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Nanostructured Films of Amphiphilic FluorinatedBlock Copolymers for Fouling Release Application

Elisa Martinelli, Serena Agostini, Giancarlo Galli, Emo Chiellini, Antonella Glisenti,Michala E. Pettitt, Maureen E. Callow, James A. Callow, Katja Graf, and Frank W. BartelsLangmuir, 2008, 24 (22), 13138-13147 • DOI: 10.1021/la801991k • Publication Date (Web): 18 October 2008

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Nanostructured Films of Amphiphilic Fluorinated Block Copolymersfor Fouling Release Application

Elisa Martinelli,† Serena Agostini,† Giancarlo Galli,*,† Emo Chiellini,† Antonella Glisenti,‡

Michala E. Pettitt,§ Maureen E. Callow,§ James A. Callow,§ Katja Graf,| andFrank W. Bartels|

Dipartimento di Chimica e Chimica Industriale and UdR Pisa INSTM, UniVersita di Pisa,Via Risorgimento 35, 56126 Pisa, Italy, Dipartimento di Scienze Chimiche, UniVersita di PadoVa,

35131 PadoVa, Italy, School of Biosciences, The UniVersity of Birmingham, B15 2TT, United Kingdom,and Polymer Research, BASF SE, 67056 Ludwigshafen, Germany

ReceiVed June 25, 2008. ReVised Manuscript ReceiVed August 29, 2008

New amphiphilic block copolymers SnSzm consisting of blocks with varied degrees of polymerization, n and m,of polystyrene, S, and polystyrene carrying an amphiphilic polyoxyethylene-polytetrafluoroethylene chain side-group,Sz, were prepared by controlled atom transfer radical polymerization (ATRP). The block copolymers, either aloneor in a blend with commercial SEBS (10 wt% SEBS), were spin-coated in thinner films (200-400 nm) on glass andspray-coated in thicker films (∼500 nm) on a SEBS underlayer (150-200 µm). Angle-resolved X-ray photoelectronspectroscopy (XPS) measurements proved that at any photoemission angle, φ, the atomic ratio F/C was larger thanthat expected from the known stoichiometry. Consistent with the enrichment of the outer film surface (3-10 nm) inF content, the measured contact angles, θ, with water (θw g 107°) and n-hexadecane (θh g 64°) pointed to thesimultaneous hydrophobic and lipophobic character of the films. The film surface tension γS calculated from the θvalues was in the range 13-15 mN/m. However, the XPS measurements on the “wet” films after immersion in waterdemonstrated that the film surface underwent reconstruction owing to its amphiphilic nature, thereby giving rise toa more chemically heterogeneous structure. The atomic force microscopy (AFM) images (tapping mode/AC mode)revealed well-defined morphological features of the nanostructured films. Depending on the chemical compositionof the block copolymers, spherical (ca. 20 nm diameter) and lying cylindrical (24-29 nm periodicity) nanodomainsof the S discrete phase were segregated from the Sz continuous matrix (root-mean-square, rms, roughness ≈ 1 nm).After immersion in water, the underwater AFM patterns evidenced a transformation to a mixed surface structure, inwhich the nanoscale heterogeneity and topography (rms ) 1-6 nm) were increased. The coatings were subjected tolaboratory bioassays to explore their intrinsic ability to resist the settlement and reduce the adhesion strength of twomarine algae, viz., the macroalga (seaweed) UlVa linza and the unicellular diatom NaVicula perminuta. The amphiphilicnature of the copolymer coatings resulted in distinctly different performances against these two organisms. UlVaadhered less strongly to the coatings richer in the amphiphilic polystyrene component, percentage removal beingmaximal at intermediate weight contents. In contrast, NaVicula cells adhered less strongly to coatings with a lowerweight percentage of the amphiphilic side chains. The results are discussed in terms of the changes in surface structurecaused by immersion and the effects such changes may have on the adhesion of the test organisms.

IntroductionThe design and the control of polymer surface structure and/

or nanostructure are topical subjects. Understanding the cor-relation between the structure and the properties of the surfaceof a material and the tuning of appropriate chemical-physicalproperties at a molecular level may lead to novel developmentsin a number of fields where interfacial interactions, operatingwithin a few nanometers of a surface, are critical. Foulingorganisms, such as larvae of invertebrates and spores of algae,are highly selective in their preferences for certain surfaces.Several interfacial properties of the surface, including wettability,1

topography,2,3 and chemical heterogeneity,4 have been shown toact either as “cues” that moderate initial settlement of the fouling

organism or as factors that determine adhesion strength.1 In thelatter context, the fouling-release paradigm, based on fracturemechanics, is that the stress required to detach a micro-organismfrom the coating is given by (WEc/a)1/2, where W is the work ofadhesion or the energy per unit area needed to separate theinterface, a is the radius of the contact region, and Ec is thecomposite modulus of the adhesive matrix and the coating.5

Silicone- and fluoropolymer-based elastomers possess theproperties required for good fouling release, namely, a relativelylow surface energy (22 mN/m or less) and low modulus (3-1.4MPa).6,7 Silicone elastomers are the basis of current commerciallyavailable “fouling release” coatings, so-called because foulantsadhere weakly and can be released under the hydrodynamic forcesgenerated as the vessel moves.8 The relatively high sailing speed(20-30 knots), at which the macrofouling generally drops off,makes these coatings unsuitable for most vessels. Moreover,

* To whom correspondence should be addressed. Email gallig@dcci.unipi.it.† Universita di Pisa.‡ Universita di Padova.§ The University of Birmingham.| Polymer Research, BASF SE.(1) Schilp, S.; Kueller, A.; Rosenhahn, A.; Grunze, M.; Pettitt, M. E.; Callow,

M. E.; Callow, J. A. Biointerphases 2007, 2, 143–150.(2) Schumacher, J. F.; Aldred, N.; Callow, M. E.; Finlay, J. A.; Callow, J. A.;

Clare, A. S.; Brennan, A. B. Biofouling 2007, 23, 307–317.(3) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson,

L. H.; Callow, M. E.; Callow, J. A.; Finlay, J. A.; Brennan, A. B. Biofouling 2007,23, 55–62.

(4) Finlay, J. A.; Krishnan, S.; Callow, M. E.; Callow, J. A.; Dong, R.; Asgill,N.; Wong, K.; Kramer, E. J.; Ober, C. K. Langmuir 2008, 24, 503–510.

(5) Chaudhury, M. K.; Finlay, J. A.; Chung, J. Y.; Callow, M. E.; Callow, J. A.Biofouling 2005, 21, 41–48.

(6) Brady, R. F.; Bonafede, S. J.; Schmidt, D. L. JOCCA-Surf. Coat. Int. 1999,82, 582–585.

(7) Brady, R. F.; Singer, I. L. Biofouling 2000, 15, 73–81.(8) Kavanagh, C. J.; Quinn, R. D.; Swain, G. W. J. Adhes. 2005, 81, 843–868.

