10.1586/14789450.4.4.565 © 2007 Future Drugs Ltd ISSN 1478-9450
Nanobiotechnology: quantum ...
Zhang, Kaji, Tokeshi & Baba
566 Expert Rev. Proteomics 4(4), (2007)
surface traps. In order to enhance the fluorescence ef...
Nanobiotechnology: quantum dots in bioimaging 567
Chan et al. modified the surface of CdSe/ZnS QDs wi...
Zhang, Kaji, Tokeshi & Baba
568 Expert Rev. Proteomics 4(4), (2007)
aggregation and fluorescence loss in physiological buf...
Nanobiotechnology: quantum dots in bioimaging 569
Figure 3. Cancer therapy on the dot? (A) Photodynam...
Zhang, Kaji, Tokeshi & Baba
570 Expert Rev. Proteomics 4(4), (2007)
may perturb cell motility. Parak et al. deposited thin...
Nanobiotechnology: quantum dots in bioimaging 571
Key issues
• Quantum dots (QDs) were prepared for f...
Zhang, Kaji, Tokeshi & Baba
572 Expert Rev. Proteomics 4(4), (2007)
24 Voura EB, Jaiswal JK, Mattoussi H,
Simon SM. Tracki...
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nanobiotechnology QD in bioimaging

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  • 1. Review 10.1586/14789450.4.4.565 © 2007 Future Drugs Ltd ISSN 1478-9450 Nanobiotechnology: quantum dots in bioimaging Yong Zhang† , Noritada Kaji, Manabu Tokeshi and Yoshinobu Baba † Author for correspondence Graduate School of Pharmaceutical Sciences, University of Tokushima, 1-78, Shomachi, Tokushima 770-8505; Nagoya University, Department of Applied Chemistry, Graduate School of Engineering, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Tel.: +81 527 894 666 Fax: +81 527 894 666 KEYWORDS: biological application, modification, nano, quantum dot, synthesis, toxicity Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots. Expert Rev. Proteomics 4(4), 565–572 (2007) With the completion of the Human Genome Project and the cataloging of all gene sequences [1], biological and biomedical inves- tigations are now focusing on how the tens or hundreds of thousands of proteins in a single cell function and interact with each other. Proteomics evokes the set of all protein iso- forms and modifications, the interactions between them and the structural description of proteins and their higher-order complexes [2]. By studying global patterns of protein con- tent and activity and how these change during development or in response to disease, pro- teomics research is poised to boost our under- standing of systems-level cellular behavior. Clinical research also hopes to benefit from proteomics by both the identification of new drug targets and the development of new diagnostic markers. The scanning probe microscopies have limi- tations in observing intracellular structures with high selectivity and following the dynamic behavior of these structures. Fluores- cence is a widely used tool to address this problem. However, conventional organic fluorophores have two significant limitations: they can not fluoresce continuously for long periods and they are not optimized for multi- color applications. The latter limitation stems from two factors: each fluorophore can be optimally excited only by the light of a defined wavelength (which usually makes it necessary to use as many excitation sources as types of fluorophore) and each fluorophore has a rela- tively broad emission spectrum (which often causes the signals from different fluorophores to overlap) [3]. Nanoparticles are microscopic particles with at least one dimension less than 100 nm. They are usually made of materials such as metals, dielectrics and semiconductors. Great scientific interest is focused on nanoparticles as they are, effectively, a bridge between bulk materials and molecular structures. A quan- tum dot (QD) is made of a semiconductor and has a discrete, quantized energy spectrum. Properly modified QDs were largely used for single-molecule probing. Compared with con- ventional fluorophores, the nanocrystals have a narrow, size-tunable, symmetric emission spectrum and are photochemically stable (FIGURE 1) [4,5]. Synthesis of quantum dots The most common QD system is a CdSe/ZnS core/shell semiconductor nanocrystal system. The surface-to-volume ratio of CdSe cores is very high. There are many vacancies and trap sites on the surface, such that the fluorescence spectrum of bare QDs has a broad tail due to CONTENTS Synthesis of quantum dots Modification of quantum dots Biological application of quantum dots Concern regarding the toxicity of quantum dots Conclusion Expert commentary Five-year view Financial disclosure Key issues References Affiliations For reprint orders, please contact
