SURFE²R N1 - トランスポーターの詳細な研究に

SURFE²R (SURFace Electrogenic Event Reader：サーファー) テクノロジーは、従来のパッチクランプ法に比べて起電性トランスポーター（シンポーター、エクスチェンジャー、ユニポーター）、ポンプの測定用に最適化されています。
通常これらの膜輸送体は、イオンチャネルに比べて輸送率が低くなるため測定が困難です。SURFE²R テクノロジーはそれを補うため、最大10⁹個に及ぶトランスポーターを同時に測定可能できる大きなセンサーサイズを採用し、最高のS/N比を実現しました。
SURFE²R N1 は基礎研究、アカデミック向けにデザインされています。より高いスループットが必要な場合は、SURFE²R 96SE による96ch センサーの完全同時測定も可能です。

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SURFE²R N1の特徴

• 蛍光プローブや放射標識リガンドを用いず測定可能
• 最大 52 種類の溶液による自動測定
• 150 データポイント / 日
• 分注機、測定部、コンピュータを統合したオールインワンデバイス
• 教育ツールとしても最適な簡単操作

SSM法による電気生理学測定の特徴

• 100以上のターゲットがバリデート済み, SSM-電気生理学を使用した100以上の査読付き論文
• トランスポーター、ポンプ、リガンド依存性チャネル
• 非起電性トランスポーターや糖類の活性評価にも適用可能
• 精製したトランスポーター、細胞から調製したベシクル、プロテオリポソームなど
• ラベルフリーの電気生理学的測定法
• 0.1 – 1 µg / センサーの少サンプル量で100実験まで実施可能
• 高分解能のリアルタイムデータ（シングルポイントの検出ではありません）
• パッチクランプ法より高いシグナル増幅率
• 律速な結合キネティクスの評価
• EC₅₀、IC_50、速度定数、トランスポーター遺伝子多型の比較など

当測定技術では、1990年後半に確立した Solid-Supported Membrane- (SSM法) に基づく電気生理学的測定手法を採用しています。SSM-電気生理学測定法の詳細は Technology section に示しています。試料調製の手順は、まず再利用可能な金コートされたセンサー上に簡単なピペッティング操作でSSMを形成させます。その後、トランスポーターを含む試料をセンサー上に加えてSSMに物理的に吸着させます。細胞破砕後の膜ベシクルや精製タンパクを再構成させたプロテオリポソームなど、測定対象となるタンパクを含むあらゆる膜調製が使用可能です。また、これらの試料は長期間の凍結保存が可能であるため、細胞培養も必要ありません。
SSM-電気生理学では溶液交換で基質またはリガンド添加によりトランスポーターを活性化し、発生した電荷の転移を検出、解析可能とします。SSMの高い安定性により、同一試料で100回にも及ぶ連続測定が可能になり、EC₅₀ や IC₅₀.等のパラメーター決定を容易に行うことができます。また、最高水準の溶液交換の時間分解能により、結合反応の遅いトランスポーターから速いトランスポーター測定まで適用でき、速度定数を決定することができます。

装置の取り扱いは極めて簡便で、1日で操作を習得できます。また、全自動のロボット測定により研究者の負担は大幅に削減されます。筐体には実験で必要となる分注機、測定部からコンピューターまで、すべて統合されています。

詳細情報：

Add-Ons and Features

The SURFE²R Stimulating Optical Lid (SOL)

SURFE2R SOL

The SURFE²R Stimulating Optical Lid (SOL) replaces our standard lid for the measurement chamber. The optical lid contains a plate with an integrated LED ring - exchangeable within seconds and available in eight different wavelengths. The LED may be used to activate light gated channels or light driven pumps such as Bacteriorhodopsin. But it can be also applied to generate a membrane voltage through the action of Channelrhodopsin or release caged compounds.

Usually the SURFE²R applies a solution exchange providing a substrate to activate the transporter and record the resulting transient transporter current. Now substrate and light activation can be used individually; or a combination of light and substrate stimuli enables experimental designs which were never seen before.

