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Novel Molecular Recognition via Fluorescent Resonance Energy Transfer Using a Biotin-PEG/Polyamine Stabilized CdS Quantum Dot Yukio Nagasaki,*,† Takehiko Ishii,† Yuka Sunaga,† Yousuke Watanabe,† Hidenori Otsuka,‡,§ and Kazunori Kataoka*,‡ Department of Materials Science, Tokyo University of Science, Noda 278-8510, Japan, and Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received October 29, 2003. In Final Form: March 23, 2004 A novel functionally PEGylated quantum dot (QD) was prepared by a coprecipitation method in the presence of the biotin-PEG/polyamine block copolymer. When CdCl2 and Na2S were mixed in aqueous media in the presence of the biotin-PEG-b-poly(2-(N,N-dimethylamino)ethyl methacrylate) [biotin-PEG/ PAMA], a CdS QD with a size of ca. 5 nm was prepared. The polyamine segment was anchored on the surface of the formed CdS nanoparticle, whereas the PEG segment was tethered on the surface to form a hydrophilic palisade, thus improving the dispersion stability in aqueous media even under a high salt concentration condition. An effective fluorescent resonance energy transfer (FRET) was observed by the specific interaction of the biotin-PEG/PAMA stabilized CdS QD with TexasRed-labeled streptavidin of the physiological ionic strength of 0.15 M. The extent of the energy transfer was in proportion to the concentration of the TexasRed-streptavidin. This FRET system using the PEGylated CdS QD coupled with fluorescent-labeled protein can be utilized as a highly sensitive bioanalytical system.
Introduction The optical properties of quantum dots (QDs) have been intensively studied for the past several decades. Since the use of ligand-conjugated QDs as fluorescent biolabeling reagents was reported in 1998 by the groups of Alivisatos and Nie,1,2 many approaches to QD applications have been done in the bioanalytical field such as DNA sequencing, tissue immunodiagnostics, and single molecular imaging.3-6 Several advantages of QDs as biolabeling agents are (i) tunable fluorescent wavelength by size, (ii) sharp and symmetrical fluorescent peak, (iii) strong and long life emission, and (iv) wide excitation wavelength. Though QDs are expected to be one of the important nanomaterials in the bioanalytical field, their dispersion stability in liquid significantly decreases with their decreasing size, especially in the range of nanometers due to an increased surface area. In aqueous media, the nanosized QDs are mainly dispersed by the ionic repulsion force of the adsorbed ions on their surfaces. In a physiological environment such as serum and cell extracted * To whom correspondence should be addressed. Prof. Yukio Nagasaki: phone, +81-4-7124-1501 (ext 4310); fax, +81-4-71238878; e-mail,
[email protected]. Prof. Kazunori Kataoka: phone, +81-3-5841-7139; fax, +81-3-5841-7139; e-mail,
[email protected]. † Tokyo University of Science. ‡ University of Tokyo. § Present address: Artificial Organ Materials Research Group, Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. (1) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (2) Chan, W. C.; Nie, S. Science 1998, 281, 2016. (3) Sondi, I.; Siiman, O.; Koester, S.; Matijevic, E. Langmuir 2000, 16, 3107. (4) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21 (4), 452. (5) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21 (1), 47. (6) Taylor, J. R.; Fang, M. M.; Nie, S. Anal. Chem. 2000, 72, 1979.
