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Langmuir 2001, 17, 5082-5084
Development of Uniform Chitosan Thin-Film Layers on Silicon Chips Frances S. Ligler,*,† Brian M. Lingerfelt,‡ Ronald P. Price,† and Paul E. Schoen† Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, D.C. 20375-5348 and Furman University, Greenville, South Carolina 29613 Received January 29, 2001. In Final Form: June 1, 2001 Nature creates selectively refractive materials in butterfly wings and crab shells by alternating layers of chitin with different refractive indices. To mimic this construction, control of both thickness and refractive index of chitosan films is required. Methods were developed for adhering the chitosan layer to glass and silicon, polymerizing the chitosan, stabilizing the chitosan films against environmental changes in temperature and humidity, and depositing a uniform chitosan layer with defined thickness. First, commercial chitosan was hydrolyzed to obtain lower molecular weight polymers and further purified. Then chitosan layers were prepared on clean silicon chips pretreated with polyvinyl butyral resin for adhesion. The chitosan solution with a polymerizing component and a plasticizer consisted of 2.5% purified chitosan/2.5% Resimene/0.25% tetraethylene glycol. Spin-coating at varying speeds after an incubation period of 1 h at room temperature produced chitosan layer thicknesses in the ideal range of 1200-2000 Å. The different thicknesses were reproducible, with a standard deviation across the film between 3 and 5%. The experimental index of refraction calculated for these layers was 1.59.
Introduction Multiple layers of chitin are responsible for the diversity of color found in animals.1-7 The remarkable colors result from a combination of optical phenomena, such as reflection, refraction, and thin-film interference of light, from alternating layers of high and low refractive index materials.7 The examples of vertical diffraction gratings found in nature employ layers that are 1000-2500 Å in depth.6,8 The layers can be arranged in a regular stack which alternates layers of equal thickness, in a chirped stack with layers of decreasing thickness, or in a chaotic stack with layers of random thickness.9 The first step toward making any of these stacks is to make chitosan films which are uniform over relatively large areas and which are of defined thickness. In addition, the chitosan film must be optically transparent (nonabsorbing and nonscattering) and resistant to swelling or cracking during changes in ambient humidity or moderate alterations in temperature. In a pioneering paper documenting the use of a chitosan film for an optical application, Jiang et al.10 fabricated waveguides out of chitosan. The authors demonstrated that light loss in the visible region was a function of the scattering by contaminants in the film and that films made from low molecular mass chitosan (70 000 Da) were less lossy (1.5 dB/cm) than films made from medium molecular * To whom correspondence should be addressed. Tel: 202-4046002. Fax: 202-404-8897. Email:
[email protected]. † Center for Bio/Molecular Science & Engineering. ‡ Furman University. (1) Ghiradella, H. Appl. Opt. 1991, 30, 3492. (2) Monroe, E. A.; Monroe, S. E. Science 1967, 159, 97. (3) Neville, A. C.; Caveney, S. Biol. Rev. 1969, 44, 531. (4) Caveney, S. Proc. R. Soc. London, Ser. B 1971, 178, 205. (5) Neville, A. C. Biology of Fibrous Composites: Development Beyond the Cell Membrane; Cambridge University Press: London, 1993. (6) Parker, A. R. Am. Sci. 1999, 87, 248. (7) Srinivasarao, M. Chem. Rev. 1999, 99, 1935. (8) Land, M. F. Prog. Biophys. Mol. Biol. 1972, 24, 77. (9) Parker, A. R.; McKensie, D. R.; Large, M. C. J. J. Exp. Biol. 1998, 201, 1307. (10) Jiang, H.; Su, W.; Caracci, S.; Bunning, T. J.; Cooper, T.; Adams, W. W. J. Appl. Polym. Sci. 1996, 61, 1163.
mass chitosan (750 000 Da, >2.5 dB/cm). The films were spin-coated at 30-250 rpm onto glass or silicon surfaces, and a recurring problem was documented with nonuniformity across the substrate, with variations of the order of 500 Å across film of 1 to 10 µm thick. For this study, it was critical to begin with fully soluble, relatively shortchain chitosan in solution to deposit uniform films. The solubility of chitosan is a function of chain length; the length of the carbohydrate polymer was shortened using acid cleavage. Additional deacetylation was accomplished using a strong base to yield more soluble chitosan. To stabilize the films against changes in response to fluctuating temperature and humidity, the chitosan was polymerized and a plasticizer was added. Finally, a spincoating method was developed which produced a film of reproducible thickness that was homogeneous across the substrate to within 3-5%. Experimental Section Materials. The chitosan supplied by Protan (Raymond, WA) was of high molecular weight (approximately 161 000 Da) and was greater than 70% deacetylated. Polyvinyl butyral resin (Butvar, also known as B-98) was obtained from Monsanto (St. Louis, MO). The solution of Butvar used to coat the silicon chips and glass slides was prepared by dissolving 1.25 g of Butvar in 500 mL of CHCl3 (HPLC grade). Resimene, a partially methylated melamine-formaldehyde cross-linking resin supplied in water, was acquired from Solutia Inc. (St. Louis, MO). All solvents used as plasticizers were purchased from either Aldrich (Milwaukee, WI) or Fischer Scientific. The silicon wafers were purchased from MEMC Electronic Materials, Inc., in St. Peters, MO (10 cm diameter wafer, crystal face of 100, n type). All water was deionized and double distilled.
