For all the hype about nanotechnology, sometimes small isn't quite small enough. Quantum dots enable imaging advances in fields from oncology to neuroscience, yet at a whopping dozen nanometers or more, sometimes they're just too big. "They're the size of proteins," says Marcel P. Bruchez, cofounding scientist at Quantum Dots Corp. "Anything you can do to minimize the size will minimize the impact on the biological system."

A quantum dot's size governs the color of light it emits, but the size that determines the optical properties is only the core-shell. The problem is that for biological applications, quantum dots must be changed from being hydrophobic as grown, to hydrophilic, without a loss in fluorescence or stability.

The solution is to create the high-tech equivalent of a peanut M&M: a semiconductor core (usually cadmium sulfide, selenide, or telluride), coated by an insulating shell, which is then given a ligand coating, sometimes called a cap. The amphiphilic ligand is hydrophobic where it interfaces with the shell and hydrophilic where it interfaces with the biological conjugate (e.g., antibodies, peptides, or oligonucleotides) and the environment.

"When you have a quantum dot, you have to put it in water," says Moungi Bawendi,[1] a chemistry professor at the Massachusetts Institute of Technology. "There are basically two parallel ways to do it. You can do a cap exchange, where you change the molecules that are initially on the surface with new molecules that have functionality that make it compatible with biology. Or, you can use a micelle-like, wrapping approach, which is that you keep the groups that are originally on the surface and you encapsulate them with the new material."

Both approaches have been proven, at least in principle, in the literature, and both have been shown to work reasonably well in biological imaging, according to Bawendi. The downside is that wrapped dots, like children in snowsuits, become bulkier with the additional layers. "Most of the commercially available dots encapsulate the metal part within a polymer shell and then you hook peptides onto that shell, so they tend to be much larger molecules," says Philip Dawson, an assistant professor of cell biology and chemistry at the Scripps Research Institute in La Jolla, Calif.

Thus the difficulty of the situation: In order to make a dot useful for biological applications, scientists may have to increase their hydrodynamic size so much that it becomes too big for the application for which it was intended. For many biological applications, of course, including live-animal imaging, the currently available sizes are fine. But for researchers working on membrane channels or using fluorescence resonance energy transfer (FRET), ongoing research to create smaller dots will likely be extremely valuable. "I love the tools, I'm eager to find an application for them," says Barbara Ehrlich, professor of pharmacology at Yale University, "But some smaller ones would be useful."

Shimon Weiss, head of the single-molecule biophysics group at the University of California, Los Angeles, recently described a potential answer to the size problem in a cover story review earlier this year.[2] Weiss' method, and an approach taken by Hedi Mattoussi of the US Naval Research Laboratory, is a slender variant on cap exchange. The peptides are attached directly to the cap, rather than as a next step after an interfacing ligand. "The advantage is in one molecule, he has something that is already biocompatible," says Bawendi.

Though earlier work has been published on peptide-coated quantum dots, Mattoussi says that Weiss has the first published results that show controlled binding and targeting. (Mattoussi's work with Dawson has a similar goal – to prepare compact quantum dot-peptide conjugates – but uses a different approach. A patent application has been submitted on this method, and a manuscript is in preparation.)

Researchers using FRET could find the resulting smaller size advantageous, says Dawson. "If you want to do a FRET study where you're looking for energy transfer between two fluorophores, one would be the dot, the other would be an organic fluorophore or quenching molecule," he explains, "With many of the very large polymer-coated dots, by the time you get to the surface of that polymer coating, you're at the very longest distance that is effective for FRET because you're a very long away from the center of the quantum dot. One of the significant things I've seen from Hedi's [Mattoussi's] group is that they've been able to put other fluorophores much closer to the dot, and then been able to use energy transfer to monitor molecular interactions using quantum dots."

Bruchez says currently available dots from QDC would likely be no more than six nanometers larger than the peptide dots, but that variation, he concedes, could be an important difference in certain situations, such as working with ion channels.

