α-1,4-linked glucose units. They are minor side products of bacterial glucanotransferases (CGTases, EC 2.4.1.19) and have previously been available only in very small amounts for studies of their properties in supramolecular complex formation reactions. We engineered a CGTase to synthesize mainly large-ring CD facilitating their preparation in larger amounts. By reversed phase chromatography, we obtained single CD samples composed of 10 to 12 glucose units (CD10, CD11, and CD12) with a purity of >90 %. Their identity was confirmed by high resolution mass spectrometry and fragmentation analysis. We demonstrated the non-toxicity of CD10-CD12 for human cell lines by a cell proliferation assay and impedimetric monitoring. We then showed that CD10 and CD11 are efficient chiral selectors for the capillary electrophoretic separation of the enantiomeric pharmaceuticals fluvastatin, mefloquine, carvedilol, and primaquine.

Induced cross-resistance of BRAFV600E melanoma cells to standard chemotherapeutic dacarbazine after chronic PLX4032 treatment.

The maximum response and 10-year survival rate for metastatic melanoma patients treated with standardised chemotherapy is still less than 15% and 10%, respectively. In contrast, oncogene targeting was found a promising tool for killing of BRAFV600 mutated melanoma cells. Nevertheless, despite improved response and survival rates, resistance acquisition remains an ongoing problem. In this context, the impact of chronic BRAF inhibition on the efficacy of commonly applied cytostatics is still unknown. In our study, human melanoma cells with BRAFV600E mutation were treated with chemotherapeutics and a BRAF inhibitor. Resistance patterns were analysed by microelectrode array-based impedance spectroscopy, XTT and flow cytometric apoptosis/proliferation assay. BRAFV600E melanoma cells acquired a time- and concentration-dependent desensitisation up to 100-fold towards oncogene-specific PLX4032 and chemotherapeutic dacarbazine after twelve months treatment. The impact of multiple drug insensitivity on molecular melanoma characteristics was elaborated via mRNA and protein quantification. Following BRAFV600E targeting, melanoma cells developed an increasingly aggressive, dacarbazine-insensitive phenotype. Thereby, hyperactivated canonical alternative MAPK and bypass PI3K/AKT signalling caused cross-resistance of differently acting drugs. With these results, we are the first to show that long-term melanoma therapy with BRAF inhibitors can prevent further therapeutic success with dacarbazine due to acquisition of cross-resistance.

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.

A multidimensional impedance platform for the real-time analysis of single and combination drug pharmacology in patient-derived viable melanoma models.

In today's development of anticancer drugs, there is an enormous demand for sensitive, non-invasive real-time screening technologies to identify pharmacodynamics/-kinetics of single and combined drugs with high precision. The combination of sophisticated drug sensitivity testing with advanced in vitro tumor models reflecting heterogeneous tumor behavior in vivo is needed to more reasonably predict therapeutic outcome in vivo. In this study, the benefits of our real-time, non-invasive multidimensional impedance platform over standard in vitro drug sensitivity assays were demonstrated quantitatively using an advanced melanoma model. Detailed pharmacological profiles of clinically established targeted therapeutics in single and combination treatment have been identified in patient tissue and isolated 2D/3D cell line cultures. Impedance spectroscopy revealed significant differences in tissue structure responsible for BRAF inhibitor pharmacokinetics in BRAFV600E tumor microfragments and cell lines. Remarkably, BRAF-/MEK inhibitor combination treatment of direct patient-derived tissue, but not melanoma cell lines, resulted in short-term antagonistic effects consistent with in vivo findings. In contrast, the clinically validated resistance delay and thus long-term synergy of targeted therapeutics in advanced melanoma models has been demonstrated using impedance technology. The results demonstrate limited clinical transferability of 2D/3D cancer cell line-based chemosensitivity data and underline the importance of in vivo-like direct patient-derived tissue for predictive drug studies. Our non-invasive and highly sensitive multidimensional impedance platform offers great potential for quantifying short- and long-term drug kinetics and synergies to identify the most effective drug combinations in advanced cancer models, thereby improving personalized drug development and treatment planning and ultimately, overall patient outcomes.

