A bisulfite treatment and PCR-free global DNA methylation detection method using electrochemical enzymatic signal engagement

In this paper we report on a bisulfite treatment and PCR amplification-free method for sensitive and selective quantifying of global DNA methylation. Our method utilizes a three-step strategy that involves (i) initial isolation and denaturation of global DNA using the standard isolation protocol and direct adsorption onto a bare gold electrode via gold-DNA affinity interaction, (ii) selective interrogation of methylation sites in adsorbed DNA via methylation-specific 5mC antibody, and (iii) subsequent signal enhancement using an electrochemical-enzymatic redox cycling reaction. In the redox cycling reaction, glucose oxidase (GO _x ) is used as an enzyme label, glucose as a substrate and ruthenium complex as a redox mediator. We initially investigated the enzymatic properties of GO _x by varying glucose and ruthenium concentration to delineate the redox cyclic mechanism of our assay. Because of the fast electron transfer by ruthenium (Ru) complex and intrinsic signal amplification from GO _x label, this method could detect as low as 5% methylation level in 50 ng of total DNA input. Moreover, the use of methylation-specific 5mC antibody conjugated GO _x makes this assay relatively highly selective for DNA methylation analysis. The data obtained from the electrochemical response for different levels of methylation showed excellent interassay reproducibility of RSD (relative standard deviation) < 5% for n = 3. We believe that this inexpensive, rapid, and sensitive assay will find high relevance as an alternative method for DNA methylation analysis both in research and clinical platforms.