Planar Patch-clamp Research Articles

Planar Patch-Clamp Electrodes for Single Cell and Neural Network Studies

The traditional glass pipette patch-clamp technique has contributed greatly to fundamental and pharmacological ion channel studies. The success of this serial technique has driven an effort to create wafer-based patch-clamp platforms using materials with inferior dielectric properties than glass and/or using exotic processing techniques to avoid the difficulties inherent to parallel processing of glass. We have developed a material processing scheme that generates ultrasmooth, high aspect ratio pores in fused quartz wafers. These devices are demonstrated here to be superior planar patch-clamp electrodes achieving gigaohm seals in nearly 80% of trials with a mammalian cell line, with the majority of seals over 10 GΩ and as high as 80 GΩ, competing with the best pipette-based patch-clamp measurements. Our method, amenable to batch fabrication technologies, will enable the acquisition of low noise, ion channel measurements in high throughput.We are currently merging the abovementioned devices with voltage sensitive dye (VSD) imaging and multi-electrode array (MEA) recordings in order to study multisensory integration in the medicinal leech. Initially, the planar pores will function to provide precise placement of neurons in the leech ganglion over the MEA's. The excellent spatial resolution of the VSD's combined with the temporal resolution of MEA's will provide much information of all the neurons that respond to visual stimuli in the ganglion. Further studies may employ the planar pores as intracellular electrodes, allowing voltage control and intracellular recordings of individual neurons in the ganglion.

A PDMS microfluidic system with poly(ethylene glycol)/SU-8 based apertures for planar whole cell-patch-clamp recordings

Poly(dimethylsiloxane) (PDMS) has been used as an attractive material for planar patch-clamp system fabrication. However, the hydrophobic property of PDMS prevents the establishment of a high-resistance seal with cell membrane. Here, we describe the first use of poly(ethylene glycol) (PEG)/SU-8 mixture for patch-clamp partition electrode fabrication. This PEG/SU8 mixture with weight ratio of 1:1 has been proved to have better properties compared with PDMS for cell-patch interface, such as improved mechanical property, permanent hydrophilic surface property, and easiness to achieve high-resistance cell sealing. The PEG/SU-8 partitions with 2 μm apertures were successfully fabricated by employing a two-step process of soft lithography for PDMS mold (thermal curing) and PEG/SU-8 chip (UV light curing). The partitions could be reversibly sealed to the PDMS microfluidic system including electrode solution inlet/outlet, cell manipulation inlet/outlet, cell reservoir, microchannels and microfabricated Ag/AgCl electrodes. The integrated chip was successfully used to trap Hela cells onto 2 μm aperture by a negative pressure. We demonstrated that a planar PDMS patch-clamp system with PEG/SU-8 based cell-patch interface for successful patch-clamp recording on Hela cells.

Cell placement and guidance on substrates for neurochip interfaces

Interface devices such as integrated planar patch-clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly-d-lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo-preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch-clamp interfaces on the chip.

Design and fabrication of a planar patch-clamp substrate using a silicon-on-insulator wafer

The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In this study, a planar patch-clamp substrate was designed and fabricated using a silicon-on-insulator (SOI) wafer. The access resistance and capacitance of SOI-based planar patch-clamp substrates are smaller than those of bulk silicon-based planar substrates, which will reduce the distributed RC noise.

Blockade of the I Ks potassium channel: An overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I Ks channel, which along with the transient outward current (I to) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I Ks blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I Ks channel blockade and describe the discovery of a potent I Ks blocker whose activity was not detected by standard hERG or in vitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) in vivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I Ks blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I Ks blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity. Assay Drug Development Technology 5, 617–627]). Because of the presence of I Ks channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I Ks blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I Ks liability before testing in humans. The possibility of undetected I Ks blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427–1438] in the ICH S7B regulatory guidelines.

