Biology Reference
In-Depth Information
The charge of a biolayer immobilized onto an electrode will
create an electrostatic barrier to, e.g., the negatively charged
[Fe(CN)
6
]
3
−
/
4
−
redox couple in solution, which is reflected in the
value of the charge transfer resistance. Upon interaction, the charge
distributionofthebiolayerwillchange,causingamodificationinthe
electrostaticbarrierand,therefore,inthevalueof
R
ct
.Anincreasein
R
ct
can be related to an increase in negative charge or a decrease in
positive charge at the biolayer. Reverse charge changes will cause a
decrease in
R
ct
. Another important factor to take into account when
interpretingcharge-transferresistancechangesisthefactthatsome
areasontheAusurfacewhichareaccessibletotheredoxcouple,will
be blocked upon the biomolecular interaction due to the relatively
large volumes of target molecules, such as proteins. This effect will
result in an increased
R
ct
.
On the other hand, a change in capacitance is expected upon
biomolecular interactions. When a large target biomolecule inter-
acts with the immobilized probe, the biolayer thickness increases,
causing a decrease in the total capacitance of the system.
In the case of DNA, hybridization at the electrode results in
a significant increase in the negative charge of the DNA layer.
Therefore, the electrostatic barrier to the negatively charged redox
couple becomes stronger upon hybridization, causing an increase
in the charge transfer resistance. A typical Nyquist plot (
−
Z
imag
vs.
Z
real
) is shown in Fig. 6.7 for a Au electrode after immobilization
of single-stranded DNA probe and after hybridization with its
complementary strand. The charge transfer resistance corresponds
tothediameterofthesemi-circleintheNyquistplot.Forthesample
inFig.6.7,a5k
increasein
R
ct
isobserveduponhybridization[37].
The technique is robust and large signal discrimination upon
hybridization can be obtained with optimization of the DNA
probe density and the measurement conditions. Keighley
et al.
[37] report on the optimization of co-immobilization of thio-
lated oligonucleotides and mercaptohexanol to form mixed self-
assembledmonolayersongold.Specifyingthesolutionmoleratioof
the thiol components provides an effective and easily implemented
method to accurately control the oligonucleotide surface density.
A linear relationship between mole ratio and probe density was
observed for the range (1.3-9.1)
×
10
12
probes/cm
2
. With this
