Thermosensitive Copolymer Coatings with Enhanced Wettability

Flinders University, GPO Box 2100, Bedford Park, South Australia 5042, Australia ... Maximum switching in terms of the contact angle change around...
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Thermosensitive Copolymer Coatings with Enhanced Wettability Switching Mahaveer D. Kurkuri, Matthew R. Nussio, Alec Deslandes, and Nicolas H. Voelcker* School of Chemistry, Physics and Earth Sciences, Flinders UniVersity, GPO Box 2100, Bedford Park, South Australia 5042, Australia ReceiVed NoVember 24, 2007. In Final Form: January 9, 2008 Expanded cross-linked copolymers of poly(N-isopropylacrylamide) (PNiPAAm) and poly(acrylic acid) (PAAc) of varying monomer ratios were grafted from a crystalline silicon surface. Surface-tethered polymerization was performed at a slightly basic pH, where electrostatic repulsion among acrylic acid monomer units forces the network into an expanded polymer conformation. The influence of this expanded conformation on switchability between a hydrophilic and a hydrophobic state was investigated. Characterization of the copolymer coating was carried out by means of X-ray photoelectron spectroscopy (XPS) ellipsometry, and diffuse reflectance IR. Lower critical solution temperatures (LCSTs) of the copolymer grafts on the silicon surfaces were determined by spectrophotometry. Temperature-induced wettability changes were studied using sessile drop contact angle measurements. The surface topography was investigated by atomic force microscopy (AFM) in Milli-Q water at 25 and 40 °C. The reversible attachment of a fluorescently labeled model protein was studied as a function of temperature using a fluorescence microscope and a fluorescence spectrometer. Maximum switching in terms of the contact angle change around the LCST was observed at a ratio of 36:1 PNiPAAm to PAAc. The enhanced control of biointerfaces achieved by these coatings may find applications in biomaterials, biochips, drug delivery, and microfluidics.

1. Introduction Biomaterial scientists have faced continuing challenges to develop chemically engineered surfaces for emerging biomedical applications. Materials which can attract molecules of biological importance in one condition and resist adsorption of the same molecules in another condition are of prime relevance for important applications including in controlled drug delivery,1,2 chemical sensing,3,4 bioassays,5,6 separation science,7 enzymatic activity control,8 and microactuators.9 Prime candidates for such materials and material surfaces are polymers, which alter their physicochemical properties reversibly with respect to the surrounding conditions such as pH,1,10 temperature,11-15 ionic strength,16,17 light,18,19 electric field,20 and solvent.21 * To whom correspondence should be addressed. Fax: +61 (08) 8201 2905. Phone: +61 (08) 8201 5338. E-mail: [email protected]. (1) Kurkuri, M. D.; Aminabhavi, T. M. J. Controlled Release 2004, 96, 9-20. (2) Zhang, J. L.; Srivastava, R. S.; Misra, R. D. K. Langmuir 2007, 23, 63426351. (3) Davis, J.; Glidle, A.; Cass, A. E. G.; Zhang, J.; Cooper, J. M. J. Am. Chem. Soc. 1999, 121, 4302-4303. (4) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942-1943. (5) Lai, J. J.; Hoffman, J. M.; Ebara, M.; Hoffman, A. S.; Estournes, C.; Wattiaux, A.; Stayton, P. S. Langmuir 2007, 23, 7385-7391. (6) Alarcon, C. H.; Farhan, T.; Osborne, V. L.; Huck, W. T. S.; Alexander, C. J. Mater. Chem. 2005, 15, 2089-2094. (7) Song, X.; Wang, H-I.; Shi, J.; Park, J- W.; Swanson, B. I. Chem. Mater. 2002, 14, 2342-2347. (8) Nagel, B.; Warsinke, A.; Katterle, M. Langmuir 2007, 23, 6807-6811. (9) Harmon, M. E.; Tang, M.; Frank, C. W. Polymer 2003, 44, 4547-4556. (10) Yang, S. Y.; Rubner, M. F. J. Am. Chem. Soc. 2002, 124, 2100-2101. (11) Cheng, X.; Canavan, H. E.; Stein, M. J.; Hull, J. R.; Kweskin, S. J.; Wagner, M. S.; Somorjai, G. A.; Castner, D. G.; Ratner, B. D. Langmuir 2005, 21, 7833-7841. (12) Sun, T.; Wang, G.; Feng, L.; Liu, B.; Ma, Y.; Jiang, L.; Zhu, D. Angew. Chem., Int. Ed. 2004, 43, 357-360. (13) Xia, F.; Feng, L.; Wang, S.; Sun, T.; Song, W.; Jiang, W.; Jiang, L. AdV. Mater. 2006, 18, 432-436. (14) Xiao, X.; Fu, Y. Q.; Zhou, J. J.; Bo, Z. S.; Li, L.; Chan, C. M Macromol. Rapid Commun. 2007, 28, 1003-1009. (15) Cho, E. C.; Kim, Y. D.; Cho, K. J. Colloid Interface Sci. 2005, 286, 479-486. (16) Yang, J.; Fang, L.; Wang, F.; Tan, T. W. J. Appl. Polym. Sci. 2007, 105, 539-546.

