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Gold Nanoparticle Embedded, Self-Sustained Chitosan Films as Substrates for Surface-Enhanced Raman Scattering David S. dos Santos, Jr.,†,‡ Paul J. G. Goulet,† Nicholas P. W. Pieczonka,† Osvaldo N. Oliveira, Jr.,‡ and Ricardo F. Aroca*,† Materials & Surface Science Group, School of Physical Sciences, University of Windsor, Windsor, ON, Canada, N9B 3P4, and Instituto de Fı´sica de Sa˜ o Carlos, CP 369, 13560-970 Sa˜ o Carlos, SP, Brazil Received July 5, 2004. In Final Form: August 27, 2004 In this work, self-sustained, biocompatible, biodegradable films containing gold nanostructures have been fabricated for potential application in nanobioscience and ultrasensitive chemical and biochemical analysis. We report a novel synthesis of gold nanoparticles mediated by the biopolymer chitosan. Selfsupporting thin films are formed from the resultant gold-chitosan nanocomposite solutions and characterized by UV-visible surface plasmon absorption, transmission electron microscopy, atomic force microscopy, infrared absorption, and Raman scattering measurements. Results demonstrate control over the size and distribution of the nanoparticles produced, which is promising for several applications, including the development of biosensors. As a proof of principle, we demonstrate that gold-chitosan films can be employed in trace analysis using surface-enhanced Raman scattering.
Introduction A wide range of materials, including those classified as organic, inorganic, and biological, are now used in the synthesis, fabrication, and processing of nanostructures with unique physical properties.1 This multiplicity of approaches is illustrative of the expansion of nanoscience, driven by potential applications in fundamental research and nanotechnology. It is also a result of the desire to control the size, shape, structure, and morphology of the nanostructures produced. In a field experiencing such rapid development, it may seem surprising that gold, which has been used since ancient times, plays such a prominent role.2 With their unique chemical and physical properties, gold nanoparticles have found widespread use in fundamental research,2 as well as in catalytic,3 biological,4 and sensing applications.5,6 They possess excellent biocompatibility, low toxicity, and relatively low reactivity, thus providing benefits for use within living systems. Moreover, the unique optical properties that result from surface plasmon resonance in the visible range of the electromagnetic spectrum make them particularly attractive for optical applications. The enhancements of absorption, fluorescence emission, and Raman scattering from analytes positioned near the surface of appropriate noble metallic nanoparticles are widely known.7 Surface* To whom correspondence should be addressed. E-mail:
[email protected]. † University of Windsor. ‡ Instituto de Fı´sica de Sa ˜ o Carlos. (1) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem 2000, 1, 18-52. (2) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (3) Mohr, C.; Hofmeister, H.; Radnik, J.; Claus, P. J. Am. Chem. Soc. 2003, 125, 1905-1911. (4) Hone, D. C.; Walker, P. I.; Evans-Gowing, R.; Fitzgerald, S.; Beeby, A.; Chambrier, I.; Cook, M. J.; Russell, D. A. Langmuir 2002, 18, 29852987. (5) Cao, Y. W. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 15361540. (6) Natan, M. J.; Lyon, L. A. In Metal Nanoparticles: Synthesis, Characterization and Applications; Feldheim, D. L., Colby, A. F., Jr., Eds.; Marcel Dekker: New York, 2002; pp 183-205. (7) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783-826.
enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS), as the most significant of these phenomena, permit single molecule detection at the surface of gold and silver nanoparticles,8-10 and can be employed as analytical tools for the investigation of single living cells.11 Several physical and chemical methods have been employed to fabricate gold nanoparticles,12,13 producing a wide variety of shapes including spheres, rods, prisms,14 and wires.15 Also, thin solid films containing gold nanostructures have also found wide application. Gold is electropositive and can be reduced by various agents such as borohydrate, amines, alcohols, and carboxylic acids. The most common methods, however, are based on sodium citrate, sodium borohydrate, and ascorbic acid. Typically, nanoparticles tend to aggregate during their synthesis, and stabilizers, such as small organic molecules or polymers, must be used. The latter protect nanoparticles after their formation through steric hindrance, thereby preventing aggregation. Polymers also offer control over the rate of the reduction process, and thus enable the production of nanoparticles of different shapes and sizes.16 In this report, we broaden the scope of materials used in producing gold nanoparticles, by employing dilute acetic acid as a reducing agent in a reaction mediated by the biopolymer chitosan to generate nanoparticles embedded in self-sustained films. The use of chitosan as a stabilizer for borohydrate-reduced Au nanoparticles in solution has (8) Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106. (9) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667-1670. (10) Constantino, C. J. L.; Lemma, T.; Antunes, P. A.; Aroca, R. F. Anal. Chem. 2001, 73, 3674-3678. (11) Kneipp, K.; Haka, A. S.; Kneipp, H.; Badizadegan, K.; Yoshizawa, N.; Boone, C.; Shafer-Peltier, K. E.; Motz, J. T.; Dasari, R. R.; Feld, M. S. Appl. Spectrosc. 2002, 56, 150-154. (12) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (13) Bunge, S. D.; Boyle, T. J.; Headley, T. J. Nano Lett. 2003, 3, 901-905. (14) Liz-Marza´n, L. M. Mater. Today 2004, 7, 26-31. (15) Hassenkam, T.; Norsgaard, K.; Iversen, L.; Kiely, C.; Brust, M.; Bjornholm, T. Adv. Mater. 2002, 14, 1126-1130. (16) Toshima, N.; Yonezawa, T. New J. Chem. 1998, 22, 1179-1201.
