Development of planar patch clamp with potentiometric calcium ion-selective electrode

Ion channels are proteins in cell lipid bilayer membranes and act as pores which can adopt closed and open states, thus gating the flow of ions in and out of the cell. Patch clamp technology has been the proven standard for fundamental studies of ion channel activities. However, the technique has some basic limitations: low throughput, time consuming nature of its process, need of highly skilled personnel and inability to identify ionic composition of electrophysiological events. Many different materials and fabrication methods have been introduced to replace traditional patch clamp setup to overcome limitations.^ In this dissertation, a planar patch clamp device with calcium ion-selective electrode is developed in miniaturized form for high throughput cell electrophysiology, and screening of ion channel modulators as potential drug targets in an in vitro format. Femtosecond laser-drilling technique is newly introduced to fabricate the patch-pore and new design of planar ion-selective electrode is suggested for calcium ion measurement. By integrating a standard patch clamp electrophysiological interface with calcium ion-selective electrode on a single platform, it is possible to directly identify the ionic component of a whole-cell potential recording. This system is innovative because the focus is not entirely on increasing experimental throughput, but instead offers information on user specified target ion activities through ion channels

Microfluidic platform for bilayer experimatation from a research tooltowards drug screening

The aim of this thesis, which is the development of a microfluidic platform for bilayer experimentation with the potential for drug screening on ion channels, is introduced in this chapter. After a short presentation of the field of drug screening, an outline of this thesis is given, together with a brief summary of the different chapters

Integration of single-cell electropermeabilization together with electrochemical measurement of quantal exocytosis on microchips

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from quantal exocytosis of oxidizable transmitter from individual vesicles. Using electroporation we have developed an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration can reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization have similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Robust quantal release is elicited upon electroporation in 0 Ca2+/5 mM EGTA in the bath solution. Electropermeabilization-induced transmitter release requires Cl- in the bath solution--bracketed experiments demonstrate a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl- ] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane impermeant substances into a cell.Includes bibliographical references (pages 110-128)

Developing Instrumentation for Multi-parametric Investigation of Mechanisms of Mechanosensitivity in Ion Channels

Mechanosensitive (MS) channels are implicated in pathologies of the renal and pulmonary systems. Abnormal activity in MS channel reduces cell viability causing a variety of pathologies. MS channels are also responsible for sensation of pain and hearing. Despite the vital importance of MS channels, very little is known about the gating mechanisms of these channels. Attempts to study the mechanisms are severely limited by the lack of suitable instrumentation. A better understanding of the structure-function interaction of MS channels is necessary to find pharmacological leads for the pathologies. Activation data based on indirect activation of MS channels using hypo- or hyper-osmotic solutions or viscous drag is confounded by factors like membrane stretch and cytoskeletal stress. Traditional patch clamp does not allow direct access to the cell by other probes. While a planar patch clamp chip may allow for such access, most of the existing planar patch clamp chips are focused on high throughput screening for pharmaceutical targets and have designs that limit multi-parametric studies. We present here instrumentation that combines atomic force microscopy with cellular electrophysiology based on planar patch clamp approach. The instrumentation allows multi-parametric studies on single cells and provides unique insights into mechanisms of activation of not just MS channels, but ion channels in general by combining cellular electrophysiology, optical microscopy and atomic force microscopy. Using HaCaT cells as our model system we have obtained functional maps of distribution MS channels across cell surface. The maps reveal that the distribution of MS channels on HaCaT cells is highly non-uniform and that the channels are present in small clusters instead of dispersed as single entities. Our results using direct mechanical stimulation of single cells reveal that threshold stress level is required in order to activate MS channels and that the stress has a limited spatial range. Investigation of kinetics of the electrical response to direct mechanical stimulation reveals that the MS channels respond to the mechanical signal after a small time lag, which we attribute to the conformational changes necessary while the channel is being gated. We hope that the insights gained from studying the mechanosensitive channels of HaCaT cells will also advance the understanding of MS channels in general. Apart from opening new avenues in MS channel research, the instrumentation can also be useful in studying the dynamics and gating of ligand gated channels by appropriately tagging the AFM cantilever. With further improvements in the speed of AFM imaging, it will also be possible to observe the gating of channels in real time at molecular scale by imaging the channel on the cell while the channel is being gated

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed “blind,” meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum

High-Throughput Automated Patch Clamp Investigations on Ion Channels in Erythrocytes

