Ionovation Compact人工脂质膜重组电生理分析系统
德国Ionovation公司生产的Ionovation Compact人工脂质膜重组电生理分析系统，是该领域最新的产品之一，该产品克服了传统膜片钳的一系列缺点，无论从产品的技术含量还是从产品的应用领域上来看，在电生理分析技术中始终处于领先的地位，代表着电生理分析技术发展的方向，是国内外细胞电生理分析实验室首选实验仪器。
系统介绍
中文名称：Ionovation Compact人工脂质膜重组分子自定义环境电生理分析系统；
Innovation Compact是人工脂质膜定义环境中重组分子进行电生理分析的可靠工具。这种高度灵活的桌面检测系统可适应多种实验条件，目前已经被用于对细胞膜离子通道、多种动物和植物转运蛋白和它们的细胞器进行研究。（详见参考文献可案例部分）
 产品背景：
细胞膜离子通道是最古老的功能蛋白之一，广泛存在于从细菌到植物到动物包括人类在内的生物界，是许多基本生物活动如电活动、离子转运和细胞分泌等的基础。对于人类而言，由离子通道参与的功能(如电活动)是神经及心血管等系统生理功能的最基本形式之一。对离子通道功能调节机制的深入研究是了解其生理学和生理病理学意义的关键所在。
膜片钳技术是用玻璃微电极吸管把只含1-3个离子通道、面积为几个平方微米的细胞膜通过负压吸引封接起来，由于电极尖端与细胞膜的高阻封接，在电极尖端笼罩下的那片膜事实上与膜的其他部分从电学上隔离，因此，此片膜内开放所产生的电流流进玻璃吸管，用一个极为敏感的电流监视器（膜片钳放大器）测量此电流强度，就代表单一离子通道电流。膜片钳技术发展至今，已经成为现代细胞电生理的常规方法。但是随着研究的深入，目前发现膜片钳主要有以下缺点： 测量非常耗时 需要高度熟练的操作者来进行实验 需要建立千兆欧姆的封阻，但是千兆欧姆的封阻在测试时不稳定 并不是所有离子通道都可以被测试，一般是配体或者在一侧可以交换缓冲液的才可以被测量 膜片钳无法对细胞器进行分析 在测量过程中的细胞往往形成穿孔 结果在稳定背景下经过多次测量形成平均值，测量值离散度大
表1目前膜片钳能够与不能够进行检测的疾病（细胞）
可以被膜片钳检测的疾病（细胞） | 无法被膜片钳检测的疾病（细胞） | 心律失常 | 骨质疏松 | 焦虑 | 哮喘 | 癫痫 | 过敏 | 偏头痛 | 癌症 | 耳鸣 | 自身免疫性 | 失眠 | 慢性阻塞性肺病 | 失禁 | 囊性纤维化 | 精神分裂症 | 肾结石 | 抑郁症 | 糖尿病 | 高血压 | | 记忆障碍 | | 肌强直 | |
Ionovation Compact比膜片钳的优势主要在于： 不需要建立千兆欧姆的封阻 是唯一一种采用双层封阻的测量方式 可用于检测各种细胞膜上的离子通道、囊泡、配体 容易实现对单个离子通道进行分析检测 可以在膜的两侧改变条件，形成双信道进行分析
系统应用原理
Ionovation Compact采用了人工生物脂质膜来构建离子通道的测量方法。该系统使用两个独立的电生理测量室进行测量。该双层室由一个25μm厚、直径120微米的聚四氟乙烯隔膜隔开（a）。两个聚碳酸酯小室的体积分别为1.2 ml。聚碳酸酯小室顶部有孔，方便接入的Ag / AgCl电极和注入缓冲液（a）。
当两个腔室中填充有合适的缓冲液时，溶解在正癸烷的脂质被分散到直径120微米的特氟隆隔膜的微孔（ b）中。缓冲液的液面经过连续地降低和升高过程，直到特氟隆隔膜上的过量脂质被除去，特氟隆隔膜上形成脂双层自组织形成的人工生物脂质膜（C）。
图1 聚碳酸酯小室及聚四氟乙烯隔膜
图2 正癸烷缓冲液以及形成的形成的人工生物脂质膜 
图 3传统膜片钳与Ionovation Compact的测试原理对比
然后根据具体的目的蛋白或者细胞器等进行操作。比如类似于α-溶血素的毒素，可以直接加入聚碳酸酯小室中进行测试。而需要重组的离子通道则需要将其与脂肪酸结合为重组脂蛋白，然后将这些脂蛋白或者从生物膜上“剪下”囊泡与人工生物脂质膜通过离子梯度或融合肽方法进行融合。 准备好样品后，开始进行电生理学测试。通过施加不同的电压，来监测细胞单离子通道的电流，其中在5-10 kHz带宽的信号会存储在系统的硬盘上，测试通常采用的模式是电压钳模式（voltage clamp mode），这种模式下应用不同的电压来得到目的信号。 
图4 ?Ionovation Compact测试系统工作状态
图5 ?Ionovation Compact测试系统得到的离子通道电流信号
图6Ionovation Compact测试系统测量界面
系统功能亮点 ??? 自动膜生成技术以及结合运用预制室保证了每个双脂质层试验成功。 ??? 系统设计时兼顾考虑到专家和初学者的使用水平，为专家和初学者均提供简单可靠的最有效实验应用工具。 所有实验目的均是得到理想的生物学分析结果，而实现生物测量中的膜准备工作任务交由本系统直接自动处理。 实验创新性好，解决了传统膜片钳的测量耗时、需要建立千兆欧姆的封阻等一系列缺点，相比于传统方法，Ionovation Compact测量方法高度灵活，满足不同的Idea。 ???? 高度灵活的桌面系统整合了大量通道、根孔、转运体或膜囊膜的制备经验。有很多高水平文章可做参考。
图7 Ionovation Compact具有测量方法高度灵活的设计 根据现有的已出版科研文献报道，本系统主要科研应用领域包括神经科学，脑科学，心肌细胞，心血管，药物学，药理学，生理学，细胞生物学，生殖生理，病理生理，中药学，植物细胞生理学等领域的研究，并可作为细胞生物学与分子生物学研究的桥梁。  
图8 Ionovation Compact测试系统可用于多种离子通道的分析
系统主要性能参数
测量对象：可实现对上万种离子通道、转运体和孔隙活动的精确测量；
软件：电生理数据采集分析软件Patchmaster，采用用户界面友好的软件实现测量全部控制；具备预定义协议式的用户定义实验工作流程；