Comprehensive human stem cell differentiation in a 2D and 3D mode to cardiomyocytes for long-term cultivation and multiparametric monitoring on a multimodal microelectrode array setup.

Human pluripotent stem cell derived cardiomyocytes are a promising cell source for research and clinical applications like investigation of cardiomyopathies and therefore, identification and testing of novel therapeutics as well as for cell based therapy approaches. However, actually it´s a challenge to generate matured adult cardiomyocyte-like phenotype in a reasonable time. Moreover, there is a lack of applicable non-invasive label-free monitoring techniques providing quantitative parameters for analysing the culture stability and maturation status. In this context, we established an efficient protocol based on a combined differentiation of hiPSC in 2D cultures followed by a forced reaggregation step that leads to highly enriched (>90% cardiomyocytes) cardiomyocyte clusters. Interestingly, 3D cultures revealed an accelerated maturation as well as phenotype switch from atrial to ventricular cardiomyocytes. More strikingly using combined impedimetric and electrophysiological monitoring the high functionality and long-term stability of 3D cardiomyocyte cultures, especially in comparison to 2D cultures could be demonstrated. Additionally, chronotropic as well as QT-prolongation causing reference compounds were used for validating the cardio specific and sensitive reaction over the monitored time range of more than 100 days. Thus, the approach of multiparametric bioelectronic monitoring offers capabilities for the long-term quantitative analysis of hiPS derived cardiomyocyte culture functionality and long-term stability. Moreover, the same multiparametric bioelectronic platform can be used in combination with validated long-term stable cardiomyocyte cultures for the quantitative detection of compound induced effects. This could pave the way for more predictive in vitro chronic/repeated dose cardiotoxicity testing assays.

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.

Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging.

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (µFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of µFFE as continuous purification technique for micro flow synthesis.

Microfluidic Free-Flow Electrophoresis Based Solvent Exchanger for Continuously Operating Lab-on-Chip Applications.

For miniaturization and integration of chemical synthesis and analytics on small length scales, the development of complex lab-on-chip (LOC) systems is in the focus of many current research projects. While application specific synthesis and analytic modules and LOC devices are widely described, the combination and integration of different modules is intensively investigated. Problems for in-line processes such as solvent incompatibilities, e.g., for a multistep synthesis or the combination of an organic drug synthesis with a cell-based biological activity testing system, require a solvent exchange between serialized modules. Here, we present a continuously operating microfluidic solvent exchanger based on the principle of free-flow electrophoresis for miscible organic/aqueous fluids. We highlight a proof-of-principle and describe the working principle for the model compound fluorescein, where the organic solvent DMSO is exchanged against an aqueous buffer. The DMSO removal performance could be significantly increased to 95% by optimization of the microfluidic layout. Moreover, the optimization of the inlet flow ratio resulted in a minimized dilution factor of 5, and we were able to demonstrate that a reduction of the supporting instrumentation is possible without a significant decrease of the DMSO removal performance. Finally, the compatibility of the developed solvent exchanger for cell based downstream applications was proven. The impedimetric monitoring of HEK293A cells in a continuously operating microfluidic setup revealed no adverse effects of the residual DMSO after the solvent replacement. Our solvent exchanger device demonstrates the power of micro-free-flow electrophoresis not only as a powerful technique for separation and purification of compound mixtures but also for solvent replacement.

A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode.