Trotz ihrer morphologischen Einfachheit ist die Membran der roten Blutkörperchen (Erythrozyten) mit einer Reihe von Transportern und Ionenkanälen ausgestattet, die bisher nicht vollständig charakterisiert sind und deren biologische Rolle noch wenig verstanden ist. Die meisten Techniken zur Untersuchung von Ionenkanälen messen summierte Effekte großer Zellpopulationen und verbergen so jede mutmaßliche Variabilität von Zelle zu Zelle. Die Patch-Clamp-Technik hat sich als effektives Werkzeug zur Entdeckung und Charakterisierung von Ionenkanälen auf Einzelzellenebene erwiesen. Dies besonders wichtig für Erythrozyten von Säugetieren, die eine hohe Heterogenität der Leitfähigkeit zwischen verschiedenen Spendern, und auch zwischen Zellen desselben Spenders aufweisen (Kaestner et al., 2004; Minetti et al., 2013). Die Entwicklung des automatisierten Patch-Clamps ermöglichte es, eine hohe Anzahl von Zellen gleichzeitig unter identischen experimentellen Bedingungen zu untersuchen, wodurch Zellheterogenität erstmals umfassend bestimmt wurde. In dieser Arbeit wurden Gárdos- und Piezo1-Kanäle als Hauptuntersuchungsziele ausgewählt, da sie eine prominente Rolle in erythrozytären Erkrankungen, im Einzelnen Gárdos-Kanalopathie (Fermo et al., 2017) und hereditäre Xerozytose (Zarychanski et al., 2012; Bae et al., 2013), spielen. Ziel dieser Arbeit war es, automatisierte Patch-Clamp-Assays zur Charakterisierung dieser Kanäle in Erythrozyten zu entwickeln. Es gibt bisher nur vereinzelte Publikationen zu whole-cell Patch-Clamp-Messungen von Gárdos-Kanälen in Erythrozyten (Grygorczyk et al., 1984; Wolff et al., 1988), wahrscheinlich aufgrund der geringen Expression des Proteins in zirkulierenden Erythrozyten. Der hochparallelisierte Ansatz der automatisierten Patch-Clamp-Technologie ermöglicht zuverlässig die Identifizierung von Gárdos-Strömen in Zelltypen mit einer oft geringen Anzahl von Kanälen und einer großen Heterogenität der Expression, wie bei Erythrozyten. Bisherige Piezo1-Kanaluntersuchungen zeigen, dass die Substanz Yoda1 Piezo1-Ströme bewirken kann, die empfindlich auf GdCl3 (unspezifischer Inhibitor dehnungsaktivierter Kanäle), nicht jedoch auf TRAM-34 (spezifischer Gárdos-Kanalinhibitor) reagieren. Die Anwendung dieses Assays auf Erythrozyten von Patienten mit einer neuartigen PIEZO1 R2110W-Mutation zeigte eine erhöhte Anzahl der Yoda1-empfindlichen Zellen und eine stärkere Antwort auf Yoda1 bei Patienten im Vergleich zu Kontroll-Erythrozyten. In Kombination mit der Untersuchung der Proteinstruktur, die den R2110W-Rests in einem gating-sensitiven Bereich des Kanals lokalisiert, deuten die Patch-Clamp-Ergebnisse darauf hin, dass die neue Piezo1-Mutation eine gain-of-function-Mutation ist (Rotordam et al., 2019). Zusammenfassend zeigt diese Arbeit, dass die automatisierte Patch-Clamp-Methode robuste Assays zur Untersuchung von Ionenkanälen (Gárdos und Piezo1) in Primärzellen liefert. Die Hochdurchsatztechnologie ermöglichte die Entwicklung eines zuverlässigen Assays für gering exprimierte Ionenkanäle bei hoher Heterogenität der Zellen. So war es möglich, eine neuartige Kanalmutation auf funktioneller Ebene direkt in Patientenzellen zu charakterisieren, ohne die Mutation in einem heterologen Expressionssystem exprimieren zu müssen. Dieser Ansatz kann zum Nachweis und zur Charakterisierung weiterer Kanalopathien verwendet werden, die nicht auf Erythrozyten beschränkt sind, und kann generell als zur Gensequenzierung komplementärer Routine-Screening-Assay für Krankheiten dienen, die mit Ionenkanalstörungen zusammenhängen.Despite the morphological simplicity, the Red Blood Cell (RBC) membrane is endowed with a number of transporters and ion channels, yet not fully characterized and whose biological role is still poorly understood. Most of the techniques used to investigate ion channels are addressed to large populations of cells, thus concealing any putative cell-to-cell variability. The patch clamp technique has proven to be a valid tool for the discovery and characterization of ion channels at a single-cell level. This is of particular relevance for mammalian RBCs, which present a high heterogeneity of conductance not only between different donors but also among cells of the same donor (Kaestner et al., 2004; Minetti et al., 2013). The advent of automated patch clamp allowed to probe an increased number of cells at the same time under identical experimental conditions, thus tackling cell heterogeneity issues. In this thesis, Gárdos and Piezo1 channels were selected as main targets of investigation due to their relevance in RBC-related diseases, i.e. Gárdos channelopathy (Fermo et al., 2017) and hereditary xerocytosis (Zarychanski et al., 2012; Bae et al., 2013). The aim of this work was to develop automated patch clamp assays for characterizing those channels in RBCs. As for Gárdos channels, whole cell recordings reported so far are fragmentary probably due to the low expression of the protein in circulating RBCs (Grygorczyk et al., 1984; Wolff et al., 1988). By increasing the number of cells recorded at the same time, the automated patch clamp technology allowed to identify Gárdos-mediated currents in primary cells with a low-copy number of channels and a large heterogeneity of conductance as RBCs. Piezo1 channels investigations confirmed that application of Yoda1 alone is able to elicit currents sensitive to GdCl3 (non-specific stretch-activated channels inhibitor) but not TRAM-34 (specific Gárdos channel blocker). When transferred to patients carrying a novel PIEZO1 R2110W mutation, the assay revealed that the number of responders and the magnitude of the response to Yoda1 increased in patient compared to control RBCs. This result, combined with structural studies identifying the R2110W residue in a gating sensitive area of the channel, suggested that the novel Piezo1 mutation is gain-of-function (Rotordam et al., 2019). Altogether, this work demonstrates that automated patch clamping provides robust assays to investigate ion channels (Gárdos and Piezo1) in primary cells. The high-throughput technology allowed to tackle issues as response heterogeneity and low expression of the channels, and to characterize a novel channel mutation at a functional level directly from patient cells, without having to express the mutation in a heterologous expression system. This approach may be used to detect other channelopathies not limited to RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing

