Electrogenerated chemiluminescence biosensing method for the detection of DNA demethylase activity: Combining MoS2 nanocomposite with DNA supersandwich

We report an ultrasensitive electrogenerated chemiluminescence (ECL) biosening method for the detection of DNA demethylase activity based on the site-specific cleavage of the HpaII endonuclease in conjunction with signal amplification by a Thionin-MoS2 (Th-MoS2) nanocomposite and supersandwich DNA. The Thionin (Th) served as an effective mediator for the immobilization of the capture probe onto the surface of MoS2, after which the long concatamers containing the methylated site 5′-CmCGG-3′ were hybridized with the capture probe, creating a supersandwich DNA structure. Ru(phen)32+ ECL reagents can be intercalated into the grooves of the formed supersandwich DNA structure with high affinity leading to enhanced signaling. At the biosensing electrode (Ru(phen)32+/supersandwich DNA/Th-MoS2/glassy carbon electrode) after demethylation with demethylase, the demethylated supersandwich DNA could be specifically cleaved using HpaII, resulting in a decreased ECL signal from Ru(phen)32+. An extremely low detection limit of 0.16 pg mL−1 was achieved for the detection of DNA demethylase activity. This work demonstrates that the combination of dual signal amplification using a MoS2-thionin nanocomposite and supersandwich with an inherently sensitive ECL method is a promising tactic for the detection of DNA demethylation and assay of the activity of demethylase.