pubs.acs.org/Langmuir © 2010 American Chemical Society
Cell Adhesion Properties on Chemically Micropatterned Boron-Doped Diamond Surfaces Lionel Marcon,*,†,‡ Corentin Spriet,† Yannick Coffinier,†,‡ Elisabeth Galopin,†,‡ Claire Rosnoblet,† Sabine Szunerits,†,‡ Laurent Heliot,† Pierre-Olivier Angrand,† and Rabah Boukherroub†,‡ †
Interdisciplinary Research Institute (IRI ), USR-CNRS 3078, Universit e de Lille 1, Parc de la Haute Borne, 50 avenue de Halley, 59658 Villeneuve d’Ascq, France, and ‡Institute of Electronics, Microelectronics and Nanotechnology (IEMN), UMR-CNRS 8520, Universit e de Lille 1, Cit e Scientifique, 59655 Villeneuve d’Ascq, France Received May 3, 2010. Revised Manuscript Received July 21, 2010 The adhesion properties of living cells were investigated on a range of chemically modified boron-doped diamond (BDD) surfaces. We studied the influence of oxidized, H-, amine- (NH2-), methyl- (CH3-), trifluoromethyl- (CF3-) and vinyl- (CH2dCH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells behavior. Cell-surface interactions were analyzed by fluorescence microscopy in terms of cell attachment, spreading and proliferation. U2OS cells poorly adhered on hydrophobic surfaces and their growth was blocked. In contrast, L929 cells were mainly influenced by the presence of perfluoroalkyl chains in regard to their morphology. The results were subsequently applied to selectively micropattern U2OS cells on dual hydrophobic/hydrophilic surfaces prepared by a UV/ozone lithographic approach. U2OS cells colonized preferentially hydrophilic (oxide-terminated) motifs, forming confluent arrays with distinguishable edges separating the alkyl regions.
Introduction In recent years, diamond has become a widely investigated material for its remarkable properties like its hardness, high Young modulus, thermal conductivity, dopability, and the variety of substrates onto which it can be deposited.1 The diamond-based surfaces are expected to be good candidates for number of bioapplications.2-5 Indeed, the unique combination of the mechanical, chemical, and biocompatible properties with semiconducting properties makes diamond an attractive material for various applications, in particular for biological and chemical sensing.6 Recently, boron-doped diamond (BDD) has gained remarkable interest due to its high chemical stability, good electrical conductivity, large potential window in aqueous electrolytes (about -1.35 to 2.3 V/NHE) and biocompatibility.7,8 The next step toward applying BDD to engineered tissue therapies or cell-based biosensors is to characterize its interaction with cells. To date, such studies made on chemically modified diamond-based surfaces concerned undoped homogeneous substrates with restricted chemical surface terminations. The adhesion properties and viability of osteoblasts and *To whom correspondence should be addressed. E-mail: Lionel.Marcon@ iri.univ-lille1.fr. Telephone: þ33 (0)3 62 53 17 23. Fax: þ33 (0)3 62 53 17 01.
(1) Kohn, E.; Gluche, P.; Adamschik, M. Diamond Relat. Mater. 1999, 8, 934–940. (2) Carlisle, J. A. Nat. Mater. 2004, 3, 668–669. (3) Hamers, R. J.; Butler, J. E.; Lasseter, T.; Nichols, B. M.; Russell, J. J. N.; Tse, K.-Y.; Yang, W. Diamond Relat. Mater. 2005, 14, 661–668. (4) Hartl, A.; Schmich, E.; Garrido, J. A.; Hernando, J.; Catharino, S. C. R.; Walter, S.; Feulner, P.; Kromka, A.; Steinmuller, D.; Stutzmann, M. Nat. Mater. 2004, 3, 736–742. (5) Shenderova, O. A.; Gruen, D. M. Ultrananocrystalline Diamond - Synthesis, Properties, and Applications; William Andrew Publishing: Norwich, NY, 2006. (6) Rezek, B.; Shin, D.; Nebel, C. E. Langmuir 2007, 23, 7626–7633. (7) Granger, M. C.; Witek, M.; Xu, J.; Wang, J.; Hupert, M.; Hanks, A.; Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.; Swain, G. M. Anal. Chem. 2000, 72, 3793–3804. (8) Szunerits, S.; Boukherroub, R. J. Solid State Electrochem. 2008, 12, 1205–1218. (9) Lechleitner, T.; Klauser, F.; Seppi, T.; Lechner, J.; Jennings, P.; Perco, P.; Mayer, B.; Steinm€uller-Nethl, D.; Preiner, J.; Hinterdorfer, P.; Hermann, M.; Bertel, E.; Pfaller, K.; Pfaller, W. Biomaterials 2008, 29, 4275–4284. (10) Rezek, B.; Michalı´ kova, L.; Ukraintsev, E.; Kromka, A.; Kalbacova, M. Sensors 2009, 9, 3549–3562.
