Diacetylene-Containing Ligand As a New Capping Agent for the

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Diacetylene-Containing Ligand As a New Capping Agent for the Preparation of Water-Soluble Colloidal Nanoparticles of Remarkable Stability Dorota Bartczak and Antonios G. Kanaras* School of Physics and Astronomy, University of Southampton, Southampton SO17 1BJ, U.K. Received November 20, 2009. Revised Manuscript Received January 3, 2010 A new type of strategically designed functional ligands was used to cap gold nanocrystals and form robust colloidal nanoparticles, resistant to pH changes, temperature, and ionic strength variations as well as ligand-exchange reactions. The nanoparticles are coated with ligands that polymerize upon UV-irradiation, consequently embedding the particles in a stable organic shell. The ligand consists of an anchoring thiol group, which binds directly to the nanocrystal surface and two units, one hydrophobic and one hydrophilic. The hydrophobic alkyl unit contains a diacetylene group, which undergoes a 1,4-topochemical polymerization leading to a poly(enyne) structure during UV-irradiation. The hydrophilic unit contains an oligo-ethylene glycol chain, which ensures water solubility, and a terminal carboxylic group. Derived particles were characterized by transmission electron microscopy, surface enhanced Raman spectroscopy, and visible spectroscopy. Their stability was investigated and compared to particles capped with nonpolymerized ligands.

Introduction The intrinsic properties of inorganic colloidal nanoparticles make them very important candidates for cutting-edge applications in physical and biomedical sciences.1-3 In recent years, an increasingly intense research interest has been developed, regarding the behavior of nanoparticles in biological systems and optoelectronics.4-6 For example, nanocrystals are used to dope oligomers and liquid crystals to fabricate nanocomposites with novel optical, mechanical, and electronic properties.7 Moreover, strategically designed synthetic nanoparticles are employed in imaging, therapy, and in fundamental research to study their behavior in vitro and in vivo and to develop novel ways of tuning their properties.8-14 Because of the complexity of physical and biological systems and the urgency to fully realize the properties of nanocrystals in advanced applications, the demand for the fabrication of robust *To whom correspondence should be addressed. E-mail: a.kanaras@ soton.ac.uk. Fax: þ44 (0)23 8059 3910. Tel: þ44 (0)23 8059 2466. (1) Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Nano Today 2007, 2, 18. (2) Wang, Y.; Tang, Z.; Kotov, N. A. Nano Today 2005, 8, 20. (3) Milliron, D. J.; Gur, I.; Alivisatos, A. P. MRS Bull. 2005, 30, 41. (4) Sperling, R. A.; Gil, P. R.; Zhang, F.; Zanella, M.; Parak, W. J. Chem. Soc. Rev. 2008, 37, 1896. (5) Verma, A.; Uzun, O.; Hu, Y.; Han, H. S.; Watson, N.; Chen, S.; Irvine, D. J.; Stellacci, F. Nat. Mater. 2008, 7, 588. (6) You, C. C.; Chompoosor, A.; Rotello, V. M. Nano Today 2007, 2, 34. (7) Gur, I.; Fromer, N.; Chen, C. P.; Kanaras, A. G.; Alivisatos, A. P. Nano Lett. 2007, 7, 409. (8) Sepulveda, B.; Angelome, P. C.; Lechuga, L. M.; Liz-Marzan, L. M. Nano Today 2009, 4, 244. (9) Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Angew. Chem., Int. Ed. 2009, 48, 309. (10) Ashcroft, J. M.; Gu, W.; Zhang, T.; Hughes, S. M.; Hartman, K. B.; Hofmann, C.; Kanaras, A. G.; Kilcoyne, D. A.; Le Gros, M.; Yin, Y.; Alivisatos, A. P.; Larabell, C. A. Chem. Commun. 2008, 2471. (11) Nativo, P.; Prior, I. A.; Brust, M. ACS Nano 2008, 2, 1639. (12) Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W. Nat. Nanotechnol. 2008, 3, 145. (13) Henkel, A.; Jakab, A.; Brunklaus, G.; Sonnichsen, C. J. Phys. Chem. C 2009, 113, 2200. (14) Smith, A. M.; Mohs, A. M.; Nie, S. Nat. Nanotechnol. 2009, 4, 56. (15) Lin, Y. S.; Wu, S. H.; Hung, Y.; Chou, Y. H.; Chang, C.; Lin, M. L.; Tsai, C. P.; Mou, C. Y. Chem. Mater. 2006, 18, 5170. (16) Sukhorukov, G. B.; Rogach, A. L.; Garstka, M.; Springer, S.; Parak, W. J.; Mu~noz-Javier, A.; Kreft, O.; Skirtach, A. G.; Susha, A. S.; Ramaye, Y.; Palankar, R.; Winterhalter, M. Small 2007, 3, 944.

