Antibacterial Inorganic−Organic Hybrid Coatings on Stainless Steel

Dec 10, 2009 - §CEWIC, Thule Institute, University of Oulu, P.O. Box 7300, FI 90014, Finland. Received October 27, 2009. Revised Manuscript Received ...
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Antibacterial Inorganic-Organic Hybrid Coatings on Stainless Steel via Consecutive Surface-Initiated Atom Transfer Radical Polymerization for Biocorrosion Prevention S. J. Yuan,†,‡ S. O. Pehkonen,§ Y. P. Ting,‡ K. G. Neoh,‡ and E. T. Kang*,‡ †

College of Chemical Engineering, Sichuan University, Chengdu 610065, China, ‡Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260, and § CEWIC, Thule Institute, University of Oulu, P.O. Box 7300, FI 90014, Finland Received October 27, 2009. Revised Manuscript Received November 19, 2009

To enhance the corrosion resistance of stainless steel (SS) and to impart its surface with antibacterial functionality for inhibiting biofilm formation and biocorrosion, well-defined inorganic-organic hybrid coatings, consisting of a polysilsesquioxane inner layer and quaternized poly(2-(dimethyamino)ethyl methacrylate) (P(DMAEMA)) outer blocks, were prepared via successive surface-initiated atom transfer radical polymerization (ATRP) of 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). The cross-linked P(TMASPMA), or polysilsesquioxane, inner layer provided a durable and resistant coating to electrolytes. The pendant tertiary amino groups of the P(DMAEMA) outer block were quaternized with alkyl halide to produce a high concentration of quaternary ammonium groups with biocidal functionality. The so-synthesized inorganic-organic hybrid coatings on the SS substrates exhibited good anticorrosion and antibacterial effects and inhibited biocorrosion induced by sulfate-reducing bacteria (SRB) in seawater media, as revealed by antibacterial assay and electrochemical analyses, and they are potentially useful to steel-based equipment under harsh industrial and marine environments.

1. Introduction Microorganisms have a strong tendency to populate surfaces, giving rise to a complex and strongly adhering microbial community, termed ‘‘biofilm”.1 Biofilms are detrimental to the underlying substrates, causing physical degradation or biodeterioration of metal surfaces.2-5 This phenomenon has been widely recognized as biocorrosion or microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and maritime industries. At least 20% of all corrosions are associated with biocorrosion, at a direct cost of 30-50 billion dollars annually worldwide.6 Among the various microorganisms that induce corrosion or degradation of metallic materials, sulfate-reducing *To whom correspondence should be addressed. Telephone: þ65-65162189. Fax: þ65-6779-1936. E-mail: [email protected].

(1) Costerton, W. J.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318– 1322. (2) Costerton, W. J. In Biofouling and Biocorrosion in Industrial Water System; Fleming, H. C., Geesey, G. G., Eds.; Springer-Verlag: New York, 1991. (3) Borenstein, S. W. In Microbiologically Influenced Corrosion Handbook; Hirsch, M., Ed.; Woodhead Publishing Ltd.: Cambridge, U.K., 1994. (4) Geesey, G. G.; Gillis, R. J.; Avci, R.; Daly, D.; Hamilton, M.; Shope, P.; Harkin, G. Corros. Sci. 1996, 38, 73–95. (5) Mehanna, M; Basseguy, R.; Delia, M. L.; Bergel, A. Electrochem. Commun. 2009, 11, 568–571. (6) Fleming, H. C. In Microbially Influenced Corrosion of Materials; Heitz, Z., Fleming, H. C., Sand, K., Eds.; Spring-Verlag: New York, 1996. (7) Thierry, D.; Sand, W. In Corrosion Mechanisms in Theory and Practice; Marcus, P., Oudar, J., Eds.; Marcel Dekker Inc.: New York, 1995. (8) Castaneda, H.; Benetton, X. D. Corros. Sci. 2008, 50, 1169–1183. (9) Antony, P. J.; Singh Raman, R. K.; Mohanram, R.; Kumar, P.; Raman, R. Corros. Sci. 2008, 50, 1858–1864. (10) Hamilton, W. A. Annu. Rev. Microbiol. 1985, 39, 195–217. (11) Lee, W.; Lewandowski, Z.; Nielsen, P. H.; Hamilton, W. A. Biofouling 1995, 8, 165–194. (12) Little, B. J.; Lee, J. S.; Ray, R. I. Electrochem. Acta 2008, 54, 2–7. (13) Hamilton, W. A.; Lee, W. C. In Biocorrosion in Sulfate Reducing Bacteria; Barton, L. L., Ed.; Plenum Press: New York, 1995. (14) Iverson, W. O. Science 1966, 151, 986–988. (15) Landoulsi, J.; El Kirat, K.; Richard, C.; Feron, D.; Pulvin, S. Environ. Sci. Technol. 2008, 42, 2233–2242.

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bacteria (SRB) are the most problematic and are the principal cause of failure in many steel-based structures.7-17 It is therefore of great importance to inhibit biocorrosion of SRB for prolonging the service life of maritime structures and equipment. Various strategies have been developed to combat biocorrosion.18-30 Biocidal treatment has been widely used to mitigate biocorrosion in steel pipelines and in closed systems.18-23 The practice is less than satisfactory due to potential environmental hazards, high cost, and low efficiency against biofilms. Despite their effectiveness in the marine environment, protective antifouling coatings, such as tributyltin (TBT)-based paints, have been completely phased out since 2008 owing to their detrimental effect on nontargeted marine organisms.24 In view of the complex environmental, ecological, and economical issues, more recent (16) Beech, I. B.; Sunner, J. Curr. Opin. Biotechnol. 2004, 15, 181–186. (17) King, R. A.; Wakerley, D. S. Br. Corros. J. 1973, 8, 41–45. (18) Franklin, M. J.; Nivens, D. E.; Vass, A. A.; Mittelman, M. W.; Jack, R. F.; Dowling, N. J. E.; White, D. C. Corrosion 1991, 47, 128–134. (19) El-Shamya, A. M.; Sororb, T. Y.; El-Dahana, H. A.; Ghazyc, E. A.; Eweasd, A. F. Mater. Chem. Phys. 2009, 114, 156–159. (20) Cetin, D.; Bilgic, S.; D€onmez, G. ISIJ Int. 2007, 47, 1023–1028. (21) Raman, V.; Tamilselvi, S.; Rajendran, N. Mater. Corros. 2008, 59, 329–334. (22) Gonzalez-Rodrı´ guez, C. A.; Rodrı´ guez-Gomez, F. J.; Genesca-Llongueras, J. Electrochim. Acta 2008, 54, 86–90. (23) Videla, H. A. Int. Biodeterior. Biodegrad. 2002, 49, 259–270. (24) Yebra, D. M.; Kiil, S.; Dam-Johansen, K. Prog. Org. Coat. 2004, 50, 75– 104. (25) Miyanaga, K.; Terashi, R.; Kawai, H.; Unno, H.; Tanji, Y. Biotechnol. Bioeng. 2007, 97, 850–857. (26) de Mele, M. F. L.; de Saravia, S. G. G.; Videla, H. A. In Proceedings of the 1995 International Conference on Microbially Influenced Corrosion; Borenstein, S. W., Angell, P., Eds.; NACE: New Orleans, 1995. (27) Al-Darbi, M. M.; Muntasser, Z. M.; Tango, M.; Islam, M. R. Energy Sources 2002, 24, 1009–1018. (28) Akid, R.; Wang, H. M.; Smith, T. J.; Greenfield, D.; Earthman, J. C. Adv. Funct. Mater. 2008, 18, 203–211. (29) Yuan, S. J.; Xu, F. J.; Pehkonen, S. O.; Ting, Y. P.; Neoh, K. G.; Kang, E. T. Biotechnol. Bioeng. 2009, 103, 268–281. (30) Yuan, S. J.; Pehkonen, S. O.; Ting, Y. P.; Neoh, K. G.; Kang, E. T. ACS Appl. Mater. Interfaces 2009, 1, 640–652.

