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Quantum DotsLeave the Light On

KATIE COTTINGHAM

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NNot long ago, semiconductor quantum dots(QDs) were the exclusive playthings of physi-

cists, who developed the spheres for optical and elec-tronics applications, such as LED-based systems andflat panel displays. But ever since QDs were first usedfor cellular imaging in 1998, biologists and chemistshave been abuzz about the colorful orbs. QDs “arereally neat little things,” says Erkki Ruoslahti at theBurnham Institute. “They are intensely fluorescent,so they are a great tool.”

QDs are semiconductor nanoparticles that are~20× brighter and much more photostable than mostfluorophores. According to Sangeeta Bhatia at theMassachusetts Institute of Technology (MIT), “Inbiology, the problem has been that as you’re looking

at a cell that contains a fluorophore, it’s bleaching asyou’re watching it.” Instead of photobleaching with-in seconds or minutes, QDs remain bright for severalhours under constant irradiation, and this makesthem attractive labels for intracellular tracking studies.

The size-tunable emission properties of QDs en-able researchers to create entire libraries of probesbased on one parent structure. For example, themost commonly used QDs are composed of a Cd/Secore. By varying the size of these cores, one can cre-ate Cd/Se QDs that emit at nearly any wavelength inthe visible region of the optical spectrum because ofa phenomenon known as the quantum confinementeffect. When semiconductor material is chopped intonanoscale chunks, each chunk becomes smaller thanits Bohr radius, explains Warren Chan at the Univer-sity of Toronto (Canada). All of the particle’s elec-trons become confined to a small space. This forcesthe entire particle to take on quantum propertiesthat are similar to those of an atom. Hence, QDs arealso known as “artificial atoms”.

Hundreds of different colors are possible fromQDs generated from the same material with the samesynthesis protocols. Small Cd/Se dots emit bluelight, whereas larger ones can emit green, yellow, orred light. Depending on the particular semiconductormaterial used in their cores, some QDs can emit inthe UV or near-IR range. “With the near-IR region,you can penetrate deeper into tissues,” says Chan.These optical properties are in contrast to those of or-ganic fluorophores, which are not tuned by size. If aresearcher desires a different color of an organic dye,such as fluorescein, then he or she must synthesize abrand-new molecule. And a limited number of colorvariations are currently available for the widely usedgreen fluorescent protein.

One of the most striking advantages of QDs is thatall of the colors can be seen simultaneously with afluorescence microscope (Figure 1). This is useful formultiplexing in both imaging applications and invitro assays. According to Sanford Simon at Rocke-feller University, organic dyes have narrow absorptionspectra, so a different laser must excite each one. Also,the emission spectra of dyes can overlap, so a signalfrom one fluorophore can bleed into the signal of an-other fluorophore. Researchers switch back and forthbetween lasers and filters to visualize different fluo-rophores and often must overlap images with a com-puter to determine the locations of multiple dyes.

QDs, however, have broad, overlapping absorptionspectra and very narrow emission spectra. Only oneexcitation source is needed to excite a rainbow of dots,which emit light within distinct wavelength ranges.Researchers can also visualize QDs and a fluorophoresimultaneously (Figure 2). These optical propertieslower the instrumentation cost and allow researchersto see all the QD colors at once, says Chan.

Quantum dots are brighter and

more photostable than other

fluorescent molecules, but will they

ever replace conventional labels?

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Not a panaceaDespite the numerous advantages of QDs, they are still not ascommonplace as organic fluorophores and fluorescent proteins.Chan says that part of the reason is that biocompatible QD tech-nology is not yet mature. “Quantum dots weren’t really in theball game for biologists until 1998,” he says. And the labels haveonly been commercially available for ~2 years.

According to Chan, the number of published papers on QDshas increased over the years, but one roadblock to biological ap-plications is that physical scientists conduct most of the studies.“I highly doubt [that] a lot of physical scientists would startusing quantum dots in studying biolog-ical or clinical problems or use them inperforming animal surgery unless theyfind good collaborators,” he says. “Sim-ilarly, a biologist may not understandthe intricacies of the physical propertiesof quantum dots and how these physi-cal properties may behave differently inbiological environments.” Biologists,chemists, physicists, and materials scien-tists need to share their knowledge ifQDs are to be more widely adopted aslabels, he explains.