13138 Langmuir 2008, 24, 13138-13147

10.1021/la801991k CCC: $40.75 2008 American Chemical SocietyPublished on Web 10/18/2008

their inefficiency against the formation and release of diatombiofilms9-11 necessitates the development of additional novelenvironmentally friendly coatings, which resist all forms of foulingand/or provide for ease of removal by self-cleaning at moderatespeeds.12

Amphiphilic polymer coatings are designed to preventbiofouling by providing a surface with a compositional,topological, and morphological complexity, which either reducessettlement of the motile spores or larvae4 or makes energeticallyunfavorable the hydrophobic or the hydrophilic interactionsbetween the organisms’ adhesives and the substratum. Networksprepared from hydrophobic polyisobutylene and hydrophilicpoly(2-hydroxyethyl methacrylate) or poly(N,N-dimethyl acry-lamide) were found to exhibit reduced protein adsorption andcell adhesion.13,14 In another work, microphase-separated surfacestructures of an amphiphilic interpenetrating polymer networkof polystyrene and polyurethane have been used to preparebiomaterials resistant to platelet adhesion.15 “Thermoresponsive”amphiphilic graft copolymers consisting of poly(N-isopropylacrylamide) and poly(ethylene glycol) cross-linked on a fluo-ropolymer substrate were used for the control of fibroblast celldetachment.16 Moreover, it has been found that amphiphilicpolymer networks containing hyperbranched fluoropolymerscross-linked with poly(ethylene glycol) (PEG) are characterizedby a marked phase segregation between the fluoropolymer andPEG, which results in a higher release of UlVa sporelings (youngplants) compared to poly(dimethyl siloxane) (PDMS).17 Krishnanet al. prepared an SEBS-based two-layer coating containing apoly(ethoxylated fluoroalkyl acrylate)-polystyrene amphiphilicblock copolymer, which was able to simultaneously reduce theadhesion of both UlVa sporelings and cells of the diatomNaVicula.18 The use of PEG and fluoroalkyl segments for theamphiphilic side chains was justified by the experimental resultsobtained in previous studies, which showed that diatoms werereleased more efficiently from surfaces of polymer with PEGside chains compared to that with semifluorinated side chains,whereas UlVa sporelings showed the opposite behavior.19,20

The present study focuses on the wettability and surfaceproperties and their correlation with antifouling/fouling releaseperformances of elastomeric poly(styrene-b-(ethylene-co-buty-lene)-b-polystyrene) (SEBS) based two-layer coatings containingan amphiphilic fluorinated polystyrene-based block copolymer.The synthesis by atom transfer radical polymerization (ATRP)and some characterization of this type of amphiphilic block

copolymers have recently been discussed in detail.21 They containa block carrying PEGylated-fluoroalkyl side chains similar tothat used by Krishnan et al.18 However, in that case the synthesisof the amphiphilic copolymer was based on the grafting of theside chains on a preformed poly(styrene-b-acrylic acid) copoly-mer. Such polymer-analogue synthetic routes, although versatile,can lead to a number of chemical structural defects in the finalcopolymer, which normally does not occur when well-controlledradical polymerization is achieved. In addition, block copolymerswith a polystyrene backbone appeared more suitable than didpolystyrene-polyacrylate block copolymers to promote blendingwith the thermoplastic SEBS elastomer to create better com-patibilized coatings. The two-layer strategy has the advantageof allowing the preparation of low surface energy and low elasticmodulus materials, while using a relatively low amount of thefluorinated active copolymer.

For evaluations of the antifouling and fouling-release per-formance of the experimental coatings, two marine algae wereused as test organisms, namely, the macroalga UlVa (syn,Enteromorpha) and the diatom NaVicula. The green algal genusUlVa (formerly Enteromorpha) is the most common macroalga(“seaweed”) contributing to “soft” fouling of manmade surfacesthroughout the world and has been extensively used as a modelsystem for experimental studies of biofouling and adhesion22

and the evaluation of novel marine coatings.17-20 Fouling isinitiated by the settlement and subsequent adhesion of motile,quadriflagellate zoospores (approximately 7-8 µm in length),which form the starting point of the assays. The swimming sporessettle and adhere through discharge of a glycoprotein adhesive,22

then rapidly germinate into sporelings (young plants). The strengthof attachment of sporelings to experimental coatings is evaluatedunder hydrodynamic shear in a calibrated flow channel.23

The second test alga was the diatom NaVicula. Diatoms areunicellular algae that form biofilms (slimes) on surfaces.24 UnlikeUlVa spores that are motile and therefore able to “select” whereto settle, diatom cells are not motile in the water column andreach a surface through transport in currents and gravity. Inlaboratory assays, the cells sink rapidly and form an even coveringon the test surfaces. Again, the flow channel is used to measurehow strongly the cells adhere to a surface. The reason for usingboth NaVicula and UlVa to evaluate test coatings is that apartfrom having different settlement characteristics the adhesionbiology of the two organisms is different. For example, in contrastto UlVa, diatoms adhere more strongly to hydrophobic coatings,including silicone elastomers and fluorinated block copolymersand, conversely, adhere more weakly to hydrophilic sur-faces.10,11,20,25

Experimental SectionMaterials. Anisole and diethylene glycol dimethyl ether (diglyme)

were kept at 100 °C over sodium for 4 h and then distilled underreduced pressure. 2,2′-Bipyridine (Bipy), copper(I) bromide, and1-phenylethyl bromide (1-(PE)Br) were purchased from Aldrichand used without further purifications.

The monomer Sz was synthesized according to the procedurepreviously reported from 4-vinylbenzoic acid (from Aldrich) andthe PEGylated-fluoroalkyl alcohol Zonyl FSO-100 (registered

(9) Terlizzi, A.; Conte, E.; Zupo, V.; Mazzella, L. Biofouling 2000, 15, 327–342.

(10) Holland, R.; Dugdale, T. M.; Wetherbee, R.; Brennan, A. B.; Finlay,J. A.; Callow, J. A.; Callow, M. E. Biofouling 2004, 20, 323–329.

(11) Casse, F.; Stafslien, S. J.; Bahr, J. A.; Daniels, J.; Finlay, J. A.; Callow,J. A.; Callow, M. E. Biofouling 2007, 23, 121–130.

(12) Genzer, J.; Efimenko, K. Biofouling 2006, 22, 339–360.(13) Keszler, B.; Kennedy, J. P.; Ziats, N. P.; Brunstedt, M. R.; Stack, S.; Yun,

J. K.; Anderson, J. M. Polym. Bull. 1992, 29, 681–688.(14) Park, D.; Keszler, B.; Galiatsatos, V.; Kennedy, J. P.; Ratner, B. D.

Macromolecules 1995, 28, 2595–2601.(15) Kim, J. H.; Kim, S. H.; Kim, H. K.; Akaike, T.; Kim, S. C. J. Biomed.

Mater. Res. 2002, 62, 613–621.(16) Schmaljohann, D.; Oswald, J.; Jorgensen, B.; Nitschke, M.; Beyerlein,

D.; Werner, C. Biomacromolecules 2003, 4, 1733–1739.(17) Gudipati, C. S.; Finlay, J. A.; Callow, J. A.; Callow, M. E.; Wooley, K. L.

Langmuir 2005, 21, 3044–3053.(18) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry,

R.; Ober, C. K.; Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A.Langmuir 2006, 22, 5075–5086.

(19) Youngblood, J. P.; Andruzzi, L.; Ober, C. K.; Hexemer, A.; Kramer, E. J.;Callow, J. A.; Finlay, J. A.; Callow, M. E. Biofouling 2003, 19, 91–98.

(20) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow,J. A.; Hexemer, A.; Sohn, K. E.; Kramer, E. J.; Fischer, D. A. Biomacromolecules2006, 7, 1449–1462.

(21) Martinelli, E., Menghetti, S.; Galli, G.; Glisenti A.; Krishnan, S.;. Paik,M. Y; Ober, C. K.; Smilgies, D. M.; Fischer D. A, manuscript in preparation.

