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Conjugate disperses quantum dots in live cells

Oct 2007
Michael A. Greenwood

Quantum dots continue to prove themselves as biological imaging agents, but the task of getting the minuscule crystals into tiny cells remains daunting.

One technique conjugates quantum dots with the molecule polyarginine, which quickly binds to the cell membrane. The problem is that these nanocrystals tend to stick together and do not disperse evenly throughout the cell. This bunching is apparent on the cell surface and results in irregular labeling and unequal distribution throughout the cytoplasm upon internalization. This makes it exceptionally difficult to track a cell’s progress through several generations.

A host of other techniques, such as microinjection and electroporation, also have been used to label living cells with quantum dots. Although many of these techniques avoid the need to prepare conjugates, many require other complex and time-consuming manipulations.

Researchers at Carnegie Mellon University in Pittsburgh have found a possible alternative. They conjugated quantum dots with the molecule cholera toxin B, the nontoxic subunit of the toxin released by the pathogen that causes cholera. The nanocrystals did not clump together, and they dispersed evenly throughout the cell cytoplasm. Lead researcher Byron T. Ballou said that quantum dots conjugated with cholera toxin B exhibit noticeably different behavior from those conjugated with polyarginine. After binding to a cell’s surface, they are internalized in small vesicles. The conjugates’ ability to bind to nanoscopic lipid rafts on the cell surface, to avoid clumping and to disperse evenly once inside cells potentially makes the conjugate suited for many types of research, including long-term or multigenerational cell tracking.

The research team conjugated commercially available carboxyl quantum dots that emitted at 605, 655 and 705 nm. The 605- and 655-nm quantum dots had a cadmium-selenium core with a zinc-sulfur shell. The 705-nm quantum dots consisted of a cadmium-tellurium-selenium core and a zinc-sulfur shell.

Before the quantum dots were tested in the cellular environment, they were characterized by fluorescence correlation spectroscopy, allowing the team to identify changes in their size upon conjugation with cholera toxin B. For these studies, a Coherent Ti:sapphire laser operating at 800 nm was coupled into a Carl Zeiss inverted microscope. Two PerkinElmer avalanche photodiodes detected the fluorescence emission from the quantum dots. “It’s really quite incredible,” said researcher James A.J. Fitzpatrick. “We can actually see the differences in size between the starting quantum dot and the cholera toxin B conjugates. The actual change in diameter is less than 5 nm, which you would never be able to see in conventional fluorescence microscopy.”

An M21 human melanoma cell was labeled using 655-nm quantum dots conjugated with cholera toxin B. This image represents an overlay of a differential image contrast and a Z projection of the confocal fluorescence stack. Reprinted with permission of Nano Letters.

The prepared quantum dots were applied to five types of mammalian cells — NIH 3T3 fibroblasts, human mesenchymal stem cells, mouse-muscle-derived stem cells, M21 human melanoma and MH15 teratocarcinoma mouse tumor cells — to gauge their behavior and effectiveness. All images were taken with a Carl Zeiss inverted microscope, with excitation provided by a 405-nm diode laser. Fluorescence was detected by the Meta spectral detector inside the microscope’s scan head.

The researchers found that quantum dots conjugated with cholera toxin B were dispersed throughout the cytoplasm of each of the cells, in sharp contrast to quantum dots conjugated with polyarginine. Still, after several days, some quantum dots conjugated with cholera toxin B began to form large aggregates inside individual cells. This accumulation, however, was limited. Other cholera toxin quantum dots remained dispersed throughout the cells’ cytoplasm.

One disadvantage to the cholera toxin B molecule is that labeling occurs at a slower pace than with polyarginine conjugates. The quicker labeling offered by polyarginine makes it a better choice for some applications.

Nano Letters, September 2007, pp. 2618-2626.

quantum dots
Also known as QDs. Nanocrystals of semiconductor materials that fluoresce when excited by external light sources, primarily in narrow visible and near-infrared regions; they are commonly used as alternatives to organic dyes.
biological imagingBiophotonicsMicroscopyNews & Featuresquantum dotsSensors & Detectors

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