Electrochemiluminescence biosensing of gene-specific methylation through magnetic capture and functional [Ru(byp)3]2+-doped silica

This work proposed an electrochemiluminescence (ECL) biosensing strategy for sensitive detection of 5-methylcytosine (5mC) DNA methylation. The signaling probe was prepared by encapsulating [Ru(byp)3]2+ in silica nanoparticle (Ru@SiO2) to label the secondary antibody (Ab2), which provided 3.1 × 103 [Ru(byp)3]2+ for signal amplification. Biotinylated DNA was immobilized on streptavidin-modified magnetic microbeads (S-MBs) to capture sequentially methylated ssDNA, 5mC antibody and Ru@SiO2 labeled Ab2. S-MBs enable the rapid separation of the capture product from other species in complex samples. With tripropylamine as coreactant, the concentration of methylated DNA was read out with ECL signal of Ru@SiO2. The developed biosensor showed the dynamic range of 0.5–50,000 pM and a limit of detection of 0.1 pM for the methylated DNA sequence of the promoter region of RASSF1A. This proposed method possessed the advantages of high sensitivity, convenient operation, easy automation and excellent versatility, and enormous potential for the detection of important gene-specific methylation without nucleic acid amplification and sample pretreatment.