NANO LETTERS
β-Cyclodextrin-Modified CdSe/ZnS Quantum Dots for Sensing and Chiroselective Analysis
2009 Vol. 9, No. 5 2073-2076
Ronit Freeman, Tali Finder, LiLy Bahshi, and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel Received February 13, 2009; Revised Manuscript Received March 29, 2009
ABSTRACT β-Cyclodextrin (β-CD)-functionalized CdSe/ZnS quantum dots (QDs) are used for optical sensing and chiroselective sensing of different substrates using a fluorescence resonance energy transfer (FRET) or an electron transfer (ET) mechanisms. The FRET between the QDs and Rhodamine B incorporated in the β-CD receptor sites is used for the competitive analysis of adamantanecarboxylic acid and of p-hydroxytoluene. Also, the dye-incorporated β-CD-modified QDs are used for the chiroselective optical discrimination between D,L-phenylalanine and D,L-tyrosine. The receptor-functionalized QDs are also implemented for the optical detection of p-nitrophenol using an ET quenching route.
The use of semiconductor quantum dots (QDs) as optical labels for biosensing was extensively advanced in the recent years.1 Semiconductor QDs were used as fluorescent labels for biorecognition events.2 Fluorescence resonance energy transfer (FRET) or electron transfer (ET) using QD/dye or QD/electron-acceptor conjugates were used to probe DNA hybridization,3 to follow the formation of immunocomplexes,4 and to analyze biocatalytic transformations.5 Also, QD-dye conjugates were used to follow the activities of NAD+-dependent enzymes, and the incorporation of the modified QDs in cancer cells enabled the probing of intracellular metabolism and the screening of anticancer drugs.6 While the use of QDs for biosensing is quite advanced, their application as optical probes for molecular sensing is undeveloped and scarce. Only a few examples that apply QDs for the detection of pH changes7 or nonselective analysis of ions8 were reported. Recently, boronic acid-functionalized QDs were implemented to analyze monosaccharides or dopamine, using a competitive FRET assay that included dye-labeled monosaccharides or dye-labeled dopamine as FRET acceptors.9 In the present study we report on the preparation of β-cyclodextrinfunctionalized CdSe/ZnS QDs and their use as a versatile sensing platform. β-Cyclodextrins, β-CDs, are cyclic receptors consisting of seven glucose units linked one to another by 1-4 glycoside bonds. The selective association of organic molecules to the hydrophobic cavities of cyclodextrins was used to develop different sensors10 and separation matrices.11 Specificially, since cyclodextrins are chiral, different chro* Corresponding author:
[email protected]; tel, +972-2-6585272; fax, 972-2-6527715. 10.1021/nl900470p CCC: $40.75 Published on Web 04/08/2009
2009 American Chemical Society
matographic cyclodextrin-based chiral separation processes were accomplished.12 In the present study, we present a FRET-based competitive assay using β-CD-modified CdSe/ ZnS QDs as sensors and chiroselective sensors. We also demonstrate the application of the β-CD-functionalized QDs for the direct sensing of organic substrates exhibiting electron acceptor or electron donor properties via electron transfer quenching of the luminescence of the QDs. The CdSe/ZnS QDs (d ) 3.4 nm, λem ) 520 nm) were modified with a glutathione capping monolayer, and (3aminophenyl)boronic acid was covalently tethered to the capping layer, using bis(sulfosuccinimidyl)suberate, BS3, as a bifunctional coupling reagent. The β-CD units were, then, linked to the boronic acid ligands via the secondary vicinal hydroxyl groups of the sugar units. The quantum yield of the resulting modified QDs corresponded to ca. 0.2. Scheme 1A outlines the principle for the competitive analysis of a substrate by the β-CD-functionalized QDs. The dye, Rhodamine B (1) (λabs ) 540 nm), was selected as an optical label. It binds to the β-CD cavity, Ka ) 5900 M-1, its absorption overlaps the luminescence of the QDs, and by itself is not excited at λ ) 400 nm (the wavelength of excitation of the QDs). As a result, the excitation of the QDs is accompanied by a FRET process, leading to the emission of the dye at λ) 590 nm. Using the photophysical properties of the functionalized QDs and those of the Rhodamin dye acceptor, we calculated a Ro value of 53.0 Å for the FRET process between the components. Using the luminescence emission of the QDs before and after the association of Rhodamine B, and using the calculated Ro value, we estimated the R value between the components to be 51.9
Scheme 1. (A) Sensing of Substrates by a Competitive FRET Assay Using β-Cyclodextrin-Modified CdSe/ZnS QDs with Receptor-Bound Rhodamine B (1) and (B) Direct Analysis of Substrates by the β-Cyclodextrin-Modified CdSe/ZnS QDs Using an Electron Transfer Quenching Route
