Temperature-Reversible Ultrathin Films of N-Isopropylacrylamide

Oct 27, 2007 - The concentration required to reach full coverage was found to be higher when adsorption occurred below the phase transition temperatur...
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Langmuir 2007, 23, 12159-12166

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Temperature-Reversible Ultrathin Films of N-Isopropylacrylamide Terpolymer Adsorbed at the Solid-Liquid Interface Lei Wan,† Harender S. Bisht,‡ Ye-Zi You,‡ David Oupicky,‡ and Guangzhao Mao*,† Departments of Chemical Engineering and Materials Science and Pharmaceutical Sciences, Wayne State UniVersity, Detroit, Michigan 48202 ReceiVed June 19, 2007. In Final Form: September 4, 2007 This article describes the stability and reversibility of ultrathin films of N-isopropylacrylamide (NIPA)-vinylimidazole (VI)-poly(ethylene glycol) (PEG) graft terpolymer adsorbed at the solid-liquid interface upon temperature cycling from below to above its phase transition temperature. The coil-to-globule and globule-to-coil phase transitions were captured by in situ fluid tapping atomic force microscopy (AFM). The film thickness of 1 nm was determined by AFM, X-ray photoelectron spectroscopy, and X-ray reflectivity. The concentration required to reach full coverage was found to be higher when adsorption occurred below the phase transition temperature. From 23 to 42 °C, the adsorbed NIPA terpolymer film was observed to be molecularly smooth, corresponding to the close-packed structure of flexible polymer coils. Particles containing between one and a few globules appeared abruptly at the interface at 42-43 °C, the same temperature as the solution phase transition temperature, which was determined by dynamic light scattering. The size of the particles did not change with temperature, whereas the number of particles increased with increasing temperature up to 60 °C. The particles correspond to the collapsed and associated state of the globules. The film morphological changes were found to be reversible upon temperature cycling. Subtle differences were observed between dip-coated and spin-coated films that are consistent with a higher degree of molecular freedom for spin-coated films. The study contributes to the fundamental understanding and applications of smart ultrathin films and coatings.

Introduction Smart temperature-responsive polymers (STRPs) that undergo large and abrupt physical or chemical changes in response to small changes in temperature1,2 are attractive materials for drug delivery,3-5 biosensing and immunoassays,6-8 biocatalysis,9-11 and size-selective separations.12-14 The near-physiological lower critical solution temperatures (LCSTs) of N-isopropylacrylamide(NIPA-) based polymers15 make them suitable for bioconjugation. For example, precipitation of genetically engineered elastin-like polypeptides and NIPA copolymers, induced by a local hyperthermia at 40-42 °C, has been used to selectively increase their accumulation in solid tumors.16 It is well-known that, when the solution temperature is raised above the LCST, the NIPA chain collapses from a fully hydrated random coil to a dehydrated compact globule and that the globule goes back to the coil below * Corresponding author. E-mail: [email protected]. † Department of Chemical Engineering and Materials Science. ‡ Department of Pharmaceutical Sciences. (1) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (2) Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173. (3) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1990, 13, 21-31. (4) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291-293. (5) Qiu, Y.; Park, K. AdV. Drug DeliVery ReV. 2001, 53, 321-339. (6) Monji, N.; Hoffman, A. S. Appl. Biochem. Biotechnol. 1987, 14, 107-120. (7) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G. H.; Harris, J. M.; Hoffman, A. S. Nature 1995, 378, 472-474. (8) Kuroda, K.; Swager, T. M. Macromolecules 2004, 37, 716-724. (9) Takeuchi, S.; Omodaka, I.; Hasegawa, K.; Maeda, Y.; Kitano, H. Makromol. Chem.-Macromol. Chem. Phys. 1993, 194, 1991-1999. (10) Bhattacharya, S.; Moss, R. A.; Ringsdorf, H.; Simon, J. Langmuir 1997, 13, 1869-1872. (11) Bergbreiter, D. E.; Case, B. L.; Liu, Y. S.; Caraway, J. W. Macromolecules 1998, 31, 6053-6062. (12) Ito, Y.; Kotera, S.; Inaba, M.; Kono, K.; Imanishi, Y. Polymer 1990, 31, 2157-2161. (13) Iwata, H.; Oodate, M.; Uyama, Y.; Amemiya, H.; Ikada, Y. J. Membr. Sci. 1991, 55, 119-130. (14) Park, Y. S.; Ito, Y.; Imanishi, Y. Langmuir 1998, 14, 910-914. (15) Heskins, M.; Guillet, J. E.; James, E. J. Macromol. Sci. A: Chem. 1968, 2, 1441. (16) Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. J. Controlled Release 2001, 74, 213-224.

