and Diblock Polystyrene-block-poly(methyl methacrylate) - American

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Biomacromolecules 2009, 10, 1004–1012

Water-Stable Diblock Polystyrene-block-poly(2-vinyl pyridine) and Diblock Polystyrene-block-poly(methyl methacrylate) Cylindrical Patterned Surfaces Inhibit Settlement of Zoospores of the Green Alga Ulva Claudia M. Grozea,† Nikhil Gunari,‡ John A. Finlay,§ Daniel Grozea,| Maureen E. Callow,§ James A. Callow,§ Zheng-Hong Lu,| and Gilbert C. Walker*,† Department of Chemistry, University of Toronto, Toronto M5S 3H6, Canada, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom, and Department of Materials Science and Engineering, University of Toronto, Toronto M5S 3E4, Canada Received January 16, 2009

Nanopatterned surfaces with hydrophobic and hydrophilic domains were produced using the diblock copolymer polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl methacrylate) (PS-bPMMA). The PS-b-P2VP diblock copolymer, mixed with the cross-linker benzophenone and spin-coated onto silicon wafers, showed self-assembled cylindrical structures, which were retained after UV treatment for crosslinking. The thin films displayed cylindrical domains after immersion in water. This study shows that pattern retention in water is possible for a long period of time, at least for two weeks in pure water and three weeks in artificial seawater. The PS-b-PMMA diblock showed self-assembled cylindrical structures. PS-b-P2VP and PSb-PMMA cylindrical patterned surfaces showed reduced settlement of zoospores of the green alga UlVa compared to unpatterned surfaces. The copolymers were investigated using atomic force microscopy and X-ray photoelectron spectroscopy.

Introduction Biofouling of surfaces is a problem for both the biomedical field, concerned with making novel devices such as implants, and various industries, including the marine coatings industry, concerned with making novel coatings for structures immersed in water such as ship hulls. To prevent biofouling without the use of biocides, materials must be developed that have antifouling properties, that is, inhibit the settlement of the colonizing species such as spores and larvae, and/or have fouling release properties, that is, exhibit low adhesion of the fouling organisms so that they are “released” by hydrodynamic forces.1 Zoospores of the fouling macroalga UlVa respond to a number of surface properties including wettability,2,3 charge,4 surface chemistry,5,6 and topography.7,8 Schumacher et al. fabricated a range of topographies in polydimethylsiloxane and depending on the design and length-scale of the pattern, settlement of zoospores of UlVa was either promoted or inhibited.7,8 However, the response of spores to both nanoscale topographic features and variation in local chemistry has not been explored. Nanoscale patterned surfaces show promise for many applications and studies involving cell-surface interactions,9,10 and also as scaffolds for making nanowires,11 quantum dots,12 and organic optoelectronic applications.13 Self-assembly is becoming a powerful “bottom-up” method for engineering nanostructured materials. In confined geometries, diblock copolymers can self* To whom correspondence should be addressed. Email: gwalker@ chem.utoronto.ca. † Department of Chemistry, University of Toronto. ‡ University of Pittsburgh. § University of Birmingham. | Department of Materials Science and Engineering, University of Toronto.

assemble in a variety of ordered structures with domains, on the nanoscale length such as spheres, cylinders, lamellae, or double gyroids.14-17 The phase behavior of copolymers depends on the volume fraction of each block (f) and on the product of the degree of polymerization (N) and the segment-segment (Flory-Huggins) interaction parameter (χ).18-20 In thin films, the structure is also influenced by the film thickness and the interfacial interactions.21-23 On the other hand, a random copolymer of the same molecular fraction of each monomer does not phase segregate or show nanoscale structures. Previous studies have focused on controlling the orientation and the lateral ordering of the domains. For example, Bodycomb et al. used temperature gradients to induce a lamella orientation in polystyrene-block-polyisoprene films.24 Alternatively, a chemically patterned surface approach has been used by Kim et al. to produce lamella orientated polystyrene-block-poly(methyl methacrylate).25 Recently, solvent vapor annealing has become widespread. Kim et al. showed that nearly defect-free cylinder orientated polystyrene-block-poly(ethylene oxide) can be obtained by controlling the rate of solvent annealing.26 However, the properties of these patterned surfaces, in particular the ability of these patterns to survive in environments different than air, have not been examined in detail. In biological studies, in situ measurements are preferred to mimic the actual environment of the species of interest. For applications such as antifouling coatings, studies should therefore be conducted in an aqueous environment because it is the hydrated surface with which the settling cell, spore, or larva interacts. Thus, nanoscale copolymer patterns geared toward these studies must be stable for a long period submerged in water. A regular spacing of the features is desirable to probe for biological responses on specific length scales, which is a

