Biomedical Engineering Reference
In-Depth Information
two short complementary stem sequences, which force the entire
sequence to form a stem-loop structure in the absence of a target. To
allow the monitoring conformation changes in MBs upon reactions
with targeted DNA, a luorophore and a quencher are covalently
conjugated at the termini of each MB strand. MBs act as FRET-based
switches that are normally in the closed or luorescence off state but
that switch to the open or luorescence on state in the presence of
target (complimentary) DNA strands. However, a drawback of MBs
is the limited quenching eficiency of the molecular quencher. When
the molecular quencher is replaced with Au NPs, the quenching is
much more eficient, resulting in more sensitive probes. As shown in
Fig. 10.10, detection of target DNA can be performed by using a hybrid
system containing a 1.4 nm Au NP, a single-stranded DNA molecule,
and a luorophore (rhodamine 6G) that is highly quenched by the NP
through a distance dependent process.
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When the single-stranded
DNA (ssDNA) binds to complementary DNA, the luorescence of this
system increases by a factor of more than several thousand, resulting
in sensitivity improvements up to 100-fold. The ability of this
approach to detect a single mismatch within a strand of DNA is 8-fold
greater than that of conventional MBs. Detection of DNA using DNA
functionalized 2.5 nm Au NPs has been realized; the Au NP functions
as both a nanoscaffold and a nanoquencher (an eficient energy
acceptor).
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Attached to this core are oligonucleotide molecules
labeled with a thiol group at one end and a luorophore at the other.
This hybrid bioinorganic interaction spontaneously assembles into
a constrained arch-like conformation on the particle surface. The
binding of the target molecules results in a conformational change,
which restores the luorescence of the quenched luorophore. Unlike
conventional MBs with stem-and-loop structures, the NP probes do
not require a stem, and their background luorescence increases
little with temperature. Researchers have demonstrated a novel
luorescent assay for DNA hybridization using Au NPs based on
the electrostatic properties of DNA.
98
This assay takes advantage
of the fact that ssDNA is adsorbed on negatively charged Au NPs
while double-stranded DNA (dsDNA) is not. The luorescence of
dye-tagged (rhodamine red or Cy-5) probe sequences is eficiently
quenched when they are mixed with Au NPs unless they hybridize
with components of the analyte. This assay can detect sub-femtomole
quantities of untagged target molecules in minutes.
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