quantum dots as cellular probes

30 Jun 2005 19:12 AR AR248-BE07-03.tex XMLPublishSM(2004/02/24) P1: KUV10.1146/annurev.bioeng.7.060804.100432

Annu. Rev. Biomed. Eng. 2005. 7:55–76doi: 10.1146/annurev.bioeng.7.060804.100432

Copyright c© 2005 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on April 4, 2005

QUANTUM DOTS AS CELLULAR PROBES

A. Paul Alivisatos,1,2 Weiwei Gu,2,3 and Carolyn Larabell2,4

1Department of Chemistry, University of California, Berkeley, California 94720;email: [email protected] Science Division, Lawrence Berkeley National Laboratory, Berkeley,California 94720; email: [email protected], [email protected] of Anatomy, University of California, San Francisco, California 941434Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley,California 94720

Key Words semiconductor nanocrystals, nanoparticles, fluorescent probing,imaging, biological application

■ Abstract Robust and bright light emitters, semiconductor nanocrystals [quan-tum dots (QDs)] have been adopted as a new class of fluorescent labels. Six yearsafter the first experiments of their uses in biological applications, there have beendramatic improvements in understanding surface chemistry, biocompatibility, and tar-geting specificity. Many studies have shown the great potential of using quantum dotsas new probes in vitro and in vivo. This review summarizes the recent advances ofquantum dot usage at the cellular level, including immunolabeling, cell tracking, insitu hybridization, FRET, in vivo imaging, and other related technologies. Limitationsand potential future uses of quantum dot probes are also discussed.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56QUANTUM DOTS: FROM PHYSICAL CHEMISTRY TO BIOLOGY . . . . . . . . . . 56

Synthesis and Optical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Bio-Molecule Conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

QUANTUM DOTS AS IN VITRO PROBES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Immunolabeling—Molecular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Immunolabeling—Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Live Cell Markers and Cell Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Cell Motility Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66Fluorescence Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

QUANTUM DOTS AS IN VIVO PROBES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68OTHER APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69PROSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1523-9829/05/0815-0055$20.00 55

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30 Jun 2005 19:12 AR AR248-BE07-03.tex XMLPublishSM(2004/02/24) P1: KUV

56 ALIVISATOS � GU � LARABELL

INTRODUCTION

Fluorescence probes are widely used in cell biology. Organic fluorophores, themost commonly used probes, suffer from fast photobleaching and broad, overlap-ping emission lines, and therefore are limited in applications involving long-termimaging and multicolor detection.

Advances in synthesis and biofunctionalization of colloidal semiconductornanocrystals during the past decade have generated an increasingly widespreadinterest among investigators in the fields of biology and medicine. These nano-meter-sized crystalline particles, also called quantum dots (QDs), are composedof periodic groups of II–VI (e.g., CdSe) or III–V (e.g., InP) materials. They arerobust fluorescence emitters with size-dependent emission wavelengths. Their ex-treme brightness and resistance to photobleaching enables the use of very lowlaser intensities over extended time periods, making them especially useful forlive-cell imaging, such as consecutive acquisition of z-stacks for high-resolutionthree-dimensional (3-D) reconstructions over time [four-dimensional (4-D) imag-ing]. The intense brightness is also particularly helpful for single-particle detectionand an increasing number of biomedical assays. The tunable emission wavelengthand distinct emission spectra of QDs facilitates data acquisition and analysis ofmultiple tagged molecules of interest.

Quantum dot chemistry and its early biological applications have been reviewedelsewhere (1–7). In the past six years, the progress in synthesis and optimization ofQDs for biological environments has opened the doors to an expanding variety ofbiological applications, such as serving as specific markers for cellular structuresand molecules, tracing cell lineage, monitoring physiological events in live cells,measuring cell motility, and tracking cells in vivo. Therefore, in this review wesummarize recent progress in QD biological and biomedical applications.

QUANTUM DOTS: FROM PHYSICAL CHEMISTRYTO BIOLOGY

Synthesis and Optical Properties

The synthesis of monodisperse semiconductor nanocrystals, such as CdSe, CdS, orCdTe, can be achieved by injecting liquid precursors into hot (300◦C) coordinatingorganic solvent (8, 9). Adjusting the amount of precursors and crystal growth timegenerates QDs of specific sizes (9). The quantum yield of the nanocrystal coresynthesized as above is relatively low (less than 10%) (8, 10). Usually, a shell ofhigh band-gap semiconductor material, such as ZnS, is epitaxially grown aroundthe core to achieve the quantum yield of up to 80% (10, 11).

QDs, which are only a few nanometers in diameter, exhibit discrete size-dependent energy levels. As the size of the nanocrystal increases, the energy gapalso increases, yielding a size-dependent rainbow of colors. Extensive tunabil-ity, from ultraviolet to infrared (12), can be achieved by varying the size and the

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QUANTUM DOTS AS CELLULAR PROBES 57

composition of QDs (Figure 1), enabling simultaneous examination of multiplemolecules and events. For example, small nanocrystals (∼2 nm) made of CdSeemit in the range between 495 to 515 nm, whereas larger CdSe nanocrystals (∼5nm) emit between 605 and 630 nm (8, 12).

