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Environmentally Friendly Synthesis of Highly Monodisperse Biocompatible Gold Nanoparticles with Urchin-like Shape Lehui Lu,*,† Kelong Ai,† and Yukihiro Ozaki*,‡ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022 Changchun, China, and Department of Chemistry, School of Science and Technology, Kwansei Gakuin UniVersity, Sanda, Hyogo 669-1337, Japan ReceiVed September 18, 2007. In Final Form: NoVember 5, 2007 We report a facile and environmentally friendly strategy for high-yield synthesis of highly monodisperse gold nanoparticles with urchin-like shape. A simple protein, gelatin, was first used for the control over shape and orientation of the gold nanoparticles. These nanoparticles, ready to use for biological systems, are promising in the optical imaging-based disease diagnostics and therapy because of their tunable surface plasmon resonance (SPR) and excellent surface-enhanced Raman scattering (SERS) activity.
Introduction Biocompatible gold nanoparticles have received considerable attention in recent years because of their promising applications in bioimaging, biosensors, biolabels, and biomedicine.1-4 In almost all the cases, the success relies strongly on the availability of biocompatible gold nanoparticles with high monodispersity, good water solubility, and high stability against salt-induced aggregation. Conventional synthetic strategies for such nanoparticles are based on the preparation of monodisperse spherical gold nanoparticles according to classical citrate or NaBH4 reduction methods, followed by surface modification with biomolecules.5,6 However, general disadvantages of these synthetic strategies are multiple-step experimental procedures and the aggregation of nanoparticles induced by the addition of biomolecules. More recently, Zhao et al. reported that biocompatible gold nanoparticles could be successfully prepared in one step by reducing HAuCl4 with NaBH4 in the presence of nucleotides.7 The resulting gold nanoparticles were monodisperse, water soluble, and highly stable against NaCl salt-induced aggregation. Nevertheless, this method is applied to produce only small spherical gold nanoparticles with sizes between 2 and 5 nm, which, to great extent, limits the wide use of these nanoparticles, particularly in the optical properties-based biological applications. For instance, tunable surface plasmon resonance (SPR) enhancement light scattering and absorption of gold nanoparticles make them novel and highly effective contrast agents for in vivo cancer diagnosis and therapy,4 but for roughly spherical gold nanoparticles in the size range of 2-40 nm changing their sizes offers much limited tunability of the SPR wavelength.8 In contrast, * Corresponding authors. Phone: +86-43185262418 (L.L.); +81795658349 (Y.O.). Fax: +86-43185262406 (L.L.); +81-795659077 (Y.O.). E-mail:
[email protected] (L.L);
[email protected] (Y.O.). † Chinese Academy of Sciences. ‡ Kwansei Gakuin University. (1) Sokolov, K.; Follen, M.; Aaron, J.; Pavlova, I.; Malpica, A.; Lotan, R.; Richards-Kortum, R. Cancer Res. 2003, 63, 1999. (2) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547. (3) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115. (4) Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Nano Today 2007, 2, 18. (5) Niemeyer, C. M.; Burger, W.; Peplies, I. Angew. Chem., Int. Ed. 1998, 37, 2265. (6) Le´vy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Ferning, D. G. J. Am. Chem. Soc. 2004, 126, 10076. (7) Zhao, W.; Gonzaga, F.; Li, Y.; Brook, M. A. AdV. Mater. 2007, 19, 1766.
