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Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots
Xingyong Wu1*, Hongjian Liu1, Jianquan Liu1, Kari N. Haley1, Joseph A. Treadway1, J. Peter Larson1, Nianfeng Ge2, Frank Peale2, and Marcel P. Bruchez1
Published online 2 December 2002; doi:10.1038/nbt764

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.

Quantum dots (QDs) have the potential to become a new class of fluorescent probes for many biological and biomedical applications113, especially cellular imaging2,3,10,11. As fluorescent probes, QDs have several advantages over conventional organic dyes. Their emission spectra are narrow, symmetrical, and tunable according to their size and material composition, allowing closer spacing of different probes without substantial spectral overlap. They exhibit excellent photostability. They display broad absorption spectra, making it possible to excite all colors of QDs simultaneously with a single excitation light source and to minimize sample autofluorescence by choosing an appropriate excitation wavelength2,3. Some important technical problems had yet to be solved, howeverin particular the surface coating chemistry. Therefore, the reported labeling of cellular targets with QD-based probes has been far less effective than labeling with organic dyes with regard to specificity, brightness, and spatial resolution2,3. In one previous study, cellular actin fibers were labeled with QDs conjugated to biotin. Although this was the first reported successful labeling of a specific target in the cell with a QD probe, the labeling signal was relatively weak, and the probes bound nonspecifically to the nuclear membrane2. In another study, QDs linked to the protein transferrin were transported into cultured cells, presumably by means of receptormediated endocytosis, but no specific cellular molecule was labeled3. IgG molecules conjugated to QDs showed binding capacity to specific antibodies in solution, but the binding efficiency of the QD-IgG complex to cellular targets in situ was unknown3. It was unclear from these studies whether QD-based probes are both specific enough to recognize cellular targets of interest and bright enough for effective detection in real applications.

Recently we have developed methods for the surface functionalization of QDs. We synthesized specific immunofluorescent probes by linking these QDs to streptavidin and IgGs, and conducted comprehensive investigations into the labeling efficiency of these probes at the subcellular level. Here we describe the specific labeling of different types of target (cell surface receptors, cytoskeleton components, and nuclear antigens) at different subcellular locations (surface, intracellular, and intranuclear) and with different types of specimens (cultured live cells, fixed cells, and tissue sections). Additionally, we detected two different targets in the same cell simultaneously with different colors of QDs or combinations of QDs and organic dyes. These results demonstrate the practicality of QDs as an attractive class of fluorescence labels for biological and biomedical cellular imaging.

Results and discussion

IgG molecules are widely used for bioconjugation in fluorescence labeling. It was earlier demonstrated that QDs can be linked to IgG and that the resulting QD-IgG complex is capable of binding specific antibodies to form aggregates in solution3. However, it has not been shown that QD-IgG probes can label cellular targets. To investigate the ability of QD-IgG probes to label a specific cellular target, we attempted the detection of Her2, a cancer marker overexpressed on the surface of some breast cancer cells1416, with QD 535 (QDs with emission maximum at 535 nm) and QD 630 conjugated to antimouse IgG. The QD-IgG probes successfully labeled Her2 on the surface of human SK-BR-3 breast cancer cells after the cells were incubated with a monoclonal anti-Her2 antibody that binds to the external domain of Her2 (Fig. 1A, 1C). When cells were incubated

1Quantum

Dot Corporation, 26118 Research Rd., Hayward, CA 94545. 2Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. *Corresponding author (xwu@qdots.com).
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Figure 1. Detection of cancer marker Her2 with QD-IgG. (A, C) Fixed breast cancer SK-BR-3 cells were incubated with monoclonal anti-Her2 antibody and goat anti-mouse IgG conjugated to QDs. Her2 was clearly labeled with (A) QD 535IgG and (C) QD 630IgG (B, D). When cells were incubated with normal mouse IgG and QD-IgG, there were no detectable or very weak nonspecific signals on the cell surface. The nuclei were counterstained with Hoechst 33342 (blue). Filter sets ex 480 20 nm/em 535 10 nm and ex 560 27.5 nm/em 635 10 nm were used for QD 535 and QD 630, respectively. Scale bar, 10 m.