10.1021/bm900065b CCC: $40.75  2009 American Chemical Society Published on Web 03/18/2009

Diblock Surfaces Inhibit Settlement of Zoospores

promise of diblock systems. However, to the best of our knowledge no diblock system durable to long-term exposure to water has been reported. In this study, we report a method of obtaining cylindrical nanopatterned surfaces of polystyreneblock-poly(2-vinyl pyridine) (PS-b-P2VP) that are stable in water. These surfaces provide both physical and chemical patterning due to the hydrophobic and hydrophilic nature of the domains. By photo cross-linking the cylindrical domains, a more durable material that can withstand immersion in water for extended periods of time is produced. We also show the response of the patterned surface to a biological system, by quantifying the settlement (i.e., initial attachment) of zoospores of the green alga UlVa. The pyrifom spores are 7-8 µm in length and are motile up the point at which they commit to settlement, hence, they can “choose” whether to settle on a surface.27

Experimental Section Materials. Hydroxy terminated polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) diblock copolymer (Polymer Source) and the crosslinker benzophenone (BP; Sigma Aldrich) were used in this experiment. PS-b-P2VP polymer has a polydispersity index of 1.06 and a number average molecular weight for PS of 75000 g/mol and for P2PV of 21000 g/mol. Thin films were prepared by spin coating 0.3 wt % toluene solutions of the diblock copolymer or the diblock mixed with BP (1:1 w/w) on silicon substrates at 2000 rpm for 45 s. The silicon substrates were prepared by cleaning in piranha solution (3:1 v/v concentrated H2SO4/30% H2O2) for 10 min. Caution: Piranha is a Very strong oxidant. The thin films were solvent vapor-annealed using toluene and chloroform (1/1 v/v) for 3 h. The PS-b-P2VP with BP films were UV irradiated using a Mercury Arc Lamp (Pen-Ray, 90-0012-01) with an intensity of 15 mW/cm2 for 5 min in air. Polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer (Polymer Source) with a polydispersity index of 1.10 was used in this experiment. The number average molecular weight for PS is 130000 g/mol and for PMMA is 133000 g/mol. Thin films were prepared by spin coating 0.3 wt % toluene solutions of the diblock copolymer on piranha-cleaned silicon substrates at 2000 rpm for 45 s. The thin films were solvent vapor-annealed using acetone for 5 h. Polystyrene (PS; Polymer Source) with a polydispersity index of 1.05 was used. The number average molecular weight is 131000 g/mol and the weight average molecular weight is 138000 g/mol. Poly(2vinyl pyridine) (P2VP; Polymer Source) with a polydispersity index of 1.09 was used. The number average molecular weight is 22000 g/mol and the weight average molecular weight is 24000 g/mol. Polystyreneco-2-vinyl pyridine random copolymer (P(S-r-VP); Polymer Source) with a polydispersity index of 1.7 was used. The number average molecular weight is 75000 g/mol and the weight average molecular weight is 128000 g/mol. Thin films were prepared by spin coating 0.3 wt % toluene solutions of PS, P2VP, or P(S-r-VP) on piranha cleaned silicon substrates at 2000 rpm for 45 s. Characterization of Morphology in Thin Films. The patterned diblock copolymer thin films were imaged using Atomic Force Microscopy (AFM) to examine the surface topography. The AFM (Digital Instruments, Dimension 5000) operated in Tapping Mode was used to perform the measurements in air. Rectangular shaped silicon probes (NanoWorld, NCH) with resonance frequencies in the range 280-320 kHz and a spring constant of 40 N/m were used. All measurements in solution were obtained using the Molecular Force Probe AFM (Asylum Research, MFP-3D). V-shaped, silicon nitride cantilevers (Asylum Research, AR-iDrive) exhibiting a nominal spring constant of 100 pN/nm were used. AFM images in solution were obtained in the iDrive mode in which the cantilever is actuated magnetically. A small oscillating current flows through the cantilever legs in the presence of a magnetic field causing it to vibrate. The iDrive mode simplifies in fluid imaging by reducing the multitude of resonance

