Nanotechnology, a field few people had heard of a decade ago, is now the focus of a $750 million-plus U.S. government program, not to mention the subject of Michael Crichtons latest bestseller. Scientists in this multidisciplinary field are creating new materials and devices with dimensions of 1 to 100 nanometers (billionths of a meter) and exploring a host of potential uses for these nanoscale objects.
Among the nanomaterials in the spotlight are fluorescent semiconductor nanocrystals, also known as quantum dotsclusters of a few hundred to a few thousand atoms that emit light in rainbow hues. When quantum dots were first produced in the early 1980s, researchers envisioned their use in computing, optics, and electronics. But the first practical applications of these tiny bits of semiconductor may actually occur in biology and medicine, where they show promise as an alternative to fluorescent organic dyes and proteins for labeling and imaging biological molecules in vitro and in vivo.
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There are many potential cancer-related applications, said Min Song, Ph.D., a program director at the National Cancer Institute. They include sensitive in vitro diagnostic tests; high-throughput, multiplex analysis of gene and protein expression in clinical specimens; and long-term observation of biomolecules during malignant transformation of cells in culture.
Until about 5 years ago, quantum dots were not suitable for biological applications. The dots, as synthesized, have a water-repelling outer layer that makes them insoluble in watery biological milieus. A key advance came in September 1998, when two research groups reported in Science that they had not only prepared water-soluble quantum dots but also had devised ways to conjugate, or chemically join, the dots to biological molecules such as antibodies so they could be used to label specific biological targets. To make the dots water soluble and permit conjugation to biomolecules, researchers either replace the dots hydrophobic outer layer with a different substance or coat the dots with an additional layer of material.
But many technical limitations remained. The first generation of water-soluble quantum dots fluoresced weakly in biological environments and tended to clump together and stick to things other than their intended targets. Some were toxic to living cells.
A series of recent papers, however, demonstrates that these problems have been surmounted through new approaches for modifying the surface chemistry of quantum dots. "We are seeing a surge of [biological] applicationsits great!" said Shimon Weiss, D.Sc., of the University of California at Los Angeles, who spearheaded one of the milestone 1998 studies.
Another notable development is that some bioconjugated quantum dots are now commercially available, said Shuming Nie, Ph.D., senior author of the other key 1998 study. "That basically opens the whole fieldso its no longer restricted to just a few research groups," said Nie, who holds positions at the Georgia Institute of Technology and Emory University in Atlanta.
In the January 2003 issue of Nature Biotechnology, a team led by cell biologist Xingyong Wu, Ph.D., and chemist Marcel Bruchez, Ph.D., of Quantum Dot Corp., Hayward, Calif., reported the use of quantum dot-based probes to specifically label the breast cancer marker Her2, a cell-surface receptor, on fixed and live cancer cells and tissue sections from mouse mammary tumors. Wu and his colleagues also showed that probes of two different colors could simultaneously light up distinct cellular components, including Her2 on the cell surface and proteins in the nucleus. The quantum dot-based probes were much brighter and considerably more fade-proof than comparable organic dyes.
This study, done with collaborators at Genentech in San Francisco, is the first step toward using quantum dot-based diagnostics to help guide treatment decisions for cancer patients, said Bruchez. Genentech markets Herceptin, a monoclonal antibody that targets Her2 and is used in combination with chemotherapy to treat metastatic breast cancer. But before using Herceptin, doctors need to know whether a patient has a high level of Her2, because only the 25% or so of patients with abnormally high Her2 levels on tumor cells are likely to respond to the treatment, explained Wu.
"One of the real challenges in cancer ... is diagnosing the state of a tumor and the potential for therapeutic treatment of that tumor," said Bruchez. Ultimately, he and others hope to exploit the sensitivity and multiplexing capability of quantum dots for in vitro detection of multiple protein or nucleic acid tumor cell markers that are altered at various stages of cancer. According to NCIs Song, "this capability potentially may aid physicians in making improved decisions on diagnosis, prognosis, prediction of individuals responses to therapies, selection of appropriate therapeutic regimens, and [in] monitoring recurrence of cancer." However, she noted, a great deal more work is needed to identify and clinically validate markers associated with the development and therapeutic outcomes of cancer, and this is an active area of research.
About 2 years ago, Nie and his coworkers developed an approach that vastly increases the multiplexing potential of quantum dots for detecting cancer markers. By filling tiny polymer beads with multiple colors and intensities (i.e., quantities) of dots in various combinations, the researchers created "quantum beads" with distinct optical signatures analogous to merchandise barcodes. When linked to different antibodies, peptides, or oligonucleotide probes, the barcoded beads should enable sensitive, high-throughput detection of tens of thousands of different proteins or gene sequences in clinical specimens or other samples.
