» Sign in / Register

Ferrocenylethynyl-labeled nucleotides for ratiometric redox labeling and coding of DNA

visit product page

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[3]. It has been used as an oxidizable electrochemical label for nucleotides and DNA[4-12] and has been exploited in sensors[13].

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[12]. Combining several orthogonal redox labels, an attractive option of redox coding of DNA bases[14] 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[12].

Figure 1: The electrochemical properties of ferrocene-labeled 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 [12].

Product Cat.-No. Amount Price
5-Ferrocene-dCTP (dCFcTP) NU-918 0.5 mg 400 €
7-Ferrocene-7-Deaza-dATP (dAFcTP) NU-919 0.5 mg 400 €

Questions or inquiries?

Dr. Jana Balintova

Please contact Jana with all questions or inquiries you may have!

Selected References:

[1] Hocek et al. (2011) Nucleobase modification as redox DNA labelling for electrochemical detection. Chem. Soc. Rev. 40 (12):5802.
[2] Paleček et al. (2012) Electrochemistry of nucleic acids. Chem. Rev. 112 (6):3427.
[3] Kang et al. (2009) Comparing the properties of electrochemical-based DNA sensors employing different redox tags. Anal. Chem. 81 (21):9109.
[4] Wlassoff et al. (2002) Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA. Nucleic Acids Res. 30 (12):e58.
[5] Patolsky et al. (2002) Redox-Active Nucleic-Acid Replica for the Amplified Bioelectrocatalytic Detection of Viral DNA. J. Am. Chem. Soc. 124 (5):770.
[6] Van Staveren et al. (2004) Bioorganometallic Chemistry of Ferrocene. Chem. Rev. 104 (12):5931.
[7] Di Giusto et al. (2004) Multipotential Electrochemical Detection of Primer Extension Reactions on DNA Self-Assembled Monolayers. J. Am. Chem. Soc. 126 (13):4120.
[8] Di Gusto et al. (2004) Enzymatic Synthesis of Redox‐Labeled RNA and Dual‐Potential Detection at DNA‐Modified Electrodes. Angew. Chem. Int. Ed. 43 (21):2809.
[9] Yeung et al. (2006) Electrochemical Real-Time Polymerase Chain Reaction. J. Am. Chem. Soc. 128 (41):13374.
[10] Brazdilova et al. (2007) Ferrocenylethynyl Derivatives of Nucleoside Triphosphates: Synthesis, Incorporation, Electrochemistry, and Bioanalytical Applications. Chem. Eur. J. 13 (34):9527.
[11] Ménová et al. (2013) Polymerase synthesis of oligonucleotides containing a single chemically modified nucleobase for site-specific redox labelling. Chem. Commun. 49 (41):4652.
[12] Simonova et al. (2020) Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA. Chem. Eur. J. 26 (6):1286.
[13] Magriñá et al. (2019) Electrochemical genosensor for the direct detection of tailed PCR amplicons incorporating ferrocene labelled dATP. Biosens. Bioelectron. 134:76.
[14] Debela et al. (2016) Electrochemical primer extension for the detection of single nucleotide polymorphisms in the cardiomyopathy associated MYH7 gene. Chem. Commun. 52:757.