the LCST, i.e., reversible coil-to-globule and coil-to-globule phase transitions occur.17,18 Subsequently, the solution phase separates into a NIPA-rich phase consisting of aggregated globules and a dilute solution phase. The LCST is driven by entropy to free the bound water molecules and to promote intramolecular hydrogen bonding and hydrophobic interactions. The LCST of NIPA-based polymers can be precisely varied in synthesis by introducing hydrophilic or hydrophobic co-monomers.19,20 The LCST is also sensitive to the ionic strength21 and the presence of surfactants.22,23 This article focuses on the thin film structure and properties of a new class of NIPA terpolymers that incorporate 1-vinylimidazole (VI) and poly(ethylene glycol) (PEG) grafted chains.24 It describes the reversible changes in film morphology as a function of solution temperature, as captured by in situ atomic force microscopy (AFM). The positive charges of the VI monomers below its pKa allow the polymer to adsorb onto the negatively charged mica. The weak base VI adds pH sensitivity to the polymer near its pKa (which is 6). The phase transition temperature increases to 45 °C with increasing VI content and with decreasing pH because the charged VI units perturb the hydrogen-bonding structure of the hydrating water molecules.24 The LCST of the terpolymer increases from temperatures below to temperatures above body temperature with a slight decrease of pH from 7 to 6, rendering it a promising material for biomedical applications. The PEG chains prevent aggregation of the polymer particles in solution. The presence of PEG chains in the structure (17) Goldstein, R. E. J. Chem. Phys. 1984, 80, 5340-5341. (18) Kubota, K.; Fujishige, S.; Ando, I. J. Phys. Chem. 1990, 94, 5154-5158. (19) Chen, G. H.; Hoffman, A. S. Nature 1995, 373, 49-52. (20) Neradovic, D.; Hinrichs, W. L. J.; Kettenes-van, den Bosch, J. J.; Hennink, W. E. Macromol. Rapid Commun. 1999, 20, 577-581. (21) Inomata, H.; Goto, S.; Saito, S. Langmuir 1992, 8, 1030-1031. (22) Inomata, H.; Goto, S.; Otake, K.; Saito, S. Langmuir 1992, 8, 687-690. (23) Meewes, M.; Ricka, J.; Desilva, M.; Nyffenegger, R.; Binkert, T. Macromolecules 1991, 24, 5811-5816. (24) Bisht, H. S.; Wan, L.; Mao, G.; Oupicky, D. Polymer 2005, 46, 7945.

10.1021/la701819q CCC: $37.00 © 2007 American Chemical Society Published on Web 10/27/2007

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of the terpolymers provides them with pH-independent colloidal stabilization to prevent phase separation. In this work, the interfacial structure was characterized by AFM, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity (XRR), and contact angle goniometry. The temperatureresponsive interfacial structure was compared to the polymer behavior in solution as studied by dynamic light scattering (DLS). The NIPA terpolymer was deposited by dip coating and spin coating onto mica. A thermal unit capable of varying the temperature in an AFM fluid cell from ambient temperature to 60 °C enabled the real-time observation of molecular structural changes in the adsorbed film at the solution-mica interface. Real-time AFM captured a structural transition from smooth to particulate topography in 1-nm-thick films when the temperature was raised above 42-43 °C. This interfacial transition was found to be reversible and to coincide with the LCST of the polymer in solution.