QDs have several dramatically different properties compared to organic fluo-rophores, one of which is their unique optical spectra. As illustrated in Figure 2,organic dyes typically have narrow absorption spectra, which means they canonly be excited within a narrow window of wavelengths. Furthermore, they haveasymmetric emission spectra broadened by a red-tail. In contrast, QDs have broadabsorption spectra, enabling excitation by a wide range of wavelengths, and theiremission spectra are symmetric and narrow. Consequently, multicolor nanocrystalsof different sizes can be excited by a single wavelength shorter than their emissionwavelengths, with minimum signal overlap.

Figure 2 Excitation (dotted line) and fluorescence (solid line) spectra of fluores-cein (top) and a typical water-soluble QD (bottom). The excitation wavelength was476 nm and 355 nm for fluorescein and QD, respectively. The figure is reprinted withpermission from Reference 12, copyright 1998 AAAS.

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Figure 3 Comparison of fluorescence intensities between QD 608 (emission at608 nm) and Alexa 488 after continuous illumination. The figure is reproduced withpermission from Reference 14.

QDs are also very stable light emitters owing to their inorganic composition,making them less susceptible to photobleaching than organic dye molecules (4, 12).This feature has been demonstrated in a number of biological labeling experimentswhere the photostability of QDs was compared with commonly used fluorophores,such as rhodamine, fluorescein, and Alexa-Fluor (Figure 3) (12–19). This extremephotostability makes QDs very attractive probes for imaging thick cells and tissuesover long time periods—a challenging task that necessitates collection of multipleoptical sections without damaging the specimen. In addition, the two-photon cross-section of QDs is significantly higher than that of organic fluorophores (3, 20, 21),making them quite well suited for examination of thick specimens and in vivoimaging using multiphoton excitation.

Another interesting characteristic of QDs is their fluorescence lifetime of 10to 40 ns (4, 22, 23), which is significantly longer than typical organic dyes orauto-fluorescent flavin proteins that decay on the order of a few nanoseconds (4).Combined with pulsed laser and time-gated detection, the use of QD labels canproduce images with greatly reduced levels of background noise.

There are also some photophysical properties of QDs that can, in some cases,be disadvantageous. One of these is the property referred to as blinking, that is,QDs randomly alternate between an emitting state and a nonemitting state. Thisintermittence in emission of QDs is universally observed from single dots, whichimposes some limitations in QD applications requiring single-molecule detection.However, there is limited evidence suggesting that QD blinking can be suppressedon some timescale by passivating the QD surface with thiol moieties (24), or whenusing QDs in free suspension (20). It has also been reported that QD fluorescenceintensity increases upon excitation, an event referred to as photobrightening (25,

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26). Although in most cases this property can be advantageous, it is problematic influorescence quantization studies. Both blinking and photobrightening are linkedto mobile charges on the surfaces of the dots, and although the prospects are goodthat they can be eliminated, for the time being these should be considered aslimitations of QDs.

Biocompatibility

The core and core-shell QDs synthesized as described are only soluble in nonpolarsolvents because of their hydrophobic surface layer. For QDs to be useful probesfor examination of biological specimens, the surface must be hydrophilic. Severalstrategies have been used to stabilize core-shell nanocrystals in aqueous solutions.The easiest approach is to exchange the hydrophobic surfactant molecules withbifunctional molecules, which are hydrophilic on one side and hydrophobic onthe other side, which bind to the ZnS shell. Most often, thiols (-SH) are used asanchoring groups on the ZnS surface and carboxyl (-COOH) groups are used asthe hydrophilic ends. Many biological applications of QDs have been achieved byusing mercaptohydrocarbonic acids (SH-. . .-COOH) to make QDs water soluble(13, 27–32). The long-term stability of the QDs depends on the bond betweenthiol- and ZnS, which is not strong although it can be enhanced by using two thiolgroups instead of one (33, 34). Therefore, the water solubility of the core-shellQDs capped in mercaptocarbonic acids is limited.

An alternative approach is to grow a silica shell around the particle, also calledsurface silanization (12, 25). The first step of this process involves exchanging thesurface ligand with thiol-derived silane such as mercapto-trimethoxysilane (MPS).The trimethoxysilane groups can be cross-linked by the formation of siloxanebonds. During further silica shell growth, other types of silanes can be added torender a different charge and provide functional groups on the surface. Those mostfrequently used are aminopropyl-silanes (APS), phospho-silanes, and polyethyleneglycol (PEG)-silane (2, 25). Because the silica shells are highly cross-linked,silanized QDs are extremely stable. This method’s drawbacks are that it is morelaborious and the silica shell may eventually be hydrolyzed (4).

Recently other solubilization methods have been reported, one of which involvescoating the surface with amphiphilic polymers (14, 35). Instead of exchanging thehydrophobic surfactant, the particles in this case are coated with a cross-linkedamphiphilic polymer. The hydrophobic tails of the polymer intercalate with thesurfactant molecules and the hydrophilic groups stick out to ensure water solubilityof the particle. Although this is a general method for nanocrystals grown in differentorganic solvents, the final size of the particles after coating is rather large. ForCdSe/ZnS QDs, the diameter is between 19 and 25 nm (35), which could placerestrictions on many biological applications. Other approaches, such as coatingthe QDs with phospholipid micelles (19), dithiothreitol (36), organic dendron (37,38), and oligomeric ligands (39), have also been reported.

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Bio-Molecule Conjugation

To make QDs more useful for probing live cells and other biological applications,QDs need to be conjugated to biological molecules without disturbing the biolog-ical function of these molecules. Several successful approaches have been used tolink biological molecules to QDs, including adsorption, electrostatic interaction,mercapto (-SH) exchange, and covalent linkage.

