A nanopore counter for highly sensitive evaluation of DNA methylation and its application in in vitro diagnostics

DNA methylation has been considered an essential epigenetic biomarker for diagnosing various diseases, such as cancer. A simple and sensitive way for DNA methylation level detection is necessary. Inspired by the label-free and ultra-high sensitivity of solid-state nanopores to double-stranded DNA (dsDNA), we proposed a nanopore counter for evaluating DNA methylation by integrating a dual-restriction endonuclease digestion strategy coupled with polymerase chain reaction (PCR) amplification. Simultaneous application of BstUI/HhaI endonucleases can ensure the full digestion of the unmethylated target DNA but shows no effect on the methylated ones. Therefore, only the methylated DNA remains intact and can trigger the subsequent PCR reaction, producing a large quantity of fixed-length PCR amplicons, which can be directly detected through glassy nanopores. By simply counting the event rate of the translocation signals, the concentration of methylated DNA can be determined to range from 1 aM to 0.1 nM, with the detection limit as low as 0.61 aM. Moreover, a 0.01% DNA methylation level was successfully distinguished. The strategy of using the nanopore counter for highly sensitive DNA methylation evaluation would be a low-cost but reliable alternative in the analysis of DNA methylation.