fluid, however, the electrostatic repulsion force is easily shielded by the appreciably high ionic strength, causing nonspecific coagulation of the QD particles. In this regard, polymer-supported stabilization of the QDs is one of the effective methods.7-9 Qi et al.10 reported the preparation of CdS QD in aqueous media in the presence of the poly(ethyleneimine)-g-poly(ethylene glycol) graft copolymer (PEI/PEG).11 The preparation of CdS was the simple coprecipitation method of Cd2+ with S2-. The graft copolymer stabilized the obtained CdS QD in aqueous media. On the other hand, the coordinated polymer prevents direct installation of biotag molecules on the QD surface. To utilize QDs as a biolabeling agent, it is essential to achieve both dispersion stability in physiological solution and facile installation of bio-tag molecules on the outer layer of the QDs. Recently, we have succeeded in preparing functionalized nanosized particles stabilized by our newly molecularengineered water soluble polymers based on poly(ethylene glycol) (PEG). For example, heterobifunctional CHOPEG-SH remarkably stabilized gold nanoparticles in aqueous media even in a high salt concentration.12 The mercapto group in the polymer coordinates on the gold surface to stably anchor the polymer chains on the surface. On the other hand, the aldehyde group at the distal end of the heterobifunctional PEG chain can be utilized as a ligand-installing moiety. Alternatively, the PEG/polyamine block copolymer, CHO-PEG-b-poly(2-(N,N-dimethylamino)ethyl methacrylate) (CHO-PEG/PAMA), can also be (7) Huang, J.; Sooklal, K.; Murphy, C. J.; Ploehn, H. J. Chem. Mater. 1999, 11, 3595. (8) Sooklal, K.; Hanus, L. H.; Ploehn, H. J.; Murphy, C. J. Adv. Mater. 1998, 10 (14), 1083-1087. (9) Kumbhojkar, N.; Mahamuni, S.; Leppert, V.; Risbud, S. H. Nanostruct. Mater. 1998, 10 (2), 117-129. (10) Qi, L.; Colfen, H.; Antonietti, M. Nano Lett. 2001, 1, 61. (11) Though they mentioned PEG/PEI as a block copolymer in the literature, it is not a block but a graft copolymer. (12) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226.
10.1021/la036034c CCC: $27.50 © 2004 American Chemical Society Published on Web 06/18/2004
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utilized for the stabilization of gold nanoparticles.13 Though the interaction of each tert-amino group with the gold surface is weaker than that of the mercapto group, multivalent coordination of gold with an integrated number of tert-amino groups in the polyamine segment of the block copolymer appreciably enhances the coordination ability. The other segment, PEG, forms the hydrophilic shell layer of tethered chains on the gold surface, increasing the dispersion of the particle stability in aqueous media via a steric repulsion effect. Similar to the CHO-PEG-SH stabilized gold nanoparticles, the aldehyde end group in the block copolymer can be utilized as a ligand installation site. The CHO-PEG/PAMA block copolymer is, therefore, a promising material to achieve both stabilization and functionalization of the nanoparticles. Here, we have utilized the CHO-PEG/PAMA block copolymers for the preparation of CdS QD by the coprecipitation method in aqueous solution. The thus-prepared CdS QD coated with the CHO-PEG/PAMA block copolymer was fairly stable in aqueous media even in a high salt concentration. The biotin moiety was installed at the PEG chain end as a ligand molecule, allowing it to specifically recognize streptavidin. By the use of streptavidin with a fluorescent probe, TexasRed in this study, an effective fluorescent resonance energy transfer (FRET) was observed, indicating the high utility of this method for a highly sensitive assay of biological components. This type of FRET system allows us to detect conventional fluorescent dyes at the same time with a variety of excitation wavelengths due to the wide photoluminescence of the CdS QD. It may also enable a high throughput detection of specific proteins in solution. This paper describes the preparation, the physicochemical characteristics, and the molecular recognition ability of the CdS QD stabilized by the CHO-PEG/PAMA block copolymer. Experimental Section 1. Preparation of CHO-PEG/PAMA Block Copolymers. The CHO-PEG/PAMA block copolymer was synthesized by a previously reported method.14 Briefly, after potassium 3,3diethoxypropanolate (PDP, 1 mmol) was prepared by the reaction between the corresponding alcohol and potassium naphthalene in THF (45 mL), 113.5 mmol of condensed ethylene oxide (EO; Sumitomo Seika, Japan) was added via a cooled syringe to the PDP solution. After a 2-day reaction of EO, 60 mmol of AMA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the reaction mixture and stirred for an additional 60 min at ambient temperature for the block copolymerization. The block copolymer was recovered by precipitation into a large excess of 2-propanol. The small amount of remaining PEG prepolymer was removed by Soxhlet extraction with THF after the protonation of the PAMA segment. The molecular weights of both segments in the resulting block copolymer, PEG and PAMA, were 4200 and 15 800, respectively (Mw/Mn ) 1.35). The functionality of the end acetal group was almost quantitative, which was analyzed by 1H NMR. To convert the end acetal group to an aldehyde group, the block copolymer was dissolved in an acetic acid/water mixture (10:1 v/v) and then stirred for 5 h at 35 °C. After the reaction, the mixture was neutralized by NaOH and dialyzed against water. To introduce the biotin molecule, biocytin hydrazide (Pierce, U.S.A.; biotin with hydrazyl group) was added before the dialysis and reacted for 2 h followed by the addition of NaBH4 to reduce the formed Schiff base. 2. Preparation of CHO-PEG/PAMA Stabilized CdS QD by the Coprecipitation Method in Aqueous Media. One of (13) Ishii, T.; Nagasaki, Y.; Otsuka, O.; Kataoka, K. Langmuir 2004, 20 (3), 561. (14) (a) Nagasaki, Y.; Sato, Y.; Kato, M. Macromol. Rapid Commun. 1997, 18 (9), 827. (b) Kataoka, K.; Harada, A.; Wakebayashi, D.; Nagasaki, Y. Macromolecules 1999, 32 (20), 6892.