Automated planar patch clamp: Multiple applications for miniaturized devices

The Port‐a‐Patch is a miniaturized patch clamp rig which uses a planar borosilicate glass chip for performing electrophysiological recordings. Suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. The Port‐a‐Patch has been successfully used for whole cell, perforated patch, and cell attached recordings of a variety of ion channels with high success rates (typically 60‐80%). Fast application of ligand is possible using the external perfusion system. Recordings from recombinant GABAA receptors (α1β2γ2) expressed in HEK293 cells will be shown. Examples of voltage‐gated ion channels recorded using the Port‐a‐Patch will also be shown, including NaV1.5 stably and Shaker K+ transiently expressed in HEK293 cells. The effect of temperature on the amplitude and kinetics of hERG channels will also be presented.One of the newest and most exciting aspects of planar patch clamping using the Port‐a‐Patch is the ability to perfuse the internal membrane of the cell, whilst maintaining a continuous recording during the solution exchange. The internal perfusion system for the Port‐a‐Patch allows the exchange of up to 8 different solutions. Exemplar traces will be shown for regulation of TRP V1 by internal as well as external regulators. Such ease with which the internal solution may be exchanged is a unique feature to Nanion products.

An Integrated Patch-Clamp Potentiostat With Electrode Compensation

We present the first fully integrated implementation of a patch-clamp measurement system with series-access resistance and parasitic capacitive compensation capability. The system was implemented in a 0.5- mum silicon-on-sapphire process and is capable of recording cell membrane currents up to plusmn20 nA, with an rms noise of 5 pA at 10-kHz bandwidth. The system can compensate for the capacitance and resistance of the electrode, up to 20 pF and up to 70% of the series-access resistance, respectively. The die size is 1150 by 700 mum. The power consumption is 300 muW at 3.3 V. The integrated patch-clamp system will be used to fabricate high-throughput planar patch-clamp systems.

Optimization of Ca v1.2 screening with an automated planar patch clamp platform

Introduction: Ca v1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca v1.2 block is very important in developing drugs without cardiac liability. Ca v1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca v1.2 screening assay in PatchXpress ® 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca v1.2 antagonists and an agonist were tested. Methods: HEK-293 cells stably transfected with hCa v1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba 2+ and low magnesium intracellular recording solution to minimize rundown. Ca v1.2 currents were elicited from a holding potential of − 60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. Results: Averaged peak Ca v1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 °C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg 2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca 2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC 50 values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. Discussion: This assay can be used as a reliable pharmacological screening tool for Ca v1.2 block to assess compounds for cardiac liability during lead optimization.

Use Of Isolated Vesicles From Xenopus Oocytes For Planar Patch Clamp Recordings

Oocytes from the Xenopus laevis toad represent an efficient expression system. The two-electrode voltage-clamp technique (TEVC) as well as patch clamp recordings from excised patches are powerful tools to study ion channels following expression in Xenopus oocytes. The TEVC technique allows the measurement of macroscopic currents from whole cells. It is widely used in basic research and also applied for compound screening in pharmaceutical industry. Recordings from excised patches enable the analysis of ion channels under cell free conditions and even single channel analysis. In contrast to the TEVC technique, its application requires highly skilled electrophysiologists.A publication by Yong Zhang et al. ∗ describes a technique to prepare isolated membrane vesicles from Xenopus oocytes by incubating them, after removal of the vitelline membrane, in a hypertonic “blebbing” solution. This treatment induces the formation of plasma membrane blebs that eventually detach from the oocyte surface to form isolated plasma membrane vesicles (PMVs).Here we used these PMVs to create macro patches onto a planar borosilicate glass surface using the Port-a-Patch (Nanion) automated patch clamp device. The PMVs formed readily gigaseals and showed small capacities as expected for macro patches. Currents could be recorded from voltage-gated as well as ligand-gated ion channels.In conclusion, we present an application that has the potential to overcome the practical limitations associated with the generation of excised patches and might enable the automated analysis of such patches.∗Mechanically gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes.Yong Zhang, Feng Gao, Vsevolod L. Popov,∗ Julie W. Wen,∗ and Owen P. Hamill J Physiol. 2000 February 15; 523(Pt 1): 117-130.

Robotic multiwell planar patch-clamp for native and primary mammalian cells.

