Biology Reference
In-Depth Information
9.4.1.2 Analytical signal recording
Two strategies are performed to detect the hybridization event:
enzymatic detection and electrocatalytic detection. The following
steps are carried out:
Enzymatic detection:
Reaction with antibody anti-FITC AP conju-
gate(Ab-AP):analiquotof40
μ
LofAb-APsolution(1/100dilution)
isdroppedonthegenosensordevicefor60minutes.Thenawashing
stepwith0.1MTrisbufferpH9.8,containing1%BSA,iscarriedout.
Enzymatic reaction:
An aliquot of 30
μ
L of 6 mM 3-IP is deposited
on the electrode surface for 20 minutes. After that, the reaction
is stopped by adding 4
μ
L of fuming sulphuric acid and 10
μ
Lof
ultra-pure water. In this step, the corresponding indigo product is
converted to itsparent hydrosolublecompound IC.
Analytical signal recording:
The SPCEs are held at a potential of
−
0.25V for 25 s, and then, a cyclic voltammogram is recorded from
0.25to
+
0.20Vatascanrateof50mV/s.Theanodicpeakcurrentis
measured in all experiments.
Electrocatalytic detection:
A50
μ
L portion of 0.2 M HCl solution
is dropped on the electrode surface and the electrode is held at a
potential of
+
1.35V for 1 minute. Then, the chronoamperometric
detection is performed at
−
1.40 V, recording the electric current
generated for 5minutes.
Figure 9.10 shows the scheme of the genosensor device and
the analytical signals obtained with electrocatalytic detection (Fig.
9.10A) and enzymaticdetection (Fig. 9.10B).
Moreover, the significance of the attachment of biotinylated
oligonucleotide probes through the streptavidin/biotin interaction
has been tested in a previous work [15]. When a double-labeled
(biotinandfluorescein)poly-Twasattachedtotheelectrodesurface
through the streptavidin/biotin interaction, the peak currents were
much higher than those obtained when it was accumulated on
the electrode surface by physical adsorption. This fact means that
streptavidin/biotin interaction allows to attach and orient the
oligonucleotide strands on electrode surface, whereas the direct
adsorption of the oligonucleotide on the electrode surface results