Much attention has been paid recently to poly(N-isopropylacrylamide) (PNiPAAm)-based polymers for the development of temperature-stimulus-sensitive surfaces,11-15,22-26 since neat PNiPAAm exhibits a lower critical solution temperature (LCST) of 32 °C27 at which this polymer switches reversibly its wettability due to an entropically driven change in the hydrogen-bonding pattern. Efforts are under way to improve the switchability of PNiPAAm by copolymerization with other monomers,22,24,25 grafting,26 monomer modification,28 and incorporation of surface roughness.12,13 However, there is a critical copolymer content in PNiPAAm above which thermosensitivity of the PNiPAAm is reduced or eliminated.29-31 This can be attributed to the influence of these monomer units on the subtle balance of hydrogen bonding in PNiPAAm. Zhang et al.24 have prepared hydrogels by (17) Ezell, R. G.; McCormick, C. L. J. Appl. Polym. Sci. 2007, 104, 28122821. (18) Shimoboji, T.; Ding, Z. L.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 2002, 13, 915-919. (19) Nosova, G. I.; Solovskaya, N. A.; Romashkova, K. A.; Lukyahina, V. A.; Sidorovich, A. V.; Gofman, I. V.; Aleksandrova, E. L.; Abalov, V. I.; Kudryavtsev, V. V. Polym. Sci., Ser. A 2006, 48, 569-577. (20) Kurkuri, M. D.; Lee, J-R.; Han, J. H.; Lee, I. Smart Mater. Struct. 2006, 15, 417-423. (21) Minko, S.; Muller, M.; Motornov, M.; Nitschke, M.; Grundke, K.; Stamm, M. J. Am. Chem. Soc. 2003, 125, 3896-3900. (22) Ebara, M.; Yamato, M.; Hirose, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Biomacromolecules 2003, 4, 344-349. (23) Jones, D. M.; Smith, J. R.; Huck, W. T. S.; Alexander, C. AdV. Mater. 2002, 14, 1130-1134. (24) Zhang, X-Z.; Yang, Y-Y.; Wang, F-J.; Chung, T-S. Langmuir 2002, 18, 2013-2018. (25) Cunliffe, D.; Alarcon, C. H.; Peters, V.; Smith, J. R.; Alexander, C. Langmuir 2003, 19, 2888-2899. (26) Schmaljohann, D.; Oswald, J.; Jorgensen, B.; Nitschke, M.; Beyerlein, D.; Werner, C. Biomacromolecules 2003, 4, 1733-1739. (27) Taylor, L. D.; Cerankowski, L. D. J. Polym. Sci., Part A: Polym. Chem. 1975, 13, 2551-2569. (28) Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Ed. 2000, 11, 101-110. (29) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496-2500. (30) Gutowska, A.; Bae, Y. H.; Feijian, J.; Kim, S. W. J. Controlled Release 1992, 22, 95-104. (31) Beltran, S.; Baker, J. P.; Hooper, H. H.; Blanch, H. W.; Prausnitz, J. M. Macromolecules 1991, 24, 549-551.

