Seal Resistance Research Articles

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F − containing internal solution together with a Ca 2+ containing external solution — so that a CaF 2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F − ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Honeybee Brain Oscillations Are Generated by Microtubules. The Concept of a Brain Central Oscillator.

Microtubules (MTs) are important structures of the cytoskeleton in neurons. Mammalian brain MTs act as biomolecular transistors that generate highly synchronous electrical oscillations. However, their role in brain function is largely unknown. To gain insight into the MT electrical oscillatory activity of the brain, we turned to the honeybee (Apis mellifera) as a useful model to isolate brains and MTs. The patch clamp technique was applied to MT sheets of purified honeybee brain MTs. High resistance seal patches showed electrical oscillations that linearly depended on the holding potential between ± 200 mV and had an average conductance in the order of ~9 nS. To place these oscillations in the context of the brain, we also explored local field potential (LFP) recordings from the Triton X-permeabilized whole honeybee brain unmasking spontaneous oscillations after but not before tissue permeabilization. Frequency domain spectral analysis of time records indicated at least two major peaks at approximately ~38 Hz and ~93 Hz in both preparations. The present data provide evidence that MT electrical oscillations are a novel signaling mechanism implicated in brain wave activity observed in the insect brain.

Inflammatory Foreign Body Response Induced by Neuro-Implants in Rat Cortices Depleted of Resident Microglia by a CSF1R Inhibitor and Its Implications.

Inflammatory encapsulation of implanted cortical-neuro-probes [the foreign body response (FBR)] severely limits their use in basic brain research and in clinical applications. A better understanding of the inflammatory FBR is needed to effectively mitigate these critical limitations. Combining the use of the brain permeant colony stimulating factor 1 receptor inhibitor PLX5622 and a perforated polyimide-based multielectrode array platform (PPMP) that can be sectioned along with the surrounding tissue, we examined the contribution of microglia to the formation of inflammatory FBR. To that end, we imaged the inflammatory processes induced by PPMP implantations after eliminating 89–94% of the cortical microglia by PLX5622 treatment. The observations showed that: (I) inflammatory encapsulation of implanted PPMPs proceeds by astrocytes in microglia-free cortices. The activated astrocytes adhered to the PPMP’s surfaces. This suggests that the roles of microglia in the FBR might be redundant. (II) PPMP implantation into control or continuously PLX5622-treated rats triggered a localized surge of microglia mitosis. The daughter cells that formed a “cloud” of short-lived (T1/2 ≤ 14 days) microglia around and in contact with the implant surfaces were PLX5622 insensitive. (III) Neuron degeneration by PPMP implantation and the ensuing recovery in time, space, and density progressed in a similar manner in the cortices following 89–94% depletion of microglia. This implies that microglia do not serve a protective role with respect to the neurons. (IV) Although the overall cell composition and dimensions of the encapsulating scar in PLX5622-treated rats differed from the controls, the recorded field potential (FP) qualities and yield were undistinguishable. This is accounted for by assuming that the FP amplitudes in the control and PLX5622-treated rats were related to the seal resistance formed at the interface between the adhering microglia and/or astrocytes and the PPMP platform rather than across the scar tissue. These observations suggest that the prevention of both astrocytes and microglia adhesion to the electrodes is required to improve FP recording quality and yield.

Numerical Investigations on the Leakage Flow Characteristics of Brush Seal Based on the Three-Dimensional Staggered Tube Bundle Model