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peaks mechanically coupled from the holder and fluid. The mechanical properties of the thin film were measured by AFM imaging and nanoindentation using rectangular shaped silicon tips (NanoWorld, NCH) with nominal a spring constant of 40 N/m. In short, a 1 × 1 µm AFM image was obtained in tapping mode. After imaging, the AFM stylus was positioned over the P2VP region and then the PS region and two indentation curves were obtained over each specific region of interest. Similarly different areas of the thin film were scanned and indentation plots were obtained. The resulting force-indentation curves were analyzed with custom-programmed analysis software (Wavemetrics, Igor Pro). PS, P2VP, and P(S-r-VP) films were imaged in air using an AFM (Digital Instruments, Dimension 5000) operated in Tapping Mode. Rectangular shaped silicon probes (NanoWorld, NCH) with resonance frequencies in the range 280-320 kHz and a spring constant of 40 N/m were used. The measurements in solution were obtained using the Molecular Force Probe AFM (Asylum Research, MFP-3D) operated in Contact mode. V-shaped, silicon nitride cantilevers (Veeco, DNP) exhibiting a nominal spring constant of 0.12 N/nm were used. Ellipsometry was used to measure the thickness of the films. A contact angle meter (KSV Instruments, Cam101) was used to measure the advancing contact angle of the films using ultrapure water (Mili-Q 18 MΩ). X-ray photoelectron spectroscopy was used to obtain the chemical composition of the polymer films. An ESCA (Phi, 5500) system with an Al KR (1486.7 eV) monochromated X-ray source was used to obtain the spectra at a takeoff angle of 45°. FT-IR spectroscopy (Perkin-Elmer, Spectrum BX FT-IR) was performed to obtain structural information on the films. UlWa Zoospore Settlement Assay. Attachment experiments were performed using zoospores released from mature UlVa linza plants using standard methods.2-8 In brief, zoospores were settled in individual dishes containing 10 mL of zoospore suspension, in the dark at ∼20 °C. Each dish contained one silicon wafer (size 2.5 cm × 2.5 cm) coated in polymer. After 60 min the slides were washed in seawater to remove unsettled zoospores. Slides were fixed using 2.5% glutaraldehyde in seawater. The density of zoospores attached to the surface was counted on each of the replicate silicon wafers using an image analysis system (Imaging Associates Ltd.) attached to an epifluorescence microscope (Zeiss, Aksioskop 2). Spores were visualized by autofluorescence of chlorophyll. Counts were made for 30 fields of view (each 0.17 mm2) on each wafer.

Results and Discussion 1. Ordering in Block Copolymer Thin Films. The morphology of the copolymer thin films was characterized using AFM. In Figure 1A the height image of a spin-cast and solvent annealed PS-b-P2VP film can be seen. The lattice spacing of the hexagonal packing domains is 48 ( 2 nm. The weight ratio between PS and P2VP is about 3:1, thus, in this system it is expected that the matrix would be PS and the cylinders would be P2VP. The brighter areas in the image correspond with the PS matrix, while the darker areas correspond to the P2VP domains. The solubility of the blocks and the solvent evaporation rate are responsible for the orientation of the cylinders normal to the surface. During annealing, the copolymer swells with the solvent. The solvent imparts mobility to the copolymer, thus the block can reorganize. However, some defects such as holes can be present as can be seen in the Figure 1A. The larger darker area in the image correspond to a hole in the film. The formation of holes is due to the mismatch between the thickness of the copolymer layers and the periodicity of the bulk.28 The cylindrical domains are orientated normal to the surface, however, the layer adjacent to the silicon surface has the domains orientated parallel to the surface because the P2VP block is more polar than the PS block. Liu et al. found that the film thickness for an asymmetric PS-b-P2VP block copolymer