Å. This value fits well the estimated distance between the FRET components calculated based on geometrical bonds lengths under consideration, 52.4 Å. In the presence of the analyte substrate, competitive association to the β-CD occurs. This results in the displacement of the dye, a process that is reflected by the decrease in the FRET emission of the dye, and the concomitant enhancement of the luminescence of the QDs. Control experiments indicate that the luminescence of D-glucose-modified boronic acid-functionalized QDs (that lack the β-CD receptor sites) was unaffected by the addition of the dye, implying that the association of the dye to the β-CD cavity is essential to stimulate the FRET process. Curves a to j of Figure 1A show the time-dependent luminescence features of the QDs/dye equilibrated system, upon the addition of 1-adamantanecarboxylic acid (2). Addition of 2 results in the time-dependent decrease in the FRET emission of the dye, and the concomitant increase in the luminescence intensity of the QDs. After 9 min, the luminescence features of the system level off to a constant value that represents the equilibrium achieved by the competitive binding of 2 to the β-CD cavities, and the displacement of the dye 1 from the receptor sites. The equilibrium established by the displacement of 1 and the competitive association of 2 to the β-CD is controlled by the concentration of the analyte. Figure 1B shows the calibration curve of the equilibrated QD/dye system in the presence of variable concentrations of 2. As the concentration of 2 increases, the FRET emission of 1 decreases, and the luminescence of the QDs is intensified, consistent with the competitive displacement of the dye. Similar results are observed upon analyzing 4-hydroxytoluene (3), Figure 2. It should be noted that the sensing platform is general and can be implemented for many substrates, provided that two criteria are fulfilled: (i) The analyzed substrate associates to 2074
β-CD with a binding constant that allows the displacement of the probing dye. (ii) The analyte does not quench the luminescence of the QDs. Under conditions where the analyte quenches the luminescence of the QDs, the concomitant depletion of fluorescence of the dye and the increase in the luminescence of the QDs are eliminated. This perturbs the
Figure 1. (A) Time-dependent luminescence spectrum of the Rhodamine B/β-cyclodextrin-modified CdSe/ZnS QDs system: (a) In the absence of 1-adamantanecarboxylic acid (2). (b-j) Upon interaction with 2, 1 × 10-3 M. Spectra recorded at time intervals of 1 min. (B) Calibration curve corresponding to the analysis of variable concentrations of 2 by the Rhodamine B/β-cyclodextrinmodified CdSe/ZnS QDs system. The luminescence was recorded after equilibration of the system in the presence of different concentrations of the analyte for 10 min. Nano Lett., Vol. 9, No. 5, 2009
Figure 2. (A) Time-dependent luminescence spectrum of the Rhodamine B/β-cyclodextrin-modified CdSe/ZnS QDs system: (a) In the absence of 4-hydroxytoluene (3). (b-j) Upon interaction with 3, 1 × 10-3 M. Spectra recorded at time intervals of 1 min. (B) Calibration curve corresponding to the analysis of variable concentrations of 3 by the Rhodamine B/β-cyclodextrin-modified CdSe/ ZnS QDs system. The luminescence was recorded after equilibration of the system in the presence of the different concentrations of analyte for 10 min.
competitive assay mechanism (but enables an alternative sensing route, vide infra). The specific analytes 2 and 3 were selected as model compounds to develop the sensor configuration due to their well-established association to β-CD.13 From the competitive fluorescence curves corresponding to the analysis of 2 and 3, we determined the association constants of the two substrates to be 2.07 × 104 M-1 and 5.7 × 103 M-1, respectively. The detection limits for analyzing 2 and 3 were similar, 1.0 × 10-6 M. The β-CD-functionalized QDs/dye system was also used for the chiroselective detection of substrates, using the competitive FRET assay. Figure 3A depicts the timedependent luminescence features of the QDs/dye equilibrated system upon interaction with the L-phenylalanine (4) analyte. L-Phenylalanine associates competitively to the chiral cavity of the β-CD receptors, a process that is reflected by the decrease in the FRET emission of the dye and an enhancement in the CdSe/ZnS QDs luminescence. On the other hand, D-phenylalanine (5) has very little effect on the luminescence of the β-CD QD/dye system, indicating that this enantiomer does not associate to the β-CD sites. This allows the impressive chiroselective analysis of the enantiomers by the system, Figure 3A inset. Similar results were observed upon analyzing L-tyrosine (6) and D-tyrosine (7) by the β-CDQD/dye system, Figure 3B. In this system, D-tyrosine lacks the affinity for the β-CD and affects to a low degree the FRET process between the CdSe/ZnS QDs and the dye. On the other hand, L-tyrosine exhibits affinity for the β-CD and displaces the dye from the receptor sites. This results in the decrease in the FRET fluorescence of the dye and an increase in the luminescence of the QDs. Figure 3B inset shows the derived calibration curve. Thus, the functionalized QDs can be used Nano Lett., Vol. 9, No. 5, 2009
Figure 3. (A) Time-dependent spectral changes of the Rhodamine B/β-cyclodextrin-modified CdSe/ZnS QDs system, (a) In the absence of 4. (b) to (f) Upon addition of 4, 5 × 10-4 M. Inset: Calibration curve corresponding to the analysis of variable concentrations of 4, curve a, and 5, curve b, by the Rhodamine B/βcyclodextrin-modified CdSe/ZnS QDs system. The luminescence was recorded after equilibration of the system in the presence of the different concentrations of the analytes for 5 min. (B) Timedependent spectral changes of the Rhodamine B/β-cyclodextrinmodified CdSe/ZnS QDs system. (a) In the absence of 6. (b) to (f) Upon addition of 6, 5 × 10-4 M. Inset: Calibration curve corresponding to the analysis of variable concentrations of 6, curve a, and 7, curve b, by the Rhodamine B/β-cyclodextrin-modified CdSe/ZnS QDs system. The luminescence was recorded after equilibration of the system in the presence of different concentrations of the analytes for 5 min.