shape control provides a greater versatility for tuning optical properties of the gold nanoparticles than can be achieved otherwise.9,10 Especially, if control is achieved for highly monodisperse biocompatible gold nanoparticles with complex shapes, this is of importance since it may enable high optical detectability down to an individual nanoparticle level.9,10 In previous reports,10,11 cetyltrimethylammonium bromide (CTAB), poly(vinylpyrrolidone) (PVP), and mercaptopropanic acid are generally used as the capping ligands to prepare monodisperse gold nanoparticles with complex shapes, but their use often raises environmental issues. Furthermore, effective replacement of these capping ligands with biomolecules for biological applications still faces challenges. In this article, we report a facile and environmentally friendly strategy for high-yield synthesis of highly monodisperse gold nanoparticles with urchin-like shape. These water-soluble nanoparticles are highly stable against salt-induced aggregation and exhibit tunable SPR bands directly related to their geometric shape and excellent surface-enhanced Raman scattering (SERS) activity relative to that of spherical gold nanoparticles with the approximately same diameter. Experimental Section Materials. Silver nitrate, sodium citrate, adenine, L-ascorbic acid, and potassium carbonate were obtained from Wako Pure Chemical Industries, Ltd. Hydrogen tetrachloroaurate(III) trihydrate, sodium tetrahydroborate, and gelatin were purchased from Sigma-Aldrich. All chemicals were used without further purification. Synthesis of Silver Seed. Briefly, 1 mL of an aqueous solution of sodium citrate (30 mM) was added to 100 mL of a 0.25 mM aqueous solution of silver nitrate, and the mixture was stirred for 1 min. Subsequently, 3 mL of ice-cooled aqueous solution of sodium tetrahydroborate (10 mM) was added under vigorous stirring. The mixture was stirred for an additional 30 s and then was allowed to age without agitation for 2 h at 4 °C before use. A transmission electron microscopy (TEM) examination showed that the resulting silver nanoparticles were spherical in shape with an average diameter of 4 nm. (8) (a) Jana, N. R.; Gearheart, L.; Murphy, C. J. Langmuir 2001, 17, 6782. (b) Jana, N. R.; Gearheart, L.; Murphy, C. J. Chem. Mater. 2001, 13, 2313. (9) Jin, R. C.; Cao, Y. W.; Mirkin, C. A.; Kelly, K. L.; Schatz, G. C.; Zheng, J. G. Science 2001, 294, 1901. (10) (a) Murphy, C. J.; Sau, T. K.; Gole, A. M.; Orendorff, C. J.; Gao, J. X.; Gou, L. F.; Hunyadi, S. E.; Li, T. J. Phys. Chem. B 2005, 109, 13857. (b) Orendorff, C. J.; Sau, T. K.; Murphy, C. J. Small 2006, 2, 636. (c) Sun, Y. G.; Xia, Y. N. Science 2002, 298, 2176. (11) Bakr, O. M.; Wunsch, B. H.; Stellacci, F. Chem. Mater. 2006, 18, 3297.
10.1021/la702886q CCC: $40.75 © 2008 American Chemical Society Published on Web 01/05/2008
Gold Nanoparticles with Urchin-like Shape Synthesis of Urchin-like Gold Nanoparticles. Gold precursor solution was prepared as follows. An amount of 25 mg of K2CO3 solid was dissolved in 100 mL of distilled water, followed by addition of 1.5 mL (20 mmol) of an aqueous solution of HAuCl4. The resulting solution was aged at least 1 day in the dark, during which it changed color from yellow to white due to the formation of gold hydroxide.12 A 1% aqueous solution of gelatin was prepared by dissolving 0.2 g of gelatin solid in 20 mL of water at 50 °C. For the synthesis of urchin-like gold nanoparticles (sample 1), typically, to a 10 mL volume of the aged HAuCl4/K2CO3 solution was added 1 wt % gelatin protein (3 mL) and silver seed (0.8 mL) at room temperature. Two minutes later, a 100 mM aqueous solution of ascorbic acid (0.8 mL) was added. Without stirring, the color of the mixed solution changed from yellow, to red, and then to the final purple-red within 1 h, indicating that the nanoparticle growth was complete during 1 h. For the synthesis of other samples, the amount of silver seed was simply decreased to 0.2 mL (sample 2) and 0.05 mL (sample 3), respectively, while keeping other experimental conditions fixed. Characterization. TEM images were obtained on a TECNAI F2OG2 TWIN transmission electron microscope operating at 200 kV. TEM images and analytical data were further processed by using ES Vision 4.0 (Emispec system, Inc.). X-ray diffraction (XRD) patterns of the samples were recorded on a high-resolution X-ray diffractometer (Rigaku, SLX-2000). Cu KR radiation (wavelength, 1.5405 Å) was used as an incident X-ray source (50 kV, 300 mA). UV-vis extinction spectra were taken using a UV-3101PC UVvis-NIR scanning spectrophotometer. Infrared spectra were recorded by averaging 128 scans at a 4 cm-1 resolution with a Thermo Nicolet Magna 870 spectrometer equipped with an MCT detector. Raman spectra were taken using a confocal microprobe Raman system (LabRam I from Dilor). The microscope attachment is based on an Olympus Bx40 system. An air-cooled 1024 × 256 pixels CCD was used as the detector. The excitation wavelength was 632.8 nm from an air-cooled He-Ne laser. The laser power at the sample position was typically 2.5 mW. Data acquisition time was 10 s with two accumulations.
