Biomedical Engineering Reference
In-Depth Information
sequence causing a FRET signal response, which was monitored by a homemade
optical sensor comprising a single 400-nm UV light-emitting diode (LED), optical
fibers, and a miniature 16-bit spectrophotometer [
10
].
In a competitive system, as shown in Fig.
3.1
a, two specific DNA probes are
required, too. They are labeled with donor and acceptor as the same as those in a
sandwich structure. The difference from a sandwich system is that these two DNA
probes are complementary to each other and one of them is complementary to the
target DNA. In the presence of target DNA, it can compete with probe A to hybrid-
ize with probe B to form a more stable duplex. The donor is thus far away from
acceptor producing a signal response to the target DNA. Krull demonstrated the
use of red-emitting streptavidin-coated QDs (QD(605)) as donors in FRET to intro-
duce a competitive displacement-based assay for the detection of oligonucleotides.
QD-DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23
were prepared as capture probe. The hybridization between capture probes and dye-
labeled (Alexa Fluor 647) reporter sequences which were partly complementary to
capture probe provided a FRET-sensitized emission signal due to proximity of the
QD and dye. After competition with target sequence, fully complementary recogni-
tion motif embedded within a 98-mer displacer sequence, a nM level for competi-
tive displacement hybridization assays for in vitro DNA analysis was achieved [
11
].
Molecular beacons are oligonucleotide hybridization probes that can report
the presence of specific nucleic acids in homogenous solutions. They are hairpin-
shaped molecules with a fluorescent label and quencher at each end. QDs are usually
employed in MBs as the donor for nucleic acid detection. As shown in Fig.
3.1
c, the
probe sequence is complementary to target DNA sequence. In the absence of target
DNA, a hairpin-like stem-loop structure is formed, causing FRET took place. Once
target DNA exists, such hairpin structure is changed into a duplex by the hybridiza-
tion between probe sequence and target DNA. This causes the separation of QD donor
from the quencher, giving a “signal on” response to the target DNA. Mattoussi et al.
described the synthesis and characterization of a thiol-reactive hexahistidine peptidic
linker that could be chemically attached to thiolated-DNA MB and mediated their self-
assembly to CdSe/ZnS core-shell QDs. The hairpin DNA stem structure brought the
dye acceptor into close proximity r of the QD establishing efficient FRET. The pres-
ence of DNA complementary to the MB would unwind the stem-loop structure alter-
ing the donor-acceptor distance to r′ and changing the FRET efficiency (Fig.
3.4
a)
[
12
]. Similarly, Chen et al. described a hybrid fluorescent nanoprobe composed of a
nuclease-resistant MB backbone, CdSe/ZnS core-shell QDs as donors, and gold nan-
oparticles (Au NPs) as quenchers, for the real-time visualization of virus replication
in living cells (Fig.
3.4
b). A hexahistidine-appended Tat peptide self-assembled onto
the QD surface was employed for the noninvasive delivery of the nanoprobe. Upon
the existence of the target sequence, coxsackievirus B6 (CVB 6) genome, a 7.3-fold
increase in fluorescent signal could be achieved and the real-time detection of infec-
tious viruses as well as the real-time visualization of cell-to-cell virus spreading could
be realized [
13
]. For multiple applications, QDs with different fluorescent emissions
have been linked to different MBs, allowing for simultaneous detection within one
solution [
14
]. MBs are extremely target specific, primarily because of the competition