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epithelial cells were reported on nanocrystalline diamond coated with O- and H-terminations.9,10 It was found that H-terminated areas were less favorable for cell adhesion, spreading, and viability, as compared to oxidized regions. Another study included diamond surfaces modified with carboxylic acid groups and showed that hydrophilic terminations supported the growth and attachment of neuronal cells.11 The aim of the present work was to study cell adhesion properties on BDD surfaces modified with a broader range of chemical functionalities. Various chemical terminations, differing in their wettability from hydrophilic to hydrophobic surfaces, were prepared. We investigated the impact of oxidized, H-, amine(NH2-), alkyl- (CH3-), trifluoromethyl- (CF3-) and vinyl(CH2dCH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells adhesion, morphology, and growth. L929 cells were selected because they are very useful in biocompatibility and adhesion studies due to their remarkable properties. Indeed, fibroblasts are anchorage-dependent cells and constitute the most common cells of all tissues. In this context, biological tests with L929 cell cultures are regarded as reference bioassays providing informations on cell response.12 For comparison, we employed cells from another species, namely U2OS cells. Because of their fast growth, their absence of contact inhibition and their easy handling, U2OS cells are ideal models for the present report. The cell behavior on modified surfaces was visualized by fluorescence microscopy. These data were subsequently used to guide U2OS cell growth in a spatially controlled manner onto chemically patterned surfaces. To this goal, we designed a modified surface combining oxide-terminated micropatterns with alkyl surroundings. The different wetting properties of each region allowed us to generate precise cellular patterns with the aim of using BDD-based patterned surfaces for cell-based bioassays. (11) Chong, K. F.; Loh, K. P.; Vedula, S. R. K.; Lim, C. T.; Sternschulte, H.; Steinm€uller, D.; Sheu, F.-s.; Zhong, Y. L. Langmuir 2007, 23, 5615–5621. (12) Amaral, M.; Gomes, P. S.; Lopes, M. A.; Santos, J. D.; Silva, R. F.; Fernandes, M. H. Acta Biomater. 2009, 5, 755–763.
Published on Web 08/18/2010
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Experimental Section Materials. Hexane, methanol, chloroform, and isopropanol were obtained from Sigma-Aldrich. Octadecyltrichlorosilane (OTS), 10-undecenyltrichlorosilane (UTCS), perfluorooctyltrichlorosilane (FOTS), and perfluorodecyltrichlorosilane (FDTS) were purchased from ABCR GmbH. Polycrystalline boron-doped diamond films (1.5-2 μm thick) deposited on silicon substrates in a hot filament-assisted chemical vapor deposition reactor supplied with diborane and methane in hydrogen were provided by CSEM (Neuchatel, Switzerland). The doping level of boron was determined to be NA ∼3 1019 B cm-3 by SIMS measurements. BDD Surface Functionalization. Hydrogenation. Hydrogenation of the BDD samples was performed in an ultrahigh vacuum chemical vapor deposition (CVD) chamber using the hotfilament chemical vapor deposition (HF-CVD) mode described elsewhere.13 The conditions were the following: 100 sccm H2 for 10 min and P = 15 mbar with tungsten filaments (two pairs of tungsten filaments placed 5 and 10 mm above the substrate, respectively) at 180 W (around 2450 K). The surface of the substrate was heated on the back side by using an infrared heater to keep a constant temperature of 973 K. Following this treatment, the sample was cooled to room temperature under gaseous hydrogen. Oxidation. A low pressure mercury arc lamp (UVO cleaner, Nr. 42-220, Jelight, U.S.A., P = 1.6 mW cm2, distance from sample: 3 mm, t = 60 min) was used to photochemically oxidize as-received BDD samples, as reported previously.14 Amination. Amination was performed using a plasma treatment generated by an EUROPLASMA setup, as described elsewhere.15 Silanization. Octadecyl termination was obtained by immersion of the oxidized substrates into a 10-3 M OTS solution in hexane for 16 h at room temperature in a dry-nitrogen-purged glovebox. Likewise, silanization with UTCS, FOTS, and FDTS was performed in 3.0, 