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Surface-Grafted Viologen for Precipitation of Silver Nanoparticles and Their Combined Bactericidal Activities Zhilong Shi, K. G. Neoh,* and E. T. Kang Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260 Received April 5, 2004. In Final Form: May 18, 2004
A viologen, N-hexyl-N′-(4-vinylbenzyl)-4,4′-bipyridinium dinitrate (HVVN), was synthesized and subsequently graft-copolymerized on poly(ethylene terephthalate) (PET) films. Silver nanoparticles can be deposited on the surface of the HVVN-PET film through photoinduced reduction of the silver ions in salt solution. The size and distribution of the silver nanoparticles can be varied by changing the reaction time. The pyridinium groups of the HVVN graft-copolymerized on the surface of the substrate possess bactericidal effects on Escherichia coli, and this antibacterial effect can be very significantly enhanced by the incorporation of silver nanoparticles on the HVVN-PET film. The dual functionalities of HVVN and silver remain stable after prolonged immersion in phosphate buffer solution and after aging in a weathering chamber.
Introduction The adherence of bacteria to material surfaces results in biofilm formation. Because biofilms provide a protected structure for bacteria growth, the bacteria become less susceptible to antimicrobial agents than their free counterparts.1-3 Hence, a number of recent studies have focused on the development of antibacterial surfaces to inhibit the biofilm formation. Surface functionalization of materials with silver,4-9 quaternary ammonium group,10-12 and chitosan13 offers possible solutions. Silver as a powerful antibacterial agent has been used for many years.14-17 It has the advantage of a very broad spectrum of antibacterial activity and low toxicity to human beings, which is especially important in the antibacterial treatment of wounds.4,18 A number of researchers have investigated the coating of silver on material surfaces. Ignatova et al. have immobilized silver * To whom correspondence should be addressed. Tel.: +65 68742176. Fax: +65 67791936. E-mail:
[email protected]. (1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (2) Parsek, M. R.; Singh, P. K. Annu. Rev. Microbiol. 2003, 57, 677. (3) Brown, M. R.; Barker, J. Trends in Microbiology 1999, 7, 46. (4) Gray, J. E.; Norton, P. R.; Alnouno, R.; Marolda C. L.; Valvano, M. A.; Griffiths, K. Biomaterials 2003, 24, 2759. (5) Ignatova, M.; Labaye, D.; Lenoir, S.; Strivay, D.; Jerome, R.; Jerome, C. Langmuir 2003, 19, 8971. (6) Klueh, U.; Wagner, V.; Kelly, S.; Johnson, A.; Bryers, J. D. J. Biomed. Mater. Res. B 2000, 53, 621. (7) Dai, J. H.; Bruening, M. L. Nano Lett. 2002, 2, 497. (8) Lee, H. J.; Yeo, S. Y.; Jeong, S. H. J. Mater. Sci. 2003, 38, 2199. (9) Jansen, B.; Kohnen, W. J. Ind. Microbiol. 1995, 15, 391. (10) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981. (11) Nakagawa, Y.; Hayashi, H.; Tawaratani, T.; Kourai, H.; Horie, T.; Shibasaki, I. Appl. Environ. Microbiol. 1984, 47, 513. (12) Cen, L.; Neoh, K. G.; Kang, E. T. Langmuir 2003, 19, 10295. (13) Huh, M. W.; Kang, I. K.; Lee, D. H.; Kim, W. S.; Lee, D. H. J. Appl. Polym. Sci. 2001, 81, 2769. (14) Friedenthal, H. Biochem. Z. 1919, 94, 47. (15) Silver, S. FEMS Microbiol. Rev. 2003, 27, 341. (16) Riley, D. M.; Classen, D. C.; Stevens, L. E.; Bruke, J. P. Am. J. Med. 1995, 98, 349. (17) Kawashita, M.; Tsuneyama, S.; Miyaji, F.; Kokubo, T.; Kozukam, H.; Yamamoto, K. Biomaterials 2000, 21, 393. (18) Mozingo, D. W.; McManus, A. T.; Kim, S. H.; Pruitt, B. A., Jr. J. Trauma: Inj., Infect., Crit. Care 1997, 42, 1006.
