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Nanionの技術: How it Works

2003年の 世界最小のパッチクランプ装置「Port-a-Patch」発売がすべての始まりでした。その後、当社は Port-a-Patch での成功と経験をもとに、スループットとアッセイ柔軟性を向上するために製品のポートフォリオを拡大していきました。当社は現在、ミディアム~ハイスループットの自動パッチクランプ装置のほか、脂質平面膜、SSM法による電気生理学、インピーダンス/EFP測定を行う各種装置を提供するに至りました。当社は今後もお客様の声に耳を傾け続け、研究と創薬開発のニーズに合った技術開発を精力的に行って参ります。


オートパッチクランプ

平面パッチクランプによるオートパッチクランプ

パッチクランプ法はリアルタイムでのイオンチャネル研究のゴールドスタンダードです。極めて高い時間分解能で,イオンチャネルの生物物理学的特性や化合物などによる修飾を研究できます。

チップベースの平面パッチクランプ法の開発により,パッチクランプ法の自動化が可能になりました。オートパッチクランプはスループット向上と古典的パッチクランプ法に比べて簡便な取扱いが可能であり,どなたでも容易にお使いいただけます。

Planar Patch Clamp

ナニオンは2003年に '市場初'のセミオートの平面パッチクランプ装置「Port-a-Patch」を発売しました。以降,ナニオンが次々に開発した装置の全ては "真のギガシール" 装置であり,古典法の枠を超える,平面パッチクランプ法に基づく広範な実験の可能性を提案しています。


ナニオンの平面パッチクランプ装置

icon pap   Port-a-Patch(ポートアパッチ) は,セミオートの卓上型オートパッチクランプ装置であり,細胞と脂質二分子膜実験のイオンチャネル研究に使用可能です。

icon pl   Patchliner(パッチライナー)は,極めて柔軟なアッセイデザインが可能なミディアムスループットのオートパッチクランプロボットです。

icon sp96   SyncroPatch 384PE(シンクロパッチ384PE)は,SyncroPatch 768PEにアップグレード可能なハイスループットのパッチクランプ装置であり,スループットは20,000または40,000データポイント/日にも及びます。


patch clamp instrument comparison


平面パッチクランプ法に関する論文例
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脂質二分子膜実験

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.

Bilayer Recordings


ナニオンのチップ式脂質二分子膜実験装置

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

icon orbit mini   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.

icon orbit   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.

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


bilayer instrument comparison


チップ式脂質二分子膜実験に関する論文例
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SSM-電気生理

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

icon n1   The SURFE²R N1 is a medium throughput device, designed for basic research and Universities.

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

SSM Instrument Comparison


Recommended Readings and Movies
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細胞外電位/インピーダンス測定

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

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


Recommended publications
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Recommended oral presentations
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オプトジェネティクス

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

icon CE   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
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巨大単層膜リポソーム (GUV)

GUVs: Vesicles for Multiple Applications

A vesicle is a lipid bilayer rolled up into a spherical shell, 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.

Products Vesicles bea 330


Nanion's Giant Unilamellar Vesicles Device

icon vpp   The Vesicle Prep Pro is a device for the fomration of GUVs by means of electro-swelling.


GUVs can be used for the following devices:

icon pap   Port-a-Patch: Bilayer Recordings

Icon N1   SURFE²R: SSM-based Electrophysiology

Icon 96SE   SURFE²R: SSM-based Electrophysiology

 

 

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