2.5, and 10 mM hexane solutions, respectively. The resulting surfaces were finally rinsed with chloroform, methanol, and milli-Q water and dried under a gentle stream of nitrogen. Patterning. Two types of motifs were prepared. (1) An aluminum foil was placed on one-half of a CH3-(CH2)17-terminated BDD surface and subsequently exposed to UV/ozone for 60 min at 5 mW/cm2, thus creating oxidized areas. (2) Using the same approach, a Cu-grid with 400 μm2 square openings spaced by 250 μm was sealed tightly against the octadecyl-terminated surface and exposed to UV/ozone for 60 min, generating 400 μm2 oxide patterns surrounded by C18 chains.16 The surface was thoroughly washed with isopropyl alcohol and deionized water. Cell Culture. Human osteosarcoma U2OS cells expressing nuclear yellow fluorescent protein (YFP) and mouse fibroblast L929 cells expressing nuclear green fluorescent protein (GFP) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% of a penicillin/streptomycin mix. Cells were seeded at 1.5 105 cells/cm2 and cultured at 37 °C in a humidified atmosphere containing 5% CO2. L929 cell membranes were stained with Vybrant DiI (Molecular Probes) according to the manufacturers instructions. Briefly, staining medium was prepared by adding 5 μL of the supplied dye solution to 1 mL of normal growth medium. Cells were then incubated with staining medium for 20 min, washed 3 times with warmed growth medium, and transferred in Leibovitz15 L15 (Gibco) medium for microscopy observations. The viability of cells was assessed using trypan blue die exclusion. Cells were removed by trypsinisation and suspended in 0.2% (w/v) trypan blue (13) Arnault, J. C.; Demuynck, L.; Speisser, C.; Le Normand, F. Eur. Phys. J. B 1999, 11, 327–343. (14) Boukherroub, R.; Wallart, X.; Szunerits, S.; Marcus, B.; Bouvier, P.; Mermoux, M. Electrochem. Commun. 2005, 7, 937–940. (15) Szunerits, S.; Jama, C.; Coffinier, Y.; Marcus, B.; Delabouglise, D.; Boukherroub, R. Electrochem. Commun. 2006, 8, 1185–1190. (16) Marcon, L.; Wang, M.; Coffinier, Y.; Le Normand, F.; Melnyk, O.; Boukherroub, R.; Szunerits, S. Langmuir 2010, 26, 1075–1080.
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(Invitrogen) for 5 min. Stained and unstained cells were counted using Kova Glasstic slides with grids (Hycor). Cell Counting Method. Cells were automatically counted using Image J (NIH, Bethesda, MD) software. Fluorescent microscopy images, showing the cells on modified BDD surfaces, were routinely opened and treated in the software, resulting in an accuracy of (10 cells per image. Five images per surface were analyzed. Instrumentation. Contact Angle Measurements. Milli-Q water contact angles were measured at room temperature using a remote computer-controlled goniometer system (DIGIDROP by GBX, France). The accuracy was (2°. Optical Microscopy. BDD surfaces were observed using an optical microscope (Eclipse 80i, Nikon Instruments, Tempe, AZ) equipped with a Coolsnap ES2 camera (Photometrics, Tucson, AZ), a Nikon Ph1 DLL 10/0.30 Plan Fluor objective and a super highpressure mercury arc lamp (Southern Micro Instruments). Fluorochromes were imaged with the following excitation/emission filters: YFP (514 nm/580 ( 50 nm), GFP (488 nm/560 ( 50 nm), and DiI (543 nm/590 ( 25 nm). High-magnification images were obtained using a Leica Microsystems AF6000LX fluorescence microscope (Mannheim, Germany). Time-lapse recording was performed using the Biostation IM (Nikon Instruments), which consists of an incubator incorporating a fluorescence microscope equipped with a digital CCD camera and a PC computer for data acquisition and analysis (experimental details provided in the Supporting Information). X-ray Photoelectron Spectroscopy (XPS). Measurements were performed on an Axis Ultra DLD spectrometer from Kratos (Manchester, UK) using a monochromatic Al KR X-ray source (1486.6 eV). The hemispherical analyzer was used in constant analyzer energy mode for all spectra (100 eV for survey spectra and 40 eV for high-resolution spectra). No flood gun source was needed due to conducting character of the substrates. The angle between the incident X-rays and the analyzer is 54.7°. The detection angle of the photoelectrons is 90°, as referenced to the sample surface.