Researchers predict that QDs willnot replace other fluorescent molecules.Rather, they will remain complementa-ry. “I think each has its own place, andthey are useful for different things,” says Simon. Shuming Nie atEmory University points out that only QDs can enable re-searchers to track molecules in cells for hours at a time, but al-most any type of label can be used for finding the location of oneprotein in a fixed cell. According to Simon, “People should use[QDs] if they see a very specific need or [if there’s] somethingthey can do with quantum dots that they can’t do with [othermolecules], not to use them for the sake of using them.” Headds, “If an organic fluorophore will do what you want it to do,there’s nothing wrong with [that].”

But why are organic fluorophores and fluorescent proteinsstill the default for most scientists? Although QDs enable highdegrees of multiplexing and are very bright and photostable,they have their drawbacks.

QDs are very difficult to make, and as a result, few researchlabs prepare their own. Biologists typically either buy them orcollaborate with labs that are equipped to perform organometal-lic synthesis. According to Nie, the cost of commercially pre-pared QDs is ~2–3× that of organic fluorophores. Chan says thatcompanies don’t disclose all of the details about their QDs’ sur-face chemistries. “You get this solution that glows upon UV ex-citation,” he explains. “Because it’s proprietary, if somethinggoes wrong with the experiment, it would be difficult for a re-searcher to determine the cause.”

Even when researchers synthesize their own QDs, they can’tprecisely control the surface compositions. “I think [QDs’] mainlimitation as of now is still the surface chemistry,” says Hedi Mat-toussi at the Naval Research Laboratory. “People haven’t de-

signed the . . . magical functional group that allows one to put[ligands] anywhere they want [on a QD].” If knowing the num-ber and locations of fluorescent molecules on a protein is impor-tant for an experiment, Bhatia says that genetically encoded fluo-rescent proteins, not QDs, are ideal.

Although a common complaint about QDs is that they tendto aggregate, Mattoussi says that aggregation is not an inherentproperty of the dots, as some researchers imply. Instead, aggre-gation is due to interactions between the QDs’ surfaces and theirenvironment. “If you take colloidal particles—they could bepolystyrene, silica, or gold—put them in a solution, and make

the surface interact unfavorably withthe surrounding solvent, then theywill aggregate,” says Mattoussi. “Ag-gregation is proof that you have aproblem with what you are workingwith.”

Researchers must modify QDs tomake them biocompatible, but thesemodifications result in a large, bulkysphere. And according to experts, sizeis an issue for some applications. “Dotswith a polymer coating and [other at-tachments] can be pretty big,” saysNie. Although cores are ~3–4 nm indiam, fully assembled QDs with a Zn/Sshell, a polymer or amphipathic-mole-cule coating, and antibodies or other

proteins attached to the surface can be >30 nm in diam. Nie saysthat these QDs are “much larger than organic dyes or the fluo-rescent proteins.”

QDs, particularly those that emit in the near-IR and can pen-etrate tissues, could potentially be a useful imaging tool for clini-cians. But first, QDs must overcome another major hurdle—their possible toxicity. Cadmium, one of the most commonlyused core components, is a heavy metal toxin. “The concernscome up when you start thinking about [using QDs] in an im-aging application in vivo,” says Bhatia. “I think there’s a sensi-tivity about putting anything that’s cadmium-based into pa-tients. That’s a limitation in terms of how well they are acceptedby the medical community.” Nie says, “The toxicology issue is aproblem for long-term, in vivo human applications because westill don’t understand how these dots would get out of thebody.” According to Bhatia, a lot will depend on the specific ap-plications, doses, and locations of the QDs. Interestingly, shepoints out that toxic radioisotopes are currently used in clinics ona routine basis for short-term nuclear medicine scans.

The alternativesSome researchers are developing alternatives to semiconductorQDs to overcome the size, surface chemistry, and toxicity issues.They are experimenting with different materials and fluorescencephenomena to obtain labels that are nearly as bright and photo-stable as QDs but without their limitations.

Gold is the material of choice for Robert Dickson at the Geor-gia Institute of Technology. He says that gold QDs are water-sol-

FIGURE 1. QD-tagged beads emit at different wave-lengths simultaneously. Scale bar = 5 µm. (Adaptedwith permission. Copyright 2001 Nature PublishingGroup.)