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and sophisticated multifunctional nanoparticles is growing rapidly.15,16 For instance, nanoparticles employed in biomedical applications should be stable in a large range of pH, ionic solutions, and ligand exchange reactions. A key parameter in the preparation of very stable colloidal nanoparticles is the selective choice of the molecules (in terms of chemical composition, polarity, charge, and hydrodynamic diameter) to cap the surface of nanocrystals. Additionally, it is essential to create a fine monolayer of ligands around the inorganic core that would prevent any aggregation. In recent years, a number of different types of molecules have been employed as capping agents for nanocrystals. Examples are oligonucleotides,17 peptides,18,19 lipids,20 polyethylene glycols,21-23 alkylthiols and mercaptoacids,24,25 silica shells,26 and other amphiphilic molecules such as poly(acrylic acid)27 and poly(maleic anhydride alt-1-tetradecene).28 The produced capped particles exhibit relatively good stability, but in many cases their preparation is extremely challenging. Furthermore, under stringent treatments (e.g., temperature and pH variations), particles may agglomerate or lose their functionality. To overcome these issues and construct preparatively undemanding and extremely stable colloidal nanoparticles (even under (17) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535. (18) Krpetic, Z.; Nativo, P.; Porta, F.; Brust, M. Bioconjugate Chem. 2009, 20, 619. (19) Levy, R. ChemBioChem 2006, 7, 1141. (20) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759. (21) Liu, Y.; Shipton, M. K.; Ryan, J.; Kaufman, E. D.; Franzen, S.; Feldheim, D. L. Anal. Chem. 2007, 79, 2221. (22) Kanaras, A. G.; Kamounah, F. S.; Schaumburg, K.; Kiely, C. J.; Brust, M. Chem. Commun. 2002, 2294. (23) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 12696. (24) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (25) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (26) Mine, E.; Yamada, A.; Kobayashi, Y.; Konno, M.; Liz-Marzan, L. M. J. Colloid Interface Sci. 2003, 264, 385. (27) Zhang, T.; Ge, J.; Hu, Y.; Yin, Y. Nano Lett. 2007, 7, 3203. (28) Di Corato, R.; Quarta, A.; Piacenza, P.; Ragusa, A.; Figuerola, A.; Buonsanti, R.; Cingolani, R.; Manna, L.; Pellegrino, T. J. Mater. Chem. 2008, 18, 1991.

Published on Web 01/15/2010

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Article colloidal crystal-templated “inverse opal” nanostructured gold films, as described elsewhere.30

Synthesis of Photocross-Linked DA-PEG-Coated Gold Nanoparticles. Gold nanoparticles of two different size ranges

Figure 1. The chemical structure of the capping ligand (A). A schematic illustration of photocross-linked DA-PEG ligands on the gold nanoparticle (B). The ethylene glycol unit with the terminal carboxylic group is colored blue.

harsh conditions and for a longer term), we developed a new type of coating based on ligands that can be attached to the particles as monomers and then photopolymerized to create a very stable photocross-linked ligand shell. The derived nanoparticles are soluble in water and stable in pH and temperature variations, ligand exchange reactions, and high ionic strength solutions. The capping ligand [46-mercapto-22,43-dioxo-3,6,9,12,15,18-hexaoxa21,44-diazahexatetraconta-31,33-diyn-1-oic acid, (DA-PEG), Figure 1] consists of a thiol group that binds to the gold nanoparticle surface, a hydrophobic part that contains an alkyl chain with a diacetylene group, and a hydrophilic part with an oligo-ethylene glycol chain and a terminal carboxylic group. The alkylthiol unit of DA-PEG provides a uniform hydrophobic layer, where carbon chains of DA-PEG monomers are closely packed due to van der Waals interactions. The hydrophilic unit ensures water solubility and the terminal carboxylic group offers a possibility of biomodification, such as conjugation of amine-terminated biomolecules via several coupling chemistry methods.29