Besides the stimulating optical lid itself, the addon comes with new internal electronics and a powerful software upgrade to control the LEDs: We developed a new workflow editor which can be used to set several experimental parameters such as light intensity and to switch between continous and pulse modes.

SURFE²R N1 装置およびソフトウェア

The SURFE²R N1 device

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The SURFE²R (surface electrogenic event reader) technology is the only available commercial solution for SSM-based electrophysiology on the market. Nanion Technologies developed the SURFE²R 96SE, a high-throughput system mainly used in pharmaceutical industry for drug screening purposes and the SURFE2R N1 designed for basic research.

The SURFE²R N1 device contains electrophysiological hardware, liquid handling components and the computer running Windows, plus the data recording and analysis software SurfControl. It’s an easy-to-learn, all-in-one robotic workstation which can measure 150 data points a day in a fully automated manner.

The SURFE²R N1 works with reusable sensor chips on which the membranes containing the protein of interest are adsorbed. The measurement is initiated by a substrate concentration jump. The solution exchange is controlled by the Ionjet, a robot-controlled pipette which loads solution from storage containers and injects them to the sensor enclosed by a Faraday cage. The Ionjet allows the measurement of fast kinetics with low solution consumption. An autosampler on top of the device allows placing up to 53 solutions for automated sequential measurements. During the measurement, the transport current can be viewed, compared and analyzed within the SurfControl software. The main characteristics of the device include a low-noise amplifier and a large sensor surface which both ensure a superior signal to noise ratio.

SURFE²R N1 Control Software and Automatization

The device comes with the SURFE²R N1 Control software pre-installed on the internal computer. This forms both the recording and analysis software. The functions of SurfControl include the managing and coding of workflows which represent protocols for SURFE²R assays. Workflows can contain multiple measurements, e.g. using different substrate concentrations or the comparison of transport before and after inhibition. The workflow includes parameters like duration, speed and volume of solution flow during the experiment, the number and sequence of different buffers, the number of repetitions per measurement, the incubation times between experiments and the volume used for rinsing the sensor after an experiment. During the run of one workflow no interference by the researcher is required.

Beside the design of the experiment itself, SurfControl enables the researcher to view and compare recorded traces. The graphical window allows peak detection and the calculation of the peak integrals including baseline subtraction. An additional results window is used for automated documentation of the traces including file name, values for peaks and integrals and the time of measurement. For further analysis, e.g. the fitting of kinetic parameters or the subtraction of negative control currents, the software is capable of exporting the data to the standard file format ASCII.

消耗品

SURFE²R N1 Single Sensor Chips

N1 Round V2 400x237

The SURFE²R N1 sensor chip is a proprietary innovative product by Nanion Technologies, developed for the SURFE²R N1. It is produced by an external partner, quality-assured in-house at Nanion headquarters and shipped from Munich to our international customers.

Material

The core structure of SURFE²R N1 sensor chip is a gold-coated sensor with 3 mm diameter. This structure is incorporated into a screw cap for easy handling. The SURFE²R N1 single sensor chips can be reused after an appropriate cleaning protocol.

Available chip type

"SURFE²R N1 Single Sensor Chip" (Order # 161001)

バリデート済ターゲット一覧: SSM-電気生理学的測定

インタビュー & ケーススタディ

Dr. Arnaud Javelle - Statement about the SURFE²R N1 Device

Icon N1 “Using the SURFE2R N1 we have recently obtained high quality data in a very short period of time. We have developed an assay to measure the activity of ammonium transporters from the Amt protein family. There has been considerable controversy over the mechanism of ammonium transport by Amt proteins and the controversy was due to the lack of quantitative kinetic data characterizing the activity of the proteins at the single channel level. The SSM technologies allows to overcome this hurdle and we are now capable of answering very challenging functional questions concerning the mechanisms and the energetics of these transporters."

