Facile Fabrication of Uniform Silica Films with ... - ACS Publications

Dec 15, 2009 - School of Science and Technology, Nottingham Trent University, Clifton ... uniform silica films was achieved by immobilization of silic...
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Facile Fabrication of Uniform Silica Films with Tunable Physical Properties Using Silicatein Protein from Sponges Akhilesh Rai and Carole C. Perry* School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG 11 8NS, United Kingdom Received September 9, 2009. Revised Manuscript Received November 12, 2009 We report an elegant and simple method to fabricate uniform silica films with controlled thickness, roughness, and hydrophilicity using nanogram quantities of silicatein, a protein involved in silica synthesis in sponges. The formation of uniform silica films was achieved by immobilization of silicatein on gold-coated surfaces that had been functionalized with amines. Specifically, the amines, cystamine and cysteamine having disulfide and thiol groups, respectively, were bound to surfaces and treated with a cross-linking agent, glutardialdehyde (GDA) before protein immobilization. Silica was formed on the silicatein bound surfaces under environmentally benign conditions using tetramethoxysilane (TMOS). The thickness (20-100 nm), roughness (1.2-5.2 nm), and water contact angle (48°-16°) of the silica films could be controlled by varying the amount of silicatein adsorbed (10-30 ng/cm2) and time of exposure of protein-coated surfaces (30-120 min) to silica precursors. The silicatein protein retained around 90% of its intrinsic activity when attached to the functionalized surfaces with similar activity being observed for silica films formed from TMOS or tetraethoxysilane (TEOS). This simple route to prepare silica films of controlled physical properties could have potential application in membrane fabrication, biomedical devices, biosensors, and next generation electronic components.

Introduction The fabrication of composite coatings based on silica with welldefined physical properties represents a promising new approach to fine-tune mechanical, optical, electronic, and permeability properties for use in sensor,1 membrane,2 transducer,3 optical,4 and biomedical applications.5 The fabrication methods typically used require harsh conditions of high temperature, pressure, and pH that are not suitable for processes using biomolecules to promote the synthesis of networks of nanosized particles on the surface. In contrast, biological organisms can synthesize silica-based materials over many length scales (tens of nanometers to tens of meters) under environmentally benign conditions.6 For example, diatoms,7 sponges,8 and grasses9 use the biosilicification process to sequester soluble silicon species from the environment and synthesize and assemble silica particles into intricate three-dimensional structures. This interesting biological phenomenon has led scientists to isolate and purify proteins that are responsible for the synthesis of silica *To whom correspondence should be addressed. E-mail: carole.perry@ ntu.ac.uk. (1) (a) Grant, S. A.; Glass, R. S. IEEE Trans. Biomed. Eng. 1999, 46, 1207. (b) Schmidt, H. J. Sol-Gel Sci. Technol. 2006, 40, 115. (2) Stein, A.; Melde, B. J.; Schroden, R. C. Adv. Mater. 2000, 12, 1403. (3) Sakka, S.; Yoko, T. Struct. Bonding (Berlin, Ger.) 1992, 77, 89. (4) Yeatman, E. M.; Green, M.; Dawnay, E. J. C.; Fardad, M. A.; Horowitz, F. J. Sol-Gel Sci. Technol. 1995, 2, 711. (5) (a) Lin, T. J.; Huang, K. T.; Liu, C. Y. Biosens. Bioelectron. 2006, 22, 513. (b) Huang, H.; Chen, Y. Biosens. Bioelectron. 2006, 22, 644. (6) Aizenberg, J. Adv. Mater. 2004, 16, 1295. (7) Wetherbee, R. Science 2002, 298, 547. (8) Muller, W. E. G.; Krasko, A.; Pennec, G. L.; Steffen, R.; Ammar, M. S. A.; Wiens, M.; Muller, M. I.; Schroder, H. C. Prog. Mol. Subcell. Biol. 2003, 33, 195. (9) Lux, A.; Luxova, M.; Morita, S.; Abe, J.; Inanaga, S. Can. J. Bot. 1999, 77, 955. (10) (a) Shimizu, K.; Cha, J.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6234. (b) Kroger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14133. (c) Zhou, Y.; Shimizu, K.; Cha, J. N.; Stucky, G. D.; Morse, D. E. Angew. Chem., Int. Ed. 1999, 38, 779. (d) Cha, J.; Shimizu, K.; Zhou, Y.; Christiansen, S. C.; Chmelka, B. F.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 361. (e) Brutchey, R. L.; Morse, D. E. Chem. Rev. 2008, 108, 4915. (f) Muller, W. E. G.; Schroder, H. C.; Lorenz, B.; Krasko, A. U.S. Patent 7169589.

