Electrochemical immunosensor for DNA methyltransferase activity assay based on methyl CpG-binding protein and dual gold nanoparticle conjugate-based signal amplification

Herein, we developed a new electrochemical immunoassay for detecting of DNA methylation, analyzing DNA methyltransferase (MTase) activity and evaluating the inhibition effects of pesticides on DNA MTase. The detection strategy was based on the advantages of methyl binding domain protein (MBD1) on specifically recognizing symmetrical methylated cytosine, immune recognition reaction between antibody and antigen, and antibody functionalized gold nanoparticles (AuNPs) with signal amplification effects. For improving the detection sensitivity, dual signal amplification was employed with the first of anti-his-tag antibody tagged AuNPs and the second of alkaline phosphatase (ALP) labeled IgG tagged AuNPs. The ALP converted 1-naphthyl phosphate into 1-naphthol and the electrochemical signal of 1-naphthyl showed a wider linear relationship of 0.5-500 pM with methylated DNA and 0.03-50 unit/mL with M. SssI concentration. The detection limits were 0.17 pM and 0.01 unit/mL, respectively. In addition, we also investigated the inhibition effects of chlorpyrifos and methyl parathion on MTase activity, which might provide useful information on carcinogenic mechanism of pesticide. This research also verified that the developed method could be applied to screen the inhibitors of DNA MTase and develop new anticancer drugs.