Dr. Arnaud Javelle, Chancellor's fellow, Strathclyde Institute of Pharmacy and Biomedical Sciences

Dr. Thomas Seeger & Karin V. Niessen - Statement about the SURFE²R Technology

Icon N1 “The SURFE²R makes extremely challenging electrophysiological targets accessible to robust routine analyses. For example, we have developed an assay for investigating nicotinic acetylcholine receptor ion channels, using membranes from the Pacific electric ray, Torpedo California. In this way, we overcome many of the well-known difficulties associated with this ion channel, which allows us to efficiently obtain information regarding compound pharmacology. Obtained pharmacology values match those of patch clamp recordings exceptionally well. We find that the SURFE²R is an excellent platform for characterization of electrogenic processes in isolated membranes.”

Karin V. Niessen, Dr. Thomas Seeger
Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

Nathan Thomas - Statement about the SURFE²R N1 Device

Icon N1 “We started collaborating with Nanion after receiving the Nanion´s SURFE²R N1 grant in 2018. The SURFE²R N1 has generated terrific functional data from the very first day it was installed in our lab. In general, it's very reliable and easy to use. Also, whenever we have run into issues or had questions about results, Nanion has provided prompt and helpful support”

Nathan Thomas
5th Year PhD student at University of Wisconsin-Madison, USA (Dr. Henzler-Wildman's lab)

Prof. Dr. Klaus Fendler - Statement about the SURFE²R N1 Device

Icon N1 “SSM-based electrophysiology is a well-established technique developed in our lab in the early 1990s for the investigation of electrogenic membrane transporters. It works with a wide range of samples from mammalian cell membranes over microbial membrane vesicles to proteoliposomes. SSM-based electrophysiology is especially useful in cases where conventional electrophysiology cannot be applied, e.g. for transporters residing in intracellular membranes or plasma membranes of small microbial cells. The SURFE²R N1 is a commercial instrument for SSM based electrophysiology which facilitates basic research in the field of membrane transport.“

Prof. Dr. Klaus Fendler
Max-Planck Institute of Biophysics, Frankfurt, Germany

Prof. Dr. Klaus Fendler and Co-Authors - Interview about the SURFE²R Technology

Icon N1 ““SSM-based electrophysiology is a suitable technique to study bacterial ion-translocating membrane- systems which are otherwise difficult to approach using conventional electrophysiological techniques, like patch clamp.
Additionally, the mechanical stability of SSMs facilitates the transport activation through fast solution exchange, a condition that is technically challenging in a setup of a bilayer separating two solution-containing compartments.“

Dr. Miyer Fabián Patiño-Ruiz,
Faculty of Science and Engineering, University of Groningen, the Netherlands

Dr. Andre Bazzone, Application Scientist for the SURFE²R product family interviewed the authors of the publication:
"Mutation of two key aspartate residues alters stoichiometry of the NhaB Na⁺/H⁺ exchanger from Klebsiella pneumoniae"
Published 2019 in Nature Scientific Reports.

Read the publication here and download the full case study here:

Icon N1 SURFE²R Technology Customer Case Study (2020) (1.3 MB)
Nature Scientific Reports: Characterization of the Na⁺/H⁺ exchanger NhaB on the SURFE²R N1

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Prof. Dr. Randy Stockbridge - Interview about the SURFE²R Technology

Icon N1 “We wanted to characterize the electrophysiological characteristics of our new family of transporters to find out properties such as K_m and V_max of the substrate, and ideally to screen a set of substrates for transporter activity. However, given our difficulties in developing a transport assay, I was pessimistic that we were going to be able to move beyond radiolabeled substrates.
Contrary to my expectations, electrophysiology with the SURFE²R N1 platform worked right away. We saw very robust currents in our pilot experiments during the initial training session on the instrument.“

Prof. Dr. Randy Stockbridge, Assistant Professor
University of Michigan, USA

Download the full case study here:

Icon N1 SURFE²R Technology Customer Case Study (2019) (1.8 MB)
Replacing Radiolabeling Techniques with the SURFE²R N1

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