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Improved Early Detection of HERG Channel Liability in Lead Optimization Programs with Automated Patch-Clamp Technology

The HERG channel is the molecular entity directly involved in the majority of Long QT Syndrome cardiopathology cases, constituting a possible risk of life threatening arrhythmias (Torsades de Pointes). Avoidance of HERG liability is a central regulatory issue aiming to prevent costly withdrawals of potential or existing therapeutic agents. HERG safety pharmacology is an indispensable step and a high quality assay (patch-clamp electrophysiology considered as the gold standard) must accompany IND applications. Analysis of ion channel activity has been potentiated by automated planar patch-clamp within the drug discovery industry. At Exelixis we have significantly improved the early detection of HERG liability at the stage of lead optimization programs. For this report perforated-patch whole-cell recordings from cryopreserved CHO cells heterologously expressing HERG channels were obtained with IonWorks Quattro™. Channel expression was unaltered for >3 months time (>450 pA/well). Population patch-clamp wells with seal test >35 MOhms and peak currents >100 pA were included for analysis. Overall success rate consistently obtained was >95%. These improvements enabled a timely implementation of qSAR for lead compound selection and prioritization, thus avoiding potential QT prolongation liabilities at early stages.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Guided growth of neurons and glia using microfabricated patterns of parylene-C on a SiO 2 background

This paper describes a simple technique for the patterning of glia and neurons. The integration of neuronal patterning to Multi-Electrode Arrays (MEAs), planar patch clamp and silicon based ‘lab on a chip’ technologies necessitates the development of a microfabrication-compatible method, which will be reliable and easy to implement. In this study a highly consistent, straightforward and cost effective cell patterning scheme has been developed. It is based on two common ingredients: the polymer parylene-C and horse serum. Parylene-C is deposited and photo-lithographically patterned on silicon oxide (SiO 2) surfaces. Subsequently, the patterns are activated via immersion in horse serum. Compared to non-activated controls, cells on the treated samples exhibited a significantly higher conformity to underlying parylene stripes. The immersion time of the patterns was reduced from 24 to 3 h without compromising the technique. X-ray photoelectron spectroscopy (XPS) analysis of parylene and SiO 2 surfaces before and after immersion in horse serum and gel based eluant analysis suggests that the quantity and conformation of proteins on the parylene and SiO 2 substrates might be responsible for inducing glial and neuronal patterning.

Sodium Channel Inhibitor Drug Discovery Using Automated High Throughput Electrophysiology Platforms

Voltage dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders such as epilepsy and pain as well as cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic and antiarrhythmic sodium channel blockers. While patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery in which there is often a need to evaluate many thousands to hundreds of thousands of compounds. Fortunately over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms like the PatchXpress and IonWorks, which are now widely used in drug discovery. This review highlights the strengths and weaknesses of these two high throughput devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpress and IonWorks electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state dependent modulation of sodium channels. IonWorks has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpress has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery.

Patch clamping on plane glass—fabrication of hourglass aperture and high-yield ion channel recording

Planar patch-clamp has revolutionized ion-channel measurement by eliminating laborious manipulation from the traditional micropipette approach and enabling high throughput. However, low yield in gigaseal formation and/or relatively high cost due to microfabricated processes are two main drawbacks. This paper presents patch clamping on glass substrate-an economical solution without sacrificing gigaseal yield rate. Two-stage CO(2) laser drilling methodology was used to generate an hourglass, funnel-like aperture of a specified diameter with smooth and debris-free surfaces on 150 microm borosilicate cover glass. For 1-3 microm apertures as patch-clamp chips, seal resistance was tested on human embryonic kidney, Chinese hamster ovary, and Jurkat T lymphoma cells with a gigaseal success rate of 62.5%, 43.6% and 66.7% respectively. Results also demonstrated both whole-cell and single channel recording on endogenously expressed ion channels to confirm the capability of different patch configurations.

Port-a-Patch and Patchliner: High Fidelity Electrophysiology for Secondary Screening and Safety Pharmacology

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Production of a specific extracellular inhibitor of TRPM3 channels

Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca(2+) measurement. Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca(2+) entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.

Seal Formation in Silicon Planar Patch-Clamp Microstructures

This paper presents a microfabricated planar patch-clamp electrode design and looks at the impact of several physical characteristics on seal formation. The device consists of a patch aperture, 1.5-2.5 mum in diameter and 7-12 mum in depth, with a reverse-side deep-etched 80-mum well. The patch aperture was coated with either thermal oxide or plasma-enhanced chemical vapor deposited (PECVD) SiO2. Some of the thermal oxide devices were converted into protruding nozzle structures, and some were boron-doped. Seal formation was tested with cultured N2a neuroblastoma cells. The PECVD oxide devices produced an average seal resistance of 34 MOmega(n = 24), and the thermal oxide devices produced an average seal resistance of 96 MOmega(n = 59). Seal resistance was found to positively correlate with patch aperture depth. Whole-cell recordings were obtained from 14% of cells tested with the thermal oxide devices, including a single recording where a gigaohm seal was obtained.

