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Highly Fluorescent Streptavidin-Coated CdSe Nanoparticles: Preparation in Water, Characterization, and Micropatterning Monika Ba¨umle, Dimitrios Stamou,† Jean-Manuel Segura,† Ruud Hovius,† and Horst Vogel* Laboratory of Physical Chemistry of Polymers and Membranes (LCPPM), Institute of Chemical Sciences and Engineering (ISIC), Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received December 18, 2003. In Final Form: March 18, 2004 CdSe quantum dots (QDs) with a high fluorescence quantum yield of 25% and a narrow size distribution were synthesized in a single step in water using glutathione as a stabilizing molecule. The exceptional optical properties enabled for the first time the detection of in-water-prepared single quantum dots at room temperature. For application as fluorescent bioanalytical probes, the QDs were coated with streptavidin. These QDs self-assemble with high contrast on micropatterned biotin while preserving their optical properties and their capability to bind in addition biotinylated molecules, a prerequisite for the development of novel supramolecular structures and bioassays.
Introduction Nanometer-sized, fluorescent semiconductor particles, also named quantum dots, gain increasing importance for labeling and functional screening of biomolecules in vitro and in vivo.1-9 They show high photostability which enables long-term imaging. Their narrow emission spectra can be tuned by variation of the particle size and chemical composition, allowing multiple color imaging.1-4 The best established approach for the production of semiconductor QDs (mainly CdS and CdSe) is synthesis in organic solvents in the presence of suitable stabilizers. Subsequent transfer into biologically relevant aqueous media is achieved by exchange of the stabilizers, yielding highly fluorescent QDs with narrow emission peaks and quantum yields (QYs) of up to 60%.1-9 Synthesis directly in water, an alternative strategy introduced by Weller et al.,10-12 is less demanding and less toxic than synthesis in organic solvents and also less time-consuming, avoiding exchange of stabilizers. However, QDs prepared in water, with the exception of some Hg and CdTe QDs, have been reported to show low * Corresponding author. E-mail:
[email protected]. Phone: +41 21-6933155. Fax: +41 21-6936190. † These authors contributed equally to this work. (1) Parak, W. J.; Gerion, D.; Pellegrino, T.; Zanchet, D.; Micheel, C.; Williams, S. C.; Boudreau, R.; Le Gros, M. A.; Larabell, C. A.; Alivisatos, A. P. Nanotechnology 2003, 14, R15. (2) Jaiswal, J. K.; Mattoussi H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47. (3) (a) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (b) Certificate of Analysis, Qdot 525 Streptavidin Conjugate, Qdot Corp., 2003 (www.qdot.com). (4) Chan, W. C. W.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Curr. Opin. Biotechnol. 2002, 13, 40. (5) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (6) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, M. Nat. Mater. 2003, 2, 630. (7) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378. (8) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Annu. Rev. Mater. Sci. 2000, 30, 545. (9) Alivisatos, A. P. J. Phys. Chem. 1996, 100, 13226. (10) Weller, H. Adv. Mater. 1993, 5, 88. (11) Gao, M.; Kirstein, S.; Mo¨hwald, H.; Rogach, A. L.; Kornowski, A.; Eychmu¨ller, A.; Weller, H. J. Phys. Chem. B 1998, 102, 8360. (12) Rogach, A. L.; Kornsowski, A.; Gao, M.; Eychmu¨ller, A.; Weller, H. J. Phys. Chem. B 1999, 103, 3065.