Results and Discussion The as-grown boron-doped diamond substrates used in this work (purchased from CSEM) were hydrogen-terminated. The polycrystalline diamond was deposited from a B2H6/CH4/H2 source gas mixture by hot filament-assisted chemical vapor deposition (HFCVD). The samples were then functionalized to obtain a wide variety of chemical terminations. Hydrogenation was performed using the HF-CVD technique in H2.13 Amine termination -NH2 was obtained by NH3 plasma treatment of the hydrogenated surface.15 In parallel, a UV/ozone treatment led to the incorporation of hydroxyl (C-OH), ether (C-O-C), and carbonyl (CdO) functional groups on the BDD surface.17,18 The presence of hydroxyl groups allowed covalent coupling of trichlorosilane-based molecules. The wetting properties of the resulting modified BDD surfaces were evaluated using contact angle measurements. The measured water contact angles of surfaces bearing -OH, -NH2, -H, -CHdCH2, -CH3, and -CF3 functionalities were in accordance with published data (Table 1). Oxide- and amine-terminated surfaces formed moderately wettable surfaces (θ < 55°) while those terminated with hydrogen, alkyl, and perfluoroalkyl chains generated hydrophobic surfaces (θ > 90°). The NH2- and CF3-terminated BDD surfaces were further characterized using XPS (Figures S1 and S2, Supporting Information). After NH3 plasma treatment, a signal due to N1s at 399 eV was observed, consistent with conversion of the hydrogentated surface into an amine-terminated surface. Additionally, the coupling of the hydroxyl surface groups with the perfluorotrichlorosilanes was evidenced by a peak at 687 eV due to F1s. (17) Wang, M.; Simon, N.; Charrier, G.; Bouttemy, M.; Etcheberry, A.; Li, M.; Boukherroub, R.; Szunerits, S. Electrochem. Commun. 2010, 12, 351–354. (18) Wang, M.; Simon, N.; Decorse-Pascanut, C.; Bouttemy, M.; Etcheberry, A.; Li, M.; Boukherroub, R.; Szunerits, S. Electrochim. Acta 2009, 54, 5818–5824.
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Figure 1. Fluorescent microscopy images of U2OS cells cultivated for 48 h on modified BDD. The images display the (a) oxidized, (b) aminated, (c) hydrogenated, (d) vinyl, (e,f) perfluorodecyl, and (g) octadecyl-terminated surfaces. A red color was attributed to the cells grown in the culture dish (as a reference) and a green color to the cells grown on BDD. Table 1. Water Contact Angles of Modified BDD Surfaces and Corresponding Cell Densitya
surface
U2OS cell L929 cell contact ratio (BDD/ref) ratio (BDD/ref) angle, ° (ref) per area, % per area, %
100 100 oxide-terminated 414 5328 100 100 -NH2 14 70 94 -H 92 9529 30 91 -(CH2)9-CHdCH2 10314 26 92 -(CH2)2-(CF2)7-CF3 11130 10 92 -(CF2)7-CF3 31 114 6 91 -(CH2)17-CH3 a Values represent the mean (4% of three independent experiments.
