26
miRNAs catalyzed the reduction of silver ions to metallic silver, which further
autocatalyzed the reduction of silver ions to form metallic silver precipitation on
gold, resulting in a signal enhancement [52]. This process allowed straightforward
detection of the microarray with an ordinary charge-coupled device (CCD) camera
mounted on a microscope.
They found that QD probes offered decent sensitivity down to sub-femtomolar
concentrations and dynamic range above several orders of magnitude. This was
significantly better than other dye-based methods and further hindered the use of
amplification while allowing a semi-quantitative comparison of the amount of
miRNA in different samples [53].
Figure 2.11 displays a set of images for various concentrations of miRNA (21 nt
siRNA) detected by QD [53]. It should be noted that the signals become gradually
weaker with the decrease in miRNA concentration (Fig. 2.11a). When the miRNA
concentration was as low as 39 pM, the fluorescence signal could be detected,
indicating that the lower detection limit of miRNA microarrays is at least 0.4 fmol.
As shown in Fig. 2.11b, the fluorescence intensity of the spots is linear to the model
miRNA in a logarithmic fashion from 156 to 20,000 pM, and the dynamic range is
about two orders of magnitude. This implies that this method can be used to quantify
miRNAs with a broad concentration range [53].
2.3.4.3 Sensing Based on FRET with Quantum Dot Bioconjugates
Fluorescence Resonance Energy Transfer (FRET) is a physical process in which
radiationless transmission of energy phenomena occurs depending on the distancedependent transfer of energy from a donor molecule to an acceptor molecule. FRET
has been extensively used in biophysical and biochemical studies to probe ligandreceptor binding and molecular structural changes [55–57]. In the following example, the authors incubated the dye-labeled DNA targets with biotinylated capture
DNA probes, which were allowed to be conjugated to streptavidin QDs, only when
the two DNA sequences hybridize. The resulting hybridization was then detected
RNA
A
B
RNA
RNA
HO
oligonucleotide
probes
biotin-labeled
miRNAs
hybridization
p r o c e s s e d
s il v e r
e n h a n c e m
e n t
p r o c e s s e d
w it h Q D
w it h g o ld
Base
Base
Base
Biotin-X-hydrazide
Biotin
NaIO 4
OH
O
O
O
O
O
N
Fig. 2.10 Schematic principles of the miRNA profiling microarray. (Adapted from Ref. [52])
J. H. Banoub and A. Mikhael
miRNAs catalyzed the reduction of silver ions to metallic silver, which further
autocatalyzed the reduction of silver ions to form metallic silver precipitation on
gold, resulting in a signal enhancement [52]. This process allowed straightforward
detection of the microarray with an ordinary charge-coupled device (CCD) camera
mounted on a microscope.
They found that QD probes offered decent sensitivity down to sub-femtomolar
concentrations and dynamic range above several orders of magnitude. This was
significantly better than other dye-based methods and further hindered the use of
amplification while allowing a semi-quantitative comparison of the amount of
miRNA in different samples [53].
Figure 2.11 displays a set of images for various concentrations of miRNA (21 nt
siRNA) detected by QD [53]. It should be noted that the signals become gradually
weaker with the decrease in miRNA concentration (Fig. 2.11a). When the miRNA
concentration was as low as 39 pM, the fluorescence signal could be detected,
indicating that the lower detection limit of miRNA microarrays is at least 0.4 fmol.
As shown in Fig. 2.11b, the fluorescence intensity of the spots is linear to the model
miRNA in a logarithmic fashion from 156 to 20,000 pM, and the dynamic range is
about two orders of magnitude. This implies that this method can be used to quantify
miRNAs with a broad concentration range [53].
2.3.4.3 Sensing Based on FRET with Quantum Dot Bioconjugates
Fluorescence Resonance Energy Transfer (FRET) is a physical process in which
radiationless transmission of energy phenomena occurs depending on the distancedependent transfer of energy from a donor molecule to an acceptor molecule. FRET
has been extensively used in biophysical and biochemical studies to probe ligandreceptor binding and molecular structural changes [55–57]. In the following example, the authors incubated the dye-labeled DNA targets with biotinylated capture
DNA probes, which were allowed to be conjugated to streptavidin QDs, only when
the two DNA sequences hybridize. The resulting hybridization was then detected
RNA
A
B
RNA
RNA
HO
oligonucleotide
probes
biotin-labeled
miRNAs
hybridization
p r o c e s s e d
s il v e r
e n h a n c e m
e n t
p r o c e s s e d
w it h Q D
w it h g o ld
Base
Base
Base
Biotin-X-hydrazide
Biotin
NaIO 4
OH
O
O
O
O
O
N
Fig. 2.10 Schematic principles of the miRNA profiling microarray. (Adapted from Ref. [52])
J. H. Banoub and A. Mikhael