Reconstitution of ion channels in agarose-supported silicon orifices
Reconstitution of ion channels in agarose-supported silicon orifices
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DOI：
10.1016/j.bios.2006.10.017
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发表时间：
2007-05-15
影响因子：
12.6
通讯作者：
Nadeau, Jay L.
中科院分区：
文献类型：
--
作者：
Maurer, Joshua A.;White, Victor E.;Nadeau, Jay L.
具有八个可单独寻址的微加工孔的硅晶片用于离子通道重构和单通道记录。旋涂含氟聚合物形成了比硅烷更稳定的绝缘疏水界面。膜的总电容< 10 pF，使得可以通过电容变化轻松评估双层的形成。测试了直径50-250μm的孔口双层形成的容易性和稳定性；只有那些 > 100 μ m 才会产生离子通道功能。通过用画笔将脂质涂在癸烷中，在琼脂糖支撑层上形成双层；然后可以添加第二层琼脂糖以稳定结构并防止蒸发。在玻璃板上构建微流体孔，以便于组装和流体流动的可视化，以及高分辨率显微镜，用于使用荧光脂质和通道的研究。微流体由可逆键合硅橡胶（PDMS）组成，因此整个装置可以清洗和重复使用。设备中的总电噪声足够低，足以实现单通道分辨率。使用自组装离子载体（阿拉甲辛）以及复杂的囊泡相关哺乳动物通道（人甘氨酸受体，GlyR）观察到成功的通道插入。还测试了双层形成的“免提”方法，其中通过旋涂将溶剂中的脂质施加到晶片上，干燥，然后“夹在”氮化物上方和下方的琼脂糖层之间。观察到与双层一致的电特性并获得阿拉甲辛记录，但是该方法与含有哺乳动物通道的囊泡的融合不相容。 (c) 2006 Elsevier B.V. 保留所有权利。
A silicon wafer with eight individually addressable microfabricated orifices was used for ion channel reconstitution and single-channel recording. A spin-on fluoropolymer created an insulating, hydrophobic interface that was more stable than silane. Total capacitance of the membranes was < 10 pF, making it easy to evaluate bilayer formation by capacitance change. Orifices of 50-250 mu m diameter were tested for ease and stability of bilayer formation; only those > 100 mu m resulted in ion channel function. Bilayers were formed over an agarose supporting layer by application of lipid in decane with a paintbrush; a second layer of agarose could then be added to stabilize the structure and prevent evaporation. Microfluidic wells were constructed on glass plates for ease of assembly and visualization of fluid flow, as well as high-resolution microscopy for studies using fluorescent lipids and channels. The microfluidics consisted of reversibly bonded silicone rubber (PDMS), so that the entire device could be washed and reused. Total electrical noise in the device was low enough to permit single-channel resolution. Successful channel insertions were observed with a self-assembling ionophore (alamethicin) as well as a complex, vesicle-associated mammalian channel (human glycine receptor, GlyR). A "hands-free" approach to bilayer formation was also tested, where lipid in solvent was applied to the wafer by spin-coating, dried, and then "sandwiched" between layers of agarose above and below the nitride. Electrical properties consistent with bilayers were observed and alamethicin recordings were obtained, however this method is not compatible with the fusion of vesicles containing mammalian channels. (c) 2006 Elsevier B.V. All rights reserved.