Single ion-channel recordings using glass nanopore membranes
Single ion-channel recordings using glass nanopore membranes
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DOI：
10.1021/ja073174q
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发表时间：
2007-09-26
影响因子：
15
通讯作者：
White, Henry S.
中科院分区：
文献类型：
--
作者：
White, Ryan J.;Ervin, Eric N.;White, Henry S.
蛋白质离子通道记录使用玻璃纳米孔（GNP）膜作为脂质双层膜的支持结构。GNP膜由嵌入在类似于50 μ m厚的玻璃膜中的单个圆锥形纳米孔组成，所述玻璃膜用3-氰基丙基二甲基氯硅烷单层化学改性以产生中间疏水性的表面。这种表面改性导致在玻璃表面上形成脂质单层和横跨GNP膜的小孔（100-400 nm-半径）悬浮的脂质双层，同时允许水溶液完全润湿玻璃纳米孔。GNP膜/双层结构表现出类似于70 G Ω的欧姆密封电阻和类似于0.8 V的电击穿电压，对机械干扰异常稳定，并且具有至少2周的寿命。这些有利的特性是由于跨GNP膜孔悬浮的双层的非常小的面积（10（-10）-10（-8）cm（2））。荧光显微镜和振动和频光谱表明，脂质单层形成3-氰丙基二甲基氯硅烷改性的玻璃表面上的脂质尾巴朝向玻璃。GNP膜/双层结构非常适合单离子通道记录。可再现插入的蛋白质离子通道，野生型α-溶血素（WT α HL），和随机检测的小分子，七（6-O-磺基）-β-环糊精，被证明。此外，通过在脂质双层上施加小压力（约100至350 mmHg）可重复地控制WT α HL通道的插入和去除。双层的电和机械稳定性，实现双层形成的容易性，以及控制离子通道插入的能力，再加上GNP膜基系统的小双层电容，为单离子通道记录提供了一种新的且接近最佳的系统。
Protein ion-channel recordings using a glass nanopore (GNP) membrane as the support structure for lipid bilayer membranes are presented. The GNP membrane is composed of a single conical-shaped nanopore embedded in a similar to 50 mu m-thick glass membrane chemically modified with a 3-cyanopropyldimethylchlorosilane monolayer to produce a surface of intermediate hydrophobicity. This surface modification results in lipid monolayer formation on the glass surface and a lipid bilayer suspended across the small orifice (100-400 nm-radius) of the GNP membrane, while allowing aqueous solutions to fully wet the glass nanopore. The GNP membrane/bilayer structures, which exhibit ohmic seal resistances of similar to 70 G Omega and electrical breakdown voltages of similar to 0.8 V, are exceptionally stable to mechanical disturbances and have lifetimes of at least 2 weeks. These favorable characteristics result from the very small area of bilayer (10(-10)-10(-8) cm(2)) that is suspended across the GNP membrane orifice. Fluorescence microscopy and vibrational sum frequency spectroscopy demonstrate that a lipid monolayer forms on the 3-cyanopropyldimethylchlorosilane modified glass surface with the lipid tails oriented toward the glass. The GNP membrane/bilayer structure is well suited for single ion-channel recordings. Reproducible insertion of the protein ion channel, wild-type alpha-hemolysin (WT alpha HL), and stochastic detection of a small molecule, heptakis(6-O-sulfo)-beta-cyclodextrin, are demonstrated. In addition, the insertion and removal of WT alpha HL channels are reproducibly controlled by applying small pressures (-100 to 350 mmHg) across the lipid bilayer. The electrical and mechanical stability of the bilayer, the ease of which bilayer formation is achieved, and the ability to control ion-channel insertion, coupled with the small bilayer capacitance of the GNP membranebased system, provide a new and nearly optimal system for single ion-channel recordings.