Identification and Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine on Random DNA Sequences by a Nanoconfined Electrochemiluminescence Platform

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)are twoof the most abundant epigenetic marks in mammalian genomes, and ithas been proven that these dual epigenetic marks give a more accurateprediction of recurrence and survival in cancer than the individualmark. However, due to the similar structure and low expression of5mC and 5hmC, it is challenging to distinguish and quantify the twomethylation modifications. Herein, we employed the ten-eleven translocationfamily dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification ofthe two marks based on a nanoconfined electrochemiluminescence (ECL)platform combined with the amplification strategy of a recombinasepolymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefitingfrom the TET-mediated conversion strategy, a highly consistent labelingpathway was developed for identifying dual epigenetic marks on randomsequence, which reduced the system error effectively. The ECL platformwas established via preparing a carbonized polymerdot embedded SiO2 nanonetwork (CPDs@SiO2), whichexhibited higher ECL efficiencies and more stable ECL performancecompared to those of the scattered emitters due to the nanoconfinement-enhancedECL effect. The proposed bioanalysis strategy could be employed forthe identification and quantification of 5mC and 5hmC in the rangefrom 100 aM to 100 pM, respectively, which provides a promising toolfor early diagnosis of diseases associated with abnormal methylation.