Biology Reference
In-Depth Information
based on the enzyme activity by adding H
2
O
2
and using hydro-
quinoneas a mediator (Fig.3.7D).
The chemisorbing ability of gold NPs in the nano-AuGEC was
demonstrated with an excellent LOD (9 fmol/60 pM of ssDNA) in
hybridization studies. Regarding other electrochemical transducers
previously reported, such as an avidin graphite-epoxy biocompos-
ite(Av-GEB)orproteinAgraphite-epoxybiocomposite(ProtA-GEB)
[23], the main advantages of the inorganic nanoAu-GEC electrode
compared to the biocomposite is the lack of loss of activity and that
the latter requires the temperature to be kept at 4
◦
C due to the bio-
logicalnature of themodifier, the protein avidin.
Moreover, and for the first time, a double-tagging PCR strat-
egy was performed with a thiolated primer for the detection of
Salmonella
sp. The rapid electrochemical verification of the ampli-
con coming from the pathogenic genome of
Salmonella
performed
by PCR with a set of two labeled primers was demonstrated to be
an easy way for the thiolation of the PCR product. The thiolated end
allowedtheimmobilizationoftheamplicononthenano-AuGECelec-
trodeinaneasyway.
Theprocedureconsistsbrieflyofthefollowingsteps,asschemat-
ically outlined in Fig. 3.7: (i) DNA amplification and double-labeling
of Salmonella IS200 insertion sequence; (ii) immobilization of
the doubly labeled amplicon in which the SH end of the dsDNA
amplicon was immobilized on the nanoAu-GEC nanocomposites
by chemisorption (Fig. 3.7B2); (iii) enzymatic labeling using as
enzyme label the antibody anti-DIG-HRP capable of bonding the
other labeled extreme of the dsDNA amplicon (Fig. 3.7C); and (iv)
amperometric determination (Fig. 3.7D). With this strategy, as low
as200fmolcanbeeasilydetected,withanelectrochemicalsignalof
almost3
μ
A.ThisdoubletaggingPCRstrategyopensnewroutesnot
onlyforimmobilizationpurposes,butalsoactasaneasystrategyfor
labeling withgoldor quantumdots during PCR.
The nanoAu-GEC material shows interesting properties for elec-
trochemical genosensing in hybridization experiments and very
promisingfeaturesforelectrochemicalbiosensingofawiderangeof
biomolecules, such as dsDNA, PCR products, a
nity proteins, anti-
bodies,or enzymes.