fluorescence QYs and broad fluorescence spectra.11,12 Here, we report on the synthesis of CdSe QDs in water using the oligopeptide glutathione (GSH) as a stabilizer. The QDs reveal exceptional and well-defined optical properties (QYs of up to 25%, full width at half-maximum (fwhm) of 49 nm), similar to the CdSe QDs prepared in organic solvents (those emitting in the same wavelength region as our QDs show typical QYs of 20-40%, fwhm of 30-40 nm).3,7 Any further application of QDs in bioanalytics requires their modification with biologically active molecules. Maintaining the optimal optical properties of QDs after modification has been the objective of several research groups that have exerted efforts in optimizing and investigating the properties of these systems.1-8 Along these lines, we have modified further our CdSe QDs by coupling them to streptavidin, a universal receptor protein which binds biotin selectively and with high affinity. The fluorescent properties of the protein-QD complex were investigated in solution. The ligand binding properties were assessed by self-assembling the functionalized fluorescent particles on surfaces micropatterned with the complementary ligand biotin. The latter experiments constitute the first development steps in employing fluorescent QDs as markers for bioanalytical applications, diagnostics, or high-throughput screening on microarray formats. Experimental Section Chemicals of highest purity were purchased from Fluka (GSH, poly(ethylene glycol), poly(dimethylsiloxane)), Sigma-Aldrich (Cd(ClO4)2‚6H2O, phosphate buffered saline (PBS, 80 mM NaCl, 10 mM NaHPO4 at pH 7.4), bovin serum albumin biotin (BSAbiotin)), Molecular Probes (biotin, biotin-Alexa 594), Pierce (streptavidin, dialysis cassettes), and ICN (1-(dimethylamino)5-naphthalenesulfonic acid, Al2Se3). Synthesis of Glutathione-Stabilized CdSe QDs. We modified the method of Rogach et al.12 A 4.4 mmol portion of Cd(ClO4)2‚6H2O and 5.7 mmol of GSH were dissolved in 250 mL of N2-saturated deionized water; the pH was adjusted to 11.5 with 30 mL of 1 M NaOH. To this solution, 45 mL of freshly prepared, oxygen-free 0.05 M NaHSe (generated by the reaction of Al2Se3 with 0.1 M H2SO4) was added under vigorous stirring, changing the color of the solution immediately to yellow. The
10.1021/la0363940 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/15/2004
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Figure 1. (left) Absorbance spectrum of GSH-stabilized CdSe QDs before modification by streptavidin. The spectrum shows three weak shoulders at 370, 435, and 505 nm. Solutions were at pH 7 and contained 10 wt % poly(ethylene glycol). The spectrum changed insignificantly after modification of QDs by streptavidin. Fluorescence spectra of GSH-stabilized CdSe QDs before (dotted line) and after (solid line) modification by streptavidin (λex ) 365 nm, Stokes’ shift of ∼30 nm, band edge fluorescence). Solutions were at pH 7 and contained 10 wt % poly(ethylene glycol). The coating of the CdSe QDs with streptavidin caused a broadening of the emission peak from 49 to 54 nm and an increase of the QY from 16 to 25%. solution was heated at 90 °C for 12 h, then cooled to 4 °C, and then dialyzed (3500 Da cutoff) for 12 h against water to remove nonreacted starting compounds and small molecular reaction products. The yield of QDs, determined after precipitation with 2-propanol, was 246 mg per 100 mL of reaction solution. Streptavidin Modification of CdSe QDs. Streptavidin was added to a final concentration of 0.8 µM to a 10-fold diluted QD solution at pH 7.0. After 1 h, the streptavidin-coated QDs were separated from free streptavidin by centrifugation (80 min, 13 000 rpm). After redissolving the pellet by ultrasonic treatment (15 min, 25 °C), poly(ethylene glycol) (4000 Da, 10 wt %/vol) was added to prevent aggregation of the protein-modified QDs. Fluorescence Measurements. Absorption and fluorescence spectra were recorded at room temperature using quartz cuvettes with a 10 mm optical path length on a Beckman (model DU 640) and a SPEX Fluorolog II spectrometer (Jobin Yvon, Stanmore, U.K.), respectively. The fluorescence QYs of the QDs (pH 7-8) were determined by comparing their fluorescence intensities integrated from 430 to 600 nm with those of 1-(dimethylamino)-5-naphthalenesulfonic acid in 0.1 M NaHCO3 at pH 8.5 (QY: 0.36) as a reference, both excited at 317 nm.17a The absorbances of both solutions were between 0.02 and 0.05. QYs were calculated using the following equation:
QYs )
Fs A r × × QYr Fr As
where Fs and Fr are the integrated fluorescence emissions between 430 and 600 nm of the sample and the reference, respectively, As and Ar are the absorbances at 317 nm of the sample and the reference, respectively, and QYs and QYr are the quantum yields of the sample and the reference, respectively. Single-particle measurements were performed on a modified epiluminescence wide-field microscope (Axiovert 200, Zeiss). Circularly polarized light of 457.9 nm of an Ar+ laser (Innova Sabre, Coherent) was directed by a dichroic mirror (Q495LP, Chroma) into a microscope objective (Plan-Apochromat 100x, 1.4 NA, Zeiss) to illuminate a 14 µm diameter region of the sample with an averaged intensity of 0.2-5 kW/cm2. Fluorescence was collected by the same objective, passed through a filter (HQ525/ 50, Chroma), and imaged on an intensified CCD camera (I-Pentamax 512 EFT, Roper Scientific). Glass coverslips were cleaned by plasma etching prior to experiments.