(22) Callow, J. A., Callow, M. E. In Biological AdhesiVes; Smith, A. M.,Callow, J. A., Eds.; Springer: Dordrecht, 2006; p 63.

(23) Schultz, M. P.; Finlay, J. A.; Callow, M. E.; Callow, J. A. Biofouling2000, 15, 243–251.

(24) Molino, P. J.; Wetherbee, R. Biofouling 2008, 24, 365–379.(25) Statz, A.; Finlay, J.; Dalsin, J.; Callow, M.; Callow, J. A.; Messersmith,

P. B. Biofouling 2006, 22, 391–399.

Nanostructured Films of Amphiphilic Block Copolymers Langmuir, Vol. 24, No. 22, 2008 13139

trademark of E. I. du Pont de Nemours & Co) (from Aldrich).21 TheZonyl FSO-100, F(CF2CF2)y(CH2CH2O)xCH2CH2OH, has a distri-bution of molecular weights (Mw/Mn ∼ 1.2), with x ≈ 5 and y ≈4. According to SEC and NMR measurements, the monomer Szsynthesized also presented a relatively broad distribution of molecularweights (Mw/Mn ∼ 1.3) and average degrees of polymerization x ≈5 and y ≈ 4.

Styrene (S) (from Fluka) was washed with 5% NaOH and water.After drying over Na2SO4, it was distilled under reduced pressureprior to use. (3-Glycidoxypropyl)-trimethoxysilane (GPS) (fromGelest) was used without further purification. Poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS) triblock thermoplasticelastomer (Kraton G1652M) and SEBS grafted with 1.4-2.0 wt %maleic anhydride (SEBS-MA, Kraton FG1901X) were kindlyprovided from Kraton Polymers.

Polymer Synthesis. The synthetic procedure is illustrated inScheme 1.

Br-Terminated Polystyrene Macroinitiators. In a typicalpreparation, 27.270 g (262.21 mmol) of S, 2.049 g (13.12 mmol)of Bipy, and 596 µL (4.37 mmol) of 1-(PE)Br were introduced intoa dry Schlenk flask under nitrogen. The solution was purged withnitrogen for 15 min, and then 0.625 g (4.36 mmol) of CuBr wasadded. After four freeze-thaw pump cycles, the polymerizationwas let to proceed under nitrogen for 90 min at 110 °C. When thereaction was stopped, the polymer mixture was dissolved in THFand then eluted on neutral alumina to remove the catalyst. The solventwas removed under vacuum, and the polymer was purified by repeatedprecipitations from tetrahydrofuran solutions into methanol (50%yield). The number average degree of polymerization (n) of thepolymer sample was 26, and the macroinitiator is named here S26.

1H NMR (CDCl3): δ (ppm) ) 1.0-2.2 (3H, CH2CH), 6.1-7.4(5H, aromatic). FT-IR (film): νj (cm-1) ) 3082-3026 (ν C-Haromatic), 2924 (ν C-H aliphatic), 1601 (ν CdC aromatic), 1493and 1452 (δ C-H aliphatic), 756 and 698 (δ C-H aromatic).

Polystyrene-Amphiphilic Polystyrene Diblock Copolymers.In a typical preparation, 0.444 g (0.16 mmol) of S26 and 0.077 g(0.49 mmol) of Bipy were introduced into a dry Schlenk flask, whichwas then evacuated and flushed with nitrogen three times. A solutionof 6.86 g (8.42 mmol) of Sz in 20 mL of anisole was then addedunder nitrogen. The mixture was purged with nitrogen for 30 min,and then 0.024 g (0.167 mmol) of CuBr was added. After fourfreeze-thaw pump cycles, the polymerization was allowed to proceedfor 66 h at 115 °C. When the reaction was stopped, the polymermixture was dissolved in chloroform and then washed with wateruntil discoloration of the water. The solvent was removed undervacuum, and the polymer was purified by repeated precipitationsfrom tetrahydrofuran solutions into methanol (46% yield). Thenumber average degrees of polymerization of the S and Sz blocksof the sample were 26 and 23, and the block copolymer is namedhere S26Sz23.

1H NMR (CDCl3): δ (ppm)) 0.9-2.1 (6.4H, CH2CH), 2.4 (2.0H,CH2CF2), 3.2-4.2 (20.0H, CH2O), 4.4 (2.0H, COOCH2), 6.2-8.0(9.6H aromatic). 19F NMR (CDCl3/CF3COOH): δ (ppm) )-6 (3F,CF3),-38 (2F, CF2CH2),-46 to-49 (10F, CF2),-51 (2F, CF2CF3).

FT-IR (film): νj (cm-1) ) 3080-3000 (ν C-H aromatic), 1722 (νCdO), 1400-1000 (ν C-O and ν C-F), 759 and 699 (δ C-Haromatic), 654 (ω CF2).

Preparation of Two-Layer Films. The two-layer films for testingwere prepared by using SEBS/SEBS-MA as a bottom layer andcoating the block copolymer on top of it either alone or in a blendwith SEBS, as follows (Figure 1). First, the glass microscope slides(76 × 26 mm2) were cleaned in hot piranha solution (concentratedsulfuric acid + 30 wt % hydrogen peroxide, 7/3 v/v), rinsed withdistilled water and acetone, and dried in the oven at 100 °C for 10min. A 2% (w/v) solution of GPS in 95% ethanol (pH adjusted at4.5-5 with acetic acid) was prepared by adding the silane to ethanoland stirring for 5 min. The slides were then soaked in this solutionovernight, rinsed with ethanol, and heated in an oven at 110 °C for20 min. Second, the GPS functionalized glass slides were coated bycasting on a 12% (w/v) toluene solution of SEBS-MA and SEBS(56/44 w/w). They were allowed to dry in a closed chamber for 2-3days until the solvent was evaporated.

After the slides were annealed in an oven overnight at 120 °C,a 1.5% (w/v) toluene solution of either a block copolymer alone ora blend of a block copolymer with SEBS was spray-coated on thebottom layer using a Badger model 250 airbrush (50 psi air pressure).The surfaces were vacuum-dried in an oven at 60 °C for 8 h andthen annealed at 120 °C overnight. The bottom layer (thickness150-250 µm) was the same for all the coatings, while the top layer(thickness ∼500 nm) differed in terms of either the chemicalcomposition of the block copolymer or the content of the blockcopolymer in the blend with SEBS.

The two-layer coatings are denoted as SnSzm_p where n and mindicate the degrees of polymerization of the blocks of polystyreneand PEGylated-fluorinated polystyrene, respectively, and p is theweight percentage of copolymer in the blend with SEBS in the toplayer. The control coating, denoted as SEBS, was a bottom layer ofSEBS/SEBS-MA prepared as for the fluorinated two-layer films,with a spray-coated top layer of SEBS (Table 1).

Deposition of One-Layer Films. One-layer films were preparedby spin-coating a 3 wt % solution of the neat block copolymer inCHCl3 on glass supports. The films were then vacuum-dried at 120°C overnight (thickness 200-400 nm).

Characterization. 1H NMR (vs TMS) and 19F NMR (vsCF3COOH) spectra were recorded on Varian Gemini VRX 200 andVarian Gemini VRX 300 spectrometers, respectively. Infrared spectra

Scheme 1. Synthesis of the Block Copolymers SnSzm by ATRP

Figure 1. Reaction scheme of glass functionalization by GPS and SEBS-MA anchorage.