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efforts have focused on developing environmentally benign antimicrobial coatings to prevent bacterial adhesion and biofilm formation,27-31 as the formation of biofilms is widely recognized as the key step in initiating biocorrosion. Tethering of nonleaching antimicrobial coatings on a variety of surfaces, including glass,32-34 polymers,35,36 papers,37 and metals,38-40 to prevent bacterial adhesion and biofilm formation has been well documented. Inorganic-organic hybrid coatings are of great interest in anticorrosion systems, since the organic component can impart the substrate surface with specific functionalities while the inorganic counterpart can confer mechanical strength, durability, scratch resistance, and improved adhesion.41-43 The inorganic-organic hybrid coatings are generally prepared using the sol-gel process in two ways: the organic and inorganic groups are linked by stable chemical bonds,44 or the organic component is merely embedded in the inorganic material, and vice versa.45 Recent developments in controlled/“living” radical polymerization have provided an alternative approach to the preparation of inorganic-organic hybrids materials.46 3-(Trimethoxysilyl)-propyl methacrylate with the reactive trimethoxysilyl group (-Si-(OMe)3) has been widely used in the preparation of inorganic-organic hybrid materials.32,47,48 The (-Si-(OMe)3) groups can be readily hydrolyzed into the silanol groups (-Si-(OH)3), which can subsequently condense into a durable cross-linked polysilsesquioxane network.49 In this work, environmentally benign inorganic-organic hybrid coatings with biocidal functionality are developed on stainless steel (SS) substrate, as shown schematically in Figure 1. Coupling of a sulfonyl-halide-containing silane on the active SS (SS-OH) surface provides not only a dense passivation layer but also initiation sites for the preparation of poly(3-(trimethoxysilyl)propyl methacrylate)-block-poly(2-(dimethyamino)ethyl methacrylate) (P(TMSPMA-b-PDMAEMA) copolymer via consecutive surface-initiated atom transfer radical polymerization (ATRP). The inner P(TMSPMA) block containing the reactive trimethoxysilyl groups can be readily hydrolyzed and condensed to form a cross-linked polysilsesquioxane network (31) Sreekumari, K. R.; Sato, Y.; Kikuchi, Y. Mater. Trans. 2005, 46, 1636– 1645. (32) Huang, J. Y.; Koepsel, R. R.; Murata, H.; Wu, W.; Lee, S. B.; Kowalewski, T.; Russeil, A. J.; Matyjaszewski, K. Langmuir 2008, 24, 6785–6795. (33) Madkour, A. E.; Dabkowski, J. M.; N€usslein, K.; Tew, G. N. Langmuir 2009, 25, 1060–1067. (34) Sambhy, V.; Peterson, B. R.; Sen, A. Langmuir 2008, 24, 7549–7558. (35) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 465–471. (36) Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2003, 83, 168–172. (37) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y. K.; Russell, A. J. Biomacromolecules 2004, 5, 877–882. (38) Caro, A.; Humblot, V.; Methivier, C.; Minter, M.; Salmain, M.; Pradier, C. M. J. Phys. Chem. B 2009, 113, 2101–2109. (39) Jampala, S. N.; Sarmadi, M.; Somers, E. B.; Wong, A. C. L.; Denes, F. S. Langmuir 2008, 24, 8583–8591. (40) Ignatova, M.; Voccia, S.; Gabriel, S.; Gilbert, B.; Cossement, D.; Jer^ome, R.; Jer^ome, C. Langmuir 2009, 25, 891–902. (41) Lamaka, S. V.; Shchukin, D. G.; Andreeva, D. V.; Zheludkevich, M. L.; M€ohwald, H.; Ferreira, M. G. S. Adv. Funct. Mater. 2008, 18, 3137–3147. (42) Lamaka, S. V.; Montemor, M. F.; Galio, A. F.; Zheludkevich, M. L.; Trindade, C.; Dick, L. F.; Ferreira, M. G. S. Electrochim. Acta 2008, 53, 4773– 4783. (43) Zheludkevich, M. L.; Salvado, I. M.; Ferreira, M. G. S. J. Mater. Chem. 2005, 15, 5099–5111. (44) Schottner, G. Chem. Mater. 2001, 13, 3422–3435. (45) Sanchez, U.; Soler-Illa, G. J. A. A.; Ribot, F.; Mayer, C. R.; Cabuil, V.; Lalot, T. Chem. Mater. 2001, 13, 3061–3083. (46) Pyun, J.; Matyjaszewski, K. Chem. Mater. 2001, 13, 3436–3448. (47) Cao, Z.; Du, B. Y.; Li, H. T.; Xu, J. T.; Fan, Z. Q. Langmuir 2008, 24, 5543– 5551. (48) Du, J. Z.; Chen, Y. M. Macromolecules 2004, 37, 6322–6328. (49) Du, J. Z.; Chen, Y. M.; Zhang, Y. H.; Han, C. C.; Fischer, K.; Schmidt, M. J. Am. Chem. Soc. 2003, 125, 14710–14711.