with normal mouse IgG and QD-IgG conjugates, little or no signal was detected (Fig. 1B, 1D), indicating that the QD-IgG conjugates were relatively specific for the target. Avidin (streptavidin) is another common molecule used in immunofluorescence labeling. We linked QD 560 and QD 608 to streptavidin and used the QD-streptavidin conjugates as alternative probes to detect Her2. The QD-streptavidin probes, together with a humanized anti-Her2 antibody and biotinylated goat anti-human IgG, effectively stained Her2 on the surface of fixed as well as live SK-BR-3 cells (Fig. 2A, 2B). When the cells were incubated with QDstreptavidin alone, weak or no detectable signal was observed on the cell surface (one control for QD 608 shown in Fig. 2C), indicating that the QD-streptavidin conjugates have very low nonspecific binding in a biotin-streptavidin labeling system. Fixed tissue sections are very common specimens for immunological labeling and pathological diagnosis. To investigate the compatibility of QD probes with this type of material, we incubated mouse mammary tumor tissue sections expressing human Her2 successively with rabbit antiserum reacting with the cytoplasmic domain of Her2, biotinylated goat anti-rabbit IgG, and QD 630streptavidin. QDs clearly labeled the tumor cells with the expected membranous pattern (Fig. 2D). This demonstrates that QD-based probes can be adapted to applications with tissue sections. As nanoparticles, QDs are larger (410 nm in diameter) than organic dye molecules (1 nm). Although we effectively labeled the extracellular domain of Her2 expressed on cultured cells and the cytoplasmic domain of Her2 in tissue sections with QDs, we considered that labeling proteins inside the cell might be difficult because steric factors could limit the access of QD probes to intracellular targets. To investigate this possibility, we attempted to stain microtubules in mouse 3T3 fibroblast cells using QD-streptavidin as the 42
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Figure 2. Detection of Her2 with QD-streptavidin. (A, B) SK-BR-3 cells were incubated with Herceptin (humanized anti-Her2 antibody interacting with the external domain of the antigen16), biotinylated goat anti-human IgG, and (A) QD 560streptavidin on the surface of fixed cells or (B) QD 608streptavidin on the surface of unfixed live cells. (C) Unfixed cells were incubated with anti-human IgGbiotin and QD 608streptavidin alone, and no signal was apparent (a bright-field image of a cell was inserted to indicate the presence of an unstained cell). (D) Her2 was also detected on sections of mouse mammary tumor tissue with QD 630streptavidin in combination with a rabbit antiserum recognizing the cytoplasmic domain of the antigen. Nuclei were counterstained blue with DAPI (D). Filter sets: ex 460SP/em 570 10 nm, ex 560 27.5 nm/em 605 10 nm, and ex 560 27.5 nm/em 635 10 nm were used for QD 560, QD 608, and QD 630, respectively. Scale bar, 50 m for (A), 20 m for (B) and (C), and 8 m for (D).