Both Bruchez and Clinton Ballinger, CEO of Evident Technologies, another quantum dot supplier, say their customers seem to prefer generic type dots with hydrophilic ligands that can bind to any biomolecule researchers care to attach. Evident Technologies has just released second-generation EviTags that have what Ballinger calls a "more natural" lipid coating. "The coating is so thin, you can have FRET across it, so you can tunnel between the dot and the dye that attaches to the dot. There's not a lot of distance between the dot and the dye."

Researchers would also like to harness quantum dots to monitor events in the nucleus. As with FRET, nuclear transport is bounded by size constraints, in this case the diameter of the nuclear pore, which is between 20 and 50 nm. Quantum dots typically come very close to that limit, measuring on average 12 to 30 nm in diameter when solubilized.

Chemist Fanqing Chen of Berkeley Lab and physicist Daniele Gerion of Lawrence Livermore National Laboratory published a solution to this problem last year in Nano Letters,[3] and again this past March at the American Chemical Society's national meeting in San Diego. "I think it was the first time I've seen that kind of work successfully done with the quantum dots," says Bruchez. "I thought it was a very exciting talk and a real step forward in the power of peptides in biological targeting."

Chen and Gerion based their approach on the 1998 work of Paul Alivasatas, with whom Gerion did her postdoctoral research.[4] Ironically, it should not have made for smaller dots, says Bawendi, since it involves a multilayered approach. Yet that is exactly what the pair achieved: engineering dots that are small enough (10–15 nm in diameter) to track the biological processes inside the nucleus of live HeLa cells.

Chen and Gerion attached a nuclear localization signal from the SV40 virus large T antigen to the dots to target them to the nucleus. Dots conjugated to a random peptide, in contrast, distributed about the cell, but remained locked out of the nucleus. Chen says they plan next to use HIV-TAT to deliver the dots, since it will likely be more efficient. They hope to attach protein targeting mechanisms to the quantum dots to follow other processes such as DNA replication or transcription, and then imaging them in real-time over hours, if not days. "A lot of reactions you see in vitro don't really exist in vivo," says Chen. "This will be a way to find that out."

Though smaller dots may generate the instant "ooh-aah" factor, Weiss sees the real value of his work as providing a "peptide toolkit," which will advance the potential applications of quantum dots. "We can put a mixture of different peptides on each particle," says Weiss. "It makes the dots look like proteins. If they look like proteins, you can almost give them the function of proteins. Beyond having just fluorescence of different colors, you can actually put on a dot's surface an antibody, an enzyme, a PEG [polyethylene glycol] that allows for better solubility, a chelator that can actually carry with it a radioactive tracer."

In other words, quantum dots should be viewed as a platform upon which to scaffold additional functions. The same dot that a researcher uses initially for imaging could then be enabled to perform other biological roles by adding additional peptides. "We have that adhesive sequence that is the same," says Weiss, "but then the hydrophilic part of that peptide can take all the functions that we want, so in one step you can have all these different functions."

Which doesn't sound so different from what's already available, say both Bawendi and quantum dot expert Shuming Nie[5] of the Winship Cancer Institute at Emory University in Atlanta. "I think it's interesting, but it doesn't offer any real advantages over the materials from [Quantum Dot Corp.]," says Nie. "It's difficult to control the number of ligands per dot and the orientation of ligands on the dot. Some orientations are better than others. These problems can determine the bioactivity of the quantum dot. I don't think the peptides offer better specificity or control."

But Weiss' graduate student, Fabien Pinaud, says his group's method does allow for higher avidity than competing technologies. Multiple peptides mean multiple binding sites on the dot's surface, potentially overcoming the occasional problem of ligands "falling off" the quantum dots.

Although Bawendi says Weiss' work is "great," he is even more impressed with the review paper and its suggestions for extending the uses of quantum dots. (Mattoussi has also written a comprehensive review on quantum dot solubilization[6] ; both his and Weiss' articles detail several solubilization methods in addition to the peptide-based approach.)

"The review itself is beautiful," says Bawendi. "It's a review paper that everybody's going to study, because he [Weiss] really shows what's possible to do with quantum dots, [such as] the different ways to use quantum dots to target things, to label with radionucleotides. It's really timely because those are all things that are possible to do with dots today, and you can do them with dots other than [Weiss'] dots."