Abstract
In this study, a novel electrochemical biosensor was constructed for ultrasensitive and locus-specific detection of N(6)-Methyladenine (m(6)A) in DNA using double-hindered replication and nucleic acid-coated methylene blue (MB)@Zr-MOF. Based on the combination of m(6)A-impeded replication and Ag(I)-mediated mismatch replication, this mode could effectively stop the extension of the strand once DNA polymerase encountered m(6)A site, which specifically distinguish the m(6)A site from natural A site in DNA. Also, Zr-MOF with high porosity and negative surface potential features was carefully chose to load cationic MB, resulting a stable and robust MB@Zr-MOF electrochemical tag. As a result, the developed biosensor exhibited a wide linear range from 1 fM to 1 nM with detection limit down to 0.89 fM. Profiting from the high sensitivity and selectivity, the biosensing strategy revealed good applicability, which had been demonstrated by quantitating m(6)A DNA at specific site in biological matrix. Thus, the biosensor provides a promising platform for locus-specific m(6)A DNA analysis.