Advanced electrochemical detection of nitrogenous bases, synthetic oligonucleotides, and single-stranded DNA through flow injection analysis and catalytic oxidation on Prussian Blue

Abstract

The effect of Prussian Blue (PB, ferric hexacyanoferrate) on oxidation of free nucleobases, synthetic oligonucleotides, single- and double-stranded DNA (ssDNA and dsDNA) was evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA) on carbon screen printed electrodes, both bare (SPE) and PB modified (SPE/PB). It has been found that electrocatalytic oxidation of nucleobases, namely guanine, adenine, thymine, and cytosine, or nucleobase residues takes place via electrochemically generated Berlin Green (BG), fully oxidized form of PB. The constant potential FIA allowed one to register electrooxidation of all DNA nitrogenous bases at 0.95 V and three nitrogenous bases (except for cytosine) at 0.70 V on SPE and SPE/PB. The modification of electrode surface with PB resulted in a shift of oxidation potential to less positive values and enhanced (up to 8 times) detection sensitivity for DNA nucleobases. The pronounced catalytic effect of electrogenerated BG on their oxidation has also been observed for synthetic oligonucleotides and degraded ssDNA of natural origin. By fractionating ssDNA, it has been found that the main contributor to the total oxidation signal is the fraction of lowest molecular weight (< 3 kDa). At the same time, no oxidation signal was observed for large molecular weight native dsDNA in the potential range from 0 to 1 V on bare SPE and SPE/PB in both CV and FIA measurement modes. Therefore, the advanced electrochemical detection by FIA with SPE/PB was achieved for all nucleobases, synthetic oligonucleotides, and ssDNA of natural origin. Moreover, the obtained results expand the contemporary understanding of DNA electrochemistry.