Results and Discussion Considering practical applications envisaged for biocompatible gold nanoparticles, simple, low-cost, and environmentally friendly strategies appear to be the most promising. In light of this consideration, we took the experimental measures as follows. First, we chose gelatin, a simple protein, as a template in the present case. Gelatin is the denaturation product of collagen, the most abundant protein in animal skin and bone, and has been extensively used for food, pharmaceutical, and medical applications.13 Importantly, due to the presence of functional groups including -NH2 and -COOH, gelatin can be further modified with other biomolecules for different purposes. Second, a HAuCl4/ K2CO3 solution rather than HAuCl4 was used as a precursor. As mentioned above, mixing HAuCl4 and K2CO3 solution led to the formation of more stable gold hydroxide and an approximate pH ∼ 6.0 reaction medium, thus making it possible for the reduction reaction to proceed in a controllable way.12-14 Third, ascorbic acid (vitamin C), a weak reducing agent, was chosen to reduce the gold salts. As will be discussed later, the use of gelatin and ascorbic acid provides great advantage over other methods in terms of environmentally friendly synthesis, good biocompatibility, and high stability. Figure 1 shows typical TEM images of sample 1, sample 2, and sample 3. Their high-resolution TEM images (HRTEM) are (12) Graf, C.; van Blaaderen, A. Langmuir 2002, 18, 524. (13) Olsen, D.; Yang, C.; Bodo, M.; Chang, R.; Leigh, S.; Baez, J.; Carmichael, D.; Perala, M.; Hamalainen, E. R.; Jarvinen, M.; Polarek, J. AdV. Drug DeliVery ReV. 2003, 55, 1547. (14) Xiong, Y.; McLellan, J. M.; Chen, J.; Yin, Y.; Li, Z. Y.; Xia, Y. J. Am. Chem. Soc. 2005, 127, 17118.
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also presented in the same figure. These samples consist of a large quantity of highly monodisperse nanoparticles, and the corresponding high-magnification images (see the insets) reveal that these nanoparticles possess a studded quasi-spherical shape resembling sea urchins (yield >90%). Their three-dimensional structure was verified by tilting the samples at 60° to the beam axis (Figure 2). The branch dimension of these urchin-like nanoparticles can be controlled by varying the amount of silver seed. As seen from Figure 1, parts a, c, and e, with the decrease in the amount of silver seed the dimension of the branches of the nanoparticles increases while the mean diameter of the nanoparticles changes a little (43 ( 5 nm; note that the diameter of the nanoparticle is defined as its biggest dimension). Previous reports on seeding methods showed that the mean diameter of the resulting nanoparticles should increase and their particle number should decrease as the amount of seed decreased.8 Surprisingly, we also found by using TEM measurement that the particle number for sample 3 was more than that for sample 1 and sample 2. The above results can be rationalized as follows. In this case, the role of silver seed is twofold. First, it can serve as seed to catalyze the formation of initial small gold nanocrystals. Second, besides ascorbic acid, it can also reduce gold salts by the so-called replacement reaction to produce a part of the small gold nanocrystals,10c which may be the reason why the diffraction lines corresponding to silver metal cannot be detected in the XRD measurement mentioned below. The resulting small gold nanocrystals can further grow to form the final urchin-like gold nanoparticles, evidenced by the color change from red to the final purple-red of the reaction solution. As shown in Figure 1, sample 3 possesses a more open nanostructure compared with that of sample 1 and sample 2, although their average diameters change a little. That is, the required number of gold atoms in forming a single urchin-like gold nanoparticle for sample 3 should be less than that for sample 1 and sample 2. In our experiment, the volume of HAuCl4/K2CO3 solution is the same for all three samples, and thus it is not surprising to observe