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Synthesis, Characterization, and Antifouling Potential of Functionalized Copper Nanoparticles Kelechi C. Anyaogu, Andrei V. Fedorov, and Douglas C. Neckers* Center for Photochemical Sciences, Bowling Green State UniVersity, Bowling Green, Ohio 43403 ReceiVed January 11, 2008 The synthesis, characterization, and antimicrobial properties of functionalized copper nanoparticle/polymer composites are reported. Copper nanoparticles (Cu NPs) are stabilized by surface attachment of the acrylic functionality that can be copolymerized with other acrylic monomers, thus, becoming an integral part of the polymer backbone. Biological experiments show that Cu NP/polymer composites exhibit antimicrobial activity similar to that of conventional copperbased biocides. Atomic absorption spectroscopy shows the smallest amount of copper ions leaching from chemically bound acrylated Cu NPs compared to the nonfunctionalized biocides. These composites have a strong potential for use in antibacterial or marine antifouling coatings.
Introduction In recent years, nanoparticle/polymer composites have become important owing to their small size and large surface area, and because they exhibit unique properties not seen in bulk materials. As a result, nanoparticles (NPs) have useful applications in photovoltaic cells, optical and biological sensors, conductive materials, and coating formulations.1 The antibacterial properties of copper, silver, and zinc have been widely utilized in advanced coating technologies, such as the design of materials for biomedical devices, hospital equipment, food processing and storage equipment, household materials, and antifouling paints.2 There have also been several reports on the antimicrobial activities of metal NP/polymer composites.3-9 However, there are still challenges such as the instability of the NPs, control of their size and shape, uniform dispersity in a matrix, and control of the release rate.10 Biofouling prevention remains a major challenge, and there is a need for antifouling systems that exhibit minimal/no eco* To whom correspondence should be addressed. E-mail: neckers@ photo.bgsu.edu. Telephone: 419-372-2034. Fax: 419-372-0366. (1) (a) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27. (b) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293. (c) Hasobe, T.; Imahori, H.; Kamat, P. V.; Ahn, T. K.; Kim, S. K.; Kim, D.; Fujimoto, A.; Hirakawa, T.; Fukuzumi, S. J. Am. Chem. Soc. 2005, 127, 1216. (2) (a) Mann, E. L.; Nathan, A.; James, W. M.; Sallie, W. C. Limnol. Oceanogr. 2002, 47, 976. (b) Avery, S. V.; Howlett, N. G.; Radice, S. A. Appl. EnViron. Microbiol. 1996, 62, 3960. (c) Antonietta, Z. M.; Stefania, Z.; Rebecca, P.; Riccardo, B. J. Inorg. Biochem. 1996, 35, 291. (d) Stoimenov, P. K.; Klinger, R. L.; Marchin, G. L.; Klabunde, K. J. Langmuir 2002, 18, 6679. (e) Yoichi, Y.; Hiroshi, Y.; Chikara, K.; Kei, I. Prog. Org. Coat. 2001, 42, 150. (3) Sambhy, V.; MacBride, M. M.; Peterson, B. R.; Sen, A. J. Am. Chem. Soc. 2006, 128, 9798. (4) Ho, C. H.; Tobis, J.; Sprich, C.; Thomann, R.; Tiller, J. C. AdV. Mater. 2004, 16, 957. (5) (a) Cioffi, N.; Torsi, L.; Ditaranto, N.; Sabbatini, L.; Zambonin, P. G.; Tantillo, G.; Ghibelli, L.; D’Alessio, M.; Bleve-Zacheo, T.; Traversa, E. Appl. Phys. Lett. 2004, 85, 2417. (b) Cioffi, N.; Torsi, L.; Ditaranto, N.; Tantillo, G.; Ghibelli, L.; Sabbatini, L.; Bleve-Zacheo, T.; D’Alessio, M.; Zambonin, P. G.; Traversa, E. Chem. Mater. 