10.1021/la703668s CCC: $40.75 © 2008 American Chemical Society Published on Web 03/15/2008

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Table 1. Sample Codes Used in This Study, Composition of the Corresponding Polymerization Solution, Ellipsometric Coating Thickness, and LCST (Cloud Point) of the Obtained Polymer monomer content

ellipsometric sample NiPAAm AAc bisAAm polymer cloud code (mmol) (mmol) (µmol) thickness (nm) point (°C) S-1 S-2 S-3 S-3Ta S-4 S-5 a

2.194 2.164 2.134 2.134 2.104 2.074

0 0.03 0.06 0.06 0.09 0.12

6.5 6.5 6.5 6.5 6.5 6.5

11.2 ( 0.4 12.4 ( 0.7 12.0 ( 0.7 26.2 ( 0.8 11.6 ( 0.7 12.5 ( 0.6

26.7 32.3 34.2 b 35.8 37.1

S-3T polymerization was performed for 18 h. b Not determined.

polymerizing N-isopropylacrylamide (NiPAAm) and acrylic acid (AAc) monomers in slightly alkaline medium (Tris/HCl solution, pH 8.8), which, as a result of electrostatic repulsion among AAc monomer units during polymerization, resulted in the formation of expanded hydrogels. Thus formed hydrogels have shown temperature stimulus sensitivity and improved oscillating swelling and deswelling properties in water in comparison to PNiPAAm. In this study, polymerization of NiPAAm and AAc is performed under conditions similar to those of Zhang et al.24 However, in contrast to this earlier study, in the present work polymer chains are cross-linked and tethered to a silicon surface, resulting in thermosensitive poly(N-isopropylacrylamide)-co-acrylic acid coatings with expanded conformation. A NiPAAm to AAc ratio was identified where the expanded polymer network shows improved temperature-induced wettability switching. 2. Materials and Methods 2.1. Substrate Preparation. P-type silicon wafers having an orientation of 〈100〉 with a resistivity and thickness of 10-20 Ω cm and 381 ( 25 µm, respectively, were obtained from Silicon Quest International. Rectangle-shaped silicon wafers were cut into 1 × 2 cm2 pieces and cleaned by sonication for 1 h in surfactant solution (2% Triton X-100, Aldrich, Australia) in Milli-Q water. After sonication, the wafers were washed with Milli-Q water and subsequently dried in a stream of dry nitrogen. They were then placed in a 1 M NaOH solution for 5 min, washed thoroughly in Milli-Q water, and placed in a 0.1 M HNO3 solution for 10 min followed by a final wash in Milli-Q water and drying with a stream of dry nitrogen gas. Cleaned silicon wafers were silylated by placing them in 5% 3-(trimethoxysilyl)propyl methacrylate (98+%, Aldrich) in acetone (Ajax Finechem, Australia) for 2 h at room temperature. The samples were then washed in copious amounts of acetone followed by drying in a stream of dry nitrogen gas. Silylated silicon wafers were subjected to surface-tethered polymerization reactions as follows: the inhibitor was removed from AAc (99%, Sigma-Aldrich) monomer by vacuum distillation at 16.5 mmHg and 39 °C. NiPAAm (97%, Aldrich) was recrystallized twice in n-hexane (analytical grade, Aldrich) and dried under vacuum. Tris buffer (0.5 M) of pH 8.8 was charged into a custom-made two-neck flat-bottomed flask fitted with a condenser, and the required quantities of NiPAAm, AAc, and cross-linker N,N′-methylenebisacrylamide (bisAAm; 99+%, Aldrich) were charged into the flask (see Table 1), and a temperature of 25 °C was maintained throughout the reaction. The mixture was stirred for 30 min under continuous purging with nitrogen. Then the silylated silicon wafers were placed carefully at the bottom of the flask. A 50 µL portion of ammonium persulfate (g98%, Sigma) (5 wt %, prepared in Milli-Q water) was added, followed by the injection of the accelerator N,N,N′,N′tetramethylethylenediamine (99%, Sigma) (12.5 µL) under a nitrogen atmosphere. The polymerization was allowed to proceed for 45 min or 18 h, after which the silicon wafers were removed and placed in copious amounts of Milli-Q water for 1 h at room temperature and washed with Milli-Q water to remove any remaining unbound

