Ratiometric Electrochemical Biosensing of Methyltransferase Activity

Abstract

In this work, a novel ratiometric electrochemical readout platform was proposed and developed for the fast and flexible analysis of M.SssI methyltransferase (MTase) activity. In this platform, two hairpin DNAs (H1 and H2) were designed. H1 contains the palindromic sequence of 5′-CCGG-3′ in its stem which could be methylated and hybridize with H2 labeled by methylene blue (MB) as one of the signal reporters on a gold electrode (GE) in the presence of M.SssI MTase. Additionally, a specific immunoreaction was introduced by conjugating an anti-5-methylcytosine antibody, a DNA CpG methylation recognition unit, with 1,3-ferrocenedicarboxylic acid (Fc) as the second signal reporter. The results showed that when the Fc tag approaches, the MB tag was far from the gold electrode surface, resulting in a decrease in the oxidation peak current of MB (IMB) and an increase in the oxidation peak current of Fc (IFc). The ratiometric electrochemical method above shows the linear range of detection was 0 U/mL 40 U/mL with a detection limit of 0.083 U/mL (the mean signal of blank measures þ3s).