4152 DOI: 10.1021/la903366a

particles in vivo. Specific proteins called silicatein10 and silaffins11 from sponges and diatoms, respectively, have been isolated and used for the rapid synthesis of biosilica particles in vitro via a traditional sol-gel process when added in silica precursor solutions under ambient conditions of temperature and neutral pH. As mimics of the biosilicification process in vitro, a number of polypeptides,12 diblock copolypeptides,13 and polyamines14 have been used for the synthesis of silica particles from solution. The structure, composition, and molecular weight of the biomolecules and the buffer conditions used are reported to affect the kinetics of the condensation and precipitation processes as well as affecting the shape of the silica particles so formed.12-14 The mechanism of biomolecule catalyzed silica formation from solutions containing such additives involves electrostatic interaction between positively charged amines and negatively charged silicic acids. This interaction facilitates the condensation of silica around amine containing templates and promotes the synthesis of silica particles.10,11b,15 For many applications, however, it is necessary to synthesize silica on solid surfaces rather than in solution and to control the (11) (a) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129. (b) Kent, M. S.; Murton, J. K.; Zendejas, F. J.; Tran, H.; Simmons, B. A.; Sajita, S.; Kuzmenko, I. Langmuir 2009, 25, 305. (12) (a) Bellomo, E. G.; Deming, T. J. J. Am. Chem. Soc. 2006, 128, 2276. (b) Coradin, T.; Livage, J. Colloids Surf., B 2001, 21(4), 329. (c) Knecht, M. R.; Wright, D. W. Chem. Commun. 2003, 3038. (d) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci. Nanotechnol. 2002, 2, 95. (e) Patwardhan, S. V.; Mukherjee, N.; SteinitzKannan, M.; Clarson, S. J. Chem. Commun. 2003, 1122. (f) Tomczak, M. M.; Lawrence, C.; Drummy, L. F.; Sowards, L. A.; Glawe, D. C.; Stone, M. O.; Perry, C. C.; Pochan, D. J.; Deming, T. J.; Naik, R. R. J. Am. Chem. Soc. 2005, 127, 12577. (13) (a) Brott, L. L.; Naik, R. R.; Pikas, D. J.; Kirkpatrick, S. M.; Tomlin, D. W.; Whitlock, P. W.; Clarson, S. J.; Stone, M. O. Nature 2001, 413, 291. (b) Menzel, H.; Horstmann, S.; Behrens, P.; Barnreuther, P.; Kruger, I.; Jahns, M. Chem. Commun. 2003, 2994. (14) (a) Patwardhan, S. V.; Clarson, S. J. Silicon Chem. 2002, 1, 47. (b) Patwardhan, S. V.; Clarson, S. J. Silicon Chem. 2002, 1, 207. (c) Patwardhan, S. V.; Clarson, S. J. Inorg. Organomet. Polym. 2003, 13, 193. (d) Belton, D.; Paine, G.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2004, 14, 2231. (e) Belton, D.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2005, 15, 4629. (f) Belton, D.; Patwardhan, S. V.; Perry, C. C. Chem Commun. 2005, 3475. (15) (a) Sumper, M. Science 2002, 295, 2430. (b) Sumper, M.; Kroger, N. J. J. Mater. Chem. 2004, 14, 2059. (c) Sumper, M. Angew. Chem., Int. Ed. 2004, 43, 2251.

Published on Web 12/15/2009

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pattern and shape of the deposited inorganic materials over more than the micrometer length scale. Several methods have been used for the fabrication of patterned films of polypeptides and polyamines prior to templated silica formation by methods such as electrostatic deposition,16 direct write assembly,17 holographic patterning,13a photolithography,18 and surface initiated polymerization.19 However, the deposition of uniform silica coatings on solid surfaces has not been achieved under ambient conditions despite there being many potential applications for such materials in fields such as sensors,1 membranes,2 and structural materials.20 Approaches to the generation of silica films using proteins such as silicatein from sponges and lysozyme have been developed. Tahir and co-workers have demonstrated the binding of His-tagged recombinant silicatein on nitrilotriacetic acid (NTA) terminated alkanethiol functionalized gold surfaces with the formation of heterogeneous silica, zirconia, and titania films.21 The synthesis procedure to generate the thioalkane terminated NTA molecules is a many step process that is not practically accessible to most technologists and is not suitable for the immobilization of natural or recombinant silicateins that do not have His tags. They were also not able to control the thickness/roughness of the fabricated silica films and in many cases used much more silicatein than is required for the procedure described below. In other studies, Luckarift and co-workers used lyszoyme functionalized gold surfaces for the preparation of silica-coated surfaces,22 but heterogeneous coatings showing no control over the thickness and roughness of the silica films were found to form. In a recent publication from our group,23 we have shown that tunable thickness silica films (ca. 2-50 nm thick) with tolerable uniformity can be fabricated on solid surfaces using a combination of readily available proteins such as serum albumins (BSA) or lysozyme, not originally designed to foster silica formation, in combination with simple amines such as poly(allylamine) (PAH), poly(ethyleneimine) (PEI), and octadecylamine (ODA) using environmentally friendly reaction conditions. Silica formation on the protein-coated surfaces was suggested to be related to the amount of charge available at the surface, whether from the protein alone (lysozyme) or from a combination between the protein and the amine (BSA). There was a limit to the thickness of films that could be generated using this approach, and thus, we have investigated the ability of nanomolar levels of silicatein attached to solid surfaces to form uniform silica films over multiple length scales with controlled physical properties (thickness, roughness, and wettability). For the first time, the specific activity of silicatein attached to solid surfaces has also been quantified and compared with its behavior in solution. The approach used in our previous (16) (a) Glawe, D. D.; Rodriguez, F.; Stone, M. O.; Naik, R. R. Langmuir 2005, 21, 717. (b) Pogula, S. D.; Patwardhan, S. V.; Perry, C. C.; Gillespie, J. W.; Yarlagadda, S.; Kick, K. L. Langmuir 2007, 23, 6677. (c) Laugel, N.; Hemmerle, J.; Porcel, C.; Voegel, J.-C.; Schaaf, P.; Ball, V. Langmuir 2007, 23, 3706. (17) Xu, M.; Gratson, G. M.; Duoss, E. B.; Shepherd, R. F.; Lewis, J. A. Soft Matter 2006, 2, 205. (18) Coffman, E. A.; Melechko, A. V.; Allison, D. P.; Simpson, M. L.; Doktycz, M. J. Langmuir 2004, 20, 8431. (19) (a) Kim, D. J.; Lee, K. B.; Chi, Y. S.; Kim, W. J.; Paik, H. J.; Choi, I. S. Langmuir 2004, 20, 7904. (b) Kim, D. J.; Lee, K. B.; Lee, T. G.; Shon, H. K.; Kim, W. J.; Paik, H. J.; Choi, I. S. Small 2005, 1, 992. (20) Caruso, R. A.; Antonietti, M. Chem. Mater. 2001, 13, 3272. (21) (a) Tahir, M. N.; Eberhardt, M.; Therese, H. A.; Kolb, U.; Theato, P.; Muller, W. E. G.; Schroder, H. C.; Tremel, W. Angew. Chem., Int. Ed. 2006, 45, 4803. (b) Tahir, M. N.; Theato, P.; Muller, W. E. G.; Schroder, H. C.; Borejko, A.; Faiss, S.; Janshoff, A.; Huth, J.; Tremel, W. Chem. Commun. 2005, 5533. (c) Tahir, M. N.; Theato, P.; Muller, W. E. G.; Schroder, H. C.; Janshoff, A.; Jhang, J.; Huth, J.; Tremel, W. Chem. Commun. 2004, 2848. (22) Luckarift, H. R.; Balasubramanian, S.; Paliwal, S.; Johnson, G. R.; Simonian, A. L. Colloids Surf., B 2007, 58, 28. (23) Rai, A.; Perry, C. C. Silicon 2009, 1, 91.