to analyze different chiral compounds. From the competitive fluorescence curves for analyzing the amino acids 4-7, we estimated the association constants of 4 and 5 to β-CD to be 3.05 × 103 and 3.5 × 102 M-1, respectively, and of 6 and 7 to β-CD to be 2.2 × 103 and 100 M-1, respectively. It should be noted that the determination of the optical purity of amino acids is important in the drug industry.14 The specific selection of aromatic chiral amino acid is due to the favored interactions of the phenyl ring with the β-CD receptor. The β-CD-functionalized QDs can also be used as direct luminescence sensors, provided that the analyte binds to the receptor sites, and it acts as an electron transfer quencher of luminescence of the particles, Scheme 1B. Under these conditions the association of the analyte to the β-CD cavities concentrates the analyte at the semiconductor surface and brings the analyte-quencher into close proximity to the luminescent QDs. Figure 4A shows the time-dependent 2075
Acknowledgment. This research is supported by the Israel Science Foundation, Converging Technologies Project, and by the Israel Ministry of Defense. Supporting Information Available: Details of the preparation of the modified QDs. This material is available free of charge via the Internet at http://pubs.acs.org. References
Figure 4. (A) Time-dependent luminescence quenching of the β-cyclodextrin-modified CdSe/ZnS QDs. (a) Prior to the addition of p-nitrophenol (8). (b-g) Upon interaction with 50 µM 8. Spectra recorded at time intervals corresponding to 2 min. (B) Calibration curve corresponding to the luminescence quenching of the β-cyclodextrinmodified CdSe/ZnS QDs at variable concentrations of 8. The luminescence was recorded after equilibration of the system in the presence of different concentrations of the analyte for 12 min.
luminescence changes of the QDs upon their interaction with 50 µM 4-nitrophenol (8). The luminescence of the QDs decreases, consistent with the ET quenching of the QDs. The luminescence reaches a constant value after 12 min, corresponding to the equilibration of the analyte in the β-CD receptor sites. Accordingly, the degree of luminescence quenching of the QDs is controlled by the concentration of 8. Figure 4B, shows the resulting calibration curve that corresponds to the degree of quenching of the QDs at variable concentrations of 8. For comparison, the glucose-functionalized QDs (these QDs lack the receptor sites) were reacted with 50 µM of 8. Only a minute degree of quenching (5%) of the QDs is observed. These results imply that the concentration of the analyte at the QDs surface, by means of the β-CD receptor sites, is essential to analyze 8 by the electron transfer quenching mechanism. From the fluorescence quenching curves of the QDs by 8, we estimated the association constant of 8 to β-CD to be 2.3 × 103 M-1. The detection limit for analyzing 8 corresponded to 1 × 10-6 M. In conclusion, the present study has introduced the β-CDmodified QDs as a versatile sensing and chiroselective sensing platform using a competitive FRET assay or a direct electron transfer quenching. The β-CD-functionalized CdSe/ ZnS QDs revealed optical and physical stabilities for weeks. Further variation of the sensor system could be achieved by modifying the quantum dots with R-cyclodextrin or γ-cyclodextrin, thus enabling the design of sensors for other substrates. 2076
