基于平板膜片钳技术的全自动膜片钳设备

膜片钳技术是实时研究离子通道的金标准。 With its exceptional signal resolution, complex biophysical properties and effectors of ion channels can be studied.

The automation of the patch clamp method was made possible with the development of the chip-based planar patch clamp technology. Automated patch clamp increases throughput and ease of use compared to the conventional patch clamp technique making it accessible to a wider audience.

Nanion has been the 'first on the market' with the launch of the semi-automated planar patch clamp device, the Port-a-Patch, in 2003. Since then, we have developed further instruments which are all "true Giga-seal" devices and offer a wide variety of experimental possibilities based on the planar patch clamp technology, expanding on the capabilities of conventional patch clamp.

Nanion的平板膜片钳设备

    Port-a-Patch 是一款自动膜片钳设备，拥有小巧的体积，可以记录离子通道与脂双层。

   Patchliner 是一款全自动膜片钳，可用于中等通量的灵活实验。

   SyncroPatch 384PE 是一款高通量的膜片钳设备，可以升级到SyncroPatch 768PE实现最高20,000 和 40,000数据量/天的高通量记录。

推荐的平板膜片钳文献

Artificial Bilayer Recordings:
Analysis of Channels and Pores free from other Proteins

One way to monitor ion channel activity is to use the artificial lipid bilayer recording method, in which many types of reconstituted ion channels and pores can be measured. In contrast to experiments on whole living cells, artificial bilayers provide a different approach for the investigation of ion channels and other integral membrane proteins. The main advantage lies in the complete absence of any unwanted interfering species as well as the convenient and reproducible investigation of target molecules down to a single channel level. This is achieved by the introduction of purified proteins or fusion of proteo-liposomes with precisely controlled composition to the otherwise highly insulating model membrane devoid of any contaminants. Further advantages of this technology are an excellent electrical sealing which provides an extremely sensitive monitoring (down to the single-molecule level), while the compositions of membrane and incorporated proteins can be precisely controlled. In addition, only a small fraction of membrane extract from cells expressing target ion channels is necessary.

Nanion's Chip-based Planar Bilayer Devices

   The Port-a-Patch is a semi-automated device with a small footprint for the analysis of ion channels in cells and lipid bilayers.

   The Orbit Mini combines state of the art amplifier technology with the easy and reproducible investigation of four planar lipid bilayers in parallel. The optional automated temperature control enables experiments at physiological temperatures or on temperature-sensitive species such as TRP channels.

   The Orbit 16 has a higher throughput than the Orbit Mini and comprises automated membrane generation which maximizes the system's ease of use and the reproducibility of the experiments.

   A helpful device to generate giant unilamellar vesicles (GUVs) for your bilayer recordings is the Vesicle Prep Pro.

Recommended publications on Chip-based Bilayer Recordings

The Solid Supported Membrane (SSM): Measuring transporter currents

Solid supported membrane (SSM)-based electrophysiology differs from conventional electrophysiology such as patch-clamp, since no living cells are required, but rather diverse native or artificial membrane vesicles. The samples used range from reconstituted protein in proteoliposomes to membrane preparations from organelles or plasma membrane. For the sample preparation bacterial cells, eukaryotic cell culture or native tissue can be used. The membrane sample is added to an SSM in advance of the experiment. This leads to the stable adsorption of the membranes to the SSM and the formation of a capacitively coupled compound membrane. The SSM itself consists of a lipid monolayer on top of a thiolated gold coated sensor chip. One important advantage compared to patch-clamp is the large sensor size of up to 3 mm. This allows the measurement of about 10^9 transporters at the same time and yields a significant improvement of signal to noise ratio. Therefore low-turnover targets become accessible to electrophysiological characterization.

The Difference to Conventional Electrophysiology

An important difference between SSM-based electrophysiology and conventional electrophysiology is the principle of measurement. In patch-clamp electrophysiology, stationary currents can be obtained due to the fact that the voltage and, therefore, the driving force is clamped during the measurement. In SSM-based electrophysiology a substrate gradient established by a fast solution exchange is the main driving force. The transport of charged substrates or ions into the liposomes or vesicles generates a membrane potential. This potential can be detected via capacitive coupling between the membrane and the SSM on the gold layer of the sensor. In short: The change in membrane potential due to electrogenic transport is measured. At some point the membrane potential equals the chemical driving force and the transport process comes to a halt. This is why any current measured with SSM-based electrophysiology is transient. The peak current amplitude reflects the transporter activity under steady-state conditions.

