Biomimetic materials processing

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A biomimetic super-hydrophobic/super-hydrophilic micro-patterned surface was ... Materials processing based on biomimetics has attracted attention from many ...
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Biomimetic materials processing Takahiro Ishizakia, Junko Hiedab, Maria A. Bratescub, Ngahiro Saito*b,c, Osamu Takaib a National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan b Department of Materials, Physics and Energy Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan c EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ABSTRACT A biomimetic super-hydrophobic/super-hydrophilic micro-patterned surface was successfully fabricated by microwave plasma enhanced chemical vapor deposition (MPECVD) and vacuum ultraviolet (VUV) light lithography. On the micropatterned surface, various site-selective immobilizations were carried out. The fluorescent polystyrene spheres and copper were deposited site-selectively on super-hydrophobic regions using electrostatic interactions. The micropatterned surface brought the discrete adhesions of E. coli and B. subtilis specifically on super-hydrophobic regions. On the other hand, NIH 3T3 fibroblast cells attached to the super-hydrophilic regions in a highly selective manner. Keywords: biomimetics, super-hydrophobicity, super-hydrophilicity, fibroblast cell culture

1. INTRODUCTION Materials processing based on biomimetics has attracted attention from many researchers because it is a green process that produces highly functional and novel self-organized structures.1 In nature, there are super water-repellent plant leaves such as lotus and taro. Studies of these leaves have revealed that an ultra water repellent surface with a large contact angle requires the cooperation of micro- and nano-structures.2 These surfaces are actually covered with hydrophobic micropapilla. The presence of hydrophobic groups on such a structure leads to ultra-water-repellency.3-5 These results obtained from the natural world provide a guide for constructing artificial ultra-water-repellent surfaces and designing surfaces with controllable wettability. A biomimetic approach is based on this concept. Using the biomimetic approach, super-hydrophobic films have been successfully created artificially.6-8 These super-hydrophobic surfaces control their self-cleaning properties, which is known as the lotus effect. Particles adhere to the surfaces of water droplets formed on rough surfaces and can be removed easily from the leaves when the droplets roll off. Superhydrophilic surfaces also show self-cleaning properties that work as well as those of super-hydrophobic surfaces, although in an opposite manner from a wettabilility point of view. By combining the super-hydrophobic and superhydrophilic properties, it is possible to selectively control the direction in which a water drop tends to move, because water droplets would be formed on only super-hydrophilic regions when water is spilled over an artificial superhydrophobic/super-hydrophilic micropattern. In this paper, we report on the fabrication of biomimetic super-hydrophobic/super-hydrophilic micropattern using the microwave plasma-enhanced chemical vapor deposition (MPECVD) and vacuum ultra violet (VUV) lithography. In addition, the micropatterned surface is applied to chemical reaction fields for site-selective immobilization of nanoparticles, electroless plating of copper, and biomolecules.

2. EXPERIMENTAL PROCEDURES 2.1 Fabrication of Super-hydrophobic/Super-hydrophilic micropatterned template p-type Si(100) wafers or glass plate were used as substrates. The substrates were ultrasonically cleaned in ethanol for 10 min. The cleaned substrate was put on the substrate stage in MPECVD system as shown in Figure 1. Superhydrophobic films were then prepared on the substrates. The raw materials were a gas mixture of trimethylmethoxysilane

Nanostructured Thin Films II, edited by Geoffrey B. Smith, Akhlesh Lakhtakia, Cheng-Chung Lee, Proc. of SPIE Vol. 7404, 74040M · © 2009 SPIE · CCC code: 0277-786X/09/$18 · doi: 10.1117/12.829207