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Article Scheme 1. Schematic Representation of the Stepwise Coating Process (Note That the Scheme Is Not to Scale)

study23 using amines such as PAH, PEI, and ODA was not successful, and a new approach using bifunctional amines was developed. Cystamine and cysteamine having disulfide and thiol groups, respectively, were used as bifunctional agents to functionalize gold surfaces. Both molecules react with gold via their -SH group, producing free amine groups on the surface which were further modified with glutardialdehyde (GDA) to achieve a uniform and strong adsorption of silicatein via covalent interaction between amine group(s) of the protein and the free carbonyl group of GDA (Scheme 1). The presence of glutardialdehyde as a cross-linking agent helped to maintain the activity of the protein by avoiding direct contact of the protein with the gold surface. Our studies described below show that the thickness, roughness and wettability of the films formed can be controlled by varying the amount of adsorbed protein on the surface and/or by varying the exposure time to silica precursors.

Experimental Details Reagents. Tetramethyl orthosilicate (TMOS), tetraethylorthosilicate (TEOS), cystamine dihydrochloride, cysteamine hydrochloride, and glutardialdehyde (GDA) were purchased from Sigma-Aldrich (Minneapolis, MN). Recombinant silicatein-R protein was purchased from BIOTECmarin (Germany) and was used as received. The biochemical characterization of recombinant silicatein-R protein was performed by BIOTECmarin, and data are not presented in this paper although data certifying the activity of the enzyme itself are presented. Phosphate buffer (0.1M) at pH 7.2 was freshly prepared at 25 °C using sodium salts of NaH2PO4 and Na2HPO4 obtained from Aldrich. Distilled deionized water with a conductivity of ∼1 μS was used for all experiments. Protein Immobilization Study. Glass slides were washed thoroughly with acetone and piranha solution (7 mL/3 mL mixtures of concentrated H2SO4 and 30% H2O2, respectively) for 30 min to remove any organic material and then rinsed with deionized water and dried before coating with gold using an Edwards sputter coater model S150B. Gold slides were dipped in 10-2 M cystamine/cysteamine solution for 12 h followed by washing with copious amounts of water to remove unbound amines before treatment with 2% GDA at 60 °C for 2 h and then thoroughly washed immediately with water to prevent crosslinking between GDA molecules. Silicatein (200 ng/mL, 8 nM) dissolved in 0.1 M phosphate buffer was used for immobilization on the gold surface, cystamine, cysteamine, cystamine-GDA, and cysteamine-GDA bound gold surfaces over time periods up to 6 h. Aliquots of protein solution (50 μL) were removed at different DOI: 10.1021/la903366a

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Article times to measure the amount of unadsorbed protein via the fluorescamine assay24 using a Tecan spectrafluor plate reader (360 nm excitation and 465 nm emission energy filters). The protein adsorbed onto different surfaces and concentration per unit surface area (ng/cm2) were calculated. Infrared (ATR-FTIR) analysis of amines, amine-GDA functionalized surfaces, and silicatein bound surfaces was performed using a golden Gate attenuated total reflection (ATR) accessory in a Nicolet Magna IR-750 spectrophotometer continuously purged with dry air. All surfaces were properly dried using N2 gas prior to Fourier transform infrared (FTIR) measurements. Spectra were recorded at 4 cm-1 resolution with 1026 scans being averaged and then smoothed by 11 point adjacent averaging.