2005, 17, 5255. (6) Trapalis, C. C.; Kokkoris, M.; Perdikakis, G.; Kordas, G. J. Sol-Gel Sci. Technol. 2003, 26, 1213. (7) Esteban-Cubillo, A.; Pecharroma´n, C.; Aguilar, E.; Santare´n, J.; Moya, J. S. J. Mater. Sci. 2006, 41, 5208. (8) Cioffi, N.; Ditaranto, N.; Torsi, L.; Picca, R. A.; De Giglio, E.; Sabbatini, L.; Novello, L.; Tantillo, G.; Bleve-Zacheo, T.; Zambonin, P. G. Anal. Bioanal. Chem. 2005, 382, 1912. (9) (a) Gu, C.; Sun, B.; Wu, W.; Wang, F.; Zhu, M. Macromol. Symp. 2007, 254, 160. (b) Ramstedt, M.; Cheng, N.; Azzaroni, O.; Mossialos, D.; Mathieu, H. J.; Huck, W. T. S. Langmuir 2007, 23, 3314. (10) Rong, M. Z.; Zhang, M. Q.; Ruan, W. H. Mater. Sci. Technol. 2006, 22, 787.
toxicity, active durability, and easy affordability.3 Copper and its oxides are common biocides in a large number of commercial antifouling marine paints. Incorporation of Cu-based biocides is mainly achieved by mechanical doping into paint matrixes. Recent reports have shown that Cu NP/polymer composites exhibit antifungal and antibacterial properties.5-9 However, these systems are not optimum because of the poor control of the leaching of the active biocide. This is crucial considering the growing concern on the amount of potentially toxic metals, particularly copper, leached to the marine environment from diverse sources that include antifouling paints.11 This paper reports the synthesis and characterization of Cu NPs functionalized with an acrylic group. These stabilized NPs are less prone to aggregation within a matrix. The acrylic functionality of the NPs can be copolymerized with other acrylates.12 Through this, the Cu NPs become chemically incorporated into a polymer matrix, providing a tighter control of the release of the particles. The biological activity of the nanocomposites was tested against selected microorganisms that are common constituents of the biofilm formed during the early stages of marine biofouling.13 Experimental Section Materials. 6-Mercapto-1-hexanol (97%) was acquired from Fluka. Copper(II) chloride (99.99%), 1-hexanethiol (95%), 1-decanethiol (96%), 1-dodecanethiol (98%), 12-bromo-1-dodecanol (99%), potassium thioacetate (98%), sodium thiomethoxide (95%), sodium borohydride (98%), acryloyl chloride (96%), and propionyl chloride (96%) were all acquired from Aldrich. Bis(2,4,6-trimethylbenzoyl)phenylphosphineoxide (Irgacure 819) was obtained from Ciba Specialty Chemicals, Inc. 2-Phenoxyethyl acrylate (SR 339) was obtained from Sartomer. All reagents and solvents were used without further purification. Methods. Synthesis of 12-Thioacetyldodecan-1-ol. The synthesis of 12-mercapto-1-dodecanol was carried out according to previously reported procedures14 with modifications. In a 100 mL round-bottom flask, 12-bromo-1-dodecanol (0.137 g, 0.5 mmol) was dissolved in 30 mL of dimethylformamide (DMF) and potassium thioacetate (3 (11) (a) Anderson, C.; Atlar, M.; Callow, M.; Candries, M.; Ine, A.; Townsin, R. L. J. Mar. Des. Oper. 2003, 4, 11. (b) Schiff, K.; Brown, J.; Diehl, D.; Greenstein, D. Mar. Pollut. Bull. 2007, 54, 322. (c) Schiff, K.; Diehl, D.; Valkirs, A. Mar. Pollut. Bull. 2004, 48, 371. (12) Park, J. J.; Jeong, E. J.; Lee, S. Y. U.S. Patent 0,253,536, December 16, 2004. (13) Clarkson, N. AdV. Mar. Biotechnol. 1999, 3, 87.
