An electrochemical strategy with tetrahedron rolling circle amplification for ultrasensitive detection of DNA methylation

Sensitive and specific detection of DNA methylation in genomic DNA is imperative for rapid epigenetic evaluations. Here, a novel sensitive electrochemical strategy was developed for ultrasensitive detection of DNA methylation in genomic DNA via padlock probe primer generating rolling circle amplification (RCA). Typically, after bisulfite treatment of methylated DNA, the methylation-specific linear padlock is only circularized in the presence of methylated DNA and subsequently serves as a template containing a DNA tetrahedron for RCA. The DNA tetrahedron is utilized as a nanocarrier that can be immobilized on a gold electrode to generate RCA product to load hemin, an iron-containing porphyrin with chlorine, forming the G-quadruplex as a horseradish peroxidase like DNAzyme, which reduces methylene blue (MB) in the presence of H2O2 to yield a distinct current signal. Using the developed DNAzyme with the RCA signal amplification strategy, the DNA biosensor can achieve a detection limit as low as 0.1 fM for the ultrasensitive electrochemical detection of methylated DNA sequence with a detection range from 10(-15) M to 10(-9) M. At the same time, the satisfactory specificity, reproducibility, stability and recovery performances indicated its satisfied potentials for clinical diagnosis. Most importantly, this method can be further applied to analyse other genomic DNA also.