Preparation and Analysis of Silica Coated Surfaces. Silicatein-coated surfaces were washed with phosphate buffer and deionized water to remove loosely attached protein prior to the deposition of silica. In a typical experiment, 100 mM TMOS solution was hydrolyzed in 1 mM HCl for 15 min. The silicateincoated surfaces were placed vertically in the hydrolyzed TMOS solution (to prevent adventitious precipitation of silica) for different times (0-2 h), washed with deionized water, and dried using N2 gas. ATR-FTIR analysis was used to investigate our ability to produce films with different coatings. The cystamineGDA surface was treated with 200 ng/mL silicatein at different time intervals (0-180 min) to achieve different amounts of protein adsorption. These slides were then treated with the prehydrolyzed 100 mM TMOS solution for 2 h to fabricate silica films. A limited number of silica-coated surfaces were prepared using TEOS for comparison of the silicatein activity with that detailed in the literature.10d-f Assessment of Silicatein Activity in Silica Formation. To assess the activity of silicatein alone, silica particles were synthesized in solution using 100 mM prehydrolyzed TMOS (1 mM HCl for 15 min) and 200 ng/mL silicatein (control with no silicatein) using a reaction time of 2 h (2 h reaction times were routinely found to generate good films). Samples were centrifuged and the precipitated silica particles washed three times to remove free hydrolyzed TMOS. Aliquots of the unreacted hydrolyzed TMOS remaining in solution after silica film fabrication and silica particle formation were treated with 2 M NaOH for 1 h at 80 °C to ensure all silica species remaining in suspension were broken down to monomer/dimer. The concentration of silicic acid in solution was then estimated by the molybdenum blue colorimetric method described by Iler.25 Aliquots of 10 μL were removed from different samples and added to solutions containing 15 mL of water and 1.5 mL of an acidic solution of ammonium molydate, and the resulting solutions incubated for 20 min at room temperature. Reducing agent containing Metol (8 mL) was then added to each solution, and the absorbance of the blue silicomolybdate complex was measured after 2 h at 810 nm using a Unicam UV2 UV-vis spectrophotometer. The concentrations of silicic acid condensed into silica particles or condensed on the surfaces explored as well as that remaining in solution were calculated. Within the time frame of the experiments, 16.2 ( 2 mM TMOS condensed and precipitated to form silica particles in solution. Results are expressed as the relative specific activity of free and immobilized silicatein. The specific activity was considered as 100% for silica particles synthesized using free silicatein in solution. Additional control experiments were performed with TEOS as the silica substrate using conditions as reported in the studies by Cha et al. and in the patent by Muller et al., whereby in our experiments, for an average incubation of 60 min, 337 nmol silica was generated from 100 μg of silicatein.10d,10f For a surface (Au-cystamine-GDA-silicatein-silica (TEOS)), 310 nmol silica was generated using 100 μg of silicatein. (24) Underfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871. (25) Iler, R. K. The Chemistry of Silica; John Wiley & Sons: New York, 1979.

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Scanning Electron Microscopy Study. Silica-coated surfaces were mounted on aluminum stubs with double-sided adhesive carbon tape and coated with palladium/gold before analysis using a JEOL JSM 7400F FE instrument operated at an accelerating voltage of 20 kV. Energy-dispersive X-ray analysis (EDXA) was used to obtain compositional information on the silica-coated gold slides. Atomic Force Microscopy Study. Amine, amine-GDA, protein, and silica-coated surfaces were analyzed by atomic force microscopy (AFM) in noncontact mode using a Pacific Nanotechnology Nano-R2 instrument with SiN probes at a scan rate of 0.5 Hz, in air. The line profile analysis of different surfaces was performed to estimate the thickness of protein and silica films. The roughness of the surface was assessed by measuring roughness parameters (Rrms, root-mean-square roughness) using Nanorule.exe software supplied with the instrument. Contact Angle Measurement. Contact angle measurements were performed by taking images of 5 μL water drops on surfaces using a Kruss DSA 10. A picture of the drop was taken after a few seconds to avoid any problems related to drying of the drop. Three images were taken from different areas of the surface, and tangent measurements at the drop surface interface were made and the contact angle calculated. The relationship between roughness and water contact angle was assessed using the Wenzel equation26 where the apparent contact angle of a liquid on a surface that depends on the roughness and chemical composition of the surface is expressed as cos θa ¼ r cos θ where θa is the apparent water contact angle on a rough surface and θ is the intrinsic contact angle as measured on a smooth surface; r is the surface roughness ratio of apparent to projected surface areas. The ratio of apparent surface to projected surface is always equal to or greater than unity; therefore, for a hydrophilic surface (θ < 90°), the contact angle would decrease with increasing surface roughness, leading to enhanced wetting.