Human osteosarcoma U2OS cells were plated at densities of 1.5 105 cells/cm2 onto the modified surfaces immersed in appropriately supplemented medium. Figure 1 displays fluorescent microscopy images of U2OS cells cultivated for 48 h onto BDD surfaces of different chemical composition. The ratio between the Langmuir 2010, 26(19), 15065–15069
number of cells adhered on BDD and culture dish (as a reference) per area is given in Table 1. It appears that U2OS cell attachment is related to the wettability of the BDD interface. A few cells adhered onto the alkylated surfaces while the hydrophilic samples (oxide and amine-terminated) induced cell adhesion and proliferation comparable to the standard culture dishes. Previous studies demonstrated that cells cultured on hydrophobic surfaces attach slowly and weakly, spread less, multiply slowly, and have high apoptosis rates compared to oxidized surfaces.19,20 More specifically, cells produce extracellular matrix (ECM) proteins to mediate cell adhesion onto surfaces. It is likely that the hydrophobic chemical terminations prevent the adsorption of ECM proteins. A time-lapse recording was performed to (19) Chang, E.-J.; Kim, H.-H.; Huh, J.-E.; Kim, I.-A.; Seung, Ko, J.; Chung, C.-P.; Kim, H.-M. Exp. Cell. Res. 2005, 303, 197–206. (20) Kalbacova, M.; Kalbac, M.; Dunsch, L.; Kromka, A.; Vanecek, M.; Rezek, B.; Hempel, U.; Kmoch, S. Phys. Status Solidi B 2007, 244, 4356–4359.
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Figure 2. Fluorescent microscopy images of L929 cells incubated for 24 h on octadecyl (top panels) and perfluorodecyl-terminated surfaces (lower panels). Cells express nuclear green fluorescent protein (green) and membranes were stained with DiI (red).
Figure 3. Cultures of (a,b) U2OS and (c,d) L929 cells on 1 cm2 octadecyl-modified BDD. The left panels show the 50 mm2 pattern and the
right panels show the 400 μm2 patterns. White lines indicate the edge of the hydrophobic/hydrophilic regions. Cells were plated at a density of 1.5 105 cells/cm2 and cultivated for 24 h.
assess the cell proliferation kinetics by comparing cell growth on -(CH2)9-CHdCH2, -(CH2)2-(CF2)7-CF3, -(CF2)7-CF3 and -(CH2)17-CH3 terminated BDD and culture dish. In the latter, cells divided once per 18 h while their growth and motility was blocked in the former (Figure S3, Supporting Information). The low number of cells did not allow performing a viability assay so it can be hypothesized that cells are either dead or quiescent. Further investigations will be necessary to determine the intracellular mechanisms when in contact with hydrophobic surfaces. Similar experiments were conducted with a different cell line, that is, mouse fibroblasts L929. Cell morphological properties were examined after membrane staining with DiI. This fluorochrome is a lipophilic carbocyanine dye binding to intracellular phospholipid bilayer membrane. We observed a variability in cell shape and proliferation between perfluoroalkyl-terminated surfaces and the other surfaces. As an example of this contrast, Figure 2 shows images of the octadecyl and perfluorodecyl terminated surfaces incubated for 24 h. In the first case, L929 cells showed a homogeneous membrane 15068 DOI: 10.1021/la101757f
spreading, an elongated shape, and a typical parallel alignment on confluent regions. Fibroblasts are known to continuously synthesize ECM and collagen thus maintaining a strong structural framework for animal tissues. It explains the low impact of the modified surfaces used in this study on cell adhesion predominantly observed. In the second case, on perfluorooctyl- and perfluorodecylterminated surfaces cells adopted a rounded shape and no membrane elongation. We hypothesize that formation of focal contacts and stress fibers was largely inhibited by the surfaces, as previously observed.21 This phenomenon did not prevent cell division, though, and initially seeded cells connected to form core clusters after 48 h. Neighboring cells formed connections with each other and proliferated subsequently exhibiting a morphology similar to cells grown in culture dishes. These surrounding cells mask the unsuitable properties of the surface due to support from the core cells probably by (21) Kalbacova, M.; Michalikova, L.; Baresova, V.; Kromka, A.; Rezek, B.; Kmoch, S. Phys. Status Solidi B 2008, 245, 2124–2127.