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uble and are an order of magnitude smallerthan those made of semiconductor materi-als. Gold is also less toxic than the heavymetals required for conventional QDs. Butsome researchers are hesitant to call thesenanoparticles QDs because the physics oftheir fluorescence is different. “Each goldatom contributes one electron to the con-duction band,” says Dickson, and a golddot “is really a multi-electron artificialatom, instead of a single-electron artificialatom, which semiconductor quantum dotsare.” He sees gold QDs as complementaryto conventional QDs. Semiconductor dotsare more suited to multiplexing, whereasgold QDs are more amenable to fluores-cence resonance energy transfer (FRET).Dickson says that gold QDs are in the earlystages of development and that his groupmust still work on several aspects, such asfiguring out how to attach molecules to thegold QD surfaces.

Other groups encase multiple organicand inorganic fluorophores in a silica shell; this produces a labelthat closely resembles a QD. Silica-based dots are easy to func-tionalize because many researchers have previously characterizedmolecular attachments to silica substrates. Weihong Tan at theUniversity of Florida calls his creation “FloDots”. According toTan, FloDots are much brighter than QDs and are extremelyphotostable. Most recently, he and his co-workers encapsulated

two or three differenttypes of fluorophores ina single silica shell, andthe ratio of the intensi-ties was used to distin-guish analytes in multi-plexed immunoassays.In addition, the Tan re-search group has devel-oped magnetic encap-sulated nanoparticles,which they have usedfor cell and biomole-cule separations in com-plex matrixes.

Ulrich Wiesner and colleagues at Cornell University have alsodeveloped silica-encapsulated fluorophores, which they call “Cor-nell dots”. According to Wiesner, Cornell dots are about the samesize and brightness as traditional QDs, without the toxicity con-cerns or complicated synthesis protocols. Although one would ex-pect that bunching fluorophores so closely together would causeFRET-induced quenching, silica-based dots fluoresce brightly.“We actually show that these particles are 30× brighter than theindividual chromophores in solution,” says Wiesner.

Ruoslahti’s group is also eyeing silica-based particles in addi-tion to magnetic particles. “We’re trying to build more functions

into nanoparticles, and that’s hardto do with quantum dots becausethey are just fluorescent,” he says.Hollow silica particles, for example,could carry a drug and be respon-sive to a signal.

A bright future for QDs?QDs have unique properties and,with a bit of work, they could be-come even more useful, say re-searchers. Attempts are under wayto make semiconductor QDs lessbulky and less toxic. Researchersare also developing new ways tofunctionalize QD surfaces.

Many groups are refining andoptimizing QD in vitro assays. Nieand other researchers are excitedabout using QDs as bar codes forhigh-throughput genomics andproteomics studies. QDs that emitat different wavelengths can be em-

bedded in beads at precise ratios. By varying color and intensity,researchers can perform highly multiplexed assays. Mattoussi isdeveloping QD-based sensors and fluoroimmunoassays in addi-tion to his cellular imaging work. He has used fluoroimmunoas-says to detect multiple toxins both separately and in multiplexedconditions. His group and others have designed sensors in whichthe addition of an analyte, such as the explosive TNT (2,4,6-trinitrotoluene), disrupts FRET-based quenching of a QD.

Researchers predict that in vitro QD assays will become im-portant for diagnostics. “I think one real practical [application]is in histopathology, in the clinical screening of human tissuespecimens,” says Nie. Once a sample is removed from a patient,toxicity concerns are allayed, he says.

Perhaps the most challenging application that remains is invivo imaging in humans. Several researchers are developing im-aging methods in animals as an initial step toward this goal. Nie’sgroup recently targeted QDs to human prostate tumors thatwere grown in mice (Figure 3). In another study, John Frangioniand his co-workers at MIT, the Beth Israel Deaconess MedicalCenter, and Brigham and Women’s Hospital used QDs to pin-point the lymph nodes into which tumors drain in mice and pigs.But Nie points out that QDs have a long road ahead before theywin approval for human use by the U.S. Food and Drug Ad-ministration (FDA). A QD “may not disrupt any processes, andmaybe it’s a perceived problem, but even that perceived problemis going to prevent the FDA from approving it,” he says.

Biological applications of QDs are driving much of the re-search in the field, and scientists have plenty of questions to in-vestigate in the coming years. Researchers are overcoming chal-lenges and finding new applications for QDs. The future forQDs is wide open and getting brighter every day.

Katie Cottingham is an associate editor of Analytical Chemistry.

FIGURE 2. Red QDs coupled to transferrinstain the cytoplasm of HeLa cells, and ablue fluorophore stains the nuclei.

Tumors

Injectionsite

FIGURE 3. QDs with antibodies to human prostate-specific membrane antigen light up murine tumorsthat developed from human prostate cells. (Adapt-ed with permission. Copyright 2004 Nature Pub-lishing Group.)

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