Highly Sensitive and Diversified Electrochemiluminescence DNA Methylation Biosensing Platform Based on Self-Assembly of Nanotags

This work proposes a highly sensitive, simple, and reliable electrochemiluminescence (ECL) DNA methylation biosensing platform by employing DNA-functionalized magnetic beads (DNA-MBs) for target capture and nanotag self-assembly aggregation for signal amplification. The target methylated DNA was first captured on DNA-MBs through base pairing recognition, and then its methylation sites were recognized by antibody-5mC (Ab-5mC). Afterward, a pair of antibodies functionalized [Ru(byp)3]2+-doped silica nanoparticles (Ab2-Ru@SiO2 and Ab3-Ru@SiO2) was layer-by-layer assembled on Ab-5mC for amplified signal transduction. The sensing beads could be transferred to screen-printed carbon electrodes (SPCEs) for ECL curve detection via photomultiplier tube or to gold-coated indium tin oxide (Au/ITO) arrays for high-throughput imaging detection. As the nanotag assembly layers increased from 1 to 3, the detection sensitivities of SPCE-based curve detection and Au/ITO-based imaging detection were enhanced 7-fold and 3-fold, achieving detection limits down to 0.8 pM and 0.9 fM, respectively. The nanotags showed good stability, with storage times of 300 days for Ru@SiO2 and 60 days for Ab-Ru@SiO2, respectively. This method is universal and could be applied to detect different methylated DNAs by using their corresponding DNA-MBs. The proposed ECL biosensing platform possessed advantages of high sensitivity, good diversity, and practicality, showing potential for high-throughput DNA methylation detection in clinical diagnosis.