Voltammetric determination of 5-methylcytosine at glassy carbon electrode

The electrochemical oxidation of 5-methylcytosine (5-mCyt) was studied at glassy carbon electrode (GCE) in aqueous solution, over a wide pH range, using cyclic, square wave and differential pulse voltammetry, as well as compared with the anodic behaviour of other similar bases, Cyt, thymine (Thy) and uracil (Ura). The results revealed that the electrode reaction of 5-mCyt occurs in a single, one-electron one-proton, irreversible step controlled by diffusion, generating intermediate radicals that quickly react with water and/or dimerize. Moreover, in general, it was observed that the C5 methylated pyrimidines are more easily oxidized and thus are more reactive in relation to the unmethylated bases, Cyt and Ura. A mechanism for the electro-oxidation of 5mCyt is proposed. Using differential pulse voltammetry (DPV) experimental conditions, such as the electrode size, supporting electrolyte composition, pH and influence of possible interferents (guanine, 7-methylguanine, adenine and Cyt), were also investigated for determination of 5-mCyt with low detection limit. Under the best conditions, the DPV method proposed, with a pre-treated 3.0 mm diameter GCE, provided a linear analytical curve to 5-mCyt in phosphate buffer solution (pH 7.0) in the concentration range from 3 up to 15 mu mol L-1, with a detection limit of 0.11 mu mol L-1 anda high correlation coefficient (r = 0.997). A new simple and sensitive electroanalytical method using an unmodified GCE for the quantification of DNA methylation is then presented.