It has been reported that simple small molecules, such as oligonucleotides (40,41) and various serum albumins (18), are readily adsorbed to the surface of water-soluble QDs. This adsorption is nonspecific and depends on ionic strength, pH,temperature, and the surface charge of the molecule.

Mattoussi and coworkers presented a method of conjugating proteins to QDsurfaces using electrostatic interactions. The protein of interest was engineered tofuse with a positively charged domain, which in turn interacted electrostaticallywith the negatively charged surface of the QD. The protein-QD conjugates preparedin this way were very stable and the fluorescence yield was even higher than thatfrom the nonconjugated dots (33, 34).

Biological molecules containing thiol groups can be conjugated to the QDsurface through a mercapto exchange process (28, 31, 32, 42–44). Unfortunately,the same problem of using thiol as anchoring group on a ZnS surface occursbecause the bond between Zn and thiol is not very strong and is dynamic. As aresult, biomolecules can readily detach from the surface, causing QDs to precipitatefrom the solution.

A more stable linkage is obtained by covalently linking biomolecules to thefunctional groups on the QD surface using cross-linker molecules (6, 12, 13, 25,36, 38, 45). If the surface of the nanocrystal bears -COOH, -SH, or -NH2, it is easyto link it to biological molecules that also have these reaction groups. For example,the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is com-monly used to link -NH2 and -COOH groups, whereas 4-(N-maleimidomethyl)-cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) can be used tocross-link -SH and -NH2 groups (46).

Using the above methods, there have been numerous reports of conjugatingQDs with various biological molecules, including biotin (12); oligonucleotides(19, 40, 41, 45); peptides (42, 44, 47); and proteins, including avidin/streptavidin(14, 38), albumin (48), and antibodies (13, 14, 17, 44, 49, 50). In most cases, thebiological functions of these molecules have not been affected by linkage of QDs.

Toxicity

Concerns have been raised about the toxicity of QDs, especially when they are usedto study live cells and animals because they contain elements such as cadmiumand selenium.

When properly capped by both ZnS and hydrophilic shells, no acute and obviousCdSe QD toxicity has been detected in studies of cell proliferation and viabilityin live cells (17, 44, 51, 52) and animal (20, 42) models. The most sensitive

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environment in which to test QD toxicity is that of the live embryo because evenslight cellular perturbances are manifested as measurable biological phenotypes.The toxicity of QDs was examined in Xenopus embryos (19), where injection of lowconcentrations of QDs had no negative effects. Although some abnormalities werenoted with increasing concentrations, it was not clear whether they resulted fromthe QDs or changes in the osmotic equilibrium of the cell. In another case, primaryhepatocyte cultures were examined because the liver is the major target of cadmiuminjury (52). Under standard conditions of synthesis and water solubilization, QDswere not toxic. However, cytotoxicity was observed when Cd2+ was released (52).This happened when the QD surface coating was not stable, exposing the CdSe tooxidization by air or UV damage. Therefore, strategies to protect the QD surfacesfrom oxidative environments are critical for live cell and animal experiments.Coating the QD surface with ZnS eliminates toxicity owing to air exposure butprovides only partial protection of the core from UV exposure. Larger molecules,such as bovine serum albumin (BSA) and polymer/streptavidin, further slow downthe photooxidation process. However, the potential effect of Cd2+ release over timehas not been examined. There remains a pressing need for further investigationsinto QD toxicity in live animals and safety for diagnostic applications.

Specificity

Specificity is one of the most critical criteria for measuring the value of cellularprobes. When QDs were used in early applications, nonspecific binding was re-ported (12, 36). However, as the surface chemistry has been refined, QD specificityhas greatly improved and the number of biological applications has dramaticallyincreased. Because the surface molecule and surface charge of the QDs play animportant role in their binding properties (14, 36, 38, 51), manipulating QD coatingand surface charge affects their “stickiness.” Both silica- and mercaptohydrocar-bonic acid–coated particles bind nonspecifically to cells (12, 36). Alternatively,the use of hydroxyl groups, which have low nonspecific binding to biomolecules,has improved specificity of QD-tagged probes (36, 38). Coating the QDs with amixture of phospholipids and PEG polymer also prevents aggregation of dots andnonspecific binding, because of the low nonspecific adsorption offered by PEG(19).

QUANTUM DOTS AS IN VITRO PROBES

Immunolabeling—Molecular Localization

Fluorescence immunolabeling is widely used in cell biology for probing structureand locating signal transduction–related molecules. Owing to their robust opticalproperties, QDs are ideal probes in this area.

