结合等温扩增技术用于核酸定量检测的电化学生物传感器

Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids.

作者信息

Tabata Miyuki, Yao Bo, Seichi Ayaka, Suzuki Koji, Miyahara Yuji

机构信息

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan.

Department of Chemistry, Zhejiang University, Hangzhou, China.

出版信息

Methods Mol Biol. 2017;1572:135-151. doi: 10.1007/978-1-4939-6911-1_10.

DOI:10.1007/978-1-4939-6911-1_10

PMID:28299686

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7122880/

Abstract

In recent years, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). The integration of isothermal amplification with electrical or electrochemical devices has enabled high-throughput nucleic acid-based assays with high sensitivity. We performed solid-phase rolling circle amplification (RCA) on the surface of a Au electrode, and detected RCA products in situ using chronocoulometry (CC) with [Ru (NH)] as the signaling molecule. Detection sensitivity for DNA and a microRNA (miR-143) was 100 fM and 1 pM, respectively. Furthermore, we conducted potentiometric DNA detection using an ethidium ion (Et)-selective electrode (EtISE) for real-time monitoring of isothermal DNA amplification by primer-generation RCA (PG-RCA). The EtISE potential enabled real-time monitoring of the PG-RCA reaction in the range of 10 nM-1 μM of initial target DNA. Devices based on these electrochemical techniques represent a new strategy for replacing conventional PCR for on-site detection of nucleic acids of viruses or microorganisms.

摘要

近年来，已开发出各种等温扩增技术作为聚合酶链反应（PCR）的替代方法。等温扩增与电学或电化学装置的结合，实现了具有高灵敏度的高通量基于核酸的检测。我们在金电极表面进行了固相滚环扩增（RCA），并使用[Ru(NH)]作为信号分子，通过计时库仑法（CC）原位检测RCA产物。对DNA和微小RNA（miR-143）的检测灵敏度分别为100 fM和1 pM。此外，我们使用乙锭离子（Et）选择性电极（EtISE）进行电位DNA检测，以实时监测引物生成RCA（PG-RCA）介导的等温DNA扩增。EtISE电位能够在10 nM至1 μM的初始靶DNA范围内实时监测PG-RCA反应。基于这些电化学技术的装置代表了一种替代传统PCR用于现场检测病毒或微生物核酸的新策略。