secondary label reagent. Microtubules were clearly labeled with QD 630streptavidin (Fig. 3A). When cells were incubated with QDstreptavidin alone, very weak or no apparent signals were detected (Fig. 3B), indicating that QD-streptavidin labeling of the microtubules was specific. Bruchez et al. reported labeling of actin filaments with QDbiotin2. The fluorescent signal was weak even after two rounds of biotin-avidin amplification. In addition to the specific staining of the actin filaments, the probe also showed some nonspecific binding to the nuclear membrane2. To investigate whether our QD probes could label the fine cellular structure of actin filaments, we stained fibroblasts with QD 535streptavidin after the cells were incubated with biotinylated phalloidin, a reagent that specifically binds to filamentous F-actin but not to globular G-actin17. F-actin filaments were clearly labeled with QD 535streptavidin with only one round of biotin-streptavidin interaction (Fig. 3C); moreover, there was no nonspecific staining of other parts of the cell. When cells were incubated with QD-streptavidin alone, no or very weak signals were detected (Fig. 3D). These results indicate that the QD-based probes can be bright enough and specific enough for effective labeling of fine cellular structures, a considerable improvement in performance over previously reported QD probes. Although there is a report of staining the nucleus with QDs coated with urea and acetate groups2, it remained unclear whether QDs conjugated with biomolecules could label specific antigens inside the nucleus. To investigate the labeling efficiency of QD-based bioprobes for nuclear targets, we incubated fixed human epithelial
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Figure 3. Staining of cytoskeleton fibers in 3T3 mouse fibroblast cells with QD-streptavidin. (A) Microtubules were labeled with monoclonal anti-tubulin antibody, biotinylated anti-mouse IgG and QD 630streptavidin (red). (B) Control for (A) without primary antibody. (C) Actin filaments were stained with biotinylated phalloidin and QD 535streptavidin (green). (D) Control for (C) without biotin-phalloidin. The nuclei were counterstained with Hoechst 33342 blue dye. Filter sets ex 480 20 nm/em 535 10 nm and ex 560 27.5 nm/em 635 10 nm were used to observe signals of QD 535 and QD 630, respectively. Scale bar, 10 m for (A), 24 m for (B) through (D).

cells with human anti-nuclear antigen (ANA) antibodies, followed by biotinylated anti-human IgG and QD-streptavidin. The nuclear antigens were clearly labeled with red QD 630streptavidin (Fig. 4A). In control cells incubated with secondary antibodies and QDstreptavidin alone, no detectable signal was observed (Fig. 4B). We have noticed that whereas staining of cell surface antigens in whole cells and tissue sections is reliable and effective, staining of cytoplasmic and nuclear structures is more variable. Some QD probes showed strong positive signals with low background; others exhibited weaker positive staining or higher background. This variation might be caused by inconsistencies in the surface coating or bioconjugation of QDs, and we are investigating the underlying mechanism. To further assess the specificity of the QD-streptavidin conjugates, we labeled nuclear antigens with QD 630streptavidin and microtubules with QD 535streptavidin in the same experiment by means of sequential biotin-streptavidin binding. When examined under an epifluorescence microscope with a 460 nm short-pass excitation filter (ex 460SP) and a 500 nm long-pass emission filter (em 500LP),
Figure 4. Detection of nuclear antigens and double labeling. (A) Nuclear antigens in the nuclei of human epithelial cells were labeled with ANA, antihuman IgGbiotin and QD 630streptavidin. (B) When normal human IgGs were used in place of ANA, no detectable stain was observed. (C) The nucleus of a 3T3 cell was stained with ANA, anti-human IgGbiotin, and QD 630streptavidin (red). The microtubules were labeled with mouse anti-tubulin antibody, anti-mouse IgGbiotin, and QD 535streptavidin (green). (D) Her2 on the surface of SK-BR-3 cells was stained green with mouse anti-Her2 antibody and QD 535IgG (green). Nuclear antigens were labeled with ANA, anti-human IgGbiotin and QD 630streptavidin (red). Filter sets ex 480 20 nm/em 535 10 nm and ex 560 27.5 nm /em 635 10 nm were used for detecting QD 535 and QD 630 and capturing images; ex 460SP/em 500LP was also used for observation. Scale bar, 50 m for (A), (B), and (D), and 10 m for (C).
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both colors were clearly visible and spectrally resolved to the eye. The nuclear antigens in the nucleus appeared to be labeled with the red QD 630streptavidin, and the microtubules in the cytoplasm with the green QD 535streptavidin (Fig. 4C). These results demonstrate that the QD-streptavidin conjugates are specific for their intended targets and that they can be effectively used in two-color fluorescence labeling of distinct cellular components. Although we successfully detected two cellular targets in the same cell with green and red QDs conjugated to the same molecule (streptavidin), two-color labeling is typically conducted with two distinct fluorochromes conjugated to different biomolecules. To carry out this type of double-label detection, we used QD 535IgG and QD 630streptavidin to detect, respectively, Her2 on the cell surface and nuclear antigens in the nucleus of SK-BR-3 cells. When the sample was observed under a fluorescence microscope with ex 460SP/em 500LP filter set, both QD 630-labeled (red) nuclear antigens and QD 535-labeled (green) membrane-associated Her2 were visible simultaneously (Fig. 4D). Therefore, QDs conjugated to different secondary detection reagents are effective in common fluorescent double labeling. It has been reported that the fluorescence intensities of individual QDs are much brighter than those of wavelength-matched organic dye molecules3. However, that comparison was done with nonaqueous, unconjugated QDs in a nonbiological environment3. The observed brightness could be less for QDs that are conjugated and used in staining experiments. Also, in real labeling applications, the detection sensitivity is determined by the collective emission intensity of an assembly of QDs rather than individual QDs. We compared the brightness of QD 608streptavidin and Alexa Fluor 568 streptavidin in labeling applications. We selected Alexa 568 as the benchmark for this study because Alexa dyes are reported to be brighter than any other known organic dyes18. In addition, because Alexa 568 and QD 608 have a similar emission peak, we can neglect wavelength-dependent camera sensitivity. For the same excitation wavelength (560 27.5 nm), which is optimal for Alexa 568, the positive signal of QD 608streptavidin was about four times as strong as that of Alexa 568streptavidin, whereas the background signal was only slightly higher (Fig. 5A). When QD 608streptavidin was excited with the optimal excitation filter (ex 460SP), the positive signal was more than nine times stronger than that of Alexa 568, and the