Since the first experiment where semiconductor CdSe/ZnS nanocrystals wereused to stain F-actin in fixed cells (12), investigators have performed a variety of

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Figure 4 Immunofluorescence labeling of microtubules for mouse embryo fibroblastcells (NIH/3T3) using streptavidin-coated QD 605 (left) and streptavidin conjugatedAlexa 568 (right). The microtubules were first incubated with mouse biotinylated anti-α-tubulin antibody and then streptavidin conjugated QDs or Alexa. The excitationwavelength 488 nm was used to excite QD 605, whereas 543 nm was used for Alexa568. The emission filter was LP 560 nm for both. As compared to Alexa, QD labelingshows the same labeling pattern of microtubules. The scale bar is 20 µm.

experiments in which QDs have been used to localize molecules in cells andtissues, both in live and fixed specimens (Figure 4). Kaul and colleagues re-ported immunofluorescence labeling of the heat shock 70 protein, mortalin, usingQDs to show different staining patterns in normal and transformed cells (53).Consequently, this protein has been suggested as a reliable marker for normalversus transformed cells. Taking advantage of the high photostability of QDs,Tokumasu & Dvorak were able to collect 40 consecutive optical sections usingconfocal microscopy and generated a 3-D reconstructed, high-resolution image ofthe membrane domain band 3 in erythrocytes (54). Ness et al. developed an im-munohistochemical (IHC) protocol that combines conventional enzymatic signalamplification and QD labeling to detect intracellular antigens in rat and mouse braintissue sections. Their study showed that QD IHC labeling had greater sensitivitythan similar IHC approaches using conventional dyes (16). Wu and coworkers de-veloped reliable and specific QD probes to localize the breast cancer cell surfacemarker Her2, cytoskeleton fibers, and nuclear antigens in fixed cells, live cells, andtissue sections, with a substantial increase in brightness and photostability as com-pared to organic dyes (14). Minet and colleagues also examined breast tumor cells,using QDs to label membrane glycoproteins to study heat stress effect (55). Theyfound alterations of plasma membrane organization and integrity upon heat stress.

All these studies show that QDs have moved beyond the demonstration stage andare excellent probes with enhanced signal-to-noise ratio, extremely high stability,and improved specificity suitable for studying important biological problems.

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Immunolabeling—Signaling Pathways

Research of signaling pathways between and within cells also relies heavily onbright and sensitive fluorophores. QDs have begun to play a major role in this field.

Rosenthal et al. reported using serotonin-linked QDs to target the neurotrans-mitter receptor on the cell surface. The QD probes not only recognized and labeledserotonin-specific neurotransmitters on cell membranes, but also inhibited the sero-tonin transportation in a dose-dependent manner (43). Although one to two ordersof magnitude less potent at inhibiting the receptor than free serotonin, the behaviorof QD conjugates was similar to that of free serotonin, making QDs a valuableprobe for exploring the serotonin transportation mechanism.

Dahan and coworkers studied the dynamics of individual glycine receptors(GlyRs) in neuronal membranes using QDs as fluorescent probes (56). The GlyRswere linked to QDs through primary antibody and secondary F(ab′) fragmentbridges. The QD labeling enabled the collection of sequential images for up to 20min, whereas a comparable Cy3 probe lasted only approximately 5 s. By trackingindividual dots and analyzing the trajectories, three different locations of GlyRswere characterized and corresponding diffusion coefficients were obtained. It isnot surprising that the diffusion coefficients obtained are much larger than thosemeasured with fluorescent beads because the QDs are extremely small (10–15 nm)compared with the beads (1 micron). The difference in size can significantly affectthe molecule’s motion. Being more photostable than organic dyes and much smallerthan beads, QDs proved to be a suitable probe for single-molecule experiments inliving cells, bridging the gap between large beads and small organic fluorophores.

Other signaling pathways, such as erbB/HER receptor-mediated signal trans-duction, have also been examined using QDs (57). Epidermal growth factor (EGF)conjugated to QDs is still capable of binding to and activating its receptor, theerbB1 receptor, which triggers internalization of both EGF-QD and its receptorvia endocytosis. In this case, examination of single QDs enabled discovery of aretrograde transport process in which the EGF-QD, after binding to the filopodiumof the cell, moves toward the cell body at a velocity of 10 nm/s. Owing to the pho-tostability of QD, EGF-QD binding and internalization kinetics were obtained, thelatter being the rate-limiting process (57). Such quantitative understanding of thetransduction mechanism is essential for receptor-targeted therapeutics. QDs willbe a valuable reagent for this kind of investigation.

The process of cell endocytosis has also been studied using QDs (58). In thisexperiment, the endocytosis efficiency of 15 nm QD conjugated sugar balls wascompared with the efficiency of 5 nm and 50 nm particles, revealing that endo-cytosis is highly size dependent. The 15 nm QDs were less effective at triggeringendocytosis than were 50 nm particles, but more effective than 5 nm particles(58). The QD conjugated sugar ball was an excellent size marker for studying thesize effect of endocytosis in the viral size region, and thus provided useful infor-mation for the design of artificial molecule delivery systems for gene and drugdelivery.

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Live Cell Markers and Cell Lineage

Because QDs are extremely bright and photostable, they can be used as cell markersfor long-term studies such as cell-cell interactions, cell differentiation, and celllineage tracking. Most experiments conducted to date have shown that QDs do notinterfere with normal cell physiology and cell differentiation (19, 51, 59).

The idea to use QDs as cell markers is based on the discovery that QDs canbe internalized by cells, by either receptor-mediated (13) or nonspecific endocy-tosis (4, 17, 18, 51). The exact pathway of incorporating QDs into cells underendocytosis is not understood. However, after QDs enter cells, they accumulatein vesicles in the perinuclear region (17, 18, 51). Perinuclear vesicles filled withnanocrystals were first seen by multiphoton microscopy (51). Later, with the use ofan endosome-specific marker, such as fluorescein-labeled dextran (18) or pECFP(17), it was confirmed that QDs were indeed accumulated in the endosomes orlysosomes and not coating them on the basis of colocalization of QDs and en-dosome markers. Once internalized, QDs divided with the daughter cells at celldivision (18, 19, 51).