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Figure 1. Schematic diagram illustrating the process of coupling CTCS to the SS-OH surface (the SS-CTCS surface), surfaceinitiated ATRP of TMSPMA from the SS-CTCS surface (the SS-gP(TMSPMA) surface), interchain cross-linking of the trimethoxysilyl groups (-Si-(OMe)3) after hydrolysis and subsequent condensation of the silanol groups (-Si-OH) to form the cross-linked polysilsesquioxane layer (the SS-g-CP(TMSPMA) surface), surface-initiated ATRP of DMAEMA from the SS-g-CP(TMSPMA) surface to form the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface, and subsequent N-alkylation to produce the quaternized and bactericidal SS-g-CP(TMSPMA)-b-QP(DMAEMA) surface.

with enhanced corrosion resistance and durability. The pendant tertiary amino groups of the outer P(DMAEMA) block can be quaternized with hexyl bromide to generate the biocidal quaternary ammonium polycations. The antibacterial and anticorrosion properties of the surface-functionalized SS coupons in Desulfovibrio desulfuricans inoculated simulated seawater-based modified Barr’s (SSMB) media were evaluated by viable cell assays and electrochemical analyses, respectively. For comparison purposes, pristine SS coupons were used as controls in the sterile and D. desulfuricans inoculated SSMB media under the same experimental conditions.

2. Experimental Section 2.1. Materials. Type 304 stainless steel (3 mm in thickness, nominal composition: 71.376% Fe, 8.18% Ni, 0.053% C, 18.08% Cr, 0.06% Cu, 1.68% Mn, 0.05% Mo, 0.047% N, 0.037% P, 0.007% S, and 0.43% Si) was purchased from Metal Samples Co. (Munford, AL). 2-(4-Chlorosulfonylphenyl)ethyl trichlorosilane (CTCS, 50% in toluene solution) was obtained from ABCR GmbH (Karlsruhe, Germany) and was used as received. N,N, N0 ,N00 ,N000 -Pentamethyldiethylenetriamine (PMDETA, 99%), 2,20 -bipyridines (Bpy), triethylamine (TEA, 98%), 2-(dimethyamino)ethyl methacrylate (DMAEMA, 97%), 3-(trimethoxysilyl)propyl methacrylate (TMSPMA, >98%), CuCl (99%), CuCl2 (98%), and hexyl bromide (98%) were purchased from DOI: 10.1021/la904083r

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Article Sigma-Aldrich Chemical Co. (St. Louis, MO) and were used without further purification. Solvents, such as N,N0 -dimethylformamide (DMF, >99.8%), 2-propanol, methanol (anhydrous, >99.8%), tetrahydrofuran (THF), ethanol, and acetone were of analytical grade and were used as received. Water in toluene and TEA was removed by reaction with metallic sodium, followed by distillation. All other chemical reagents and solvents were used as received. Yeast extract and agar were purchased from Oxoid (Hampshire, U.K.). Desulfovibrio desulfuricans (ATCC, No. 27774) was obtained from the American Type Culture Collection. The Live/Dead BacLight bacterial viability kit was purchased from Molecular Probes, Inc. (Eugene, OR). 2.2. Surface-Initiated ATRP. The silane coupling agent, CTCS, was introduced onto the active or hydroxyl-enriched SS (i.e., SS-OH) substrates via self-assembly to give rise to the SSCTCS surface. Details for the preparation of SS-OH and SSCTCS surfaces are described in the Supporting Information. For grafting of the TMSPMA polymer (P(TMSPMA)) brushes from the SS-CTCS surface, 1.8 mL (7.4 mmol) of TMSPMA, 15.4 μL (0.074 mmol) of PMDETA, 7.4 mg of CuCl (0.074 mmol), 2.0 mg of CuCl2 (0.015 mmol), and 3.6 mL of dried DMF were introduced into a dried Pyrex tube.32 After degassing with argon for 30 min, the SS-CTCS substrate was introduced under an argon atmosphere. The reaction tube was sealed and placed in a temperature-controlled oil bath at 60 °C for 6 h to produce the SS-g-P(TMSPMA) surface. After removal from the reaction mixture, the SS-g-P(TMSPMA) substrate was washed sequentially with copious amounts of acetone and ethanol prior to being dried under reduced pressure overnight. To form the cross-linked polysilsesquioxane layer, the SS-g-P(TMSPMA) substrate was immersed in 15 mL of deionized water, containing 0.5 mL of TEA as the catalyst, at 60 °C for 3 h to hydrolyze trimethoxysilyl groups and to subsequently condense the silanol groups to produce the cross-linked SS-g-P(TMSPMA) surface (referred to as the SS-gCP(TMSPMA) surface).32,47 The substrate was then cured in a vacuum oven at 70 °C for 1 h. The baking step under mild conditions promoted the interchain condensation of the -Si(OMe)3 groups to yield the Si-O-Si covalent network. For the preparation of CP(TMSPMA)-b-P(DMAEMA) diblock copolymer brushes on the substrate surface, the CP(TMSPMA) chain ends on the SS-g-CP(TMSPMA) surface (Figure 1) were used as the macroinitiators for the surfaceinitiated ATRP of DMAEMA to produce the diblock copolymer brushes. Typically, the reaction was carried out under the following conditions: DMAEMA (4 mL, 23.7 mmol), CuCl (23.7 mg, 0.237 mmol), CuCl2 (6.4 mg, 0.048 mmol), and Bpy ligand (35.8 mg, 0.237 mmol) were added to 4 mL of methanol in a Pyrex tube. The SS-g-CP(TMSPMA) substrate was introduced into the reaction mixture after the latter had been stirred and degassed with argon for 30 min. The reaction was allowed to proceed at 35 °C for 2-8 h to produce the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface.29 The copolymer-grafted SS surface was subsequently washed and extracted thoroughly with excess deionized water and ethanol to ensure the complete removal of the physically adsorbed polymers, if any.

2.3. Quaternization of the SS-g-CP(TMSPMA)-b-P(DMAEMA) Surfaces. The SS-g- CP(TMSPMA)-b-P(DMAEMA) substrate was immersed in a 10 mL solution of 2-propanol containing 20 vol % hexyl bromide and 0.1 mL of TEA in a Pyrex tube at 70 °C for 8 and 24 h to produce the respective SS-gCP(TMSPMA)-b-QP(DMAEMA) surfaces.29,39 After the quaternization reaction, the coupons were washed sequentially with copious amounts of acetone and deionized water to remove the unreacted hexyl bromide, prior to being dried under reduced pressure and stored in a vacuum desiccator. 2.4. Antibacterial Surface Property. The cultivation and inoculation of D. desulfuricans are described in detail in the Supporting Information. In the viability assays from fluorescence microscopy (FM) images, the Live/Dead Baclight bacterial 6730 DOI: 10.1021/la904083r