Self-Assembly of Streptavidin-Modified QDs on Microcontact-Printed BSA-Biotin. PDMS stamps for microcontact printing were inked with 30 µL of BSA-biotin (0.1 mg/mL in PBS). After washing the stamps with PBS and blowing off residual buffer under a N2 stream, the BSA-biotin was printed on glass as described elsewhere.15,16,18 The micropatterned substrate was then incubated with streptavidin-modified QDs, 10-fold diluted in PBS. Images of the fluorescent patterns of nanoparticles were recorded using a confocal microscope (LSM 510, Zeiss, excitation at 488 nm, dichroic mirror HFT 488, filter LP 505). The micropatterned streptavidin-modified QDs were then incubated for 30 min with biotin-Alexa 594 (1 µM, in PBS). After washing with PBS, images of the fluorescent patterns were recorded (excitation at 633 nm, dichroic mirror HFT 633, filter LP 650).
Results and Discussion Modification of QDs with GSH is an example of a successful imitation of a natural process: Many living cells use GSH in metal detoxification by formation and stabilization of semiconductor nanoparticles.19 We synthesized CdSe QDs by mixing aqueous solutions of cadmium(II) salt and NaHSe in the presence of GSH. The resulting solution of QDs is stable for several months at 4 °C. The fluorescence is maximal at a pH between 7 and 8, while acidification increasingly leads to decomposition of the QDs. The absorption spectrum (Figure 1) of QDs at pH 7 displays three weak shoulders at 370, 435, and 505 nm. The fluorescence spectral band (Figure 1) around 533 nm with a 49 nm fwhm indicates a narrow size distribution of the QDs. Transmission electron microscopy reveals an average size of the CdSe core of 2 nm with a size distribution of (0.2 nm.20 (13) La Van, D. A.; Lynn, D. M.; Langer, R. Nat. Rev. 2002, 1, 77. (14) Niemeyer, C. M. Angew. Chem., Int. Ed. 2003, 42, 5796. (15) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550. (16) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225. (17) (a) Li, Y.; Cha, L.; Tyer, L.; Moody, R. T.; Himel, C. M.; Hercules, D. M. J. Am. Chem. Soc. 1975, 97, 3118. (b) Standards in Fluorescence Spectrometry; Miller, J. N., Ed.; Chapman & Hall: 1981. (18) Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angew. Chem., Int. Ed. 2003, 42, 5580. (19) Ahmad, A.; Mukherjee, P.; Mandal, D.; Senapati, S.; Islam Khan, M.; Kumar, R.; Sastry, M. J. Am. Chem. Soc. 2002, 124, 12108.
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Figure 2. (a) Wide-field image of single GSH-stabilized CdSe QDs spin-coated from aqueous solution on a glass slide. Scale bar ) 5 µm. λex ) 457.9 nm, Iex ) 50 W/cm2, integration time ) 1 s. (b) Time trace of a single QD. λex ) 457.9 nm, Iex ) 500 W/cm2, integration time per point ) 25 ms, measurement frequency ) 10 Hz.
A QY of 16% for the QDs was found at the Cd/GSH ratio of 1:1.3; higher stabilizer concentrations yielded lower QYs. The chemical structure of GSH might be responsible for the unexpected high QY, as photophysical properties of QDs are strongly dependent on their local environment.11,12 GSH is likely to be attached to the surface of the CdSe QDs via its thiol group, as indicated by Fourier transform infrared spectroscopy,21 in agreement with reports for other thiol compounds.11,22 The high photostability and high QY allowed us to perform measurements on single GSH-stabilized QDs at room temperature. Figure 2a depicts a typical image of a low surface concentration of QDs spin-coated on a glass slide. Individual hydrated QDs can be recognized as diffraction-limited spots. The GSH-stabilized CdSe QDs are very photostable and emit over minutes fluorescence with a very pronounced blinking (Figure 2b), as has been often observed for individual QDs synthesized in organic solvents.22 To our knowledge, these are the first reported room-temperature images of single QDs synthesized in aqueous solution. The similarity in the fluorescence dynamics of the GSH-stabilized QDs and those synthesized in organic solvents can be related to comparable QYs. The QYs measured on ensembles of QDs are primarily influenced by two factors, the nonradiative pathways due to surface defects and the photoionization processes causing the blinking observed on individual QDs.23 The exceptionally high QY of the GSH-stabilized QDs in water is likely to be due to a reduction of the photoionization efficiency. This effect is even more pronounced at the level of single QDs due to the high excitation intensities used. For comparison, thiolactic acid-stabilized CdSe QDs, investigated under similar conditions, emit very dimly (20) Samples were prepared by dropping a 100-fold diluted QD solution on a carbon-coated copper grid. The images were recorded with a Philips CM 300 FEG/UT microscope. (21) FTIR measurement was done on a Bruker IFS 28 instrument. A droplet of the samples was dried on an ATR crystal. The spectra showed that the S-H band of GSH at 2550 cm-1 disappeared upon synthesis of the QDs. (22) (a) Nirmal, N.; Dabbousi, B. O.; Bawendi, M. G.; Macklin, J. J.; Trautman, J. K.; Harris, T. D.; Brus, L. E. Nature 1996, 383, 802. (b) Michalet, X.; Pinaud, F.; Lacoste, T. D.; Dahan, M.; Bruchez, M. P.; Alivisatos, A. P.; Weiss, S. Single Mol. 2001, 2, 261. (23) Kuno, M.; Fromm, D. P.; Johnson, S. T.; Gallagher, A.; Nesbitt, D. J. Phys. Rev. B 2003, 67, 125304.