There are several other approaches for delivering QDs into live cells. Oneof these was developed by Matteakis and coworkers (59), who used a peptide-mediated transportation method to incorporate different colored QDs into a varietyof live mammalian cells, generating a unique and spectrally resolvable code foreach cell type. The carrier for delivering QDs into the cells is the protein transloca-tion domain Pep-1, an engineered 21-residue peptide composed of a hydrophobicsequence for QD binding and a hydrophilic lysine-rich sequence from the nuclearlocalization sequence for penetrating cells (59).

Cells can also be loaded with QDs by microinjection. Dubertret et al. (19)microinjected phospholipid-coated QDs into early-stage Xenopus embryos andtracked the embryogenesis using fluorescence visualization. Compared to nonspe-cific endocytosis or peptide delivery, microinjection is more laborious and lessefficient. However, it is worthwhile noting that microinjected QDs were homoge-nously distributed throughout the cell and were not compartmentalized (19).

Internalized QDs are powerful probes for long-term studies of cell-cell interac-tions. Our group examined the interactions of human mammary epithelial tumorcells with normal cells growing in a 3-D culture system using QDs as cell-typeidentification markers. With different color-emitting QDs, we were able to un-equivocally identify the tumor cells and normal cells in a coculture system. Whencultured in the 3-D matrix, human mammary epithelial cells form acini, polarizedclusters of cells that resemble natural glandular tissue. Each acinus develops froma single cell during a 10–14 day time span. If the original cell was preloaded withQDs, all cells in the acinus will subsequently contain a subset of those QDs. Thisenabled us to add tumor cells tagged with QDs of a different emission wavelengthto the acini and clearly distinguish normal cells from tumor cells during two-weekculture periods. We observed that tumor cells migrate toward the acini, extend in-vadopodia to contact the acini, and subsequently undergo programmed cell death(60) (Figure 5). Death is always contact dependent and is induced by polarized

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cells, those organized into acini, but not individual unpolarized cells. The highphotostability of QDs enabled us to track and image these cocultures for up to twoweeks, which cannot be achieved by organic dyes.

Cell Motility Assay

Motility and migration of cancer cells are measurable properties that are associ-ated with metastases and the formation of secondary tumors. Present methods formeasuring cell patterns and the extent of cell movement suffer from a numberof practical limitations. We developed an assay based on our finding that cellsnonspecifically incorporate QDs as they crawl over them, leaving behind QD-freezones representing the pattern of phagokinetic uptake of QDs (Figure 6) (51). Simi-lar phagokinetic cell motility assays using gold colloids or polystyrene micro beadshave been used with some degree of success (61, 62), but typically require fixationof the cells. The use of QDs facilitates monitoring live cells, both before and afterperturbations, such as the addition of potential chemotherapeutic agents to monitor

Figure 6 QDs were used to study cell motility. Human mammary epithelial tumorcells, MDA-MB-231, were grown on top of a collagen layer that had been coated witha thin layer of silanized QDs. While cells migrated across the layer, they engulfed thenanocrystals and left areas that were fluorescence-free. The scale bar is 200 µm. Thefigure was reproduced with permission from Reference 51.

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changes in motility. We also found that the quantity of QDs incorporated variesamong different cell types. For example, MDA-MB-231 tumor cells uptake moreQDs than nontumorigenic MCF 10A cells (51). The QD assay provides a usefulmeasure of the invasive potential of cells (63), and it is much more sensitive than theconventional Boyden chamber assay with significantly reduced specimen process-ing (64–66). Unlike the Boyden chamber assay, which is time-consuming and usesfixed cells, the QD-based assay requires no fixation, using fluorescent detection inlive cells. These features make it a powerful new tool for cell motility studies.

In Situ Hybridization

Fluorescent in situ hybridization, also called FISH, utilizes fluorescently labeledDNA probes for gene mapping and the identification of chromosomal abnormal-ities. FISH provides researchers with a method to visualize and map the geneticmaterial in cells, including specific genes or portions of genes, and a way to quan-tify gene copy numbers within tumor cells that have abnormal gene amplification.However, use of the rapidly photobleaching organic probes compromises the ex-periments and requires immediate imaging. The use of organic dyes in multicolorFISH also has limitations, including color overlap and differential intensity of thefluorophores. These drawbacks can be resolved with the use of QDs.

The in situ hybridization procedure requires DNA-linked probes. Typical probesthat have been used include DNA conjugated to fluorescent molecules (67), gold(45, 68), and microbeads (69, 70). As might be expected, DNA or oligonucleotidescan also be conjugated to QDs (19, 28, 36, 40, 41, 45, 51) and in vitro testingdemonstrates that these conjugates retain their ability to form complementarysequences of Watson-Crick base pairs (51).

In 2001, Pathak and coworkers used QD-based FISH labeling to detect Y-chromosome-specific repeats in fixed human sperm cells. Although the resultsshow the success of hybridization using QD probes, high salt conditions insidethe cells caused partial aggregation of the nanocrystals, which gave rise to variedsignal intensities (36). Later in 2004, Xiao et al. reported using a QD FISH probeto analyze human metaphase chromosomes. The experiment was performed withtotal genomic DNA as the probe, human metaphase lymphocytes as the hybridiza-tion substrate, and the ebrB2/Her2/neu gene as the target of hybridization. Theycompared QD, Texas-Red, and FITC probe detection, demonstrating that the QDprobe was more photostable and provided higher signal-to-noise ratio than theorganic probes.

Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is a process in which energy istransferred from an excited donor to an acceptor via a resonant, near-field dipole-dipole interaction (71, 72). FRET is very sensitive to the distance between donorand acceptor and has been used to study biomolecule conformation, dynamics,and interactions. Some problems of using conventional dyes for FRET include

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fast photobleaching and significant emission overlap between donor and acceptor.QDs provide a potential solution to these problems. In 1996, resonance energytransfer between closely packed CdSe QD solids was reported by Kagan et al.(73, 74). Quenching the luminescence of small dots was accompanied by an en-hancement of the luminescence of large dots. This was consistent with resonanceenergy transfer from the small dots to the large dots. Later experiments demon-strated resonance energy transfer between QD donor/acceptor and organic dyeacceptor/donor (75, 76). One potential limitation of using QDs for FRET involvesthe physical dimensions of nanocrystals. Owing to the size of QDs, the distancebetween donor and acceptor is usually greater than 3 nm (77). In addition, cautionmust be taken with single-molecule experiments that might be complicated by theQDs blinking.

There are several examples of QDs used for FRET in biological systems. Willardet al. reported using QDs as a FRET donor in a protein-protein binding assay (31).QDs were conjugated to BSA as the FRET donors and tetramethylrhodamine(TMR) was linked to the protein as the acceptor. Enhanced TMR fluorescencewas observed as energy transferred between the QDs and the TMR. Medintz andcoworkers engineered a histidine tag on maltose binding protein (MBP) that finallybound QDs, which served as the FRET donor (77). With a quencher bound to themaltose-binding site, QD fluorescence was inhibited but was recovered by addingmaltose to displace the quencher. In 2004, the same group did an in-depth studyusing the MBP system by varying the QD size and the quantity of acceptor dye(78). Increasing the amount of acceptor dye, or the degree of spectral overlap (bychanging QD size), caused a substantial enhancement in energy transfer efficiency.This study demonstrated that QDs can be used as efficient energy donors in theFRET system and showed that by tuning their size, QDs can transfer energy toa number of organic dye molecules. In another paper, these same researchersdemonstrated reversible modulation of QD fluorescence using FRET with thephotochromic molecule BIPS (1,2,3-dihydro-1 2-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro-[2H-1-benzopyran-2,2,2-(2H)-indoline]) (79). BIPS was converted tocolored merocyanine when exposed to UV light and quenched QD emission byacting as a FRET acceptor. When exposed to white light, BIPS was convertedback to colorless spiropyran and QD emission was recovered. By switching theQD emission on and off, it is possible to use this type of system as a nanosensordevice for sensing photochromical changes.

In addition to protein binding assays, other processes like DNA replicationand telomerization can also be probed by QD-based FRET as reported by Pa-tolsky and coworkers (80). In DNA telomerization, QDs were conjugated to aDNA template molecule and mixed with dNTP (N = A, C, G) and Texas-Red-dUTP. After adding telomerase, FRET transition occurred between the QD donorand Texas-Red acceptor. A similar FRET process occurs when using QD con-jugated primers to initiate DNA replication. These results indicate the potentialuse of the QD FRET process in fast and sensitive DNA detection and DNA arrayanalyses.

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QUANTUM DOTS AS IN VIVO PROBES

Colloidal semiconductor nanocrystals have a two-photon cross-section that is twoto three orders of magnitude greater than organic dyes (20). A two-photon pro-cess requires about twice the excitation wavelength of a single-photon excitation.Longer wavelengths, extending into the infrared region, can be utilized to ex-cite chromophores in a single quantum event. Therefore, multiphoton microscopyenables the imaging of structures deep within biological tissues with minimumphotobleaching and photodamage. The high two-photon cross-section of QDs al-lows for more efficient probing of thick specimens and in vivo imaging when usingmultiphoton excitation microscopy.

Two in vivo experiments appeared almost simultaneously in 2002. In one paper,Akerman and colleagues used QDs to target tumor vasculature in mice (42). Thenanoparticles, conjugated to several peptides that differentially recognized bloodvessels located in the lung, tumor blood vessels and tumor lymphatic vessels, wereinjected into mice. Histological staining revealed that QDs were delivered to theappropriate site in vivo, guided by the peptides. In the other paper, Dubertret etal. microinjected phospholipid-coated QDs into early-stage Xenopus embryos tostudy the behavior of specific cells during embryogenesis (19). In both cases, QDsprovided a stable, robust fluorescent probe that could be used in vivo over extendedperiods of time.

Live animal imaging using QD fluorescence with multiphoton microscopy wasachieved by Larson et al. in 2003 (20). QDs, intravenously injected into mice,were detectable through intact skin at the base of the dermis (∼100 micron) usingan excitation wavelength of 900 nm. To optimize the conditions of in vivo ex-periments, Ballou et al. tested QDs with different polymer coating in vivo usingvarious imaging techniques, including light and electron microscopy on tissue sec-tions and noninvasive whole-body fluorescence imaging (81). Amazingly, theseQDs maintained their fluorescence even after four months in vivo. Although theQDs showed no deleterious effects upon the animals in these studies, a more de-tailed evaluation of potential QD toxicity in the body is warranted prior to theirlong-term usage in higher organisms.