Yuan et al. viability kit (L131152), consisting of a mixture of SYTO 9 green fluorescent nucleic acid dye and propidium iodide (PI) red fluorescent nucleic acid dye, was used. The SYTO 9 is membrane permeable and therefore stains both viable and nonviable bacteria, whereas PI, which has a higher affinity for nucleic acids, is rejected from viable bacterial cells by membrane pumps.33 When both dyes are present, PI competes with the SYTO 9 for nucleic acid binding sites. Thus, viable bacteria (which appear green) and dead bacteria (which appear red) can be distinguished under the fluorescence microscope. After the prescribed exposure time, the coupons were removed from the D. desulfuricans inoculated SSMB medium and subsequently stained by 0.1 mL solution of the Live/Dead Baclight kit L131152 on the substrate surface for 15 min. The stained coupons were imaged under a green filter (excitation/emission wavelengths, 420-480 nm/520-580 nm) or a red filter (excitation/emission wavelengths, 480-550 nm/ 590-800 nm) with a Leica DMLM microscope equipped with a 100 W Hg lamp. At least three different surface locations on each substrate were randomly chosen for FM imaging. To assess the antibacterial property of the surface-functionalized coupons in a more quantitative manner, the viable cells adhered on each substrate surface was enumerated using the 3-tube most probable number (MPN) method as a function of exposure time.29,30 The bacteria cells on the substrate surface were introduced into 10 mL of sterile PBS by sonication in an ultrasonic bath (Cole-Parmer, Vernon, IL) for 3 min at a frequency of 40-50 kHz. Each initial bacterial suspension was diluted with sterile PBS using the decimal serial dilution methods. One mL of each diluted suspension was inoculated in triplicate in separate tubes containing 9 mL of the sterile SSMB medium. The bacterial growth of each tube was examined after 72 h of incubation at 30 °C. The result, after multiplication of the dilution factor, was expressed as the mean viable cell counts for each substrate.

2.5. Anticorrosion Behavior of the Surface-Functionalized Coupons. Tafel polarization curves and electrochemical impedance spectra (EIS) were obtained to assess the anticorrosion behavior of the surface-functionalized coupons. After a predetermined exposure time, the coupon was removed from the culture medium and embedded in a poly(vinylidene difluoride) (PVDF) holder, with a circular open area of 0.785 cm2, to serve as the working electrode. An Ag/AgCl electrode was used as the reference electrode, and a platinum rod as the counter electrode. Details on the procedures and parameters used for the electrochemical studies had been described previously.50 The inhibition efficiency (IE) of the surface-functionalized coupons was calculated using the following equation:29,30 IE % ¼

io -icorr io

ð1Þ

where io and icorr were the corrosion current densities of the pristine SS and the surface-functionalized coupons, respectively, as determined from the analysis of Tafel polarization curves.

3. Results and Discussion A uniform layer of ATRP initiator immobilized on the substrate surface is essential for tethering polymer brushes on the substrates via surface-initiated ATRP.51 Silanization of the active stainless steel (SS-OH) surface with 2-(4-chloro-sulfonylphenyl)ethyl trichlorosilane (CTCS) provides the active sulfonyl halide initiator for the subsequent ATRP process. Successful immobilization of the CTCS initiator on the SS-OH surface (SS-CTCS surface) was ascertained from the X-ray photoelectron spectroscopy (XPS) results (Supporting Information, Figure S1). (50) Yuan, S. J.; Xu, F. J.; Pehkonen, S. O.; Ting, Y. P.; Neoh, K. G.; Kang, E. T. J. Electrochem. Soc. 2008, 155, C196–C210. (51) Fan, X. W.; Lin, L. J.; Dalsin, J. L.; Messersmith, P. B. J. Am. Chem. Soc. 2005, 127, 15843–15847.

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Figure 2. C 1s and Cl 2p core-level spectra of (a,b) the SS-gP(TMSPMA) surface and (c,d) the SS-g-CP(TMSPMA) surface. C 1s, Cl 2p, and N 1s core-level spectra of (e,f) the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface from 2 h of ATRP and (g,h) the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface from 8 h of ATRP.

3.1. Surface-Initiated ATRP of 3-(Trimethoxysilyl)propyl Methacrylate (TMSPMA) from the SS-CTCS Surface. Figure 2a and b shows the respective C 1s and Cl 2p spectra of the resulting SS-g-P(TMSPMA) surface. In comparison with those of the SS-CTCS surface, the disappearance of the S signals and the decrease in intensity of the Cl signals in the wide scan spectrum of the SS-g-P(TMSPMA) surface indicate that the thickness of P(TMSPMA) brushes is larger than the probing depth of the XPS technique (about 8 nm in an organic matrix52) after 6 h of ATRP (Supporting Information, Figure S2a). The thickness of the P(TMSPMA) brushes grafted on the SS substrate surface is about 37 nm (Supporting Information, Table S1). The C 1s core-level spectrum of the SS-g-P(TMSPMA) surface can be curve-fitted into four peak components with binding energies (BEs) at 283.9, 284.6, 286.2, and 288.4 eV, attributable to the C-Si, C-H, C-O, and OdC-O species, respectively (Figure 2a).53 The area ratio of the four peak components is (52) Wagner, C. D.; Moulder, J. F.; Davis, J. E.; Riggs, W. M. Handbook of X-Ray Photoelectron Spectroscopy; Perkin-Elmer Corp.: Eden Prairie, MN, 1992; pp 31, 216, 230, 231, 236. (53) Xu, F. J.; Wuang, S. C.; Zong, B. Y.; Kang, E. T.; Neoh, K. G. J. Nanosci. Nanotechnol. 2006, 6, 1458–1453.

