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

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

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

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

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

Authors

Miyuki Tabata¹, Bo Yao², Ayaka Seichi³, Koji Suzuki³, Yuji Miyahara⁴

Affiliations

¹ Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan.
² Department of Chemistry, Zhejiang University, Hangzhou, China.
³ Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University, Yokohama, Japan.
⁴ Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan. miyahara.bsr@tmd.ac.jp.

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 (NH₃)₆]³⁺ 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 (Et⁺ISE) for real-time monitoring of isothermal DNA amplification by primer-generation RCA (PG-RCA). The Et⁺ISE 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.

Keywords: Biosensor; Chronocoulometry; Ion selective electrode; Isothermal nucleic acid amplification; PCR; microRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biosensing Techniques* / instrumentation
Biosensing Techniques* / methods
DNA / analysis
DNA / genetics
Electrochemical Techniques* / instrumentation
Electrochemical Techniques* / methods
Electrodes
Gold
MicroRNAs
Nucleic Acid Amplification Techniques* / instrumentation
Nucleic Acid Amplification Techniques* / methods
Polymerase Chain Reaction
Temperature

Substances

MicroRNAs
Gold
DNA