10.1021/la800102f CCC: $40.75 © 2008 American Chemical Society Published on Web 03/15/2008
Functionalized Cu NPs as a Biocide equiv) was added. The yellowish mixture was stirred at 60 °C overnight under argon, after which the mixture was cooled to room temperature followed by DMF removal under reduced pressure. The crude product was dissolved in EtOAc (20 mL) followed by addition of H2O (50 mL). The mixture was added to a separatory funnel containing EtOAc (50 mL) and H2O (150 mL). The organic layer was washed twice with H2O and then with saturated aqueous NaHCO3 and brine. The organic layer was later dried over anhydrous Na2SO4 and concentrated to yield white crystals of 12-thioacetyldodecan-1-ol (0.127 g, 98%). 1H NMR (300 MHz, CDCl3): δ (ppm) 3.62 (t, 2H, J ) 15 Hz), 2.86 (t, 2H, J ) 15 Hz), 2.31 (s, 3H), 2.81 (s, 1H), 1.55 (m, 4H), 1.3 (m, 16H). 13C NMR (300 MHz, CDCl3): δ (ppm) 196.00, 63.00, 32.81, 30.62, 29.60, 29.59, 29.56, 29.50, 29.48, 29.40, 29.19, 29.10, 28.80, 25.75. Synthesis of 12-Mercapto-1-dodecanol. A solution of 12-thioacetyldodecan-1-ol (76 mg, 0.3 mmol) in 50 mL of MeOH and a solution of sodium thiomethoxide (77 mg, 1.1 mmol) in 50 mL of MeOH were purged with argon for 60 min. The two solutions were then combined, and the reaction was stirred for 1 h. This was followed by quenching with 1 M NH4Cl solution (100 mL). The resulting slurry was extracted twice with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure to produce a clear liquid (0.061 g, 93%). 1H NMR (300 MHz, CDCl3): δ (ppm) 3.58 (t, 2H, J ) 12 Hz), 2.44 (q, 2H, J ) 9 Hz), 1.5 (m, 4H), 1.2 (m, 17H). 13C NMR (300 MHz, CDCl3): δ (ppm) 63.00, 34.10, 32.91, 29.61, 29.59, 29.41, 29.22, 29.05, 28.52, 28.35, 25.76, 24.65. IR: υ 3426, 3022, 2942, 2552 cm-1. Preparation of Thiol Functionalized Copper Nanoparticles. The method used to functionalize the copper nanoparticles was a modification of Brust’s procedure.15 Copper(II) chloride (1.2 g, 9 mmol) was first dissolved in 200 mL of methanol. To this solution was added a solution of the thiol alcohol (18 mmol) in methanol (20 mL), and the mixture was stirred for 30 min under argon followed by addition of glacial acetic acid (160 µL) with stirring. After that, 5 mL of a 0.5 M solution of sodium borohydride (NaBH4) was added dropwise with constant stirring. The brownish reaction mixture was allowed to stir for 4 h. After removal of the solvent, the resulting residue was dispersed in ethanol and washed three times (centrifugation at 6000 rpm, 15 min, Sorvall RC 5C Plus centrifuge) to remove unreacted thiol ligands. The nanoparticles were then vacuumdried to yield a dark brown powder. Other alkanethiol modified copper nanoparticles were prepared following reported procedures.15c For convenience, the prepared Cu NPs are designated as CuC6 for hexanethiol-capped (Cu NP-S-(CH2)5-CH3), CuC10 for decanethiol-capped (Cu NP-S-(CH2)9-CH3), CuC12 for dodecanethiol-capped (Cu NP-S-(CH2)11-CH3), CuC6OH for mercaptohexanol-capped (Cu NP-S-(CH2)6-OH), CuC6Ac (Cu NPS-(CH2)6-O-(CO)-CHdCH2)/CuC12Ac (Cu NP-S-(CH2)12O-(CO)-CHdCH2) for acrylated, and CuC6Pr (Cu NP-S(CH2)6-O-(CO)-CH2-CH3) for propionated. Preparation of Acrylic Functionalized Copper Nanoparticles. Mercaptoalcohol-capped nanoparticles (100 mg) were dispersed in dichloromethane (200 mL) and stirred under argon for 30 min. To the