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、古典的な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、これらの輸送系タンパクはイオンチャネルに比べて発現は低くなるため評価が困難です。SURFE²R テクノロジーは、大きなセンサー径を採用することで109 個に及ぶトランスポーターを一度に測定可能であり、最高のS/N比を得ることができます。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。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。
SURFE²R N1の特徴
• 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、電気生理学測定部、コンピュータを統合したオールインワン装置
• 教育ツールとしても最適な簡便操作
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, IC50, 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の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、電気生理学的ハードウェア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：
SURFE²R N1 装置およびソフトウェア
The SURFE2R N1 device
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
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
“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
“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
Prof. Dr. Klaus Fendler - Statement about the SURFE²R N1 Device
“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. Randy Stockbridge - Interview about the SURFE²R Technology
“We wanted to characterize the electrophysiological characteristics of our new family of transporters to find out properties such as Km and Vmax 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:
SURFE²R Technology Customer Case Study (2019) 
(1.8 MB)
Replacing Radiolabeling Techniques with the SURFE²R N1
データ ＆ アプリケーション
NCX1 - Block by KB-R7943
SURFE2R N1 data and applications:
Cardiac NCX1 expressed in iCell® Cardiomyocytes2, Cor.4U® Cardiomyocytes or HEK cells was blocked by increasing concentrations of KB-R7943.
NCX1 - Block by SEA0400
SURFE2R N1 data and applications:
Cardiac NCX1 expressed in iCell® Cardiomyocytes2 or HEK cells was blocked by increasing concentrations of SEA0400, a specific blocker of NCX.
AmtB - Activation by ammonium
SURFE2R N1 data and applications:
Purified AmtB from E.Coli was incorporated into liposomes and used on the SURFE2R N1. Shown are transient currents measured after a 100 mM ammonium jump in empty liposomes (green) or proteoliposomes containing AmtB at a lipid protein ratio (LPR) of 50 (black), 10 (red) or 5 (blue). Insert: Normalized current measured in proteoliposomes containing AmtB at an LPR of 50 (black), 10 (red) or 5 (blue).
Data from Mirandela et al, 2018
AmtB - Substrate specificity
SURFE2R N1 data and applications:
Purified AmtB from E.Coli was incorporated into liposomes and used on the SURFE2R N1. AmtB substrate specificity. (A) Transient current measured after a 100 mM substrate jump. Ammonium (red), methylammonium (black), potassium (green) or sodium (purple). Potassium and sodium do not act as substrates for AmtB and methylammonium translocates but at a much reduced rate compared with ammonium. Insert: Normalised current after a 100 mM substrate jump. Ammonium (red), methylammonium (black).
Data from Mirandela et al, 2018
EAAT3 - Current Traces at different Sodium Chloride Concentrations
SURFE2R N1 data and applications:
EAAT3 current traces were recorded after application of different Sodium Chloride concentrations. The EC50 of the peak current was determined as 42.2 mM.
Organic Cation Transporter 2 (OCT2) - Pharmacology
SURFE2R N1 data and applications:
EC50 values and relative Vmax for three different substrates are shown.
The IC50 value for Choline was determined as 4.6 mM, for TEA as 0.33 mM and for Metformin as 8.6 mM.
Sodium-Potassium ATPase - Analysis
SURFE2R N1 data and applications:
The Sodium-Potassium ATPase, also known as Na+/K+ pump is responsible for the active transport of Na+ and K+ in the cells containing relatively high concentrations of K+ ions but low concentrations of Na+ ions. The Na+/K+-ATPase helps maintain resting potential, avail transport, and regulate cellular volume. Here, we demonstrate the conductance of the pump, in the presence of ATP, Na+ and K+, or in the presence of ATP and Na+.
Respiratory Chain Complex V - Action of the ATP Synthase after Application of ATP
SURFE2R N1 data and applications:
The Respiratory Chain Complex V uses the transmembrane proton gradient (produced by Complex I, III and IV) to generates ATP from ADP plus phosphate. One component of ATP synthase acts as an ion channel that provides for a proton flux back into the mitochondrial matrix. This reflux releases free energy, which is used to drive ATP synthesis, catalyzed by the other component of the complex. Experiments on the SURFE2R N1 show the action of the ATPase after application of ATP.