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A
Figure 5. Fluorescence intensity and emission spectra of QDstreptavidin conjugates. (A) Fluorescence intensity comparison between Alexa 568streptavidin and QD 608streptavidin. Mouse kidney sections were stained with ANA, anti-human IgG-biotin, and Alexa 568streptavidin or QD 608streptavidin to label nuclear antigens. Filter set ex 560 27.5 nm /em 605 10 nm was used to detect both Alexa 568 and QD 608. Filter set ex 460SP/em 605 10 nm was also used to optimize detection of QD 608. Images were captured from positively stained and control specimens with the same gain and exposure time. The mean fluorescence intensities were analyzed with Scion Image software (Scion Corporation, Frederick, MD) and plotted with Microsoft Excel. (B) Emission spectra of QDs used in the labeling experiments of this report.

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background signal was only about three times that of Alexa 568. It should be emphasized that other influences could contribute to these observed intensity differences. We also carried out similar intensity comparisons with QD 535, QD 560, and QD 630 (see emission spectra in Fig. 5B). Although their fluorescence intensities were different, all of the tested QD probes were brighter (by two to four times under optimal excitation) than spectrally similar Alexa dyes. Another feature that is critical for most fluorescent applications is photostability. Although there are some reports on the photostability of QDs, the QDs used in these studies either had different surface coatings2 or were not conjugated to biomolecules3. Taking advantage of the excitation flexibility of QD probes, we designed an experiment allowing us to monitor the fluorescence intensity changes of QDs and the organic dye Alexa Fluor 488 simultaneously in the same cell with the same excitation wavelength for 3 min. We chose Alexa 488 for this experiment because of a report that Alexa dyes are more photostable than other organic dyes18. In these observations, we used a 100 oil-immersion objective, which is optimal for examining fine cellular structures such as microtubules but requires high photostability of the label because of the high excitation light intensity at the focus of the objective. Regardless of which
Figure 6. Photostability comparison between QDs and Alexa 488. (A) Top row: Nuclear antigens were labeled with QD 630streptavidin (red), and microtubules were labeled with Alexa 488 conjugated to anti-mouse IgG (green) simultaneously in a 3T3 cell. Bottom row: Microtubules were labeled with QD 630streptavidin (red), and nuclear antigens were stained green with Alexa 488 conjugated to anti-human IgG. The specimens were continuously illuminated for 3 min with light from a 100 W mercury lamp under a 100 1.30 oil-immersion objective. An excitation filter (ex 485 20 nm) was used to excite both Alexa 488 and QD 630. Emission filters em 535 10 nm and em 635 10 nm on a motorized filter wheel were used to collect Alexa 488 and QD 630 signals, respectively. Images were captured with a cooled CCD camera at 10 s intervals for each color automatically. Images at 0, 20, 60, 120, and 180 s are shown. Whereas labeling signals of Alexa 488 faded quickly and became undetectable within 2 min, the signals of QD 630 showed no obvious change for the entire 3 min illumination period. Scale bar, 10 m. (B) Quantitative analysis of changes in intensities of QD 608streptavidin (stained microtubules) and Alexa 488streptavidin (stained nuclear antigens) using specimens mounted with glycerol or antifade mounting medium Vectashield. All experimental conditions were the same as in (A) except that the emission filter for QD 608 was 605 10 nm. Mean fluorescence intensity was automatically measured every 10 s for 3 min.

