Biology Reference
In-Depth Information
in which the biotin-labeled capture probe is immobilized on the
streptavidin magnetic beads, while the hybridization with the tar-
get and with a second complementary probe—in this case labeled
with digoxigenin—is occurring at the same time (30 min at 42
◦
C)
(Fig. 3.5, A1); (ii) enzymatic labeling using as enzyme label the
antibody anti-DIG-HRP (Fig. 3.5, B); (iii) magnetic capture of the
modified magnetic particles (Fig. 3.5, C); and (iv) amperometric
determination based on the enzyme activity by adding H
2
O
2
and
using hydroquinone as a mediator (Fig. 3.5, D) [97]. When the
DNA target immobilized on the magnetic beads was detected by
the intrinsic DNA oxidation signal coming from the guanine moi-
eties, the procedure consists of the following steps, as previously
described in detail [96]: (i) electrochemical pre-treatment of the m-
GECtransducer;(ii)biotinylatedinosine-substitutedprobeimmobi-
lization on streptavidin magnetic beads; (iii) hybridization with the
target; (iv) magnetic capture of the modified magnetic particles, fol-
lowedbydryadsorption,wasperformedduring45minat80
◦
C;and
(v) electrochemical determination based on DPV.
The procedure for electrochemical DNA biosensing based on
magnetic beads was also used for the detection of IS200 element
specific for
Salmonella
spp.
This new electrochemical genomagnetic strategy using magneto
electrodesinconnectionwithmagneticparticlesoffersmanypoten-
tialadvantagescomparedtomoretraditionalstrategiesfordetecting
DNA.
This new strategy takes advantages of working with magnetic
particles, such as improved and more effective biological reactions,
washing steps, and magnetic separation after each step. This elec-
trochemicalgenomagneticassayprovidesmuchsensitive,rapid,and
cheaper detection than other assays previously reported. This sen-
sitivity of the GEC with respect to other electrochemical transducer
andselectivityconferredbythemagneticseparationwerealsoused
for the detection ofPCR amplicons coming from real samples.
The rapid electrochemical verification of the amplicon com-
ing from the
Salmonella
IS200 element [97] as well as the
eaeA
gene, related with
Escherichia coli O157:H7
[75] was performed
by double-labeling the amplicon during PCR with a set of two
labeled PCR primers—one of them with biotin and the other one