  • 2. Zhang, Kaji, Tokeshi & Baba 566 Expert Rev. Proteomics 4(4), (2007) surface traps. In order to enhance the fluorescence efficiency, ZnS was directly grown onto CdSe cores to passivate the sur- face. The fluorescence efficiency of the ZnS-capped CdSe clus- ters was dramatically enhanced [6,7]. A two-step procedure is involved in the synthesis of CdSe/ZnS core/shell nanocrystals. In the first step, CdSe core particles are formed. The core parti- cles are then overcoated by ZnS in the second step. Tempera- ture plays a critical factor in controlling the procedure in both steps. The synthesis is based on Ostwald ripening, where fewer and larger crystals, which have smaller surface-to-volume ratios compared with small particles, form the solid, thus making the entire system more stable [6,8]. QD that emits blue light is elusive owing to a lack of appro- priate core-shell materials. Although blue emission can be obtained from CdSe particles, the size of CdSe particles is lim- ited within 2 nm. The small size makes synthesis and other operations difficult. Steckel et al. developed a synthesis method that was more facile to operate. They substituted CdS (4.7 ± 0.4–5.2 ± 0.4 nm) for CdSe core. Blue light-emitting (CdS)ZnS core-shell nanocrystals (460–480 nm) showed quantum efficiencies in the range of 20–30% [9]. Bailey et al. reported a procedure for preparing large quanti- ties of alloyed semiconductor quantum dots (CdSeTe) for con- tinuous tuning of quantum confinement without changing the particle size. Their results demonstrated that, besides par- ticle size, composition and internal structure of QDs were available for tuning the optical and electronic properties of alloyed semiconductor quantum dots [10]. Zhong et al. discov- ered that the composition-tunable emission across the visible spectrum could be formed over the composition of ZnxCd1-xSe nanocrystals (the emission wavelength blue shifts gradually with the increase in Zn content). The high lumines- cence efficiency and stability of the resulting alloy nanocrystals were attributed to the larger particle size, higher crystallinity, higher covalency, lower interdiffusion and spatial composition fluctuation [11]. They also successfully synthesized high-quality alloyed ZnxCd1-xS nanocrystals with high luminescent quan- tum yields and extremely narrow emission spectral widths of 14–18 nm. The obtained narrow spectral width stems from the uniform size and shape distribution, the high composition homogeneity and the relatively large particle radius [12]. A wavelength range of particular interest for biomedical imaging is the near-infrared (NIR) between 800 and 900 nm, where absorption in tissue is minimal. Kim et al. developed QDs with a core/shell/shell structure consisting of an alloy core of InAs1-xPx, an intermediate shell of InP and an outer shell of ZnSe. Alloyed core dots of InAs1-xPx show tunable emission in the NIR region and the InP shell leads to a red shift and an increase in the quantum yield [13]. Modification of quantum dots Hydrophobic surface of QDs leads to aggregation and non- specific adsorption, which hiders their application as biolabels. To make QDs water soluble, their surface species were exchanged with polar species. Figure 1. Spectrum comparison between fluorescein (A) and a typical quantum dot (QD) (B). QDs have a much broader adsorption spectrum (dashed line) than fluorescein. The emission spectrum (solid line) of QDs is relatively narrow and symmetrical compared with that of fluorescein (adapted from Figure 1 of [4]). Size-tunable QDs are shown in (C). The average sizes of the QDs are 4.2 (0.5), 4.4 (0.4), 5.6 (0.4) and 6.4 (0.4) nm for green, yellow, red and dark red, respectively. Adapted from [18] with permission from the American Association for the Advancement of Science. Normalizedintensity 1.0 0.8 0.6 0.4 0.2 0.0 400 500 600 700 Normalizedintensity 1.0 0.8 0.6 0.4 0.2 0.0 400 500 600 700 Wavelength (nm) Wavelength (nm) Intensity(arb.units) Wavelength (nm) 1.0 0.8 0.6 0.4 0.2 0.0 480 520 560 600 640 680 720 B C A
