- Improving FRET efficiency for quantum dots
Technique could be useful for DNA detection without PCR
Investigators at the City University of New York have developed a
method for making quantum dots more effective in nanosensors that are based on Förster
resonance energy transfer (FRET). It could be used to detect RNA and DNA without
polymerase chain reaction (PCR) amplification, which could help improve clinical
laboratory detection of pathogens and cancer markers.
Because quantum dots can be engineered to have
specific excitation and emission spectra, they have become an important part of
genomic analysis, fluorescence imaging and other biological applications. These
qualities also make them desirable for detecting individual proteins, DNA and RNA
When the quantum dots are not in
a capillary flow, FRET efficiency for the single-stranded DNA/quantum dot complexes
increases more rapidly than for the double-stranded DNA/quantum dot complexes, most
likely because single-stranded DNA is more flexible (A). A capillary flow, however,
bends the double-stranded DNA complexes and increases FRET efficiency in a manner
similar to single-stranded DNA complexes (B). (E = FRET efficiency.)
FRET occurs when a receptor absorbs
light and transfers that energy to a nearby fluorophore, which then fluoresces.
The technique, however, presents some challenges. Its efficiency falls off with
the sixth power of the distance between the donor and the acceptor. This is a particular
problem with quantum dots because of their size and the short Förster distance
between the quantum dot and the fluorescent dye pairs.
To overcome these challenges, researchers
Chun-yang Zhang and Lawrence W. Johnson used streptavidin-biotin binding to attach
DNA labeled with the fluorescent dye Cy5 to individual quantum dots. This type of
binding is strong and stable over a wide range of temperatures. Also, each streptavidin
can bind four biotins, which theoretically allows the researchers to attach up to
45 individual strands of labeled DNA to a single quantum dot. Then, to increase
FRET efficiency, they detected it using single-molecule detection in a capillary
flow. The force of a capillary flow bent the strands of DNA, putting the Cy5 molecule
closer to the quantum dot. The work was published in the June 27 Web edition of
By using capillary flow, researchers increased the efficiency of quantum-dot-based
With this strategy, the investigators
tested the method using 25 base single-stranded DNA and 25-mer double-stranded DNA.
The single strand is much more flexible than the double, and the researchers examined
how the difference in flexibility affected the spatial distance between the quantum
dot and the Cy5 in the flow.
First, they tested how the binding
of each type of DNA affected the fluorescence of the quantum dots and the Cy5. They
found that, as the amount of DNA bound to each quantum dot increased, the quantum
dot fluorescence at 605 nm decreased and the fluorescence of the Cy5 at 675 nm increased.
At all levels of DNA binding they studied, the single-stranded DNA complexes responded
with greater increases in Cy5 fluorescence and with greater decreases in quantum
dot fluorescence than the double-stranded DNA. The increased flexibility of the
single strand allowed it to move closer to the quantum dot.
Using a single-molecule-detection strategy,
researchers measured bursts of fluorescence from the acceptor channel (top) and
the donor channel (bottom) created by the quantum dot and double-stranded DNA complexes
as they moved through the laser focal volume.
To conduct these measurements, the
scientists housed the bulk solution in a 700-ml quartz cuvette and collected the
spectra with a PerkinElmer luminescence spectrometer. They used 488 nm as an excitation
wavelength and detected fluorescence from 550 to 725 nm. They measured absorption
spectra using a PerkinElmer UV/VIS/NIR spectrometer and recorded the spectral range
of 550 to 700 nm at a rate of 600 nm/min.
To further investigate the potential
for this method to improve FRET-based nanosensors, Zhang and Johnson tested it in
a capillary flow using a single-molecule-detection setup. The flow brings the DNA
closer to the quantum dot, and the speed of the flow movement prevents photobleaching
of the fluorescent dyes attached to the DNA complexes.
They used a Coherent argon laser at
488 nm for excitation. After collimating the beam and passing it through a dichroic
mirror, they focused it on the center of a 50-mm capillary with a 100x oil-immersion
Olympus lens. A syringe pump moved the solution through the capillary. The same
objective collected fluorescence, which passed through the first dichroic mirror,
and a 50-mm Melles Griot pinhole. Then it was separated by a dichroic mirror, and
a bandpass filter filtered out the Cy5 signal. An EG&G avalanche photodiode
detected the Cy5 signal. The quantum dot signal at 605 nm was filtered by another
bandpass filter and detected by another avalanche photodiode.
By detecting the Cy5 and the quantum
dot fluorescence separately, the researchers could correlate bursts of fluorescence
from the quantum dot with the Cy5 fluorescence. Interestingly, they observed more
bursts of fluorescence from the double-stranded DNA complexes than from the single-stranded.
When they calculated the average distance between the Cy5 fluorophore in both ensemble
solution and in the capillary flows, they found no appreciable difference for the
single-stranded DNA complexes.
However, the capillary flow had a significant
effect on the stiffer, double-stranded DNA complexes. The separation distance went
from 120 ±4.3 Å to 95.9 ±8.5 Å. Further investigation revealed
that the single-stranded complexes were quenched by Cy5 dimer formations and the
inner-filter effect more readily than the double-stranded complexes.
Zhang and Johnson say that this work
could lead to more rapid detection of nucleotides without amplification. “It
can find wide applicability in the sensitive detection of bacteria, viruses and
cancer markers in clinical diagnosis,” Johnson explained. He added that observation
techniques had limited the study of DNA deformation in a fluid flow to large molecules,
such as λ-DNA.
“This procedure also may offer
a promising approach to study the deformation of small nucleic acids in fluid flow,”
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