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about 1.0:4.9:4.1:0.98, comparable to the theoretical molar ratio of 1:5:4:1 for the molecular structure of P(TMSPMA) (Supporting Information, Table S1). Furthermore, the XPSderived [Si]/[C] molar ratio of 1.0:9.7 is also in fairly good agreement with the theoretical ratio of 1:10 for P(TMSPMA). These results indicate that the reactive trimethoxysilyl groups (-Si-(OMe)3) on the P(TMSPMA) chains remain intact during the ATRP process. The increase in surface hydrophobicity, as indicated by the increase in static water contact angle from about 50° for the SS-CTCS surface to about 74° for the SSP(TMSPMA) surface (Support Information, Table S1), suggests that hydrolysis of the reactive -Si-(OMe)3 groups did not occur to a significant extent during the ATRP process. The persistence of chloride species (with the Cl 2p3/2 peak component at the BE of about 200 eV) is consistent with the fact that the “living” chain end from the ATRP process involves a dormant alkyl halide group, which can be reactivated to initiate the subsequent block copolymerization.37,46 After 3 h of TEA-catalyzed hydrolysis of the SS-gP(TMSPMA) surface at 60 °C, the pendant trimethoxysilyl groups (-Si-(OMe)3) on neighboring P(TMSTPA) chains have reacted with one other to form a cross-linked polysilsesquioxane network. The cross-linked SS-g-P(TMSPMA) surface is thus referred as the SS-g-CP(TMSPMA) surface. The SS-g-CP(TMSPMA) surface becomes more hydrophilic, and its static water contact angle decreases to about 41° (Support Information, Table S1). Figure 2c and d shows C 1s and Cl 2p core-level spectra of the SS-g-CP(TMSPMA) surface, respectively. The XPS-derived [Si]/[C] molar ratio of about 1.0:7.4 (Supporting Information, Figure S2b and Table S1), which is comparable to the theoretical molar ratio of 1:7 for completely hydrolyzed P(TMSPMA), indicates that most of the -Si-(OMe)3 groups have condensed irreversibly to form the polysilsesquioxane network. The observed area ratio of the C-Si, C-H, C-O, and OdC-O peak components for the SS-g-CP(TMSPMA) surface in Figure 2c (about 1.0:4.8:1.7:1.0) is comparable to the theoretical value of 1:5:1:1 for the polysilsesquioxane network. The persistence of the covalently bonded chloride species (with the Cl 2p3/2 peak component at the BE of about 200 eV) on the SS-gCP(TMSPMA) surface suggests that the hydrolysis and condensation reactions of the pendant -Si-(OMe)3 groups on the P(TMSPMA) chains have little effect on the terminal alkyl halide groups. The [Cl]/[C] ratio (determined from the sensitivity factor corrected Cl 2p and C 1s core-level spectral area ratio) of the P(TMSPMA)-grafted surface even undergoes a slight increase from 5.64  10-3 to 6.07  10-3 after the hydrolysis and condensation reaction (Figure 2b and d), arising from the loss of trimethoxyl groups. Thus, the SS-g-CP(TMSPMA) surface with the cross-linked polysilsesquioxane network is used in the subsequent block copolymerization, as well as in evaluating the antibacterial and anticorrosion properties of the surface-functionalized coupons. 3.2. Block Copolymer Brushes via Consecutive SurfaceInitiated ATRP and Quaternization of the P(DMAEMA) Chains. The alkyl halide chain ends on the SS-g-CP(TMSPMA) surface were used as the macroinitiators for the consecutive surface-initiated ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) to produce the SS-g-CP(TMSPMA)-bP(DMAEMA) surfaces (Figure 1). Figure 2e-h shows the respective C 1s and N 1s core-level spectra of the SS-g-CP(TMSPMA)-b-P(DMAEMA) surfaces from 2 and 8 h of ATRP. The appearance of the N signal and the decrease in intensity of the Si signal in the wide scan spectrum (Supporting Information, Figure S2c), as well as the predominance of neutral amine species DOI: 10.1021/la904083r

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Figure 3. C 1s and N 1s core-level spectra of (a,b) the SS-gCP(TMSPMA)-b- QP(DMAEMA) surface from 8 h of quaternization reaction and (c,d) the SS-g-CP(TMSPMA)-b-QP(DMAEMA) surface from 24 h of quaternization reaction.

(with BE at 399.4 eV) in the curve-fitted N 1s core-level spectrum (Figure 2f), indicates that DMAEMA has been successfully block copolymerized on the SS-g-CP(TMSPMA) surfaces after 2 h of ATRP, albeit the thickness of the P(DMAEMA) block (about 6 nm) is still less than the probing depth of the XPS technique (about 8 nm in an organic matrix52). Upon prolonging the ATRP time to 8 h, the disappearance of the Si signal in the wide scan spectrum of the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface (Supporting Information, Figure S2d) indicates that the thickness of the P(DMAEMA) block is larger than 8 nm. The thickness of the P(DMAEMA) block has increased to about 11 nm after 8 h of ATRP (Supporting Information, Table S1). For the surfaceinitiated ATRP process, the increase in thickness of the grafted polymer brushes is approximately linear with the polymerization time.54 The C 1s core-level spectrum can be curve-fitted into four peak components with BEs at 284.6, 285.5, 286.2, and 288.4 eV, attributable to the C-H, C-N, C-O, and OdC-O species,52 respectively. The [C-H]:[C-N]:[C-O]:[OdC-O] peak component area ratio is about 3.2:2.9:1.1:1.0 (Supporting Information, Table S1), which is consistent with the chemical structure of P(DMAEMA) with a theoretical component molar ratio of 3:3:1:1. The appearance of an additional minor peak component at the BE of about 402.6 eV in the N 1s core-level spectrum (Figure 2h), attributable to the positively charged nitrogen (Nþ) species, is ascribed to a small degree (around 7%) of quaternization of the P(DMAEMA) block by the terminal alkyl halide groups upon increasing the ATRP time.55 The self-quaternization effect is further ascertained by the appearance of an anionic chloride species (Cl-, with a Cl 2p3/2 peak component at the BE of about 197 eV52) in the Cl 2p core-level spectrum (inset of Figure 2h). Due to thicker coverage of the P(DMAEMA) blocks, the SS-g-CP(TMSPMA)-b-P(DMAEMA) surface from 8 h of ATRP is used in the subsequent N-alkylation reaction to generate the antibacterial surface. The pendant tertiary amino groups of P(DMAEMA) in the diblock copolymer brushes can be quaternized directly with hexyl bromide to produce the polycationic chains and the desired antibacterial functionality (referred to as the SS-g-P(TMSPMA)b-QP(DMAEMA) surface). Figure 3 shows the respective C 1s (54) Prucker, O.; R€uhe, T. Macromolecules 1998, 31, 602–613. (55) Wang, J. S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614–5615.