13140 Langmuir, Vol. 24, No. 22, 2008 Martinelli et al.

were recorded with a Spectrum One Perkin-Elmer Fourier Transforminfrared spectrophotometer with 4 cm-1 resolution. Polymer filmswere cast on a KBr crystal plate.

The number and weight average molecular weights of the polymers,Mn and Mw, respectively, were determined by size exclusionchromatography (SEC) with a Jasco PU-1580 liquid chromatographequipped with two PL gel 5 µm Mixed-D columns, a Jasco 830-RIrefractive index detector, and a Perkin-Elmer LC75 UV detector.Polystyrene standards ((4.0 × 102)-(4.0 × 105) g/mol) were usedfor calibration.

Static contact angles (θ) were measured on one-layer and two-layer coatings using the sessile drop technique with a FTA200 Camtelgoniometer (water (J. T. Baker, HPLC grade) and n-hexadecane(Sigma-Aldrich, 99%)). The measured values of θ were then usedto extract the surface tension of the polymer films referring to theso-called Owens-Wendt-Kaelble approach.26,27 This regardsthe surface tension as being composed of two additive components,the dispersion (γd) and the hydrogen bonding and dipole-dipole(γp) components (eq 1). A geometric mean relationship is postulatedboth of the solid-liquid and liquid-liquid interfacial tensions(eq 2).

γ) γd + γp (1)

γ12 ) γ1 + γ2 - 2(γ1dγ2

d)0.5 (2)

Combination with the well-known Young’s equation leads to

γL(1+ cos θ)) 2[(γSdγL

d)0.5 + (γSpγL

p)0.5] (3)

where γS is the surface tension of the solid tested and γL is that ofthe wetting liquid. Since there are two unknowns (γS

d and γSp) of

the solid, it is necessary to use contact angle measurements of atleast two different liquids with known γL

d and γLp on one and the

same surface and solve the two simultaneous equations.X-ray photoelectron spectroscopy (XPS) spectra were recorded

by using a Perkin-Elmer PHI 5600 spectrometer with a standard AlKR source (1486.6 eV) operating at 350 W. The working pressurewas less than 10-8 Pa. The spectrometer was calibrated by assumingthe binding energy (BE) of the Au 4f7/2 line to be 84.0 eV withrespect to the Fermi level. Extended spectra (survey) were collectedin the range 0-1350 eV (187.85 eV pass energy, 0.5 eV step, 0.025s/step). Detailed spectra were recorded for the following regions:C (1s), O (1s), and F (1s) (11.75 eV pass energy, 0.1 eV step, 0.1eV s/step). The standard deviation in the BE values of the XPS linewas 0.10 eV. The atomic percentage, after a Shirley-type backgroundsubtraction,28 was evaluated using the PHI sensitivity factors.29 Totake into account charging problems, the C(1s) peak was consideredat 285.0 eV and the peak BE differences were evaluated. Polymerfilms were prepared by spin-coating a 3 wt % polymer solution onglass supports, then vacuum-dried and finally annealed at 120 °Covernight.

Scanning electron microscope (SEM) images were recorded withan electronic microscope JEOL 5600 LV operating at 13 kV and 20kV. The films (170-200 µm thickness) were prepared by hot-pressingSEBS at 230 °C. A 1.5% (w/v) toluene solution containing a blendof the amphiphilic fluorinated block copolymer and SEBS was thenspray-coated on top of the SEBS film. Then, the two-layer filmswere dried under vacuum and either annealed at 120 °C overnightor not annealed. Before observation, the film was fractured in liquidnitrogen and then vacuum-metallized.

Atomic force microscopy (AFM) experiments under ambientconditions (dry state) were done in the tapping mode on a commercialMultimode system equipped with a Nanoscope IIIa controller (VeecoInstruments) using silicon cantilevers with a nominal force constantof 42 N/m from Olympus (type OMCL-AC160TS) at a resonancefrequency of about 320 kHz. To analyze the behavior of the surfacesin a marine environment, artificial seawater (ASW) was preparedfrom Tropic Marin salt (Dr. Biener GmbH). Surfaces were storedin ASW for 7 days, then transferred into the experimental chamberof the AFM filled with ASW, avoiding dry-out or surfacereorganization. All in situ (in solution) experiments were conductedin the AC mode on a commercial MFP-3D system from AsylumResearch using silicon cantilevers with an Al reflex coating and anominal force constant of 2.8 N/m from nanosensors (type PPP-FMR) at a resonance frequency of about 24 kHz. The scan rate waskept at 1 Hz in all experiments, while the tip-sample forces werecarefully minimized to avoid artifacts. To characterize the (sub-)microscale structure of the coatings, root-mean-square (rms)roughness was determined over regions of 1 × 1 µm2 size andaveraged over at least 5 measurements.

Algal Assays. Coated slides were immersed in racks placed in30 L tanks of deionized water that were continuously circulatedthrough a carbon filter for 7 days. Samples were equilibrated inartificial seawater (ASW) for 1 h before the start of the assays.Zoospores of the green macroalga UlVa linza were released fromreproductive thalli into artificial seawater (ASW) at pH 8.0 and 32per thousand (“Tropic Marin”, Aquarientechnik GmbH). A suspen-sion containing 1.5 × 106 zoospores/mL was used for settlementassays following the principles outlined in ref 30. Three replicatesof each surface were placed in a separate compartment of aQuadriperm dish (Greiner Bio-one Ltd.) to which 10 mL of zoosporesuspension were added. After 45 min in the dark, the slides werewashed to remove unsettled (swimming) spores. Slides were fixedin 2.5% (v/v) glutaraldehyde, washed, and air-dried as described inref 30. The density of settled (adhered) spores was determined usinga Zeiss Kontron 3000 image analysis system attached to a Zeissepifluorescence microscope and video camera. Thirty fields of view,each 0.17 mm2, were counted at 1 mm intervals along the length ofeach of 3 replicate slides.

Experiments were also conducted on sporelings of UlVa, i.e.,young plants that develop from attached spores. A PDMS elastomer(Silastic-T2, Dow Corning) was also included in this assay in orderto allow the sporeling release data to be compared directly with thatfor a coating with established sporeling release properties. Fivereplicates were treated as above, and at the end of the settlementperiod, the washed slides were replaced in Quadriperm dishes towhich 10 mL of growth medium were added.31 Sporelings weregrown for 7 days at 18 °C with a 16 h/8 h light/dark cycle, priorto the determination of biomass. The medium was refreshed every2 days. Sporeling biomass was measured in situ on slides by theautofluorescence of chlorophyll (430 nm/630 nm excitation/emission,respectively) using a multiwell plate reader (Tecan Genios Plus) asdescribed in ref 30. Fluorescence was recorded as relative fluorescenceunits (RFU) from direct readings.

To assess the strength of adhesion of the sporelings, the slideswere exposed to a wall shear stress of 20 Pa in a flow channel.23

Percentage removal of sporeling biomass was determined from theRFU values recorded before and after exposure to flow using 216

(26) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741–1477.(27) Kaelble, D. H. J. Adhes. 1970, 2, 66–81.(28) Shirley, D. A. Phys. ReV. B 1972, 5, 4709–14.(29) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D., Handbook

of X-ray Photoelectron Spectroscopy; Physical Electronics: Eden Prairie, MN,1992.

(30) Beigbeder, A.; Degee, P.; Conlan, S. L.; Mutton, R.; Clare, A. S.; Pettitt,M. E.; Callow, M. E.; Callow, J. A.; Dubois, P. Biofouling 2008, 24, 291–302.

