Voltammetric analysis of 5-(4-Azidophenyl)-2′-deoxycytidine nucleoside and azidophenyl-labelled single- and double-stranded DNAs

Voltammetric determination of a redox labeled nucleoside 5-(4-azidophenyl)-2′-deoxycytidine (dC^AZP) and various polymerase-synthesized dC^AZP-labeled DNAs in aqueous buffers is presented. Influence of: i) pH (2–12), ii) scan rates (0.02–10 V s⁻¹), and iii) dC^AZP concentration (0.02–10 μmol l⁻¹), on voltammograms of dC^AZP were systematically studied for the first time using CV at a hanging mercury drop electrode. Electrode potential-controlled adsorption driven process allowed sensitive determination of dC^AZP at nanomolar concentrations using adsorptive stripping voltammetry. Transfer stripping voltammetry (TSV) was used for the detection of dC^AZP-labeled DNA in femtomole quantities. Precise sequence-specific incorporation of dC^AZP into DNA by primer extension was used to demonstrate a perfect correlation between the number of incorporated AZP moieties and TSV responses. In addition, for the first time we used polymerase chain reaction to prepare an about 350-bp double-stranded DNA fragment globally modified with dC^AZP, and of terminal deoxynucleotidyl transferase tailing reaction to generate end-labeled single stranded oligonucleotides. Effects of DNA structure on the AZP-modified DNA TSV responses are discussed.