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Materials. Deionized water with resistivity equal to or greater than 18 MΩ cm (Nanopure System, Barnstead) was used in solution preparation. Grade V5 muscovite mica was purchased from Ted Pella and hand cleaved just before use. Mica was used as the main substrate in all measurements except XRR where silicon wafer was used. One-sided polished N-type silicon (100) wafers (test grade with a resistivity of 1-2 Ω cm and a thickness of 500 ( 25 µm) were purchased from Wafer World. The precut silicon was rinsed with ethanol and acetone and then oxidized by the RCA method.25,26 The samples were cleaned at 60 °C under ultrasonication in 1:2:8 HCl/H2O2/deionized water (by volume) for 30 min and then in 1:2:7 NH4OH/H2O2/deionized water (by volume) for another 30 min. Phosphate buffer was purchased from Fisher Scientific and used as received. N-Isopropylacrylamide (NIPA), 1-vinylimidazole (VI), poly(ethylene glycol) methyl ether methacrylate with average molecular weight 2080 (PEGMA, 50% solution in water), and azobisisobutyronitrile (AIBN) were purchased from Aldrich. NIPA Terpolymer Synthesis. NIPA (0.67 M), VI (0.078 M), PEGMA (15.3 mM), and AIBN (7 mM) were dissolved in acetone, transferred into a glass ampule, and deoxygenated by a stream of N2. The ampule was sealed, and polymerization was carried out in a water bath at 60 °C for 24 h. The reaction mixture was then added dropwise to cold diethyl ether. The precipitated polymer was isolated by filtration, reprecipitated from methanol solution, and dried overnight in a vacuum before analysis. The composition of the terpolymer was determined in D2O solutions by 1H NMR analysis using a Varian 400 MHz NMR spectrometer. Areas of -N-CH proton of NIPA (∼4 ppm), protons of the VI cycle (-CH, ∼7.2 ppm), and methylene protons of PEG (3.6 ppm) were used to calculate the composition of the terpolymer. The VI content was also confirmed by titration with 0.01 M NaOH. The number-average (Mn) and weightaverage (Mw) molecular weights and the polydispersity (Mw/Mn) of the polymer were determined by size-exclusion chromatography (SEC) using a Shimadzu LC-10ADVP liquid chromatograph equipped with a Shimadzu CTO-10ASVP column oven and a Waters Styragel HR-4E 7.8 × 300 mm column. The system was also equipped with a seven-angle BIMwA static light-scattering detector and a BIDNDC differential refractometer (both from Brookhaven Instruments). The BIMwA detector was equipped with a 30 mW vertically polarized solid-state laser (660 nm) as a light source. Tetrahydrofuran was used as the eluent at a flow rate of 1 mL/min at 30 °C. GPC data were analyzed using PSS WinGPC Unity software from Polymer Standards Services. Film Preparation. The NIPA terpolymer was dissolved in 50 mM phosphate buffer (pH 5) to obtain 1 g/L stock solution. The stock solution was diluted with deionized water to obtain different