(1) (a) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (b) Chan, W. C. W.; Maxwell, D. J.; Gao, X. H.; Bailey, R. E.; Han, M. Y.; Nie, S. M. Curr. Opin. Biotechnol. 2002, 13, 40–46. (c) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. ReV. 2007, 36, 579–591. (2) (a) Goldman, E. R.; Balighian, E. D.; Mattoussi, H. M.; Kuno, K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378–6382. (b) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (c) Costa-Fernandez, J. M.; Pereiro, R.; Sanz-Medel, A. TrAC, Trends Anal. Chem. 2006, 25, 207–218. (d) Klostranec, J. M.; Chan, W. C. W. AdV. Mater. 2006, 18, 1953– 1964. (e) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.; Charles, P. T.; Mauro, J. M. Anal. Chem. 2002, 74, 841–847. (3) Zhang, C. Y.; Johnson, L. W. Angew. Chem., Int. Ed. 2007, 46, 3482– 3485. (4) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.; Clapp, A. R.; Uyeda, H. T.; Deschamps, J. R.; Lassman, M. E.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 6744–6751. (5) (a) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 13918–13919. (b) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat. Mater. 2006, 5, 581–589. (c) Shi, L. F.; Rosenzweig, N.; Rosenzweig, Z. Anal. Chem. 2007, 79, 208–214. (d) Gill, R.; Freeman, R.; Xu, J. P.; Willner, I.; Winograd, S.; Shweky, I.; Banin, U. J. Am. Chem. Soc. 2006, 128, 15376–15377. (6) (a) Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Angew. Chem., Int. Ed. 2009, 121, 315–319. (b) Freeman, R.; Willner, I. Nano Lett. 2009, 9, 322–326. (7) Snee, P. T.; Somers, R. C.; Nair, G.; Zimmer, J. P.; Bawendi, M. G.; Nocera, D. G. J. Am. Chem. Soc. 2006, 128, 13320–13321. (8) Chen, C. Y.; Cheng, C. T.; Lai, C. W.; Wu, P. W.; Wu, K. C.; Chou, P. T.; Choub, Y. H.; Chiub, H. T. Chem. Commun. 2006, 3, 263–265. (9) Freeman, R.; Bahshi, L.; Finder, T.; Gill, R.; Willner, I. Chem. Commun. 2009, 7, 764–766. (10) (a) Ueno, A. AdV. Mater. 1993, 5, 132–134. (b) Corradini, R.; Dossena, A.; Galaverna, G.; Marchelli, R.; Panagia, A.; Sartor, G. J. Org. Chem. 1997, 62, 6283–6289. (c) Haider, J. M.; Pikramenou, Z. Chem. Soc. ReV. 2005, 34, 120–132. (d) Furukawa, S.; Mihara, H.; Ueno, A. Macromol. Rapid Commun. 2003, 24, 202–206. (e) Pagliari, S.; Corradini, R.; Galaverna, G.; Sforza, S.; Dossena, A.; Montalti, M.; Prodi, L.; Zaccheroni, N.; Marchelli, R. Chem.sEur. J. 2004, 10, 2749–2758. (11) (a) Armstrong, D. W.; DeMond, W. J. Chromatogr. Sci. 1984, 22, 411–415. (b) Hinze, W. L.; Riehl, T. E.; Armstrong, D. W.; Mond, W. D.; Alak, A.; Ward, T. Anal. Chem. 1985, 57, 237–242. (c) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science 1986, 232, 1132–1135. (d) Sternitzke, K. D.; Fan, T. Y.; Dunn, D. L. J. Chromatogr. 1992, 589, 159–164. (e) Pavel, J.; Simona, B.; Josef, P. J. Chromatrogr., A 2000, 871, 139–152. (f) Wang, M.; Kan, W.; He, X.; Tan, T. J. Liq. Chromatogr. Relat. Technol. 2005, 28, 1509–1518. (12) (a) Stalcup, A. M.; Williams, K. L. J. Liq. Chromatogr. 1992, 15, 29–37. (b) Camilleri, P.; Reid, C. A.; Manallack, D. T. Chromatographia 1994, 38, 771–775. (c) Risley, D. S.; Strege, M. A. Anal. Chem. 2000, 72, 1736–1739. (d) Schumacher, D. D.; Mitchell, C. R.; Xiao, T. L.; Rozhkov, R. V.; Larock, R. C.; Armstrong, D. W. J. Chromatogr., A 2003, 1011, 37–47. (e) Han, X.; Zhong, Q.; Yue, D.; Della Ca`, N.; Larock, R. C.; Armstrong, D. W. Chromatographia 2005, 61, 205–211. (f) Sun, P.; Wang, C.; Armstrong, D. W. J. Liq. Chromatogr. Relat. Technol. 2006, 29, 1847–1860. (13) Ogoshi, T.; Harada, A. Sensors 2008, 8, 4961–4982. (14) Izake, E. L. J. Pharm. Sci. 2007, 96, 1659–1676.
NL900470P Nano Lett., Vol. 9, No. 5, 2009
