Biology Reference
In-Depth Information
hold the electrode at a positive potential. AFM studies of ssDNA
immobilized on a pyrolytic graphite electrode showed that holding
the electrode at 300 mV (vs. Ag wire) increased the film thickness
of the adsorbed ssDNA film from 0.98
±
0.40 nm (open-circuit
adsorption) to 2.37
±
0.4 nm, which suggests that at a positive
potential more than a single monolayer was adsorbed [124]. The
electrode was almost completely covered, with very few holes.
In the case of screen-printed carbon electrodes, we have used a
mildly positive potential (100 mV vs. AgCl screen-printed track
for 30s) which produced a strong adsorption, such that the DNA
remained adsorbed after washing. This method is attractive since
the electrodes are disposable and, as noted earlier, it means only a
small solutionvolume isneeded.
8.6.2
Probe Attachment
A convenient method of attaching latex-based labels to DNA is the
avidin (or streptavidin)-biotin system, which has been widely used
[125-130]. DNA sequences with a biotin tag at the 5' end are
commercially available. Avidin and streptavidin are proteins which
possess a high binding a
nity for biotin (
K
a
=
10
15
M
−
1
)andcan
be adsorbed onto labels by incubating the labels in an appropriate
solution (e.g., in 3 mg mL
−
1
of protein for at least 15 min). Uptake
of the protein can be monitored by centrifuging down the solid and
then decanting off the liquid. A reduction in protein absorbance
at 280 nm confirms uptake onto the label. The main difference
between the two proteins is in the value of the isoelectric point (5
forstreptavidinand10.5foravidin),andinthefactthatstreptavidin
is much more expensive. In labeling hollow capsules and latex we
used pH values that would render avidin positive. This facilitated
adsorptiontothenegativePSSouterlayerofthecapsules.Inthecase
of adsorption to gold-modified latex, we expect the main location of
the avidin to be on the negatively charged gold particles, since the
PAHlatex outer layer is positive.
8.6.3
Detection Sequence
The simplest detection scheme is to use a single probe to detect
the target, with either the target or the probe being labeled. This