(31) Starr, R. C.; Zeikus, J. A. J. Phycol. 1989, 23, S1–S27.(32) Pettitt, M. E.; Henry, S. L.; Callow, M. E.; Callow, J. A.; Clare, A. S.

Biofouling 2004, 20, 299–311.

Table 1. Composition of the Top Layer of the Two-LayerCoatings SnSzm_p

two-layer coating composition (wt %) na mb

S26Sz23_100 S26Sz23 (100%) 26 23S26Sz23_90 S26Sz23/SEBS (90%/10%) 26 23S81Sz19_100 S81Sz19 (100%) 81 19S81Sz19_90 S81Sz19/SEBS (90%/10%) 81 19SEBS SEBS (100%) - -

a Degree of polymerization of the polystyrene block b Degree ofpolymerization of the PEGylated-fluorinated polystyrene block.

Nanostructured Films of Amphiphilic Block Copolymers Langmuir, Vol. 24, No. 22, 2008 13141

paired (before and after) readings per replicate slide. Removal wascalculated for each of these individual points in the sporeling Biofilm.Percentage removal data were arcsine-transformed and the normalityassessed using the Anderson-Darling test for conformity. Differencesbetween surfaces were tested using a one-way ANOVA followedby Tukey’s test for pairwise comparisons.

The diatom assay followed the methods described in ref 32. Inbrief, cells of NaVicula perminuta were resuspended in ASW to aconcentration of 0.3 µg chlorophyll a/mL. The surfaces (6 replicates)were placed in Quadriperm dishes to which 10 mL of the diatomculture were added. After 2 h, the slides were washed to remove anynonattached cells. Three replicate slides were then fixed and processedas described for UlVa zoospores. The remaining three slides wereexposed to a wall shear stress of 51 Pa in a flow channel,23 beforefixation and processing. Removal data are expressed as a percentageof the density of cells prior to exposure in the flow channel.

Results and Discussion

Synthesis of Block Copolymers. The A-B type blockcopolymers consisted of a block of polystyrene (block A) anda block of polystyrene carrying a PEGylated-fluoroalkyl sidechain (block B). This dual hydrophilic-hydrophobic natureprovided an amphiphilic character to the materials, and the thinfilms derived therefrom were anticipated to respond to contactwith water and seawater.

The block copolymers, SnSzm, were prepared by a two-stepprocedure based on two sequential atom transfer radicalpolymerizations (ATRP) (Scheme 1). In the first step, bromo-terminated polystyrene macroinitiators, Sn, having molecularweights of 8.4 × 103 and 2.7 × 103 g/mol (number averagedegree of polymerization n ) 81 and 26) were prepared fromstyrene, S, in the presence of 1-phenylethyl bromide (1-(PE)Br)/CuBr/2,2′-bipyridine (Bipy) (1/1/3 molar ratio) at 110 °C. In thesecond step, the bromo-terminated polystyrenes were used toinitiate the polymerization of the PEGylated-fluoroalkyl 4-vi-nylbenzoate monomer, Sz, at 115 °C, which was thus linked asthe amphiphilic block. The average numbers (x and y) of repeatunits in the polyoxyethylene and polytetrafluoroethylene segmentsof the Sz side chains were found to be 5 and 4, respectively, by1H NMR and 19F NMR.

The two copolymers prepared differ in the average degreesof polymerization of the polystyrene block (n) and the PEGylated-fluorinated polystyrene block (m) and their relative lengths. Inparticular, m was 23 and 19 for S26Sz23 and S81Sz19, andaccordingly, the number average molecular weights (Mn) of thetwo block copolymers were 21.4 × 103 g/mol and 23.9 × 103

g/mol, respectively. The SEC elution curves of the blockcopolymers were monomodal and characterized by a polydis-persity Mw/Mn ≈ 1.5. In a previous paper, we have discussed thedetailed synthesis of the block copolymers and pointed out thepossibility of varying the composition, and consistentlythe morphology, of the copolymers by tailoring the reactionconditions, due to the controlled nature of the ATRP.21 Controlledradical polymerizations have been widely used for the homo andblock copolymerization of semi- and highly fluorinated mono-mers,33 since such techniques allow for the development ofadvanced well-defined polymeric architectures (star, dendritic,graft, or block copolymers) having predictable molecular weightsand narrow molecular weight distributions. The present blockcopolymers provide one more example in which the versatilityof the ATRP is exploited to prepare novel amphiphilic blockcopolymers.

Preparation of Polymer Films. The films for studies ofwettability and biological performance of the amphiphilic

polymers were prepared according to two different methods: (i)a thin layer of the active fluorinated block copolymer wasdeposited on a glass slide by spin-coating (one-layer geometry),and (ii) a thin layer of the active fluorinated block copolymereither alone or blended with SEBS in different proportions p (p) 100 and 90 wt %) was spray-coated on a SEBS/SEBS-MAbased bottom layer (two-layer geometry). The films were annealedat 120 °C for 12-15 h to promote the formation of an equilibriumstructure.

According to the latter strategy, the films SnSzm_p consistedof a low surface energy top layer deposited on a low elasticmodulus bottom layer. The surface of the glass substrates hadpreviously been chemically modified with glycidyl groups by(3-glycidoxypropyl)-trimethoxysilane (GPS)-functionalization,which were then reacted with the maleic anhydride (MA) groupsof SEBS-MA (Figure 1). This ensured covalent anchorage of thebottom layer to the glass substrate and prevented delaminationof the polymer in water during the assays.

SEM images on cryogenic fracture sections confirmed theactual deposition of a thinner top film of fluorinated polymer(∼500 nm thick) on the thicker bottom layer of SEBS (150-200µm thick) (Figure 2a). The two layers adhered well to each othernotably in the annealed samples (Figure 2b).

EDX elemental analysis carried out on two-layer filmsevidenced the uniform presence of fluorine at the outerpolymer-air interface throughout the film, whereas this elementwas completely absent in the SEBS bottom layer. Therefore, thefluorinated polymer was actually confined in the top layer withoutdiffusing significantly out of it into the layer underneath.

(33) Hansen, N. M. L.; Jankova, K.; Hvilsted, S. Eur. Polym. J. 2007, 43,255–293.

Figure 2. SEM images of a two-layer film before (a) and after (b)annealing at 120 °C overnight.

13142 Langmuir, Vol. 24, No. 22, 2008 Martinelli et al.

Isothermal annealing promoted enrichment in fluorinated chainsat the outermost surface, owing to their low surface energy.

XPS Analysis. A more quantitative analysis of the blockcopolymer surfaces was performed by X-ray photoelectronspectroscopy (XPS) measurements on thin films of the copoly-mers. Spectra were recorded at different photoemission angles(the angles between the surface normal and the paths taken bythe electrons toward the detector) φ ) 70°, 50°, and 20°,corresponding to increasing sampling depths in the range 3-10nm.

The results are discussed here in detail for S26Sz23 as a typicalexample. The survey spectra showed the signals due to theelements constituting the repeat units only: C (∼290 eV), O(∼533 eV), and F (∼689 eV) (Figure 3).