Recently, Gao et al. reported in vivo cancer targeting and imaging using QDs(21). They conjugated QDs to the antibody specific for the prostate cancer cellmarker PSMA. After injection into mice that had been transplanted with humanprostate cancer cells, the QD-tagged PSMA antibodies recognized and bound atthe tumor site and were clearly imaged in vivo. Owing to the QDs’ large absorptioncoefficient and long lifetime, in vivo imaging of QDs was much brighter and moresensitive than imaging with green fluorescence protein (GFP) (Figure 7), whichwas totally buried in autofluorescence and background.

The use of QDs during surgical procedures was demonstrated by Kim andcoworkers (82), who mapped sentinel lymph nodes (SLN) with near-infrared fluo-rescent QDs in mouse and pigs. QDs were injected intradermally into the animal,

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entered the lymphatic system, and were followed using an intraoperative imagingsystem. The surgeon followed the flow of QDs in real time with NIR QD imageguidance, and quickly identified the position of the SLN in a precise and rapidsurgical procedure. The use of QDs as cell tracers after transplantation into micehas also been reported (83).

The above studies have shown the great potential of using QDs as in vivo probesfor cancer studies, drug delivery, and noninvasive whole-body imaging. It is worthnoting that the degradation and metabolism of QDs in the body remains to beinvestigated and there are reports that QDs injected can accumulate in the kidney,liver, and spleen (21, 42). Whether the QDs can ultimately be cleared from thebody is not known. More research in this area needs to be completed before QDscan be used as probes in the human body.

OTHER APPLICATIONS

QDs have also been found to be useful in the study of microorganisms. Kloepferet al. reported targeting QD conjugates to surface glycoproteins of bacteria andfungi (26). QDs were also used as cell membrane permeable indicators forEscherichia coli (84).

The luminescence of QDs can be affected by ionic environments and pH(85–88). Cadmium, zinc, and manganese ions increase the luminescence of CdSnanocrystals in basic solution, whereas copper ions quench their emission (89).Chen et al. reported that by changing the capping layer, the luminescence of CdSQDs selectively responded to the presence of zinc or copper ions.

Combined with magnetic nanoparticles, QDs have been demonstrated to be use-ful for cell detection and separation, as reported by Wang et al. The nanocompositeparticles, made up of magnetic Fe2O3 superparamagnetic cores and CdSe/ZnS QDshells, were used to magnetically separate breast cancer cells, which were then de-tected by fluorescence (90). With some technical improvements, this new techniquemight be useful for in vitro cancer diagnosis in the future.

QDs have also been investigated for use in drug delivery. Lai et al. used surface-modified CdS QDs as chemically removable caps to keep pharmaceutical drugmolecules and neurotransmitters inside a mesoporous silica nanosphere-based sys-tem. The CdS cap ensures the drug is inside the system until triggered by disulfidebond–reducing reagents. It is interesting to note that QDs here play a role as asize-defined cap and not as a fluorescent molecule (91).

Localization of specific biomolecules in cells and tissues at a high resolutionprovides both structural and quantitative information for molecular cell biology.However, popular immunofluorescence labeling is limited in spatial resolution. Be-cause QDs are composed of heavy elements such as cadmium, it is possible to usethem as contrast or labeling probes in transmission electron and X-ray microscopyfor high-resolution imaging of cells. Nisman et al. used CdSe QDs as probesin both conventional and energy-filtered TEM, demonstrating the feasibility of

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Figure 8 (A) Transmission electron microscopy detection of nuclear promyelocyticleukemia protein (PML) within a Hep-2 PML I cell section with QD labeling. QDsare highlighted with arrows. (B) Electron spectroscopic imaging shows the distribu-tion of discrete QDs within the PML body. The image was generated by dividing acadmium postedge (510 eV) image by its preedge (415 eV) image after alignment bycross-correlation. The scale bar is 50 nm. A and B were reproduced with permissionfrom Reference 92. (C) Soft X-ray microscopy image of QDs in liposomes. QDs arehighlighted with arrows. The X-ray image was taken as described in Reference 93.

labeling the nuclear promyelocytic leukemia (PML) protein on cell sections to ob-tain correlative fluorescence and TEM images. To acquire chemical and structuralinformation and to get better signal-to-noise ratio, electron spectroscopic imaging(ESI) was used to identify the elemental map of cadmium without interferencefrom other elements such as nitrogen (Figure 8). Also, we have demonstratedthe ability to detect liposome-encapsulated QDs using soft X-ray microscopy (C.Larabell, unpublished data).

PROSPECTIVE

There are numerous unexplored possibilities to expand the repertoire of QD label-ing in the future. One area of biological research that has not yet been addressedwith these novel nanostructures is molecular polarity. Semiconductor nanorods(quantum rods), which demonstrate polarized emission properties, might be po-tent tools for such experiments.

The multilevel imaging on both the medical and cellular scales demonstratesthe need for development of nanoparticles that can be used in all kinds of imagingtechniques, such as magnetic resonance imaging, PET or SPECT, optical confocalmicroscopy, X-ray microscopy, and TEM.

With the completion of the Human Genome Project, it is timely to try to iden-tify specific genes involved in disease and the relationship of gene regulation tocell function. Cellular probes that can identify a single gene, RNA, or protein at aspecific time and location within a cell will facilitate an understanding of cellularfunction at the molecular level. QDs, with their intense luminosity and high pho-tostability, are the best candidate for this research. Tuning the chemistry to avoid

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sample aggregation within cells and optimizing imaging and detection conditionsfor single QD experiments will be the key to achieve this goal.

