基于MspA蛋白质纳米孔传感器的主客体化学研究

A Single-Molecule Mycobacterium Smegmatis Porin A Protein Nanopore Sensor for Host-guest Chemistry

以大肠杆菌原核系统表达的方法制备了MspA蛋白质纳米孔,并利用表面活性剂高温提取获得高活性MspA蛋白纳米孔。实验结果表明,0.5%的提取剂90℃提取30 min为最佳提取条件。利用制备的MspA纳米孔在单分子水平上研究了其与多种环糊精的相互作用,结果表明,在众多环糊精分子中,全-6-氨基-β-环糊精(am_7-β-CD)与MspA蛋白纳米孔相互作用较强且阻塞平台较一致,其滞留时间随电压增大而逐渐减小,但阻塞电流受电压影响不大。筛选使用am_7-β-CD作为适配体非共价键装配MspA纳米孔,在单分子水平上研究主客体化学反应。加入am_7-β-CD后,当盐酸金刚烷胺小分子浓度为50 nmol/L时,即出现二级阻塞平台,其滞留时间随电压增大而逐渐减小。这种新型蛋白质纳米孔单分子传感器可用于有机小分子的检测识别,极大拓宽了MspA纳米孔分析技术的的应用范围。

A mycobacterium smegmatis porin A （ MspA） protein nanopore was prepared by E. coil prokaryotic expression system. The extraction experiments of MspA nanopore using surfactants showed that an extraction agent concentration of 0 . 5%, a temperature of 90℃ and an extraction time of 30 min were the optimum extraction conditions. The interactions of MspA nanopore with a variety of cyclodextrins were examined at single-molecule level. The interaction between MspA and Per-6-amino-β-cyclodextrin （ am7-β-CD ） was stronger and consistent with blocking platform. Its retention time in the MspA nanopore decreased with the increasing voltage, whereas the blocking current was almost not affected by voltage. So the am7-β-CD was selected as a non-covalent adapter and lodged within single MspA nanopore to study the single-molecule host-guest chemistry. After adding am7-β-CD to form a first blocked platform, a small concentration of 50 μmol/L for the amantadine hydrochloride showed a second blocked platform and its residence time decreased with the increasing voltage. This new protein nanopore sensor can be used as a single-molecule detector and identifier for small organic molecules, which will greatly broaden the application range of MspA nanopore.