concentrations with pH values between 4.8 and 5.0. The NIPA terpolymer films were prepared using either dip coating or spin coating at room temperature (23 °C). Some films were made at 55 °C, which is above the LCST. In dip coating, freshly cleaved mica was immersed in the terpolymer solution for 30 min, rinsed with deionized water, and dried with N2. In spin coating, 100 µL of terpolymer solution was placed on the substrate, and after a 1-min standing time, the substrate was spun at 3000 rpm for 1 min (PM101DT-R485 photo resist spinner, Headway Research). The spin-coated film was rinsed with deionized water to remove excess polymer and dried with N2. In XRR experiments, an RCA-cleaned silicon wafer was used instead of mica. Dynamic Light Scattering (DLS). DLS was used to determine the hydrodynamic diameter and ζ potential of the terpolymer in solution as a function of temperature. A ZetaPlus Particle Size and Zeta Potential Analyzer (Brookhaven Instruments) equipped with a 35 mW solid-state laser (658 nm) was used. Scattered light was detected at 90°. The LCST of the terpolymer was estimated from the onset temperature in the scattering intensity versus temperature curve using a polymer concentration of 0.5 g/L (pH 5). Mean hydrodynamic diameters were calculated for the size distribution by weight, assuming a log-normal distribution using the supplied algorithm, and the results are expressed as mean ( standard deviation of three runs. ζ potential values were calculated from measured velocities using the Smoluchowski equation, and the results are expressed as mean ( standard deviation of 10 runs. X-ray Photoelectron Spectroscopy (XPS). XPS analysis of the thin film samples on mica was conducted with a PHI 5500 spectrometer (Perkin-Elmer) equipped with an aluminum KR X-ray radiation source (1486.6 eV) and an AugerScan system control (RBD Enterprises). The pressure in the chamber was below 2 × 10-9 Torr before the data were taken, and the voltage and current of the anode were 15 kV and 13.5 mA, respectively. The takeoff angle was set at 45°. The pass energies for survey and multiplex scans were 117.40 and 23.50 eV, respectively. The binding energy scale was referenced by setting the C 1s peak maximum at 285.0 eV. X-ray Reflectivity (XRR). Polymer films deposited on silicon wafers were studied with a SmartLab high-resolution θ/θ XRD system using Cu KR radiation (λ ) 1.54 Å) (Rigaku). The scan range was 0-10° with a step size of 0.01° and a speed of 1°/min. The incident and receiving slit sizes were 0.05 and 0.25 mm, respectively. The reflectivity data were fitted with established algorithms to obtain the film thickness.27,28 Contact Angle Goniometry. The contact angle was measured with an NRL contact angle goniometer (model 100, Rame-Hart) in the laboratory atmosphere. A 20-µL droplet of the sample was placed on the substrate, and contact angles were read on both sides of the droplet. Five droplets were placed at various spots near the center of the substrates, and contact angles were averaged (with a typical error of measurement of (3°). AFM. AFM imaging was conducted using a Nanoscope III MultiMode atomic force microscope equipped with a type E piezoelectric scanner with maximum scan ranges of 10 µm (X and Y directions) and 2.5 µm (Z direction) from VEECO/Digital Instruments. Ex situ AFM imaging of samples was conducted in tapping mode (oscillation frequency ≈ 250-300 kHz) in ambient atmosphere using etched silicon probes (TESP, VEECO) with a nominal radius of curvature of less than 10 nm. In situ AFM imaging was conducted in liquid tapping mode (oscillation frequency ≈ 8 kHz, line scan rate ) 2-3 µm/s) using silicon nitride probes (NP, VEECO) with a radius of curvature of 20 nm and a cantilever spring constant of 0.38 N/m as provided by the manufacturer. In situ AFM imaging of the phase transition was conducted using a temperature controller (model 2216e, Eurotherm) capable of controlling the fluid temperature in the AFM fluid cell from room temperature to 60 °C. The AFM scanner was calibrated separately when used with the temperature controller. The heating unit, placed directly under the

(25) Kern, W. J. Electrochem. Soc. 1990, 137, 1887-1892. (26) Wu, B.; Mao, G.; Ng, K. Y. S. Colloids Surf. A: Physicochem. Eng. Aspects 2000, 162, 203-213.

(27) Klappe, J. G. E.; Fewster, P. F. J. Appl. Crystallogr. 1994, 27, 103-110. (28) Dane, A. D.; Veldhuis, A.; de Boer, D. K. G.; Leenaers, A. J. G.; Buydens, L. M. C. Physica B 1998, 253, 254-268.

