Electrochemical detection of redox-labeled DNA is a less expensive alternative to fluorescent techniques offering comparable sensitivity for diverse applications in bioanalysis (DNA hybridization, SNP typing, minisequencing) and diagnostics such as genetic-related diseases[1,2]. Ferrocene is a classical electrochemical standard due to its reversible redox chemistry, convenient redox potential and commercial availability. It has been used as an oxidizable electrochemical label for nucleotides and DNA[4-12] and has been exploited in sensors.
Ferrocenylethynyl-labeled nucleotides dNFcTPs (dAFcTP, dCFcTP) (Figure 1B) are efficient DNA polymerase substrates in primer extension (PEX) reactions. Square-wave voltammetry of nucleotides dNFcTPs (dAFcTP, dCFcTP) indicates that the ferrocenyl moieties display reversible redox behaviour. Combining several orthogonal redox labels, an attractive option of redox coding of DNA bases can be envisaged for applications in sequencing. The orthogonality requires labeling of each nucleobase by a different redox-active group, and each label should be “readable” in the presence of all the other labels and give a ratiometric signal intensity.
The ferrocene can be distinguished and electrochemically quantified by using the approach based on the synthesis of tailed-PEX products bearing the ferrocene and ferrocenecarboxamide labels and their capture on gold electrodes (Figure 1A). Agarose gel electrophoresis shows the formation of the full-length double-redox-labeled DNA (Figure 1C). The labeled DNA products, hybridized to the complementary capture probe immobilized on gold electrode, display clearly distinguishable, fully orthogonal and ratiometric peaks (Figure 1D), demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.
Figure 1: The electrochemical properties of ferrocene-labeled DNA. A) Modified DNA bearing ferrocene labels and its capture on electrode. B) Structures of the modified nucleotides dNFcTPs. C) Agarose gel electrophoresis of PEX products using a combination of dAFcTP, dCFcPaTP with different ratios. D) Square wave voltammograms of the oxidation peaks of dAFc and dCFcPa on PEX products for dAFc/dCFcPa ratios (8:2, 2:8 and 4:4). Modified according to .
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