Biomedical Engineering Reference
In-Depth Information
and coated onto the microbeads to generate a nanobarcoded microbead termed as
QBeads. Gene-specific oligonucleotide probes are conjugated with the surface of
each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated
cRNAs are generated from sample total RNA and hybridized to the gene probes on
the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin
on the cRNA, acting as a quantification reporter. Target identity was decoded based
on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and
585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA
captured on the beads and provides the quantification for the corresponding target
gene. It provides increased flexibility, convenience, and cost-effectiveness in com-
parison with conventional gene expression profiling methods.
3.2.2 Foster (or Fluorescence) Resonance Energy
Transfer System
Several studies have demonstrated the effective use of QD FRET donors to detect
small analytes by utilizing a common strategy that relies on conjugating QDs with
target-binding receptors, which can be either proteins [
36
,
37
], antibody fragments
[
38
,
39
], or DNA aptamers [
40
,
41
]. FRET and QDs were also employed for RNA
analysis. For example, a single-stranded siRNA conjugated with QD was designed
and used as a hybridization probe for the development of a comparatively sim-
ple and rapid procedure for preliminary screening of highly effective siRNA
sequences for RNA interference (RNAi) in mammalian cells (Fig.
3.11
) [
42
].
The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and
Cy5-labeled mRNA served as a hybridization sample. The accessibility and affin-
ity of the siRNA sequence for the target mRNA site were determined by FRET
between a QD (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at
an appropriate distance from each other when hybridization occurred. The FRET
signal was observed only when there was high accessibility between an antisense
siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs.
This method can markedly facilitate the screening of truly effective siRNAs and
significantly shorten the time-consuming siRNA screening procedures.
To improve the detection sensitivity, flexibility, and adaptability, various new
strategies have been developed, such as rolling circle amplification [
43
], isothermal
amplification [
44
], and isothermal strand-displacement polymerase reaction [
45
]. A
novel miRNA detection method based on the two-stage exponential amplification
reaction (EXPAR) and a single QD-based nanosensor was developed (Fig.
3.12
)
[
46
]. EXPAR provides high amplification efficiency, which can rapidly amplify
short oligonucleotides (10
6
-10
9
-fold) within minutes. The two-stage EXPAR
involved two templates and two-stage amplification reactions under isothermal con-
ditions [
47
]. The first-stage reaction (Fig.
3.12
a, b) was an exponential amplifica-
tion with the involvement of the X′-X′ template, which enabled the amplification of
miRNA. The second-stage reaction (Fig.
3.12
c) was a linear amplification with the