ions in polypyrrole composite coatings on stainless steel and carbon fibers via the complexation of the silver ions with the polyanionic dopants of polypyrrole.5 Recently, the formation of silver nanoparticles by reduction of silver ions on material surfaces has been reported.7,19,20 Zhang et al. have reported that silver ions reduced by a triblock copolymer on the TiO2 surface have enhanced antibacterial activity.20 In our earlier work, we have formulated a process for obtaining gold and platinum thin film coatings by utilizing the redox reaction of viologen.21 Gold and platinum ions from the respective salt solutions can be reduced and deposited on the substrates which are surface graftcopolymerized with viologen. Recently, we have also demonstrated that a viologen containing a vinyl group and a six-carbon alkyl chain, N-hexyl-N ′-vinylbenzyl-4,4′bipyridinium bromide chloride, can be graft-copolymerized on a substrate to confer the antibacterial property to its surface.22 In the present paper, we show that the antibacterial property of the viologen surface-grafted substrate can be significantly enhanced by further utilizing the unique property of viologen to form silver nanoparticles on the substrate surface. Poly(ethylene terephthalate) (PET) was chosen as the substrate in this investigation because its excellent physicochemical properties, such as good mechanical strength, good stability in the presence of body fluids, and high radiation resistance, make it an excellent candidate in biomaterial applications.23,24 Characterization of the silver nanoparticles was carried out using UV-visible absorption spectroscopy, X-ray photoelectron spectroscopy (XPS), and field-emission scanning electron microscopy (FE-SEM). The antibacterial activity of the surfaces dual functionalized with viologen and silver was assayed using Escherichia coli (E. coli). (19) Aymonier, C.; Schlotterbeck, U.; Antonietti, L.; Zacharias, P.; Thomann, R.; Tiller, J. C.; Meching, S. Chem. Commun. 2002, 24, 3018. (20) Zhang, L. Z.; Yu, J. C.; Yip, H. Y.; Kwan, Q. L.; Kwong, K. W.; Xu, A. W.; Wong, P. K. Langmuir 2003, 19, 10372. (21) Zhao, L. P.; Neoh, K. G.; Kang, E. T. Langmuir 2003, 19, 5137. (22) Shi, Z. L.; Neoh, K. G.; Kang, E. T. Biomaterials, in press. (23) Massia, S. P.; Stark, J.; David, S. Biomaterials 2000, 21, 2253. (24) Gupta, B.; Plummer, C.; Bisson, I.; Frey, P.; Hilborn, J. Biomaterials 2002, 23, 863.
10.1021/la049132m CCC: $27.50 © 2004 American Chemical Society Published on Web 07/07/2004
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Experimental Section Materials. Biaxially oriented PET films of about 100 µm in thickness were purchased from the Goodfellow, Inc., of Cambridge, U.K. Vinyl benzyl chloride (VBC), 4,4′-bipyridine (98%), hexylbromide, cetyltrimethylammonium chloride, and silver nitrate were obtained from Aldrich Chemical Co. and used as received. Peptone, yeast extract, agar, and beef extract were purchased from Oxoid. E. coli was obtained from American Type Culture Collection (ATCC DH5R). Solvents, such as acetonitrile, acetone, and other chemicals, were of reagent grade and used as received from Aldrich Chemical Co. Synthesis of Viologen. N-Hexyl-N ′-(4-vinylbenzyl)-4,4′bipyridinium bromide chloride was synthesized according to the method reported in our earlier paper.25 A two-step reaction scheme was utilized. 4,4′-Bipyridine was first reacted with hexylbromide to give N-hexyl-4-(4-pyridyl)pyridinium bromide. This intermediate product was then reacted with VBC to result in N-hexyl-N ′-(4-vinylbenzyl)-4,4′-bipyridinium bromide chloride. The elemental analysis of the as-synthesized final product gave the following results: C, 63.39 wt %; H, 6.20 wt %; and N, 5.99 wt %. These values compare well with the theoretical values expected for C25H30N2BrCl (C, 63.36 wt %; H, 6.38 wt %; N, 5.91 wt %). Fourier transform infrared and XPS analyses were used to confirm the structure of the product.22,25 A stoichiometric amount of silver nitrate (0.717 g in 10 mL of H2O) was reacted with 1 g of as-synthesized N-hexyl-N ′-(4-vinylbenzyl)-4,4′bipyridinium bromide chloride to facilitate anion exchange resulting in N-hexyl-N ′-(4-vinylbenzyl)-4,4′-bipyridinium dinitrate.26 After filtration of the silver halide precipitate, the viologen was recovered from the filtrate by evaporation of the water. This viologen derivative will be denoted as HVVN in the subsequent discussion. Argon Plasma Pretreatment of the PET Surface. The PET film was cut into strips of 2.0 cm × 3.0 cm in size, washed with ethanol for 10 min using an ultrasonic bath to remove surface impurities, and then washed with a copious amount of doubly distilled water. They were then dried under a dynamic vacuum in a desiccator. Argon plasma pretreatment of the substrate was carried out by placing it between two electrodes and subjecting to glow discharge for 30 s in an Anatech SP100 plasma system. This treatment time was earlier found to result in an optimal condition for grafting on PET substrates.27 The substrate was then exposed to air for 5-10 min to facilitate the formation of surface oxide and peroxide groups before graft copolymerization was carried