resulting suspension was added 1.7 mL of triethylamine, and it was stirred for 15 min. Subsequently, 1 mL of acryloyl chloride was added dropwise, and the reaction mixture was allowed to stir overnight under inert atmosphere. The mixture was then washed twice with 2 M ammonium hydroxide solution and water. The organic layer was washed twice with 1.8 M sodium bicarbonate solution. After the removal of the solvent, the brownish-orange residue was redispersed in ethanol and washed two times (centrifugation at 6000 rpm, 15 min, Sorvall RC 5C Plus centrifuge). The acrylated copper nanoparticles were then vacuum-dried. (14) (a) Wijtmans, M.; Rosenthal, S. J.; Zwanenburg, B.; Porter, N. A. J. Am. Chem. Soc. 2006, 128, 11720. (b) Paulini, R.; Frankamp, B. L.; Rotello, V. M. Langmuir 2002, 18, 2368. (c) Robinson, A.; Fang, J.; Chou, P.; Liao, K.; Chu, R.; Lee, S. ChemBioChem 2005, 6, 1899. (15) (a) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Chem. Soc., Chem. Commun. 1995, 1655. (b) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Nano Lett. 2002, 2, 3. (c) Ang, T. P.; Wee, T. S. A.; Chin, W. S. J. Phys. Chem. B 2004, 108, 11001.
Langmuir, Vol. 24, No. 8, 2008 4341 Solution Photopolymerization of Acrylic Functionalized Copper Nanoparticles. The acrylated Cu NPs were subjected to radical polymerization using bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide (Irgacure 819). Owing to the poor solubility of the nanoparticles,16-18 they were ultrasonically dispersed in toluene (or benzene-d6). The resulting suspension was filtered through a membrane filter (PTFE 0.45 µm), and the particles were washed several times with CHCl3 to remove any free or desorbed ligands. They were then redispersed in toluene and filtered to yield a solution of the nanoparticles. This was followed by dissolving the photoinitiator (0.01%) in the solution. A RMR-600 Rayonet photochemical reactor equipped with seven lamps (λ ) 350 nm) was used for irradiation. Preparation of Copper Nanoparticle/Polymer Composites. The copper nanoparticles (at designated wt %) were uniformly dispersed in 2-phenoxyethyl acrylate by ultrasonic treatment. This acrylic monomer was chosen because of its low volatility, good adhesion properties, and wide applicability.19 Irgacure 819 (0.2wt %) was used as a photoinitiator. Formulations were handled in the dark until use. A Teflon mold (7 mm diameter × 7 mm thick) was used to cast the prepared formulations. This was followed by irradiation using a 395 UV LED light source (UV Process Supplies Inc.; output: 29.6 mW cm-2 at 2 cm working distance) for a given period of time. A blanket of N2 gas was applied to eliminate excess oxygen that can inhibit polymerization.20 The tack-free polymer pellets were then used for biological and analytical experiments. Characterization. Transmission electron microscopy (TEM) was performed using a ZEISS EM10 transmission electron microscope operating at 80 kV. NP suspensions in toluene were dropcast onto 300-mesh copper grids coated with Formvar and carbon and airdried before viewing. Size and morphological analysis of the NPs was done manually on no less than 200 particles. A diffraction grating replica was used to calibrate the TEM images. Fourier transform infrared (FTIR) spectra were recorded on a ThermoNicolet IR200 spectrometer to confirm the attachment of the thiol ligand and identify the acrylic functionality. The spectra of mercaptoalcohol-capped Cu NPs and the acrylated species were recorded from KBr pellets prepared by mixing the nanoparticles with KBr (Aldrich, 99%) in a 1:100 (wt/wt) ratio. UV-vis spectra were recorded on a Shimadzu MultiSpec-1501 spectrometer. 