Result and Discussion The goal of this experimental study was to use silicatein, the protein found in sponge spicules, to produce uniform silica films that could then be used for the fabrication of electronic devices on a range of materials such as silicon, glass, gold, and polymer surfaces. Use of a “gentle” method developed in the group for immobilization of proteins such as lysozyme and serum albumins where large quantities of protein were available was not suitable for silicatein in part due to the very limited amounts of material available.23 Our aim was to develop a straightforward fabrication method utilizing silicatein at sub-10 nM concentration, which is much lower than what others have used, and to quantify the activity of the bound protein. The data given below show the outcomes of our present study. Following the procedures depicted in Scheme 1 and described in the Experimental Details section, FTIR spectroscopy was used to identify whether molecules (amines, glutardialdehyde, and silicatein) were bound to the gold-coated surfaces (Figure 1). Very weak FTIR signals were observed for cystamine and cysteamine before and after modification with GDA. The presence of the amines on the surfaces was identified by bands centered at 1595, 1510, and 1450 cm-1 which correspond to the deformation vibration (δNH of NH2), the N-H bending mode, and the C-H bending mode of cystamine and cysteamine, respectively (Figure 1A and B, line 2 in each panel).27 After (26) Wenzel, R. N. Ind. Eng. Chem. 1936, 28, 988. (27) (a) Pradier, C.-M.; Salmain, M.; Zheng, L.; Jaouen, G. Surf. Sci. 2002, 502-503, 193. (b) Huang, I.-Y.; Lee, M.-C. Sens. Actuators, B 2008, 132, 340. (c) Merland, F. T.; Methivier, C.; Pasquinet, E.; Hairault, L.; Pradier, C. M. Sens. Actuators, B 2006, 114, 223.

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Figure 1. (A) FTIR spectra of a bare gold surface (curve 1), cystamine-Au (curve 2), cystamine-GDA-Au (curve 3), and silicatein adsorbed on cystamine-GDA-Au surface (curve 4). (B) FTIR spectra of a bare gold surface (curve 1), cysteamine-Au (curve 2), cysteamine-GDA-Au (curve 3), and silicatein adsorbed on cysteamine-GDA-Au surface (curve 4). Panels C and D show representative AFM images of cystamine and cysteamine functionalized gold surfaces, respectively.

modification with glutardialdehyde (GDA), the band at 1595 cm-1 disappeared due to reaction with the carbonyl group of GDA and new bands at 1650 and 1730 cm-1 appeared, that could be assigned to the CdN stretch and free carbonyl groups of GDA, respectively (curves 3). Curves 4 in Figure 1A and B show the FTIR spectra of silicatein adsorbed on these two surfaces. Curves labeled 4 show the characteristic amide-I (CdO stretching mode) and amide-II bands (N-H bending mode) at 1650 and 1520 cm-1, respectively, indicating the presence of adsorbed silicatein on the different surfaces. The adsorption behavior of silicatein on amine- and amineGDA-coated surfaces is presented in Figure 2. Little silicatein was adsorbed on the gold surfaces and amine-coated gold surfaces even after 6 h of incubation in solution (curves 1-3, Figure 2 and Table 1). Any adsorption of silicatein on the bare gold surface was due to nonspecific physical interactions. Cystamine and cysteamine having disulfide and thiol groups with short carbon chains, respectively, gave rise to poorly organized monolayers and weak adsorption of the protein (Figures 1C, D and 2).28 The presence of ionic functional groups (for example, amine) on the cystamineand cysteamine-coated surfaces is also known to destabilize monolayer formation in a high dielectric constant solvent such as water.29 AFM studies show the formation of monolayers of cystamine and cysteamine on gold surfaces, although the thickness of the cystamine (0.4 nm) and cysteamine (0.4 nm) layers was not sufficient for the strong adsorption of protein (Figures 1 and 2). A large amount of silicatein was adsorbed on the Aucystamine-GDA (Figure 2, curve 5) and Au-cysteamine-GDA (Figure 2, curve 4) surfaces compared with adsorption on the Au and Au-amine treated surfaces (Figure 2, curves 1-3). Cystamine reacts with the gold surface on cleavage of the sulfur-sulfur disulfide bond, while cysteamine reacts via the thiol group, (28) (a) Wagner, P.; Hegner, M.; Guntherodt, H. J.; Semenza, G. Langmuir 1995, 11, 3867. (b) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12, 3604. (c) Wirde, M.; Gelius, U.; Nyholm, L. Langmuir 1999, 15, 6370. (29) Doblhofer, K.; Figura, J.; Fuhrhop, J. H. Langmuir 1992, 8, 1811.

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Figure 2. Time dependent adsorption of silicatein on a bare gold surface (curve 1), cystamine-Au (curve 2), and cysteamine-Au (curve 3) surfaces. Freundlich adsorption isotherm of silicatein adsorption on Au-cystamine-GDA (curve 5) and Au-cysteamineGDA (curve 4) is also presented. Table 1. Amount Adsorbed and Rate Constant (k) for Silicatein on Various Surfaces

surface

maximum adsorbed from a 200 ng/mL initial solution (ng/cm2)

rate constant k (min-1)