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(TMMOS) and Ar. The microwave discharge was maintained by introducing Ar gas. The partial pressures of TMMOS and Ar were kept constant at 93 and 40 Pa, respectively. Prior to the preparation, the chamber of the MPECVD system was evacuated down to 6.7 Pa. A 2.45 GHz generator supplied microwave power of 300 W. The substrate temperature was remained below 333 K during the preparation for 5 min. Figure 2 shows a schematic illustration for the fabrication process of micropatterned super-hydrophilic/hydrophobic surface. After preparation of super-hydrophobic film, vacuum ultraviolet (VUV) light with a wavelength of 172 nm was selectively irradiated for 20 min at the pressure of 10 Pa through a TEM mesh to fabricate super-hydrophilic region. The VUV irradiation in the presence of O2 and H2O produces atomic oxygen and OH radical. The atomic oxygen promotes oxidization and decomposition of organic compounds, and the OH radical introduces hydroxyl group onto sample surface as a termination reaction, leading to that the region irradiated by VUV becomes super-hydrophilic.

Figure 1 Schematic illustration of MPECVD system. 2.2 Characterization Water contact angles of prepared surfaces were measured with a contact angle meter (KRÜSS, DSA10-Mk2) based on a sessile drop measuring method with a water droplet of 2 mm in diameter. Their measurements were conducted in air at 298 K. Behaviors of water droplets on the micropatterned super-hydrophilic/hydrophobic surface were observed using an environmental scanning electron microscope (ESEM: Nikon Co., ESEM-2700), equipped with a differential pumping system, and gaseous secondary electron detector.9-12 The ESEM can image a hydrated sample, and even water drops condensed on a sample surface, without drying them. Water vapor was introduced into the specimen chamber of the ESEM to maintain a pressure of 650 Pa. The sample holder was cooled down to a temperature of 275.5 K, below the dew point (276.1 K) at this water vapor pressure. Consequently, water vapor condensed on the sample surface and formed a number of drops. The sample holder was tilted 60o from the normal. Root mean square roughness (RMS) of the films was acquired in dynamic force mode with an atomic force microscope (AFM; Seiko Instruments, SPA300HV + SPI3800N) using a Si probe (Seiko instruments, SI-DF3; force constant: 1.7 N/m). Zeta-potential of carboxylate-terminated

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polystyrene fluorescent sphere was measured by an electrophoretic light scattering spectrophotometer (ELSS; ELS-600, Otsuka Electronics). A solution containing 1 mM KCl as supporting electrolyte was used, adjusting its pH over the range of 2.5 to 8 by adding HCl or NaOH. 2.3 Site-selective immobilization The micropatterned super-hydrophilic/hydrophobic substrates were immersed in a solution dispersed with carboxylate-terminated polystyrene fluorescent spheres with a mean diameter of 200 nm. The procedure of site-selective immobilization of the polystyrene spheres was as follows. Firstly, a mixture aqueous solution of ultra pure water and the fluorescent polystyrene spheres was prepared. The volume ratio mixed (fluorescence spheres/H2O) was 20. The mixed aqueous solution was adjusted to pH 2.6 with a HCl and then stirred ultrasonically for 20 min to disperse the fluorescent polystyrene spheres. Next, the micropatterned super-hydrophilic/hydrophobic substrate was immersed for 90 min in the mixed solution. Finally, the substrate was stored for 30 min in air and dehydrated by air-drying. Assembling of the fluorescent polystyrene spheres on the template was observed with an optical fluorescence microscope (Olympus, IX71ARCEVA). The procedures of site-selective electroless plating of copper were performed as follows. The micropatterned surface was immersed in a pH=1.1 solution containing Sn (High Purity Chemicals, Co., Ltd.). Such solution is commonly used in order to activate a surface. Next, the Sn adsorbed substrate was immersed in a pH = 1.6 electroless plating solution containing Pd ions (High Purity Chemicals, Co., Ltd.). The sample was then immersed in a pH = 13.0 electroless plating solution containing Cu ions (High Purity Chemicals, Co. Ltd.). The site-selective electroless plating of copper on super-hydrophilic/hydrophobic micropatterned template was observed using a field emission scanning electron microscopy (FE-SEM: JEOL Ltd., JSM-6330F) with an accelerating voltage at 10 keV. NIH 3T3 fibroblast cells (American Type Culture Collection, Manassas, VA, USA) were cultured using Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml streptomycin sulfate, and 100 U/ml potassium penicillin G. The cells were cultured at 37 °C in a humidified atmosphere of CO2 and 95 % air. The cultured cells were observed with a phase-contrast microscopy. Four bacterial strains, Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Pseudomonas stutzeri (P. stutzeri), and Pseudomonas aeruginosa (P. aeruginosa), were used for site-selective immobilization of bacteria. E. coli and B. subtilis have been defined as a gram-negative model bacterium and gram-positive model bacterium, respectively.13,14 P. subtilis and P. aeruginosa bacteria belong to gene Pseudomonas and have extracellular polysaccharide (EPS) composed by an algin acid.15 The difference between P. stutzeri and P. aeruginosa consists in growth rate and gene arrangement.16 Put it all together, the four bacteria were selected from viewpoint of peptidoglycan cell wall (E.coli vs B. subtilis), EPS (E.coli vs P. stutzeri, P. aeruginosa), and growth rate (P. stutzeri vs P. aeruginosa). Four kinds of bacteria were sowed on a micropatterned super-hydrophobic/super-hydrophilic surface at 4.0×104 cell/dish and incubated under 5 % of CO2 concentration at 37 oC for 24 hours. The culture medium was conformed to the condition of OXOID CM3. After the incubation, the substrate was rinsed with Dulbecco’s phosphate buffered saline (D-PBS) (Nacalai Tesque Inc.) and the number of bacteria adhered on the micropatterned surface was counted by a phase-contrast microscope (Olympus, CKX41N-31PHP). The bacteria counting was made for at least 10 fields of view for each sample.