Langmuir, Vol. 20, No. 15, 2004 6397 the representative procedures for the preparation of CdS QD stabilized by the CHO-PEG/PAMA block copolymer is described. To an 8 mL aqueous solution of the block copolymer (3.08 × 10-4 mol/L as amine concentration in the block copolymer) in a glass vial, CdCl2 (Wako, Japan; 2.5 × 10-3 mol/L) and Na2S (Wako, Japan; 2.5 × 10-3 mol/L) were added in this order and stirred for 1 h at ambient temperature. The purification was carried out by dialysis against water. The biotin-installed CdS QD was prepared in a similar manner using the biotin-PEG/PAMA block copolymer. 3. Measurements. UV-vis spectra were recorded using a Shimadzu UV-2400PC spectrometer with a 1 cm quartz cell. Fluorescence spectra were obtained using a Hitachi F-2500 spectrometer with an excitation wavelength of 400 nm. The excitation and emission bandwidths were both 2.5 nm. The ζ-potential over a pH range of 2-11 was measured using a LEZA600 (Otsuka Electric Co., Japan) in 7.5 mM NaCl solution. The pH was adjusted with 7.5 mM HCl or NaOH.
Results and Discussion A water soluble polyamine, such as poly(ethyleneimine), is known to stabilize an aqueous dispersion of CdS QD.7 Indeed, as reported here, the polymethacrylic ester possessing the tert-amino group in the side chains has a stabilizing effect on CdS QD in aqueous solution. Figure 1 shows the results of the coprecipitation of CdCl2 with Na2S in the absence and presence of several water soluble polymers. In the absence of any polymer, CdS precipitated due to the increase in the size of the crystals. Even in the presence of commercially available PEG, the phenomenon was the same as in the absence of any polymer. In the presence of the PAMA homopolymer with the molecular weight of 5000, no precipitation was observed in the low salt concentrations. The solution was transparent with a pale yellow color. With the increasing salt concentration, however, the CdS particles immediately precipitate, indicating the low dispersion stability of the QD for the PAMA homopolymer. In addition, almost no emission spectrum was observed by excitation at 400 nm. When CdS QD was prepared in the presence of the CHO-PEG/ PAMA block copolymer, on the contrary, a strong emission spectrum was observed at 540 nm using the same excitation wavelength. The CdS QD thus prepared was fairly stable. Actually, no precipitation was observed in the 0.3 M NaCl solution for several days. The photoluminescent (PL) emission of CdS DQ is affected by the surface charge state.15 Several approaches to the PL activation were reported, which were based on the confinement of charge carriers within the QD cores.16 For example, coordination of electron-donating compounds such as an amine and phosphine oxide significantly improves the fluorescent performance.17,18 In the case of the QD stabilization by the PAMA homopolymer, the amino groups in the side chain of PAMA were used not only for coordination on the CdS surface but also for the solubilization in the aqueous phase. Thus, PAMA homopolymer is softly bound on the surface of the CdS QD. For the CHO-PEG/PAMA block copolymer stabilization, on the contrary, the PAMA segments strongly coordinated on the surface of the CdS QD, which is probably due to the immiscibility of the block copolymer segments to each (15) Kuno, M.; Lee, J. K.; Dabbousi, B. O.; Mikulec, F. V.; Bawendi, M. G. J. Chem. Phys. 1997, 106, 9869. (16) (a) Moore, D. E.; Patel, K. Langmuir 2001, 17, 2541. (b) Zhang, Z.; Dai, S.; Fan, X.; Blom, D. A.; Pennycook, S. J.; Wei, Y. J. Phys. Chem. B 2001, 105 (29), 6755-6758. (c) Elbaum, R.; Vega, S.; Hodes, G. Chem. Mater. 2001, 13 (7), 2272. (d) Farmer, S. C.; Patten, T. E. Chem. Mater. 2001, 13 (11), 3920. (17) Fogg, D. E.; Radzilowski, L. H.; Blanski, R.; Schrock, R. R.; Thomas, E. L. Macromolecules 1997, 30 (3), 417. (18) Veinot, J. G. C.; Galloro, J.; Pugliese, L.; Bell, V.; Pestrin, R.; Pietro, W. J. Can. J. Chem. 1998, 76 (11), 1530.