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Figure 1. AFM height images of (A) solvent annealed PS-b-P2VP film, (B) solvent annealed PS-b-P2VP and BP film, (C) solvent annealed and UV irradiated PS-b-P2VP and BP film, and (D) solvent annealed PS-b-PMMA film. Image size: 1000 × 1000 nm. Z range: 20 nm. Table 1. Advancing Water Contact Angles (CA)

Table 2. XPS Analysis of Three Different Samples

CA after solvent CA after UV CA after 2 h CA after 8 annealing irradiation in water days in water PS-b-P2VP PS-b-P2VP and BP PS-b-PMMA

94 ( 3° 94 ( 3°

not applicable 61 ( 3°

72 ( 3° 64 ( 4°

63 ( 3° 62 ( 3°

84 ( 3°

not applicable

80 ( 3°

70 ( 3°

can be quantized by t ) (n + R)L + β where, t is the film thickness, n is the number of bulk cylindrical layers, R is the fractional thickness of the top cylindrical layer, L is the layer period, and β is the thickness of the lamellar layer adjacent to the surface.29 When the thickness of the film is different from t by L/2, holes and islands will appear. In addition, these holes can also be produced by nucleation from small impurities present on the surface. The advancing water contact angle of these surfaces is 94 ( 3°, as shown in Table 1, which corresponds to a hydrophobic surface. This contact angle for the copolymer sytem is not surprising since the advancing water contact angle of pure PS is 95 ( 3° and of pure P2VP is 65 ( 3°. The advancing water contact angle of the random copolymer P(Sr-VP) is 88 ( 3°. The thickness of the films used throughout this study is 20 ( 3 nm. In Figure 1B, the height image of a spin-cast and solvent annealed film of PS-b-P2VP mixed with BP is shown. The brighter areas in the image correspond to the PS matrix, while the darker areas correspond to the P2VP domains, cylinders orientated normal to the surface. The advancing water contact angle of the thin films remains 94 ( 3°, as shown in Table 1, which implies that BP is either not present at the surface or it does not have a great effect on the surface energy of the film. The height of the grains in this image is 3 ( 1 nm. In Figure 1C the height image of a spin-cast and solvent annealed PS-b-P2VP mixed with BP film after it was photo cross-linked is shown. The brighter areas in the image correspond to the P2VP domains, while the darker areas correspond

PS-b-P2VP PS-b-P2VP and BP PS-b-P2VP and BP (UV in air 5 min)

carbon 1s %

oxygen 1s %

nitrogen 1s %

93.9 92.6 65.2

4.9 6.4 33.1

1.2 0.9 1.7

to the PS matrix. In this case, a micelle-type structure is observed. The advancing water contact angle decreases to 61 ( 3° as shown in Table 1, which indicates that these new films are more hydrophilic than the noncross-linked films. The UV irradiation helps to introduce more oxygen-containing surface groups. The height of the grains is 3 ( 1 nm. In Figure 1D the height image of a spin-cast and solvent annealed film of PS-b-PMMA is shown. The brighter areas in the image correspond to the PS matrix, while the darker areas correspond to the PMMA domains, cylinders orientated normal to the surface. The lattice spacing of the hexagonal packing domains is 110 ( 2 nm. The advancing water contact angle of this thin film is 84 ( 3° as shown in Table 1. UV irradiation can induce both chain scission and crosslinking in polymer films. However, direct UV irradiation can cause thin film polymers to dewet, especially PS on Si wafers.30 Carroll et al. showed that by adding a BP containing molecule to the PS solution and then spin-coating to obtain thin films, the dewetting process is inhibited.30 In addition, BP helps in the cross-linking process. Upon UV irradiation, BP forms a radical, which abstracts a hydrogen atom from PS31 or P2VP. When the sample is exposed to atmospheric oxygen, this BP radical forms a highly reactive hydroperoxide radical, which can react with the polymer radical to form a polymer hydroperoxide. This can further break down to produce a shorter chain polymer radical and carbonyl species, leading to an increase in polarity of the polymer chains. Afterward, the radical species recombine to give rise to cross-linked chains. UV irradiation


Figure 2. High-resolution XPS of (A) C 1s, (B) O 1s, and (C) N 1s spectra of (a) PS-b-P2VP film after solvent annealing, dotted line and (b) PS-b-P2VP and BP after UV in air, solid line.