1H NMR spectra were recorded using a 300 MHz Bruker Avance 300 spectrometer. Solid state 13C NMR was performed using a 400 MHz Varian Unity Plus system. Powder X-ray diffraction was performed on a Scintag XDS-2000 diffractometer operating at 40 kV and 40 mA. Biological Activity Tests. Strains of green algae, cyanobacteria, and diatoms were used as test organisms. A freshwater green algal representative (Chlamydomonas sp. strain CD1 Red) was obtained from the collection of Professor G. S. Bullerjahn (BGSU), whereas cyanobacterial strains Synechocystis sp. PCC 6803 (freshwater) and Synechococcus sp. PCC 7002 (marine) were obtained from the Pasteur Culture Collection. The marine diatom Phaeodactylum tricornutum CCMP 1327 was obtained from the National Center for Culture of Marine Phytoplankton. Media Preparation and Cell Growth Measurements. The freshwater strains were cultured in BG-11 medium,21 whereas marine strains were grown in Medium A22 or f/2 seawater.23 Cultures were maintained on a rotary shaker (120 rpm) at 25 °C for 5 days and continuously illuminated with white fluorescent light with a constant light intensity of 40 µmol quanta m-2 s-1 (Biospherical Instruments Inc., 001257FB). For the cell growth measurements, aliquots (1.0 mL) of the cultures were poured into 100 mL Erlenmeyer flasks (16) Zhang, J.; Whitesell, J. K.; Fox, M. A. Chem. Mater. 2001, 13, 2323. (17) Chen, S.; Sommers, J. M. J. Phys. Chem. B 2001, 105, 8816. (18) Chen, S.; Huang, K.; Stearns, J. A. Chem. Mater. 2000, 12, 540. (19) Sartomer Company, Inc., Exton, PA. (20) Hoyle, C. E. Technical Conference Proceedings, UV & EB Technology Expo & Conference; RadTech International North America: Chevy Chase, MD, 2004; pp. 892-899. (21) Allen, M. M. J. Phycol. 1968, 4, 1. (22) Stevens, S. E., Jr.; Patterson, C. O. P.; Myers, J. J. Phycol. 1973, 9, 427. (23) Guillard, R. R. L.; Ryther, J. H. Can. J. Microbiol. 1962, 8, 229.
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Figure 1. Preparation of acrylic functionalized copper nanoparticles. for each strain were taken in triplicate, and the experiments were repeated three times. The controls and treated cultures were grown under the same conditions of temperature, photoperiod, and agitation. Atomic Absorption Spectroscopy (AAS). Analysis of copper release from the composites was carried out using a Buck Instruments 210VGP spectrophotometer (det. limit: 0.02 mg/L) equipped with a copper cathode lamp and an open flame furnace. Solutions of CuSO4 were prepared in doubly distilled water in acid-rinsed (10% HCl) Teflon flasks as standards for calibration. To monitor leaching, the Cu NP/polymer composite pellets were placed in acid-rinsed polycarbonate flasks containing 50 mL of doubly distilled water on a rotary shaker (120rpm) and maintained under the same conditions used for the cell growth experiments. Small samples for Cu analysis were removed at 24 h intervals. All manipulations to monitor Cu leaching were conducted in a laminar flow hood to minimize contamination.