out. UV-Induced Surface Graft Polymerization. An aqueous 40 wt % solution of the HVVN monomer was placed on each surface of the argon plasma-pretreated substrate, which was sandwiched between two pieces of quartz plates. The assembly was exposed to UV irradiation from a 1-kW high-pressure Hg lamp in a rotary photochemical reactor (RH400-10W Riko Denki Kogyo of Chiba, Japan) at 25-28 °C for 30 min on each surface. The HVVN graft-copolymerized substrate was extracted from the quartz plates after prolonged immersion in water and again subjected to thorough washing with water to remove viologen, which was not graft-copolymerized on the substrate. An overall scheme of argon plasma pretreatment and the graft copolymerization of HVVN with the PET is shown in Scheme 1. This substrate will be referred to as the HVVN-PET substrate in the subsequent discussion. Silver Deposition on HVVN-PET. The HVVN-PET film was then inserted into a Pyrex tube that contained 20 mL of silver nitrate solution at a concentration of 200 mg/L. The solution was degassed for 20 min with argon and then sealed under an argon atmosphere. The substrate in the silver nitrate solution was subsequently exposed to UV irradiation on each surface in the rotary photochemical reactor at 25-28 °C for various periods of time. After irradiation, the substrates were washed with doubly distilled water and dried under a reduced pressure. (25) Liu, X.; Neoh, K. G.; Zhao, L. P.; Kang, E. T. Langmuir 2002, 18, 2914. (26) Nambu, Y.; Yamamoto, K.; Endo, T. J. Chem. Soc., Chem. Commun. 1986, 7, 574. (27) Ying, L.; Yin, C.; Zhuo, R. X.; Leong, K. W.; Mao, H. Q.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2003, 4, 157.
Shi et al. Scheme 1
Tests and Characterization. The surface compositions were measured using XPS on an AXIS HSi spectrometer (Kratos Analytical, Ltd.) with an Al KR X-ray source (1486.6-eV photons). The detailed process for XPS measurements was similar to that reported earlier.21 All binding energies were referenced to the C(1s) hydrocarbon peak at 284.6 eV. In the peak synthesis, the line width (full width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. FE-SEM imaging and composition determination of the surface were carried out on a JEOL JSM 6700F scanning electron microscope with energy-dispersive X-ray (EDX) accessory. The UV-visible absorption of the HVVN-PET films before and after reaction with silver nitrate was monitored on a Shimadzu UV-3101 PC scanning spectrophotometer. The amount of N+ immobilized on the surface of the PET film was determined by the modified dye interaction method.10,28 The PET films after surface functionalization were immersed in a 1 wt % fluorescein (Na salt) solution in distilled water for 10 min with constant shaking, followed by thorough rinsing with doubly distilled water. The stained film was then placed in an aqueous solution of 0.25 wt % cetyltrimethylammonium chloride, and the mixture was shaken for 10 min to desorb the dye. An aqueous 0.1 M phosphate buffer, pH 8.0, was then added in a ratio of one part buffer to nine parts cetyltrimethylammonium chloride solution, and the absorbance of the resultant solution was measured at 501 nm. The amount of dye bound to N+ on the film surface was calculated on the basis of a standard calibration. The corresponding N+ concentration was then calculated on the basis of the assumption of one dye molecule per seven N+ units.10 The amount of silver immobilized on the surface of the HVVNfunctionalized PET film was determined from the difference in the concentration of silver ions before and after the photoinduced reaction of the silver nitrate solution with the HVVN-PET film. The silver concentration in the solution was measured using an inductively coupled plasma (ICP) emission spectrometer (PerkinElmer Optima 3000DV) at a wavelength of 328.068 nm. The detection limit of this instrument is specified to be 0.9 ppb. Determination of the Antibacterial Effect of HVVNFunctionalized PET Films. E. coli, a gram-negative bacteria, was cultivated in 50 mL of a 3.1% yeast-dextrose broth (containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride, 5 g/L glucose, and 3 g/L yeast extract at a pH of 6.8)28 at 37 °C. All glassware and polymer samples were sterilized in an autoclave at 120 °C for 20 min or with UV irradiation before experiments. The E. coli containing broth was centrifuged at 2700 rpm for 10 min, and after the removal of the supernatant, the cells were washed twice with a sterile phosphate buffer solution, PBS (containing 5.4 g of sodium dihydrogen phosphate monohydrate and 8.66 g of anhydrous disodium hydrogen phosphate in 1 L of distilled water, adjusted to pH ) 7.0) and resuspended in PBS at a concentration of 107 cells/mL.22 One piece of the PET film (either pristine or functionalized) of 2 cm × 3 cm in size was immersed in 30 mL of this suspension in an Erlenmeyer flask and shaken at 200 rpm at 37 °C. The viable cell counts of the E. coli were measured by the surface spread-plate method. At the predetermined time, 1 mL of bacteria culture was taken from (28) Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Biotechnol. Prog. 2002, 18, 1082.
