Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap’s ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

随着时间的推移，已经采取了许多策略来捕获和监测单个蛋白质，以检测这些基本纳米机器的分子机制。具有纳米孔的单蛋白传感特别有吸引力，因为它允许基于离子电流的无标记高带宽检测。在这里，我们展示了纳米孔电渗阱 (NEOtrap)，它允许以亚毫秒时间分辨率捕获和观察单个蛋白质数小时。NEOtrap 是通过将 DNA 折纸球连接到钝化的固态纳米孔上而形成的，该纳米孔密封了用户定义尺寸的纳米腔，并产生了一种电渗流，可以捕获附近的粒子，而不管它们的电荷如何。我们展示了 NEOtrap 根据大小和形状敏感地区分蛋白质的能力，并区分核苷酸依赖性蛋白质构象，如伴侣蛋白 Hsp90 所示。鉴于在广泛的生物相关时间范围内进行无标记单蛋白检测的实验简单性和能力，NEOtrap 开辟了研究蛋白质功能分子动力学的新途径。