The elemental analysis data for the different angles φ aresummarized in Table 2 and compared with the correspondingvalues calculated from the known stoichiometric ratios of theblock components. The atomic percentage changed with φ, thusshowing that there was a composition gradient along the polymersurface normal. In particular, the C percentage increased withincreasing sampling depth from 45.0% to 54.2% in going fromφ) 70° to φ) 20°, while the F percentage followed the oppositetrend, decreasing from 42.8% to 29.5%. In addition, theexperimental F/C ratio was much higher than the theoretical one(0.46) at any given photoemission angle, e.g., 0.95 at φ ) 70°.These findings indicate that the topmost surface was enrichedin fluorine with respect to the bulk. Many other examples havebeen reported in the literature about the selective segregation ofsemifluorinated chains of a polymeric structure at the polymer-airinterface, because of their low surface energy.34-36

The results on the atomic composition as a function of φ wereconfirmed by closer inspection of the C(1s) peak (Figure 4). Itrevealed a complex shape, due to the presence of at least fiveoverlapping contributions at ∼285 eV (CH, CH2, and CdC),∼287 eV (CH2CF2 and CH2O), ∼289 eV (COO), ∼292.0 eV(CF2), and ∼294 eV (CF3). Two trends were particularly clear:(i) the contribution of the CF2 groups was much more evidentat high φ, e.g., the integrated area under the CF2 peak decreasedfrom 23.4% to 12.2% with decreasing φ from 70° to 20°, and(ii) the contribution of the CF3 groups was more intense at largeφ, e.g., the integrated area under the CF3 peak decreased from3.5% to no more detectable values with decreasing φ from 70°to 20°. Therefore, the perfluorinated segments of the side chainsappeared to be stretched out at the polymer-air interface, withthe terminal CF3 groups pointing outward.

The angle-dependent XPS analysis was also carried out on thesame samples after 9 days of immersion in water (wet sample),with the aim of ascertaining whether the surface could undergoreconstruction as a consequence of its amphiphilic nature. Thesurface composition of the wet films is expected to be thatcorresponding to a kinetically trapped condition, rather than theequilibrium state when in contact with water. However,reorganization of blocks occurs rather slowly at room temperature.The XPS spectra of the wet surface can, therefore, be consideredindicative of chemical composition when the surface is in contactwith water. The atomic compositions of the wet surface are alsocollected in Table 2. The elemental composition varied with thephotoemission angle, and both of the carbon and fluorine atomicpercentages followed the same trends discussed for the dry surface.Furthermore, the C(1s) signal of the wet films also exhibited thesame shape and dependence on photoemission angle as for thedry films (Figure 5). In fact, the integrated area associated tothe CF2 peak decreased from 18.5% to 9.3% in going from φ )70° to φ ) 20°, and the CF3 contribution decreased from 2.9%at φ ) 70° to no more detectable values at φ ) 20°. However,by comparing the elemental analysis values obtained for the dryand wet polymer surfaces, it was found that, at any investigatedφ, the F percentage decreased, e.g., from 42.8% to 35.4% at φ

) 70°, after contact with water, whereas the O percentageincreased, e.g., from 12.2% to 14.3% at φ ) 70°. These resultssupport the hypothesis of a surface reorganization and agreewith those already reported for analogous amphiphilic blockcopolymers.18,21 There the surface reorganization was basically

(34) Tsibouklis, J.; Graham, P.; Eaton, P. J.; Smith, J. R.; Nevell, T. G.; Smart,J. D.; Ewen, R. J. Macromolecules 2000, 33, 8460–8465.

(35) Nishino, T.; Urushihara, Y.; Meguro, M.; Nakamae, K. J. Colloid InterfaceSci. 2005, 283, 533–538.

(36) Saıdi, S.; Guittard, F.; Guimon, C.; Geribaldi, S. Macromol. Chem. Phys.2005, 206, 1098–1105.

Figure 3. XPS survey spectra for S26Sz23 at three different photo-emission angles: 70° (a), 50° (b), and 20° (c).

Table 2. XPS Atomic Compositions for S26Sz23 at DifferentPhotoemission Angles after Annealing at 120 °C (Dry) and after

Immersion in Water for 9 Days (Wet)

dry wet

φ (°) C (%) O (%) F (%) C (%) O (%) F (%)

stoichiometrica 61 11 28 61 11 2870 45.0 12.2 42.8 50.3 14.3 35.450 49.3 14.8 35.9 54.8 16.3 28.920 54.2 16.3 29.5 63.8 17.5 18.7

a Calculated on the basis of the known composition of the block copoly-mer.

Figure 4. Area-normalized C(1s) XPS signals at photoemission anglesof 70°, 50°, and 20° for S26Sz23 after annealing at 120 °C (dry).

Nanostructured Films of Amphiphilic Block Copolymers Langmuir, Vol. 24, No. 22, 2008 13143

due to the flipping of the PEGylated-fluoroalkyl side chains,which made the surface more hydrophilic by exposing theoxyethylene segments to contact with water and hiding thehydrophobic fluorinated segments in the underlying layers. Thus,the film surface could react to the external environment owingto its responsive, amphiphilic nature, thereby giving rise to amore chemically heterogeneous structure.

Static Contact Angles and Surface Energy. The static contactangles of water (θw) and n-hexadecane (θh) on the two-layercoatings, the SEBS control, and the pristine block copolymerswere measured by the sessile drop technique. The two-layercoatings turned out to be both hydrophobic and lipophobic at thesame time, being θwg107° and θhg64° (Table 3). By comparingthe contact angle values of the two-layer coatings with those ofSEBS films on one hand and those of the neat block copolymerfilms on the other hand, two findings are noteworthy. First, bothθw and θh are significantly larger for the fluorinated coatingsthan for the SEBS, even though the difference is more markedfor θh than for θw. This suggests that the inclusion of the fluorinatedblock copolymer in the top layer affected the hydrophobic andespecially the lipophobic character of the coatings. Second, thetwo-layer coatings generally showed water and n-hexadecanecontact angles similar to and even higher than those of therespective pristine block copolymers, independent of the toplayer composition. This was a further confirmation of thepreferential and selective segregation of the fluoroalkyl chainsat the polymer-air interface, even when the block copolymerwas blended with SEBS. Chemical incompatibility was possiblyenhanced and lipophobicity was dramatically improved withrespect to SEBS.

Measurements of liquid-solid contact angles are commonlyused to evaluate solid surface tension (γS). To extract the solid

surface tension from experimental θ values, we used eq 3 afterthe Owens-Wendt-Kaelble approach.26,27 The surface tensionsγS and the related components calculated for the fluorinated films,the respective block copolymers, and the SEBS controls are alsopresented in Table 3. All the fluorinated two-layer films exhibitedlow surface tensions (13.5 mN/m e γS e 15.3 mN/m), as aconsequence of both substantial hydrophobicity and lipophobi-city. These values were similar to those of the one-layer blockcopolymer films and markedly lower than those of the controlSEBS coating. As expected of nonpolar, non-hydrogen-bondingsurfaces such as fluorinated surfaces, the dispersion contribution(γS

d ≈ 13-14 mN/m) to γS was largely dominant, with the polarcontribution being minimal (γS

p ≈ 0.5-2 mN/m). Suchcontribution was mainly due to the PEGylated segments, whichpermitted polar interactions with the test liquids.17 Surfaceenergies of the fluorinated two-layer coatings slightly increasedas the weight content of Sz units decreased, thus lending furthersupport to the occurrence of microphase separation of theincompatible blocks, with the PEGylated-fluorinated styrene blockbeing segregated and exposed at the outer interface.

