TdT-assisted DNA elongation amplification enables highly sensitive label-free electrochemical detection of DNA methyltransferase.

Aberrant DNA methylation has emerged as a critical epigenetic biomarker closely associated with bacterial pathogenicity and human tumorigenesis. In this study, a highly sensitive and label-free electrochemical biosensor has been developed for the detection of Dam MTase activity. 3'-aminated hairpin DNA probes (HP1) containing Dam MTase-specific recognition sites are immobilized onto the surface of a gold electrode. In the presence of Dam MTase, HP1 is methylated and subsequently cleaved by DpnI, thereby exposing free 3'-hydroxyl groups. These hydroxyl groups serve as primers and are extended by terminal deoxynucleotidyl transferase (TdT) to generate long poly-thymine (polyT) tails. As an electrochemical redox indicator with numerous positive charges, ruthenium hexaammine trichloride (RuHex) can be captured by negatively charged DNA extension products. Accordingly, a remarkably enhanced electrochemical signal is produced, and the signal response is proportional to methyltransferase activity. This biosensor exhibits a good linear relationship in the range of 0.1 U/mL to 20 U/mL, and the limit of detection (LOD) is as low as 0.57 U/mL. Furthermore, its application value has been verified through the successful screening of existing MTase inhibitors (5-fluorouracil and gentamicin), indicating its potential application prospects in clinical diagnosis and antibacterial drug screening.