Quantum beads may provide a faster, more flexible, and cheaper alternative to chip-based microarray technologies now used for multiplex analyses, Nie said. The beads seek out and bind their targets in solution rather than on the surface of a silicon chip or glass slide, cutting reaction times from many hours to a few minutes. Furthermore, Nie said, "if you want to detect a new gene or protein, we can basically just make a new bead ... and add it to the pool," rather than having to produce a completely new chip. Pittsburgh-based Crystalplex Corp., for which Nie is a consultant, and Quantum Dot Corp. are both developing bead-based barcoding technologies for commercialization.
Follow the Dancing Dots
Quantum dots also show promise for long-term, real-time imaging of living cells. "I was surprised at how much biology is done with optical microscopy," and at how many of these studies are limited by rapid fading of the organic dyes and proteins used to tag molecules of interest, said David Norris, Ph.D., a chemist at the University of Minnesota in Minneapolis. "You can shine light on [quantum dots] for a very long time and they just keep fluorescing," which opens the possibility for many biological studies that couldnt be done before, he said.
For instance, as described in the November 29, 2002, issue of Science, Norris, Benoit Dubertret, Ph.D., of the Laboratoire DOptique Physique at Centre National de la Recherche Scientifique in Paris, and several colleagues injected quantum dots encapsulated in a synthetic phospholipid bubble into Xenopus frog embryo cells and watched the embryos develop into tadpoles over a 5-day period. The dots were stable, nontoxic, and highly resistant to fading under constant illumination in vivo.
A study in the January 2003 issue of Nature Biotechnology demonstrated the use of another version of bioconjugated quantum dots to noninvasively label cultured human HeLa cells and slime mold cells for long periods without affecting cell growth or development. The researchers also used the dots for long-term multicolor in vivo imaging, and showed that the dots could specifically label targets on the surface of living cells.
Ultimately, scientists hope to use multicolored quantum dots to label many different molecules at a time and "follow the molecular dance inside the cell" at the level of individual molecules or molecular complexes such as the ribosome, says UCLAs Weiss, an expert on single-molecule imaging. For cancer researchers, this raises the prospect of observing the actions and interactions of multiple cellular proteinsfor example, components of an intracellular signaling cascadeduring malignant transformation in cell culture.
Researchers are also starting to use quantum dots for in vivo imaging in laboratory animals. For instance, Weisss group is developing dots to image tumors in small animals. To facilitate such studies, Weiss, Nie, and others are also developing biocompatible quantum dots that fluoresce in the near-infrared region of the spectrum, which should provide greater sensitivity and better resolution in vivo than visible dots. Indeed, Nie said his team is already using tumor cells tagged with near-infrared dots to study cancer cell migration and metastasis by imaging live animals in real time.
A Glimpse Into the (Nano)Crystal Ball
Although many opportunities for biological applications of quantum dots are now available, room for improvement remains. Researchers are continuing to explore various ways to modify quantum dot surface chemistry that will further improve the dots stability and optical properties in biological systems. "Its not clear if there will be one optimal coating," Weiss said. Instead, different coatings might be needed for different applications.
Researchers are also working on methods to target quantum dots of different colors to specific locations, structures, and, ultimately, individual molecules inside cells for multiplex in vivo imaging. One ongoing focus in Weisss laboratory is the development of new types of microscopes and detectors that are optimized to "capitalize on all the beautiful properties of quantum dots" for applications in cell imaging, he said.
For the longer term, some researchers hope to use quantum dots for in vivo imaging and detection of cancer in humans (see sidebar, p. 503). However, before quantum dots can be used for medical imaging, careful studies of the dots toxicity and clearance from the body will be needed.
A study in the JanuaryMarch issue of Molecular Imaging reveals some complexities of using quantum dots for in vivo imaging. Results of the study, led jointly by John V. Frangioni, M.D., Ph.D., of Bostons Beth Israel Deaconess Medical Center and quantum dot chemist Moungi G. Bawendi, Ph.D., of the Massachusetts Institute of Technology in Cambridge, indicate that the absorbance and scattering properties of living tissue will drastically affect the performance of quantum dots in vivo, so excitation and emission wavelengths will have to be selected carefully for each application. The researchers predict that infrared quantum dots will outperform near-infrared dots by several orders of magnitude in tissues with a high hemoglobin-to-water ratio, such as blood. This finding could be especially important for investigators developing biomedical imaging applications, they note.
Of course, quantum dots are not the only nanomaterial with potential biological and biomedical applications, but their multiplexing abilities are unique, Weiss said. "In the future, youre probably going to have a whole toolbox of different nanomaterials that will be useful in different situations," Norris said. "Its clear that if you want to do something thats fluorescence based, youre going to [reach for] a semiconductor particle."
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