Anal. Chem. 2006, 78, 321-330
Uncoated, Broad Fluorescent, and Size-Homogeneous CdSe Quantum Dots for Bioanalyses Zhivko Zhelev,† Rumiana Bakalova,*,† Hideki Ohba,*,† Rajan Jose,| Yusuke Imai,† and Yoshinobu Baba‡,§
On-site Sensing and Diagnosis Research Laboratory, AIST-Kyushu, 807-1 Shuku-machi, Tosu, Japan, Health Technology Research Center, AIST-Shikoku, Takamatsu, Japan, Toyota Technological Institute, Nagoya, Japan, and Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan
In the present study, we describe the synthesis of highly luminescent uncoated water-soluble CdSe quantum dots (QDs) possessing the following characteristics: ∼2 nm in diameter, with very good size distribution (in 95% homodispersed) accompanied by a broad-band photoluminescent spectrum. The synthetic procedure is simple, is conducted at room temperature, in the absence of the most popular coordinating ligands (as TOPO or HDA), and is highly reproducible. The obtained CdSe core QDs possessed a comparatively long fluorescence half-life (∼30-90 ns, depending on the emission wavelength) detected by time-resolved spectroscopy. These QDs were further conjugated with antibodies and applied in several biochemical analyses. The application of quantum dots (QDs) in life science research increased enormously1-8 in the last years, starting from 1998 with the reports of Chan and Nie9 and Bruchez and colleagues10 about the possibility of using them as novel fluorophores in multiplex bioanalyses. The synthesis of highly luminescent water-soluble QDs became a priority. At present, the synthetic process has several shortcomings: it is time- and cost-consuming, still com* Corresponding authors. Tel.: +81-942-813628. Fax: +81-942-813690. E-mail:
[email protected]. E-mail:
[email protected]. † AIST-Kyushu. ‡ AIST-Shikoku. § Nagoya University. | Toyota Technological Institute. (1) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (2) Medintz, I. L.; Claap, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630-638. (3) Alivisatos, P. Nat. Biotechnol. 2004, 22, 47-52. (4) Ozkan, M. Drug Discovery Today 2004, 9, 1065-1071. (5) Bakalova, R.; Ohba, H.; Zhelev, Z.; Ishikawa, M.; Baba, Y. Nat. Biotechnol. 2004, 22, 1360-1361. (6) Voura, E. B.; Jaiswal, J. K.; Mattoussi, H.; Simon, S. M. Nat. Med. 2004, 10, 993-998. (7) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W.; Nie, S. Nat. Biotechnol. 2004, 22, 969-976. (8) Zorov, D. B.; Kobrinsky, E.; Juhaszova, M.; Sollott, S. J. Circ. Res. 2004, 95, 239-252. (9) Chan, W. C.; Nie, S. Science 1998, 281, 2016-2018. (10) Bruchez, M.; Morrone, P.; Gin, S.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. 10.1021/ac0511896 CCC: $33.50 Published on Web 11/25/2005
© 2006 American Chemical Society
plicated, and conducted at high temperature; the preservation of the photoluminescent (PL) properties of the synthesized QDs requires a covering with several coatsscurrently, the most popular QDs consist of a CdSe core, followed by a ZnS shell and one or more organic coats. The encapsulation of QDs and their primary ligands with macromolecules such as lipids and polymers preserves their quantum yield (QY); however, the resulting overcoated nanocrystals become largestheir size is commensurate with or larger than the size of biomolecules that have to be conjugated.11,12 The efforts are directed to the development of procedures resulting in a synthesis of highly luminescent watersoluble QDs with diameters of 10-15 nm or lower, consisting of a thin inorganic/organic cover.13-17 The CdSe QDs are confirmed as the most useful in life science research, at least for the time being. However, a successful application of uncoated CdSe QDs in biochemical experiments has still not been shown, because the surface exchange of their primary ligands (as TOPO or HDA) and subsequent transfer in aqueous solutions results in a significat lose of their PL properties. Several groups have been described a synthesis of CdSe QDs directly in aqueous solution.18,19 The crude product contains nanocrystals with heterogeneous size in the range from 2 to 5 nm, and in all cases, the QY is too low (less than 0.1%) to be used directly for conjugation with immunocompatible molecules. Procedures, including additional coatings, are required to improve their PL characteristics before applying them in life science experiments. An excellent review, summarizing all known synthetic and capping strategies in the quantum dot field, can be (11) Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41-46. (12) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (13) Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. J. Am. Chem. Soc. 2004, 126, 6115-6123. (14) Kim, S.; Bawendi, M. G. J. Am. Chem. Soc. 2003, 125, 14652-14653. (15) Wang, Y. A.; Li, J. J.; Chen, H.; Peng, X. J. Am. Chem. Soc. 2002, 124, 2293-2298. (16) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861-8871. (17) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183-184. (18) Rogach, A. L.; Kornowski, A.; Gao, M.; Eychmuller, A.; Weller, H. J. Phys. Chem. B 1999, 103, 3065-3069. (19) Zhou, X.; Kobayashi, Y.; Romanyuk, V.; Ochuchi, N.; Takeda, M.; Tsunekawa, S.; Kasuya, A. Appl. Surf. Sci. 2005, 281-286.