Recent studies have shown the potential for the use of QDs in cancer diag-nosis and therapy. Thus, there is an urgent need to understand QD toxicity andmetabolism in the body. Although it is possible to modify QD surfaces so thatQDs are cleared from the body within a reasonable time, it will be more difficult toreplace the toxic elements of QDs while keeping their desirable optical propertiesintact.

ACKNOWLEDGMENTS

We thank Dr. Yi Cui, Aihua Fu, and Christine Micheel for helpful discussions.We are grateful to Rosanne Boudreau and Benjamin Engel for proofreading themanuscript. This work was supported (in part) by NIH National Center for ResearchResources through the University of California, Los Angeles, subaward agreement0980GFD623 through the U.S. Dept. of Energy under Contract No. DE-AC03-76SF00098.

The Annual Review of Biomedical Engineering is online athttp://bioeng.annualreviews.org

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QUANTUM DOTS AS CELLULAR PROBES C-1

Figure 1 Emission spectra of several semiconductor nanocrystals showing theirsize- and composition-dependent emission character. Red, green, and blue series re-present different-sized InAs, InP, and CdSe nanocrystals, respectively. The sizes ofthe nanocrystals are indicated above their corresponding spectra. The figure isreprinted with permission from Reference 12, copyright 1998 AAAS.

HI-RES-BE07-03-Alivisatos.qxd 6/30/05 8:02 PM Page 1

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C-2 ALIVISATOS ■ GU ■ LARABELL

Figure 5 QDs were used to study mixed cell interactions in a 3-D matrigel culturesystem. (A) Human mammalian epithelial MCF 10A cells (tagged with green-emit-ting silica coated QDs) form acini structures after growing in growth-factor reducedmatrigel for 10 days. (B) After the acini were formed, human breast tumor MDA-MB-231 cells (tagged with red-emitting silica coated QDs) were added to the culture.After 14–16 h of incubation, the tumor cells had attached to the acini’s basementmembrane. (C) The contact was fatal to the tumor cells, which were found dead sur-rounding the MCF 10A organoid. Most of the tumor cells had lysed, leaving trans-parent ghosts loosely attached to the organoid, but a few newly attached cells stillretained red-emitting QDs. (D) The MCF-10A organoid and all invading tumor cells;it is a superimposition of all sections, displaying the sharp edge of each cell followedby a projection of color-coded depth information so that red is the uninvolved lowerportion of the MCF-10A organoid and the tumor cells are shades of orange throughgreen. The scale bar is 10 �m. The figure was reproduced with permission fromReference 60.


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QUANTUM DOTS AS CELLULAR PROBES C-3

Figure 7 In vivo imaging using QDs. (a) Sensitivity comparison between QD-taggedand green fluorescence protein (GFP) transfected cancer cells. QD-labeled cells(orange) were injected on the right flank of a mouse, whereas the same number ofGFP-labeled cells (green) were injected on the left flank (circle) of the same animal.(b) Simultaneous in vivo imaging of multicolor QD-encoded microbeads, which wereinjected at three adjacent locations on a host animal. The figure was reprinted withpermission from Reference 21.


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P1: KUV

June 20, 2005 22:39 Annual Reviews AR248-FM

Annual Review of Biomedical EngineeringVolume 7, 2005

CONTENTS

FRONTISPIECE, Werner Goldsmith xii

WERNER GOLDSMITH: LIFE AND WORK (1924–2003), Stanley A. Berger,Albert I. King, and Jack L. Lewis 1

DNA MECHANICS, Craig J. Benham and Steven P. Mielke 21

QUANTUM DOTS AS CELLULAR PROBES, A. Paul Alivisatos, Weiwei Gu,and Carolyn Larabell 55

BLOOD-ON-A-CHIP, Mehmet Toner and Daniel Irimia 77

BIOCHEMISTRY AND BIOMECHANICS OF CELL MOTILITY, Song Li,Jun-Lin Guan, and Shu Chien 105

MOLECULAR MECHANICS AND DYNAMICS OF LEUKOCYTERECRUITMENT DURING INFLAMMATION, Scott I. Simonand Chad E. Green 151

DETERMINISTIC AND STOCHASTIC ELEMENTS OF AXONAL GUIDANCE,Susan Maskery and Troy Shinbrot 187

STRUCTURE AND MECHANICS OF HEALING MYOCARDIAL INFARCTS,Jeffrey W. Holmes, Thomas K. Borg, and James W. Covell 223

INSTRUMENTATION ASPECTS OF ANIMAL PET, Yuan-Chuan Taiand Richard Laforest 255

IN VIVO MAGNETIC RESONANCE SPECTROSCOPY IN CANCER,Robert J. Gillies and David L. Morse 287

FUNCTIONAL ELECTRICAL STIMULATION FOR NEUROMUSCULARAPPLICATIONS, P. Hunter Peckham and Jayme S. Knutson 327

RETINAL PROSTHESIS, James D. Weiland, Wentai Liu, and Mark S. Humayun 361

INDEXESSubject Index 403Cumulative Index of Contributing Authors, Volumes 1–7 413Cumulative Index of Chapter Titles, Volumes 1–7 416

ERRATAAn online log of corrections to Annual Review of BiomedicalEngineering chapters may be found at http://bioeng.annualreviews.org/

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