Experimental Section Materials. All reagents were purchased from the following suppliers and used without further purification: sodium tetrachloroaurate (III) dihydrate, trisodium citrate, sodium phosphate monobasic, sodium phosphate dibasic, tetrakis(hydroxymethyl)phosphonium chloride (THPC), sodium hydroxide, potassium carbonate, mercaptoethanol, and methanol were purchased from Sigma-Aldrich. Monocarboxy (1-mercaptoundec-11-yl) hexaethylene glycol (PEG) and 46-mercapto-22,43-dioxo-3,6,9,12,15,18hexaoxa-21,44-diazahexatetraconta-31,33-diyn-1-oic acid were purchased from Prochimia Surfaces. Bis(p-sulfonatophenyl)phenyl phosphine dehydrate dipotassium salt (BSPP) was purchased from Strem-Chemicals, Inc. Milli-Q water was used in all experiments. Characterization Methods. TEM images were obtained with a Hitachi H7000 transmission electron microscope operating at a bias voltage of 75 kV. All samples were deposited on carbon film 400 mesh copper (50) grids purchased from Agar Scientific Ltd. UV-visible spectra of colloidal gold nanoparticles were collected using a Cary 300 Bio UV-vis spectrophotometer over the range from 400 to 800 nm. All Raman spectra were obtained using Renishaw Raman 2000 system equipped with a 515 nm He laser with a maximum of 3 mW power and a spectral resolution of 4 cm-1. A 50 objective was used. Spectra were recorded over the range from 400 to 3400 cm-1 with a maximum accumulation time of 10 s. Raman spectra were collected from solid DA-PEG (Supporting Information). SERS spectra of colloidal gold nanoparticles were collected from air-dried samples deposited on (29) Hermanson, G. T. Bioconjugate Techniques; Academic Press: London, U.K., 1996; p 169. (30) Abdelsalam, M. E.; Bartlett, P. N.; Baumberg, J. J.; Cintra, S.; Kelf, T. A.; Russell, A. E. Electrochem. Commun. 2005, 7, 740.

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(15 ( 1.5 and 3.25 ( 1.5 nm) were prepared. Larger particles were synthesized according to the well-established citrate reduction method31 and capped with bis(p-sulfonato-phenyl)phenyl phosphine dihydrate dipotassium salt, a phosphine derivative (BSPP),32,33 while smaller ones were prepared by the Duff method.34 The surface of the gold nanoparticles was capped with the DA-PEG monomers via thiol covalent bonding. Freshly prepared DA-PEG solution in methanol (5 mg/mL, 100 μL) was added to an aqueous solution of gold nanoparticles (5 mL, 5 nM) while stirring in the dark. After 10 min of stirring at room temperature, 0.2 M phosphate buffer solution (5 mL, 0.3 M NaCl, pH 8) was introduced. The reaction mixture was kept overnight at 4 °C in the dark to ensure fine coating of DA-PEG molecules. Larger (15 ( 1.5 nm) gold nanoparticles were purified from the excess of DA-PEG ligands by 3 centrifugation steps (16 400 rpm, 15 min) followed by decantation. Smaller, 3.25 ( 1.5 nm, particles were purified by 3 steps of ultrafiltration (10 k nanosep filters, 9000 rpm, 15 min). Supernatants, from each step of purification, were combined prior to thiol quantification. Particles were suspended in 0.1 M phosphate buffer (pH 8) and UV-irradiated for 15 min (254 nm light wavelength, 35 W lamp power, 10 cm from the UV lamp) in order to photocross-link DA-PEG monomers. Quantification of Thiols on Nanoparticles’ Surface. An average number of DA-PEG ligands per nanoparticle was estimated using the Ellman’s thiol quantification method.35 Quantification buffer (0.5 mL, 0.1 M sodium phosphate pH 8, 1 mM EDTA) was mixed with 10 μL of Ellman’s reagent [solid 5,50 dithiobis(2-nitrobenzoic acid) dissolved in 1 mL of quantification buffer]. Then, 50 μL of thiol containing solution (0.1-1 mM range) was introduced and mixed well. The reaction mixture was incubated for 15 min at room temperature. The maximum absorbance was measured at 412 nm and the thiol content was calculated from a calibration curve. Stability Tests. The stability of photocross-linked DA-PEGcoated large (sol. 1) and small (sol. 2) gold nanoparticles (15 ( 1.5 and 3.25 ( 1.5 nm, respectively) was tested under various conditions (pH, ligand exchange reactions, and temperature). Sets of gold nanoparticle solutions [nonpolymerized DA-PEG coated large (sol. 3) and small (sol. 4) particles, BSPP-coated large AuNPs (sol. 5), monocarboxy(1-mercaptoundec-11-yl) hexaethylene glycol (PEG)-capped large (sol. 6) and small (sol. 7) particles, and tetrakis(hydroxymethyl)phosphonium chloride (THPC)coated small particles (sol. 8)] were prepared and used as reference samples in order to assess the stability of all colloids. Dispersity of particles was tested in the pH ranging from 1.2 to 14. All solutions were incubated at room temperature. Stability of nanoparticles in high ionic strength solutions (2 M sodium chloride) was also monitored. The stability of colloidal nanoparticles against ligand-exchange reactions was investigated in the presence of thiol-containing ligands such as mercaptoethanol, dithiothreitol, cysteamine, cysteine, R-lipoic acid, and 11-amino-1undecanethiol. In a typical experiment, solutions (1), (3), (5), and (6) were incubated with 10% mercaptoethanol (1:1 water/MeOH) at room temperature and pH 3-9. The stability of solutions (1) and (2) was also tested at elevated temperatures. Nanoparticles were heated up to 100 °C and cooled down to room temperature in five repeated cycles. Low-temperature stability tests were performed by three freezing (-20 °C) and (31) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55. (32) Schmid, G. Chem. Rev. 1992, 92, 1709. (33) Loweth, C. J.; Caldwell, W. B.; Peng, X.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. 1999, 38, 1808. (34) Duff, D. G.; Baiker, A. Langmuir 1993, 9, 2310. (35) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