the results discussed above. The HRTEM images in Figure 1, parts b, d, and f, reveal clear lattice fringes with an interplane distance of 0.204 nm corresponding to the (200) lattice space of metallic gold (see the inset in Figure 1f). Moreover, PVP was used to replace gelatin protein as a comparison with other experimental conditions unchanged. We found that nanoparticles with irregular shapes and a mean diameter of 16 ( 4 nm were obtained (Figure 3a). An interplane distance of 0.236 nm was measured from the HRTEM image for all lattice fringes, which is assigned to the (111) lattice space of metallic gold (Figure 3b). This indicated that gelatin protein played an essential role in controlling the urchin-like shape and orientation of the nanoparticles. Representative XRD patterns of the samples prepared in the presence of PVP and gelatin protein are shown in Figure 4. Four diffraction lines are observed in each XRD pattern at 2θ ∼ 38.2°, 44.5°, 64.9°, and 77.9°, corresponding to (111), (200), (220), and (311) reflections, respectively, for the face-centered cubic structure of metallic gold (JCPDF no. 04-0784). Interestingly, the (200) diffraction line shows the strongest intensity in the XRD pattern of sample 3. The ratio between the maximum intensities of the (200) and (111) diffraction lines is about 3.5, substantially larger than that described in the JCPDF card (3.5 vs 0.6). This reveals that the {100} crystal faces of sample 3 tended to be preferentially oriented parallel to the surface of the supporting substrate and the presence of gelatin molecules was responsible for this oriented growth. Furthermore, the mean diameters of the nanoparticles for sample 3 and the PVP-coated
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Figure 1. Typical TEM images of (a) sample 1, (c) sample 2, and (e) sample 3, and the corresponding high-resolution images of (b) sample 1, (d) sample 2, and (f) sample 3. The insets show the corresponding high-magnification images.
nanoparticles were determined from the width of the strongest diffraction lines (note that the (200) diffraction line is used for sample 3 and (111) is used for the PVP-coated gold nanoparticles)
by using the Debye-Scherrer formula to be about 40.5 and 14.5 nm, respectively, coinciding well with those measured from TEM images.15
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Figure 2. TEM images of sample 2, tilting the sample plane from (a) 0° to (b) 60°.
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Figure 3. (a) TEM image and (b) the corresponding HRTEM image of the gold nanoparticles prepared in the presence of PVP. The inset shows the corresponding high-magnification image.
Gelatin protein consists of one single chain of a highly repetitive sequence of amino acids [glycine-X-Y]n, where X and Y are often proline and hydroxyproline, respectively.16 In this case, the isoelectric point of the gelatin is about 9.0. The pH value used in our experiment is about 6.0, and thus the gelatin is positively charged. The positively charged gelatin could be electrostatically adsorbed on the negatively charged citrate-coated silver seeds. With the gelatin concentration and temperature of the present study, the gelatin should be in the sol state dispersed mainly as random coils.16,17 These random coils adsorbed on the silver seeds serve as structure-directing agents for the growth of the urchin-like gold nanoparticles with preferential orientation of the {100} crystal faces. The mechanism may be relevant to selective binding of gelatin molecules to certain crystal faces of initial small nanoparticles. In this case, gelatin molecules may preferentially bind to the {100} crystal planes of initial small nanoparticles. Such behavior slowed the growth rate along the 〈100〉 direction, and thus promoted a highly anisotropic growth in other directions such as 〈111〉, ultimately leading to formation of the urchin-like gold nanoparticles with preferential orientation
Figure 4. XRD patterns of the products prepared in the presence of (a) PVP and (b) gelatin (sample 3).
