Stable Converter-Powered Cascade Amplification of DNAzyme and Cas12a for Electrochemiluminescent Detection of DNA Adenine Methyltransferase

Although CRISPR-Cas12a has become a powerful analytical tool owing to its collateral cleavage activity and programmability, its application for accurate and sensitive detection of non-nucleic acid targets such as DNA adenine methyltransferase (Dam) remains hindered by the lack of stable and sensitive signal conversion strategies for Cas12a activator output. Here, we developed a stable dual-sites dumbbell-shaped converter (DDC) for reliable signal conversion coupled with a smart allosteric spherical DNAzyme (SASD) and Cas12a for cascade amplification, enabling highly accurate and sensitive electrochemiluminescent (ECL) detection of Dam activity. Specifically, the nuclease-resistant, closed-loop structure of DDC is first methylated by Dam and subsequently cleaved by DpnI, converting Dam activity into a mimic target (MT). This MT then hybridizes with SASD, triggering an allosteric transition that reconstitutes a DNAzyme. The activated DNAzyme not only releases a Cas12a activator but also regenerates the MT, initiating a cyclic reaction for robust signal amplification. Subsequently, activated Cas12a restores the ECL signal of nitrogen-doped carbon dots (N-CDs) by cleaving the ferrocene-labeled quenching probes on the ECL electrode, enabling quantitative analysis of Dam activity. The proposed biosensor displays a wide linear range from 1.0 × 10 –6 to 1.0 U mL –1 and a detection limit down to 8.61 × 10 –7 U mL –1, offering a powerful tool for early and low-abundance DNA methylation detection.