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been recently reported in a study examining the catalytic activity of Au.17 However, borohydrate is a very strong reducing agent, and thus requires precise control over experimental conditions to avoid problems of irreproducibility and clustering of nanoparticles. The present approach, on the other hand, offers a much slower reduction, and thus a greater degree of control over the nanoparticles produced, along with higher reproducibility. Moreover, it is carried out in a mild, biofriendly medium that allows for the production of films, gels, and beads. In the literature, there are reports of acetic acid being used as a reducing agent for the production of gold particles on the microscale, either by the thermal decomposition of gold acetate18 or by the optically induced reaction of gold salts in acetic acid.19 In this work, however, we present the first report of Au nanoparticles being produced through reduction by acetic acid. Chitosan is the (β-1,4)-linked D-glucosamine derivative of the polysaccharide chitin, the most abundant natural polysaccharide after cellulose, and has been extensively studied for more than 30 years. It is an inexpensive, renewable material with applications in cosmetics, pharmaceuticals, food science, wastewater treatment, biotechnology, and others.20,21 Analogous to other polysaccharides, chitosan has unique structural and physicochemical characteristics that differ considerably from typical synthetic polymers. Chitosan’s structure is similar to that of cellulose, but it has better processability due to the presence of amino groups (pKa 6.2) in its chains. In fact, its chemistry is largely determined by its amino and hydroxyl groups that act as potential sites for chemical enzyme immobilization or simply for altering the polymer’s functionalty.22 Also, chitosan is well-known as a strong chelating agent for metals and proteins,23 making it particularly useful in sensor development24 Here, a report is made of a new class of high quality, self-supported thin films consisting of biopolymeric chitosan matrices containing dispersions of gold nanoparticles with a narrow size distribution. These films possess interesting structural, chemical, and optical properties that can be tailored for a wide variety of applications including biosensors. As a proof of principle, the films were adapted for use as substrates for surface-enhanced Raman scattering measurements using a quintessential test analyte, rhodamine 6G. Trace analysis, down to 10-6 M, was established without spectral interference from the chitosan host matrix. Experimental Section Chitosan, obtained from shrimp chitin (an N-acetylglucosamine polymer), was provided by Cyrbe do Brasil Corporation. It has an average molecular weight of 500 000 g/mol, as determined by viscometry, and a 75% degree of deacetylation,
(17) Esumi, K.; Takei, N.; Yoshimura, T. Colloids Surf., B 2003, 32, 117-123. (18) Warren, L. F., Jr.; Cunningham, P. H. United States Patent 4,933,204, 1990. (19) Krynetskii, A. B.; Rukhadze, A. A.; Fadeeva, S. S. Zh. Fiz. Khim. 1999, 73, 576-579. (20) Sandford, P. A. In Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties and Applications; Skjak, G., Anthonsen, T., Sandford, P., Eds.; Elsevier: New York, 1988; pp 51-69. (21) Rabea, E. I.; Badawy, E.-T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W. Biomacromolecules 2003, 4, 1457-1465. (22) Kurita, K. Prog. Polym. Sci. 2001, 26, 1921-1971. (23) Ligler, F. S.; Schauer, C. L.; Chen, M.-S.; Chatterley, M.; Eisemann, K.; Welsh, E. R.; Price, R. R.; Schoen, P. E. Thin Solid Films 2003, 434, 250-257. (24) dos Santos, D. S., Jr.; Riul, A., Jr.; Malmegrin, R. R.; Fonseca, F. J.; Oliveira, O. N., Jr.; Mattoso, L. H. C. Macromol. Biosci. 2003, 3, 591-595.
dos Santos et al. as obtained from 1H NMR measurements.25 It was purified twice by solubilization in a 1% aqueous acetic acid solution, filtered with filter paper, precipitated with a 1% aqueous sodium hydroxide solution, and washed extensively with deionized water until neutral pH was reached. Hydrochloroauric acid was purchased from Aldrich and used without further purification. All glassware was thoroughly cleaned using detergent, aqua regia, and copious rinsing with deionized water. Solutions were prepared using deionized water. Films were prepared by casting measured volumes of the solutions onto glass slides at room temperature and allowing for evaporation of the solvent. Syntheses were carried out in a tri-necked round-bottom flask equipped with a reflux condenser and involved the addition of tetrachloroauric(III) acid and chitosan in the proportions mentioned in the text. Solutions were stirred until a temperature of 85 °C was reached and were then allowed to react for an additional 5 h. UV-visible absorption spectroscopy was used to monitor the plasmon absorption of the gold nanoparticles produced, and all spectra were collected using a Cary UV-visible spectrometer. Atomic force microscopy (AFM) was recorded using a Digital Instruments NanoScope IV, operating in non-contact tapping mode with a n+-silicon tip. A drive frequency of 214 kHz and a scan rate of 1 Hz were used for scan sizes of 5 µm2 with 256 sample lines. Topographical (height), error (amplitude), and phase images were used for analysis of the surface morphology of the films. Transmission electron microscopy (TEM) images were obtained with a Philips CM20 scanning transmission electron microscope operating with a 120 kV accelerating voltage. Infrared absorption measurements were recorded with a Bruker Equinox 55 Fourier transform infrared (FTIR) spectrometer, employing a nitrogen cooled mercury cadmium telluride (MCT) detector. Each spectrum was measured in transmission mode with 256 scans and 4 cm-1 resolution. All Raman spectra were acquired with a Renishaw inVia Raman microscope system using a 633 nm excitation line directed through a 50× objective with