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thawing (room temperature) cycles. The optical properties of nanoparticles were monitored after each cycle to determine the degree of dispersion.

Results and Discussion DA-PEG-Coated Nanoparticles. Photopolymerization of diacetylene-containing molecules has been used in the past to create self-assembled layers on planar surfaces, and to form ribbons, vesicles and Langmuir-Blodgett films.36-39 The interest regarding these polymers is based on their dramatic color change, between red and blue, that is dependent on the alignment of the ligands, influenced by various external factors (e.g., stretching, pH variation, and temperature). These attractive optical properties make polydiacetylenes promising candidates for biosensing applications.39 Research regarding the use of diacetylene-containing ligands as capping agents for gold nanoparticles is very limited and only appeared in the literature in recent years,40-42 where, for example, authors exploited the capacity to synthesize gold nanoparticles in organic solvents using thiolalkyl-diacetylene molecules.43 In this article, we report the use of a strategically designed diacetylene-containing molecule as a stabilizing agent for watersoluble spherical nanoparticles. DA-PEGs can be packed on the surface of colloids and photopolymerized by UV-irradiation. The robustness of this cross-linked layer is strongly dependent on the degree of polymerization of diacetylene groups on the nanoparticle surface. In agreement with the literature, to achieve successful polymerization, the favorable distance between two neighboring diacetylene groups is 5 A˚.44 Given the fcc lattice parameters and the fact that the radius of a gold atom is about 1.441 A˚, the distance between two diacetylene groups of neighboring gold atoms should fall into the optimum polymerization range. Therefore, to create a fine polymeric shell around nanoparticles, high loading of monomers, chemisorbed on the nanoparticle surface, is absolutely necessary. In our experiments, fine packing between neighboring monomers of DA-PEG is guaranteed by the long carbon chain, present in the hydrophobic part of the capping ligand. Nevertheless, to verify the level of loading, we have quantified the average number of monomers attached to each nanoparticle following Ellman’s method (see Experimental Section). The results of several independent measurements performed on large and small particles are shown in Table 1. Overall, it was calculated that an average of 2073 ( 250 DA-PEGs were chemisorbed on the surface of each large (15 ( 1.5 nm) particle, while an average of 67 ( 22 was found per each small (3.25 ( 1.5 nm) nanoparticle. These results show well above 50% typical surface coverage, which provides a sufficiently high packing of monomers. Characterization of Cross-Linked Ligands on the Surface of Nanoparticles by SERS. We have used Raman and surfaceenhanced Raman spectroscopy to evaluate the photopolymerization process of the monomers attached to the gold nanoparticles. (36) Stanish, I.; Santos, J. P.; Singh, A. J. Am. Chem. Soc. 2001, 123, 1008. (37) Song, J.; Cheng, Q.; Stevens, R. C. Chem. Phys. Lipids 2002, 114, 203. (38) Ahn, D. J.; Kim, J. M. Acc. Chem. Res. 2008, 41, 805. (39) Jung, Y. K.; Kim, T. W.; Kim, J.; Kim, J. M.; Park, H. G. Adv. Funct. Mater. 2008, 18, 701. (40) Demartini, A.; Alloisio, M.; Cuniberti, C.; Dellepiane, G.; Jadhav, S. A.; Thea, S.; Giorgetti, E.; Gellini, C.; Muniz-Miranda, M. J. Phys. Chem. C 2009, 113, 19475. (41) Alloisio, M.; Demartini, A.; Cuniberti, C.; Miranda-Muniz, M.; Giorgetti, E.; Giusti, A.; Dellepiane, G. Phys. Chem. Chem. Phys. 2008, 10, 2214. (42) Alloisio, M.; Demartini, A.; Cuniberti, C.; Dellepiane, G.; Muniz-Miranda, M.; Giorgetti, E. Vib. Spectrosc. 2008, 48, 53. (43) Alloisio, M.; Demartini, A.; Cuniberti, C.; Petrillo, G.; Thea, S.; Giorgetti, E.; Giusti, A.; Dellepiane, G. J. Phys. Chem. C 2007, 111, 345. (44) Kricheldorf, H. R.; Schwarz, G. Handbook of Polymer Synthesis; Marcel Dekker Inc.: New York, 1992; p 1629.

