Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695-7905, United States.
Anal Chem. 2018 Jan 2;90(1):690-695. doi: 10.1021/acs.analchem.7b03848. Epub 2017 Dec 11.
Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.
核酸（DNA 和 RNA）为各种病原体提供了重要的指纹信息，具有重要的诊断价值；然而，目前迫切需要改进方法，以便能够以简单的即时检测形式，实现具有高灵敏度和特异性的核酸的快速检测。在这里，我们提出了一种系统，该系统利用一系列引发链置换反应的链置换反应，旨在将给定数量的 RNA 分子（即分析物）转换为大量信号分子，而无需任何洗涤步骤或热循环。用于生成信号的荧光探针被设计用来消耗催化反应的产物，以推动平衡并增强检测的折叠扩增，从而提高灵敏度和反应速度。我们还对 toehold-assisted 反应系统进行了建模，以便更好地了解其性能和能力，并用两种具有诊断重要性的分析物（即流感病毒 RNA 和 micro RNA (miR-31)）来验证该方法的成功。我们还表明，扩增信号允许使用紧凑且具有成本效益的基于智能手机的荧光读取器，这是基于核酸的即时检测诊断系统的重要要求。
