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SURFE²R 96SE
SURFE²R 96SE - 高通量转运体研究设备
与 SURFE²R N1一样,SURFE²R 96SE基于固体支撑膜(SSM)电生理技术,解决转运体与离子泵电流过小的问题,同时将通量提高到平行进行96个记录。只需要将转运体 样品加到96孔记录板镀金电极上。并且为了进一步减少用户的工作量,所有电极预处理步骤都由SURFE²R 96SE自动完成。并且可以加任何底物都可以加入激活转运体,且会记录相应的转运或绑定电流。所有的以上步骤,从制备样品、测定到数据分析都是自动进行的。
• 通过内置的96通道低噪音放大器平行进行96孔记录
• 每天可获取10,000 个数据点
• 整合 CyBio® Felix 小型移液平台
• 自动化的实验流程,包括自动制备样品
• 自动数据分析与导出,例如测定EC50等
• 拥有8个放置部同溶液的位点 (支持1孔板到96孔板)
• 已在超过100 目标上通过验证,发表了超过 100篇同行评审过的文献
• 可测量转运体、离子泵与配体门控通道
• 也可测量电中性交换体、糖结合蛋白
• 可使用从细胞提纯的生物膜与囊泡或蛋白脂质体
• 非标记电学测量手段
• 每孔仅需要0.1 – 1 µg蛋白样品,并足够进行最多100次实验
• 实时数据与5 ms的高时间分辨率,非单点读取
• 相对膜片钳更高信号放大率
• 可完成快速绑定动力学测量
• 可完成EC50、 IC50、转运速度常数、转运体亚型比较...
基于固体支撑膜技术(solid-supported membrane,SSM) 的电生理技术建立于90年代末,关于SSM电生理技术的详细资料请参考技术细节。SURFE²R 96SE相对于SURFE²R N1 拥有更高的通量。更适用于诸如待测物或转运体筛选项目。SURFE2R 96SE也适用于需要较高通量的基础研究。其数据获取速度相较于 SURFE²R N1快了将近100倍。
The SURFE2R 96SE 设备
SURFE2R(表面致电事件阅读器)技术是市场上唯一可用的基于SSM的电生理学商业解决方案。Nanion Technologies开发了SURFE2R N1,作为大学和基础研究的易学一体式设备,以及高通量设备SURFE2R 96SE。
SURFE2R 96SE集成在CyBio®Felix液体处理平台中。Nanion Technologies提供电生理硬件和运行数据记录和分析软件包的计算机。该装置不仅可用于电生理测量,还可用于样品制备和移液。该设备每天可以以完全自动化的方式测量10,000个数据点。与SURFE2R N1相比,SURFE2R 96SE还配备了专门的数据分析软件,可以自动分析你每天生成的大量数据。
SURFE2R 96SE采用标准的96孔板形式的传感器。含有感兴趣蛋白质的膜被吸附到这些传感器上。测量是由底物浓度跳变引起的。溶液交换由CyBio®Felix液体处理平台控制。移液头包含96个移液管,从溶液位置加载溶液并将其注入到传感器板上。传感器板上所有96个传感器同时测量,但每次测量可以使用不同的解决方案或传送器。
传感器板由法拉第笼包围,并连接96通道放大器和96个作为参考电极的金针。CyBio®Felix可容纳8个井板,适用于8到8 × 96种不同的解决方案,在每个自动化工作流程中,可在不同条件下进行多达7次连续测量。CyBio®移液器允许在低溶液消耗的情况下测量快速动力学,每次测量只使用50µl的复合溶液,时间分辨率可降至5 ms。
软件包
我们的软件部门专门为SURFE2R 96SE开发了一个软件包。
•SurfControl作为记录软件工作,实时显示数据
•DataControl用作分析软件,可配置为自动化数据分析
•此外,CyBio Composer plus预装工作流操作液体处理平台和与录音软件通信
SURFE²R 96SE 芯片板
SURFE²R 96SE传感器板是Nanion Technologies为SURFE²R 96SE开发的专有创新产品。它由一家外部合作伙伴生产,由Nanion总部内部质量保证,并从慕尼黑运往我们的国际客户。
SURFE²R 96SE传感器板的核心结构是直径为3mm的镀金传感器。该结构与96孔板结合,便于操作。在实验过程中,传感器板由法拉第笼封闭,并连接到96通道放大器。
"SURFE²R 96SE Sensor Plate": 96-well recording plate for the SURFE²R 96SE (Order # 182001)
数据与应用
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.
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
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
ChR2 - Cell-based assay optimization
SURFE2R N1 data and applications:
For assay optimization we checked the influence on cell suspension volume we have attached to one single sensor on the signal-to-noise ratio during the SURFE2R experiment.