Au Au-cystamine Au-cysteamine Au-cystamine-GDA Au-cysteamine-GDA

4.5 ( 0.2 4.4 ( 0.4 4.38 ( 0.1 28 ( 1.3 19.5 ( 1.2

2.45  10-3 4.86  10-3 5.23  10-3 2.4  10-2 1.25  10-2

leading to the presence of two free amine groups per molecule on the cystamine-coated surface compared to one amine group per molecule on the cysteamine-coated surface as shown schematically (Scheme 1). This observation is supported by calculations of the “area under the peak” arising from the free amine group (range 1620-1570 cm-1), which shows that the cystamine-coated surface has a ca. 1.8 times larger amine band area than the cysteaminecoated surface, indicating that more amine groups would be available for interaction with other chemically compatible molecules. Modification of the surfaces with 2% glutardialdehyde (GDA) introduced a spacer between the surface and the protein that improved the mobility and the strength of adsorption of the silicatein. This treatment moved the protein further from the gold surface, thereby reducing interactions between the protein and the surface with maintenance of protein activity. It is possible that glutardialdehye reacts via a Schiff base reaction more strongly with the cystamine-coated surface than the cysteamine-coated surface due to the presence of more free amine groups on the cystamine surface.30 This proposal is supported by the relative peak areas of the free carbonyl group (range 1750-1710 cm-1) of GDA for the two surfaces which shows a ca. 2.4 times larger area for the GDAcystamine-Au surface as compared with the GDA-cysteamine-Au surface. This reaction introduces carbonyl groups to the glutardialdehyde modified surfaces that were then available to react with amino groups on the surface of silicatein via a covalent coupling, thereby promoting the strong adsorption of silicatein. The adsorption of silicatein on the Au-cystamine-GDA, Aucysteamine-GDA, Au-cystamine, Au-cysteamine, and bare gold (30) Benesch, J.; Tengvall, P. Biomaterials 2002, 23, 2561.

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Figure 3. (A) Representative AFM image of silicatein adsorbed on Au-cystamine-GDA. AFM images of silica film deposited on silicatein bound surface after 30 min (B), 60 min (C), and 120 min (D) incubation in 100 mM TMOS solution.

Figure 4. (A) Representative AFM image of silicatein adsorbed on Au-cysteamine-GDA. AFM images of silica film deposited on silicatein bound surface after 30 min (B), 60 min (C), and 120 min (D) of incubation in 100 mM TMOS solution.

surfaces exhibited Freundlich adsorption behavior (Figure 2). The Freundlich isotherm was used to fit the data (R2 = 0.99890.9889 for curves 1-5), as one or more interactions between adsorbed proteins or adsorbed silicatein and the surface may be present during the adsorption process. A poorer fit of the data to the Langmuir isotherm (R2 = 0.9341-0.9631 for curves 1-5), that assumes monolayer surface coverage with no interactions between proteins adsorbed on the surface, was found and was not therefore used for calculation of amounts adsorbed or rate constants for adsorption. The rate constant (k) for adsorption on the different surfaces is given in Table 1. Silicatein adsorbed more strongly and rapidly onto the Au-cystamine-GDA surface than on the Au-cysteamine-GDA surface due to the presence of a larger number of GDA molecules on the former surface as discussed above (Table 1, Figure 2). The adsorption behavior of silicatein on both surfaces showed an initial rapid and extensive adsorption to probably form a surface saturated with protein followed by a further phase of more limited adsorption, which could have arisen through reorganization of protein on the surface.31 AFM images obtained from surfaces (gold alone and with amines) before and after silicatein adsorption gave heterogeneous films of silicatein (Supporting Information Figure SI1) most probably due to the adsorption that was detected (Figure 2 and Table 1). Silicatein deposited uniformly on GDA functionalized cystamine and cysteamine surfaces as observed by AFM (Figures 3A and 4A). Measurement of the thickness, roughness, and contact angle of the films at each stage of the treatment process showed an increase in both thickness and roughness commensurate with a distinct layer being formed at each stage (Table 2). The trend of contact angle reduction probably reflects the varying wettability of the differing chemical functionalities on the surface after each stage of the deposition process (Figure 1A and B). The combined thickness of the Au-cystamine-GDA and Au-cysteamine-GDA surfaces was 5.1 nm. The deposition of silicatein increased the film

thickness to 9.5 nm on both surfaces. The theoretical diameter of a ca. 24 kDa globular protein is approximately 3.2 nm, possibly indicating the adsorption of slightly more than a monolayer of silicatein on both surfaces. The thickness of the films after silicatein deposition is the same, but the adsorbed amounts are different on the two surfaces (Table 1). This is probably due to the fact that the higher loading of GDA on the cystamine surface could promote more adsorption and a more dense packing by rearrangement of protein on the Aucystamine-GDA surface compared to the Au-cysteamine-GDA surface.31 There is perhaps a suggestion of this from the slightly rougher surface obtained for the Au-cystamine-GDA-silicatein film as compared to the Au-cysteamine-GDA-silicatein film (Table 2). The roughness of the bare gold surface was 0.38 nm, which is the atomically flat surface, but the roughness at every stage of the coating process increased, illustrating the transformation of atomically flat gold surfaces to molecularly flat surfaces (Table 2). In order to verify that the activity of the adsorbed silicatein was maintained on the various surfaces, silica films were fabricated at room temperature by treating all surfaces, held vertically, with 100 mM prehydrolyzed TMOS. The specific activity of immobilized silicatein on cystamine-GDA-Au and cysteamine-GDA-Au surfaces was calculated following the procedures described in the Experimental Details section and estimated to be 90 ( 2% as compared to free silicatein with the reduction most likely being due to the dense packing and unavailability of a few silicatein protein molecules to the silica precursors. A similar maintenance of activity for silica surfaces prepared with TEOS was also obtained (data not shown). Analysis of silica deposited on amine-GDA surfaces (no silicatein) and amine-GDA-silicatein surfaces was also performed by AFM (Figures 3 and 4). No coating of silica particles was observed on amine-GDA surfaces without silicatein even after 2 h of treatment with hydrolyzed TMOS (Supporting Information Figure SI2), which is in good agreement with FTIR spectra that do not show any signals involving Si in the characteristic 1100-800 cm-1 region (curves 2 and 3, Figure 5). Additionally, we observed melting of these films when exposed to the electron beam during scanning electron

(31) (a) Hylton, D. M.; Klee, D.; Fabry, M.; Hocker J. Colloid Interface Sci. 1999, 220, 198. (b) Kleijn, M.; Norde, W. Heterog. Chem. Rev. 1995, 2, 157.

