Biology Reference
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5' biotinylated aptamer
Streptavidin-coated magnetic bead
Thrombin
5' biotinylated secondary aptamer
Streptavidin-alkaline phosphatase conjugate
Working electrode
Magnetic bar
Figure 2.2.
Schematic representation of the electrochemical sandwich
assay performed forthe detection of thrombin.
1-methoxyphenazine methosulfate (m-PMS). Using this approach,
10 nMof thrombin was detected.
Centi
et al.
[13] developed an electrochemical aptamer-based
sandwich assay for analysis of thrombin in complex matrices, using
a simple-target capturing step by aptamer functionalized magnetic
beads. The assay was carried out by immobilizing the 15-mer
biotinylated aptamer on streptavidin-coated magnetic beads and
thenincubatingthemodifiedbeadswiththetargetanalyteandwith
the 29-mer biotinylated aptamer (Fig. 2.2). At this point, a solution
oftheconjugatestreptavidin-alkalinephosphatasewasaddedtothe
beads and, after streptavidin-biotin recognition the enzymatic sub-
strate (1-naphthyl phosphate) solution was added: the enzymatic
substrate was converted by AP into 1-naphthol, which was oxidized
at the working electrode surface. The amount of oxidized naphthol
was quantified by differential pulse voltammetry. The assay was
applied to the analysis of thrombin in buffer [detection limit (DL)
found was 0.45 nM], spiked serum, and plasma with similar ana-
lytical performances. Moreover, thrombin was generated
in situ
in
plasma by the conversion of its precursor prothrombin, and the for-
mation of thrombin wasfollowed at different times.