Materials and Methods

Temperature-ReVersible Films of NIPA Terpolymer

Figure 1. Chemical structure of the NIPA terpolymer and its molecular weight distribution as determined by size-exclusion chromatography. fluid cell but isolated from the scanner by a spacer block, allowed for rapid changes (approximately 4 °C/min for heating and 2 °C/min for cooling) of the solution temperature. Only height images are shown. Height images were plane-fit in the fast scan direction with no additional filtering operation. The surface roughness of the films was determined using the root-mean-square surface roughness Rq ) x(∑zi2/n), where zi is the height value and n is the number of pixels in the image.

Results and Discussion Temperature-Responsive Structure in Solution. The terpolymer used here contained 12 mol % VI and 1 mol % PEG grafts (Figure 1) according to 1H NMR analysis. The VI content was also confirmed by titration with 0.01 M NaOH. Although gel formation due to unwanted cross-linking during the copolymerization of NIPA and PEG methacrylates has been reported,29 our SEC analysis showed a monomodal distribution of molecular weights with no evidence of gel formation (Figure 1). The weightaverage molecular weight (Mw) of the prepared terpolymer was 2.06 × 105, and the polydispersity (Mw/Mn) was 2.51. The relatively high polydispersity of molecular weights was due to significant differences in the reactivity of PEG macromonomer and NIPA and VI monomers.30 The phase transition temperature of the terpolymer was determined from the temperature dependence of the scattering intensity measured at a 90° scattering angle from 0.5 g/L solution at pH 5 in a glass cuvette. The temperature was increased stepwise in 1 °C increments in the range of 25-55 °C, and scattering intensity and mean hydrodynamic radius were measured after 3 min of equilibration at each temperature. The phase transition temperature at 40-43 °C was determined by the sharp increase of the scattering intensity in Figure 2a. Whereas the terpolymer exists as a unimer solution below the transition with an average hydrodynamic diameter of around 25 nm, it forms particles with sizes in the range of 44-55 nm. The size decrease above the phase transition temperature was not accompanied by a corresponding decrease in scattering intensity, suggesting further association of more densely packed particles. Measurement of the ζ potential (Table 1) revealed that the particles are positively charged, suggesting localization of the charged VI units on the surface of the coils and associated globules. The positive charges enable the adsorption of the NIPA polymer on negatively charged mica. Film Thickness Measurements. The previously studied NIPA polymer films generally had film thicknesses above 10 nm.31-37 (29) Virtanen, J.; Baron, C.; Tenhu, H. Macromolecules 2000, 33, 336-341. (30) Gramm, S.; Komber, H.; Schmaljohann, D. J. Polym. Sci. A: Polym. Chem. 2005, 43, 142-148.

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Figure 2. Hydrodynamic radius dependence on temperature (pH 5). Table 1. Dependence of ζ Potential in 0.5 g/L NIPA Terpolymer Solution (pH 5) on Temperature temperature (°C)

ζ potential (mV)