target was stained with QD-streptavidin, the fluorescent signal was very stable against photobleaching (Fig. 6A). In contrast, Alexa 488 was bleached very quickly. We compared further the photostability of QD 608streptavidin and Alexa 488streptavidin with specimens mounted with glycerol or the antifade mounting medium Vectashield (Fig. 6B). Without antifade medium, the fluorescence intensity of Alexa 488 decreased to 50% of the initial intensity at 10 s, and to 10% at 60 s. With the protection of antifade reagents, Alexa 488 retained 80% of the initial intensity at 60 s and 55% at the end of 3 min of illumination. For QD 608streptavidin, the intensity was 104% of the initial intensity for glycerol-mounted specimens and 97% for antifade mediummounted specimens at the end of the illumination. The results indicate that even with anti-bleaching protection, Alexa 488streptavidin is still much less photostable than the QD-streptavidin. Although antifade mounting medium can provide conventional organic dyes some protection against photobleaching, for some applications, such as live cell imaging, antifade medium cannot be used. Therefore photostable QD probes will be particularly valuable for quantitative fluorescent molecular detection and live cell imaging. With the completion of the Human Genome Project and the cataloging of all gene sequences, biological and biomedical investigations are now focusing on how the tens or hundreds of thousands of proteins in a single cell function and interact with each other. A promising approach is fluorescence microscopy, which has exquisite sensitivity down to the single-molecule level together with high spatial and sub-