Abstract To accurately predict the leakage flow and resistance characteristics of brush seals, the multiblock structured mesh and the mesh motion technique are applied to the three-dimensional (3D) staggered tube bundle model of brush seals. The multiblock structured mesh can easily add nodes and set boundary layers in the interbristle gap between adjacent bristles, which can ensure good mesh quality (orthogonal angle and expansion ratio). The mesh motion technique realizes the overall axial compactness of the bristle pack. The effects of pressure ratio Rp, sealing clearance c, and bristle pack compactness on the leakage flow and resistance characteristics are investigated. To analyze the aerodynamic resistance of the brush seals, Euler number (Eu) is applied in this study. The numerical results are in good agreement with the experimental data. Thus, the accuracy of the presented numerical method is validated. For the contacting brush seal, ΔSx, i has a significant effect on the leakage flow rate reduction. For the clearance brush seal, ΔSx, i has little effect on the leakage flow rate reduction. The leakage flow passing through the sealing clearance keeps almost constant. As for aerodynamic resistance, the presence of the sealing clearance can effectively convert the pressure energy of the leakage flow into the kinetic energy. As a result, the leakage flow velocity exiting the bristle pack of the clearance brush seal is 1.5 to 2.0 times larger than that of the contacting brush seal. Although the existence of the sealing clearance obviously increases the leakage flow rate, it effectively reduces the aerodynamic forces acting on the bristles. The developed numerical approach based on the three-dimensional staggered tube bundle model and multiblock structured mesh can serve as a technical method for analysis of the sealing mechanisms of brush seals.

Fabrication of Glass Micropipette Device Using Reflow Processes and Its Integration With Microfluidic Channels for Patch Clamp Recording of Cell

This paper presents a borosilicate glass micropipette device that has a high-aspect-ratio pore for patch clamp recording applications. The device was fabricated by carrying out glass reflow processes on a silicon substrate. The shape of the micropipette was determined by the width and thickness of the reflowed glass structure, which matches well with the rounded tip of a conventional glass pipette. The diameter of the micropipette pore was first defined by deep reactive ion etching (DRIE) of a silicon pillar, which was positioned at the center of the glass structure. Then, the silicon pillar was removed, and the pore diameter was additionally reduced to the submicron scale by depositing a parylene-C film. The fabricated device was integrated with microfluidic channels for patch clamp recording experiments: microfluidic PDMS layers were assembled on the top and bottom surfaces of the micropipette chip for the medium exchange and the application of negative pressure, respectively. The quality of seal between a cerebral cortex cell of a rat and the glass micropipette was characterized by the amplitude of the current produced by an applied voltage pulse. A negative pressure applied through the bottom microfluidic channel pulled down the cell and increased the seal resistance from 9.82 $\text{M}\Omega $ to 2.06 $\text{G}\Omega $ . This indicates that the gigaseal was successfully formed around the micropipette pore. In addition, the action potentials of the patched cortical neuron were recorded using the fabricated device.

Study of the mechanical behavior of gamma-irradiated single-use bag seals

• Seal resistance under strain maintained whatever the gamma irradiation dose. • Seal integrity unchanged in PE and EVA bags whatever the gamma irradiation dose. • Same tensile behavior of all the PE bag seals. • Seal mechanical behavior depending on the seal location on the EVA bag. To protect from the external environment the solutions stored in the single use plastic bags used in the biopharmaceutical industry, perfect sealing of the bag is necessary to maintain confinement integrity. These single use plastic bags are manufactured from two different multilayer films, an EVA (ethyl vinyl acetate)/EVOH (ethyl vinyl alcohol)/EVA film and a PE (polyethylene)/EVOH/PE film. The EVA or PE contact layer provides chemical biocompatibility with the bag content (biological solution, food, etc.) in contact with the plastic while the EVOH layer provides oxygen barrier properties. Our first objective was to study the impact of the gamma irradiation dose (from 0 kGy to 270 kGy) on the mechanical tensile behavior of the seals. Our second objective was to evaluate the impact of the location of the seals on their tensile properties. The study showed that the seals are never impacted during tensile testing: this evaluation reveals that in fine cracking of the film occurs before any modification of the seal. Its function of closure and content protection from the external environment is fully achieved, whatever the gamma irradiation dose and the seal location. Only the multilayer film on both sides of the seal is altered after 100 % elongation strain. The EVA/EVOH/EVA and the PE/EVOH/PE multilayer films showed no degradation for a y-irradiation dose up to 115 kGy whereas they were altered at 270 kGy. The seals at different locations on the EVA bag showed different film mechanical behaviors, in correlation with the orientation of the polymer film extrusion process.