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and N 1s core-level spectra of the SS-g-P(TMSPMA)-bQP(DMAEMA) surface from 8 and 24 h of the quaternization reaction. The N 1s core-level spectra can be curve-fitted into two peak components with BEs at 399.4 and 402.6 eV,52 attributable to the neutral amine and positively charged nitrogen (Nþ) species, respectively (Figure 3b and d). The surface concentration of positively charged (Nþ) species, or the extent of quaternization, can be expressed as the [Nþ]/[N] ratio (determined from the corresponding Nþ peak component and total nitrogen spectral area ratio within the probing depth of the XPS technique). The [Nþ]/[N] ratio is about 0.37 after 8 h of the N-alkylation reaction (Figure 3b), indicating that about one-third of the DMAEMA repeat units have been quaternized. A higher extent of quaternization of about 0.58 can be achieved upon prolonging the reaction time to 24 h (Figure 3d). The corresponding Br 3d core-level spectrum has a spin-orbit split doublet, Br 3d5/2 and Br 3d3/2 with BEs at 67.3 and 68.4 eV, respectively, attributable to the ionic bromide (Br-) species52 (Supporting Information, Figure S2e and f). The quaternized SS-g-CP(TMSPMA)-b- QP(DMAEMA) surface becomes more hydrophilic, and its static water contact angle decreases from about 52° to about 36° (Supporting Information, Table S1). The SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupon from 24 h of quaternization reaction is chosen to evaluate the bactericidal and anticorrosion properties of the inorganicorganic hybrid coatings. 3.3. Antibacterial Characteristics of the Functionalized SS Surfaces. The bactericidal effect of the functionalized SS surface was investigated by comparing of the number of viable D. desulfuricans cells after being in contact with these substrate surfaces. The distribution of viable and dead bacteria on various substrate surfaces, after 3 and 28 days of exposure in the D. desulfuricans inoculated SSMB medium, was observed by staining with combined fluorescent dyes of SYTO 9 and PI. Parts a-h of Figure 4 show the respective fluorescence microscopy (FM) images, including green fluorescent (viable cells) and red fluorescent (dead cells) images, of the pristine SS and SS-g-CP(TMSPMA)-b-QP(DMAEMA) surfaces after 3 days of exposure in the D. desulfuricans inoculated SSMB medium. The corresponding FM images of the SS-CTCS and SS-g-CP(TMSPMA) surfaces were also obtained (Supporting Information, Figure S3). Numerous bacterial cells with green fluorescence distributed, either individually or in small clusters (Figure 4a), and only a few single cells with red fluorescence (Figure 4b) can be observed on the pristine SS surface, indicating that most of the bacterial cells are viable with their cell membranes intact on the pristine SS surface. The presence of a high concentration of viable cells can also be seen on the SS-CTCS and SS-g-CP(TMSPMA) surfaces (Supporting Information, Figure S3a, b, e, and f), although the density of bacteria cells on the SS-g-CP(TMSPMA) surface appears to be lower that those on the corresponding pristine SS and SS-CTCS coupons at the same initial exposure period. The phenomenon is probably associated with the low surface energy of the cross-linked polysilsesquioxane layer. It has been reported that a variety of silicone coatings can be used as “fouling-release” coatings to protect the substrate surface from fouling and biofilm growth.56 For the SS-g-CP(TMSPMA)-b-QP(DMAEMA) surface, only very few viable cells are sparsely distributed over the entire surface (stained green, Figure 4e), and the vast majority of the bacterial cells are dead cells (stained red, Figure 4f), indicating that the quaternary ammonium groups on the QP(DMAEMA) polycationic chains are effective in killing the bacteria. (56) Berglin, M.; Wynne, K. J.; Gatenholm, P. J. Colloid Interface Sci. 2003, 257, 383–391.

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Figure 5. Number of viable cells adhered to the pristine SS, SSCTCS, SS-g-CP(TMSPMA), and SS-g-CP(TMSPMA)-b-QP(DMAEMA) surfaces as a function of exposure time in the D. desulfuricans inoculated SSMB medium.

Figure 4. Representative fluorescence microscopy images of (a-d) the pristine SS and (e-h) the SS-g-CP(TMSPMA)-b-QP(DMAEMA) surfaces under the green filter (a,c,e,g) and the red filter (b,d,f,h) after exposure to the D. desulfuricans inoculated SSMB medium for 3 and 28 days. Scale bars: 50 μm.

Upon prolonging exposure time to 28 days, the clusters of viable cells become dense and aggregate to form thick and heterogeneous biofilms on the pristine SS and SS-CTCS surfaces (Figure 4c and Supporting Information, Figure S3c). In spite of possessing the “fouling-release” characteristics, the SS-g-CP(TMSPMA) surfaces are also covered with colonies and patchy biofilms of viable cells (stained green, Supporting Information, Figure S3g) after 28 days of exposure. On the other hand, a good number of dead cells (stained red) are discernible on the pristine SS (Figure 4d), SS-CTCS (Supporting Information, Figure S3d), and SS-g-CP(TMSPMA) (Supporting Information, Figure S3h) surfaces. These dead cells are mainly due to natural apoptosis during the bacteria growth process. These results confirm that normal material surfaces are good substrates for the proliferation of microorganisms and biofilm formation will occur readily on such materials in contact with bacteria.1,29,30 In the case of the SSg-CP(TMSPMA)-b-QP(DMAEMA) surface, there are only a few single viable cells (stained green, Figure 4g) and a high concentration of bacterial cells, large-sized colonies, and even patchy Langmuir 2010, 26(9), 6728–6736

biofilms with red fluorescence bestrewed on the coupon surface (Figure 4h), indicating that almost all bacterial cells are killed by the surface bearing a high concentration of quaternary ammonium (Nþ) groups. The high antibacterial efficiency of the surface-attached QP(DMAEMA) polycationic chains is thus ascertained. Interactions between quaternary amine groups and bacterial cell membranes are believed to have disrupted the plasmic membranes of cells, resulting in the release of intracellular materials.35-37 The fluorescence microscopy (FM) images, however, can only provide qualitative information on the bactericidal properties of the functionalized surfaces. The enumeration of viable cells adhered on different surfaces, a more quantitative approach to evaluate the antibacterial properties, was also carried out upon detaching the bacterial cells from the surfaces. The viable cell counts on the pristine SS and SS-CTCS surfaces were about 105 cells/cm2 after 3 days of exposure and increased rapidly by more than 2 orders of magnitude after 14 days of exposure (Figure 5). The surface density of viable cells on the SS-g-CP(TMSPMA) surface also remained above 106 cells/cm2 throughout the exposure periods (Figure 5). On the other hand, the number of viable bacterial cells on the SS-g-CP(TMSPMA)-b-QP(DMAEMA) surface remained at less than 103 cells/cm2 throughout the exposure period of 42 days (Figure 5), indicative of a low survival rate of the bacterial cells on this surface. The significant decrease in the number of viable cells adhered on the SS-g-CP(TMSPMA)b-QP(DMAEMA) surface further confirms the effectiveness of the quaternary ammonium groups of the QP(DMAEMA) polycationic chains in killing the D. desulfuricans cells. Thus, the viable cell enumeration results are in good agreement with the qualitative trend observed in the FM images. 3.4. Anticorrosion Behavior of the Functionalized SS Surfaces. Polarization curves are suitable means for the determination of instantaneous corrosion rates of metal coupons in the presence of inhibitors,19,22 bacteria,7,8 biocides,23 and coatings,29,30,57,58 as well as for monitoring electrochemical reactions at the functionalized surfaces.58 Figure 6 shows the Tafel polarization curves of the pristine SS and surface-functionalized coupons after various exposure periods in the sterile and (57) Karpagavalli, R.; Zhou, A. H.; Chellamuthu, P.; Nguyen, K. J. Biomed. Mater. Res. A 2007, 83A, 1087–1095. (58) Li, Y. S.; Ba, A.; Mahmood, M. S. Electrochim. Acta 2008, 53, 7859–7862.