Respiratory Chain Complex IV - Current Traces
SURFE2R N1 data and applications:
The Respiratory Chain Complex IV, known as Cytochrom c oxidase, reduces oxygen by cunsumption of electrons from cytocrome c and transport of two protons to the intermembrans space. Here the reaction runs backwards.
Respiratory Chain Complex II and III - Current Traces
SURFE2R N1 data and applications:
Respiratory Chain Complex II is a succinate dehydrogenase. In this complex, succinate is oxidated to fumarate. The electrons are transfered to the quinone pool, the Complex III (cytochrome bc1 complex) and cytochrome c, while four protons are transported to the intermembrane space.
Respiratory Chain Complex I and III - Current Over Mitochondrial Membrane
SURFE2R N1 data and applications:
In the respiratory chain Complex I and III, oxidation of NADH leads to transport of 4 protons across the membrane. The electrons are transferred to cytochrome c. Because of the electron leakage to oxygen, both complex are the main sites of production of harmful superoxide. In these experiments using the SURFE2R N1, application of 100µM NADH results in a current over the mitochondrial membrane, where Complex I and III are expressed.
Adenine Nucleotide Translocator (ANT) - Recorded on the Surfer
SURFE2R N1 data and applications:
The Adenine Nucleotide Translocator (ANT) is located in the inner membrane of mitochondria and transports, as the name suggests, either ADP or ATP alone or against each other. Recordings were done on mitochondria inner membrane preparations from pigs heart. Example traces display the action of ANT either in the presence of ATP, in an other experiment in the presence of ADP.
PepT1 - Pharmacology of Lys[Z(NO2)]-Val
SURFE2R N1 data and applications:
Representive traces of PepT1 overexpressed in CHO cells. PepT1 (SLC15A1) is expressed mainly in intestine and kidney, it enables uptake of oligopeptides. The PepT1 signal is evoked by glycylglycin. The transport of oligopeptides can be inhibited by addition of Lys[Z(NO2)]-Val. The IC50 of Lys[Z(NO2)]-Val was 380µM.
ウェビナー ＆ 動画
2018 - Electrophysiological Characterization of Sugar Transporters using SSM-based Electrophysiology
SURFE²R 96SE and SURFE²R N1 Oral Presentation
Presenter:
Dr. Andre Bazzone, Application Scientist, Nanion Technologies GmbH, Germany
Andre is an expert in the field of SSM-based electrophysiology: He made his PhD at the Max-Planck-Institute of Biophysics in Frankfurt, Germany on the electrophysiological characterization of sugar transporters using the SSM-based electrophysiology in 2016. Right afterwards, he started as Application Scientist at Nanion Technologies and today he is an important member of the Nanion SURFE²R team.
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 - Variations on the Coupling Theme in Secondary Active Transport
SURFE²R N1 Oral Presentation
Presenter:
Gregor Madej, University of Regensburg, Germany
ダウンロード：
アプリケーションノート
CNT1 - "Electrophysiological recordings of CNT1 (SLC28A1) activity on Nanion’s SURFE²R N1"
SURFE2R N1 application note (0.4 MB)
EAAT3 - "Electrophysiological recordings of EAAT3 (SLC1A1) activity on Nanion’s SURFE²R N1"
SURFE²R application note (0.4 MB)
GAT1 - "Electrophysiological recordings of hGAT1 (SLC6A1) activity on Nanion’s SURFE²R N1"
SURFE²R application note (0.4 MB)
Neurons - "Electrophysiological recordings of LGIC and AA transporters in iCell® GlutaNeurons"
Patchliner, 
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i) and SURFE2R N1 application note: (0.5 MB)
Cells were kindly provided by FUJIFILM Cellular Dynamics, Inc.
PepT1 - "Electrophysiological recordings of PepT1 (SLC15A1) activity on Nanion’s SURFE²R"
SURFE²R N1 application note: (0.9 MB)
製品カタログ
簡易マニュアル
SURFE²R N1 - Quickstart Guide "Protocols"
SURFE²R N1 Quickstart Guide "Protocols"
論文
2019 - Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic
SURFE²R ONE (a predecessor model of SURFE²R N1) publication in PNAS (2019)
Authors:
Tadini-Buoninsegni F., Mikkelsen S.A., Mogensen L.S., Molday R.S., and Andersen J.P.