can also produce radical centers on the PS or P2VP chains without the use of BP. If the chains are close to each other they can recombine to produce a cross-linked system. Thus, there are two pathways responsible for cross-linking the diblock copolymer. XPS analysis was performed on three different samples, PSb-P2VP film after solvent annealing, PS-b-P2VP and BP after solvent annealing, and PS-b-P2VP and BP after UV in air. As shown in Table 2, the relative chemical composition of the films is carbon, oxygen and nitrogen, which was expected for our phase separated block copolymer. The compositions of PS-bP2VP, PS-b-P2VP, and BP are similar in C, O, and N. The difference between these two can be attributed to variations between samples that occur due to self-assembly because XPS has a finite depth of penetration into the surface of around 5-6 nm. Analysis of unpatterned PS-b-P2VP films show that at the surface only PS is present; no N peak was observed in the spectra. The UV treated sample has a larger fraction of O and a lower fraction of C, which is due to oxidation. Nitrogen chemical deviations can be attributed to sample variations. High-resolution XPS analysis was performed on these three different samples for C, O, and N, which are shown in Figure 2. The PS-b-P2VP and PS-b-P2VP with BP samples had similar spectra, thus, for clarity only one was included in the figure. In Figure 2A the C 1s spectrum shows another peak at about 289 eV binding energy for the UV irradiated sample. This peak


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Figure 3. (A) Force-indentation curve of the PS-b-P2VP film. Dots are the data point, while the solid line is the fit by a paraboloidal tip shape; (B) the elastic modulus distribution for PS-b-P2VP film; (C) the elastic modulus distribution for PS-b-P2VP and BP film after UV irradiation.

corresponds to the carbonyl group. The O 1s peak of the UV sample is broader, which indicates an increase in the type of O bonds. In addition, more peaks are present in the N 1s spectrum for the UV treated sample. These peaks that appear at a higher binding energy can be attributed to pyridone and to pyridineN-oxide. Both the matrix and the cylinders survive the UV treatment. FT-IR spectroscopy showed an extra peak at 1712 cm-1 for the UV irradiated sample, which corresponds to the carbonyl group. We investigated the mechanical properties of the PS-b-P2VP copolymer film and the PS-b-P2VP and BP film after UV treatment in air using AFM indentation measurements. Figure 3A shows the force-indentation curve for PS-b-P2VP. The PSb-P2VP and BP film after UV has a similar force curve. The Young’s modulus was determined by considering load-indentation dependence for a paraboloidal tip shape32 given by eq 1

F)

4E√R 3/2 δ 3(1 - V2)

(1)

where F is the loading force in nN, E is Young’s modulus in Pa, R is the radius of curvature of the tip in nm, δ is the

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Figure 4. Force-extension curve of (A) PS-b-P2VP film and (B) PSb-P2VP and BP film after UV irradiation. The gray curve is the trace data, while the black curve is the retract data.

indentation in nm, and ν is the Poisson’s ratio (0.5). The forceindentation curves obtained on the darker and brighter regions did not show different moduli. PS and P2VP have been previously shown to have similar mechanical properties such as glass transition temperature and elastic modulus, thus the average elastic modulus of the PS-b-P2VP surface is a good approximation.33,34 Figure 3B shows a histogram of the moduli obtained by fitting the force-indentation curves using equation (1). The elastic modulus for PS-b-P2VP is 3.3 ( 1 GPa. This value is in good agreement with the previously reported value for PS of 3.5 GPa.35 The elastic modulus for the UV irradiated film is 4.1 ( 1 GPa. The UV irradiated film was not statistically