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Article Table 2. Roughness, Thickness, and Contact Angle Measurements of the As Prepared Films on Gold Surfaces

surface

Au

Au cystamine

Au cystamine-GDA

Au cystamine-GDA-silicatein

Au cystamine-GDA-silicatein-Si

roughness (nm) thickness (nm) contact angle (deg)

0.38 4 67

0.67 4.4 59

1.17 5.1 54

3.17 9.5 48

5.2 105 15

surface

Au

Au cysteamine

Au cysteamine-GDA

Au cysteamine-GDA-silicatein

Au cysteamine-GDA-silicatein-Si

roughness (nm) thickness (nm) contact angle (deg)

0.38 4 67

0.91 4.4 58

1.1 5.1 54

2.69 9.5 47

3.12 48 14

Figure 5. (A) FTIR spectra of a bare gold surface (curve 1), silica films deposited on Au-cystamine-GDA (curve 2), Au-cysteamineGDA (curve 3), Au-cysteamine-GDA-silicatein (curve 4), and Au-cystamine-GDA-silicatein (curve 5). (B) EDXA of Au-cystamine-GDA-silicatein (curve 3) and Au-cysteamine-GDA-silicatein (curve 4) as well as after silica deposition on Au-cysteamineGDA-silicatein (curve 2) and Au-cystamine-GDA-silicatein (curve 1).

microscopy analysis, indicating the absence of silica. Measurement of the levels of silicic acid after film formation demonstrated that 14 ( 1.2 and 9 ( 1 mM silicic acid were condensed to form uniform silica coatings on cystamine-GDA-silicatein and cysteamine-GDA-silicatein functionalized gold surfaces after 2 h of reaction (Figures 3D and 4D). This is in comparison to previous reports that have shown the formation of heterogeneous films of silica, titania, and zirconia after a much longer period of reaction (8 h) and without quantification of the amount of material deposited on silicatein chemically tethered to gold surfaces.21 AFM images clearly show interconnected structures of silica particles of size from 20 to 40 nm on both surfaces. The total thickness of the films after silica deposition on the Au-cystamineGDA-silicatein surface was estimated to be 105 and 48 nm on the Au-cysteamine-silicatein bound surface in line with the differences in bound silicatein. These results confirm that the activity of silicatein was maintained after immobilization on the amineGDA modified surfaces. The wettability of the silica films was characterized using water contact angle measurements. The high roughness silica films on both surfaces promoted very hydrophilic (∼15°) behavior due to the combined effect of the hydrophilic silica and the network of nanoporous structures present on the silica films (Table 2).32 In another set of control experiments, silicatein bound gold surfaces were used to fabricate silica films. FTIR analysis of silicatein bound to a gold surface (Supporting Information Figure SI3, curve 1) showed a significant broadening of the amide-I band compared to the band observed for silicatein-cystamine-GDA and silicatein-cysteamine-GDA surfaces, indicating some denaturation of adsorbed silicatein protein on the gold surface. Furthermore, AFM analysis also showed heterogeneous silica (32) (a) Sun, T.; Feng, L.; Gao, X.; Jiang, L. Acc. Chem. Res. 2005, 38, 644. (b) Sato, O.; Kubo, S.; Gu, Z.-Z. Acc. Chem. Res. 2009, 42, 1. (c) Liu, X.; He, J. J. Colloid Interface Sci. 2007, 314, 341.

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films, demonstrating the importance of the surface modification of the gold surface prior to the immobilization of silicatein to form uniform silica films (Supporting Information Figure SI1C). FTIR and EDXA were additionally used to confirm the presence of silica on the surfaces. No signature of silica was observed on a bare gold surface nor GDA modified amine treated surfaces after treatment with hydrolyzed TMOS for 2 h (curves 1-3, Figure 5A). Peaks arising from vibrations of Si-O containing species were identified at ∼1040, 950, and 800 cm-1, corresponding to Si-O-Si (asym), Si-OH, and Si-O-Si (sym) bonds, respectively, after the silica films were formed on the Au-amine-GDA-silicatein surfaces (curves 4 and 5 in Figure 5A).33 Furthermore, EDXA showed (Figure 5B) a strong silicon signal at 1.75 keV only for the Au-amine-GDA-silicateinsilica-coated surfaces (curves 1 and 2). To understand the time required to complete silica coatings on different surfaces, AFM and SEM analysis was performed after a range of incubation periods (30-120 min) (Figures 3 and 4 and Supporting Information Figure SI4). The coverage (roughness) of the silica films on the different surfaces increased with increasing reaction time with surface coverage being faster on the cystamineGDA surface as compared to the cysteamine-GDA surface, most likely due to the higher loading of silicatein achieved (Figures 3 and 4). SEM images are shown at the same magnification to highlight the increment of silica particle coverage on these surfaces with respect to time (Supporting Information Figure SI4). It was observed that the smaller silica particles slowly transformed into rougher coatings via the coalescence of particles (Figures 3 and 4). The initially formed silica layers (1 h of reaction) appear to act as a template for the growth of thicker and continuous silica films after 2 h of reaction (Figures 3 and 4).34 A small amount of cracking was observed on the thickest silica films (Figure 3D) due to drying effects. Low magnification AFM and SEM images show featureless and smooth surfaces over large areas, demonstrating the uniformity of the silica films over length scales larger than 1 cm2 (Figure 6). A few large silica particles were observed on the surfaces (Figure 6), that had simply settled down on the surfaces during synthesis in solution. We further investigated the possibility of fine-tuning the thickness, roughness, and wettability of silica films using the Au-cystamine-GDA system by immobilizing different amounts of silicatein on these surfaces (Figure 7). By adsorbing between 10 and ca. 30 ng protein/cm2, we were able to generate silica films between 20 and ca. 100 nm thickness along with roughness from ca. 1.2 to 5.2 nm. Figure 7C shows that there is a direct relationship between the roughness and thickness of the film. The roughness increased with increasing thickness of the deposited (33) Fidalgo, A.; IIharco, L. M. J. Non-Cryst. Solids 2001, 283, 144. (34) Vrieling, E. G.; Hazelaar, S.; Gieskes, W. W. C.; Sun, Q.; Beelen, T. P. M.; Santen, R. A. V. In Silicon biomineralization towards mimicking biogenic silica formation in diatoms; Muller, W. E. D., Ed; Progress in Molecular and Subcellular Biology, Springer-Verlag, Berlin, 2003.