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Figure 6. Tafel polarization curves of the pristine SS, SS-CTCS, SS-g-CP(TMSPMA), and SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupons in the D. desulfuricans inoculated SSMB medium, as well as the pristine SS coupon in the sterile SSMB medium, for (a) 3, (b) 14, (c) 28, and (d) 42 days.

D. desulfuricans inoculated SSMB medium. These curves were analyzed quantitatively, similarly to those described previously.59 The analysis results are summarized in Table S2 of the Supporting Information. The corrosion potential, Ecorr, of the pristine SS coupons remains fairly constant throughout in the sterile SSMB medium, whereas it undergoes a negative shift with exposure time in the D. desulfuricans inoculated SSMB medium and remains below -500 mV after 14 days of exposure (Supporting Information, Table S2). The active (negative) shift of the corrosion potential in the presence of SRB has been widely reported,8-10,19,29 and it is usually associated with the enhanced anodic dissolution process in terms of the mixed potential theory.60 The values of Ecorr of the surface-functionalized coupons are more noble than those of the corresponding pristine SS coupon in the D. desulfuricans inoculated SSMB medium. For the SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupon, in particular, its Ecorr values remain 300 mV higher than those of the pristine SS coupons throughout the exposure periods (Supporting Information, Table S2). The ennoblement in Ecorr is a common phenomenon observed for the polymer-coated coupons over the bare metals.61 The corrosion current density, icorr, of the pristine SS coupons undergoes a slight decrease in the sterile SSMB medium, arising from surface passivation by the conditioning layer of yeast extract and the formation of Cr-enriched oxide films. On the contrary, the icorr value of the pristine SS coupon increases gradually with exposure time in the inoculated medium and reaches about 136 μA 3 cm-2 after 42 days of exposure (Supporting Information, Table S2), indicative of the substantially enhanced corrosion rate under the effect of D. desulfuricans bacteria. Thus, the corrosion rate of the (59) Yuan, S. J.; Liu, C. K.; Pehkonen, S. O.; Bai, R. B.; Neoh, K. G.; Ting, Y. P.; Kang, E. T. Biofouling 2009, 25, 109–125. (60) Huang, G. T.; Chan, K. Y.; Fang, H. H. P. J. Electrochem. Soc. 2004, 151, B434–439. (61) Spinks, G. M.; Dominis, A. J.; Wallcace, G. G.; Tallman, D. E. J. Solid State Electrochem. 2002, 6, 85–100.

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pristine SS coupon increases to more than 1.3 mm 3 year-1, which is much higher than the expected corrosion rates (about 0.03-0.06 mm 3 year-1) of steel structures in marine environments.62 In the case of the SS-CTCS coupon, the icorr value also undergoes a noticeable increase with exposure time in the D. desulfuricans inoculated medium, indicating that the silane monolayer cannot effectively resist the synergistic effect of active Cl-, biogenic S2-, and bacterial cells of D. desulfuricans. As for the SSg-CP(TMSPMA) and SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupons, the magnitudes of icorr are reduced significantly by about 15- and 30-fold, respectively, from that of the corresponding pristine SS coupon after 28 days of exposure in the D. desulfuricans inoculated SSMB medium, indicating that the inorganic-organic hybrid coatings possess the desired capability to prevent corrosion and biocorrosion by D. desulfuricans. As a consequence, the inhibition efficiencies (IE) of the SS-g-P(TMSPMA) and SS-g-P(TMSPMA)-b-QP(DMAEMA) coupons remained higher than 91% and 95%, respectively, after 28 days of exposure (Supporting Information, Table S2). On the other hand, the IE values of the SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupon are always higher than those of the corresponding SS-g-CP(TMSPMA) coupon, suggesting that the anticorrosion capability of the cross-linked polysilsesquioxane layer in the D. desulfuricans inoculated medium is further enhanced by the grafted QP(DMAEMA) outer block. Electrochemical impedance spectra (EIS) analysis provides an effective method for measuring the resistance against transfer of ionic species to the underlying metal surface, and it has been widely used to evaluate the barrier properties of inorganic and organic coatings.58,63 To gain better insights into the anticorrosion behavior of the surface-grafted organic-inorganic coatings, the impedance spectra (including Nyquist plots and phase angle Bode plots) were obtained for the pristine SS and surfacefunctionalized coupons after various exposure periods in the sterile and D. desulfuricans inoculated SSMB medium. The results are shown, respectively, in Figures S4 and S5 of the Supporting Information. The results were further fitted with appropriate equivalent electrical circuits (EECs) using the program EQUIVCRT by Boukamp.64 Three types of EECs were proposed to model the respective impedance spectra of the pristine SS and surface-functionalized coupons (Supporting Information, Figure S6). Among the circuit elements in these EECs, the charge transfer resistance, Rct, of a coating on the substrate is a measure of the coating’s ability to act as a barrier to the corrosion process, the pore resistance, Rpo, represents the extent of ionic conduction through a polymeric coating in an electrolyte environment, and it is widely used as a criterion for assessing the extent of corrosion protection derived from the organic coatings, while the constant phase angle element (CPE) of coating, Qc, used in place of the coating capacitance (Cc) by taking into account surface heterogeneity and diffusion processes,65 provides information on the extent of water uptake and the stability of the coatings. The fitted parameters of the impedance spectra are summarized in Table S3 of the Supporting Information. The goodness of fitting process can be deduced from the chi-square (χ2) test (Supporting Information, Table S3). For the pristine SS coupon in the sterile SSMB medium, the values of the charge transfer resistance, Rct, and the resistance of surface film, Rf, remain rather steady and even undergo a slight (62) (63) (64) (65) 742.