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different than the regular PS-b-P2VP film due to the presence of some uncross-linked chains. The role of finite sample thickness and the stiffness of the underlying substrate has not been taken into detailed account in the modeling of the film modulus, and which could lead to a ca. 50% error in the estimated moduli.36 Figure 4 shows the force vs extension curves for PS-b-P2VP copolymer films and for PS-b-P2VP and BP film after UV treatment in air. Figure 4A shows a large hysteresis between the trace and retrace data. The PS-b-P2VP film shows a plastic deformation after the tip indents into the surface. Also, there is a strong repulsive force present in the trace curve starting at about 20 nm. The onset of the repulsive force coincides with the thickness measured by ellipsometry. The onset of the repulsive force ranges from 10 to 20 nm. Figure 4B shows a lower hysteresis between the trace and retrace data. The crosslinked PS-b-P2VP film displays a decrease in this plastic deformation behavior, because the cross-linking restrains the flow of the copolymer chains. 2. Block Copolymer Behavior Underwater. When the noncross-linked PS-b-P2VP films were immersed in ultrapure water, the pattern was preserved up to 1.5 h, with the measurement performed in water. This can be seen from the AFM height images in Figure 5A and B. The brighter areas in the image correspond with the PS matrix, while the darker areas correspond to the P2VP domains. This surface does not promote the formation of nanobubbles, since the available PS surface between the cylinders is not enough for these hundreds of nanometer sized features to develop.37 The advancing water contact angle of these hydrated films is 72 ( 3°, which is much lower than the PS-b-P2VP film after solvent annealing. The pKa of the conjugate acid for P2VP is 4.5, which is smaller than the one for ultrapure water, pKa ) 5.5.38 Thus, P2VP proton uptake will not be a problem in this study. When the film is immersed in water, the hydrophobic PS matrix tries to minimize

Figure 5. AFM height images of solvent annealed (A) PS-b-P2VP film in water after 1 h and (B) PS-b-P2VP film in water after 1.5 h. Z range: 8 nm; (C) PS-b-P2VP film in air after 2 h in water and (D) PS-b-P2VP film in air after 8 days in water. Image size: 1000 × 1000 nm. Z range: 20 nm.



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Figure 6. AFM height images of (A) cross-linked PS-b-P2VP and BP film in water after 1 h and (B) cross-linked PS-b-P2VP and BP film in water after 48 h. Z range: 12 nm; (C) cross-linked PS-b-P2VP and BP film in air after 2 h in water and (D) cross-linked PS-b-P2VP and BP film in air after 8 days in water. Z range: 40 nm; (E) random copolymer film P(S-r-VP) in air, Z range: 30 nm, (F) random copolymer film P(S-r-VP) in water after 1 h, Z range: 4 nm. Image size: 1000 × 1000 nm.

the contact area exposed to the water. The copolymer starts to move, until the pattern disappears from the surface. When the films are taken out of the water, dried under nitrogen gas and measured in air, a cylindrical pattern is still present after 2 h of water immersion. However, there is not enough water present in the system to stop the rearrangement of the film when it is brought back to air. After 24 h, the pattern is no longer present, however, some isolated cylinders can still be seen (see Supporting Information, Figure S1). The pattern does not reappear after the film is immersed in water for more that 8 days. The AFM height images of these films in air are shown in Figure 5C and D. The advancing water contact angle of these films is again much lower than before, 63 ( 3°. When the film is immersed in water for a longer period of time, water has time to penetrate into the film and cause a more permanent restructuring of the copolymer. Drying under nitrogen gas is not enough to cause the pattern to return. The cross-linked PS-b-P2VP mixed with BP films were also immersed in water for various periods of time. The micellelike structure changed into cylinders orientated normal to the surface when placed in water. The AFM height images in Figure 6A and B show the cylindrical structure after 1 and 48 h in

water. The same pattern was observed when the film was immersed in artificial seawater (salinity 35 ppt) for 1 h (see Supporting Information, Figure S2). The darker areas in the image correspond with the P2VP domains, while the brighter areas correspond to the PS matrix. Even after 48 h some cylinders are still present on the surface. When the copolymer is cross-linked, it is harder for the water to infiltrate the film. In addition, the oxidation of the film, which decreases the hydrophobicity of the film, results in retention of the pattern. After the copolymer film was dried with nitrogen gas after 2 h and after 8 days in water the pattern reappeared. Figure 6C and D shows the copolymer film in air after 2 h in water and 8 days respectively. The advancing water contact angle of these films is about 64 ( 4° and 62 ( 3°, which indicates that water does not infiltrate significantly into the film. Thus, the UV crosslinking stabilizes the surface groups. The pattern also reappeared upon drying after two weeks in water (see Supporting Information, Figure S3A). In addition, the copolymer film showed pattern retention after immersion in artificial seawater for three weeks and then dried in nitrogen gas (see Supporting Information, Figure S3B). In contrast, the random copolymer P(S-r-

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Figure 7. AFM height images of solvent annealed (A) PS-b-PMMA film 1 h in water and (B) PS-b-PMMA film 1.5 h in water. Z range: 8 nm; (C) PS-b-PMMA film in air after 2 h in water and (D) PS-b-PMMA film in air after 8 days in water. Image size: 1000 × 1000 nm. Z range: 40 nm.

