Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification

In this work, we fabricated a novel electrochemical immunosensor for detection of DNA methylation , analysis of DNA MTase activity and screening of MTase inhibitor. The immunosensor was on the basis of methyl binding domain protein of MeCP2 as DNA CpG methylation recognization unit, anti-His tag antibody as “immuno-bridge” and horseradish peroxidase labeled immuneglobulin G functionalized gold nanoparticles (AuNPs–IgG–HRP) as signal amplification unit. In the presence of M. Sss I MTase, the symmetrical sequence of 5′-CCGG-3′ was methylated and then recognized by MeCP2 protein. By the immunoreactions, anti-His tag antibody and AuNPs–IgG–HRP was captured on the electrode surface successively. Under the catalysis effect of HRP towards hydroquinone oxidized by H 2 O 2 , the electrochemical reduction signal of benzoquinone was used to analyze M. Sss I MTase activity. The electrochemical reduction signal demonstrated a wide linear relationship with M. Sss I concentration ranging from 0.05   unit/mL to 90   unit/mL, achieving a detection limit of 0.017   unit/mL (S/N=3). The most important advantages of this method were its high sensitivity and good selectivity , which enabled the detection of even one-base mismatched sequence. In addition, we also verified that the developed method could be applied for screening the inhibitors of DNA MTase and for developing new anticancer drugs.