Results and Discussion
Figure 2. FTIR spectra of (a) mercaptohexanol-capped copper nanoparticles (dashed line) and free mercaptohexanol ligand (solid line), and (b) acrylated copper nanoparticles. containing 50 mL of sterilized culture medium. The Cu NP/polymer composite pellets were immersed into the flasks containing the growing cells at the start of the culture period. Cell growth of the cyanobacteria and green algae was correlated with absorbance measured at 24 h intervals using a Spectronic 20 Genesis spectrophotometer. The optimal wavelength for monitoring the culture growth was 680 nm.24,25 The cell growth of the diatoms was monitored daily by measuring in ViVo chlorophyll fluorescence (λex ) 436 nm; λem ) 680 nm) using a Turner TD-700 fluorometer. Measurements
The strategy employed in the functionalization of copper nanoparticles is similar to that developed by Brust et al. for gold NPs.15a First, a self-assembled monolayer (SAM) of thiol alcohol (6-mercapto-1-hexanol and 12-mercapto-1-dodecanol) on spherically shaped Cu NPs was prepared by reducing CuCl2 using NaBH4. This was followed by esterification of the -OH functionality with acryloyl chloride to yield the acrylated Cu NPs (Figure 1). The absence of the S-H stretching mode at 2560 cm-1 in the FTIR spectrum of the modified nanoparticles (Figure 2a) provides strong evidence for the attachment of the thiol ligand onto the surface of the NPs and formation of SAMs by the thiol functionality.15a,c The spectrum of the esterified nanoclusters shows complete disappearance of the -OH group and appearances of a carbonyl stretch at ∼1730 cm-1 and characteristic acrylic wag at 811 cm-1 (Figure 2b). TEM images do not show a significant change in size or shape of the nanoparticles after coupling the acrylic functionality (Figure 3). The particles were also characterized using NMR spectroscopy. 1H NMR spectra of the nanoparticles showed some broadened
Figure 3. Transmission electron microscopy images of the prepared NPs. Inset displays the typical size distribution histogram of the particles. The NPs fall mainly within the 5-15 nm size range and are spherical in shape. Scale bar represents 40 nm.
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Figure 4. Powder X-ray diffractograms of (a) mercaptohexane- and mercaptohexanol-capped Cu NPs and (b) acrylated Cu NPs. Insets show the selected area electron diffraction (SAED; TEM) images of the corresponding particles. The acrylated species display less degree of order (symmetry) of the crystal lattice.
peaks and changes in chemical shifts compared to the free thiol molecules (see the Supporting Information). This is typical for chemisorbed functional groups on metal NP surfaces.1a, 16, 26-28 Figure 4 shows the powder X-ray diffractograms of the copper nanoparticles. The thiol alkyl-capped Cu NPs display low angle Bragg peaks, similar to those previously reported.15c,29,30 These peaks can be indexed to face-centered cubic unit cells, and such (24) Ma, J.; Xu, L.; Wang, S. Weed Sci. 2002, 50, 555. (25) Bogdanova, A.; Piunova, V.; Berger, D.; Fedorov, A. V.; Neckers, D. C. Biomacromolecules 2007, 8, 439. (26) Terrill, R. H.; Postlethwaite, T. A.; Chen, C.; Poon, C.; Terzis, A.; Chen, A.; Hutchison, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.; Superfine, R.; Falvo, M.; Johnson, C. S., Jr.; Samulski, E. T.; Murray, R. W. J. Am. Chem. Soc. 1995, 117, 12537. (27) Dong, T.-Y.; Wu, H.-H.; Lin, M.-C. Langmuir 2006, 22, 6754. (28) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17. (29) Aslam, M.; Gopakumar, G.; Shoba, T. L.; Mulla, I. S.; Vijayamohanan, K.; Kulkarni, S. K.; Urban, J.; Vogel, W. J. Colloid Interface Sci. 2002, 255, 79. (30) Chen, L.; Zhang, D.; Chen, J.; Zhou, H.; Wan, H. Mater. Sci. Eng., A 2006, 415, 156.
