Dual-mode ratiometric biosensor based on self-feedback amplification strategy for the sensitive evaluation of methylated DNA

A novel ratiometric biosensor was developed for the sensitive and reliable monitoring of DNA methylation levels based on the “turn-on” electrochemiluminescence (ECL) signal and the “turn-off” electrochemical (EC) response. It integrated three effective signal amplification techniques: entropy-driven strand-displacement reactions, DNA walking machine, and target analogue-guided self-feedback system. EC probes (S-Fc) and signal amplification units (D:L:B duplex) were anchored on Ru@COF-LZU1/GCE. With the help of fuel DNA (F), the methylated DNA interacted with D:L:B duplex in an entropy-driven manner, which activated the target recycling and produced abundant output D (DNA walking strand). Then, the DNAzyme in the releasing D began to cleave S-Fc with the addition of Mn 2+ . The cleaved target analogues initiated the self-feedback circuit via the same entropy-driven cascade amplification technique, which resulted in the significant suppression of EC signal and the recovery of ECL signal. The ratiometric biosensor displayed exceptional selectivity for discriminating DNA with different methylation sites. It was envisaged that this self-calibration biosensor would be an appealing tool for site-specific analysis of methylation in clinical diagnosis. • A dual-mode ratiometric biosensor was developed for DNA methylation detection. • A spatial-confinement Ru@COF-LZU1 effectively improved ECL emission of Ru(bpy) 3 2+ . • Multi-integrated DNA amplification strategies led to a low detection limit of 18 aM. • The opposite trend of EC and ECL signals achieved an enhanced ECL/EC ratio. • Dual-mode output strategy offered self-verification to ensure accuracy and reliability.