Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition

Detection of DNA methylation in real human serum is of great importance to push the development of clinical research and early diagnosis of human diseases. Herein, taking advantage of stable pore structure of aerolysin in a harsh environment, we distinguish methylated cytosine from cytosine using aerolysin nanopore in human serum. Since wild-type (WT) aerolysin enables high sensitivity detection of DNA, the subtle difference between methylated cytosine and cytosine could be measured directly without any specific designs. Methylated cytosine induced a population of I/I₀ = 0.53 while cytosine was focused on I/I₀ = 0.56. The dwell time of methylated cytosine (5.3 ± 0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms), which improves the accuracy for the discrimination of the two oligomers. Moreover, the pore-membrane system could remain stable for more than 2 h and achieve the detection of methylated cytosine with zero-background signal in the presence of serum. Additionally, event frequency of methylated cytosine is in correspondence with the relative concentration and facilitate the quantification of methylation.