Anal. Chem. 2008, 80, 1988-1994
Separation of Bioconjugated Quantum Dots Using Capillary Electrophoresis Glorimar Vicente and Luis A. Colo´n*
Department of Chemistry, Natural Science Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000
Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. Under a given set of experimental conditions, the fluorescence intensity of QDSt and QDBi emitting light at 655 nm indicated that about 90% of QDBi complexed with 70% of QDSt. A two-color experiment that made use of two different sizes of QD (i.e., 585 and 655 nm) indicated that 30% of the 655 nm QDBi complexed with 53% of the 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This, in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared. Bioconjugation of nanoparticles has recently gained popularity in bioanalysis since the nanoparticles can provide unique optical and/or magnetic properties that can facilitate detection and/or identification of biomolecules. One type of such nanoparticles includes semiconductor nanocrystals, also known as quantum dots (QDs), which have been studied widely because of their sizedependent optical properties. These nanoparticles are typically less than 10 nm in diameter and have shown tremendous potential as sensors,1 optical devices,2 and as biological labels.3-6 Furthermore, molecular targets can be encoded with QDs for their specific * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Nazzal, A. Y.; Qu, L. H.; Peng, X. G.; Xiao, M. Nano Lett. 2003, 3, 819822. (2) Brus, L. Appl. Phys. A 1991, 53, 465-474. (3) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434-1436. (4) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (5) Cognet, L.; Tardin, C.; Boyer, D.; Choquet, D.; Tamarat, P.; Lounis, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11350-11355. (6) Gao, X. H.; Nie, S. M. Trends Biotechnol. 2003, 21, 371-373.
1988 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
identification.3-6 QDs, in principle, could be used in many applications in which organic fluorophores are used. These fluorescent nanoparticles are good candidates for molecular labeling because they are extremely photostable, show broad absorption spectra, narrow emission bands, and are brighter than traditional organic fluorophores.7,8 Because of the broad absorption spectral characteristics, a single excitation source can be used to excite multiple QDs with different emission spectra, hence, offering the potential of detecting multiple targets simultaneously in a single sample. One can also envision the use of highly efficient separation technology for the separation of nanoparticles bonded to the target molecules from the nonbonded ones. Capillary electrophoresis (CE) is a powerful separation technique that has been very useful in the analysis of biological samples as it has provided for methods with high selectivity, low detection limits, small sample consumption, ease of use, high separation efficiencies, and short analysis times.9 CE has found great applicability in the analysis of a variety of biologically important molecules.10 CE has also been utilized to separate nanomaterials having different compositions and sizes, including latex,11 inorganic oxide,12 gold,13 and silver14 nanoparticles. Despite the potential advantages of employing QDs as fluorescent labels in combination with CE, just a few reports have appeared in the literature. Huang et al. separated CdTe QDs conjugated to bovine serum albumin from the unconjugated QD using CE/LIF.15 In a similar study, the authors probed the surface modification of CdTe QDs with two different proteins (plant lectin and anti-von Willebrand factors antibody) using CE.16 In another study, CdTe QDs of different sizes (1.9 and 4.5 nm) were separated by capillary gel electrophoresis.17 More recently, Feng et al. developed an immunoassay for the determination of immunoglobulin M using an (7) Smith, A. M.; Nie, S. M. Analyst 2004, 129, 672-677. (8) Murphy, C. J. Anal. Chem. 2002, 74, 520A-526A. (9) Weinberger, R. Practical Capillary Electrophoresis; Academic Press: San Diego, CA, 2000. (10) Khaledi, M. G. High Performance Capillary Electrophoresis; Wiley: New York, 1998. (11) Jones, H. K.; Ballou, N. E. Anal. Chem. 1990, 62, 2484-2490. (12) Petersen, S. L.; Ballou, N. E. J. Chromatogr., A 1999, 834, 445-452. (13) Liu, F. K.; Wei, G. T. Anal. Chim. Acta 2004, 510, 77-83. (14) Liu, F. K.; Ko, F. H.; Huang, P. W.; Wu, C. H.; Chu, T. C. J. Chromatogr., A 2005, 1062, 139-145. (15) Huang, X. Y.; Weng, J. F.; Sang, F. M.; Song, X. T.; Cao, C. X.; Ren, J. C. J. Chromatogr., A 2006, 1113, 251-254. (16) Weng, J. F.; Song, X. T.; Li, L. A.; Qian, H. F.; Chen, K. Y.; Xu, X. M.; Cao, C. X.; Ren, J. C. Talanta 2006, 70, 397-402. (17) Song, X. T.; Li, L.; Chan, H. F.; Fang, N. H.; Ren, J. C. Electrophoresis 2006, 27, 1341-1346. 10.1021/ac702062u CCC: $40.75
© 2008 American Chemical Society Published on Web 02/16/2008
antibody labeled with a CdSe/ZnS QD.18 Pereira et al. used CE to characterize the charge of two commercially available (sodium mercaptopropionate CdTe/ZnS and carboxylic acid CdSe/ZnS) QDs.19 All of these reports have shown separation of bioconjugated QDs from nonconjugated ones. The separation of different QDs conjugated to different biomolecules, however, has not been reported. CE separation of differently bioconjugated QDs would be useful since it can provide experimental conditions that would allow the development of CE-based assays (e.g., immunoassays) for the determination of specific analytes. Herein, we report on the development of CE conditions for the separation of QDs conjugated to different biomolecules. We explore the possibility of following reactions between biomolecules attached to QDs. In order to prove the feasibility of our approach, we monitored the reaction of streptavidin and biotin, both conjugated to CdSe/ZnS QDs. EXPERIMENTAL SECTION Chemicals and Materials. The bioconjugated CdSe/ZnS quantum dots (655 nm QD streptavidin, 585 nm QD streptavidin, and 655 nm QD biotin conjugates) were obtained from Quantum Dot Corporation (Howard, CA). The concentration of the QD nanoparticles, as received, was reported by the manufacturer to be 2 µM. The QDs were received diluted in a pH 8.3 buffer composed of 50 mM borate, 0.5% bovine serum albumin (BSA), and 0.05% sodium azide (preservative). Stock solutions of QDs were prepared using the QD incubation buffer obtained from Quantum Dot Corporation. The composition of QD incubation buffer was 97% deionized water, 2% BSA,
