Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.
Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.
复制标题
DOI：
10.1021/nn506606e
复制
发表时间：
2015-02-24
期刊：
影响因子：
17.1
通讯作者：
Chen M
中科院分区：
文献类型：
--
作者：
Fahie M;Chisholm C;Chen M
具有刚性结构的寡聚蛋白质纳米孔已经被工程化用于感测广泛的分析物的目的，包括小分子和生物物种，如蛋白质和DNA。我们选择单体β-桶孔蛋白OmpG作为衍生纳米孔传感器的平台。OmpG装饰有七个灵活的环，当离子电流通过孔时，这些环动态移动以创建独特的门控模式。生物素被化学地拴在这些环中最灵活的一个上。分析物蛋白结合显著改变了环进出孔蛋白的门控特性。具有结合靶的孔的门控特征对分子身份非常敏感-甚至提供区分抗体混合物内的同源物的能力。对每种分析物总共分析了5个门控参数，以创建每种生物素结合蛋白的独特指纹图谱。我们利用门控噪声作为分子标识符可以允许更复杂的传感器设计，而OmpG的单体结构大大简化了纳米孔的生产。
Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop’s movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity – even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design while OmpG’s monomeric structure greatly simplifies nanopore production.