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mouse IgG conjugated to QD 535 (80 nM) or QD 630 (40 nM) for 30 min each. For detection of Her2 with QD-streptavidin, the fixed and BSAblocked cells were incubated sequentially with 2 g/ml of a humanized anti-Her2 monoclonal antibody binding to the external domain of Her2 (Herceptin; Genentech, Inc., S. San Francisco, CA) for 30 min, 1.5 g/ml biotinylated goat anti-human IgG (Vector) for 30 min, then 40 nM QDstreptavidin for 30 min. Nuclei were counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR). To label Her2 on unfixed cells, cultured live SK-BR-3 cells were washed with cool PBS, incubated sequentially with 5 g/ml Herceptin (Genentech) for 1 h, 1.5 g/ml biotinylated goat antihuman IgG (Vector) for 30 min, and 40 nM QD-streptavidin for 30 min. All antibodies and QD-streptavidin were diluted in PBS containing 10% (wt/vol) BSA, and all steps were carried out at 4 C. Labeled cells were suspended in 10% (wt/vol) BSAPBS. A drop of cell suspension was placed on a glass slide, covered with a coverslip, examined, and imaged immediately under an upright fluorescence microscope. Detection of Her2 in mouse mammary tumor tissue sections. Mammary tumors were identified in transgenic mice expressing the human gene encoding Her2 under the control of the mouse mammary tumor virus promoter (Sharon Erickson, personal communication). Formalin-fixed, paraffinembedded tumors were sectioned at 5 m and treated with Target Retrieval (DAKO Corporation, Carpinteria, CA), Avidin Biotin Blocking Solution (Vector) and 10% (vol/vol) normal goat serum plus 3% (wt/vol) BSA before being incubated sequentially with 5 g/ml of a rabbit anti-Her2 polyclonal antibody directed against the cytoplasmic domain of Her2 (catalog no. A0458; DAKO Corporation) for 30 min, biotinylated goat anti-rabbit IgG for 30 min, and finally with 40 nM QD 630streptavidin for 30 min. Slides were mounted with Vectashield containing DAPI (Vector). Labeling of actin and microtubules. To label actin, fixed and BSA-blocked 3T3 cells were incubated sequentially with 0.5 M of biotinylated phalloidin (Molecular Probes) and 40 nM QD-streptavidin. To label microtubules, fixed and blocked 3T3 cells were incubated sequentially with monoclonal anti--tubulin (1:200 dilution of ascites fluid) overnight at 4 C, 15 g/ml biotinylated goat anti-mouse IgG (Vector) for 30 min, and finally with 40 nM of QD-streptavidin for 30 min. Nuclei were counterstained with 2 g/ml Hoechst 33342 (Molecular Probes). Nuclear antigen detection. Slides containing acetone-fixed human epithelial cells (INOVA Diagnostics, San Diego, CA) were blocked with 1% (wt/vol) BSA0.05% (vol/vol) Triton X-100 for 20 min and incubated overnight with human polyclonal anti-nuclear antigen antibodies (ANA; from INOVA). The next day the cells were incubated with 3 g/ml biotinylated goat anti-human IgG (Vector) for 30 min and then with 40 nM of QDstreptavidin for 30 min. Double labeling of nuclear antigens and Her2. Fixed and BSA-blocked SK-BR-3 cells were incubated with a mixture of ANA and mouse anti-Her2 antibody (Zymed Laboratories Inc.) overnight at 4 C, followed by incubation in 3 g/ml biotinylated goat anti-human IgG. Then the nuclear antigens and Her2 were labeled, respectively, by incubating cells with a mixture of red QD 630streptavidin (40 nM) and green QD 535 conjugated to anti-mouse IgG (80 nM) for 30 min. Double labeling of nuclear antigens and microtubules. Fixed and BSAblocked 3T3 cells were incubated sequentially with a mixture of ANA and mouse anti--tubulin overnight at 4 C, 3 g/ml biotinylated anti-human IgG and 40 nM red QD 630streptavidin for 30 min each. The cells then were blocked with 50 g/ml biotin for 20 min, rinsed, and incubated sequentially with 15 g/ml biotinylated anti-mouse IgG and 40 nM green QD 535streptavidin for 30 min each. Double labeling with Alexa 488 and QD-streptavidin. Fixed and BSAblocked 3T3 cells were incubated sequentially with a mixture of ANA and mouse anti--tubulin overnight at 4 C, with 3 g/ml biotinylated antihuman IgG for 30 min, and then with a mixture of Alexa 488 conjugated to anti-mouse IgG (68 nM; Molecular Probes) to label microtubules and red QD-streptavidin (40 nM) to label the nucleus. Alternatively, after overnight incubation with primary antibodies, cells were incubated with goat anti-mouse IgGbiotin and then exposed to a mixture of 68 nM Alexa 488 conjugated to anti-human IgG (to label the nucleus) and 40 nM QDstreptavidin (to stain microtubules).
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cellular resolution19,20. Because most cellular functions are accomplished by many proteins working together, there is a clear need to study multiple biological targets simultaneously in the cell. This goal has driven the development of new fluorescent labels that permit multiplexed labeling of cellular targets. However, the number of available fluorescent dyes that have distinguishable emission spectra is limited because each dye typically displays a broad emission width. Moreover, each dye typically requires a different wavelength of light for effective excitation, making it extremely difficult, if not impossible, to detect multiple targets simultaneously. In contrast, the emission widths of QDs are narrower than those of organic dyes2,3, allowing multiple colors of QDs to be used without spectral overlap. More importantly, all of the possible colors can be simultaneously excited with a single wavelength of light, just as we showed the simultaneous detection of cancer marker Her2 and nuclear antigens in this report. At this stage, QD-based probes still have some limitations, including their dependency on specific buffer conditions to obtain optimal signal-to-noise ratio, the scarcity of optimal filter sets, and the difficulty in defining and characterizing coating conditions. Yet these limitations are being rapidly resolved. We expect that the exceptionally bright, highly photostable, and multiplexing-compatible QD-based labels will be ideal fluorescence probes for multiplexed target analysis in biological and biomedical applications.

