By David N. Leff

If the mother of a child with Down's syndrome becomes pregnant again, she will likely undergo prenatal diagnosis to find out whether the fetus she is now carrying also harbors an extra chromosome 21.

There is an off-the-shelf DNA probe that can detect the redundant chromosome by hybridizing with its genomic look-alike. The probe is hitched to a light-emitting protein called fluorescein, which turns bright green in water. (Such organic dyes may be radioactive.) If the probe lights up three times rather than two, obviously it's found that third copy.

The technique is called FISH — fluorescence in situ hybridization. FISH can hunt down multiple chromosomal anomalies by applying more than one differently color-coded dye.

"Our non-isotopic quantum dots would be very attractive for multicolor FISH," observed analytical chemist Shuming Nie, at Indiana University, in Bloomington. "They might replace the organic dyes now used in that detection technique.

"A quantum dot [QD] is a very small, nanometer-scale, crystalline semiconductor particle, which glows in different, very bright colors, depending on its size," he explained.

"When we make it bigger, it goes to shorter wavelengths. For example, a very small QD, one to two nanometers [nm] in diameter, is missing the light wavelength for blue, so it lights up blue. At three to four nm," he said, "it's greenish-yellow; [at] five to six, red."

QDs can be pictured as ultra-tiny flakes or grains off conventional semiconductors, used in computer chips. Like these, they consist typically of cadmium, selenium and zinc compounds.

"We make QDs," Nie described, "through a chemical synthesis procedure in an airtight hood — a glove box — in which we add some of the components. For example, the QD we study consists of cadmium and selenide, two precursor compounds, which form the core of the particle.

"In the next step," he continued, "we have to cap this core particle, which has certain common defects. So we coat it with an outer shell of arsenic, zinc and sulfur. The outer shell confines the electrons on the inside, something like insulation."

Nie is senior author of a paper in today's Science, dated Sept. 25, 1998, titled, "Quantum dot bioconjugates for ultrasensitive nonisotopic detection."

An adjacent article by chemist A. Paul Alivasatos, at the University of California, in Berkeley, is headed, "Semiconductor nanocrystals as fluorescent biological labels."

Biology Wins One From Physical Chemistry

"I think our paper in Science," Nie told BioWorld Today, "together with the one from the UC/Berkeley group, represents the first application of QDs in the biological world. Most people in the QD field believed that the main or early use would be in the optical electronics field, in transistors, before thinking they could probably make a QD laser, or high-density memory.

"No one ever predicted," Nie went on, "that the first application of this kind of QD research would actually be in the biological sciences. That's really a surprising element."

Both groups reported experimental demonstrations of QD performance in biological settings.

"We used it to study receptor-mediated endocytosis [cell engulfment] in cell biology," Nie recounted. "We conjugated QDs to transferrin, the iron-transfer protein required by all cells to bring in [iron] from the outside. We incubated that conjugate in cell culture for some time, then found that the cell actually took in the transferrin with the QDs."

Nie went on: "It demonstrated that these kinds of quantum dots are biocompatible, non-toxic and very stable. We could illuminate them with a laser beam for a long time, and they'd still be active.

"A typical organic dye," he pointed out, "can be damaged by that treatment very quickly. It would be photochemically decomposed — photo-bleached."

Alivasatos and his co-authors carried out a labeling experiment on mouse fibroblast cells, demonstrating dual emission from single excitation. Their QDs, too, consisted of a cadmium selenide core nanocrystal, coated with a shell of cadmium sulfur, to boost fluorescence and reduce photo-bleaching. Both layers were wrapped in a shell of silica for water solubility.

Small (2 nm) green-glowing QDs penetrated the mouse cells' nuclei; larger (4 nm) ones, emitting red light, attached to actin filaments along the outer cell membrane. Both labels were visible to the naked eye, and could be photographed with a Polaroid camera.

Will Quantum Dots Replace Organic Marker Dyes?

"The development of semiconductor nanocrystals for biological labeling," Alivasatos commented, "gives biologists an entire new class of fluorescent probes for which no small organic molecule equivalent exists. In some cases, they may be superior to existing fluorophores."

Nie said that his quantum-dot work is "completely independent" from that of the Science paper's California team.

"The two groups have used very different approaches," he pointed out. "The one key difference is the way we make these QDs water-soluble, so as to be compatible with the biological environment.

"We used a bifunctional compound, mercaptoacetic acid," he related. "One end is a sulfur group that binds with the QD. The other is a carboxylic acid, which makes it water-soluble.

"In their case," he added, "they use a silica layer, and do not attach the QD to any biological molecule covalently."

Nie sees the biomedical uses of QDs as "primarily in the diagnostic area."

"This kind of QD," he surmised, "would be used to detect a label in immunoassays for cardiovascular diseases and cancer. Whereas," he observed, "infectious-disease diagnostics are mainly based on nucleic-acid detection, also very suitable for this kind of quantum dot."

He and his lab are "now in the process of preparing large quantities of these QDs, at different colors, to make them commercially available for biological people, as well as for the chemical industry," Nie said. *