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Figure 2 Schematic illustration of fabrication processes of micropatterned super-hydrophilic/hydrophobic surface.

3.

Results and Discussion

Water contact angle measurements showed that the water contact angles of water droplets on super-hydrophobic and super-hydrophilic surfaces were over 150 and less than 5 degrees, respectively. The wettability of the VUV irradiated super-hydrophobic surface was dramatically changed from super-hydrophobic to super-hydrophilic property with an increase in irradiation time. Figure 3 (a) shows an ESEM image of water droplets behaviors on superhydrophobic surface. All the water droplets appear to be almost spherical. AFM studies revealed that the RMS roughness on super-hydrophobic surface was in the ranges of 70 to 100 nm. Water droplets condensed on the micropatterned super-

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hydrophobic/hydrophilic surface were also observed with an ESEM. The ESEM image of water droplets behaviors is shown in Figure 3 (b). As clearly indicated by the water droplets on the surface, water vapor selectively condensed on only super-hydrophilic region. The super-hydrophilic regions were filled with condensate instantly. The superhydrophilic surface had a RMS roughness of c.a. 80 nm. XPS measurements revealed that peak areas corresponding to C-O and C=O bonds increased with VUV irradiation duration whereas that originating from C-C bond decreased considerably, indicating that VUV irradiation induces chemical conversion of hydrophobic functional groups (–CH3) into hydrophilic groups such as –COOH, –CHO, and Si–OH. These results strongly support that the super-hydrophobic surface was chemically modified to be super-hydrophilic without any physical damage. These results suggest that the micropatterned surface with super-hydrophilic/hydrophobic regions was successfully fabricated.

