Fc-labeled DNA

A number of Fc-DNA conjugates were reported in the literature, and a selected number of the reports are summarized in Table 1. Fc-labeled DNA constructs have emerged as an effective tool to investigate DNA hybridization and offer a convenient method for DNA mismatch detection by differential hybridization. 

Importantly, King succeeded in the formation of a Fc-labeled 998-bp (base-pair) construct by PCR using T4 DNA polymerase in the presence of Fcl-dUTP. Incorporation of the redox label shows that Fcl-dUTP is suitable as a substrate for PCR. In contrast, Fc2-dUTP acts predominantly as a terminator in the PCR. The melting behavior of a 37-mer duplex containing five Fcl-dU residues reveals that the labeled nucleotide induces only a modest helix destabilisation, with T ,=71°C for a labeled duplex versus 75°C for the corresponding nonlabeled ds-DNA construct. King reports that the Fc-labeled DNA is detected at femtomolar levels by HPLC using a coulometric detector. Thus, it must be emphasized that the incorporation of the Fc label by PCR and its facile and cost-effective electrochemical detection should promote the use of this technique in nucleic acid analysis and may replace the more costly fluorescence-based detection systems. 

Based on earlier results by Barton and coworkers, who reported that the electron transfer kinetics will exhibit a directional dependence, Kraatz and coworkers reported a comparative study of the ET through two configurations of ds-DNA. For this purpose, two Fc-labeled DNA conjugates and their complementary strand were prepared by solid phase synthesis ... 

The Fc label was introduced as an amide using Fc-OBt, an active ester that readily reacts with available amino groups. One construct had the Fc group at the 5 terminus of the DNA, whereas the other had the Fc at the 3 terminus. The DNA constructs were purified by RP-HPLC and characterized by MALDI-TOF MS and UV-vis spectroscopy. Typical UV-vis spectra for Fc-labeled ds-DNA and Fc-labeled ss-DNA are shown in Figure 8. After hybridization of the Fc-labeled DNA strand to its complementary strand in 20 mM Tris buffer (pH 8.7) for 24 h at room temperature, the Fc-labeled ds-DNA was exposed to gold microelectrodes of 50 om... 

Quenching effect of ferrocene (Fc) is proved very practical for the development of ECL sensors. A reagentless signal-on ECL biosensor for DNA hybridization detection was developed (see Fig. 6.2). In the development of this biosensor, the quenching effect of Fc on intrinsic cathodic ECL at thin oxide-covered glassy carbon (C/C Oi-x) electrodes was employed. The main advantage of the present sensor lies in the fact that ECL is generated from the electrode itself and no luminophore or luminophore-labeled DNA probe is needed. The detection limit was ca. 5.0 pM (S/N = 3) [18]. 

Mt/SECM-AFM was also used to explore the conformation and motional dynamics of end-grafted DNA oligonucleotides [16]. Force and current approach curves were recorded at a gold surface bearing a layer of thiol-end grafted Fc-labeled (dT)2o chains, both before and after hybridization... 

Figure 7 Umek s detection system using a SAM containing 3 -thiol terminated DNA capture probes and Fc-labeled signaling probes. ...

An excellent example of an innovative labeling strategy is provided by Willner and coworkers,94 who used Fc-conjugated uridine (dUTP) as electron transfer mediators between the redox enzymes and the electrode for the amplified bioelec -trocatalytic detection of viral DNA. 

Figure 8.6 Schematic representation of a biomolecular-nanoparticle supramolecular assembly consisting of double-stranded DNA (ds-ODN) modified electrodes for the capture of p53 protein. Electrochemical signal generation and amplification occurred by subsequent labeling of the p53 tetramer with biotin (Biotin-Mi) and capture of streptavidin-modified Fc-capped AuNPs.26 (Reprinted with permission from J. Wang et al., Anal. Chem. 2008,80,769 -774. Copyright 2008 American Chemical Society.)...

Fig. 5 Immobilized nucleic acid assays utilizing redox-active moieties, a Amplified detection of viral DNA by generation of a redox-active replica and the bioelectrocatalyzed oxidation of glucose (Reprinted with permission from [200]. Copyright(2002) American Chemical Society), b Alternative formats for the capture on a gold electrode SAM of solution-extended primers or direct surface extension of primer with electrotides (adapted from [185]). c Ferrocene-labelled hairpin for electrochemical DNA hybridization detection. A Fc-hairpin-SH macromolecule is immobilized on a gold electrode. When a complementary DNA target strand binds to the hairpin, it opens and the ferrocene redox probe is separated from the electrode, producing a decrease in the observed current (Reprinted with permission from [203], Copyright(2004) American Chemical Society)...

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