Use of a Planar Patch Electrode to Measure NBCe1‐A Function in Mammalian Cells

Standard whole cell‐patch clamping is labor‐intensive whereas automated planar patch clamp devices offer the possibility of higher throughput. Planar patch clamp electrodes are also attractive because their geometry facilitates obtaining electrical and optical recordings simultaneously. Previous studies have utilized the Port‐a‐Patch system (Nanion Technologies GmbH), to study ion channel activity however there are no studies which have demonstrated the utility of this approach with electrogenic cotransporters, which in general have a lower conductance. In this study, we utilized a modified Port‐a‐Patch system to measure the functional activity of the electrogenic sodium bicarbonate cotransporter, NBCe1‐A. HEK‐293T cells permanently expressing NBCe1‐A were generated. Cell monolayers were detached using 0.025% Trypsin/0.22 mM EDTA in PBS and were re‐suspended at a concentration of 500,000 cells/ml in 50% DMEM/PBS. The cells were kept at room temperature until measurements were made. Approximately 2,500 cells were placed on a planar electrode chip from which a single cell was automatically selected. Planar electrode chips with variable resistances were assessed. Using chips with a resistance of 2 – 3.5 MOhm, we routinely obtained GOhm seals using this approach. Whole cell currents were elicited by voltage ramps from −100 mV to +30 mV from a holding potential of −100 mV. Cell membrane capacitance for the HEK‐293T cells was 7.14 ± 0.67 pF (n = 21) and access resistance was 8.6 ± 0.5 MOhm (n =21). The DIDS‐inhibitable Na+‐and HCO3−‐dependent NBCe1‐A‐mediated average whole cell current was 29.3 ± 3.2 pA (n =21) at +30 mV. These results represent the first reported use of a planar patch‐clamp device to study ionic currents mediated by a heterologously expressed electrogenic cotransporter in mammalian cells.Supported by the NIH

Planar patch‐clamp on synoviocytes from rheumatoid arthritic joints

Patch clamp is still accepted as the best technique for precise and direct characterization of ion channel properties. The development of automated planar patch‐clamp technologies overcomes the problems of low throughput, laborious, manual processing associated with conventional patch‐clamping techniques. Our Patchliners Octo and Quattro (Nanion Technologies GmbH) are robotic multi‐channel patch clamp systems that enable recordings with increased throughput capabilities. They utilise planar glass chips embedded in a micro‐fluidic perfusion system for rapid solution exchange. Protocols have been developed for cell lines, but we were also interested in the potential benefits for studies of endogenous channels of primary cells. We find that routine giga‐seals and whole‐cell recordings can be obtained with various primary cell types. One such cell type is the CD55‐positive fibroblast‐like synoviocytes, which have previously proved difficult to patch‐clamp by conventional methodology. The success rate in producing high quality recordings, maintained for 10 min, was surprisingly greater than for conventional patch‐clamp methodology (~70 % compared with <10 %). The results suggest there is potential for automated planar patch‐clamp devices in the study of ionic currents through channel proteins of primary cells.The research is funded by the Wellcome Trust and we thank Nanion for customer support.

Probing whole cell currents in high-frequency electrical fields: Identification of thermal effects

An open-end coaxial probe is combined with a planar patch-clamp system to apply electric fields with GHz frequencies during conventional patch-clamp measurements. The combination of pulsed microwave irradiation and lock-in detection allows for the separation of fast and slow effects and hence facilitates the identification of thermal effects. The setup and the influence of radiation on the patch-clamp current are thoroughly characterized. For the independent optical verification of heating effects, a temperature microscopy technique is applied with high spatial, temporal and temperature resolution. It is shown that the effect of radiation at GHz frequencies on whole cell currents is predominantly thermal in nature in the case of RBL cells with an endogenous K ir 2.1 channel.