a superlattice formation can be attributed to the interdigitation of the ligand chains chemisorbed on the copper particles.15c Interestingly, no sharp diffraction peaks were observed for the acrylated Cu NPs (Figures 4b and S10c in the Supporting Information). As may be observed with an amorphous material, the diffractogram displays less crystallinity of the particles indicated by the broad peaks.31 The acrylic functionality most likely induces some flexibility to the lattice structure. However, more experiments are necessary for further elucidation. Figure 5 shows the TEM images of the NPs before and after UV light irradiation in the presence of photoinitiator. The particles undergo polymerization through the acrylic functionality. This is attributed to the coalescence of the particles (Figure 5c,d). In a recent study, we observed that metal nanoparticle/ligand (31) (a) Erlacher, A.; Ambrico, M.; Capozzi, V.; Augelli, V.; Jaeger, H.; Ullrich, B. Semicond. Sci. Technol. 2004, 19, 1322. (b) Erlacher, A.; Ambrico, M.; Perna, G.; Schiavulli, L.; Ligonzo, T.; Jaeger, H.; Ullrich, B. Appl. Surf. Sci. 2005, 248, 402.
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Figure 5. TEM images of (a) CuC6Ac plus Irgacure 819 without irradiation, (b) CuC6Ac with 30 min irradiation, and (c and d) CuC6Ac plus Irgacure 819 with 30 min irradiation. Scale bar represents 50 nm.
Figure 6. 1H NMR (in benzene-d6) results for polymerization of acrylated (CuC6Ac) particles. Conditions: (a) no PI, no irradiation; (b) no PI, 15 min irradiation; (c) PI, no irradiation; and (d) PI, 15 min irradiation. (PI: photoinitiator.)
intermediates can yield such clusters following charge recombination.32 The NP polymerization was also confirmed by the disappearance of the 1H NMR acrylic double bond signal (δ 5.27 (32) Cai, X.; Anyaogu, K. C.; Neckers, D. C. J. Am. Chem. Soc. 2007, 129, 11324.
ppm) after several minutes of UV light irradiation (Figure 6). This signal could not be assigned to any traces of acryloyl chloride (δ 4.85 ppm; IR: υ 1755 cm-1) or acrylic acid (δ 5.37 ppm), even though the IR data rule out the presence of the latter as a possible impurity. The chloroform signal comes from the residual amount left after washing of the particles. Due to the reduced
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Figure 7. Biological activity of thiol-stabilized copper nanoparticles against Synechosystis sp. PCC 6803. Points are means of triplicate cultures.
solubility of dried nanoparticle powders in most solvents, the purified particles were not subjected to prolonged drying. Biological Activity of the Cu NP/Polymer Composites. The biological activity of the different functionalized copper NP/ polymer composites was tested against freshwater cyanobacterium Synechosystis sp. PCC 6803. The loading of the NPs into the monomer matrix (wt %), dispersion, and polymerization were carried out under similar conditions. Two controls were established: the culture without any polymers or biocides and the polymer lacking any copper particles. The latter was important to probe the effects of the polymer matrix or the irradiated photoinitiator on cell growth. We have also attempted to gain insights into the effects of the nature of the NP functionalization on biological activity (Figure 7). All Cu NP species tested exhibit good antibacterial activity. There was no significant difference observed in the biological activity of the particles based on the length of the ligand (thiolalkane) chain (P > 0.05). However, there was an effect based on the chemical nature of the ligand with CuC6Pr showing a stronger negative effect on cyanobacterial growth than CuC6Ac (one-tailed t-test, p < 0.05, ANOVA). Atomic absorption spectroscopy was used to evaluate copper ion release kinetics from the Cu NP/polymer composites. To mimic the conventional ablative copper paints, we used Cu powders (Fairmount Chemical Company) and CuO nanoparticles (ca. 30 nm, Alfa Aesar) as active biocides in the polymers. Figure 8a shows release profiles of the functionalized Cu NPs when
Figure 8. Amounts of copper released from the polymer matrix determined by atomic absorption spectroscopy for (a) different Cu NP species and (b) different loads of the acrylated Cu NPs. Each point is an average of at least five values. The inset in (a) shows a histogram of copper ion release rates from the polymer matrix (1wt % load) which depicts the levels of leaching control achieved with the stabilized particles.
compared with the nonfunctionalized biocides. It is clear that the stabilized particles display a better control of the release. They are less prone to aggregation within the matrix owing to the compatibility of the chemisorbed ligand chains and, thus, display more uniform dispersity.