自动操作：全自动设备操作（双层生产和验证、双层完整性的电容控制、膜双侧灌注系统）；·双层分子可视化操作；
电流测量：稳定低噪声Ag/AgCl电极，采用盐桥记录电流范围从pA到几个nA；
耗材：一次性双分子层室可简单、快速置换；
系统组成
·双分子层室
·Ag/AgCl电极
·探头/放大器???????????
·摄像头
·灌注系统
·控制面板
·远程控制器
·数据采集分析软件系统
 ??参考文献
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案例1 Thomas Theis. Functional roles of transient receptor potential canonical channels and myristoylated alanine-rich protein kinase C substrate as novel interaction partners of the neural cell adhesion molecule NCAM and polysialic acid in Mus musculus，2013 博士论文 
Figure 5.31: Schematic drawing of the setup to measure the capacitance of an artificially lipid bilayer. The Ionovation Compact V02 system was used to build up an artificially lipid bilayer and to measure the capacitance of the bilayer. The lipid bilayer separates adjacent chambers， which were filled with electrode buffer. The artificially lipid bilayer contains a mixture of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) (1:1). In the circuit diagram the resistance circuit (red) and the capacitor circuit (cyan) is shown. The resistance of the red marked circuit is increased when the lipid bilayer is formed. As a consequence more current flows through the capacitor circuit and， thus， lead to an increase of capacitance. When colominic acid and a peptide derived from the ED of MARCKS (ED-Peptide) are added to the trans chamber and the cis chamber， respectively， and if they interact through the lipid bilayer， the lipid bilayer would be more instable from both sides and change its capacitance. Therefore the resistance would decrease and less current would flow through the capacitor circuit which would diminish the capacitance (A). Chondroitin sulfate (ChS) has similar chemical properties as colominic acid， but it is not known that neither it attaches to the membrane nor that it can interact with MARCKS. The control-peptide is also derived from the ED of MARCKS， but the phenylalanine residues were changed to alanine residues. Both were used as controls， which would not penetrate in the lipid bilayer and therefore the bilayer would be stable. The resistance in the red marked circuit would be higher in the controls and therefore more current would flow through the cyan marked circuit followed by an increase of the capacitance (B， C， D).?
案例2 Citation: Schmidt F， Levin J， Kamp F， Kretzschmar H， Giese A， et al. (2012) Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by a-Synuclein Oligomers in Lipid Membranes. PLoS ONE 7(8): e42545. doi:10.1371/journal.pone.0042545  ?