VP) does not phase segregate or show nanoscale structures as shown in Figure 6E and F. When the PS-b-PMMA films were immersed in water, the copolymer film swelled after 1 h and the patterned is almost gone after 1.5 h, with the measurement performed in water. This can be seen from the AFM height images in Figure 7A and B. The brighter areas in the image correspond to the PMMA domains, while the darker areas correspond to the PS matrix. In this case too, the hydrophobic PS matrix tries to minimize the contact area with water, which causes the pattern to disappear from the film surface. After water immersion for 2 h the film still displays a cylindrical pattern when it is dried under nitrogen gas and measured in air. The advancing water contact angle of these films is 80 ( 3°, which is slightly lower than the PS-bPMMA film after solvent annealing. There is not enough water present in the system to stop the rearrangement of the film. After 24 h the film looks more swollen and the pattern is not as clear as before (see Supporting Information, Figure S4). After immersion in water for 8 days, the pattern is less clear and the film is more swollen by the water. The AFM height images of these films in air are shown in Figure 7C and D. The brighter areas in the image correspond to the PS matrix, while the darker areas correspond to the PMMA domains. The advancing water contact angle of these films is again much lower than before, 70 ( 3°. As the copolymer is immersed for longer periods of time in water, the more swollen the film gets and the worse the pattern appears. However, because the PS-b-PMMA copolymer is more hydrophilic than the PS-b-P2VP copolymer, it retains the pattern for a longer period of time. 3. Settlement of Zoospores of the Green Alga UlWa. The density of zoospores attached to a surface indicates how hospitable that surface is for settlement. Permanent attachment is the consequence of the secretion of a hydrophilic selfaggregating glycoprotein, which anchors the spore permanently to the substratum.27 In Figure 8A the density of zoospores settled

(attached) on a variety of polymers on silicon wafers is shown. A one-way ANOVA test showed that there are significant differences present F 5534 ) 610.92 P < 0.05. Thus, all the comparisons are significantly different. Visual observations of settled spores did not reveal any differences in the pattern of distribution on any of the surfaces. The settlement density of spores was greatest on the two control surfaces, that is, PS and P2PV; however, it was slightly higher on P2VP. Although settlement density on smooth, uncharged surfaces generally correlates with increasing hydrophobicity,2,3 this trend is not seen here. However, neither of the control surfaces are entirely smooth, holes and small bumps are present at the surface and are maintained when the surfaces are immersed in water (see Supporting Information, Figure S5 and S6). The advancing water contact angle for PS decreases after 2 h immersion in water from 95 ( 3° to 91 ( 3°, while the one for P2VP remains the same at 63 ( 2°; thus, no major reorganization of the polymers takes place on this time scale. The lowest settlement density was on the PS-b-P2VP and BP after UV treatment and PS-b-PMMA surfaces, both of which are comprised of cylindrical patterns. The advancing water contact angle after 2 h in water was 64 ( 4° for PS-b-P2VP and BP after UV treatment and 80 ( 4° for PS-b-PMMA. The density of spores was significantly lower on PS-b-PMMA and the high degree of swelling associated with this surface after immersion in seawater may have enhanced its efficacy. It is interesting to note that the cylindrical pattern appears to inhibit the settlement of spores. The chemical and topographical heterogeneity of these surfaces may be working independently or in tandem to discourage spore settlement. The significantly lower settlement on PS-b-P2VP and BP after UV treatment compared to PS-b-P2VP may reflect the higher degree of stability of the cylindrical pattern as shown by underwater AFM. The PS-b-P2VP and BP after UV treatment film does not reorganize in water and it also maintains its advancing water