Figure 3 ESEM images of water droplets behaviors (a) on super hydrophobic surface and (b) on micropatterned superhydrophobic/super-hydrophilic surface. Water vapor selectively condensed on only super-hydrophilic region. To use the micropatterned surface as a site-selective chemical reaction field, we attempted to site-selectively immobilize carboxyl-terminated fluorescent polystyrene spheres. Figure 4 shows an image acquired by fluorescent microscopy of the micropatterned surface after immersion. The light areas and dark regions correspond to superhydrophobic and hydrophilic regions. This fluorescent microscope image indicates that carboxyl-modified polystyrene fluorescence spheres selectively adsorbed on the super-hydrophobic region, that is, -CH3 groups regions. To elucidate the mechanism for site-selective immobilization of polystyrene spheres, The surface charges were investigated by measuring the zeta-potential of the carboxyl-terminated fluorescent polystyrene spheres. Figure 5 shows the variation in zetapotential of the carboxyl-terminated fluorescent polystyrene spheres. The zeta-potential was estimated from the values averaged three times. At the pH range of 2.5 to 8.2, all the zeta-potentials of the carboxyl-terminated fluorescent polystyrene spheres show negative values. This is due to partial ionization of carboxyl groups to –COO-. On the other hand, the functional groups on super-hydrophilic regions are mainly silanol (–SiOH). It has been reported17,18 that isoelectric point (IEP) of the silanol surface was in the ranges of 1.5 to 2.2. Thus, in the solution of pH 2.6, the silanol groups show negative zeta-potential, since they are partially ionized to –SiO- due to the proton dissociation from -SiOH groups. The surfaces of super-hydrophobic regions are mainly covered with methyl groups. It was reported19,20 that IEP of the methyl-terminated SAM surface was at approximately pH 4.0. Thus, the methyl-terminated SAM surface would become positive zeta-potential at the pH less than 4.0, which was attributed to proton adsorption in the double layer. Consequently, the micropatterned surface composed of super-hydrophilic and -hydrophobic regions would be separately charged with different polarity with dependant on the surface functional groups. The carboxyl and silanol groups in the solution of pH 2.6 were converted into –COO- and –SiO- groups, respectively, so the selective adsorption of fluorescence spheres on the super-hydrophilic regions could not occurred due to their repulsive electrostatic interaction to the surface.

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On the contrary, the methyl groups on the super-hydrophobic regions were positively charged. Thus, the carboxylmodified polystyrene fluorescence spheres adsorbed onto the super-hydrophobic regions due to electrostatic attractive forces. This indicates that we demonstrated site-selective chemical reaction due to the difference of surface functional group on super-hydrophilic/hydrophobic micropatterned template.

Figure 4 A fluorescent microscopic image of fluorescence polystyrene spheres on super-hydrophilic/hydrophobic template micropatterned. The spheres selectively assembled on super-hydrophobic regions.

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Figure 5 The zeta-potentials of the carboxyl-terminated fluorescence polystyrene spheres in the pH range of 2.5 to 8.2. Next, we attempted to selectively deposit Cu through electroless plating on the super-hydrophilic/hydrophobic template micropatterned. Figure 6 shows FE-SEM images of the Cu deposited on the micropatterned surface. The bright and dark regions correspond to metal and super-hydrophilic ones, respectively. The image indicates highly selective metal deposition onto the super-hydrophobic regions. The deposition consisted of uniformly distributed large grains, typically 500 nm in size. Figure 7 shows the Cu distribution based on energy dispersive X-ray analysis (EDAX) on a line and FE-SEM image of the template surface, respectively. The EDAX analysis revealed that the deposits on the superhydrophobic regions were Cu particles. No Cu distribution was observed in the dark circular regions, that is, superhydrophilic regions. In order to evaluate the strength, the adhesion test was conducted with a Scotch tape. No peeling was observed on the sample surface.

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Figure 6 (a) FE-SEM image of site-selective Cu deposition on micropatterned super-hydrophilic/hydrophobicsurface. Cu was selectively deposited on super-hydrophobic regions through electroless plating. (b) FE-SEM image enlarged of the surface morphology.