实验方法：
Influence of a-syn on electrophysiological properties of planar lipid bilayers Planar lipid bilayers were produced in the Ionovation Compact (Ionovation，Osnabruck， Germany) by the painting technique [20]. Two bath chambers separated by a Teflon-septum were filled with 250 mM KCl， 10 mM MOPS， pH= 7.2 (Merck). In the cis-chamber， 2 ml of a 100 mg/ml-solution of purified azolectin in n-Decane (Ionovation) was applied to a pinhole of 120 mmin diameter. After 30 min incubation at RT， lipid was thinned out by repetitive lowering and re-raising of the buffer-level until a bilayer was formed. Bilayer formation was monitored optically and by capacitance- and conductance-measurements. The resulting bilayers had a typical capacitance of 60–80pF and a resistance of .100GV. The monitoring of the bilayer was performed using Ag/AgCl-electrodes (Ionovation)， an EPC 10-amplifier and Patchmaster-software (HEKA， Lambrecht/Pfalz， Germany). The electrode in the cis-chamber was directly connected to the amplifier， so all potentials are referred to this compartment. The noise was ，0.4pA (r.m.s.) at 3 kHz bandwidth. After bilayer formation， we waited for 10 min to ensure application of the protein to a stable bilayer-system. Then， a-syn aggregation samples (total assay volume: 200 ml) were added in aliquots of 20 ml close to the membrane in the trans-chamber. The electrophysiological properties were monitored using +/220 mV- squarewave-voltage pulses. Pore formation resulted in an increase in the current flow over the membrane compared to an intact bilayer (Fig. 1A). Threshold for pore detection was set to a conductance of 70pS. If no increase in bilayer conductance beyond the threshold was detected for 5 min， the next aliquot of the sample was added. Pore detection rate was defined as the probability of pore detection per a-syn aggregation sample. In the event of an increase in bilayer conductance， a standardized recording-protocol was employed， consisting of a voltage-ramp reaching from 2100 to +100 mV over 10 sec and different squarewave-voltage pulses.
案例3 Citation: Smeazzetto S， Saponaro A， Young HS， Moncelli MR， Thiel G, ?(2013) .Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity. PLoS ONE 8(1): e52744. doi:10.1371/journal.pone.0052744 
实验方法
Planar lipid bilayer and single channel measurements Experiments with planar lipid bilayers were carried out as described previously [23] using the folding method with a 10 mg/ ml solution of diphytanoylphosphatidylcholine (DPhPC) (Avanti- Polar， AL， USA) in pentane. The experimental chambers used to assemble the planar bilayer were either custom made or disposable chambers (Ionovation， Osnabruck， Germany). The measurements were performed in a buffer containing 500 mM KCl， 10 mM Mops/Tris pH 7. The Ag/AgCl electrode in the cis compartment was directly connected to the head stage of a current amplifier (EPC 7， List， Darmstadt， Germany); the trans chamber was grounded. Currents were recorded and stored by an analogue/digital-converter (LIH 1600， HEKA electronics， Lam- brecht， Germany) with a sampling rate of 3.571 kHz after low pass filtering at 1 kHz. Data were recorded by Patchmaster-Software (HEKA electronics， Lambrecht， Germany) and analyzed with the Fitmaster-Software (HEKA electronics， Lambrecht， Germany) and the KielPatch program (University of Kiel， www.zbm.uni-kiel. de/aghansen/software.html) and Origin (OriginLab. Northamp- ton， MA， USA). The apparent single channel current amplitudes (Iapp) were determined by visual inspection of the current traces using the KielPatch software. The open probability (Po) was calculated with the KielPatch software. In the case that more than one channel was present in a bilayer we estimated the number of channels from the maximal number of concomitant open events. The protein， which was dissolved in water， was added directly to the trans chamber at a final concentration of ca. 0.3 mM. Before addition of the protein the bilayer conductance was routinely recorded for approximately 1 hour in order to exclude artefacts from contaminations. Only bilayers without artefacts were used for reconstitution of PLN. To perform experiments of phosphorylated wt-PLN， the protein was added to the bilayer after 3 h incubation at 30uC under the following conditions: 1 mg/ml wt-PLN， 20 mM imidazole， 100 mM KCl， 1 mM DTT， 10 mM MgCl2， 0.5 mM EGTA， 1 mM ATP and (5 units/10 mg PLN) PKA (Sigma) diluted in storage buffer solution.
案例4：Erika Kovács-Bogdán, et al. Tic20 forms a channel independent of Tic110 in chloroplasts.BMC plant biology, 2011, 11:133—134 Results: We performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20- proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro. 
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