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Table 1. The Number of DA-PEG Ligands Per One Gold Nanoparticle, Estimated Using Ellman’s Quantification Method estimated number of DA-PEGs per particle 15 ( 1.5 nm particle 2027 2118 1944 1945 2330

3.25 ( 1.5 nm particle 82 37 64 72 80

Figure 2. A schematic illustration of the photopolymerization between diacetylene groups (I). SERS spectra of DA-PEG coated large (II) and small (III) gold nanoparticles before (red line) and after UV-irradiation (black line), where (a) [2150 cm-1] represents the stretching vibration of carbon-carbon triple bonds, (b) [2257 cm-1] a feature band of the diacetylene group and (c) [1556 cm-1] the stretching vibration of carbon-carbon double bond.

The Raman spectrum of the free DA-PEG (see Supporting Information Figure S1) and the SERS spectra of the colloidal solutions (1) and (3) (see Figure 2) clearly show the expected fingerprints of the free diacetylene group. During UV-irradiation, the diacetylene group undergoes 1,4-topochemical polymerization leading to a poly(enyne) structure, which is trans-selective and clearly presented in the SERS spectra of the DA-PEG coated particles (see Figure 2). The SERS spectrum of large photopolymerized particles shows no characteristic feature of the diacetylene stretch. Moreover, a new peak in the spectral region of 1556 cm-1, which is attributed to the double carbon-carbon bond vibrations, is present. In the case of small polymerized nanoparticles, a peak ascribed to the double-bond vibrations is Langmuir 2010, 26(10), 7072–7077

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Figure 3. TEM images and histograms representing the size distribution of DA-PEG coated gold nanoparticles after UV-irradiation. (sol. 1)

15 ( 1.5 nm; (sol. 2) 3.25 ( 1.5 nm, and before UV-irradiation (sol. 3) 15 ( 1.5 nm; (sol. 4) 3.25 ( 1.5 nm. Scale bars are 200 nm for (1) and (3) and 100 nm for (2) and (4).