A Microfluidic Device With Optically-Controlled Electrodes for On-Demand Electrical Impedance Measurement of Targeted Single Cells

Electrical impedance measurement of a live cell is important for monitoring the cell’s status. Label-free and non-invasive techniques for measuring the impedance of live cells have attracted much attention. Existing techniques are capable of measuring the impedance of entire cell populations and/or the instantaneous impedance of single cells, but an approach to track and monitor the electrical properties of single cells during their growth process has not yet been reported. This paper presents a microfluidic device integrated with optically-controlled electrodes (MOCE) for electrical impedance measurement of multiple individual cells over a time period. An equivalent circuit model to quantify the seal resistance, membrane capacitance, cytoplasmic resistance of single cells is proposed. In experiments, the adherence process of C2C12 myoblast cells was characterized by measuring individual cells’ impedance data. During cell growth, the seal resistance <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$R_{seal}$ </tex-math></inline-formula> increased gradually, while the membrane capacitance stayed at approximately <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10^{-9}$ </tex-math></inline-formula> F and the cytoplasmic resistance stayed at approximately <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10^{9} ~\Omega $ </tex-math></inline-formula> . The results demonstrate the feasibility and effectiveness of the MOCE-based method for on-demand single-cell electrical impedance measurement. [2020-0265]

Immunohistological and Ultrastructural Study of the Inflammatory Response to Perforated Polyimide Cortical Implants: Mechanisms Underlying Deterioration of Electrophysiological Recording Quality.

The deterioration of field potential (FP) recording quality and yield by in vivo multielectrode arrays (MEA) within days to weeks of implantation severely limits progress in basic and applied brain research. The prevailing hypothesis is that implantation of MEA platforms initiate and perpetuate inflammatory processes which culminate in the formation of scar tissue (the foreign body response, FBR) around the implant. The FBR leads to progressive degradation of the recording qualities by displacing neurons away from the electrode surfaces, increasing the resistance between neurons (current source) and the sensing pads and by reducing the neurons’ excitable membrane properties and functional synaptic connectivity through the release of pro-inflammatory cytokines. Meticulous attempts to causally relate the cellular composition, cell density, and electrical properties of the FBR have failed to unequivocally correlate the deterioration of recording quality with the histological severity of the FBR. Based on confocal and electron microscope analysis of thin sections of polyimide based MEA implants along with the surrounding brain tissue at different points in time after implantation, we propose that abrupt FP amplitude attenuation occurs at the implant/brain-parenchyma junction as a result of high seal resistance insulation formed by adhering microglia to the implant surfaces. In contrast to the prevailing hypothesis, that FP decrease occurs across the encapsulating scar of the implanted MEA, this mechanism potentially explains why no correlations have been found between the dimensions and density of the FBR and the recording quality. Recognizing that the seal resistance formed by adhering-microglia to the implant constitutes a downstream element undermining extracellular FP recordings, suggests that approaches to mitigate the formation of the insulating glial could lead to improved recording quality and yield.

Design and analysis of material discharge plate of microsurfacing machine

The Microsurfacing is an imposing mixture of mineral aggregate, finely crumbled latent mineral or rock that is covered for a fabricated item, polymer modified cationic emulsified asphalt, supplements and water, assorted and outspread over a properly assembled surface. It is also used for pavement of rut and surface texturing other than sealants. Rather than breaking and curing via evaporation, a chemical reaction causes the material to set-up. When applied the Microsurfacing shall have a homogeneous appearance, fill cracks, stick firmly to the plane and allot a rainproof, high resistance seal (Zalnezhad and Hesami, 2019). In this study, the main problem is that the microsurfacing mixture flows through the material discharge plate which is attached to the paver system. If the material of the tank gets empty then the machine needs to refill its microsurfacing material and in that case some amount of microsurfacing material gets sticks to the rubber plate that is material discharge plate, due to this there is a productive loss. Since, the machine needs to stopped for washing or changing the material discharge plate. In order to resolve this problem, there is need to design and analyze the Material Discharge Plate of Microsurfacing Machine so, that the plate can be utilized for prolonged period of time and cost can be minimized by using other alloys in place of rubber material discharge plate. By taking all consideration, discharge plate is analyzed in the software. Results shows that there is significant improvement in production and reduction in cost of washing or changing the material discharge plate.