We note that the coatings exhibited high values of advancingcontact angles (θa ≈ 110°) and low values of receding contactangles (θr ≈ 50°) leading to large hystereses (θa - θr ≈ 60°)when water was used as the wetting liquid. Such a phenomenonis probably due to a combination of chemical heterogeneity andrestructuring of the surface, with the latter being affected uponcontact with water.18,21 The hypothesis of surface modificationwas also confirmed by static contact angle measurementsperformed after 10 day immersion in water. In particular, it wasfound out that θw was depressed from ∼110° to ∼80°, while θh

was from ∼70° to ∼60°. Owing to the concomitant decrease inhydrophobicity and lipophobicity, the surface tension significantlyrose, reaching values of 20-30 mN/m. These findings supportthe XPS results and indirectly prove that the surface becamemore enriched in the high-surface-energy PEG component aftercontact with water, while the fluorinated segments tended to besegregated in the bulk of the film. The results of detailed studiesof contact angles with various interrogating fluids, includingunderwater measurements, will be presented in a forthcomingpaper.

Surface Morphology. The use of the phase separation behaviorof fluorinated (co)polymers and blends is well-suited to generateordered, self-assembled, low surface energy materials.17,37-41

We used AFM to investigate the morphological features of thefluorinated two-layer coatings and SEBS reference after annealingat 120 °C for 15 h. In any case, the coatings presented lowsurface roughness values (rms ≈ 1 nm) on the (sub)micrometerscale. Moreover, the surface roughness was substantiallyindependent of the chemical composition of the top layer (Table4). The phase images (from tapping mode/AC mode) of theannealed films (Figure 6 (left)) evidence the formation of complexand well-defined morphological structures resulting from thethermodynamically induced phase segregation of the differentcomponents of the coating, because of mutual chemical incom-patibility.40 The fluorinated coating morphology depended onthe amphiphilic copolymer composition. While S26Sz23_100displayed a morphology with spherical domains of ca. 20 nm

(37) Gan, D. J.; Mueller, A.; Wooley, K. L. J. Polym. Sci., Part A: Polym.Chem. 2003, 41, 3531–3540.

(38) Fujimori, A.; Shibasaki, Y.; Araki, T.; Nakahara, H. Macromol. Chem.Phys. 2004, 205, 843–854.

(39) Hikita, M.; Tanaka, K.; Nakamura, T.; Kajiyama, T.; Takahara, A.Langmuir 2004, 20, 5304–5310.

(40) Al-Hussein, M.; Serero, Y.; Konovalov, O.; Mourran, A.; Moller, M.; deJeu, W. H. Macromolecules 2005, 38, 9610–9616.

(41) Busse, K.; Peetla, C.; Kressler, J. Langmuir 2007, 23, 6975–6982.

Figure 5. Area-normalized C(1s) XPS signals at photoemission anglesof 70°, 50°, and 20° for S26Sz23 after immersion in water for 9 days(wet).

Table 3. Contact Angles and Surface Tensions for theOne-Layer and Two-Layer Films and the SEBS Control

filmcompositiona

(wt %)θw

b

(°)θh

c

(°)γS

dd

(mN/m)γS

pd

(mN/m)γS

d

(mN/m)

S26Sz23e 87 107 ( 1 66 ( 1 13.7 1.6 15.3S81Sz19e 65 106 ( 1 66 ( 1 13.7 1.3 15.0S26Sz23_100 87 113 ( 2 69 ( 2 12.9 0.6 13.5S26Sz23_90 78 112 ( 2 66 ( 1 13.7 0.5 14.2S81Sz19_100 65 110 ( 1 64 ( 1 14.1 0.8 14.9S81Sz19_90 58 107 ( 1 65 ( 1 13.9 1.4 15.3SEBS 0 102 ( 2 26 ( 1 24.8 0.6 25.4

a Content of Sz in the top layer. b Measured with water c Measured withn-hexadecane. d Calculated with the Owens-Wendt-Kaelble method: γS

d

dispersion component, γSp polar component. e One-layer film.

13144 Langmuir, Vol. 24, No. 22, 2008 Martinelli et al.

size, S81Sz91_100 showed a morphology with lying cylinderswith a periodicity of 24-29 nm. In both cases, the discretedomains of polystyrene were dispersed in the continuous matrixof amphiphilic polystyrene. These two morphologies are thesame as those of the respective neat (one-layer) block copolymersand consistent with previous AFM and GISAXS results onanalogous block copolymers.21 Very close domain size andperiodicity, ca. 20 nm and 25-27 nm, were detected for thetwo-layer coatings S26Sz23_90 and S81Sz19_90, respectively,containing 10 wt % SEBS in the top layer blend. On the otherhand, the morphology of the SEBS control films was significantlydifferent and consisted of nanophase domains arranged in awormlike fashion, in agreement with what was previously reportedfor SEBS coatings prepared by casting toluene solutions of thepolymer.42

The capability of these amphiphilic block copolymers ofundergoing surface reconstruction upon contact with water,already proven by XPS as well as contact angle measurements,is also reflected in changes of both the surface roughness andthe morphology of the two-layer films. After 7 days of immersionin ASW, the roughness increased for the fluorinated surfaces,reaching the maximum value of 6 nm for S26Sz23_100, whileit remained practically unaffected for the SEBS (Table 4). Theunderwater rms data showed a monotonic dependence on thecomposition of the fluorinated top layer, and in particular,roughness decreased with decreasing Sz content. The roughertopographies exhibited by the underwater amphiphilic films canbe explained as a result of the surface modification, which is alsowell-proven by under-ASW AFM phase images (Figure 6 (right)).These images reveal the transformation from a well-orderedsurface morphology to a mixed surface structure, in which thenanoscale heterogeneity was increased as nanoaggregates ornanoparticles overlapped onto the original cylindrical or sphericalmorphologies. The same structures emerge by storage and imagingin deionized water (data not shown), proving that they are notprecipitates of seawater salts but intrinsic features of the polymermaterial. A reduction of the block copolymer content in the toplayer seems to result in a more ordered surface patterning inaqueous environment, clearly visible for the S26Sz23 system(Figure 6b,c) A homogeneous nanopattern in the range 50-100nm length scale still existed for the surface of S26Sz23_90.

Assays with Marine Algae. The density of UlVa zoosporesattached after a 45 min settlement period was similar on all theexperimental surfaces, but slightly lower on the SEBS control(Figure 7). Spores have been shown previously to settle (attach)in high numbers to hydrophobic surfaces, including fluorinatedsurfaces.4,18,20 Sporelings (young plants) grew well on all surfaces,and after 7 days, a green lawn covered the surface of all samples.The percentage release of biomass after exposure to a wall shearstress of 20 Pa in a flow channel (Figure 8) shows that significantlymore biomass was removed from all the experimental coatingscompared to the control SEBS. Furthermore, more biomass was

removed from the experimental coatings than from the Silastic-T2 standard, which had similar removal (30%) as the SEBScontrol. UlVa sporelings adhere weakly to PDMS-based elas-tomers such as Silastic-T2.11,18,20,30,43 Decreasing the proportionof fluorinated component significantly enhanced the release

(42) Ganguly, A.; De Sarkar, M.; Bhowmick, A. K. J. Polym. Sci., Part B:Polym. Phys. 2007, 45, 52–66.

