Chemistry Reference
In-Depth Information
site. To investigate the feasibility of internal fl uorophore/quencher labelling on
DNAzyme, Li and colleagues conducted systematic studies by placing different
dye/quencher pairs at various locations on the substrate.
76
The results indicated that
generally, the closer the dye/quencher to the cleavage site, the higher the fl uores-
cence enhancement upon lead-induced enzymatic cleavage. However, the reaction
rates were also lowered as a result. Given these observations, internal dye/quencher
labelling for making DNAzyme sensors is feasible, but the design needs to be
optimized.
Instead of developing sensing strategies based on known DNAzymes, one
appealing alternative would be directly selecting DNAzymes with a built-in signal
transduction mechanism. Li and coworkers have realized this experimentally by
modifying the general
in vitro
selection scheme.
16,77
Compared to conventional
selection, these authors added a few extra steps to each round to introduce a short
nucleic acid strand to the DNAs in the pool before reaction in the presence of metal
ions. This short strand contained an adenine ribonucleotide (rA) as the cleavage
site, and a fl uorophore and quencher labelling the two nucleotides closest to rA.
When incubated with target metal ions, only those sequences that could cleave the
rA site were collected and enriched. The fi nal products of this selection were
DNAzymes that were able to function normally with the internal labels and gener-
ate greatly enhanced fl uorescence upon addition of target metal ions.
14.5.2 Colorimetric Sensors Based on DNA zymes
Fluorescence-based DNAzyme sensors are excellent for metal ion detection due to
their high sensitivity and selectivity. They also provide a solution to on-site metal
analysis when equipped with portable fl uorometers. However, to further reduce the
cost and simplify the process of metal sensing, thus making it readily accessible to
a much wider range of fi elds, including clinics, battlegrounds and even households,
new strategies need to be employed. We have made progress in this direction with
the development of colorimetric metal ion sensors.
In a colorimetric sensor, a colour change is observed in the presence of target
analytes. Compared to other techniques, colorimetric sensors can minimize or even
eliminate the need for analytical instruments, making on-site detection much more
convenient. Some common colour-reporting groups include small organic dyes,
conjugated polymers and metallic nanoparticles. Among them, metallic nanoparti-
cles display strong distance-dependent optical properties and very high extinction
coeffi cients.
78
Therefore, we designed our fi rst - generation colorimetric lead sensor
based on DNAzymes and DNA-functionalized gold nanoparticles (AuNPs).
79
Briefl y, the substrate of '8-17' DNAzyme was extended at both ends with a short
DNA strand of the same sequence. In this way, when AuNPs functionalized with the
complementary DNA (cDNA) of the short extension were introduced, the AuNPs
became literally crosslinked by the extended substrate to produce aggregated nano-
particle clusters (Figure 14.8). Such AuNP aggregates display a blue colour, due to
their strong absorption of light with wavelengths around 700nm. The '8-17'
DNAzyme was then introduced to hybridize with the remaining sequence of sub-
strate. When Pb(II) was present, the enzymatic reaction was initiated to cleave the
