Biology Reference
In-Depth Information
gives rise to non-linear calibration plots. The alternative is to use
probes in which guanine bases have been replaced by inosine.
Then the analytical signal appearing with the hybridization and
background signalsare negligible.
Another strategy to differentiate the signal of the single strand
from that of the double strand is based on the use of a protein
that binds specifically to the ssDNA, preventing the oxidation of the
guaninein singlestrands of DNA [16].
The great advantage of this type of detection is to avoid the use
ofmarksorindicatorsofhybridization,simplifyingtheexperimental
procedure. However, the detection based on the electroactivity of
bases gives rise to a lack of sensitivity. Various proposals based
on the use of oxidation products of adenine as catalysts of NADH
oxidation [17], or those based in the use of mediators for the
oxidation of bases, with ruthenium complex [18, 19] or osmium
complex [20] have been proposed in order to get an amplification
of the signalandthereby improve the sensitivity.
However, these methods induce an irreversible process prevent-
ing multiuse andare limited bythe adenineand guaninecontent.
9.3.1.2 Indirect transduction methods
Indirect transduction relies on the use of indicators or labels.
The first ones are based on the differences in the electrochemical
behavior of indicators that interact in a different extension with
dsDNA and ssDNA. The indicators for hybridization detection can
be anticancer agents, organic dyes, or metal complexes, and are not
generally covalently joined to DNA. The latter strategies include the
useoflabelscovalentlyjoinedtoDNAsuchasferrocene,enzymes,or
metal nanoparticles.
Useofindicators
Indicators are electroactive compounds that
present different a
nity for ssDNA and dsDNA; they used to be
anticarcinogenicagents, organic dyes, or metallic complexes.
Some metallic complexes like Ru(NH
3
)
3
6
, [Fe(CN
6
]
3
−
/
4
−
,
Co(phen)
3
3
,orRu(bpy)
2
3
, and some organic compounds like
methylene blue (MB) recognize the hybridization reaction. The
uniontakesplaceviaelectrostaticinteractionwiththehollowsofthe
