Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique

Abstract

We report on an electrochemical method for the determination of the activity of the enzyme methyltransferase (MTase). The methyl-binding domain-1 protein was applied to recognize symmetrically methylated cytosine in CpG (-C-phosphate-G-) islands of ds-DNA which then specifically bind to anti-His tag antibody. Hyperbranched rolling circle amplification (RCA) was used to improve sensitivity. When the dsDNA was treated with M.Sss I methyltransferase, the sequence 5′-CCGG-3′ was methylated and recognized by the methyl binding protein. In turn, the anti-His tag, biotinylated IgG, streptavidin and biotinylated oligonucleotide were captured successively on the surface of an electrode. Subsequently, the RCA reaction was initiated and streptavidin-labeled alkaline phosphatase immobilized on the surface of the electrode. ALP was able to catalyze the hydrolysis of 1-naphthyl phosphate to form 1-naphthol at pH 9.8. The oxidation peak current of 1-naphthol was used to monitor the methylation process. The response obtained by differential pulse voltammetry was linearly related to the concentration of M.Sss I MTase in the range from 0.1 to 40 unit mL−1, and the detection limit was 0.03 unit mL−1 (at an SNR of 3). The inhibitory action of paclitaxel on the activity of M.Sss I MTase also was investigated.