polymer. The samples were dried under a stream of nitrogen gas. The samples were stored for further study. The sample code and composition are tabulated in Table 1. 2.2. Characterization of Polymer Coatings. The chemical composition of the polymer-coated surface was characterized and compared with that of a bare silicon surface using a Leybold LHS11 X-ray photoelectron spectroscope using Al KR X-rays (1486.6 eV), at 14 kV acceleration voltage and an emission current of 30 mA. Gaussian-Lorentzian peaks were fitted using XPSPEAK v4.1. The thickness of the polymer coatings on the silicon wafers was measured using an SE 400 (SENTECH Instruments GmbH, Germany) with multiple angles of 40°, 45°, 50°, 55°, and 60° at room temperature, considering the refractive index of silicon and silicon dioxide as 3.85 and 1.46, respectively. A helium/neon laser was used at the characteristic wavelength of 632.8 nm. A minimum of 10 measurements were carried out on each sample. Fourier transform infrared measurements for surface analyses were carried out using a Nicolet Avatar 370 MCT and a diffuse reflectance Fourier transform (DRIFT) accessory supplied with the instrument. A total of 60 scans were averaged with a spectral resolution of 4 cm-1. The sample compartment was purged with dry air. The LCST (cloud point) of the copolymer was determined by using a Hewlett-Packard (8452A) diode array spectrophotometer measuring a change in the absorbance at 450 nm. The copolymer solution in the supernatant of the surface-tethered polymerization reaction was diluted 10 times in Milli-Q water and transferred into a glass cuvette. Heating the polymer solution placed in the cuvette was performed with an Agilent 89090A Peltier heater from 20 to 45 °C with 0.5 °C increments. The cloud point was obtained as the temperature at which the solution turned turbid and gave a sharp increase in absorbance. 2.3. Switchability Studies. Contact angle measurements were conducted on a custom-made setup32 which consists of a sample stage, a light source, a computer-controlled CCD camera with a macrolens, and a heating element placed on the sample stage to control the temperature of the silicon wafers. A drop of Milli-Q water (2 µL) was placed on the surface of the dry sample. The sample stage and camera positions were adjusted so that the image of the drop was clearly obtained on the computer monitor. With the help of Scion Image 4.0.2 software, the contact angles at both sides of the drop were measured. A minimum of five drops were measured for each sample. To study the surface topography in the wet state, atomic force microscopy (AFM) was performed using a MultiMode Nanoscope IV (Veeco Corp.) in Milli-Q water at different temperatures. The samples were imaged at 25 and 40 °C to study the surface roughness by using the temperature controller provided with the instrument. All images were obtained in tapping mode by using triangular Si3N4 cantilevers with a spring constant of 0.15 N m-1 (OTR-8, Digital Instruments) operating at their resonant frequency (generally about 9.5 kHz). The imaged areas for each sample were 3 × 3 µm2. Image processing was performed using Nanoscope v5.30 off-line software (Veeco Corp.). For fluorescence microscopy measurements, a Laborlux D fluorescence microscope (Leica, Germany) was used and images were captured using a Nikon Digital Sight DS-L1 (Nikon Corp., Japan) and a Nikon Digital Sight DS-SM camera (Nikon Corp.) head. Fluorescence was detected through a 450-490 nm excitation filter and a 515 nm suppression filter at a constant exposure time. Two freshly polymer coated silicon wafers of each sample (S-1, S-2, S-3, S-4, and S-5) were soaked independently for 15 min in a 0.1 mg/mL fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) solution in PBS (1 L of PBS contains 8 g of NaCl, 0.2 g of KCl, 1.18 g of Na2HPO4·2H2O, and 0.24 g of KH2PO4) of pH 7.4 in the dark. One solution was kept at 25 °C and the other at 40 °C. The samples were washed in Milli-Q water maintained at the respective temperatures followed by drying in a stream of nitrogen. (32) Low, S. P.; Williams, K. A.; Canham, L. T.; Voelcker, N. H. Biomaterials 2006, 27, 4538-4546.

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Figure 1. Schematic diagram of (a) the monomer solution containing deprotonated acrylic acid monomer molecules which electrostatically repel each other (dashed arrows) and (b) the expanded conformation of the cross-linked polymer network after polymerization. Spheres with a negative charge indicate -COO- groups.

PNiPAAm is a temperature-sensitive polymer with isopropyl amide side chains. This polymer is characterized by an LCST below which the side chains form hydrogen bonds with water molecules, resulting in a swollen hydrogel polymer. Above the LCST, the polymer forms inter- and intramolecular hydrogen bonds with neighboring isopropyl amide groups, resulting in cross-linking, expulsion of water from the polymer network, and collapse of the polymer chains. The polymer becomes more hydrophobic.27 Hence, PNiPAAm reversibly switches between hydrophilic and hydrophobic states around the LCST of the polymer, which is 32 °C for pure PNiPAAm. The LCST varies if PNiPAAm is modified or copolymerized with other monomers.29 Here, AAc was used as a comonomer. The pKa value of AAc is 4.2,33 and at the slightly alkaline polymerization pH of 8.8, the -COOH groups of AAc are fully dissociated. Hence, the

