Biology Reference
In-Depth Information
themodifiedmagneticbeadsandafteranincubationstep,theexcess
ofreagentswasremovedbymagneticseparation.Inaddition,forthe
signal amplification, thiocyanuric acid (3.5
·
10
−
6
μ
M) was added
before incubation and the magnetic bead-aptamerI/thrombin/Au-
NP-aptamerII conjugates were re-suspended in HCl 0.1 M solution.
A scheme of the sandwich assay is shown in Fig. 2.4.
Asignalamplificationwasobtainedbyforminganetworkofthio-
cyanuricacid/Au-NPs.TheelectrochemicaloxidationofAu-NPswas
performed at
+
1.25 V for 120 s on a glassy carbon electrode. Imme-
diatelyaftertheelectrochemicaloxidation,differentialpulsevoltam-
metry was performed resulting in an analytical signal due to the
reduction of AuCl
4
−
, which relates to the amount of the Au-NPs for
the sandwich format.
Adetectionlimitof8aMwasachieved.Todemonstratethefeasi-
bility of this approach, the aptasensor was applied to the detection
of thrombin in someplasma samples.
Hansen and coworkers [29] reported an electrochemical
aptasensorinvolvingnanocrystaltracersforthedetectionofthrom-
bin. The aptasensor was based on a displacement assay (Fig. 2.5).
Thiolated-aptamersspecificforthrombinandlysozymewereimmo-
bilized on a gold electrode. Thrombin and lysozyme were modified
with CdS and PbS quantum dots (QDs), respectively and these were
boundtotherespectiveaptamersimmobilizedonthesurface.Inthe
presence of the target protein, the QD-tagged protein was displaced
and the number of QDs left on the surface decreased. After dissolv-
ing the remaining QDs on the surface using 0.1 M HNO
3
, the metal
ions(Cd
2
+
andPb
2
+
)wereidentifiedandtheirconcentrationatmer-
cury coated glassy carbon electrode was detected by electrochemi-
cal stripping. The concentration of the metallic ions was correlated
with the concentration of the target proteins in solutions. Owing to
theamplificationeffectoriginatedbydissolvingQDsandbythehigh
sensitivity correlated to the electrochemical stripping detection, a
detection limit of 0.5 pM was achieved for thrombin. It is impor-
tanttounderlinethat,usingthisapproach,differentaptamerscould
be immobilized on the same gold substrate, since different protein
targets can be labelled with QDs with different cation compositions
(CdS,ZnS,CuSandPbS).Asdemonstratedbyauthors,thrombinand
lysozyme were labeled with CdS and PbS and both proteins were
simultaneously detected on thesame gold substrate.
