Biology Reference
In-Depth Information
In the case of the electrocatalytic detection, a linear relationship
between the recorded current and the logarithm of the concen-
tration of ply target is obtained for concentrations between 5 and
100 pg/
μ
L. These genosensors can detect 5 pg/
μ
L (24.5 fmol
in 30
μ
L) of complementary ply target, using the electrocatalytic
detection.
To improve the selectivity of the ply genosensor, more stringent
experimental conditions are tested. A concentration of 25% for-
mamide is added to the hybridization buffer. It is well known that
this molecule hampers the hybridization reaction. In these more
stringentconditionsandusingtheenzymaticdetection,alinearrela-
tionshipbetweenpeakcurrentandconcentrationofoligonucleotide
target is obtained for concentrations between 0.25 and 5 pg/
μ
L.
Genosensors can detect about 1.2 fmols of complementary target in
30
μ
L in these more stringentexperimental conditions.
In the case of electrocatalytic detection, a linear relationship
between the recorded current and the logarithm of the concen-
tration of oligonucleotide target is obtained for concentrations
between 50 and 1000 pg/
μ
L.
Usingthisstrategyofdetection,thegenosensorscandetectabout
245 fmol of complementary target in 30
μ
L in these more stringent
experimental conditions.
As expected, the sensitivity decreases in these stringent
experimental conditions for both enzymatic and electrocatalytic
detection but the detection of one-base mismatch on an oligonu-
cleotide sequence can be performed for any concentration assayed
(Fig. 9.12).
Although the sensitivity of the electrocatalytic detection is 50-
fold (under non-stringent conditions) and 200-fold (using 25%
formamide in the hybridization solution) lower than that obtained
with the enzymatic detection, the analysis time is considerably
shorter, because the analytical signal is achieved directly from
the platinum complex whereas in the enzymatic detection two
additional steps are necessary to obtain the analytical signal:
the reaction with antibody anti-fluorescein and the enzymatic
reaction.Thus,theoverallanalysistimeofthischronoamperometric
method is about the half than that resulting from the enzymatic
method.