AAc monomer anions will experience electrostatic repulsion24 as schematically explained in Figure 1a. When the polymerization process is triggered by the addition of free radical initiator in the presence of a methacrylate-functionalized silicon wafer, surfacetethered copolymerization commences, resulting in the formation of a copolymer layer in expanded conformation24 as schematically shown in Figure 1b. The copolymer layer was cross-linked during the polymerization process to help retain an expanded conformation. The concentration of the cross-linker was low enough to prevent hydrogel formation in the bulk solution. This expanded conformation would generate a free volume which would be occupied by water molecules below the LCST, enhancing the hydrophilicity of the copolymer. To test our hypothesis that polymerizing PNiPAAm in the presence of AAc and cross-linker in an alkaline medium on functionalized silicon surfaces could result in expanded conformations experiencing increased switchability, we have prepared different cross-linked coatings containing different ratios of PAAc and PNiPAAm and compared those coatings with pure cross-linked PNiPAAm coatings. To understand the role of PAAc in the coating, the overall monomer concentration was kept constant during polymerization; the silicon surface polymerized without AAc was termed S-1, while the copolymer samples of NiPAAm to AAc ratios of 72:1, 36:1, 23:1, and 17:1 were abbreviated as S-2, S-3, S-4, and S-5, respectively. The polymerization time for these samples was 45 min, while S-3T was the sample polymerized for 18 h with a 36:1 ratio of NiPAAm to AAc (see Table 1). Ellipsometric analysis was carried out to measure the thickness of the coatings (Table 1). The thickness for all samples except S-3T was in the range of 11.2 ( 0.4 (S-1) to 12.5 ( 0.6 (S-5) nm. Intermediate thicknesses of 12.4 ( 0.7, 12.0 ( 0.7, and 11.6 ( 0.7 nm were determined for S-2, S-3, and S-4, respectively. For sample S-3T, where polymerization was performed for 18 h, a more than doubled thickness of 26.2 ( 0.8 nm was determined. The low value of the standard deviation for all samples is indicative of a uniform polymer coating, which shows that our coatings made by fast free radical polymerization are equal or superior in quality to others obtained by electron beam polymerization34 and atom transfer radical polymerization (ATRP).23 X-ray photoelectron spectroscopy (XPS) analysis was then performed to confirm the presence of the copolymer coating on

(33) Tamura, T.; Uehara, H.; Ogawara, K.; Kawauchi, S.; Satoh, M.; Komiyama, J. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, 1523-1531.

(34) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506-5511.

The quantification of fluorescence intensity was performed with the GenePix Pro 3.0 fluorescence image analysis program. The images obtained by fluorescence microscopy were converted to gray scale before analysis with the GenePix program. A fluorescence spectrometer (LS 55, Perkin-Elmer Instruments) was used to study the reversibility of FITC-BSA protein adsorption on the silicon wafer surface. A freshly polymer coated silicon wafer sample of 1 × 2 cm2 dimension was dipped in a glass cuvette containing a stirred solution of 0.1 µg/mL FITC-BSA prepared in PBS of pH 7.4. The temperature of the cuvette was alternated between 20 and 40 °C by using a Lauda K-2/R (Brinkmann Instruments) thermostat. When the temperature of the cuvette was maintained at 40 °C, the suspended BSA protein molecules were expected to adsorb on the hydrophobic sample surfaces, which led to fluorescence depletion in solution. On the other hand, when the temperature of the cuvette was switched to 20 °C, the FITC-labeled protein molecules were expected to desorb from the hydrophilic sample surfaces into the surrounding solution, leading to higher fluorescence intensity in solution. This process of adsorption and desorption of protein molecules onto the sample surfaces was studied by changing the temperature of the cuvette. Fluorescence measurements were taken at each temperature point for various cycles after a 5 min holding time to allow for temperature equilibration. The fluorescence of the solution was measured using an emission wavelength of 520 nm with a photomultiplier voltage of 775 V, a scan rate of 200 nm/min, an excitation wavelength of 480 nm, and excitation and excitation/ emission slit widths of 5 nm.

3. Results and Discussion

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Figure 3. High-resolution C1s spectrum and peak fit of sample S-3.

