DISSERTATION

______________________________________________________________________ Introduction
DNA detection via redox mediators was employed using various mediators such as
[Ru(bpy)3] 2+ , [Co(phen)3] 3+ , ferrocene or [Fe(CN)6] 3-/4- (Figure 1.15, a). Catalytic oxidation of
guanine using [Ru(bpy)3] 2+ on indium tin oxide (ITO) electrodes is a well-known example 60 .
The approach involves addition of DNA into a solution containing the ruthenium complex,
while the electrode is held at a potential suitable for the oxidation of the reduced form of the
complex. The complex is regenerated by the oxidation of guanine from DNA. Consequently,
the signal is enhanced proportionally to the amount of guanine available for oxidation, since
the direct guanine oxidation is not possible at ITO electrodes.
The characteristics of non-covalently interacting indicators are a different affinity towards dsand
ssDNA. They can interact with DNA either by electrostatic binding, binding to the groove
of dsDNA or intercalate into dsDNA (Figure 1.15, b). The most widely used indicators are
daunomycin, proflavine, antraquinone, methylene blue, [Co(phen)3] 3+ and [Ru(NH3)6] 3+ . One
of the initial studies on non-covalent interaction of compounds with DNA was done by
Mikkelsen et al., using [Co(phen)3] 3+ as a dsDNA minor groove binder. The metal complex is
positively charged and attracted by negatively charged DNA, resulting in a higher current for
the more negatively charged dsDNA 61 . Furthermore, Barton and coworkers pioneered the work
on long-range charge transfer resistance through DNA, using electrochemically active
intercalators 62 (methylene blue and daunomycin). Interaction of non-electrochemically active
intercalating compounds was investigated using EIS 63 .
First covalently bound DNA markers were investigated in the beginning of the 1980s 46 .
Labelling of the DNA can be performed using the probe DNA, where the label is positioned at
the distant end of the probe (Figure 1.15, c). Due to the flexibility of the ssDNA, without the
presence of target DNA, the label is close enough to the surface and the signal is detected. Upon
hybridization the signal switches off due to the rigidity of the dsDNA. Namely, after the
hybridization the label is too far away from the surface for the electron transfer to occur.
Labelling of the target DNA is also possible, and it is usually done at the end that is close to the
surface upon hybridization (Figure 1.15, d). Therefore, after hybridization the signal is switched
on. However, this approach requires a preparation step in which the target DNA from the sample
needs to be labelled, which prolongs the assay time. In a sandwich-type assay, immobilized
probe DNA is initially hybridized with a portion of the non-labelled target DNA from the
sample, and subsequently a labelled signal DNA is hybridized with the overhang of the target
DNA (Figure 1.15, e). Enzymes are readily used as labels for this approach 64 . In this case, DNA
1.4 Hybridization detection 24