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Figure 1. Preparation of CdS QD by the coprecipitation method in the absence of stabilizing polymer (a) and in the presence of PEG-OH (Mn ) 5000) (b), poly(2-(N,N-dimethylamino)ethyl methacrylate (Mn ) 5000) (c), and CHO-PEG/PAMA block copolymer (d). Fluorescent spectra of sample c (e) and sample d (f) excited at 400 nm. CdCl2 (2.5 × 10-3 mol/L); Na2S (2.5 × 10-3 mol/L); [amino group in PAMA segment of homopolymer and block copolymer]0 ) 3.08 × 10-4 mol/L.
Figure 2. Fluorescent spectra of CdS (2.5 × 10-3 mol/L) prepared using CdCl2 (2.5 × 10-3 mol/L) with Na2S in the presence of CHO-PEG/PAMA block copolymer in aqueous media: [amino group in PAMA segment of the block copolymer]0 ) 1.16 (a), 3.08 (b), and 4.62 (c) × 10-4 mol/L excited at 400 nm. UV-vis absorption spectrum of CdS QD (d).
other, viz., the PAMA segment is segregated from the PEG segment to stably settle on the QD surface to minimize interfacial free energy. Figure 2 shows typical fluorescent spectra of CdS prepared in the presence of the CHO-PEG/PAMA block copolymer. With the increasing block copolymer concentration, the emission intensity increased along with a hypochromic shift. The solutions were transparent regardless of the block copolymer concentration in this experimental region. Thus, the hypochromic shift in the emission may be due to the decreased size of the formed CdS with the increasing block copolymer concentration. To estimate the size of the obtained CdS QD, UV-vis spectra were recorded, and the spectra are shown in the same figure. As can be seen in the figure, the edge of the absorption spectrum of the obtained CdS was 467 nm. From the band gap theory reported by Henglein,19 the size of the CdS QD was estimated to be 4.8 nm which is corresponding to the size measured from the transmission (19) Henglein, A. Chem. Rev. 1989, 89, 1961.
Figure 3. ζ-Potential variation as a function of pH, showing the same sample as in Figure 2.
electron microscopy (TEM) image, indicating that the block copolymer effectively coordinates on the CdS surface to control the crystallization growth. Note that large crystallites are obtained by the coprecipitation method of CdCl2 with Na2S in the absence of the block copolymer in aqueous media as already stated. The crystal structure of obtained CdS QD was established by X-ray powder diffraction (XRD). The XRD pattern exhibited characteristic peaks of CdS hexagonal wurtzite structure (see the Supporting Information). Figure 3 shows plots of the ζ-potential of the CdS QD versus pH change. In the acidic region, the surface charge of CdS was positive () +15 mV), while it became slightly negative () -3 mV) in the alkaline region. This can be explained by the deprotonation of the PAMA segment on the surface of the CdS QD. Regardless of the pH of the solution, the solution showed no coagulation, which is the proof that the CdS QD was stably dispersed not only by the charge repulsion but also by the steric repulsion of the PEG tethered chain on the surface. Since the block copolymer carries a functional aldehyde group at the PEG chain end, the CdS can be easily functionalized using the end aldehyde group. There are two ways to introduce a functional ligand at the PEG chain end. One is to introduce the specific ligand to the PEG chain end of the block copolymer followed by the prepara-
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Figure 5. Plots of FRET intensities versus TexasRedstreptavidin concentration, after the biotinylated CdS QD was mixed with nonlabeled streptavidin (b) and with nonlabeled BSA (O). Conditions are the same as in Figure 4.