also evident. Two smaller peaks in the triple-bond vibrations region are shown, indicating a degree of ambiguous packing of the molecules on the surface of small particles and a possible partial polymerization of the monomers. One may hypothesize that the unexpected SERS spectrum of UV-irradiated 3.25 nm nanoparticles derives from the high-surface curvature of small gold clusters. Indeed, such curvature might have a strong effect on the absolute distances between two neighboring diacetylene groups, preventing complete polymerization of the organic shell. However, the small particles are very robust and show no sign of aggregation under wide pH range and high salt concentration (see Supporting Information Figure S2). Characterization of Nanoparticles by TEM and Visible Spectroscopy. The size of nonpolymerized and polymerized nanoparticles was determined by transmission electron microscopy (TEM). Figure 3 shows TEM images and size distribution histograms of large and small UV-irradiated and nonpolymerized nanoparticles. Images show that the particles are dispersed with no sign of aggregation before and after photopolymerization. Histograms illustrate a similar average nanoparticles’ diameter before and after photocross-linking, indicating that the inorganic core remained stable under the irradiation conditions used in our experiments. After surface functionalization of large nanoparticles with DA-PEG monomers, the position of the plasmon band was slightly red shifted from 522 to 526 nm, demonstrating a small change in the refractive index (Figure 4) of the nanocrystals. Visible spectra of both small and large particles represent dispersed colloids of typical sizes. As expected from Mie’s theory45 the plasmon band of small particles occurs as a shoulder deriving from the electron surface scattering effect.46 Photopolymerized nanoparticles exhibit a slight sharpness of the absorbance peak with no significant shifting indicating that the metallic core has not been affected by the UV-irradiation (Figure 4). It is also worth highlighting here that DA-PEGcapped nanoparticles do not show any feature that can be attributed to the presence of the diacetylene-derivative or its polymeric structure. Stability Tests of DA-PEG-Coated Nanoparticles. To assess the stability of polymer-coated particles, solutions (1) (45) Mie, G. Ann. Phys. (Leipzig) 1908, 25, 377. (46) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters. Springer Series in Materials Science; Springer: Berlin, 1995.

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Figure 4. UV-visible spectra of colloidal gold nanoparticles. 15 ( 1.5 nm (I) and 3.25 nm ( 1.5 nm (II). DA-PEG-coated nanoparticles before and after photopolymerization are shown as a dashed and full line, respectively. The plasmon band of BSPP (spectrum I) or THPC (spectrum II) coated nanoparticles is shown as a dotted line. An insert (spectrum I) represents a magnification of 500-550 nm spectral region.

and (2) were exposed to various chemical conditions. In each experiment, their robustness was compared to monomer-coated nanoparticles. The polymeric nanoparticles did not aggregate in a high ionic strength environment and showed excellent stability in the presence of a high concentration of large proteins (e.g., BSA) and changes in the solvent polarity (stable in mixtures of water and isopropanol). In comparison to nonpolymerized, polyethylene glycol derivative-coated nanoparticles, polymeric DA-PEG colloids present remarkable stability in the pH range from 1.2 to 14 and temperatures as high as 100 °C. Moreover, larger polymer-coated nanoparticles show exceptional robustness against ligand-exchange reactions with thiol-terminated ligands (see Figures 5 and 7). To use colloidal nanoparticles in advanced biomedical applications, particles must be resistant to aggregation caused by pH variation or a high ionic strength environment. We have tested the stability of polymeric DA-PEG nanoparticles over a wide pH range. As an example, Figure 6 shows a colorimetric test of large colloids [solutions (1), (3), (5), and (6)] and associated visible spectra. It is evident that at pH values lower than 2, the only stable colloids are the DA-PEG coated. This notable stability, even in DOI: 10.1021/la9044013

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Figure 5. Digital pictures and associated UV-visible spectra of gold nanoparticles (15 ( 1.5 nm) at various pH (left) and also in presence of 10% mercaptoethanol (right). Nanoparticles coated with photopolymerized DA-PEG (sol. 1). Nanoparticles coated with monomers of DA-PEG (sol. 3). Nanoparticles coated with BSPP (sol. 5). Nanoparticles coated with (monocarboxy(1-mercaptoundec-11-yl) hexaethylene glycol) (sol. 6). [cl (control)] - nanoparticles in water.

Figure 6. UV-visible spectra of 3.25 ( 1.5 nm gold nanoparticles before (I) and after (II) incubation with 0.1 M of 11-mercapto11-undecanethiol. Nanoparticles coated with photopolymerized DA-PEG (sol. 2). Nanoparticles coated with monomers of DAPEG (sol. 4). Nanoparticles coated with (monocarboxy (1-mercaptoundec-11-yl) hexaetylene glycol) (sol. 7). Nanoparticles coated with THPC (sol. 8).