Figure 2. XPS overview spectra collected on the (a) bare silicon wafer and (b) sample S-3. Table 2. Elemental Composition as Determined by XPS and Theoretical Means

Figure 4. DRIFT spectrum of sample S-3 showing the characteristic vibrations of PNiPAAm.

element concentration (atom %) element

bare silicon

S-3 (measured)

S-3 (theoretical)

C O N Si

2.2 22.2 1.1 74.6

51.4 27.8 20.8 0.0

74.7 13.0 12.3 0.0

the silicon substrate surface. Figure 2 shows XPS overview spectra for bare silicon and for sample S-3, the copolymer coating formed from a 36:1 NiPAAm/AAc mixture. The carbon, nitrogen, and oxygen peaks apparent in Figure 2b are evidence of the presence of the copolymer coating. Table 2 shows the measured and theoretical elemental compositions of this surface. The oxygen content was found to be significanly higher than the nitrogen content, which may be due to a surface enrichment in the PAAc component or due to aging of the coating under incorporation of oxygen. The nitrogen level was also higher than the theoretical amount, which may be due to the incorporation of more than stochiometric amounts of cross-linker in the coating. Such preferred incorporation of the nitrogen-rich cross-linker could arise from its bifunctionality and potentially higher inherent monomer reactivity. The complete attenuation of the Si signal from the underlying silicon wafer in the case of sample S-3 confirms that the deposition of the copolymer created a stable, pinhole-free coating with a thickness of at least 10 nm (the approximate information depth of the XPS method). Figure 3 shows the high-resolution C1s spectrum obtained for sample S-3. Three different peaks were fitted within the high-resolution C1s spectrum. The location of the C1s constituent peaks at binding energies of 285.5, 286.3, 287 and 287.8 eV can be assigned to the chemical bonding environments C-C/H, C-N, NsCdO, and O-CdO, respectively.35-38 Table 3 shows the experimentally determined relative percentage of C1s binding environments

within the copolymer, which is in good agreement with the theoretical values. Similar XPS results were obtained for the other copolymer coatings (data not shown). To further chemically describe the polymer coating on the silicon surfaces, DRIFT measurements were carried out on sample S-3. The results are presented in Figure 4. The DRIFT spectrum corresponds well to the expected vibrational spectrum for PNiPAAm. For example, the peak at 3310 cm-1 was assigned to the -NH stretching, vibration and the peak at 1550 cm-1 was due to the -NH bending or deformation vibration of PNiPAAm. The peak at 1630 cm-1 was attributed to the -CdO groups of PNiPAAm. A broad -OH peak at around 3400 cm-1 was also apparent, probably due to hydrogen-bonded water. Finally, the peak at 2970 cm-1 was assigned to -CH stretching vibrations.39,40 Since it is reported in the literature that the LCST of PNiPAAm varies with the amount and type of copolymer used in the polymerization,29 we measured the LCSTs for the different copolymers used in this study. The obtained values are tabulated in Table 1. The cross-linked neat PNiPAAm polymer which was deposited for sample S-1 shows an LCST of 26.7 °C. The LCST increased with increasing PAAc content in the copolymer from 32.3, 34.2, 35.8, and 37.1 °C for copolymer compositions for S-2, S-3, S-4, and S-5, respectively. (35) LeMieux, M. C.; Peleshanko, S.; Anderson, K. D.; Tsukruk, V. V. Langmuir 2007, 23, 265-273. (36) Bullett, N. A.; Talib, R. A.; Short, R. D.; McArthur, S. L.; Shard, A. G. Surf. Interface Anal. 2006, 38, 1109-1116. (37) Tu, H.; Heitzman, C. E.; Braun, P. V. Langmuir 2004, 20, 8313-8320. (38) Cole, M. A.; Jasieniak, M.; Voelcker, N. H.; Thissen, H.; Horn, R.; Griesser, H. J. Proc. SPIE 2007, 6416, 6416061-10. (39) Yamagata, Y.; Shiratori, S. Thin Solid Films 2003, 438-439, 238-242. (40) Zhang, J. L.; Srivastava, R. S.; Misra, R. D. K. Langmuir 2007, 23, 6342-6351.

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Figure 5. Water contact angles at 20 and 40 °C as well as the contact angle difference for samples S-1 to S-5.