10.1021/la010148b CCC: $20.00 © 2001 American Chemical Society Published on Web 07/14/2001
Development of Chitosan Layers on Silicon Chips Chitosan Preparation. The commercially purchased chitosan was decolorized and further purified to achieve 90-95% deacetylation. Also, the presence of extraneous material, such as pieces of crab shell, necessitated additional purification. A 5% solution (by wt) of chitosan (yellow in color) was prepared by slowly blending 50 g of chitosan into 950 mL of water. While blending, 50 mL (5%) of glacial acetic acid was added, stirring until all of the chitosan was dissolved. Saturated NaOH was added until the chitosan formed a thick gel. The gel was washed repeatedly over Whatman No. 4 filter paper with water and methanol to remove any lipids and other contaminants. The gel was then redissolved in concentrated HCl/30% hydrogen peroxide (9:1). The volume of this solution used to redissolve the chitosan was approximately 20% of the entire volume of chitosan. When the chitosan was bleached white, saturated NaOH was added and the resulting gel was washed repeatedly over Whatman No. 4 filter paper with water. The wet, white chitosan was then lyophilized to a fluffy, white powder. For use, the lyophilized chitosan was dissolved in 2.5% glacial acetic acid containing 0.01% NaN3, filtered over two Whatman No. 3 paper filters to remove residual debris, and then filtered through a 0.45 µm cellulose nitrate filter with a Whatman No. 1 paper prefilter. Plasticizer Selection. Precleaned microscope slides were dipped in the Butvar solution for about 5 s and were allowed to air-dry. Solutions of 2.5% chitosan/2.5% Resimene/0.25% plasticizer additive (w/w/w) were prepared, and 0.5 mL of the solution was spread over the surface of each of two slides. The slides were then baked in an oven at 80 °C for 1 h and examined for clarity, peeling, and cracking. The slide was rinsed in water, baked for another hour, and reexamined. The films that remained intact were analyzed on a Bausch & Lomb Abbe refractometer to test for changes in refractive index. Preparation of Chitosan Films. The silicon chips (approximately 4 cm2) were cleaned using a modified piranha etch protocol.11 First, the chips were immersed in a solution of NH4OH:H2O2:H2O (1:1:5) at 80 °C for 5 min. The chips were then rinsed repeatedly with water and immersed in a solution of HCl: H2O2:H2O (1:1:5) at 80 °C for 5 min. After additional rinsing, the chips were dried under a N2 stream, submerged for about 5 s in the Butvar/CHCl3 solution, and air-dried. For dip-coating, Butvar-coated chips were submerged in a 2.5% chitosan/2.5% Resimene solution for varying lengths of time. Upon removal from the chitosan solution, the edges of the silicon chips were touched to tissue paper to remove the excess solution and were then baked in an oven at 80 °C for 1 h. For spin-coating, a solution of 2.5% chitosan/2.5% Resimene (w/w) was prepared with the addition of 0.25% tetraethylene glycol where indicated. About 0.4 mL of this solution was dropped onto each of the Butvar-coated silicon chips so that the majority of the chips were covered in solution. Where indicated, the chips were incubated for 1 h at room temperature before spinning at the indicated speed for 30 s and baked at 80 °C for 1 h. Ellipsometer measurements to obtain the thickness of the chitosan films were made on a single-wavelength ellipsometer (Gaertner Scientific Corp., Chicago, IL). The manually entered index of refraction for the chitosan was 1.59. A five-point scan with a measuring diameter of 10 mm was used.
Results and Discussion Initially, difficulty was experienced in obtaining adequate adhesion of the chitosan films to the glass or silicon. Jiang et al.10 note that good adhesion was obtained when substrates were processed under clean conditions but give no description of such conditions. In our hands, careful cleaning was insufficient to promote adhesion. In addition to Butvar, aminosilane monolayers and polyelectrolytes were tested for improving adhesion of the chitosan/ Resimene films, but neither approach was reliable (data not shown). Several different compounds were evaluated as plasticizers to be incorporated into the chitosan/Resimene (11) Shriver-Lake, L. C. In Immobilized Biomolecules in Analysis; Cass, T., Ligler, F. S., Eds.; Oxford University Press: Oxford, 1999; pp 1-14.
Langmuir, Vol. 17, No. 16, 2001 5083 Table 1. Addition of Plasticizers to 2.5% Chitosan/ 2.5% Resimene Filmsa cracked/peeled
additive
film appearance
first hour (%)
octane 1-decanol tetraethylene glycol triethylene glycol diethylene glycol glycerol poly(propylene) glycol none
clear rough at edges clear (few opaque spots) clear clear (few opaque spots) clear white clear
75