  • 3. Nanobiotechnology: quantum dots in bioimaging 567 Chan et al. modified the surface of CdSe/ZnS QDs with mercaptoacetic acid through the binding of the mercapto group to a Zn atom. The carboxylic acid group rendered the QDs water soluble. The free carboxyl group was also available for covalent coupling to various biomolecules by crosslinking to reactive amine groups [14]. Mattoussi et al. developed a strat- egy based on self assembly utilizing electrostatic attractions between negatively charged alkyl-COOH-capped CdSe/ZnS QDs and specific proteins consisting of positively charged attachment domains. The alkyl-COOH groups permitted dis- persion of QDs. The specific protein was in charge of fusing with desired biologically relevant domains (FIGURE 2) [15]. When the organic ligand shell of QDs was modified by lig- and exchange with thiols, the quantum yield (QY) would diminish. Encapsulating QDs and their initial ligands with macromolecules could preserve QY, but resulted in a bulky size that was not desired. Kim et al. developed oligomeric phosphine ligands to passivate QDs. The thin organic shells avoided bulky size and retained high QY [16]. Pinaud et al. found a naturally evolved interaction between organic and inorganic. Based on this interesting finding, they designed synthetic α-peptides resembling phytochelatins. These α-peptides could naturally bind on the surface of QDs and could make QDs buffer soluble, biocompatible and photostable [17]. Although priming the QD surface with a thiolated molecule that has a free car- boxyl group could make QDs soluble, the bond is dynamic, leading to the low sta- bility of QDs in water. Gerion et al. masked QDs with a robust silica shell, the procedure yielded nanocrystals encapsu- lated in a silica shell of about 2–5 nm, functionalized with thiols and/or amines on the surface. The silica-coated QDs showed a greater stability in biological buffers compared with nanoparticles primed with thiolated molecules [18]. Although polar species could be exchanged on the surface to make the QDs water soluble, both monolayers and multilayers suffered disadvantages, such as poor stability, long, difficult coating processes, nonspecific adsorp- tion and aggregation. Dubertret et al. discovered that CdSe/ZnS QDs could be encapsulated in the hydrophobic core of a micelle composed of a mixture of N-poly(ethylene glycol) phosphati- dylethanolamine and phosphatidyl- choline without any surface modifica- tion. Transmission electron microscopy images of QD–micelles were fairly mon- odisperse, indicating low aggregation. The fluorescence signal-to-background ratio of QD–micelles was greater than 150, compared with approximately four for silica-coated QDs, owing to the low nonspecific adsorption [19]. With bioconjungation, QDs could target the desired pro- teins. A three-layer strategy is often adopted: primary anti- body, followed by biotinylated secondary antibody, followed by streptavidin–QD. The size of this QD complex (∼50 nm) can affect membrane protein trafficking and can reduce accessibility to crowded locations in cells. Howarth et al. developed a method to target QDs to cell surface proteins that eliminated the bulky antibodies and provided a stable linkage between the QD and the protein of interest. Mam- malian cell-surface proteins tagged with a 15 amino acid acceptor peptide could be biotinylated by biotin ligase added to the medium, while endogenous proteins remained unmodified. The biotin group then served as a handle for targeting streptavidin-conjugated QDs [20]. The method was demonstrated in targeting QDs to surface proteins of HeLa cells (FIGURE 2). Gao et al. developed a class of polymer-encap- sulated and bioconjugated QD probes for cancer targeting and imaging in vivo. CdSe/ZnS QDs were encapsulated with a triblock copolymer, multiple poly(ethylene glycol) molecules and affinity ligands. The structural design avoided particle Figure 2. (A) CdSe–ZnS core-shell nanoparticle with dihydrolipoic acid surface capping groups. (B) S–S linked MBP–zb homodimer and detail showing nucleotide and primary amino acid sequences of the C-terminal basic leucine zipper interaction domain. The poly-Asn flexible linker is boxed with dashed lines, unique engineered cysteine is double boxed and lysine residues contributing to the net positive charge of the leucine zipper are single boxed. MBP-zb: Maltose-binding protein-basic leucine zipper. Reproduced from [15] with permission from the American Association for the Advancement of Science. Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile TCG AGC TCG AAC AAC AAC AAC AAT AAC AAT AAC AAC CTC GGG ATC Glu Gly Arg Cys Gly Gly Ser Ala Gln Leu Lys Lys Lys Leu Gln GAG GGA AGG TGC GGT GGC TCA GCT CAG TTG AAA AAG AAA TTG CAA Leu Lys Lys Lys Leu Ala Gln Gly Gly Asp *** CTC AAG AAG AAA CTC GCC CAG GGT GGG GAT TAA TCT AGA GTC GAC Xbal Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala GCA CTG AAG AAA AAG AAC GCT CAG CTG AAG TGG AAA CTT CAA GCC Flexible peptide linker Interchain dislufide bond Basic leucine zipper MBP COO– MBP + + + + + + S CdSe ZnS HS SH O– O HS HS O SHHS O– O SH SH O– O B A S O– COO–
  • 4. Zhang, Kaji, Tokeshi & Baba 568 Expert Rev. Proteomics 4(4), (2007) aggregation and fluorescence loss in physiological buffers and in live animals. Tumor targeting was achieved by bioconjuga- tion with an antibody. Combined with wavelength-resolved imaging, the QD probes allowed sensitive and multicolor imaging of cancer cells in living animals [21]. Wu et al. coated CdSe/ZnS nanocrystals with a neutralized amphiphilic poly- mer. The surface was then coupled to streptavidin or IgG. The QDs successfully labeled a specific cellular target (i.e., labeling the breast cancer marker Her2 on the surface of fixed and live cancer cells, staining actin and microtubule fibers in the cyto- plasm and detecting nuclear antigens inside the nucleus) [22]. Vu et al. conjugated the peptide ligand βNGF to the QD sur- face. These βNGF–QDs activated TrkA receptors and initiated neuronal differentiation in PC12 cells [23]. Biological application of quantum dots Organic fluorophores have limitations for multicolor imag- ing, since they require distinct excitation wavelengths and their broad emission regions overlap with each other. By con- trast, QDs can be excited by a wide spectrum of single and multiphoton excitation light and have narrow emission spec- tra. Voura et al. delivered dihydroxylipoic acid-capped QDs into cells by Lipofectamine™ 2000 to study extravasation in vivo. Five different populations of cells were simultaneously identified [24]. Monitoring the interactions of multiple proteins or cells within an organism is valuable when trying to understand the complexity and dynamics of biological interactions. Organic fluorophores are subject to photobleaching for this aim. Jaiswal et al. developed an approach to conjugate CdSe/ZnS QDs capped with dihydrolipoic acid ligands to positively charged desired proteins. These QDs were demonstrated to be suitable for simultaneous tracking of multiple proteins and live cells for long periods [25]. Multiphoton microscopy is a primary fluorescence imaging technique in thick specimens. Compared with conventional fluorophores, the cross-sections of QDs were higher by three orders of magnitude. Therefore, use of QDs may enable imaging at greater depths than standard fluorophores do. Larson et al. compared QDs with conventional methods by injecting 70 kDa fluorescein isothiocyanate dextran at its sol- ubility limit. An image acquired at the same depth with five- times as much power shows considerably less detail. Thus, QDs could be bright specific labels useful for tracking cells deep within tissue or for detecting low concentrations of antigens [26]. Targeting the nanoparticles to specific tissues and cell types is important to realize disease sensing and drug delivery. Aker- man et al. coated CdSe/ZnS core shell QDs with a site-recog- nizing peptide. The peptide-coated QDs were then injected into the tail vein of a mouse to investigate their homing abili- ties. The modified QDs exactly found their targets in the rele- vant vascular site [27]. Lidke et al. employed a QD–streptavidin conjugate for in vivo studies of transduction. The measure- ments revealed a new insight into processes and interactions that could previously only be studied on fixed cells or by bio- chemical fractionation. They discovered that erbB2, but not erbB3, heterodimerized with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling [28]. Gac et al. attached biotinylated annexin V on QD–streptavidin conju- gates for studying the apoptosis process. The time lapse of QDs and standard organic