the case of monomer coated DA-PEG nanoparticles, may be a result of enhanced packing efficiency of monomers on the nanoparticle surface. Indeed, due to the presence of the triplebonds and long carbon chains, the van der Waals interactions between ligands can be maximized and improved assemblage of monomers on the nanoparticle surface could be achieved (this is in agreement with the high-surface coverage estimated earlier). Furthermore, at pH lower than 1.6, where destabilization of monomeric particles is evident, polymeric DA-PEG particles do not show any sign of aggregation (see visible spectra, Figure 5). In fact, small and large polymer-coated particles (see Figure 5 and Supporting Information Figure S2) are extremely robust even at lower pH (down to 1.2) and in a concentration of sodium chloride up to 2 M. Indeed, the robustness of the polymeric shell enhances the stability of nanoparticles, which now seems to be less dependent on the variations in the surface charge. In many cases, colloidal nanoparticles should remain stable and retain their functionality in thiol-containing environments. 7076 DOI: 10.1021/la9044013

Figure 7. Temperature stability test of polymerized DA-PEG coated gold nanoparticles. UV-visible spectra collected after three repeated freezing and thawing cycles (left) of 15 ( 1.5 nm (sol. 1) and 3.25 ( 1.5 nm (sol. 2) nanoparticles. Five heating/cooling cycles (right) of 15 ( 1.5 nm (sol. 1) and 3.25 ( 1.5 nm (sol. 3) nanoparticles.

For this reason, we have tested the stability of the polymeric nanoparticles using a variety of thiols (e.g., mercaptoethanol, dithiothreitol, cysteamine, cysteine, R-lipoic acid, and 11-amino1-undecanethiol) and by regulating the pH of each solution. Figure 5 shows a colorimetric test of solutions (1), (3), (5), and (6) and associated visible spectra of particles incubated for 72 h in Langmuir 2010, 26(10), 7072–7077

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10% mercaptoethanol solution at a pH ranging from 3 to 9. Monomer-coated DA-PEG particles and other monomer thiolated-PEG-coated particles aggregate under these stringent conditions, whereas polymer-coated DA-PEG nanoparticles stay unaltered at all tested pH values. A firm polymeric shell surrounding the large gold nanoparticles may explain such enhanced stability against ligand-exchange reactions. Small polymer-coated particles, however, do not show similar robustness, upon treatment with 10% mercaptoethanol. As indicated earlier, this is probably due to only a partial polymerization of the monomers on the highly curved nanoparticle surface. However, under milder conditions (0.1 M 11-amino1-undecanethiol, (see Figure 6) small photopolymerized particles are more stable than the nonpolymerized ones. Advanced applications, especially biological, require excellent temperature stability of colloids. For example, biomolecules are often kept frozen to preserve them in a native form for a longterm. Therefore, stability of colloids loaded with biomolecules (e.g., peptides, DNA), against repeated freezing and thawing cycles is vital. The ability to repeatedly heat colloidal nanoparticles at elevated temperatures would also be beneficial. For example, particles that are stable in these conditions could be used in techniques such as the polymerase chain reaction. For this reason, we have monitored the stability of colloids in repeated heating/cooling and freezing/thawing cycles using visible spectroscopy (see Figure 7). We have found that larger polymeric DA-PEG particles show no shift in the position of their plasmon band even after five heating cycles up to 100 °C. Both, large and small nanoparticles can be also frozen/defrosted at least 3 times. A partial polymerization of DA-PEG ligands on the highly curved small nanoparticles or ripening could explain their spectral

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variation at high temperatures. Nonetheless, larger polymeric DA-PEG-coated particles present a clear opportunity for many applications that comprise temperature variations, including freezing/thawing and heating/cooling cycles.

Conclusions We have successfully prepared and characterized a new family of robust gold nanoparticles based on a strategically designed type of ligand that can be readily photopolymerized on the nanoparticle surface. The remarkable stability of polymeric DA-PEG particles in a vast pH range and high ionic strength solutions as well as in temperature variations and ligand-exchange reactions position them as potential candidates for many applications. It is anticipated that the new polymeric DA-PEG-coated nanoparticles could have a broad use in biomedical sciences. It is possible to expand this technology to other popular types of inorganic nanoparticles, and such experiments are in place within our group. Acknowledgment. We thank Professor Phil Bartlett and Mr. Robert Johnson for the SERS and Raman measurements and related discussions. The authors thank the University of Southampton and the Royal Society for financial support of this project. D.B. thanks EPSRC and the School of Physics and Astronomy for a financial support. A.G.K. thanks the Research Council UK (RCUK) for a fellowship. Supporting Information Available: Raman spectrum of DA-PEG ligand and pH stability tests of small gold nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

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