Biology Reference
In-Depth Information
9.4.3.1 Gold nanostructuration of screen-printed carbon
electrodes
Gold nanostructures were
in situ
generated over SPCEs (SPCnAuEs)
applying a constant current intensity of
−
5
μ
A for 2 minutes in a
0.1mMacidicsolutionofAuCl
4
.Afterthat,andinthesamemedium,
a potential of
+
0.1 V was applied during 2 minutes, in order to
desorb hydrogen.
9.4.3.2 Genosensor design
The formation of the sensing phase was performed by dropping
20
μ
L of 3'-thiolated oligonucleotide probe 10 nM for 20 minutes
and after rinsing with 0.1 M Tris-HNO
3
pH 7.2, a blocking step
with casein (2%) was carried out. Then, the electrodes were rinsed
with 2
SSC buffer pH 7.2 containing 1% BSA. After that, the
hybridization was performed at room temperature placing 40
μ
L
of 3'-biotinylated oligonucleotide target solutions in 2
×
SSC buffer
pH 7.2, containing 1% BSA, on the surface of the genosensor for 1
hourandthenrinsingwith0.1MTris-HNO
3
pH7.2buffercontaining
2mMMg(NO
3
)
2
.Then,areactionwithalkalinephosphataselabeled
streptavidin (S-AP) was performed dropping aliquots of 40
μ
Lof
S-AP solutions (5
×
10
−
10
M) on the genosensor device for 60
minutes. Finally, after a washing step with 0.1 M Tris-HNO
3
buffer
pH 9.8, containing 20 mM Mg(NO
3
)
2
, the enzymatic reaction of the
substrate,amixtureof3-indoxylphosphate(3-IP)andsilvernitrate,
was performed. In this reaction, 3-IP produces a compound able to
reduce silver ions in solution into a metallic deposit. The deposited
silver is electrochemically stripped into solution and measured by
anodic stripping voltammetry giving place to the analytical signal
Fig. 9.14.
×
9.4.3.3 Results
Adsorptionofthiolatedprobeswasstudied,inthissenseadsorption
time and probe concentration were tested. Results obtained shown
that20minuteswasenoughtimetoreachaplateauintheanalytical
signal, and probe concentration was fixed in 10 nM, because
higher concentrations resulted in a decrease in the analytical signal.