Table 4. Root-Mean-Square Roughnessaof SEBS andFluorinated Two-Layer Films after Annealing at 120 °C (Dry)

and under ASW after 7 Days of Immersion in ASW(Underwater)

filmcompositionb

(wt %)dry rms

(nm)underwaterrms (nm)

S26Sz23_100 87 ∼1 6S26Sz23_90 78 ∼1 4S81Sz19_100 65 ∼1 4S81Sz19_90 58 ∼1 3SEBS 0 ∼1 ∼1a Measured over a 1 µm × 1 µm scanning area. b Content of Sz in the

top layer.

Figure 6. AFM phase images of SEBS (a), S26Sz23_100 (b), S26Sz23_90(c), S81Sz19_100 (d), and S81Sz19_90 (e) recorded after annealing at120 °C (dry) (left) and under ASW after 7 days of immersion in ASW(wet) (right).

Nanostructured Films of Amphiphilic Block Copolymers Langmuir, Vol. 24, No. 22, 2008 13145

properties of S26Sz23. In contrast, biomass release from S81Sz19was improved when the fluorinated component was maximum.The best-performing coating overall was S26Sz23_90. Interest-ingly enough, this was the coating exhibiting the most regular(nanopatterned) surface structure in seawater (Figure 6c).

The adhesion strength of attached NaVicula cells wasdetermined by assessing the proportion of cells removed afterexposure to a wall shear stress of 51 Pa (Figure 9). Cells adheredmore strongly to the S26z23 coatings than to the S81Sz19coatings. Higher removal was seen from S26Sz23_90 thanS26Sz23_100, but there was no significant difference betweenS81Sz19_100 and S81Sz19_90. High removal was also seenfrom the control SEBS. Adhesion strength of diatoms is broadlyrelated to wettability. Diatom cells generally adhere weakly tohydrophilic surfaces compared to hydrophobic surfaces.10,11,20,25

All the experimental fluorinated test surfaces discussed in thispaper are hydrophobic with water contact angles between 106°and 113° and surface tensions in the range 13.5-15.3 mN/m.Such small differences are unlikely to explain the difference inperformance against diatoms seen for the S26Sz23 vs S81Sz19surfaces, and it is more likely that differences in surfacerestructuring behavior, such as morphology and/or chemicalheterogeneity, are responsible for the observed variations inperformance.

Concluding Remarks

The controlled nature of the atom transfer radical polymer-ization allows for the synthesis of amphiphilic block copolymersdiffering in the content and relative length of the constituentpolymer blocks. Control over the chemical constitution in turnenables formation of different surface morphologies, in whichthe minor polystyrene component was segregated into discretenanodomains, either spherical or cylindrical, embedded in theamphiphilic polystyrene matrix. The XPS and contact angleanalyses confirmed that the introduction of the oxyethylene-tetrafluoroethylene side groups provided an amphiphilic extracharacter to the otherwise hydrophobic polystyrene backbone.The different capabilities of the hydrophilic oxyethylene chainsegments and the hydrophobic/lipophobic tetrafluoroethylenechain segments to interact with the surrounding medium resultedin a responsive character of the material, which was expressedin a kind of multifold or “ambiguous” nature of the surface ofthe films. The amphiphilic behavior of the copolymer films alsoresulted in distinct performances against the different testorganisms. On the one hand, UlVa sporelings exhibited a muchweaker adhesion on the films richer in the amphiphilic polystyrenecomponent, the percentage removal being largest at moderate tohigh, though not highest, weight contents. On the other hand,NaVicula cells showed a higher removal from the films with alower weight percentage of the amphiphilic side chains, whichhad a higher surface free energy. While UlVa sporelings wereremoved more easily than from the SEBS internal control, whichperforms as well as the PDMS standard, NaVicula diatoms adheredmore strongly to the films.

Our findings also confirm that surface reconstruction afterimmersion in water of the films involves more massiverearrangement in the surface structure as probed by AFM. Wemay speculate that such changes in surface structure areresponsible for the observed differences in biological performance,since it is known that surface morphology and topographyinfluence adhesion strength of fouling organisms on polymercoatings, and differences in surface nanoroughness have beenshown to affect protein adsorption44 and attachment of bacteria45

and animal cells.46 However, the evaluated changes of surfacerugosity of the dry and underwater films as expressed by the rmsvalues were relatively minor in extentsbeing only a fewnanometers. In the absence of any detailed, systematic studies

(43) Majumdar, P.; Lee, E.; Patel, N.; Ward, K.; Stafslien, S. J.; Daniels, J.;Boudjouk, P.; Callow, M. E.; Callow, J. A.; Thompson, S. E. M. Biofouling 2008,24, 185–200.

(44) Horguard, M. B.; Rechendorff, K.; Chevallier, J.; Foss, M.; Besenbacher,F. J. Phys. Chem. B 2008, 112, 8241–8249.

(45) Kerr, A.; Cowling, M. J. Phil. Mag. 2003, 83, 2779–2795.(46) Curtis, A. S. G.; Casey, B; Gallagher, J. O.; Pasqui, D.; Wood, M. A.;

Wilkinson, C. D. W. Biophys. Chem. 2001, 94, 275–283.

Figure 7. Mean number of UlVa spores/mm2 surface attached after 45min settlement. The bars show 2 × SEM.

Figure 8. Percentage removal of UlVa sporelings after exposure to awall shear stress of 20 Pa. N ) 864 (216 readings × 4 replicate slides),error bars ) (2 × Standard Error derived from arcsine transformeddata.

Figure 9. Percentage removal of NaVicula cells after exposure to a wallshear stress of 51 Pa. N ) 90, bars show 2 × SEM derived from arcsine-transformed data.

13146 Langmuir, Vol. 24, No. 22, 2008 Martinelli et al.

on the influence of nanoscale roughness on the organisms usedin this work, such explanations must therefore remain speculative,although it may be noted that the release of sporelings of UlVawas shown to be higher for quaternized PDMS coatings thatexhibited high surface heterogeneity and nanoroughness.43

However, the relevance of this is not clear, since the topologicalcharacterizations were performed on dry coatings.

A further issue is that rms values for rugosity may notencapsulate the full extent of surface restructuring, and thequalitative differences between the coatings, as observed in theAFM phase images, may be indicative of more profound changesin surface molecular properties and the formation of domains ofdifferent surface chemistry. It is known that subtle changes inmolecular structure, order, and crystallinity of model surfacessuch as self-assembled alkanethiol monolayers can stronglyinfluence the adhesion of spores of UlVa and cells of NaVicula.47

Spores of UlVa also respond to microscale patterns of different

surface chemistry and wettability.4 One may therefore hypothesizethat the molecular and nanoscale ambiguity of the amphiphilicsurface lowers the driving forces for the adsorption of adhesivemacromolecules and hence reduces the adhesion strength of theorganisms. Unfortunately, the lack of knowledge on the chemistryof the relevant adhesives and their curing characteristics meansthat such mechanistic interpretations must remain speculative.

Acknowledgment. The work was funded by the EC Frame-work 6 Integrated Project ‘AMBIO’ (Advanced NanostructuredSurfaces for the Control of Biofouling). This article reflects onlythe authors’ views and the European Commission is not liablefor any use that may be made of information contained therein.The Italian MiUR (PRIN) is also gratefully acknowledged.

Supporting Information Available: Rms roughness of the filmswas investigated by AFM as a function of surface area. This materialis available free of charge via the Internet at http://pubs.acs.org.

LA801991K(47) Bowen, J.; Pettitt, M. E.; Kendall, K.; Leggett, G. J.; Preece, J. A.; Callow,

M. E.; Callow, J. A. J. R. Soc. Interface 2006, 22, 473–478.

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