Figure 4. Fluorescent spectra of the mixture of biotinylated CdS QD with TexasRed-streptavidin. I ) 0.15 M; excitation wavelength ) 400 nm; [CdS]0 ) 396 µmol/L. The numbers in the figure denote the concentration of TexasRed-streptavidin.
tion of CdS QD by the coprecipitation method. The other is the ligand conjugation to the PEG chain end after the CdS QD was prepared using the CHO-PEG/PAMA block copolymer. To ensure the installation of a ligand at the PEG chain end, the former method was employed in this study. A biotin molecule was introduced at the end of the PEG/PAMA block copolymer. The biotin-ended PEG/ PAMA block copolymer also worked as a stabilizer of CdS QD by the coprecipitation method. Actually, a strong emission at 540 nm was observed with excitation at 400 nm. The biotin-installed CdS QD thus prepared was then subjected to the molecular recognition test. Kotov et al.20 reported the conjugation of CdTe QD with albumin. They found that the effective resonance energy transfer took place from the tryptophan moieties in albumin to CdTe nanoparticles through the conjugation. Orden et al.21 utilized the FRET system for molecular recognition; viz., the effective FRET was observed from QD to tetramethylrhodamine when a biotinylated albumin conjugated with CdSe-ZnS QD was mixed with tetramethylrhodaminelabeled streptavidin. The QD approached tetramethylrhodamine via the streptavidin-biotin interaction, which causes the effective energy transfer. In the present study, a biotin molecule installed at the distal end of the PEG chain on the QD can be utilized for protein binding assay. To utilize the FRET between the QD and protein for molecular recognition, TexasRed-streptavidin was used as a model compound in this study. Figure 4 shows the fluorescent spectra of the mixture of biotinylated CdS QD with TexasRed-streptavidin excited at 400 nm. Note that TexasRed has almost no absorption at 400 nm. The peak at 620 nm is TexasRed luminescence, which was increasing with the increasing TexasRed-streptavidin concentration, indicating that the effective energy transfer occurred from the CdS emission to TexasRed on the streptavidin. To confirm if the observed FRET was based on the specific molecular recognition, an inhibition experiment was carried out. These results are shown in Figure 5. After the biotinylated CdS QD was (20) Mamedova, N. N.; Kotov, N. A.; Rogach, A. L.; Studer, J. Nano Lett. 2001, 1, 281-286. (21) Willard, D. M.; Carillo, L. L.; Jung, J.; Orden, A. V. Nano Lett. 2001, 1 (9), 469.
Figure 6. TexasRed-streptavidin concentration dependence of FRET intensity.
mixed with streptavidin or bovine serum albumin (BSA) (without TexasRed probe), the TexasRed-streptavidin was added and the FRET intensity was monitored. When streptavidin was used in this system, the FRET intensity significantly decreased, indicating that the streptavidin without the TexasRed probe interrupted the approach of the TexasRed probe to the biotinylated CdS. In the case of the BSA inhibition, the FRET signal slightly increased when a small amount of BSA was added to the biotinylated CdS-TexasRed-streptavidin system. A further increase in the BSA concentration did not affect the FRET intensity, meaning that the BSA molecule did not interrupt the biotin-streptavidin interaction. The slight increase in the FRET intensity by the addition of BSA was attributable to the fact that the BSA molecules in the solution make the approach of TexasRed-streptavidin to the biotinylated CdS surface rather easy due to the exclusion volume effect of BSA.22 On the basis of these results, the observed FRET luminescence can be attributed to the specific molecular recognition of the biotin molecule adjacent to the PEG tethered chain on the CdS surface with TexasRedstreptavidin. Figure 6 shows the concentration effect of TexasRedstreptavidin on the FRET intensities. The FRET peak proportionally increased with the increasing TexasRedstreptavidin. This can be utilized as a quantitative protein assay system. In conclusion, biotin-PEG/polyamine block copolymers can be used to prepare nanosized CdS by the coprecipitation method. The obtained CdS QD was stabilized enough in aqueous media by the coordination of polyamine (22) Ortega-Vinuesa, J. L.; Molina-Bolivar, J. A.; Hidalgo-Alvarez, R. J. Immunol. Methods 1996, 190, 29.
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in the block polymer on the CdS surface. The PEG segment in the copolymer formed a tethered chain on the CdS surface. The biotin molecule at the distal end of the PEG tethered chain showed an effective molecular recognition to TexasRed-streptavidin, which caused the fluorescent resonance energy transfer between CdS QD and TexasRed adjacent to the streptavidin molecule. The observed FRET system between CdS QD and the fluorescent-labeled protein can be utilized as one of the new molecular recognition systems.
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Acknowledgment. A part of this study was financially supported by the Special Coordination Funds, Ministry of Education, Science and Sports, Japan. Supporting Information Available: TEM image of PEG/PAMA CdS QD and XRD of PEG/PAMA CdS QD. This material is available free of charge via the Internet at http://pubs.acs.org. LA036034C