(15) Warren, B. E. X-ray Diffraction; Addison-Wesley: London, 1969. (16) Ward, A. G.; Courts, A. The Science and Technology of Gelatin; Academic Press: New York, 1977. (17) Liu, S. H.; Zhang, Z. H.; Han, M. Y. AdV. Mater. 2005, 17, 1862.
of the {100} crystal faces parallel to the surface of the supporting substrate. Recent studies by Murphy and co-workers on branched metal nanoparticles also showed that the preferential binding of
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Figure 5. IR spectrum of sample 3 on the CaF2 substrate.
CTAB molecules to specific crystal faces played a key role in the formation of the branched metal nanoparticles.10a,b Evidence for the presence of gelatin molecules on the surface of the nanoparticles was obtained by an infrared (IR) spectrum. Figure 5 shows the IR spectrum of sample 3 transferred onto a CaF2 substrate. The absorption bands at 1655 cm-1 (CdO stretching) and 1542 cm-1 (N-H bending/C-N stretching) originating from the polypeptide chains of gelatin protein are clearly observed.18 Before the IR experiments all the samples were thoroughly washed with water to remove residues of the reactants. Accordingly, it is reasonable to conclude that the IR spectral signals of sample 3 result from the gelatin molecules on the surface of the nanoparticles. More recently, Bauermann et al. also reported similar experimental results.19 They found that in the presence of gelatin protein ZnO crystals had the structure with a preferential {100} orientation and their specific shapes were generated only when gelatin was used. The SPR properties of gold nanoparticles are dependent on particle size and shape, and thus the changes in size and shape of the gold nanoparticles should be reflected by their UV-vis extinction spectra. Figure 6A, curves a-d, shows UV-vis extinction spectra taken from silver seed, sample 1, sample 2, and sample 3, respectively. Only one SPR band is observed in each case in the wavelength range from 350 to 1000 nm. As the amount of silver seed decreases, the SPR bands increasingly broaden and a 61 nm red shift occurs from sample 1 to sample 3. As mentioned above, the size change from sample 1 to sample 3 is less than 10 nm. Considering the effect of size on SPR bands, it is impossible for such a small size change to result in a 61 nm SPR band shift in this case. Accordingly, it is very likely that the large SPR shift originates from the specific branched structure of the urchin-like nanoparticles. Such dramatic change in SPR wavelength offers an excellent platform for the optical propertiesbased biological and medical applications of these gold nanoparticles.4 To be useful for biological applications, gold nanoparticles have to be stable against salt-induced aggregation because bioassays are generally performed in the presence of buffering and salt solutions.6 Figure 6B compares UV-vis extinction spectra of sample 2 in the absence and presence of 1 M NaCl at pH 7.0. No significant shift in the UV-vis extinction spectrum is observed within at least 1 h, indicating that the resulting gold nanoparticles are stable at the high-concentration NaCl solution. This is further (18) Krimm, S.; Bandekor, J. AdV. Protein Chem. 1986, 38, 183. (19) Bauermann, L. P.; Campo, A.; Bill, J.; Aldinger, F. Chem. Mater. 2006, 18, 2016.
Figure 6. (A) UV-vis extinction spectra of (a) silver seed, (b) sample 1, (c) sample 2, and (d) sample 3. (B) UV-vis extinction spectra of sample 2 (a) in water solution and (b) in 1 M NaCl. The spectra were taken 1 h after mixing sample 2 with salt solution. (C) SPR scattering image of sample 2.
evidenced by the direct SPR scattering image of these nanoparticles that were incubated with 1 M NaCl for 1 h and then spin-coated onto the glass substrate (Figure 6C). As can be seen from Figure 6C, the high monodispersity of the gold nanoparticles was well kept and no obvious particle aggregation occurred. Moreover, we found that these gold nanoparticles were stable in a pH range of 4.4-10. Nanoparticle-based SERS techniques have proven to be one of the most sensitive and promising approaches for bioassay applications, particularly in living cells.20,21 For example, Kneipp (20) Kneipp, K.; Kneipp, H.; Kneipp, J. Acc. Chem. Res. 2006, 39, 443. (21) Aroca, A. Surface-Enhanced Vibrational Spectroscopy; John Wiley & Sons: New York, 2006.
