Biology Reference
In-Depth Information
The utility of Av-GEB platform was demonstrated for the deter-
mination of the mecA DNA sequence related with methicillin-
resistant
S. aureus
(MRSA) [85] in a simpler and specific manner
withrespect to previous DNA biosensingdevices [25].
The genosensor design based on Av-GEB not only is able to
successfully immobilize onto the electrode surface with the mecA
biotin-labeledcaptureprobe,whilethehybridizationwiththemecA
target and the mecA digoxigenin-labeled probe is occurring at the
same time, butisalso capable ofdistinguishing SNPs.
Compared to genosensors based on GEC, the novelty of this
approach is in part attributed to the simplicity of its design, com-
bining the hybridization and the immobilization of DNA in one ana-
lytical step.
The optimum time for the one-step immobilization/
hybridization procedure was found to be 60 min [74]. The pro-
posed DNA biosensor design has proven to be successful in using
asimplebulkmodificationstep;hence,overcomingthecomplicated
pre-treatment steps associated with other DNA biosensor designs.
Additionally, the use of a one-step immobilization and hybridiza-
tionprocedurereducestheexperimentaltime.Stabilitystudiescon-
ducted demonstrate the capability of the same electrode to be used
for a 12-week period [74].
The rapid electrochemical verification of the amplicon com-
ing from the
Escherichia coli O157:H7
genome was performed by
double-labeling the amplicon during PCR with a set of two labeled
PCRprimers—oneofthemwithbiotinandtheotheronewithdigox-
igenin. During PCR, not only the amplification of the
E. coli
was
achieved but also the double-labeling of the amplicon ends with (i)
the biotinylated capture primer to achieve the immobilization on a
biosensor based on a bulk-modified avidin biocomposite (Av-GEB)
and(ii)thedigoxigeninsignalingprimertoachievetheelectrochem-
icaldetection.Theprocedureconsistedbrieflyofthefollowingsteps:
(i) DNA amplification and double-labeling of the
eaeA
gene, related
withthepathogenicactivityof
EscherichiacoliO157:H7
;(ii)immobi-
lization of the doubly labeled amplicon in which the biotin extreme
of the dsDNA amplicon was immobilized on the Av-GEB biosensor;
(iii) enzymatic labeling with anti-DIG-HRP capable of bonding with
the other labeled extreme of the dsDNA amplicon; and (iv) ampero-
metric determination [75].
