Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

中文翻译：

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使用多组氨酸标记的单价链霉亲和素提高选择性固态纳米孔传感的性能

近年来，固态（SS-）纳米孔传感受到了极大的关注，但它因其固有的选择性缺乏而受到限制。为了解决这个问题，我们之前建立了一种新型 SS-纳米孔测定法，仅当目标生物素化核酸片段与单价链霉亲和素 (MS) 结合时才会产生易位信号，单价链霉亲和素 (MS) 是一种具有单个高亲和力生物素结合域的蛋白质变体。虽然这种方法可以选择性定量不同的核酸生物标志物，但仍需要增强灵敏度以改善低丰度翻译靶标的检测。由于决定测定效果的易位动力学很大程度上取决于成分电荷特征，因此我们在此采用多组氨酸标记的 MS (hMS) 来改变成分的可检测性。我们研究了缓冲液 pH 值、盐浓度和 SS 纳米孔径对替代试剂性能的影响，显着提高了测量灵敏度和选择性，并扩大了适用于测定的设备尺寸范围。我们利用这一改进来检测掺入人血浆中的低至 1 nM 的 miRNA。总体而言，我们的研究结果提高了 SS 纳米孔在分子生物标志物定量分析中更广泛应用的潜力。