Integrated Nanoscale Tools for Interrogating Living Cells

The development of next-generation, nanoscale technologies that interface biological systems will pave the way towards new understanding of such complex systems. Nanowires – one-dimensional nanoscale structures – have shown unique potential as an ideal physical interface to biological systems. Herein, we focus on the development of nanowire-based devices that can enable a wide variety of biological studies. First, we built upon standard nanofabrication techniques to optimize nanowire devices, resulting in perfectly ordered arrays of both opaque (Silicon) and transparent (Silicon dioxide) nanowires with user defined structural profile, densities, and overall patterns, as well as high sample consistency and large scale production. The high-precision and well-controlled fabrication method in conjunction with additional technologies laid the foundation for the generation of highly specialized platforms for imaging, electrochemical interrogation, and molecular biology. Next, we utilized nanowires as the fundamental structure in the development of integrated nanoelectronic platforms to directly interrogate the electrical activity of biological systems. Initially, we generated a scalable intracellular electrode platform based on vertical nanowires that allows for parallel electrical interfacing to multiple mammalian neurons. Our prototype device consisted of 16 individually addressable stimulation/recording sites, each containing an array of 9 electrically active silicon nanowires. We showed that these vertical nanowire electrode arrays could intracellularly record and stimulate neuronal activity in dissociated cultures of rat cortical neurons similar to patch clamp electrodes. In addition, we used our intracellular electrode platform to measure multiple individual synaptic connections, which enables the reconstruction of the functional connectivity maps of neuronal circuits. In order to expand and improve the capability of this functional prototype device we designed and fabricated a new hybrid chip that combines a front-side nanowire-based interface for neuronal recording with backside complementary metal oxide semiconductor (CMOS) circuits for on-chip multiplexing, voltage control for stimulation, signal amplification, and signal processing. Individual chips contain 1024 stimulation/recording sites enabling large-scale interfacing of neuronal networks with single cell resolution. Through electrical and electrochemical characterization of the devices, we demonstrated their enhanced functionality at a massively parallel scale. In our initial cell experiments, we achieved intracellular stimulations and recordings of changes in the membrane potential in a variety of cells including: HEK293T, cardiomyocytes, and rat cortical neurons. This demonstrated the device capability for single-cell-resolution recording/stimulation which when extended to a large number of neurons in a massively parallel fashion will enable the functional mapping of a complex neuronal network.Physic

Glass reflow on 3-dimensional micro-apertures for electrophysiological measurements on-chip

We propose a new method to fabricate micro-apertures for on-chip electrophysiological measurements of living cells. Thermal reflow of phosphosilicate glass (PSG) is applied to funnel- or nozzle-type microstructures to generate very smooth surfaces on the finalized chip. Such 3-dimensional microstructures show close similarities to fire-polished glass pipette tips. Immobilized cells fit perfectly to these structures offering a large contact area for sealing between the cell membrane and the oxide surface. A tight cell/chip-aperture seal is an important requirement for the present application. We demonstrate the formation of stable gigaseals with Chinese hamster ovary (CHO) cells for both types of microstructures without the need of any post-fabrication surface treatment. By adjusting the PSG reflow parameters, the shape of the apertures can be modified and diameters down to the sub-micrometer range may be achieved. The application of PSG reflow to MEMS fabrication is an interesting new option to create unconventional microstructure