Chemistry Reference
In-Depth Information
Figure 14.6
A catalytic beacon Pb
2+
sensor: (Left) Schematics of beacon signal generation;
(Right) selectivity of the sensor, inset: kinetics of fl uorescence increase with Pb
2+
or other
divalent metal ions. (Reprinted with permission from
J. Am. Chem. Soc
., 2000,
122
, 10466-
10467.)
the lead sensor. Furthermore, a fl uorophore was used to label the 3
′
- end of the
enzyme strand and a fl uorescence quencher to label the 5
- end of the substrate.
Close proximity between the fl uorophore and quencher resulted in a low fl uores-
cence background. When lead was present, the enzyme was activated to cleave the
substrate at the cleavage site. The smaller substrate fragment could not stay hybrid-
ized to the enzyme strand. Therefore these two strands were detached, causing
separation of the fl uorophore and quencher and consequently restored fl uorescence.
This sensor turned out to be highly sensitive for lead ions with a detection limit of
10 nM. It also responded
′
80 times better to Pb(II) than to the next competitive
metal ion (Co(II) and Zn(II)) and hundreds times better to other metal ions tested.
One problem with this sensor was that it required 4 °C for reasonably low fl uores-
cence background and effective detection. To overcome this limitation, a new sensor
design was proposed, which added an additional quencher at the 5
>
- end of the sub-
strate strand.
69
This internal quencher ensured a low background in the absence of
lead even when the substrate was not effi ciently hybridized to the enzyme. Indeed,
a much improved signal-to-background ratio was obtained and sensitive lead detec-
tion could be conducted at room temperature. Two additional advantages of the
fl uorescent DNAzyme sensor are worth mentioning. First, since the detection is
based on the kinetics of fl uorescence enhancement, it is less affected by background
fl uorescence in environmental samples. Additionally, because catalytic reactions are
responsible for signal generation, a single Pb
2+
ion can catalyse the turnover of
multiple fl uorescent DNAzymes when even higher sensitivity is desired.
This successful sensing strategy has been applied to other DNAzymes and their
metal ion cofactors. One notable example is the sensor for
UO
2+
recently developed
in our lab (Figure 14.7).
43
A DNAzyme specifi c for
UO
2+
was fi rst identifi ed via
in
vitro
selection. Following that, the same one-fl uorophore - two - quencher approach
was adapted to the new DNAzyme and
UO
2+
detection was achieved with excellent
sensitivity and selectivity. Most sensors based on molecular probes are not expected
′