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observed at 730 and 1325 cm-1, assigned to the purine ring breathing mode and the CN stretching mode, respectively.25 In contrast, the 45 nm spherical gold nanoparticles exhibit only extremely weak SERS enhancement. The SERS signal intensity at 730 cm-1 from sample 3 is approximately 10 times higher than that from the 45 nm spherical gold nanoparticles, indicating that sample 3 is highly desirable for SERS applications.22,23 It is not surprising to observe such large SERS enhancement for sample 3. SERS is known to be a very local phenomenon generally occurring at crevices or rough surfaces. In our case, many crevices are produced between branches of the urchin-like nanoparticles. Excited by the laser, a collective SPR is trapped at these crevices, creating a huge local electric field, and thus leading to large SERS intensity enhancement.22,23
Conclusions Figure 7. SERS spectra of 0.1 mM adenine obtained from (a) sample 3 and (b) 45 nm spherical gold nanoparticles.
et al. transferred gold nanoparticles into a cellular interior and successfully acquired a good-quality SERS signal corresponding to cellular constituents.20 The as-prepared gold nanoparticles may be desirable for this purpose. First, as discussed earlier, these nanoparticles are ready to use in biological systems. Second, according to previous studies high SERS intensity enhancement is generally observed for the metal nanostructures with complex shapes, thus allowing for greater sensitivity for bioassay.20-23 Based on the above considerations, we examined the SERS activity of the as-prepared gold nanoparticles by using the DNA base adenine. For a comparison, spherical gold nanoparticles with approximately the same diameter (about 45 nm) were synthesized as a control, and their concentration is 1.2 times higher than that of sample 3 in order to collect a better SERS signal.24 Figure 7 compares the SERS spectra of 0.1 mM adenine obtained from sample 3 and 45 nm spherical gold nanoparticles with the 632 nm excitation. Two prominent Raman bands are (22) (a) Kim, K.; Park, H. K.; Kim, N. H. Langmuir 2006, 22, 3421. (b) Kottmann, J. P.; Martin, O. J. F. Phys. ReV. B 2001, 64, 235402. (23) (a) Lu, L. H.; Randjelovic, I.; Capek, R.; Gaponik, N.; Yang, J. H.; Zhang, H. J.; Eychmu¨ller, A. Chem. Mater. 2005, 17, 5731. (b) Lu, L. H.; Kobayashi, A.; Tawa, K.; Ozaki, Y. Chem. Mater. 2006, 18, 4894. (24) Liu, N. G.; Prall, B. S.; Klimov, V. I. J. Am. Chem. Soc. 2006, 128, 15362.
In summary, a facile and environmentally friendly strategy was presented for high-yield synthesis of highly monodisperse urchin-like gold nanoparticles. Gelatin, from the most abundant protein in animal skin and bone, was used as a template for the control over shape and orientation of the gold nanoparticles, which provides great advantage over other methods in terms of low cost and wide use. These biocompatible gold nanoparticles are water soluble, highly stable in the high-concentration salt solution and the wide pH range, and thus ready to use for biological applications. Importantly, tunable SPR properties and excellent SERS enhancement ability of such nanoparticles enable their applications in the optical imaging-based disease diagnostics and therapy. We are currently pursing such imaging studies. Acknowledgment. We thank Dr. A. Kobayashi from AIST for the TEM characterization and Dr. H. Sato from Kwansei Gakuin University for XRD examination. This work has been partially supported by the “Hundred Talents Project” (initialization support) of the Chinese Academy of Sciences and by the “Open Research Center” project for private universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2001-2008. LA702886Q (25) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. ReV. 1999, 99, 2957.