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Figure 2. Amount of silver deposited on the surface of the HVVN-PET film after reaction with a 200 mg/L AgNO3 solution under UV irradiation for various time periods.
Figure 1. XPS N(1s) (a) and C(1s) (b) core-level spectra of the PET film after HVVN graft copolymerization using a 40 wt % HVVN monomer concentration. the flask, and decimal serial dilutions with PBS were repeated with each initial sample. A drop of 0.1 mL of the diluted sample was then spread onto triplicate solid growth agar plates. After incubation of the plates at 37 °C for 24 h, the number of viable cells (colonies) was counted manually and the results after multiplication with the dilution factor were expressed as mean colony forming units per milliliter. The stability of the functionalized films was tested in a Ci3000 Xenon Weather-Ometer (Atlas Electric Device Co., Chicago, U.S.A.). During the test, the weather chamber was maintained at a relative humidity of 70% and a dry bulb temperature and black panel temperature of 40 and 70 °C, respectively. The simulated solar irradiation was directed at the film’s surface with an intensity of 0.56 W/m2 at 340 nm. A water spray was activated for 5 min in each 30-min cycle. After 48 h, the film was removed from the chamber and dried under a reduced pressure before being subjected to an antibacterial assay. The bactericidal efficiency of the film after weathering was then compared to that before the weathering test.
Results and Discussion 1. HVVN-PET Films. As shown in our earlier paper,22 the success of the UV-induced surface graft copolymerization of HVVN on the PET films can be ascertained by comparing the XPS spectra of the film before and after the grafting process. The presence of the surface-grafted HVVN polymer after the UV-induced graft-copolymerization step can be deduced from the presence of the N(1s) signal in the XPS wide scan spectra. The corresponding N(1s) core-level spectrum (Figure 1a) shows well-resolved peaks due to the nitrogen radical (N•) at 399.6 eV formed during X-ray excitation in the analysis chamber,29 positively charged nitrogen (N+) at 401.8 eV,22 and NO3- at 406.2 eV.30 The almost 1:1 peak area ratio of the nitrogen from the viologen chain backbone (N• and N+) and the NO3- counterion confirms that charge neutrality is maintained. The C(1s) core-level spectrum after HVVN (29) Sampanthar, J. T.; Neoh, K. G.; Ng, S. W.; Kang, E. T.; Tan, K. L. Adv. Mater. 2000, 12, 1536. (30) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. In Handbook of X-ray Photoelectron Spectroscopy; Chastain, J., Ed.; PerkinElmer Corp.: Eden Prairie, MN, 1992.
grafting shows a peak at 285.5 eV attributable to the Cs N species of the HVVN in addition to the CsC, CsH peak at 284.6 eV, and the CsO and CdO peak components of the pristine PET film are masked. This implies that the surface HVVN copolymer is thicker than the probing depth of the XPS technique (∼10 nm). No Cl and Br signals can be detected in HVVN-PET film, which shows that the exchange of the halide anions by NO3- anions was complete. The quantitative amount of [N+] present on the film surfaces was obtained from the titration method using fluorescein (Na salt). Previous work has shown that this dye only binds to quaternary amino groups but not the tertiary or primary groups.10,12 The amount of surface [N+] increases from 5 to 25 nmol/cm2 as the concentration of HVVN monomer used in the grafting process increases from 10 to 40 wt %. Further increase in monomer concentration beyond this value results in only a small increase in [N+]. Hence, for the present work, a 40 wt % concentration of HVVN was used in the grafting process. 2. Photoinduced Reduction of Silver on HVVNPET Films. The amount of silver deposited on the surface of HVVN-PET films was calculated from the difference in the silver ion concentration of the silver nitrate solution before and after reaction. Figure 2 shows the silver uptake by the HVVN-PET film as a function of the UV irradiation time. After a reaction time of 30 min, the uptake of silver has almost reached a plateau of 0.08 µmol/cm2. This amount of silver deposited on the HVVN-PET film represents 4% of the original silver ions from solution. The chemical state of the silver on the HVVN-PET film was investigated using the XPS technique. Figure 3 shows the XPS Ag(3d) core-level spectra of the HVVNPET film after photoinduced reaction with a 200 mg/L AgNO3 solution for 15 min. Because no Ag(3d) signal was discernible on the HVVN-PET film surface before the photoinduced reaction in AgNO3, the peaks in this spectrum are attributable to the silver uptake from the AgNO3 solution. The peaks at 368.0 eV (3d5/2) and 374.0 eV (3d3/2) attributable to the Ag0 species30-32 show that Ag+ ions from the solution have been reduced to the metallic state upon reaction with the viologen on the HVVN-PET film surface. Our previous study has postulated that upon UV irradiation the viologen dication can be readily reduced to a radical cation via the transfer (31) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers: the Scienta ESCA300 Database; John Wiley: Chichester, U.K., 1992; p 278. (32) Stathatos, E.; Lianos, P. Langmuir 2000, 16, 2398.
