DNA methylation is used to dynamically reprogram cells in the course of early embryonic development in
mammals. 5-Hydroxymethylcytosine in DNA (5-hmC-DNA) plays essential roles in the demethylation processes. 5-
Methylcytosine in DNA (5-mC-DNA) is oxidized to 5-hmC-DNA by 10−11 translocation proteins, which are relatively high abundance in embryonic stem cells and neurons. A new method was developed herein to quantify 5-hmC-DNA based on selective electrogenerated chemiluminescence (ECL) labeling with the specific oxidation of 5-hmC to 5-fC by KRuO4. A thiolated capture probe (ssDNA, 35-mer) for the target DNA containing 5-hmC was self-assembled on a gold surface. The 5-hmC in the target DNA was selectively transformed to 5-fC via oxidation by KRuO4 and then subsequently labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI-labeled target DNA was hybridized with the capture probe on the electrode,resulting in a strong ECL emission. An extremely low detection limit of 1.4 × 10−13 M was achieved for the detection of 5-hmCDNA.In addition, this ECL method was useful for the quantification of 5-hmC in serum samples. This work demonstrates that selective 5-hmC oxidation in combination with an inherently sensitive ECL method is a promising tactic for 5-hmC biosensing.