Lab-on-a-chip devices that combine, e.g. chemical synthesis with integrated on-chip analytics and multi-compartment organ-on-a-chip approaches, are a fast and attractive evolving research area. While integration of appropriate cell models in microfluidic setups for monitoring the biological activity of synthesis products or test compounds is already in focus, the integration of label-free bioelectronic analysis techniques is still poorly realized. In this context, we investigated the capabilities of impedance spectroscopy as a non-destructive real-time monitoring technique for adherent cell models in a microfluidic setup. While an initial adaptation of a microelectrode array (MEA) layout from a static setup revealed clear restrictions in the application of impedance spectroscopy in a microfluidic chip, we could demonstrate the advantage of a FEM simulation based rational MEA layout optimization for an optimum electrical field distribution within microfluidic structures. Furthermore, FEM simulation based analysis of shear stress and time-dependent test compound distribution led to identification of an optimal flow rate. Based on the simulation derived optimized microfluidic MEA, comparable impedance spectra characteristics were achieved for HEK293A cells cultured under microfluidic and static conditions. Furthermore, HEK293A cells expressing Y1 receptors were used to successfully demonstrate the capabilities of impedimetric monitoring of cellular alterations in the microfluidic setup. More strikingly, the maximum impedimetric signal for the receptor activation was significantly increased by a factor of 2.8. Detailed investigations of cell morphology and motility led to the conclusion that cultivation under microfluidic conditions could lead to an extended and stabilized cell-electrode interface.

In vitro field potential monitoring on a multi-microelectrode array for the electrophysiological long-term screening of neural stem cell maturation.

Due to the lack of appropriate cell models as well as automated electrophysiology monitoring technologies, the standardized identification of neurotoxic or protective effects in vitro remains a major problem in today's pharmaceutical ingredient development. Over the past few years, in vivo-like human pluripotent stem cell-derived neuronal networks have turned out to be a promising physiological cell source, if the establishment of robust and time-saving functional maturation strategies based on stable and expandable neural progenitor populations can be achieved. Here, we describe a multi-microelectrode array (MMEA)-based bioelectronics platform that was optimized for long-term electrophysiological activity monitoring of neuronal networks via field potential measurements. Differentiation of small molecule-based neuronal progenitors on MMEAs led to functional neurons within 15 days. More strikingly, these functional neuronal cultures could remain electrophysiologically stable on the MMEAs for more than four weeks. The observed electrophysiological properties correlated with the expression of typical neuron subtype markers and were further validated by specific neurotransmitter applications. With our established monitoring platform, we could show for the first time the long-term stability of the neural stem cell-like progenitor population to differentiate to electrophysiologically active dopaminergic neuronal networks for more than 80 passages. In conclusion, we provide a comprehensive long-term stable field potential monitoring platform based on stem cell-derived human neuronal networks that can be automated and up-scaled for standardized high-content screening applications e.g. in the field of neurotoxic and neuroprotective therapeutics identification.

Novel 96-well quantitative bioelectrocatalytic analysis platform reveals highly efficient direct electrode regeneration of cytochrome P450 BM3 on indium tin oxide.

Enzymes are the most effective catalysts for a broad range of difficult chemical reactions e.g. hydroxylation of non-activated C-H Bonds and stereoselective synthesis. Nevertheless, a lot of enzymes are not accessible for the biotechnological applications or industrial use. One reason is the prerequisite of expensive cofactors. In this context, we developed a bioelectrocatalytic analysis platform for the electrochemical and photonic quantification of the direct electron transfer from the electrode to redox enzymes and therefore, bypass the need of soluble cofactors that had to be continuously exchanged or regenerated. As reference enzyme, we chose cytochrome P450 BM3 that is restricted by NADPH dependence. We optimized the substrate spectrum for aromatic compounds by introduction of the triple mutation A74G/F87V/L188Q and established a sensitive fluorimetric product formation assay to monitor the enzymatic conversion of 7-ethoxycoumarine to 7-hydroxycoumarine. Gold and indium tin oxide electrodes were characterized with respect to surface morphology, charge-transfer resistance and P450 BM3 immobilization as well as activity. Using gold electrodes, no significant product formation by electrode mediated direct electron transfer could be detected. In contrast, P450 BM3 adsorbed on unmodified indium tin oxide electrodes revealed 36% activity by electrode mediated direct electron transfer in comparison to enzyme regeneration by NADPH. Since the reaction volumes are in the microliter range and upscaling of the measurement system is easily possible, our analysis platform is a useful tool for bioelectrocatalytic enzyme characterization and library screening based optimization for applications in the field of enzyme catalyzed chemical synthesis but also enzyme based fuel cells.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes.

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.