2019 - Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae
SURFE²R N1 publication in Nature Scientific Reports (2019)
Authors:
Patiño-Ruiz M., Fendler K., & Călinescu O.
2019 - Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems
SURFE2R N1 and 
Orbit 16 publication in Frontiers in Pharmacology (2019)
Authors:
Dondapati S.K., Lübberding H., Zemella A., Thoring L., Wüstenhagen D.A., Kubick S.
2019 - A two-lane mechanism for selective biological ammonium transport
SURFE²R N1 pre-publication in bioRxiv (2019)
Authors:
Williamson G., Tamburrino G., Mirandela G.D., Boeckstaens M., Bage M., Pisliakov A., Ives C.M., Terras E.T., Bizior A., Hoskisson P.A., Marini A.M., Zachariae U., Javelle A.
2018 - The lipid environment determines the activity of the E. coli ammonium transporter, AmtB
SURFE²R N1 publication in Faseb J. (2018)
Authors:
Mirandela G.D., Tamburrino G., Hoskisson P.A., Zachariae U., Javelle A.
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 - Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation
SURFE²R N1 publication in Structural Dynamics (2017)
Authors:
Shah N.R., Wilkinson C., Harborne S.P.D., Turku A., Li K.-M., Sun Y.-J., Harris S., Goldman A.
2017 - Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter
SURFE²R N1 publication in Frontiers in Molecular Neuroscience (2017)
Authors:
Choudhary P., Armstrong E.J., Jorgensen C.C., Piotrowski M., Barthmes M., Torella R., Johnston S.E., Maruyama Y., Janiszewski J.S., Storer R.I., Skerratt S.E., Benn C.L.
2016 - Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism
SURFE²R N1 publication in Nature Communications (2016)
Authors:
Li K., Wilkinson C., Kellosalo J., Tsai J., Kajander T, Jeuken L.J.C., Sun Y., Goldman A.
2016 - Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology
SURFE²R N1 and 
Vesicle Prep Pro publication in Journal of General Physiology (2016)
Authors:
Barthmes M., Liao J., Jiang Y., Brüggemann A., Wahl-Schott C.
2014 - Anticancer Ruthenium(III) Complex KP1019 Interferes with ATP-Dependent Ca2+ Translocation by Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA)
SURFE²R N1 publication in ChemMedChem (2014)
Authors:
Sadafi F.Z., Massai L., Bartolommei G., Moncelli M.R., Messori L., Tadini-Buoninsegni F.
2013 - Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid supported membrane for the investigation of membrane transporters
SURFE²R N1 publication in Langmuir (2013)
Authors:
Sacconi A., Moncelli M.R., Mergheri G., Tadini-Buoninsegni F.
2009 - Measuring Ion Channels on Solid Supported Membranes
SURFE²R ONE (a predecessor model of SURFE²R N1) publication in Biophysical Journal (2009)
Authors:
Schulz P., Dueck B., Mourot A., Hatahet L., Fendler K.
2006 - Establishment of Cell-Free Electrophysiology for Ion Transporters: Application for Pharmacological Profiling
SURFE²R ONE (a predecessor model of SURFE²R N1) publication in Journal of Biomolecular Screening (2006)
Authors:
Geibel S., Flores-Herr N., Licher T., Vollert H.
ポスター
2018 - Label-free analysis of Na+/Ca2+- exchanger (NCX) isolated from iPSC-derived cardiomyocytes
SURFE²R N1 and 
SURFE2R 96SE poster, Europhysiology Meeting 2018 (1.5 MB)
2018 - Transported by light: optogenetic control of NCX1
SURFE²R N1 poster, Biophysics Annual Meeting 2018 (2.2 MB)
2015 - Organellar Transporters and Ion Channels - How to access their electrophysiology by using the SURFE2R technology and Planar Patch Clamp
SURFE²R N1 and SyncroPatch 96 (a predecessor model of the SyncroPatch 384PE) and 
Port-a-Patch poster, GRC - Organellar Channels and Transporters 2015 (1.6 MB)
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)
2015 - Functional Characterization of Procaryotic NCX by Solid Supported Membrane Technology
SURFE²R N1 poster, Gordon Research Conference 2015 (0.9 MB)
