Biology Reference
In-Depth Information
Centi
et al.
[18] compared the performances of several assay for-
mats based on the coupling of magnetic beads with electrochemi-
cal transduction always for the detection of thrombin. Among the
developed assays, one of the used strategies was based on the
direct measurement oftheenzymaticproduct ofthrombincaptured
by the immobilized aptamer. The main differences between this
work and the work by Mir
et al.
[17] involve the use of magnetic
beads as solid support on which the aptamer-based assay is per-
formed. Streptavidin-coated magnetic beads modified by immobi-
lization of the biotinylated thrombin aptamer were incubated with
different concentrations of thrombin in the range 100 to 600 nM
for 30 min. Bound thrombin was detected by re-suspending the
beads in the thrombin substrate,
β
-Ala-Gly-Arg-
p
-nitroaniline, for
30minat37
◦
C.Thesolutioncontainingthethrombinreactionprod-
uct was deposited onto the surface of the working screen-printed
graphiteelectrode,withoutanystirring.Theaptamer-boundthrom-
bin was detected by quantification of
p
-nitroaniline produced from
the thrombin catalyzed reaction. The DPV measurements showed a
decreaseofthepeakat
−
730mVvs.Ag/AgClpseudo-referenceelec-
trode related to the
β
-Ala-Gly-Arg-
p
-nitroaniline substrate and the
appearanceofanewpeakat
−
870mVvs.Ag/AgClpseudo-reference
electrode, indicating the formation of
p
-nitroaniline (Fig. 2.3). The
same measurements were carried out in absence of thrombin, and
only a reproducible peak at
−
730 mV was observed (16.1
±
0.4
μ
A).
A linear increase of
p
-nitroanilinepeak current was observed in the
studied concentration range of thrombin. On the contrary, a linear
decrease in thrombin substrate was observed increasing the throm-
binconcentration.Thedetectionlimit(DL)foundforthrombinusing
this approach was 175 nM.
In another direct approach, non-faradic electrochemical
impedance spectroscopy (NIS) was used for the direct detection of
platelet-derived growth factor-BB (PDGF-BB) [21]. Binding of PDGF
to its aptamer immobilized on a silicon electrode surface leads to
a decrease in capacitance measured by electrochemical impedance
spectroscopy (NIS). Because of the high sensitivity and specificity
(DL 40 nM) and the absence of reagent to be used when performing
the test, this biosensor design could be promising for
in vivo
moni-
toring.
