Electrogenerated chemiluminescence biosensing method for the detection of DNA methylation and assay of the methyltransferase activity

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the CpG methyltransferase (M. Sssl) activity was developed on basis of enzyme-linkage reactions and ruthenium complex served as an ECL tag. The ECL biosensing electrode was fabricated by self-assembling 5'-thiol modified 32-mer single-strand DNA (ss-DNA)-tagged with ruthenium bis (2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine on the surface of a gold electrode, and then hybridized with complementary ss-DNA to form duplex DNA (ds-DNA). When M. Sssl and S-adenosylmethionine were introduced, all cytosine residues within 5'-CG-3' of ds-DNA on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by HpaII endonuclease, the un-methylated cytosines were cleaved, thus led to decrease ECL signal. The ECL intensity of ECL biosensing electrode is related to the methylation level and M. Sssl activity in a fixed concentration HpaII endonuclease. The increased ECL intensity was direct proportion to M. Sssl activity in the range from 0.05 to 100 U/mL with a detection limit of 0.02 U/mL. This work demonstrates that the combination of the enzyme-linkage reactions with a highly sensitive ECL technique is a great promising approach for the detection of DNA methylation level, assay of the activity of MTase, and evaluation of the capability of inhibitors for the methyltransferase. (C) 2012 Elsevier B.V. All rights reserved.