dyes was investigated. They discov- ered that either FITC- or Alexa Fluor 647-annexin V conju- gates photobleached within 25 min, while QDs stained the cells well for several hours. Photostability of QDs enabled the visualization of the fast event occurring at the membrane of apoptotic cells. However, such events would be missed with organic dyes [29]. Protein transduction domains (PTDs) are capable of trans- ducing cargo across the plasma membrane. Whilst the size of QDs falls well within the range of cargoes; based on these considerations, Lagerholm et al. utilized a nine residue biotin- ylated L-arginine peptide as PTD for intracellular delivery of QDs. The cell uptake efficiency of QDs was greater by a mag- nitude of almost two, as compared with incubation with bare QDs. Images of transmission electron microscopy showed that QDs were concentrated in endosomes and lysosomes. This method revealed that uptake efficiency of QDs could be dramatically improved in coding cells [30]. The emission properties of QDs could be tuned to emit into the NIR region in contrast to the visible emission of the most conventional photosensitizers. Since there is minimal light scattering and absorption in the NIR region of the spectrum, light of low intensity can be used to penetrate tissue to depths of several centimeters, thereby allowing access to deep-seated tumors. In addition, their large transition dipole moment led to strong absorption, making them potential candidates for application in photodynamic processes. Bakalova et al. thus reported an exploitation of QDs energy-transfer properties to give a therapeutic effect (FIGURE 3) [31]. Conventional NIR fluorophores, such as IRDye78-CA, dissolved in serum or aqueous buffer rapidly photobleach. Kim et al. prepared NIR CdTe(CdSe) core(shell) type II QDs for sentinel lymph node (SLN) mapping. When incu- bating these QDs in 100% serum at 37°C for more than 30 min, fluorescence emission decreased by only 10%. The relatively stable QD system provided the surgeon with direct visual guidance throughout the SLN mapping procedure (FIGURE 4) [32]. Compared with gold nanoparticles (40 nm) or latex spheres (500 nm), QDs could easily access single molecules and help us to understand the dynamics of cellular organization. Dahan et al. used single-QD tracking to study the rapid lat- eral dynamics of Gly receptors. According to their observa- tion, the receptors were classified as synaptic, perisynaptic and extrasynaptic with distinct diffusion properties [33]. Phagokinetic track is a rapid and automatic method for stud- ying cell motility. The previously used marker in this method was an Au particle that had many limitations. For example, the large Au particles could not stick well to the substrate and
  • 5. Nanobiotechnology: quantum dots in bioimaging 569 Figure 3. Cancer therapy on the dot? (A) Photodynamic processes involved in photodynamic therapy. (B) Possible mechanisms for induction of photodynamic processes by quantum dots. Reproduced with permission from Macmillan Publishers Ltd: Nat. Biotechnol. [31], © (2004). ROS: Reactive oxygen species. Figure 4. NIR QD sentinel lymph node mapping in the mouse and pig. (A) Images of mouse injected intradermally with 10 pmol of NIR QDs in the left paw. Left: preinjection NIR autofluorescence image; middle: 5 min post-injection white light color video image; right: 5 min post-injection NIR fluorescence image. An arrow indicates the putative axillary sentinel lymph node. Fluorescence images have identical exposure times and normalization. (B) Images of the mouse 5 min after reinjection with 1% isosulfan blue and exposure of the actual sentinel lymph node (left: color video; right: NIR fluorescence images). Isosulfan blue and NIR QDs were localized in the same lymph node (arrows). (C) Images of the surgical field in a pig injected intradermally with 400 pmol of NIR QDs in the right groin. Four time points are shown from top to bottom: before injection (autofluorescence), 30 s after injection, 4 min after injection and during image-guided resection. For each time point, color video (left), NIR fluorescence (middle) and color–NIR merge (right) images are shown. Fluorescence images have identical exposure times and normalization. To create the merged image, the NIR fluorescence image was pseudocolored