蛋白质-核酸相互作用不仅对遗传调控和细胞代谢是基本的，还是人工生物传感器的分子基础。但是，这种相互作用通常是弱的和动态的，因此其特异性和敏感性的定量检测具有挑战性。我们采用引物交换反应（PER）扩增的蛋白质-核酸相互作用，设计一个通用而超灵敏的电化学传感器，用于血液中微小RNA（miRNA）的定量。该PER-miR传感器利用S9.6抗体和miRNA/DNA杂交体之间的特异性识别，与PER衍生的多酶催化相结合，实现超灵敏的miRNA检测。表面结合动力学分析表明，miRNA/DNA杂交体与电极附着的S9.6抗体之间存在合理的Kd（8.9纳摩尔）。基于这种有利的亲和力，可编程的PER扩增使传感器能够以高达90.5 aM的敏感度检测目标miRNA，比例常规设计中没有PER时高出三个数量级，并具有单碱基分辨率的特异性。此外，PER-miR传感器可同时检测多个miRNA，在四种细胞系的裂解物中测量目标miRNA，并通过直接分析人类血样（n = 40）区分肿瘤患者和健康个体。这些优点使传感器成为一种有前途的工具，可以实现生物分子相互作用的定量感应和精密诊断。版权所有©2023 Elsevier B.V.

Protein-nucleic acid interactions are not only fundamental to genetic regulation and cellular metabolism, but molecular basis to artificial biosensors. However, such interactions are generally weak and dynamic, making their specific and sensitive quantitative detection challenging. By using primer exchange reaction (PER)-amplified protein-nucleic acid interactions, we here design a universal and ultrasensitive electrochemical sensor to quantify microRNAs (miRNAs) in blood. This PER-miR sensor leverages specific recognition between S9.6 antibodies and miRNA/DNA hybrids to couple with PER-derived multi-enzyme catalysis for ultrasensitive miRNA detection. Surface binding kinetic analysis shows a rational Kd (8.9 nM) between the miRNA/DNA heteroduplex and electrode-attached S9.6 antibody. Based on such a favorable affinity, the programmable PER amplification enables the sensor to detect target miRNAs with sensitivity up to 90.5 aM, three orders of magnitude higher than that without PER in routine design, and with specificity of single-base resolution. Furthermore, the PER-miR sensor allows detecting multiple miRNAs in parallel, measuring target miRNA in lysates across four types of cell lines, and differentiating tumor patients from healthy individuals by directly analyzing the human blood samples (n = 40). These advantages make the sensor a promising tool to enable quantitative sensing of biomolecular interactions and precision diagnostics.Copyright © 2023 Elsevier B.V. All rights reserved.