Sampling a biomarker of the human immunodeficiency virus across a synthetic nanopore.
Sampling a biomarker of the human immunodeficiency virus across a synthetic nanopore.
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DOI：
10.1021/nn400125c
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发表时间：
2013-04-23
期刊：
影响因子：
17.1
通讯作者：
Movileanu, Liviu
中科院分区：
文献类型：
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作者：
Niedzwiecki, David J.;Iyer, Raghuvaran;Borer, Philip N.;Movileanu, Liviu
关键词：
纳米生物技术的一个主要目标是比目前更早地设计用于分子生物医学诊断的新方法，并且不使用昂贵的试剂和复杂的设备。在这项工作中，我们展示了使用合成纳米孔和电阻脉冲技术对核衣壳蛋白 7 (NCp7)（HIV-1 病毒的蛋白质生物标志物）进行单分子检测的原理证明。生物传感机制依赖于逆转录病毒RNA基因组包装域中NCp7和茎环3（SL3）适体之间的特异性相互作用。这项研究的一个关键步骤是选择最佳的纳米孔尺寸，以准确、无标记地测定 NCp7 蛋白 - SL3 RNA 适体复合物的解离常数。因此，我们系统地研究了在氮化硅膜中使用两类纳米孔的NCp7蛋白-SL3 RNA适体复合物：(i)小纳米孔，其内径小于6 nm，和(ii)大纳米孔，其内径在7至15 nm范围内。在这里，我们证明，只有使用内径小于或相当于 NCp7-SL3 适体复合物最大横截面尺寸的纳米孔，才能准确测量两个相互作用伙伴之间的解离常数。值得注意的是，这种确定可以在不需要事先纳米孔功能化的情况下完成。此外，利用小型固态纳米孔，我们通过使用 N-乙基马来酰亚胺测试案例证明了检测抑制 NCp7 和 SL3 RNA 之间结合相互作用的候选药物的能力。
One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein - SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein - SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 through 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7-SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of N-ethylmaleimide.
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DOI：
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发表时间：
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