lime green and superimposed on the color video image. The position of a nipple (N) is indicated. Reproduced with permission from Macmillan Publishers Ltd: Nat. Biotechnol. [32], © (2004). NIR: Near infrared; QD: Quantum dot. Intersystem crossing Chemicalreactions Fluorescence Internalconversion Absorptionoflight Phosphorescence S0 S1 S2 S3 3 O2 (or X) 1 O2 (or X*) Antibody Cancer cell hν hν Quantum dot hν ROS Energy transfer Classical photosensitizer Cd2+ 3 O2 3 O2 3 O2 DNA degradation Initiation of apoptosis 1 O2 Cd2+ ROS 1 O2 BA 1 cm 1 cm Preinjection autofluorescence Color video 5 min post injection NIR fluorescence 5 min post injection Color video NIR fluorescence Color video NIR fluorescence Color-NIR merge Image- guided resection 4min postinjection 30s post injection Preinjection (auto- fluorescence) B CA
  • 6. Zhang, Kaji, Tokeshi & Baba 570 Expert Rev. Proteomics 4(4), (2007) may perturb cell motility. Parak et al. deposited thin layers of colloidal semiconductor nanocrystals on collagen-coated tissue culture substrates, followed by seeding of cells. Human mam- mary epithelial tumor cells (MDA-MB-231) voraciously engulfed nanocrystals as they migrated and generated a region free of QDs that revealed their pathways. By contrast, non- tumor cells (MCF-10 A) appeared to be relatively immotile depending on the observation that the layer of nanocrystals was virtually identical to that seen around cells. By comparing these two cell types, they demonstrated the use of colloidal QD-based phagokinetic tracking [34]. Pathology scoring is the most widely used method of quan- titative immunohistochemistry in clinical settings. However, this method is on a discontinuous scale and the human eye is not capable of discerning subtle differences in the antigen expression level. Another method for protein quantification is the use of fluorescence molecules through acquisition of high- power images. Low accuracy was suffered due to photo- bleaching of organic dyes. Ghazani et al. utilized the QD-based immunoprofiling of proteins in the quantitative analysis of tissue microarrays. The new method was sensitive, accurate and on a continuous scale, and was validated in the analysis of tumor antigens [35]. Reverse-phase protein microarray (RPMA) is a high-through- put proteomic platform currently being developed for use in clinical trials. Conventional labeling techniques for RPMA detection include radioactivity, chromagens and fluorescence. However, they often have significant limitations in terms of their sensitivity, dynamic range, durability, speed, safety and ability to multiplex. Geho et al. demonstrated that the use of QD conjugated to streptavidin, QD 655 Sav, in a RPMA had advantages of multiplexed assays, detection of unamplified signals, expanded dynamic range and robustness [36]. Concern regarding the toxicity of quantum dots Although QDs have received enormous attention for their potential applications in biology and medicine, questions con- cerning their potential cytotoxicity remain unanswered. A key issue in evaluating the utility of these materials is the assessment of their potential toxicity – either due to their inherent chemi- cal composition (e.g., heavy metals) or as a consequence of their nanoscale properties (e.g., inhalation of particulate carbon nanotubes). Derfus et al. demonstrated that CdSe-core QDs were indeed cytotoxic under certain conditions. Specifically, surface oxidation through a variety of pathways led to the for- mation of reduced Cd on the QD surface and release of free cadmium ions, and correlated with cell death. However, the use of QDs in vivo must be critically examined, as their results sug- gested Cd release was a possibility over time. Surface coatings such as ZnS and bovine serum albumin (BSA) were shown to significantly reduce, but not eliminate, cytotoxicity [37]. Hoshino et al. revealed that the toxicity of QDs in biological systems was dependent on the surface molecules of the nanoc- rystal particle instead of core material [38]. Lovric et al. discov- ered that QD-induced cytotoxicity was in part dependent on QD size and was characterized by chromatin condensation and membrane blebbing. BSA–QDs conjugates were significantly less toxic than free QDs [39]. Conclusion The emission wavelength desired is available by controlling