Contact angles for all the samples above and below the LCST were measured to study their ability to switch between hydrophilic and hydrophobic states. At 20 °C, which is below the LCST, PNiPAAm swells in water by forming extensive hydrogen bonds with water molecules available in the surroundings, while above the LCST (at 40 °C), the same polymer expels water and shrinks by forming intra- and intermolecular hydrogen bonds due to entropic forces.41 For flat surfaces coated with PNiPAAm the difference in water contact angle as a measure of wettability is less than 30° according to a variety of studies.6,11,12,25,37 The literature further suggests that an increased osmotic pressure results in a rapidly discontinuous volume change during the phase transition41 and that a hydrogel with an expanded network can have greater temperature sensitivity.24 Hence, in the present study we hypothesized that wettability hysteresis below and above the LCST could be increased by forming an expanded conformation of the copolymer on the silicon wafer. The contact angles measured above and below the LCST are plotted and presented in Figure 5 for all the samples at 20 and 40 °C. For sample S-1 made only from NiPAAm and cross-linker bisAAm, contact angles of 49.5 ( 1.2° and 69.4 ( 0.8° were measured at 20 and 40 °C, respectively, showing a switchabililty in terms of a contact angle difference of around 20°. S-2 and S-3 showed slightly lower contact angles at 20 °C of 46.9 ( 0.8° and 46.2 ( 1.2°, respectively. Sample S-2 showed the same contact angle of 69.2 ( 0.3° at 40 °C as that of S-1. The contact angle difference for S-2 of 22.2° is slightly higher than that of S-1. In the case of S-3 a significant increase in the high-temperature contact angle (80.4 ( 1.4°) was observed. This gave rise to a contact angle difference of 34°. Such an increased switchability may be due to an expanded conformation of the copolymer which has enough free volume to accommodate water molecules below the LCST and an optimum copolymer composition for intra- and intermolecular isopropyl amido hydrogen bonding, enhancing hydrophobicity above the LCST. Enhanced hydrophobicity could also be explained by an increased surface roughness12 as a result of collapse of the expanded conformation which was evidenced in subsequent AFM studies. Though the low-temperature contact angles for samples S-4 and S-5 having higher AAc/NiPAAm ratios are similar to those of samples S-2 and S-3, the measured high-temperature contact angles are significantly lower with 59.2 ( 1.3° and 64.4 ( 2.4°, respectively, for S-4 and S-5. This may be due to the effect of the PAAc content in the copolymer which at these ratios may interfere with the entropic effects driving

Figure 6. AFM images obtained in Milli-Q water for (a) cleaned bare silicon at 25 °C, (b) sample S-3 at 25 °C, and (c) sample S-3 at 40 °C (z scale 10 nm).

intra- and intermolecular hydrogen bonding in the PNiPAAm structure, reducing the contact angle above the LCST. This issue of decreased or even eliminated temperature sensitivity of PNiPAAm as a result of copolymer content is reported in the literature.29-31 The surface topography in Milli-Q water with respect to temperature was studied by AFM. The bare silicon surface, which was cleaned by sonication in surfactant solution, demonstrated a root-mean-square (rms) surface roughness of 1.4 nm in Milli-Q water at 25 °C. A representative height image of this surface is presented in Figure 6a. As can be seen in Figure 6b, we observed a significant increase in surface roughness for sample S-3 at 25 °C in Milli-Q water (rms surface roughness value of 10.3 nm). At this temperature below the LCST, the coating is expected to be a water-swollen hydrogel. The surface of this coating had a granular topography with features of 50.5 ( 2.2 nm diameter. A further increase in the surface roughness for the same sample at 40 °C (rms surface roughness value of 12.7 nm) is observed. The height image of this surface is displayed in Figure 6c. The image shows that the cross-linked PNiPAAm chains have collapsed into nodules of 119.7 ( 8.4 nm diameter presumably due to intra- and intermolecular hydrogen bonding between the amide hydrogen and oxygen of the PNiPAAm above the LCST of the copolymer. This is somewhat unexpected since one might have expected the polymer layer to condense simply in the z direction (leading to a decrease of the coating thickness).

Table 3. Chemical Bonding Environments of the Polymer in S-3 as Derived from the XPS C1s High-Resolution Data and Theoretical Composition binding environment C-C/H C-N

composition (%) measured

theoretical

59.4 16.6

66.6 16.5

binding environment NsCdO O-CdO

composition (%) measured

theoretical

15.9 0.9

16.5 0.4

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Figure 8. Fluorescence microscopy images collected for sample S-3 in PBS solution maintained at (a) 40 °C and (b) 25 °C.

Figure 7. AFM image and section analysis for sample S-3T in air at 25 °C after a square was scratched at high contact force to remove the polymer layer.