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F− containing internal solution together with a Ca2+ containing external solution — so that a CaF2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Optimal Pipette Resistance, Seal Resistance, and Zero-Current Membrane Potential for Loose Patch or Breakthrough Whole-Cell Recording in vivo.

In vivo loose patch and breakthrough whole-cell recordings are useful tools for investigating the intrinsic and synaptic properties of neurons. However, the correlation among pipette resistance, seal condition, and recording time is not thoroughly clear. Presently, we investigated the recording time of different pipette resistances and seal conditions in loose patch and breakthrough whole-cell recordings. The recording time did not change with pipette resistance for loose patch recording (Rp-loose) and first increased and then decreased as seal resistance for loose patch recording (Rs-loose) increased. For a high probability of a recording time ≥30 min, the low and high cutoff values of Rs-loose were 21.5 and 36 MΩ, respectively. For neurons with Rs-loose values of 21.5–36 MΩ, the action potential (AP) amplitudes changed slightly 30 min after the seal. The recording time increased as seal resistance for whole-cell recording (Rs-tight) increased and the zero-current membrane potential for breakthrough whole-cell recording (MPzero-current) decreased. For a high probability of a recording time ≥30 min, the cutoff values of Rs-tight and MPzero-current were 2.35 GΩ and −53.5 mV, respectively. The area under the curve (AUC) of the MPzero-current receiver operating characteristic (ROC) curve was larger than that of the Rs-tight ROC curve. For neurons with MPzero-current values ≤ −53.5 mV, the inhibitory or excitatory postsynaptic current amplitudes did not show significant changes 30 min after the seal. In neurons with Rs-tight values ≥2.35 GΩ, the recording time gradually increased and then decreased as the pipette resistance for whole-cell recording (Rp-tight) increased. For the high probability of a recording time ≥30 min, the low and high cutoff values of Rp-tight were 6.15 and 6.45 MΩ, respectively. Together, we concluded that the optimal Rs-loose range is 21.5–36 MΩ, the optimal Rp-tight range is 6.15–6.45 MΩ, and the optimal Rs-tight and MPzero-current values are ≥2.35 GΩ and ≤ −53.5 mV, respectively. Compared with Rs-tight, the MPzero-current value can more accurately discriminate recording times ≥30 min and <30 min.

Voltage Imaging of Cortical Oscillations in Layer 1 with Two-Photon Microscopy.

Membrane voltage oscillations in layer 1 (L1) of primary sensory cortices might be important indicators of cortical gain control, attentional focusing, and signal integration. However, electric field recordings are hampered by the low seal resistance of electrodes close to the brain surface. To study L1 membrane voltage oscillations, we synthesized a new voltage-sensitive dye, di1-ANNINE (anellated hemicyanine)-6plus, that can diffuse into tissue. We applied it with a new surgery, leaving the dura intact but allowing injection of large quantities of staining solution, and imaged cortical membrane potential oscillations with two-photon microscopy depth-resolved (25–100 μm below dura) in anesthetized and awake mice. We found delta (0.5–4 Hz), theta (4–10 Hz), low beta (10–20 Hz), and low gamma (30–40 Hz) oscillations. All oscillations were stronger in awake animals. While the power of delta, theta, and low beta oscillations increased with depth, the power of low gamma was more constant throughout L1. These findings identify L1 as an important coordination hub for the dynamic binding process of neurons mediated by oscillations.

A stepwise approach to resolving small ionic currents in vascular tissue.

