Highly sensitive ratiometric electrochemical DNA biosensor based on homogeneous exonuclease III-assisted target recycling amplification and one-step triggered dual-signal output

Abstract

A unique and versatile ratiometric electrochemical DNA biosensor was designed for the first time by integration of homogeneous exonuclease III-assisted target recycling amplification and one-step triggered dual-signal output. The presence of the target DNA would hybridize with the specifically designed ferrocene-labeled hairpin probe (Fc-P1) and initiate homogeneous exonuclease III-assisted target recycling amplification, which led to the decrease of the local concentration of Fc-P1. The target DNA dependent amount of remaining Fc-P1 could be conveniently monitored by hybridize the remaining Fc-P1 with the methylene blue-labeled hairpin probe ((MB-PP1) on the sensor electrode to trigger the MB tags away from while the Fc tags close to the sensing interface. Therefore, such recognition event can be translated into the homogeneous amplification coupled with one-step triggered dual-signal ratiometric electrochemical readout. The proposed biosensor achieves a detection limit of 12.8 fM within a linear range of 0.02 pM to 2 nM for target DNA species related to oral cancer over expressed 1, along with a favorable specificity. More importantly, the biosensor had been applied in detection of target DNA in artificial saliva sample and provided significant potential application in clinical analysis. This strategy possessed good capacity to integrate the nuclease amplification techniques with ratiometric output modes, and would open new opportunities for sensitive detection of other biorecognition events.