We describe the quantitative nonlabel electrochemical detection of both cytosine (C) and methylcytosine (mC) in oligonucleotides using newly developed nanocarbon film electrodes. The film consists of nanocrystalline sp2 and sp3 mixed bonds formed by employing the electron cyclotron resonance (ECR) sputtering method. We successfully used this film to develop a simple electrochemical DNA methylation analysis technique based on the measurement of the differences between the oxidation currents of C and mC since our ECR nanocarbon film electrode can directly measure all DNA bases more quantitatively than conventional glassy carbon or boron-doped diamond electrodes. The excellent properties of ECR nanocarbon film electrodes result from the fact that they have a wide potential window while maintaining the high electrode activity needed to oxidize oligonucleotides electrochemically. Proof-of-concept experiments were performed with synthetic oligonucleotides including different numbers of C and mC. This film allowed us to perform both C- and mC-positive assays solely by using the electrochemical oxidation of oligonucleotides without bisulfite or labeling processes.

We have developed ultra-flat carbon film electrodes with a wide potential window and a low capacitive current by the electron cyclotron resonance (ECR) sputtering method. The film consists of sp2 and sp3 bonds (sp3/sp2 ratio = 0.702) and is sufficiently conductive for electrochemical measurements without doping. The film has average roughness of 0.7 A, which is much flatter than that of nanocrystalline diamond film. The potential limit of ECR sputtered carbon (current limit < +/-500 muA/cm2) in the positive direction is 2.0 V vs Ag/AgCl, which is slightly lower than that of boron-doped diamond (2.1 V) and much wider than that of a glassy carbon (GC) electrode (1.7 V). In contrast, a much wider potential window can be obtained in the negative direction. The capacitive current is also much lower than that of a GC electrode due to the ultra-flat surface and the low number of oxygen-containing groups at the film surface. ECR sputtered carbon film can be used to measure each base of oligonucleotides by electrochemical oxidation without any pretreatment. The ultra-flat surface and low surface oxygen concentration suppress fouling with electroactive species, such as oligonucleotides, NADH, and bisphenol A.