Arterial membrane potential (Vm) is set by an active interplay among ion channels whose principal function is to set contractility through the gating of voltage-operated Ca2+ channels. To garner an understanding of this electrical parameter, the activity of each channel must be established under near-physiological conditions, a significant challenge given their small magnitude. The inward rectifying K+ (KIR) channel is illustrative of the problem, as its outward "physiological" component is almost undetectable. This study describes a stepwise approach to dissect small ionic currents at physiological Vm using endothelial and smooth muscle cells freshly isolated from rat cerebral arteries. We highlight three critical steps, beginning with the voltage clamping of vascular cells bathed in physiological solutions while maintaining a giga-ohm seal. KIR channels are then inhibited (micromolar Ba2+) so that a difference current can be created, once Ba2+ traces are corrected for the changing seal resistance and subtle instrument drift, pulling the reversal potential rightward. The latter is a new procedure and entails the alignment of whole cell current traces at a voltage where KIR is silent and other channels exhibit limited activity. We subsequently introduced corrected and uncorrected currents into computer models of the arterial wall to show how these subtle adjustments markedly impact the importance of KIR in Vm and arterial tone regulation. We argue that this refined approach can be used on an array of vascular ion channels to build a complete picture of how they dynamically interact to set arterial tone in key organs like the brain.NEW & NOTEWORTHY This work describes a stepwise approach to resolve small ionic currents involved in controlling Vm in resistance arteries. Using this new methodology, we particularly resolved the outward component of the KIR current in native vascular cells, voltage clamped in near-physiological conditions. This novel approach can be applied to any other vascular currents and used to better interpret how vascular ion channels cooperate to control arterial tone.

Opportunities and dilemmas of in vitro nano neural electrodes.

Developing electrophysiological platforms to capture electrical activities of neurons and exert modulatory stimuli lays the foundation for many neuroscience-related disciplines, including the neuron–machine interface, neuroprosthesis, and mapping of brain circuitry. Intrinsically more advantageous than genetic and chemical neuronal probes, electrical interfaces directly target the fundamental driving force—transmembrane currents—behind the complicated and diverse neuronal signals, allowing for the discovery of neural computational mechanisms of the most accurate extent. Furthermore, establishing electrical access to neurons is so far the most promising solution to integrate large-scale, high-speed modern electronics with neurons that are highly dynamic and adaptive. Over the evolution of electrode-based electrophysiologies, there has long been a trade-off in terms of precision, invasiveness, and parallel access due to limitations in fabrication techniques and insufficient understanding of membrane–electrode interactions. On the one hand, intracellular platforms based on patch clamps and sharp electrodes suffer from acute cellular damage, fluid diffusion, and labor-intensive micromanipulation, with little room for parallel recordings. On the other hand, conventional extracellular microelectrode arrays cannot detect from subcellular compartments or capture subthreshold membrane potentials because of the large electrode size and poor seal resistance, making it impossible to depict a comprehensive picture of a neuron's electrical activities. Recently, the application of nanotechnology on neuronal electrophysiology has brought about a promising solution to mitigate these conflicts on a single chip. In particular, three dimensional nanostructures of 10–100 nm in diameter are naturally fit to achieve the purpose of precise and localized interrogations. Engineering them into vertical nanoprobes bound on planar substrates resulted in excellent membrane–electrode seals and high-density electrode distribution. There is no doubt that 3D vertical nanoelectrodes have achieved a fundamental milestone in terms of high precision, low invasiveness, and parallel recording at the neuron–electrode interface, albeit with there being substantial engineering issues that remain before the potential of nano neural interfaces can be fully exploited. Within this framework, we review the qualitative breakthroughs and opportunities brought about by 3D vertical nanoelectrodes, and discuss the major limitations of current electrode designs with respect to rational and seamless cell-on-chip systems.

Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (IK1 ). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite8 ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.

Effect of metal surface state on injection joining strength of aluminum-rubber composite part

Metal–rubber composite parts with characteristics of high elasticity and compressibility are widely used in seal and vibration resistance engineering fields. The injection technology was proposed to mold metal – rubber composite parts. With pretreatments of anodization and silane coupling agent, the morphology and composition of Al surface were changed, and then Al - rubber composite parts were fabricated by injection technology. The effects of Al surface morphologies and compositions induced by different conditions of anodization and coupling agent were discussed. The scanning electron microscopy (SEM) and X-ray energy spectrum analyser (XPS) of Al surface examination proved that microstructures and hydroxyl groups formed on the metal surface contribute significantly to the bonding strength of the Al - rubber interface. Furthermore, the effect of metal temperature on the bonding strength between the rubber and the Al was investigated. By increasing the metal temperature to 120 °C, the bonding strength improved by more than twenty folds compared to the lower temperature case. Results showed that the molded composite parts exhibit excellent tensile shear strength, exceeding the shearing strength of the rubber itself. The presented method can be promoted in related engineering fields.