Analytical Chemistry, Vol. 78, No. 1, January 1, 2006 321
found in ref 20. The major characteristic of the commonly described CdSe QDs is their narrow symmetric PL spectrum. The broadband PL spectrum of CdSe QDs is usually associated with the presence of different size nanocrystals in the crude solution. Despite this generally held view, recently, Chen et al.21 reported that CdSe nanocrystals with homodispersed and uniquely stable 2-nm size manifest also a broad photoluminescence in organic solvents. Unfortunately, the authors did not report the quantum efficiency of the synthesized nanocrystals, as well as the possibility for water solubilization with preservation of their PL properties. In the present study, we describe the synthesis of highly luminescent CdSe QDs possessing unusual characteristics: small size (∼2 nm in diameter), very good size distribution (in 95% homodispersed), broad PL spectrum, and comparatively long fluorescence half-life (∼30-90 ns, depending on the emission wavelength). The synthetic procedure was simple, was conducted at room temperature (∼22 °C), occurred in the absence of the most popular coordinating ligands such as TOPO or HDA, and was highly reproducible. The synthesized uncoated CdSe core QDs were easily subjected to water solubilization (using mercaptosuccinic acid as a surface-modifying reagent) with enhancement of their QY (from ∼40% in chloroform to ∼50% in phosphatebuffered saline, PBS). Moreover, the water-soluble CdSe core QDs showed an excellent transparency and stability after long-term storage in full lack of aggregation. These QDs were further conjugated with peptides (antibodies) and applied in several biochemical analyses using flow cytometry and immunoblotting. EXPERIMENTAL SECTION Synthesis of CdSe Core Nanocrystals. The stock solution of tri-n-octylphosphine selenide (TOP-Se) was prepared by dissolving Se shots (0.7896 g, 10 mM, Aldrich) in TOP (7.413 g, 20 mM, Lancaster) at 100 °C for 2 h in argon atmosphere and then cooled to room temperature. Cadmium acetate (1 g, 3.75 mM, Wako) was dispersed in 2 mL of TOP in a round-bottom flask in argon atmosphere by stirring intensively for 20 min at 100 °C with subsequent cooling to room temperature. Four milliliters of Se stock solution was injected into the Cd stock solution. This provided a homogeneous reaction, in which all monomers exist in a liquid phase. Volume of the flasks was 100 mL. The reaction mixture was stirred intensively during 2-4 h at room temperature. Formation of CdSe nanocrystals was monitored by UV/visible absorption and fluorescent spectroscopy. The reaction was stopped by addition of ∼30 mL of chloroform. The crude solution (dissolved in chloroform) showed a slight turbidity as a result of the insolubility of nonreacted cadmium acetate in chloroform. After centrifugation at 16 000 rpm, the crude solution showed an excellent transparency. The obtained nanocrystal solution in chloroform (supernatant) was kept at -20 °C in the dark for a long time. No changes in the absorption and PL spectra were observed after 11 months. Calculation of Photoluminescence Quantum Yield.22 For determination of room temperature fluorescence QY, the spectrally (20) Medintz, I. L.; Uyeda, T.; Goldman, E.; Mattoussi, H. Nat. Mater. 2005, 4, 435-446. (21) Chen, X.; Samia, A. C. S.; Lou, Y.; Burda, C. J. Am. Chem. Soc. 2005, 127, 4372-4375. (22) Reiss, P.; Bleuse, J.; Pron, A. Nano Lett. 2002, 2, 781-784.
322 Analytical Chemistry, Vol. 78, No. 1, January 1, 2006
Table 1. Quantum Yield (QY) of Water-Soluble CdSe Core QDs after Surface Modification with Different Substances
a
surface-modifying substance
QY (%) of watersoluble QDsa
mercaptosuccinic acid dimercaptosuccinic acid mercaptopropionic acid mercaptoacetic acid seleno-L-methionine cysteamine hydrochloride DL-cysteine
∼50 ∼8