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Figure 3. XPS Ag(3d) core-level spectrum of the HVVN-PET film after reaction with a 200 mg/L AgNO3 solution for 15 min.
Figure 4. UV-visible absorption spectra of the HVVN-PET films before and after reaction with a 200 mg/L AgNO3 solution under UV irradiation for different periods of time.
of an electron from the counteranion. The viologen radical cations can then react with the metal ions in solution or adsorbed on the film surface, and concomitantly the radical cations undergo oxidation back to the dication state.21 The N(1s) core-level spectrum of the HVVN-PET film after 15 min of photoinduced reaction with AgNO3 is nearly the same as that before reaction (as shown in Figure 1a). For this film, the Ag/N (N from viologen chain backbone) ratio as determined by XPS is 0.18. This ratio has been calculated from the ratio of the areas of the respective peaks after correction with the sensitivity factors. It has been reported that when silver particles are reduced to nanometer dimensions, they exhibit unique optical properties in the visible spectral range due to the excitation of the collective oscillations of conducting electrons known as surface plasmons.33 The wavelength of the absorption maximum depends on the size, shape, and dielectric environment of nanoparticles. The UVvisible absorption spectra of the HVVN-PET film before and after reaction with the silver nitrate solution under UV irradiation for various time periods are shown in Figure 4. No distinct absorption bands between 350 and 750 nm can be observed in the spectrum of the pristine PET and HVVN-PET films. After reaction of HVVNPET with the silver nitrate solution under UV irradiation, a new band appears in the 400-nm region. This absorption is the characteristic plasmon peak of silver nanoparticles.34 As can be seen from Figure 4, there is a red shift of the (33) Malynych, S.; Chumanov, G. J. Am. Chem. Soc. 2003, 125, 2896.
Figure 5. Scanning electron micrographs of the HVVN-PET films before and after reaction with a 200 mg/L AgNO3 solution for (a) 15 min and (b) 30 min under UV irradiation.
absorption band from 420 to 440 nm with increasing reaction time. At the same time, the full width at halfmaximum of the absorption band also increases. This result indicates that, with increasing reaction time, the size of the nanoparticles increases and the size distribution also becomes wider. The FE-SEM images of the HVVN-PET film after reaction with a 200 mg/L AgNO3 solution for 15 and 30 min under UV irradiation are shown in Figure 5. Using the software package (Smart-view) available with the FESEM, the mean diameter (Dm) of the nanoparticles was calculated from 20 particles chosen arbitrarily from each image. For a reaction time of 15 min, the Dm of the silver nanoparticles is 25 nm (Figure 5a). After a reaction time of 30 min, the silver nanoparticles have started to aggregate (Figure 5b). EDX analysis of the HVVN-PET film surface confirms the extensive presence of silver on the surface of the HVVN-PET films (not shown). 3. Antibacterial Effect of the Functionalized PET Films. The antibacterial effect of the functionalized PET films was investigated by a comparison of the number of viable cells in the suspension in contact with the different substrates. (34) Doremus, R. H. Langmuir 2002, 18, 2436. Lin, X. Z.; Teng, X. W.; Yang, H. Langmuir 2003, 19, 10081. Chen, C. W.; Chen, M. Q.; Serizawa, T.; Akashi, M. Adv. Mater. 1998, 10, 1122. Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932.
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Figure 6. Viable E. coli cell number as a function of time in contact with the different substrates: pristine PET (9), VBVPET ([), HVVN-PET (b), HVVN-PET after reaction in AgNO3 for 30 min (1), and HVVN-PET after reaction in AgNO3 for 15 min (2). The cell number was determined by the surfacespread method.
From Figure 6, it can be seen that with the pristine PET film, the number of viable cells in the suspension decreased by