Figure 3. (a) Confocal microscopy image of streptavidinmodified, GSH-stabilized CdSe QDs (in PBS) specifically bound to microcontact-printed lines of BSA-biotin on glass (λex ) 488 nm, dichroic mirror HFT 488, filter LP 505). (b) Confocal microscopy image of the micropatterned QDs shown in part a after addition of biotin conjugated to the fluorophore Alexa 594 (λex ) 633 nm, dichroic mirror HFT 633, filter LP 650). (c) Fluorescence intensity profiles along the white arrows in parts a and b. Scale bar ) 40 µm.
and photobleach very rapidly, impairing the observation of individual QDs. Possibly, high photoionization efficiency causes the thiolactic acid-coated QDs to rapidly populate dark, long-living ionized states. Many research groups pursue biological applications of semiconductor QDs.1-8,25 To be used as biomarkers, we coated our GSH-stabilized QDs with streptavidin, a wellcharacterized protein which binds selectively and with high affinity biotin and biotin-containing compounds and is therefore used as a universal building block for biosensing surfaces.24 Streptavidin binds nonspecifically to QDs via electrostatic interactions and thereby induces a slight broadening of the fluorescence band of the QDs to a fwhm of 54 nm (Figure 1) and an increase in the QY from 16 to 25%. Such changes of quantum yield upon electrostatic binding have already been observed several times.25 Optimal optical properties were obtained by adding 0.8 µM streptavidin to the QD solution, while (24) (a) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1. (b) Bieri, C.; Ernst, O. E.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105. (25) (a)Lin, Z.; Cui, S.; Zhang, H.; Chen, Q.; Yang, B.; Su, X.; Zhang, J.; Jin, Q. Anal. Biochem. 2003, 319, 239. (b) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142.
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higher protein concentrations led to a decrease in fluorescence QYs. Protein-coated QDs show a tendency to aggregate.25a Adding the hydrophilic noncharged polymer poly(ethylene glycol) prevented particle aggregation while preserving biofunctionality. To apply such fluorescent QDs for novel bioassays based on microarrays and surface-sensitive detection, it is important to investigate the biological activity of these QDs on solid supports. As demonstrated in Figure 3a, streptavidin-coated QDs specifically bind to microcontactprinted BSA-biotin. The QDs form micropatterns with a high intensity contrast to neighboring background. The formation of fluorescent micropatterns can be fully suppressed if the QDs are incubated with 1 mM biotin prior to their addition. The micropatterned QDs bind in addition fluorescently labeled biotin, indicating the presence of more than one streptavidin molecule on one QD (Figure 3b). This opens perspectives to investigate the optical and biological properties of self-organized systems confined to one or several molecular layers comprising biotinylated lipids, receptor proteins, or DNAs.24b,26,27 (26) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610.
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Conclusion Given their convenient preparation and modification in aqueous solution, the GSH-stabilized QDs might be an attractive alternative to the well-established QDs synthesized in organic solvents. We demonstrate that the novel approach of self-assembling biomodified semiconductor QDs on a prepatterned template preserves the optical and biological properties, which opens perspectives for many applications of QDs in surface science and bioanalytics. Acknowledgment. This work was financially supported by the Top NANO 21 program (Project No. 4679.1) and EPFL internal grants. We are grateful to Y. Axmann for transmission electron microscopy images and to W.-P. Ulrich for infrared spectroscopy. We would like to thank E. Delamarche for supporting us with polymer stamps for microcontact printing. LA0363940 (27) Martinez, K. L.; Meyer, B. H.; Hovius, R., Lundstrom, K.; Vogel, H. Langmuir 2003, 19, 10925.