Storage Stability and Material Compatibility of Poly(oxymethylene) Dimethyl Ether Diesel Fuel

While a large part of traffic volume can be electrified to avoid local pollutant emissions, some applications will still require an internal combustion engine as an energy converter in the future. Construction machines such as vibratory plates, excavators, or emergency generators are examples of such applications. In order to reduce or avoid the pollutants of the remaining internal combustion engines, exhaust aftertreatment systems or synthetic fuels can be implemented. One category of these literature-discussed fuels is poly(oxymethylene) dimethyl ether (OME). In this work, neat OME as well as four OME–diesel blends and conventional diesel fuel as reference were investigated according to the material compatibility of two elastomers (NBR and FKM) and long-term storage stability of the fuel itself. The material compatibility of OME according to both elastomers NBR and FKM is difficult. The tensile stress test (DIN 53504), the Shore hardness (ISO 7619-1), and the compression set (ISO 815-1) show significant changes for neat OME compared to the diesel used elastomers. In addition, the change of mass and volume of the elastomers (DIN ISO 1817) are up to 75 wt % and 140 vol % higher compared to diesel fuel. Only FKM specimens, which were submerged in an OME–diesel blend with 5 vol % OME content, show comparable properties to the diesel reference. Based on the findings of this work, it is concluded that engines with NBR or FKM seals should replace their seals with OME resistant seals. In contrast, the long-term storage stability (ASTM D4625-14) of OME has much better properties than diesel fuel. No deposits were detectable after 24 weeks accelerated fuel aging at an ambient temperature of 43 °C.

Using Impedance Measurement to Characterize Cell-Electrode Adhesion with Customized 3D-Nanostructured Multielectrode Arrays

Multielectrode array (MEA) technology is used as an extracellular recording method to investigate the electrical activity of complex excitable cell networks and tissue slices. In this context, an array of single-contactable, sensing electrodes record the electrical field potentials induced by the ion flux across the cell membranes. Therefore, MEAs are an ideal tool to investigate network activities for prolonged periods. Still, MEA technology suffers from low-signal-to-noise ratios caused by a dissatisfied cell-electrode contact, thus revealing only limited measurements. To improve cell recording, a tight cell-electrode contact is inalienable. This can be achieved by three-dimensional micro- or nanostructures on top of the sensing electrodes. Beside the fact of increasing the electrochemically active surface area, thus leading to a decrease in the overall chip impedance, the nano-sized structures can also trigger an engulfment process of the cells. By minimizing the gap between cell and electrode, a high seal resistance can be achieved which in return results in recording of higher signal amplitudes. In this work, electrochemical concepts are used for fabrication as well as for characterizing the chips and to investigate the cell-electrode interaction.The developed process allows the fabrication of half-structured MEAs with different three-dimensional nanostructure shapes, geometries and pitch lengths and is mainly based on thermal nanoimprint lithography (NIL), gold electroplating and microstructuring techniques. During thermal NIL a customized, nanostructured master mold is pressed into a polymer resist of a wafer substrate, thus creating a negative copy of the master´s nanostructures. The subsequent electroplating step can be adjusted in a way, that it fills or overgrows the NIL templates, thereby creating different shapes. Photolithography, chemical vapor deposition and etching steps are used to fabricate the overall MEA geometry.Cyclic voltammetry as well as electrochemical impedance spectroscopy have been applied to characterize the effect of different nanostructure geometries on charge transfer, double-layer capacitance and impedance of the MEA electrodes. Compared to unstructured electrodes, charge transfer and double-layer capacitance of the nanostructured electrodes are increased, whereas the impedance of the nanostructured electrodes are decreased. These results can directly be related to an increase of the electrochemically active surface area and vary with the investigated nanostructure layout.To examine the effect of the nanostructures on cell-electrode coupling in detail and to quantify cell adhesion, impedance sensing at a fixed frequency of 1 kHz has been performed using the impedance-stable human embryonic kidney cell line HEK 293. Impedance values have been measured with and without cells. Due to the chip layout where non- and nanostructured electrodes have been included on the chip, the relative change of the impedance values could directly be correlated with cell-electrode adhesion. By this, a significant increase or decrease of cell-electrode coupling depending on the nanostructure layout could be demonstrated. For verification, action potential recordings with neural stem cells of the subventricular zone of postnatal mice have been performed, showing a distinct increase of the recorded signal amplitudes of the nanostructured electrodes compared to unstructured electrodes of the same chip. The latter might be correlated to an engulfment process of the cells. Figure: Left: Impedance measurement to quantify cell-electrode adhesion. Right: SEM picture of attached SVZ cells on top of overgrown pillar structures (Inset: Magnification, scale bar: 2 µm). Figure 1