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exchanging growth factors and producing ECM. These clusters of cooperating cells probably allow them to form some focal adhesion. Viability of the cells attached to the perfluoroalkyl-modified surfaces for 48 h was 92 ( 0.9% (98 ( 1.1% for the culture dish and the other modified surfaces). Patterning cells on modified surfaces has recently received considerable attention due to its important role in fundamental cell biology, tissue engineering, cell-based biosensors and biomedical/ diagnostic microdevices.22,23 This technique constitutes one way to control the complex behavior exhibited by cells with their surrounding environment.24 It consists in selectively guiding, trapping, and attaching cells into a defined area without damaging the cells. Typical surface engineering strategies for cell patterning aim to control protein adsorption on surfaces via regions possessing fouling or nonfouling properties. One common approach is by means of localized self-assembled monolayers (SAMs) to adhere ECM proteins. In our study, patterning provides a controlled experimental system to assess the impact of the chemical functionalization of BDD surfaces toward the growth of U2OS cells. We designed two types of hydrophilic oxide-terminated patterns on 1 cm2 octadecylmodified BDD substrates: (i) one occupying one-half of the surface and (ii) 400 μm2 aligned squares. An octadecyl termination was chosen because, as discussed earlier, this alkyl chain was the less favorable for U2OS cell adhesion. Figure 3a,b display fluorescence images of each patterned surface after incubation with U2OS cells for 24 h. Hydrophilic and hydrophobic motifs are clearly defined in both cases. Cells colonized preferentially hydrophilic (oxidized) motifs forming confluent arrays after 72 h with distinguishable edges separating the octadecyl regions. The two types of patterned surfaces were then used with the less surface-dependent L929 cell line, as observed in the previous paragraph. As expected, motifs were indistinguishable and cells grew normally on the whole surface (Figure 3c,d). A previous study demonstrated that the initial density of plated cells facing similar patterns had an impact on their selective colonization.10 We tested different densities ranging from 104 to 109 cells/cm2 (22) Polla, D. L.; Erdman, A. G.; Robbins, W. P.; Markus, D. T.; Diaz-Diaz, J.; Rizq, R.; Nam, Y.; Brickner, H. T.; Wang, A.; Krulevitch, P. Annu. Rev. Biomed. Eng. 2003, 2, 551–576. (23) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107–110. (24) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573–1583.
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with no impact on their adhesion onto the patterned surfaces (Figure S4, Supporting Information).
Conclusions In this work, we characterized the adhesion properties of living cells onto diamond substrates as a function of their surface termination. If human osteosarcoma U2OS cells were sensitive to the hydrophobicity of the substrates, mouse fibroblasts L929 were influenced only by the presence of perfluoroalkyl chains. These properties were subsequently used to culture U2OS cells in predetermined patterns using chemically patterned regions. This has enormous potential for the development of diamond-based biosensors, cell-based assays, and bioengineering. In addition, the micrometer-size of the fabricated patterns is potentially amenable to the size of a single cell, thus allowing a new range of studies, for example, the physical rules of morphogenesis,25 cell polarity,26 and cell division axis.27 Acknowledgment. The Centre National de la Recherche Scientifique (CNRS) and the Nord- Pas de Calais region are gratefully acknowledged for financial support. EG thanks the CNRS for providing a postdoctoral scholarship funding. We thank Damien Schapman and the Biophotonic Core Facility of Lille (USR 3078/IRI). Supporting Information Available: XPS spectra of amineand trifluoromethyl-terminated surfaces, time-lapse cell imaging, incubation of various densities of L929 cells on patterned BDD surfaces. This material is available free of charge via the Internet at http://pubs.acs.org. (25) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–411. (26) Thery, M.; Racine, V.; Piel, M.; Pepin, A.; Dimitrov, A.; Chen, Y.; Sibarita, J.-B.; Bornens, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19771–19776. (27) Thery, M.; Racine, V.; Pepin, A.; Piel, M.; Chen, Y.; Sibarita, J.-B.; Bornens, M. Nat. Cell Biol. 2005, 7, 947–953. (28) Kulkarni, S. A.; Ogale, S. B.; Vijayamohanan, K. P. J. Colloid Interface Sci. 2008, 318, 372–379. (29) Janssen, D.; De Palma, R.; Verlaak, S.; Heremans, P.; Dehaen, W. Thin Solid Films 2006, 515, 1433–1438. (30) Gorham, J. M.; Stover, A. K.; Fairbrother, D. H. J. Phys. Chem. C 2007, 111, 18663–18671. (31) Park, K. J.; Doub, J. M.; Gougousi, T.; Parsons, G. N. Appl. Phys. Lett. 2005, 86, 051903.
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