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Figure 7 FE-SEM image of site-selective Cu deposition on micropatterned super-hydrophilic/hydrophobic surface and Cu distribution on line by EDAX. The site-selective deposition of copper would be achieved through the site-selective deposition of Sn particles on the super-hydrophobic regions. The adsorption mechanism of Sn particles to the super-hydrophobic regions is considered as follows. After immersion of the micropatterned surface, the -CH3-terminated regions are oxidized to -COOH terminated ones. This oxidation of the -CH3-terminated surface can be explained by the disproportionation of Sn as follows: 2Sn2+ = Sn0 + Sn4+. The Sn4+ ions in the acid solution act as a catalyst for oxidation. Due to their presence, the CH3-terminated surface can be oxidized to -COOH terminated one by the Sn4+ ions. Saito et al confirmed by XPS analysis that carboxyl end groups had been introduced into the -CH3-terminated surface by the disproportionation of Sn.21 Thus, Sn particles were selectively adsorbed on the carboxyl end groups, leading to the site-selective deposition of copper.

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Figure 8 Phase-contrast microscopic image of micropattering of NIH 3T3 fibroblast cells cultured on superhydrophobic/super-hydrophilic surface for 72 h. Figure 8 shows a phase-contrast microscopic image of micropattering of NIH 3T3 fibroblast cells cultured on the micropatterned super-hydrophobic/super-hydrophilic surface for 72 h. When the cells were seeded on the micropatterned super-hydrophobic/super-hydrophilic substrate, the cells attached to the super-hydrophilic regions in a highly selective manner. The cell adhesion on the super-hydrophilic region is considered to occur through selective attachment of cell adhesion molecules such as fibronectin and vitronctin. These molecules are known to play important roles in cellular function.22-24 The cellular response including adhesion and proliferation could be affected significantly by these proteins adsorbed on the surface. Thus, the differences in the proteins adsorption behaviors might affect the cell attachments. Figure 9 shows visual microscopic images of bacteria adhesion behaviors on the micropatterned superhydrophobic/super-hydrophilic surface after the incubation. On the case of super-hydrophobic/super-hydrophilic micropattern, E. coli and B. subtilis showed discrete adhesion on super-hydrophobic regions in comparison with superhydrophilic ones. For the other bacteria (P. stutzeri and P. aeruginosa), they adhered uniformly on both superhydrophobic and super-hydrophilic regions. Reasons for the different behaviors between bacteria might be resulted from difference of physical or chemical interaction of the cellular surface with the each region. As shown in Figure 8, the immobilization on specific regions regulated by wettability was not achieved well for P. stutzeri and P. aeruginosa. The differences in bacterial adhesion would be resulted from the differences of the physical or the chemical interactions of specific EPS. This is because there were no differences of adhesion between E. coli as gram negative model bacteria and B. subtilis as gram positive model bacteria. P. stutzeri and P. aeruginosa belong to gene Pseudomonas and have a characteristic EPS composed of the algin acid. This adhesion behavior excludes the effect of the hydrophilic groups on the surface and could be explained by surface roughness. These results indicate that by employing the technique presented here, we were able to successfully fabricate superhydrophilic/hydrophobic micropatterned surface and use it as site-selective chemical reaction field.

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Figure 9 Microscopic images of bacteria adhesion behaviors on micropatterned super-hydrophobic / super-hydrophilic surface; (a): E. coli, (b): B. subtilis, (c): P. stutzeri, (d): P. aeruginosa.

4. Conclusions We successfully fabricated a biomimetic super-hydrophobic/super-hydrophilic micro-patterned template. By using the micropatterned surface, various site-selective immobilizations were achieved. The fluorescent polystyrene spheres and copper were deposited on super-hydrophobic regions. The micropatterned surface brought the discrete adhesions of E. coli and B. subtilis specifically on super-hydrophobic regions. On the contrary, NIH 3T3 fibroblast cells attached to the super-hydrophilic regions in a highly selective manner. We believe that the biomimetic approach would be a key technology for achieving site-selective immobilization of organic and inorganic material.

Acknowledgement The work was partly supported by Grant-in-Aid for Young Scientists (B) (No. 20760491) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Knowledge Cluster Initiative ~Tokai Region Nanotechnology Manufacturing Cluster~.

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