Electronics and Computer Science, ‡Centre for Biological Sciences, and §Institute for Life Sciences, University of Southampton , Southampton SO17 1BJ, United Kingdom.
Anal Chem. 2017 Sep 5;89(17):8822-8829. doi: 10.1021/acs.analchem.7b01246. Epub 2017 Aug 11.
In resistive pulse sensing of microRNA biomarkers, selectivity is achieved with polynucleotide-extended DNA probes, with the unzipping of a miRNA-DNA duplex in the nanopore recorded as a resistive current pulse. As the assay sensitivity is determined by the pulse frequency, we investigated the effect of cis/trans electrolyte concentration gradients applied over α-hemolysin nanopores. KCl gradients were found to exponentially increase the pulse frequency, while reducing the preference for 3'-first pore entry of the duplex and accelerating duplex unzipping, all manifestations of an enhanced electrophoretic force. Unlike silicon nitride pores, a counteracting contribution from electro-osmotic flow along the pore wall was not apparent. Significantly, a gradient of 0.5/4 M KCl increased the pulse frequency ∼60-fold with respect to symmetrical 1 M KCl, while the duplex dwell time in the nanopore remained acceptable for pulse detection and could be extended by LiCl addition. Steeper gradients caused lipid bilayer destabilization and pore instability, limiting the total number of recorded pulses. The 8-fold KCl gradient enabled a linear relationship between pulse frequency and miRNA concentration for the range of 0.1-100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for subnanomolar miRNA quantification with bilayer-embedded porins.
在 miRNA 生物标志物的电阻脉冲感应中，通过多核苷酸扩展的 DNA 探针实现了选择性，其中 miRNA-DNA 双链体在纳米孔中的解链被记录为电阻脉冲。由于测定灵敏度取决于脉冲频率，我们研究了施加在α-溶血素纳米孔上的顺式/反式电解质浓度梯度的影响。发现 KCl 梯度使脉冲频率呈指数级增加，同时降低了双链体优先从 3'端进入孔的偏好性，并加速了双链体的解链，所有这些都是增强电泳力的表现。与氮化硅孔不同，沿孔壁的电渗流的反作用贡献不明显。值得注意的是，与对称的 1 M KCl 相比，0.5/4 M KCl 的梯度使脉冲频率提高了约 60 倍，而双链体在纳米孔中的停留时间仍然适合脉冲检测，并且可以通过添加 LiCl 来延长。更陡的梯度导致脂质双层失稳和孔不稳定，限制了记录的脉冲总数。8 倍的 KCl 梯度使脉冲频率与 miRNA 浓度在 0.1-100 nM 范围内呈线性关系。这项工作突出了生物和固态纳米孔感应之间的差异，并为使用双层嵌入的 porin 进行亚纳摩尔 miRNA 定量提供了策略。
