Biology Reference
In-Depth Information
electrostatic attraction between the sensor surface and the Fc-PNA-
DNA hybrid caused a significant increase of the electrochemical
signal, which is proportional to the amount of complementary DNA
present. Importantly, the PAH-modified sensor was found to be
more sensitive (with a detection limit of 40 fM) than the bare
ITO substrate (with a detection limit of 500 fM). The method was
further validated by discrimination of fully matched and mismatch
DNA strands at elevated temperatures and detection of unpurified
PCR amplicons with detection limit of 4.17 aM. Recently, a new
strategywasreportedthatmakesuseoftheminorgroovebindingof
singly reduced cation radical viologen (V) groups C
12
VC
6
VC
12
[62].
In the presence of complementary PNA-DNA hybrids, the
V
2
+
/
+
redox couple of C
12
VC
6
VC
12
exhibited a unique double-wave cyclic
voltammogram, with the formal potential shifted by -100 mV from
the
E
f
in the presence of single base mismatched DNA-PNA hybrids
or PNAprobes alone.
Without a doubt, unique properties make PNA an interesting,
although sometimes synthetically challenging and expensive, alter-
nativefor design of biosensors.
7.3.5
Protein Mediated DNA Biosensors
MutS is a 97 kDa protein that is part of the DNA repair “engine”
in
E. coli
. The protein binds to many of single nucleotide DNA
mismatches and has been used for label-free nucleotide mismatch
detection. There are several reports of utilizing MutS to detect
single nucleotide mismatches by a number of different analytical
tools. However, electrochemical techniques have recently been used
due to inherent sensitivity. It was observed that for alkanethiol-
diluted ds-DNA on gold, the charge transfer resistance
R
ct
increases
considerably after binding of MutS to a A-C mismatch, while no
change in
R
ct
was observed when measuring the electrochemical
impedanceofmatchedDNAduplexinthepresenceofMutSsincethe
enzymedoes notbindto fully matched ds-DNA (seeFig. 7.8) [63].
Palecek
et al.
[64] and Masarik
et al.
[65] detected a G-T
mismatch at CPE and HMDE using chronopotentiometric stripping
analysis (CSA) and squarewave voltammetry (SWV) in the presence
of MutS. Cho
et al.
[66] found that the binding a
nity of MutS for