25 40 48

8.9 ( 0.7 8.2 ( 1.4 20.4 ( 4.1

This study focuses on the structural transition exhibited in ultrathin NIPA films with thicknesses approaching 1 nm, which means that the polymer chains were confined to a 2-D adsorbed state with the majority of segments in close proximity to the substrate. The film thickness was significantly below the radius of gyration of the NIPA terpolymer coils in solution. The film thickness influences the extent of conformational transition of NIPA polymers at the interface. AFM, XPS, and XRR were used to determine the film thickness below the LCST. The concentration used for all three methods was 0.05 g/L. We estimated a minimum film thickness of 0.8-0.9 nm for the dip-coated film on mica by an AFM scratch test. The scratch test was conducted on an area of 400 × 400 nm2 by scanning with an AFM tip in contact mode at 3 Hz and a constant force between 100 and 150 nN for 10 min. The film thickness was determined by AFM sectional height analysis of the height difference between the previously untouched and the scratched area. The film thickness was also estimated by an XPS analysis method from the literature.38 The presence of the NIPA polymer was indicated by the appearance of the N 1s peak (399.5 eV). Assuming a homogeneous film, the N/Si molar ratio is a function of the film thickness and degree of surface coverage. The thickness thus computed from the variation in N/Si ratio with concentration was 0.8-0.9 nm for a dip-coated film on mica.39 The thickness of the dip-coated film on an RCA-treated silicon wafer was determined to be 1.3 ( 0.3 nm by XRR. The film thickness was found to be lower for the spin-coated films, 0.8 ( 0.2 nm, also determined by XRR. This small but finite difference in film thickness for the first layer next to the substrate between the spin-coated and dip-coated samples is opposite to what has been reported in the layer-by(31) Suzuki, A.; Yamazaki, M.; Kobiki, Y.; Suzuki, H. Macromolecules 1997, 30, 2350-2354. (32) Zhu, P. W.; Napper, D. H. Phys. ReV. E 1998, 57, 3101-3106. (33) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Langmuir 1998, 14, 4657-4662. (34) Lee, L. T.; Jean, B.; Menelle, A. Langmuir 1999, 15, 3267-3272. (35) Fu, Q.; Rao, G. V. R.; Basame, S. B.; Keller, D. J.; Artyushkova, K.; Fulghum, J. E.; Lopez, G. P. J. Am. Chem. Soc. 2004, 126, 8904-8905. (36) Sun, T.; Wang, G.; Feng, L.; Liu, B.; Ma, Y.; Jiang, L.; Dhu, D. Angew. Chem., Int. Ed. 2004, 43, 357-360. (37) Cheng, X. H.; 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. (38) Callewaert, M.; Grandfils, C.; Boulange-Petermann, L.; Rouxhet, P. G. J. Colloid Interface Sci. 2004, 276, 299-305.