ChR2 - Exposure time and frequency of light
SURFE2R N1 data and applications:
We investigated the influence of the duration and frequency of the light pulses for ChR2 activation on the stability of the ChR2 signals over time.
ChR2 - Membrane potential as driving force
SURFE2R N1 data and applications:
We investigated the effect of a membrane potential - generated by Na+/Ca2+ exchange activity of NCX1 - on the signal amplitude of light induced Na+ flux by ChR2.
ChR2 - Stability of Retinal binding
SURFE2R N1 data and applications:
We investigated the effects of retinal addition and removal on light-induced ChR2 activity using a prototypical SURFE2R N1 add-on for activation by light.
SURFE2R N1 data and applications:
Depending on the direction of the Na+ gradient, we observed positive or negative transient currents after ChR2 activation by light. A negative control without ChR2 shows no current signal upon light activation.
ChR2 - Variation of light intensity
SURFE2R N1 data and applications:
We observed saturation kinetics for the light-induced ChR2 activity with increasing LED intensities using a prototypical SURFE2R N1 add-on for activation by light.
ClC - EC50 for inward and outward directed chloride flux
SURFE2R N1 data and applications:
We determined EC50 and relative Vmax for Cl- in an Cl-/H+ exchange assay for both transport directions and found a slight asymmetry in transporter kinetics.
SURFE2R N1 data and applications:
We investigated the effect of the specific ClC inhibitor OADS on the transport activity of ClC and its reversibility.
ClC - Inward and outward directed chloride flux
SURFE2R N1 data and applications:
According to the transport direction of 2Cl-/1H+ exchange, we observed positive (Cl- efflux) or negative (Cl- influx) transient currents.
ClC - Proteoliposomes with different lipid-to-protein ratios
SURFE2R N1 data and applications:
We tested proteoliposomes with different densities of reconstituted ClC protein for their signal-to-noise ratios and compared the obtained signal amplitudes with a negative control: liposomes without reconstituted protein.
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.
NCX_Mj - EC50 for Calcium and Magnesium
SURFE2R N1 data and applications:
Measurement of the transport activity of NCX_Mj. The first solution exchange provides a Na+-free solution to establish an outward directed Na+ gradient; the second solution exchange provides the divalent cation to activate the ion exchanger. By applying different concentrations of one divalent cation on the same sensor, we determined EC50 for Ca2+, Mg2+ and Sr2+.
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.
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.
NCX1 - Current traces recorded from Cor.4U Cardiomyocytes, iCell Cardiomyocytes and HEK cells
SURFE2R N1 data and applications:
Cardiac NCX1 was activated using 30 μM Ca2+ on the SURFE2R N1. NCX1 was recorded from Cor.4U® Cardiomyocytes, iCell® Cardiomyocytes2 or overexpressed in HEK cells. The calcium affinity was determined for NCX1 recorded from iCell® Cardiomyocytes2 and HEK cells, shown on the right.
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.
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.
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.
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 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 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.
SURFE2R N1 data and applications:
Ca2+-transport in SERCA is triggered by ATP hydrolysis. Using ATP concentration jumps, the authors determined the IC50 of SERCA inhibition by cisplatin
SGLT1 - Current Traces after Application of alpha-MDG
SURFE2R N1 data and applications:
Current traces after application of 10 mM and 50 mM alpha-MDG.
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+.
发表文献:
2021 - Structure and lipid-mediated remodelling mechanism of the Na+/H+ exchanger NHA2
2021 - Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers
2021 - Structural basis for Potassium transport by KdpFABC
2021 - Functional Characterization of SLC Transporters Using Solid Supported Membranes
2021 - Engineering and functional characterization of a proton-driven β-lactam antibiotic translocation module for bionanotechnological applications
2021 - Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport
2021 - A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds
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资料下载:
Nanion_Poster_SURFE2R_96SE_FLEXcyte96_2021_BPS_NCXCIPA.pdf 附件下载 (下载 0 次)
Nanion_Poster_SP_Unattended_Runs_SPS_Virtual_2020.pdf 附件下载 (下载 0 次)
Nanion_Poster_SURFE2R_2018_Europhysiology.pdf 附件下载 (下载 0 次)
Nanion_Poster_SURFE²R_Neurons_Cardiomyocytes_BPS_2020 (1).pdf 附件下载 (下载 0 次)
Nanion_Poster_SURFE2R_PAP_384PE_2015_GRC.pdf 附件下载 (下载 0 次)
SURFE2R高通量药物转运体产品彩页.pdf 附件下载 (下载 1 次)
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