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  • Improving FRET efficiency for quantum dots

Aug 2006
Technique could be useful for DNA detection without PCR

Kevin Robinson

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 using FRET.

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 Analytical Chemistry.

By using capillary flow, researchers increased the efficiency of quantum-dot-based FRET.

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,” he said.

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