O-28 Simulated differential sensitivity of motor and sensory nerves for conduction block in immune-mediated neuropathies

Background Immune-mediated polyneuropathies affect myelinated nerve fibers, resulting in conduction slowing or block, with selective involvement of sensory and motor axons. The underlying mechanisms are not well understood. We studied the impact of putative mechanisms by focusing on nodal and paranodal dysfunction on saltatory conduction in a longitudinal myelinated axon model. Materials and methods We extended our longitudinal axon model, which consisted of 41 nodes of Ranvier, with biophysical characteristics unique for human myelinated motor or sensory axons. We studied the effects of impaired nodal sodium channel conductance, paranodal myelin loop detachment by reducing paranodal seal resistance, and their interaction on saltatory conduction in the nine middle nodes and surrounding paranodes. Results Physiological motor and sensory conduction velocities were approximately 48 m/s and 50 m/s, respectively. Progressive conduction slowing was observed when reducing maximum sodium channel conductance and reducing paranodal seal resistance. Conduction blocks eventually occurred at a 75% (motor) and 78% (sensory) reduction in maximum sodium channel conductance and at a 87% (motor) and 89% (sensory) reduction in paranodal seal resistance. A boundary of block emerged representing the severity levels where both interacting mechanisms induced a conduction block. Conclusion Using our longitudinal myelinated motor and sensory axon model, simulations support the view that biophysical differences between motor and sensory axons potentially contribute to a differential susceptibility for development of a conduction block in immune-mediated polyneuropathies.

Introducing Simulated IK1 into Human iPSC-Cardiomyocytes using Dynamic Clamp on an Automated Patch Clamp System

Dynamic clamp is a powerful tool involving injection of real-time simulated currents into patch-clamped cells, and has been employed in conventional patch-clamp to inject IK1 current into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs are attractive cell types because of their unlimited availability and human origin. However, IK1 is expressed at low levels in those cells, hence they display a more depolarized membrane potential than adult cardiomyocytes (CM). Introducing simulated IK1 into hiPSC-CMs improves resting potential and makes them a viable alternative to the scarcely available adult human CMs. Another limitation of hiPSC-CMs is that the ratio of seal resistance (Rseal) to membrane resistance (Rm) is small due to smaller capacitance and typically slightly lower Rseal in automated patch clamp (APC). Thus, membrane potential dissipation is more pronounced compared to patch-clamp recordings of adult CMs. To correct for that, we implemented a Rseal compensation mechanism to inject current compensating for the potential dissipation through Rseal. In this study, we combined dynamic clamp with APC to demonstrate IK1 conductance can be added to hiPSC-CMs, and applying automatic Rseal compensation simultaneously. Our results show virtual IK1 can be successfully injected into hiPSC-CMs in up to 4 cells simultaneously and Rseal is correctly compensated avoiding overcompensation. Our approach results in more stable resting membrane potentials and improved action potential (AP) shape. Increased IK1 resulted in AP shortening and acceleration of the upstroke. We measured native, but small, Ba2+-sensitive IK1 in voltage-clamp mode in approximately 50% of these cells. Adding a Ca2+ channel activator (BayK 8644), or blocker (Nifedipine) caused an increase and decrease of the AP duration, respectively. In conclusion, combining dynamic clamp and Rseal compensation with APC resulted in an enhanced, medium-throughput platform for safety pharmacology.