particle size, composition and the internal structure of QDs. Before their biolabel applications, the surface of QDs should be modified to make them soluble and site targetable. QDs will complement conventional organic fluorophores for applications needing better photostability, NIR emission or single-molecule sensitivity over long time scales. With the help of QDs, we can better understand the dynamics of cellular organization. Expert commentary QDs have a wide absorption range and relatively narrow emis- sion spectrum. It is possible to simultaneously probe several QD-labeled targets with one excitation source. This approach will provide us with more information to better understand the dynamics of cellular organization. The higher cross-section of QDs compared with conventional fluorophores is useful for detecting low concentrations of antigens. When QDs were modified by site-recognizing peptides, they could find and pre- cisely label target proteins. The measurement may reveal new insight into processes and interactions within cells or tissues. NIR emission QDs are potential candidates to replace conven- tional photosensitizers because the light in the NIR region shows low absorption in tissues. QD-based immunoprofiling of proteins in microarrays is more sensitive and accurate than conventional methods. Five-year view QDs have far from exhausted their biological potential. Mostly driven by cellular labeling, the effort to enable everyday research is ongoing. The future work is involved in simultane- ous tracking of multiple proteins and live cells for long periods and, therefore, in investigating a range of phenomena in cell and developmental biology that have been unexplored because of the lack of suitable fluorescent labels. In addition, an under- standing of receptor-mediated transduction mechanisms is essential for rational receptor-targeted therapeutics. Delivery and targeting of ligand compounds that surpass cell surface binding and evoke sufficient cellular responses are key require- ments for designing functional cell probes and delivery devices. The ligand-conjugated QD will play a key role in this effort in the near future. Finally, it is of great interest to develop new QDs deposited in a vertical gradient, which may lead to a 3D view of extracellular matrix media for depth contrast. Financial disclosure The authors have no relevant financial interests, including employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties related to this manuscript.
  • 7. Nanobiotechnology: quantum dots in bioimaging 571 Key issues • Quantum dots (QDs) were prepared for fluorophores. A narrow, size-tunable, symmetric emission spectrum, photochemical stability and a continuous excitation spectrum made QDs complementary to conventional fluorophores. • Different color-emitting QDs could be made through the control of constituent stoichiometries in alloy nanoparticles. The composition-tunable emission was investigated over the composition of the ZnxCd1-xSe nanocrystals. • A self-assembly method for conjugating protein molecules to CdSe-ZnS core-shell QDs was described. The conjugation utilized electrostatic attractions between negatively charged lipoic acid-capped CdSe-ZnS QDs and engineered bifunctional recombinant proteins, comprising positively charged attachment domains. • Hydrophobic CdSe/ZnS core/shell nanocrystals were embedded in a siloxane shell. The introduction of functionalized groups onto the siloxane surface would permit the conjugation of nanocrystals to biological entities. • QDs were first used as markers for phagokinetic tracks. • QDs were used for multiphoton imaging in live animals. • In vivo targeting studies of human prostate cancer growing in nude mice. • CdSe-core QDs were found cytotoxic in the case of forming reduced Cd. Surface coating could dramatically reduce the cytotoxicity. References Papers of special note have been highlighted as: • of interest •• of considerable interest 1 Venter JC, Adams MD. The sequence of the human genome. Science 291, 1304–1351 (2001). 2 Tyers M, Mann M. From genomics to proteomics. Nature 422, 193–197 (2003). 3 Jaiswal JK, Simon SM. Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504 (2004). 4 Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. 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