Cascade Dual-Signal Enhancement by Integrating Exonuclease III-Assisted Target-Recycling and Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of DNA

Based on a cascade dual-signal enhancement strategy by integrating exonuclease III (Exo III)-assisted target-recycling (EATR) with rolling circle amplification (RCA), a highly sensitive electrochemical biosensor was developed for the detection of target DNA (tDNA), a kind of Alzheimer's disease biomarkers. When tDNA was hybridized with hairpin DNA (hDNA) previously modified in the electrode surface by Au-SH bond, the subsequent introduction of Exo III triggered EATR through cleaving hDNA, releasing tDNA and further hybridizing with other hDNA. After repeated recycling, the remained DNA (rDNA) fragments of hDNA as initiators of RCA were extended, generating numerous copies of G-rich base sequence. The presence of hemin resulted in the in situ assembly of multiple hemin/G-quarduplex structures in the resultant electrode surface, which were acted as both label-free redox probes and mimicking peroxidase catalysts for the electrochemical signal readout and amplification, producing significantly enhanced electrochemical response dependent on tDNA. Thus, the developed biosensor exhibited ultrahigh sensitivity with a limit of detection of 3.3 aM. By combining EATR with subsequent RCA, the cascade dual-signal amplification strategy would be promising and potential in biological analysis and clinical diagnosis.