RESUMEN
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.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Técnicas de Amplificación de Ácido Nucleico , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , ADN/análisis , ADN/genética , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Oro , MicroARNs , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa , TemperaturaRESUMEN
An electrochemical detection system for an isothermal DNA amplification method using an ion-selective electrode (ISE) was developed as a low-cost, simple and real-time monitoring system. The system is based on potentiometry using an ethidium ion (Et(+)) selective electrode that relies on monitoring DNA amplification by measuring potential changes in the reaction solution containing ethidium bromide (EtBr) as an intercalator to DNA. With progressing primer generation-rolling circle amplification (PG-RCA) under isothermal condition at 37°C, EtBr is bound to the newly formed DNA, resulting in a lowered free EtBr concentration in the sample solution. In this case, the Et(+) ISE potential allows real-time monitoring of the PG-RCA reaction in the range of 10 nM - 1 µM initial target DNA.