Chemistry Reference
In-Depth Information
Figure 14.7
(Plate 14)
Design and performance of DNAzyme-based
UO
2+
sensor. Compet-
ing metals were tested from 10
m
M to 1 mM, and
UO
2+
(the last three bars) was tested from
0 to 10 nM. Inset: Sequence, schematics and fl uorescent detection of the DNAzyme-based
UO
2+
sensor. (Juewen Liu
et al
., A catalytic beacon sensor for uranium with parts-per-trillion
sensitivity and millionfold selectivity,
Proc. Natl. Acad. Sci. USA
,
104
, 2056-2061. Copyright
2007, National Academy of Sciences, USA.) (See colour plate section)
to compete or exceed instrumental analysis. However, the
UO
2+
DNAzyme sensor
was able to realize a detection limit of about 45 pM, even lower than that obtained
on ICP-MS (420 pM). In addition, it has a selectivity of more than one million-fold
over any other metal ions tested.
Development of fl uorescent sensors for paramagnetic metal ions (i.e., those
metal ions with unpaired electrons) such as Cu
2+
has long been a challenge, due to
their intrinsic fl uorescence quenching properties that often cause decreased emis-
sion upon metal binding. On the other hand, DNAzyme-based sensors are less
restricted by this problem since the metal-recognition core of the enzyme can
be separated from the fl uorescent signalling moiety, thus minimizing the contact-
induced quenching. Therefore our laboratory developed a fl uorescent DNAzyme
sensor for Cu(II) based on a previously reported Cu(II) DNAzyme by Breaker and
coworkers.
12,70,71
This DNA-cleaving DNAzyme was tagged with a fl uorophore and
two quenchers similar to the sensors above.
72
With this sensor, Cu
2+
was readily
detected down to 35 nM at room temperature, and the selectivity was 2000-fold better
than other tested ions.
We have recently designed a modifi ed DNAzyme sensor for Hg
2+
detection,
using the dual quencher method to construct a catalytic beacon. Five T-T mis-
matches replaced the double-strand in the loop region of the
UO
2+
DNAzyme that
is required for binding
UO
2+
, thus inactivating the enzyme. Addition of Hg
2+
could
stabilize the T - T mismatches,
73 - 75
resulting in restored activity. This sensor has a
detection limit of 2.4 nM, the lowest among known Hg
2+
sensors based on small or
macromolecules.
The examples shown above all require labelling with fl uorophores and quench-
ers on the termini of the DNA strands. However, the ideal positions to place the
fl uorophore and quencher pair would be on the two nucleotides closest to the cleav-
age site on the substrate strand. In this way, the quencher on the enzyme strand
would no longer be necessary since the quencher and fl uorophore on the substrate
could stay close enough for minimum background. Cleavage of the substrate would
then separate this pair and generate enhanced fl uorescence. One concern for this
design is possible activity decrease due to these two modifi cations near the active