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layer (LbL) multilayer film literature.40,41 In LbL films, spincoated films generally exhibit a higher film thickness than dipcoated films. It should be pointed out that, in all of these cases, similarly to dip coating, the substrate containing the spin-coated films was thoroughly rinsed with water after deposition of each layer to remove nonadsorbing polymers. In conclusion, all of the film thickness values point to a very thin layer of 2-D coils for the adsorbed NIPA terpolymer. Therefore, unlike previous studies of NIPA phase transition at an interface where the majority of the chain segments were free from the substrate, almost all of the NIPA terpolymer segments can be assumed to be in direct contact with the substrate in this case via electrostatic interactions between the positively charged VI unit and the negatively charged mica. Temperature-Responsive Wettability. The contact angle of water was measured on films spin-coated from 0.05 g/L polymer solution on mica and on a silicon wafer. The contact angles were measured below (23 °C) and above (55 °C) the LCST. For the contact angle measurements above the LCST, the films were immersed and allowed to swell in water at 55 °C for 20 min and then left in the oven to dry at 55 °C before measurements. The contact angle of the film on a silicon wafer changed from 40.2° ( 2.0° below the LCST to 54.4° ( 5.0° above the LCST. An increase in contact angle is expected as a polymer acquires a more hydrophobic nature above its LCST. However, the contact angle measured for the mica-supported film decreased from 40.4° ( 2.2° below the LCST to 33.3° ( 2.8° above the LCST, probably as a result of exposure of the bare mica substrate after the coil-to-globule transition. Temperature-Responsive Structure at the Solid-Liquid Interface. The adsorption behavior of the NIPA terpolymer on mica as a function of solution concentration and temperature was studied by ex situ and in situ AFM. The film coverage was monitored to determine the concentration range necessary for full coverage. Figure 3a-d shows adsorbed films on mica with increasing concentration from 10-4 to 10-2 g/L (pH 5) and an adsorption temperature of 23 °C. The films were rinsed, dried, and imaged by AFM in tapping mode in air. The features in Figure 3a correspond to individual polymer coils with an average diameter of 12.0 nm. The heights of the features, 0.3-0.5 nm, are similar to the thicknesses of individual polymer coils flattened by surface adsorption as reported in the literature.42-45 Occasionally, highly stretched coils or chains were observed. At 5 × 10-4 g/L (Figure 3b), the coils associated with each other to form larger domains that coexisted with remaining individual coils. The height of the domains was identical to that of the coils. Adsorption at 10-3 g/L yielded smooth and full monolayer coverage (Figure 3c). The root-mean-square surface roughness, (39) To determine the film thickness, we used a method provided by reference 38. The relative sensitivity factors were iN ) 0.477 and iSi ) 0.328, and the photoionization cross sections were σN ) 1.8 and σSi ) 0.817 as provided by the XPS manufacturer. The elemental concentrations in the adsorbed polymer film and mica substratum were calculated to be CNad ) 10.46 M and CSisu ) 16.68 M, respectively. The inelastic electron mean free paths were calculated to be λSisu ) 3.33 nm in mica and λSiad ) 3.99 nm and λNad ) 3.11 nm in the polymer film, assuming a mica density of 2.8 g/cm3 and a polymer density of 1.127 g/cm3. The N-to-Si molar ratio in the film was determined to be 4.9%:12.3%. The film thickness at full coverage was obtained from the surface coverage versus thickness plot. (40) Cho, J.; Char, K.; Hong, J. D.; Lee, K. B. AdV. Mater. 2001, 13, 10761078. (41) Chiarelli, P. A.; Johal, M. S.; Casson, J. L.; Roberts, J. B.; Robinson, J. M.; Wang, H. L. AdV. Mater. 2001, 13, 1167-1171. (42) Minko, S.; Kiriy, A.; Gorodyska, G.; Stamm, M. J. Am. Chem. Soc. 2002, 124, 3218-3219. (43) Kiriy, A.; Gorodyska, G.; Minko, S.; Jaeger, W.; Stepanek, P.; Stamm, M. J. Am. Chem. Soc. 2002, 124, 13454-13462. (44) Kumaki, J.; Hashimoto, T. J. Am. Chem. Soc. 2003, 125, 4907-4917. (45) Minko, S.; Roiter, Y. Curr. Opin. Colloid Interface Sci. 2005, 10, 9-15.

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Figure 3. AFM images (tapping mode in air) of NIPA terpolymer films made at room temperature from different concentrations: (a) 1 × 10-4, (b) 5 × 10-4, (c) 1 × 10-3, and (d) 1 × 10-2 g/L. The films were dip-coated on mica at 23 °C. The z range is 4 nm. (e) AFM height image (tapping mode in deionized water) of a spincoated film on mica at room temperature from 5 × 10-4 g/L and phosphate buffer (pH 3). The z range is 3 nm. The scan size of the inset image is 115 nm.

Rq, was 0.2 nm in a 1 × 1 µm2 area. It is evident that 1 × 10-3 g/L is close to the minimum concentration necessary for full coverage because only a few dark spots existed in the otherwise continuous film. Films adsorbed at concentrations above 1 × 10-3 g/L remained smooth and continuous (Figure 3d). Adsorption at a lower pH (i.e., 3.0) using 0.01 M phosphate buffer produced stretched chains (Figure 3e) whose contour length was estimated using WSxM software (Nanotec Electronica, version 4.0). The measured contour length of 88.0 ( 35.0 nm is significantly lower than the calculated value of 448 nm for NIPA terpolymer with a molecular weight of 2.06 × 105 g/mol.46 Such a discrepancy between AFM-determined and theoretical values is often observed and attributed to the combined effect of the necklace chain structure and the spatial resolution limit of the AFM.43,47 The side chains were invisible in these AFM images, probably because of its low percentage (