Experimental protocol
All reagents were purchased from Sigma-Aldrich (St. Louis, MO), and all experiments were done at room temperature unless indicated otherwise. QDstreptavidin conjugates are available through Quantum Dot Corporation (Hayward, CA; www.qdots.com). A range of additional colors and conjugates will be available from January 2003. Preparation of QD conjugates. Organic-soluble, CdSe/ZnS core-shell nanocrystals21,22 were isolated from hexanes and ligand solution with an equal volume of methanol, rinsed with methanol, and redispersed in CHCl3. These materials were mixed with neutralized amphiphilic polymer (40% octylamine-modified polyacrylic acid, 2,000 units/QD) in CHCl3, and the solvent evaporated. The dry film was redispersed in water and purified from excess polymer by gel filtration. The surface coating was cross-linked further by EDC (1-ethyl-3-(3-dimethylamino propyl)carbodiimide)-mediated coupling to lysine (or polyethylene glycollysine), and these materials were then coupled to streptavidin or antibodies by an EDC-mediated coupling reaction in 10 mM borate buffer, pH 8.0. Unless otherwise noted, QD bioconjugates were diluted for use in 10 mM borate buffer, pH 8.2. QD 535, QD 560, QD 608, and QD 630 were used for this report. Cell culture and fixation. The human breast cancer cell line SK-BR-3 and mouse 3T3 fibroblasts were ordered from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured (37 C, 5% CO2) on glass chamber slides (Nalge Nunc International, Naperville, IL) in McCoys 5A medium (for SK-BR-3) or Dulbeccos modified Eagles medium (DMEM; for 3T3) with 10% (vol/vol) FBS (Invitrogen Corp., Carlsbad, CA) overnight. For detection of Her2, SK-BR-3 cells were fixed with 4% formaldehyde for 10 min and blocked for 20 min in PBS containing 1% (wt/vol) BSA. For staining of actin, microtubules and double labeling, SK-BR-3 or 3T3 cells were fixed with 90% (vol/vol) methanol10 mM EGTA at 20 C for 15 min and blocked in 1% (wt/vol) BSA0.1% (vol/vol) Triton X-100 for 20 min. Fluorescence microscopy. Stained cells or tissue sections were mounted on slides in 90% (vol/vol) glycerol10% PBS or Vectashield antifade medium (Vector Laboratories, Burlingame, CA) with coverslips. Samples were examined under a Nikon E-800 epifluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Images were captured and processed with SimplePCI image analysis software (Compix, Inc., Cranberry Township, PA). Detection of Her2 on breast cancer surface. Fixed and BSA-blocked SK-BR-3 cells were incubated sequentially with 2.5 g/ml of a mouse anti-Her2 antibody that binds the external domain of Her2 (catalog no. 28-0003; Zymed Laboratories Inc., S. San Francisco, CA) and goat antiwww.nature.com/naturebiotechnology

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Acknowledgments We express our appreciation to Walt Mahoney, Ping Wu, and Charles Z. Hotz for supervising the project and for their critical reading of this manuscript. We thank Don Z. Zehnder, Edward Adams, Ted Haxo, Linh H. Nguyen, Christopher H. Ng, and Susan Palmieri for their contribution in generating QD probes and images. We also wish to acknowledge Hugh Daniels for his

participation in the early stages of the work. This work was supported in part by grant R44 CA88391-03 from the National Institutes of Health. Competing interests statement The authors declare that they have no competing financial interests.
Received 27 June 2002; accepted 25 October 2002

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JANUARY 2003

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