Beech, I. B.; Campbell, S. A. Electrochim. Acta 2008, 54, 14–21. Qui~nones, R.; Gawalt, E. S. Langmuir 2008, 24, 10858–10864. Boukamp, B. A. Solid State Ionics 1986, 20, 31–44. Mansfeld, F.; Jeanjaquet, S. L.; Kendig, M. W. Corros. Sci. 1986, 28, 735–

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increase with exposure time (Supporting Information, Table S3), attributable to the passivation of the conditioning layer of yeast extract. On the contrary, the Rct value of the pristine SS coupon decreases gradually with exposure time in the D. desulfuricans inoculated medium, indicative of the enhancement of corrosion by D. desulfuricans. The resistance of the surface film, Rf, increases initially with exposure time due to the formation of a porous iron sulfide layer in the D. desulfuricans inoculated medium and then decreases with exposure time, attributable to the initiation of localized corrosion beneath the corrosion products (Supporting Information, Table S3). These results further confirm that the sulfide-reducing bacteria are detrimental to the passivity and integrity of the passive film on the SS substrates.7-9,11,18,22 For the SS-CTCS coupon, although the magnitude of Rct is larger than that of the pristine SS coupon, it also undergoes a gradual decrease with exposure time, similar to that of the pristine SS coupon, indicative of the gradual loss of passivity of the immobilized silane monolayer. Thus, the Rct value of the SSCTCS coupon becomes comparable to that of the pristine SS coupon after 42 days of exposure in the D. desulfuricans inoculated medium (Supporting Information, Table S3). This result is further confirmed by the gradual decrease in pore resistance, Rpo, with exposure time. Thus, the durability and corrosion resistance of self-assembly monolayers are limited during prolonged exposure in harsh environments.66,67 In the case of the SS-gCP(TMSPMA) and SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupons, the Rct values are significantly larger than those of the corresponding pristine SS and SS-CTCS coupons throughout the exposure periods in the D. desulfuricans inoculated medium, indicative of the decrease in corrosion rates of the SS coupons under protection of the inorganic-organic coatings (Supporting Information, Table S3). Furthermore, the Rct and Rpo values of the SS-g-CP(TMSPMA)-b-QP(DMAEMA) coupon are both considerably larger than those of the SS-g-CP(TMSPMA) coupon throughout the exposure periods, indicative of the substantial increase in barrier capability and corrosion resistance by the grafted QP(DMAEMA) outer blocks against the penetration of active ions (Cl- and S2-), water, and the attack of D. desulfuricans. Moreover, the capacitance (Qc) of the SS-g-CP(TMSPMA) coupon undergoes a noticeable increase with time (Supporting Information, Table S3), suggesting that the water/electrolyte has penetrated into the polysilsesquioxane network during the prolonged exposure, leading to the decrease in stability and protective capability of the polysilsesquioxane network. The phenomenon has been recognized to be associated with microcracks in the silica or polysilsesquioxane coating.67 However, the Qc value of the SSg-CP(TMSPMA)-b-QP(DMAEMA) coupon remains small and stable throughout the exposure period, indicative of improved stability of the coating in the presence of an outer QP(DMAEMA) polymer layer. 3.5. Stability and Durability of the Surface-Functionalized Coupons. As the failure and delamination of the polymeric coatings have been widely recognized to enhance localized dissolution,65,67 which can be even more detrimental than that produced by biofilms, it is of great importance to assess the stability of the grafted polymeric coatings. The SEM images of pristine and surface-functionalized SS coupons, obtained after exposure to the sterile and D. desulfuricans inoculated SSMB medium for 3 and 42 days upon removal of the corrosion deposits (66) Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 9022–9028. (67) Metroke, T. L.; Parkhill, R. L.; Knobbe, E. T. Prog. Org. Coat. 2001, 41, 233–238.

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(including bacterial cells and corrosion products), are shown in Figure S7 of the Supporting Information. The pristine SS coupon remains almost unchanged after 42 days of exposure in the sterile SSMB medium, and no corrosion can be observed (Supporting Information, Figure S7a). However, extensive micropits and some macropits are observed on the pristine SS surface under the effect of D. desulfuricans for 42 days (Supporting Information, Figure S7b). The observation is consistent with the aforementioned electrochemical findings that surface passivity of the SS substrate has been compromised or disrupted under the attack of D. desulfuricans. After 3 days of exposure, the SS-CTCS coupon shows a homogeneous and compact silane passivation layer upon removal of the corrosion deposits (Supporting Information, Figure S7c). However, some inceptive micropits can be spotted over the coupon surface after 42 days of exposure in the D. desulfuricans inoculated medium (Supporting Information, Figure S7d), indicating that the integrity of the silane passivation layer has been compromised under the prolonged attack of D. desulfuricans. For the SS-g-CP(TMSPMA) surfaces, the surface coating is retained after 3 days of exposure (Supporting Information, Figure S7e). The compactness of the coating, however, decreases after 42 days of exposure (Supporting Information, Figure S7f), albeit no pitting corrosion is discernible. The changes in the polysilsesquioxane network are probably due to the detrimental effect of biofilm formation and water uptake by the microcracks in the coating. On the other hand, no change in characteristics of the SS-g-CP(TMSPMA)-b-QP(DMAEMA) surface is discernible after 3 and 42 days of exposure (Supporting Information, Figure S7g and S7h), indicating that not only does the inorganic-organic hybrid coating provide the desired passivity for preventing corrosion and biocorrosion, it also remains stable and durable during prolonged exposure in harsh marine environments.

4. Conclusions Antibacterial inorganic-organic hybrid coatings, consisting of a cross-linked polysilsesquioxane inner layer and quaternized P(DMAEMA) outer brushes, were developed on a stainless steel (SS) surface, via consecutive surface-initiated atom transfer radical polymerization (ATRP), as an environmentally friendly means for inhibiting biofilm formation and biocorrosion. The process involved (i) covalent immobilization of a sulfonyl halide initiator monolayer on the active stainless steel (SS-OH) surface via self-assembly of a trichlorosilane coupling agent, (ii) functionalization of the silane-immobilized surface via surfaceinitiated ATRP of TMSPMA and subsequent hydrolysis and condensation of the pendant trimethoxysilyl groups (-Si-(OMe)3) of the P(TMSPMA) chains to form the crosslinked polysilsesquioxane layer, (iii) grafting of P(DMAEMA) block via consecutive surface-initiated ATRP, and (iv) N-alkylation of the P(DMAEMA) block to produce a high concentration of the quaternary ammonium moieties with biocidal functionality. Not only did the quaternized SS-g-CP(TMSPMA)-bQP(DMAEMA) surface exhibit the desired antibacterial activity to inhibit biofilm formation, as revealed by fluorescence microscopy images and viable cell counts, it also exhibited enhanced anticorrosion properties over that of the polysilsesquioxane passivated surface in D. desulfuricans inoculated medium, as revealed by electrochemical studies. The good antibacterial capability, corrosion protection efficiency, and stability of this inorganic-organic hybrid coating will allow it to serve as a model system for inhibition of biocorrosion in steels in harsh environments. DOI: 10.1021/la904083r

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Acknowledgment. The authors would like to thank the National University of Singapore for the financial support of this study under the FRC Research Grant R-279-000-236-112. Supporting Information Available: Additional experimental procedures and surface characterization results,

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microorganism cultivation and inoculation, analysis of Tafel plots, parameters for fitting electrochemical impedance spectra, XPS spectra, fluorescence microscopy images, impedance spectra, equivalent circuit models, and SEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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