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Preparation of Functionally PEGylated Gold Nanoparticles with Narrow Distribution through Autoreduction of Auric Cation by r-Biotinyl-PEG-block-[poly(2-(N,N-dimethylamino)ethyl methacrylate)] Takehiko Ishii,† Hidenori Otsuka,‡,§ Kazunori Kataoka,‡ and Yukio Nagasaki*,† Department of Material Science & Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan, and Department of Materials Science & Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received September 5, 2003. In Final Form: December 6, 2003 PEGylated gold nanoparticles with biotin moieties installed at the distal end of the PEG tethered chains were prepared by the autoreduction of HAuCl4 catalyzed by R-biotinyl-PEG-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (biotinyl-PEG/PAMA) in aqueous medium at room temperature. The size of the gold nanoparticles was controllable in a range of 6-13 nm by changing the initial Au3+/polymer ratio, while retaining their narrow size distribution. The dispersion stability of the nanoparticles in aqueous medium was extremely high even under the condition of salt concentration as high as I ) 2.0. BiotinylPEG/PAMA-anchored gold nanoparticles underwent specific aggregation in the presence of streptavidin, revealing their promising utility as colloidal sensing systems applicable under biological condition.
Colloidal gold nanoparticles with a size range of several to hundreds of nanometers show a bright-pinkish color due to plasmon resonance and have been widely utilized for preparation of stained glasses for several hundred years. Recently, a novel and important application of colloidal gold nanoparticles has emerged in the bioanalytical field based on their colorimetric change in plasmon resonance absorption due to aggregation in an aqueous entity.1 Mirkin and co-workers proposed the use of a colloidal gold system for colorimetric gene detection in 1996, which opened a new way of simple sensing of single nucleotide polymorphorism (SNP). The growing interest in colloidal gold nanoparticles in the bioanalytical field lends a progressive impetus to the development of their novel preparation methods.2 Most of these methods were based on the reduction of tetrachloroauric acid (HAuCl4) in aqueous medium. Gold nanoparticles thus prepared are dispersed in the solution by ionic repulsion of adsorbed ions such as AuCl2- on their surface.3 Under physiological salt concentration, however, the ionically stabilized gold nanoparticles tend to ag* Corresponding author: tel, +81-4-7124-1501 (ext 4310); fax, +81-4-7123-8878; e-mail,
[email protected]. † Tokyo University of Science. ‡ The 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) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (b) Alivisatos, A. P.; Johnson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Nature 1996, 382, 609-611. (c) Sastry, M.; Lala, N.; Patil, V.; Chavan, S. P.; Chittiboyina, A. G. Langmuir 1998, 14, 4138-4142. (d) Connolly, S.; Fitzmaurice, D. Adv. Mater. 1999, 11(14), 1202-1205. (e) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (f) Nath, N.; Chilkoti, A. J. Am. Chem. Soc. 2001, 123, 8197-8202. (g) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509. (h) Thanh, N. T. K.; Rosenzweig, Z. Anal. Chem. 2002, 74 (7), 1624-1628. (2) (a) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (b) Brust, M.; Walker, M.; Schiffrin, D.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (c) Han, M. Y.; Quek, C. H.; Huang, W.; Chew, C. H.; Gan, L. M. Chem. Mater. 1999, 11, 1, 1144-1147. (d) Brown, K. R.; Walter, D. G.; Natan, M. J. Chem. Mater. 2000, 12 (2), 306-313.
gregate because of the charge shielding. To improve their dispersibility in the solution with appreciably high ionic strength, various types of low molecular weight stabilizers as well as of water-soluble polymers such as starches are often added in the solution.2,4,6-8 An alternative way to stabilize the dispersion is to form a brushed layer of hydrophilic polymer strands on the surface of the gold nanoparticles. Indeed, a brushed layer of poly(ethylene glycol) (PEG) was successfully prepared on gold nanoparticles using end-thiolated PEG.4 Functionalization of the R-end of ω-thiolated PEG was further accomplished to install specific ligands for molecular sensing:5 R-lactosylPEG-anchored gold nanoparticles were demonstrated to undergo sensitive and quantitative aggregation responding to a lactose-specific lectin, RCA120.6 Furthermore, water soluble polymers possessing coordination ability with metals such as poly(2-vinylpyridine) and poly(ethylenimine) have been utilized as a stabilizer for metal colloids.7,8 For example, Antonietti and coworkers reported the preparation of PEG-stabilized gold colloids through the mixing of AuCl3 with poly(ethylenimine)-poly(ethylene glycol) graft copolymer (PEI/PEG).9 The amino group in the PEI segment of the PEI-PEG graft copolymer was considered to have the ability to reduce auric cations to form a gold colloid in the AuCl3incorporated PEI/PEG micelles. Although the formed gold (3) (a) Weiser, H. B. Inorg. Colloid Chem. 1933, 1, 21-57. (b) Biggs, S.; Chow, M. K.; Grieser, F. J. Colloid Interface Sci. 1993, 160, 511. (c) Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1989. (4) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 12696-12697. (5) Akiyama, Y.; Otsuka, H.; Nagasaki, Y.; Kato, M.; Kataoka, K. Bioconjugate Chem. 2000, 11, 947-950. (6) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226-8230. (7) Mayer, A. B. R.; Mark, J. E. Eur. Polym. J. 1998, 34 (1), 103-108. (8) Bronstein, L. M.; Gourkova, S. N.; Sidorov, A. Y.; Valetsky, P. M.; Hartmann, J.; Breulmann, M.; Colfen, H.; Antonietti, M. Inorg. Chim. Acta 1998, 280 (1-2), 348-354. (9) Though they described their PEG/PEI as a block copolymer, it is actually not a block, but a graft copolymer.
