Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface-bilayers
Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface-bilayers
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微流体液滴界面双层中人氯细胞内离子通道的功能化
DOI：
10.1016/j.bios.2019.111920
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发表时间：
2020
影响因子：
12.6
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Zhang Y
使用基于活细胞的技术（例如膜片钳电生理学）分析通过人类细胞内氯离子通道的离子通量既费力又耗时。可扩展的微流体系统与基于液滴界面双层（DIB）的自动化协议的集成，其中离子通道被并入，规避了与活细胞测量相关的几个限制，并促进了在可扩展的体外条件下的测试。在这里，我们设计并测试了用于平行形成DIB阵列的新型微流体布局，并开发了用于Cl−通量的基于DIB的荧光分析的第一个例子，从而可以研究人类氯离子胞内离子通道1（CLIC 1）的功能特性。微流体方案依赖于用于液滴配对和DIB形成的被动几何形状。使用重组表达的CLIC 1，我们确定了最佳条件，以最大限度地将蛋白质整合到脂质双层中，并将蛋白质寡聚化到功能性离子通道中。最后，使用相同的Cl−通量测定，相对于α-溶血素评估CLIC 1离子通道功能性进入微流体DIB。
Profiling ion flux through human intracellular chloride ion channels using live-cell based techniques, such as patch-clamp electrophysiology, is laborious and time-consuming. The integration of scalable microfluidic systems with automatable protocols based on droplet-interface-bilayers (DIBs) within which ion channels are incorporated circumvents several limitations associated with live-cell measurements and facilitates testing in controllablein vitroconditions. Here, we have designed and tested novel microfluidic layouts for the formation of arrays of DIBs in parallel and developed the first example of a miniaturised, DIB-based, fluorescence assays for Cl−fluxing, allowing the investigation of the functional properties of the human chloride intracellular ion channel 1 (CLIC1). The microfluidic protocols relied on passive geometries for droplet pairing and DIB formation. Using recombinantly expressed CLIC1, we identified the best conditions to maximise protein integration into a lipid bilayer and the oligomerisation of the protein into functional ion channels. Finally, CLIC1 ion channel functionality was assessed relative to α-Haemolysin into microfluidic DIBs using the same Cl−fluxing assay.