Table 1. Biological Activity of Acrylated Copper Nanoparticles Compared to the Conventionally Used Copper Biocides in Antifouling Coatings optical density at 680 nm during exponential growth of organisms organism Chlamydomonas sp. Synechocystis sp. PCC 6803 Synechococcus sp. PCC 7002 Phaeodactylum tricornutum CCMP 1327
culture
culture/pol
green algaea
description
0.214 (0.033)
0.14 (0.014)
0.062c
Cu powder
0.066c
CuO NPs
0.061c
CuC6Ac
cyanobacteria b (fresh water strain)
0.608 (0.124)
0.316 (0.062)
0.127 (0.002)
0.115 (0.002)
0.139 (0.003)
cyanobacteriab (salt water strain)
0.087 (0.010)
0.070 (0.012)
0.052 (0.002)
0.048 (0.003)
0.055 (0.004)
diatoma
6.413 (0.374)
5.583 (0.165)
3.135c
3.332c
3.892c
a Eukaryotic organism. b Prokaryotic organism. c Treated cultures display a strong negative effect on cell growth. Values in parenthesis represent the cell growth rate (µ, units/day). All cultures were treated with 1 wt % load of biocide in the polymer matrix. Cell growth of the diatoms was determined by measuring in ViVo chlorophyll fluorescence using a fluorometer.
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The slowest release profile was observed for the acrylated species (CuC6Ac). By comparison, a higher amount of copper was leached from the matrix of CuC6Pr (Figure 8a). This can be explained by the acrylated copper nanoparticles being copolymerized into the polymer. As a result, leaching of the biocide was more efficiently controlled. An increase of the biocide load from 1% to 10% or even 25% did not result in significant enhancement of antimicrobial properties (Figure S22 in the Supporting Information). Rather, higher loads of the Cu NPs resulted only in a higher leaching rate (Figure 8b). Results of the biological activity trials on other microorganisms are summarized in Table 1 and in the Supporting Information (Figures S17-S22). The antimicrobial activity of the acrylated copper nanoparticles matches well with conventionally used biocides. The data clearly demonstrate that even though the NP containing composites exhibit slow and controlled leaching, biological activity is not compromised. The poorly controlled leaching of the nonstabilized Cu powders and CuO NPs might explain why most commercial paint formulations contain them in large amounts (50-60%weight). In summary, we have demonstrated the potential of functionalized Cu NPs as a biocide against biofilm formation. The large active surface area offered by the NPs makes it possible to reduce the load (1%weight or even less) and provides better
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leaching control, especially when NPs are chemically attached to the polymer backbone. Biological activity is not compromised by the controlled release of Cu ions from the nanocomposites. These materials may have value for the development of antibacterial paints and coatings for household materials, hospital and food storage equipment, as well as to reduce biofouling on ships and fouling-prone infrastructures exposed to aquatic environments. Acknowledgment. The authors are grateful to Professor George S. Bullerjahn and Professor R. Michael McKay for providing cultures and access to resources in their labs. We thank Professor McKay for very useful discussions pertaining to the biological activity assessment of the NPs. The authors thank the Office of Naval Research for financial support (Grant No. N00014-04-1-0406). Contribution number 670 from the Center for Photochemical Sciences. Supporting Information Available: Additional NMR spectra, IR spectra, TEM images, powder X-ray diffractograms, UV-visible spectra, biological activity profiles, and compositions of culture media. This material is available free of charge via the Internet at http://pubs. acs.org. LA800102F
