Khan Muhammad S, Dosoky Noura S, Berdiev Bakhrom K, Williams John D
Electrical and Computer Engineering Department, University of Alabama in Huntsville, Huntsville, AL, 35899, USA.
Biotechnology Science and Engineering Program, University of Alabama in Huntsville, Huntsville, AL, 35899, USA.
Eur Biophys J. 2016 Dec;45(8):843-852. doi: 10.1007/s00249-016-1156-8. Epub 2016 Aug 1.
Black lipid membranes (BLMs) have been used for detecting single-channel activities of pore-forming peptides and ion channels. However, the short lifetimes and poor mechanical stability of suspended bilayers limit their applications in high throughput electrophysiological experiments. In this work, we present a synthetic solid-state nanopore functionalized with BLM fused with channel protein. A nanopore with diameter of ~180 nm was electrochemically fabricated in a thin silicon membrane. Folding and painting techniques were demonstrated for production of stable suspended BLMs followed by incorporation of transmembrane protein, ENaC. Membrane formation was confirmed by employing electrochemical impedance spectroscopy (EIS) in the frequency regime of 10-10 Hz. Results show that electrochemically fabricated solid state nanopore support resulted in excellent membrane stability, with >1 GΩ of up to 72 and 41 h for painting and folding techniques, respectively. After fusion of ENaC channel protein, the BLM exhibits the stability of ~5 h. We anticipate that such a solid-state nanopore with diameter in the range of 150-200 nm and thickness <1 µm could be a potential platform to enhance the throughput of ion-channel characterization using BLMs.
黑色脂质膜（BLMs）已被用于检测成孔肽和离子通道的单通道活性。然而，悬浮双层膜的寿命短且机械稳定性差，限制了它们在高通量电生理实验中的应用。在这项工作中，我们展示了一种用与通道蛋白融合的BLM功能化的合成固态纳米孔。在薄硅膜中通过电化学方法制备了直径约为180 nm的纳米孔。展示了折叠和涂覆技术用于生产稳定的悬浮BLMs，随后掺入跨膜蛋白ENaC。通过在10 - 10 Hz频率范围内采用电化学阻抗谱（EIS）来确认膜的形成。结果表明，电化学制备的固态纳米孔支撑体具有出色的膜稳定性，对于涂覆和折叠技术，分别高达72小时和41小时，电阻大于1 GΩ。融合ENaC通道蛋白后，BLM表现出约5小时的稳定性。我们预计，这种直径在150 - 200 nm范围内且厚度小于1 µm的固态纳米孔可能成为一个潜在的平台，以提高使用BLMs进行离子通道表征的通量。