Since the current decay is fast, one measurement takes only one second. Due to the high stability of the SSM, multiple measurements can be performed using the same sensor and different buffer conditions to determine kinetic parameters such as EC₅₀, IC₅₀ or even rate constants.

Nanion's Chip-based Solid Supported Membrane Devices

   The SURFE²R N1 is a medium throughput device, designed for basic research and Universities.

   The SURFE²R 96SE is a high throughput device, integrated into the CyBio Felix liquid handling platform.

Recommended Readings and Movies

2018 - Transporters Investigated Using the SURFE²R Instruments

   SURFE²R 96SE and      SURFE²R N1 Oral Presentation

Presenter: 
Dr. Maria Barthmes, Product Manager SURFE²R product family, Nanion Technologies GmbH, Germany

2017 - SSM-Based Electrophysiology for Transporter Research

  SURFE²R N1 and     SURFE²R 96SE book chapter in Methody in Enzymology

Authors:
Bazzone A., Barthmes M., Fendler K.

2017 - An emerging technique for the characterization of transport proteins: SSM-based electrophysiology

  SURFE²R N1 and      SURFE²R SE96 poster, 19th IUPAB / 11th EBSA congress 2017     (3.3 MB)

preview the file here for download

2016 - Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology

  SURFE²R N96 (predesessor model of SURFE²R 96SE) publication in Toxicological Letters (2016)

Authors: 
Niessen K.V., Muschik S., Langguth F., Rappenglück S., Seeger T., Thiermann H., Worek F.

Impedance and Extracellular Field Potential Recordings (EFP)

Impedance and Extracellular Field Potential recordings are non-invasive "label-free" methods and ideally suited to analyze cell motility and action potentials of excitable, intact cultured cells, e. g. cardiomyocytes and neurons.

The extracellular field potential is the electrical potential produced by cells, e.g. nerve or muscle cells, outside of the cell. Electrophysiological studies investigate these potentials using extracellular microelectrodes. In these experiments the extracellular field potential is detected as an electrical potential. For individual cells, the time course of the extracellular potential theoretically is inversely proportional to the transmembrane current.

The electrical impedance is the measurement of the resistance to a current when a voltage is applied and possesses both magnitude and phase. In other words, it is the voltage–current ratio for a single complex exponential at a particular frequency ω. The impedance may be measured or displayed directly in ohms.
Practically: Cells seeded onto an electrical impedance chip or plate cover the electrodes inducing a higher resistance which can be detected. Impedance assays may be carried out at one frequency or over a range of frequencies. Depending on the applied frequency ω, the electrons cross through or in between the cells and thus different parameters of cell morphology and cover rate can be detected. Electrical impedance measurements are suitable to detect the contraction motion of cardiomyocytes, cell growth and motility as well as changes in cell morphology upon application of toxicological substances or substances effecting the cell-cell and cell-matrix contacts in long-term studies.

Nanion's Plate-based Impedance and EFP device

   The CardioExcyte 96 records action potentials (EFP) and contraction motility (impedance) of cardiomyocytes in parallel.

Recommended publications

Recommended oral presentations

Optogenetics: Optical Triggering of Action Potentials in Excitable Cells

Optogenetics is a novel technology which can be used to control excitable cells: The expression of specific light-sensitive ion channels makes it possible to depolarize cells with light pulses with millisecond-scale temporal precision and thus accurately triggering action potentials.

Optogenetics offers great potential in regard to cardiac safety studies: Compounds affecting the contraction of cardiomyocytes may affect ion channels and receptors in a use-dependent way which results in different effects for different beat rates. With this technology, its is possible to pace cardiomyocytes in a large beat frequency range simply with light pulses to discover the use-dependent effects of toxins. In iPSC-derived cardiomyocytes, the optogenetic actuator Channelrhodopsin has been proven to be well suited and accepted as a model. A transient transfection Kit for Channelrhodposin is available from iPSC cell providers, e.g. NCardia, for the use of optogenetics in cardiac safety studies.

Nanion's Optogenetics-Compatible Device

   The CardioExcyte 96 is a device for the investigation of impedance and extracellular field potentials of iPSC-derived cardiomyocytes. A specifically developed lid "SOL" enables the optogenetic pacing of the cells.