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Figure 6. Representative AFM and SEM images of silica film deposited on Au-cystamine-GDA-silicatein (A and B) and Au-cysteamineGDA-silicatein (C and D) surfaces.

film (Table 2 and Figure 7D).32 This result is in accord with those obtained by others, who have shown that surface roughness decreases the contact angle, therefore enhancing the wettability of a surface.35 In other experiments, different amounts of silicatein (6.8 and 17 ng/cm2) were immobilized on Au-cystamine-GDA surfaces using silicatein solution (50 and 100 ng/mL) followed by silica deposition on these surfaces to further confirm our ability to control the thickness and roughness of the deposited silica films. The roughness and thickness of the silica films were found to increase with increasing amount of protein adsorbed on the surface (data not shown), but the use of the more dilute solutions of silicatein did not produce films with as high a degree of homogeneity as those produced from silicatein solutions at 200 ng/mL (Figure 7). This result suggests that an efficient and uniform deposition of silicatein is necessary to obtain continuous silica films on surfaces. Figure 7. Measurements of roughness and thickness of silica films deposited on Au-cystamine-GDA surfaces with different amounts of silicatein adsorbed (A and B). The relationship between thickness and roughness as well as contact angle and roughness of the silica films is presented in Figure 6C and D, respectively.

silica films, which was also concomitant with decreasing contact angle (Figure 7C and D). The thinner films had very low roughness (ca. 1.2 nm), suggesting that they may be nonporous with normal hydrophilic behavior (Figure 7). On the other hand, the thicker films had a roughness on the order of 4-5 nm, suggesting that interstitial nanovoids between silica particles might be present on the silica films. The resulting rough silica films and the network of nanopores present on the surface led to immediate spreading of water droplets as soon as they contacted with the surface (Supporting Information Figure SI5). Therefore, the intrinsically low contact angle of the silica particles coupled with a large roughness and nanoporous structure generated a very hydrophilic 4158 DOI: 10.1021/la903366a

Conclusions We have shown that uniform silica films can be fabricated on cystamine-GDA-Au and cysteamine-GDA-Au surfaces using nanomolar amounts of silicatein by a sol-gel reaction under environmentally benign conditions. Silicatein was immobilized strongly via covalent binding on both surfaces, although more could be adsorbed on the cystamine-GDA-Au surface than on the cysteamine-GDA-Au surface due to the presence of a greater number of free carbonyl groups on the former surface. The presence of a “linker”, that is, GDA, to prevent contact between the surface and silicatein was necessary to maintain optimum activity. Confirmation that the activity of silicatein was maintained after immobilization on various surfaces was provided by the formation of uniform silica films and by (35) (a) Han, T.-Y.; Shr, J.-F.; Wu, C.-F.; Hsieh, C.-T. Thin Solid Films 2007, 515, 4666. (b) Triantafyllidis, D.; Li, L.; Stott, F. H. Mater. Sci. Eng., A 2005, 390, 271. (c) Chow, T. S. Phys. Rev. Lett. 1997, 97, 1089.

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quantification of the specific activity of silicatein, suggesting that the active site of the immobilized silicatein was available to the solution phase. Physical properties such as thickness, roughness, and wettability of the deposited silica films can be fine-tuned by varying the amount of adsorbed silicatein on the surface and exposure time to a silica precursor. Our study suggests that thickness and wettability are directly related to the roughness of the silica films. The general principles of the described method can be extended to the fabrication of a wide range of materials with controlled thickness and roughness on solid surfaces in an economical fashion under environmentally benign conditions for applications in membrane fabrication, biomedical devices, biosensors, and next generation electronic components.

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Article

Acknowledgment. The authors would like to thank the European Union framework 6 programme BIOLITHO for funding this project. Supporting Information Available: AFM images of a bare gold surface and gold surfaces modified with cystamine, cysteamine, and silicatein as well as after silica coatings on GDA-cystamine-Au and GDA-cysteamine-Au surfaces. FTIR spectra of silicatein adsorbed on GDA-cystamineAu and GDA-cysteamine-Au surfaces. SEM images of the time dependent silica coatings on different surfaces. Images of water droplets on various silica films with different roughness. This material is available free of charge via the Internet at http://pubs.acs.org.

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