10.1021/la035653i CCC: $27.50 © 2004 American Chemical Society Published on Web 12/30/2003
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colloid was stabilized by the layer of a hydrophilic PEG segment in aqueous media, the distal end of PEG has no reactive group for further functionalization, including installation of ligand molecules. Here, we would like to report a simple and effective approach for the concomitant stabilization and functionalization of gold nanoparticles using a PEG/polyamine block copolymer possessing a functional group at the end of the PEG chain: acetal-PEG-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (acetal-PEG/PAMA).10 Worth noting is that acetal-PEG/PAMA facilitates autoreduction of auric cations to form nanoparticles with very narrow size distribution only by mixing it with HAuCl4 aqueous solution at room temperature. The obtained nanoparticles were very stable even under salt concentration as high as I ) 2.0. Because the acetal group at the PEG chain end of the block copolymer is easily converted into an aldehyde group, various ligands can be anchored onto the prepared gold nanoparticles. Biotinylation as an example is shown in Scheme 1. Acetal-PEG/PAMA was synthesized by our original method based on anionic polymerization. Potassium 3,3diethoxypropyl alcoholate (PDA) initiated the ring opening anionic polymerization of ethylene oxide in THF, followed by the block copolymerization of 2-(N,N-dimethylamino)ethyl methacrylate (AMA) to form acetal-PEG/PAMA. PAMA is soluble in aqueous media even in an alkaline region; therefore, the block copolymer is water soluble over a wide pH range. The unreacted prepolymer, acetalPEG-OH, was successfully removed from the sample by Soxhlet extraction with THF after protonation of the PAMA segment in the block copolymer. The molecular weights of the PEG and PAMA segments thus prepared were 5100 (size exclusion chromatography) and 3800 (1H NMR), respectively. The quantitative introduction of the R-acetal group was confirmed by 1H NMR analysis. The R-acetal group of acetal-PEG/PAMA was quantitatively converted into an aldehyde group by immersion in 90% acetic acid aqueous solution at 35 °C for 5 h. This (10) Kataoka, K.; Harada, A.; Wakebayashi, D.; Nagasaki, Y. Macromolecules 1999, 32, 6892-6894.
was confirmed by the 1H NMR spectrum, viz., the acetal methine proton disappeared completely, while a new peak based on the aldehyde proton was clearly observed at 9.8 ppm. Biocytin hydrazide was then reacted with the R-aldehyde group to introduce a biotin group at the PEG chain end of the block copolymer (R-biotinyl-PEG/PAMA). Because an amino group is known to show coordination ability on a metal surface, R-biotinyl (or R-acetal)-PEG/ PAMA was expected to function as a stabilizer of colloidal gold nanoparticles. An unprecedented finding is that the block copolymer even facilitated autoreduction of the auric cations to obtain gold nanoparticles without using any additional reducing reagent. The tetrachloroauric acid solution was changed from colorless to bright red due to the formation of colloidal gold nanoparticles when PEG/ PAMA was added to the solution in an appropriate ratio. The tertiary amino groups in the PAMA segment play a crucial role in the reduction of auric cations as well as the anchoring of PEG on the surface of the gold nanoparticles. Figure 1 shows the change in the plasmon absorbance at 520 nm of the tetrachloroaurate aqueous solution with different polymer additives. In the presence of acetal-PEGOH (Mw 5900) at room temperature, no color change was observed. On the contrary, both PAMA homopolymer and acetal-PEG/PAMA block copolymer induced a change in the color of the solution due to a gradual increase in the absorbance at 520 nm over several hours, indicating the reduction of auric cations to form colloidal gold. Nevertheless, the gold nanoparticles obtained by the PAMA homopolymer were rather unstable under a high salt condition, and the characteristic absorption of colloidal gold nanoparticles was gradually reduced with time (data not shown). Worth noting is that the reduction process finished within ca. 3 h in the case of acetal-PEG/PAMA at room temperature to form stable nanoparticles having a narrow size distribution. The insert in Figure 1 shows a transmission electron microscopy (TEM) image of the thus-prepared gold nanoparticles. The average size and the distribution index (Dw/Dn) of the obtained gold nanoparticles, under the condition of the weight ratio of (acetal-PEG/PAMA)/(HAuCl4‚4H2O) ) 13.5, were 11.8 nm
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Figure 1. Change in the plasmon absorbance (λ ) 520 nm) of the tetrachloroaurate aqueous solution in the presence of R-acetal-PEG-OH (O), PAMA (2), and R-acetal-PEG/PAMA (9). Inset is a TEM image of gold nanoparticles prepared in the presence of R-acetal-PEG/PAMA.
and 1.01, respectively. With the increasing weight ratio from 6 to 24, the average particle size decreased from 13 to 6 nm. Note that these acetal-PEG/PAMA-stabilized gold nanoparticles have a substantially narrower distribution than those prepared by the PEI/PEG graft copolymer and comparable to conventional methods. This is obviously an advantage of using block copolymers as stabilizers because they can form a multimolecular micelle structure with a definite association number and well-defined coreshell architecture: an aurate-complex core segregated and surrounded by hydrophilic shell layer. The obtained gold nanoparticles were purified by centrifugation at 45000g, for 30 min at 20 °C. The precipitated particles were resuspended in water or in various buffer solutions with a wide range of pH. The centrifugations were repeated several times to remove free block copolymers. No color change was observed, while retaining the transparency of the solution. This behavior clearly indicates the high dispersion stability of the acetal-PEG/ PAMA-anchored gold nanoparticles. Commercially available gold nanoparticles, prepared by citrate, were dispersed in an aqueous solution through the ionic repulsion of the surface-adsorbed ions. Thus, the citrate-reduced gold nanoparticles have an appreciably negative ζ-potential (
