Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and the Zernike Institute for Advanced Materials, University of Groningen , Nijenborgh 7, Groningen, The Netherlands.
ACS Nano. 2014 Apr 22;8(4):3380-92. doi: 10.1021/nn405884a. Epub 2014 Mar 11.
Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.
难以构建脆弱的脂质双层以及难以确定反应物质位于膜的哪一侧。在这里，我们提出了一种在微结构的 Si/SiO2 芯片上生成具有生理相关脂质组成的悬浮脂质双层的方法，该方法可用于高通量筛选膜转运和病毒膜融合。同时观察数百个单分子通道可提供统计信息，揭示细菌毒素α-溶血素（αHL）的孔组装和电导率的群体异质性。在单通道水平上研究了脂质组成和离子强度对 αHL 孔形成的影响，从而解析了孔组装途径的特征。特定抗体抑制了孔的形成，证明了该平台在筛选细菌毒素和细胞穿透剂方面的适用性。此外，使用专门的芯片结构可以直接观察到 H3N2 流感病毒与悬浮脂质双层的融合。所提出的微孔阵列与使用空气物镜从下方进行荧光读出兼容，从而允许在多孔板格式中进行类似板读数器的高通量膜转运筛选。
