Biology Reference
In-Depth Information
oligonucleotide capture probes onto the transducer surface and
hybridization of single-stranded oligonucleotide target which is
labeled with haptens (biotin, fluorescent, digoxigenin, etc.) on one
end during PCR. Thus, this method can only be applied on PCR
amplicons that are labeled with haptens on one end. The two-
component complex on the transducer surface will enable binding
of a specific anti-hapten-conjugated HRP or alkaline phosphatase
(AP) reporter enzyme to the probes-target complex. Addition of the
enzyme-specific redox substrate and application of a fixed potential
between working and reference electrodes on the transducer
surface generates an enzyme-mediated redox cycle and detected
in the form of a current (Fig. 15.2A). The electroredox current
amplitude reveals the concentration ofthe probe-target complexes.
15.2.2.2 Indirect method
This approach for species-specific identification of bacterial
pathogens involves immobilization of a single-stranded oligonu-
cleotide capture probe onto the transducer surface, followed by
hybridization of single-stranded oligonucleotide targets and a
detection probe which is labeled with haptens (biotin, fluorescent,
digoxigenin, etc.) on one end. This method can be performed even
without labeling the PCR amplicons. The detection of the three-
component “sandwich” complex (capture probes-target-detection
probes) on the transducer surface is the same as using the direct
method (Fig 15.2B).
15.2.2.3 Rapid method
In this approach double-stranded oligonucleotide PCR amplicons
are labeled with a hapten on one end and another different hapten
ontheotherendduringPCR.Thetransducersurface(goldorcarbon
screen-printed chips) is treated with protein based anti-haptens
that capture one of the hapten label on the PCR amplicons. The
second hapten label on the PCR amplicons will bind to a specific
anti-hapten-conjugated HRP or AP reporter enzyme to labeled PCR
amplicons. Addition of the enzyme-specific redox substrate and
application of a fixed potential between working and reference