AFM scratching experiments were used to confirm ellipsometric thickness measurements. Here, a small area of 650 × 650 nm2 at 25 °C was repeatedly scanned with the AFM tip at high loading force on the S-3T sample. Analysis was performed in air to be consistent with ellipsometry measurements. A 3 × 3 µm2 image at low loading force together with a representative cross-section is presented in Figure 7 showing the square-shaped scratch. An average thickness of around 25.5 nm was recorded, which is in good agreement with ellipsometry measurements. Next, we investigated biomolecule adsorption to the copolymer coatings below and above the LCST. Figure 8 displays fluorescence microscopy images of two S-3 samples that had been incubated in an FITC-BSA solution separately at 25 and 40 °C. There is a significant difference in the amount of adsorbed protein. The more hydrophobic surface above the LCST gave rise to high fluorescence levels of intensity of approximately 27 500 quanta (Figure 8a). At the same time, the surface which was exposed to protein solution at a temperature below the LCST showed a very low fluorescence intensity of approximately 3300 quanta, indicating that this surface is able to resist the adsorption of even a notoriously sticky protein such as BSA (Figure 8b). The other samples (S-1, S-2, S-4, and S-5) showed a similar trend of higher fluorescence intensity for the wafers soaked in protein solution at 40 °C and low fluorescence intensity for those soaked in protein solution at 25 °C. Finally, we tested the reversibility of protein adsorption for sample S-3 using a fluorescence spectrometer. Figure 9 shows that the fluorescence of an FITC-BSA solution fluctuates when (41) Casolaro, M. Macromolecules 1995, 28, 2351-2358. (42) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379-6380.

Figure 9. Fluorescence spectrophotometric study showing the reversible behavior of sample S-3 with respect to the adsorption and desorption of BSA-FITC as a function of temperature (n ) 3).

the temperature is changed around the LCST. The solution fluorescence drops at high temperature (above the LCST), due to adsorption of protein from solution on the sample surface. Higher solution fluorescence is observed at low temperature (below the LCST) as the surface becomes hydrophilic and desorption of the protein molecules from the sample surface takes place. Over three cycles, switchability was retained.

4. Conclusions Cross-linked copolymers of PNiPAAm and PAAc of different compositions were successfully grafted from a silicon substrate by fast surface-tethered free radical polymerization, resulting in the formation of novel ultrathin stimulus-sensitive films of expanded polymer networks. Elemental analysis by XPS and chemical analysis by FTIR confirm the presence of uniform and pinhole-free copolymer coatings on the silicon surface with a uniform ellipsometric thickness of 11-12 nm. Measurement of the LCST for all the samples indicates that the LCST increases (from 26.7 to 37.1 °C) with increasing amounts of PAAc in the copolymer. Contact angle measurements were performed on all samples with Milli-Q water. The highest wettability difference (34° contact angle difference) was observed for sample S-3. The surface topography was studied by AFM in Milli-Q water on bare and coated silicon below and above the LCST. The surface roughness increased drastically when the sample was heated

4244 Langmuir, Vol. 24, No. 8, 2008

above the LCST along with significant topographical changes. The reversible adsorption of FITC-BSA was demonstrated using fluorescence microscopy and spectrometry. Preparations utilized in the current study have demonstrated certain advantages over existing surfaces, for example, uniform thickness, better switchability between hydrophilicity and hydrophobicity, and a short coating time. The coatings can in principle be developed on other substrates and may be used for biomedical applications, in particular in cell sheet engineering for ocular surfaces, (43) Yamato, M.; Okano, T. Mater. Today 2004, 5, 42-47. (44) Kushida, A.; Yamato, M.; Isoi, Y.; Kikuchi, A.; Okano, T. Eur. Cells Mater. 2005, 10, 23-30.

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periodontal ligaments, cardiac patches, and bladder augmentation,43,44 as well as in drug delivery, for biomolecular separation and process control, and finally in microfluidics.45-47 Acknowledgment. Funding from the Flinders University Science & Engineering Program Grant Scheme is kindly acknowledged. LA703668S (45) Hoffman, A. S. J. Controlled Release 1987, 6, 297-305. (46) Saitoh, T.; Suzuki, Y.; Hiraide, M. Anal. Sci. 2002, 18, 203-205. (47) Lai, J. J.; Hoffman, J. M.; Ebara, M.; Hoffman, A. S.; Estournes, C.; Wattiaux, A.; Stayton, P. S. Langmuir 2005, 10, 23-30.