Biology Reference
In-Depth Information
[55], who demonstrated that the PNA film retains its e
cient
hybridizationpropertiesunderavarietyofconditionsandtherefore
offers significant advantages over “classic” DNA based capture
probes.
Faster hybridization, minimal dependence on ionic strength,
and higher specificity and sensitivity (including discrimination
for single-base mismatches) were demonstrated. Electrochemical
detection of a single nucleotide base pair mismatch was achieved
using a mixed film composed of PNA and 6-mercapto-1-hexanol
as diluent [56]. Figure 7.6 outlines the principle of the PNA
biosensor proposed by Aoki
et al.
Binding of the complementary
oligonucleotide to the PNA probe increased the negative charge
at the electrode surface resulting in an increased electrosta-
tic repulsion between the monolayer and the redox marker
[Fe(CN)
6
]
4
−
/
3
−
present in solution. In essence, the redox reaction of
the redox probe was hindered upon hybridization with the target
DNA, Fig. 7.6b. Subsequently, Wang and coworkers reported an
electrochemicalimpedancespectroscopy(EIS)studyonthesemixed
alkanethiol/PNA films [57], providing insight into the repulsive
interactions between [Fe(CN)
6
]
3
−
/
4
−
in the presence of matched
films and those containing a single nucleotide mismatch containing
PNA/DNAhybrids.Hashimoto
et al
.usedPNAaspartofanelectrode
array sensor [58]. Synthetic PNA probes modified with the thiol-
containing amino acid cysteine were immobilized on the gold
electrodes of the array. Hoechst33258 is known as a minor groove
binder and specifically binds to ds-DNA and was exploited in this
study. In contrast to other DNA binding molecules that often bind
not only to the hybrids but also to the single strands, Hoechst33258
only binds to ds-DNA. The array was used for detection of the
PCR amplified cancer gene
ras
. The PNA showed stronger binding
a
nity for complimentary DNA than for strands with a single base
mismatch allowing the detection of point mutations. In another
investigation, nanogold-modified electrodes were used to increase
the amount of immobilized ss-PNA capture probes leading to
an increase in the electrochemical signal [59]. Fc-functionalized
polythiophene was used as a cationic hybridization indicator that
adsorbed onto the negatively charged DNA backbone, giving rise
to a clear hybridization signal in the CV and DPV. The method
