Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film.

We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp(2) and sp(3) mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (approximately 24 mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5'-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60 mers) with different methylation ratios.