Biology Reference
In-Depth Information
2.5 Electrochemical Aptasensors Based on a Direct Assay
Some papers have used the catalytic activity of thrombin for the
determination of this protein.
α
-Human thrombin is a highly spe-
cific serine protease that catalyses the hydrolysis of the throm-
bin chromogenic substrate,
β
-Ala-Gly-Arg-
p
-nitroaniline producing
p
-nitroaniline. The rate of yellow colored
p
-nitroaniline formation
can be followed by its UV absorption at 405 nm, or electrochemi-
cally by the reduction of its nitro group. The electrochemical detec-
tionoffersbenefitsintermsofsensitivityandspeed.Whensaturated
by enzyme substrate the formation rate of
p
-nitroaniline is propor-
tionalto the enzymeconcentration.
Mir et al.
[17] first reported the detection of thrombin bound
to an aptamer selective for thrombin by the quantification of
p
-nitroaniline produced by the enzymatic reaction catalyzed by
thrombin.
A mixed self-assembled monolayer was used for the aptamer
immobilization on the gold electrode. The aptamer-modified elec-
trodes were then incubated for 1 h at 37
◦
C with thrombin
(18
μ
g/mL). Electrochemical measurements were recorded in the
thin-layer cell configured to contain a total volume of 20
μ
L.
Thrombin chromogenic substrate (
β
-Ala-Gly-Arg-
p
-nitroaniline)
was injected into the cell and differential pulse voltammetry (DPV)
measurementsbetween
−
0.2and
−
1Vwithapulseheightof
−
0.05
V and pulse duration of 70 ms were carried out. The DPV measure-
ments showed that
β
-Ala-Gly-Arg-
p
-nitroaniline substrate and the
p
-nitroanilineproducthavedifferentredoxpotentials.Moreover,the
DPV experiments showed a current peak at
−
0.45 V in the pres-
ence of the thrombin substrate. After 5 min, the peak at
−
0.45 V
decreased and a new peak was detected at
0.70 V, indicating the
formationof
p
-nitroaniline.Thesamemeasurementscarriedouton
acontrolelectrodeinordertotestthespecificityoftheassay:inthis
experiment bovine serum albumin (BSA) substituted thrombin and
in this case onlythe peak at 0.45 V was measured.
The authors demonstrated a huge reduction in the assay time
considering that the optical detection of
p
-nitroaniline needed 3 h
against 5 min necessary for the electrochemical measurement.
−