Recommended Readings on Optogenetics for Cardiac Safety Studies

2017 - Cross-site comparison of myocyte phase II compounds on different iPS cardiomyocytes based on CardioExcyte 96 recordings

   CardioExcyte 96 presentation (slide deck)      (2.2 MB)

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2017 - Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation

  CardioExcyte 96 publication in International Journal of Molecular Sciences

Authors:
Rehnelt S., Malan D., Juhasz K., Wolters B., Doerr L., Beckler M., Kettenhofen R., Bohlen H., Bruegmann T., Sasse P.

2018 - Innovations for cell monitoring in safety and toxicity assays

   CardioExcyte 96 &      SyncroPatch 384PE presentation (slide deck)      (3.2 MB)

preview the file here for download

2018 - Optogenetic control of transiently transfected hiPSC-derived cardiomyocytes for the assessment of drug related cardiotoxicity

   CardioExcyte 96 presentation (slide deck)      (1.4 MB)

preview the file here for download

2018 - Optogenetic technologies enable high throughput ion channel drug discovery and toxicity screening

   SyncroPatch 384PE and      CardioExcyte 96 poster, Biophysics Annual Meeting 2018     (1.3 MB)

preview the file here for download

2018 - Transported by light: optogenetic control of NCX1

   SURFE²R N1 poster, Biophysics Annual Meeting 2018     (2.2 MB)

preview the file here for download

CardioExcyte 96 产品彩页 - Pacing

  CardioExcyte 96 产品彩页     (1.0 MB)

预览文件下载

CardioExcyte 96 产品彩页 - SOL

  CardioExcyte 96 产品彩页     (2.0 MB)

预览文件下载

Cardiomyocytes - Channelrhodopsin 2 (ChR2) transfected Cor.4U cells and optical pacing

   CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

ChR2 transfected Cor.4U cells are following the optical pace rate.

Raw data traces upon a 1 Hz, 1.5 Hz, 2 Hz., 2.5 Hz and 3 Hz stimulation rate, extracellular field potentials (top) and impedance (bottom).

 

 

 

Cardiomyocytes - Myocyte phase II study: CiPA conform analysis and arrhythmia detection

   CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

Nanion developed a CiPA conform analysis for the Myocyte phase II study. The feature comes along is included in our CiPA analysis routine. Automated arrhythmia detection is just one highlight out of many when it comes to the CardioExcyte 96 software.

Cardiomyocytes - Optogenetics meets cardiac safety

   CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

The stimulating optical lid, CardioExcyte 96 SOL, uses LEDs for spatially uniform stimulation of cells transfected with light-gated ion channels such as Channelrhodopsin2 (ChR2).

Right graph: LPM – Light pulse per minute plotted against the recorded beat rate (average of 96 wells). ChR2 transfected Cor.4U cells are following the optical pace rate.

GUV:囊泡的多种应用

囊泡是由脂双层卷成的球型外壳， enclosing a small amount of water and separating it from the water outside the vesicle. Because of this fundamental similarity to the cell membrane, vesicles have been used extensively to study the properties of lipid bilayers. Another reason vesicles have been used so frequently is that they are relatively easy to make. If a sample of dehydrated lipid is exposed to water it will spontaneously form vesicles. These initial vesicles are typically multilamellar (many-walled) and range in size from tens of nanometers to several micrometers.

Further methods are required to break these initial vesicles into smaller, single-walled vesicles of uniform diameter known as small unilamellar vesicles (SUVs) with diameters of 50 nm to 200 nm. Since artificial SUVs can be made in large quantities they are suitable for bulk material studies such as x-ray diffraction to determine lattice spacing and differential scanning calorimetry to determine phase transitions. Dual polarisation interferometry can measure unilamellar and multilamellar structures and insertion into and disruption of the vesicles in a label-free assay format. Vesicles can also be labelled with fluorescent dyes to allow sensitive FRET-based fusion assays.

In spite of this fluorescent labeling it is often difficult to perform detailed imaging on SUVs simply because they are so small. To combat this problem researchers have developed the giant unilamellar vesicle (GUV). GUVs are large enough (several tens of micrometres) to study with traditional fluorescence microscopy. Many of the studies of lipid rafts in artificial lipid systems have been performed with GUVs for this reason. Compared to supported bilayers, GUVs present a more “natural” environment since there is no nearby solid surface to induce defects or denature proteins.

Nanion的巨型单层囊泡制备设备

   The Vesicle Prep Pro 是一款通过电膨化制备GUV的设备。

GUV可以在以下设备中应用:

   Port-a-Patch: 脂双层记录

   SURFE²R: 基于SSM的电生理

   SURFE²R: 基于SSM的电生理