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value of 0 for grain size as shown in Figure 8C; however, at the film surface both PS and P2VP areas are present. Settlement densities on these surfaces were intermediate between the high settlement on the PS and P2VP and the low settlement on the PS-b-P2VP and BP after UV treatment and PS-b-PMMA surfaces. In water, the P(S-r-VP) surface restructures slightly, with mobile P2VP units migrating to the surface; the advancing water contact angle value of this film decreases from 88 ( 3° to 80 ( 3° after 2 h in water. Similarly, the PS-b-P2VP block copolymer restructures underwater to have more of the P2VP domains at the surface; the cylindrical nanopattern is not retained. The advancing water contact angle value of this film after 2 h in water decreases from 94 ( 3° to 72 ( 3°. Another factor that can influence the settlement of spores is the area of the surface that is available for contact with the spores. This contact area is slightly larger for the homopolymers and the random copolymers than for the patterned copolymer surfaces. The hardness of the adhesive produced by the spores must also be taken into account. The modulus of the adhesive is in the MPa range, 0.2 ( 0.1 MPa for the outer 600 nm thick layer and 3 ( 1 MPa for the inner layer.39 The polymer surfaces are much harder than the adhesive, in the GPa range; thus, in order to increase contact area the glue is required to conform to the surface. Roughness alone or contact area alone does not explain settlement behavior, something beyond the contact area seem to be at play.

Conclusions Thin films of PS-b-P2VP block copolymer mixed with BP show cylindrical domains. However, after photo cross-linking the film shows micelle-type structures. Immersion in water causes the structure to change to cylindrical domains and is still stable after 48 h in water. The cylindrical domains also reappear when the sample is dried after 2 weeks in water and 3 weeks in artificial seawater. In this article we present a simple route to obtain surfaces that retain their nanoscale patterns after water immersion and we report mechanical properties obtained by nanoindentation. These nanopatterned films were also shown to affect the settlement response of zoospores of UlVa. Spore settlement density was greatly reduced on the cylindrical patterned PS-b-P2VP and BP after UV cross-linking and PSb-PMMA surfaces. To further elucidate the mechanism of interaction, experiments on spore interactions with surfaces displaying a range of feature sizes are currently underway. Figure 8. (A) The density of attached Ulva spores on polymers on silicon wafers. Each point is the mean from 90 counts on three replicate slides (30 on each wafer). Bars show 95% confidence limits. (B) The advancing water contact angle for polymers on silicon wafers. (C) Average grain size analysis.

contact angle value of 64 ( 4°. The different hydrophobicity between the cylindrical portion of the pattern and the matrix may deter spores, which would have settled on a surface with uniform hydrophobicity. The overall advancing water contact angle of the surface after water immersion can be seen in Figure 8B. For example, the uniform P2VP polymer surface has the same overall advancing water contact angle, but very different spore settlement densities. Similarly, the nanoscale roughness may act as a deterrent if the spores prefer a smoother surface. Alternatively, it is possible that all of this heterogeneity simply confuses the spore by sending it conflicting signals. On the PSb-P2VP surface, which also had the patterning, the settlement density was similar to that on the random copolymer surface, P(S-r-VP). The P(S-r-VP) copolymer does not phase segregate into nanopatterned domains, thus grain size analysis gives a

Acknowledgment. We thank Dr. S. Zou, Dr. Z. Fakhraai, and A. Tanur for their discussion and help in this work. We thank J. A. Forrest for the use of an ellipsometer. The work was supported by the Natural Sciences and Engineering Research Council of Canada and the Office of Naval Research (award N00014-05-10765 to G.C.W. and award N00014-08-10010 to J.A.C. and M.E.C.). Supporting Information Available. AFM height image of PS-b-P2VP film in air after 24 h in water. AFM height image of cross-linked PS-b-P2VP and BP film in artificial seawater after 1 h. AFM height image of cross-linked PS-b-P2VP and BP film in air after 2 weeks in water and after 3 weeks in artificial seawater. AFM height image of PS-b-PMMA film in air after 24 h in water. AFM height image of PS film in air and in water after 1 h. AFM height image of P2VP film in air and in water after 1 h. This material is available free of charge via the Internet at http://pubs.acs.org.

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