Biology Reference
In-Depth Information
Electrochemicalaptasensorbasedontheuseofalabelhasfound
various applications. In the following sections, some examples are
discussed.
2.3 Electrochemical Aptasensors Based on
a Sandwich Assay
The use of a sandwich format allows detecting the target ana-
lyte with very high sensitivity and selectivity. Two conditions are
required: (1) the analyte possesses two epitopes which are so dif-
ferent that both ligands can bind to the analyte without the binding
of one affecting the binding of the other, and (2) two aptamers are
selected against such analyte. The disadvantage related to this for-
mat consists of several incubation steps that make the assay time
consuming.
This format is widely used in the case of large molecules such
as proteins and hormones; in particular it has been applied to
the detection of thrombin, which has been mostly used as model
system. Thrombin is an important serine protease in the blood
coagulation cascade. It contains a heparin-binding exosite and
fibrinogen-recognition exosite. In 1992, the first DNA thrombin
aptamer was isolated by Bock and coworkers [10] and the most
active strand was a 15-mer oligonucleotide with a
K
d
around
100 nM. This aptamer interacts with the fibrinogen-recognition
exosite.Theotherthrombin-bindingaptamerselectedbyTassetand
coworkers [11] is a 29-mer single-stranded DNA with a
K
d
around
0.5nM.Thisaptamerbindstotheheparin-bindingexositeofthrom-
bin.RNA aptamers for thrombin havealso been selected.
Ikebukuro
et al
. [12] first reported an electrochemical aptasen-
sor for the detection of thrombin based on a sandwich-based assay.
Two different aptamers specific for thrombin were used: the 29-
mer thiolated aptamer and the 15-mer aptamer labeled with glu-
cosedehydrogenase(GDH).Thethiolatedaptamerwasimmobilized
onto gold electrodes; thrombin at different concentrations and then
the enzyme-labeled aptamer was added to the aptamer-modified
electrodes. The electric current generated by the addition of glu-
cose was measured at 0.1 V vs. Ag/AgCl in a buffer containing
