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Fabrication of Thermosensitive Polymer Nanopatterns through Chemical Lithography and Atom Transfer Radical Polymerization Qiang He,† Alexander Ku¨ller,‡ Michael Grunze,‡ and Junbai Li*,† Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China, Angewandte Physikalische Chemie, UniVersita¨t Heidelberg, Im Neuenheimer Feld 253, D-69120 Heidelberg (Germany), and Institute for Molecular Biophysics, UniVersity of Maine, Orono, Maine 04469 ReceiVed September 24, 2006. In Final Form: NoVember 20, 2006 Micro- and nanopatterns of thermosensitive poly(N-isopropylacrylamide) brush on gold substrate were prepared by using chemical lithography combined with surface-initiated atom transfer radical polymerization. Self-assembled monolayers of 4′-nitro-1, 1′-biphenyl-4-thiol were structured by chemical lithography which produced cross-linked 4′-amino-1,1′-biphenyl-4-thiol monolayer within a nitro-terminated matrix. The terminal amino groups in monolayers were bounded with the surface initiator bromoisobutyryl bromide. After polymerization, the smallest size can reach to 70-nm line width and dots. The thermosensitivity of poly(N-isopropylacrylamide) brushes is demonstrated by contact angle measurement and fluid atomic force microscopy. This fabrication approach allows creating spatially defined polymer patterns and provides a simple and versatile method to construct complex micro- and nanopatterned polymer brushes with spatial and topographic control in a single step.
Introduction There is an increasing interest of preparing polymer brush patterns on solid substrates, especially in a nanoscale, to tailor their surface properties such as adsorption behavior, wettability, friction, and thermal stability.1,2 The generation of complex micropatterns in surface-grafted polymer films is typically achieved by photolithographic techniques.3,4 While successful, the usage of these patterned polymer films is restricted by their limited stability, thickness, and grafting density and the difficulties in their preparation over large areas and complicated topographies. Recently, surface-initiated atom transfer radical polymerization (SI-ATRP)5-9 has been shown to be an effective method for the controlled growth of patterned polymer brushes.10-12 Furthermore, ATRP in water media at room temperature will be beneficial to prepare grafted polymer brushes on thiol self-assembled monolayer (SAM). The resulting polymer structures should exhibit a good contrast between the functionalized and unfunctionalized regions in term of their chemical and physical properties. However, because of the intrinsic limitations of the presently * To whom correspondence should be addressed. Tel: +86-10-82614087. Fax: +86-10-82612629. E-mail:
[email protected]. † Chinese Academy of Sciences. ‡ Universita ¨ t Heidelberg and University of Maine Orono. (1) Ikkala, I.; Brinke, G. Science 2002, 295, 2407. (2) Husemann, M.; Mecerreyes, D.; Hawker, C. J.; Hedrick, J. L.; Shah, R.; Abott, N. L. Angew. Chem., Int. Ed. 1999, 38, 647. (3) Amiel, C.; Sikka, M.; Schneider, J. W.; Tsao, J. H.; Tirrell, M.; Mays, J. W. Macromolecules 1995, 28, 3125. (4) Husemann, M.; Morrison, M.; Benoit, D.; Frommer, J.; Mate, C. M.; Hinsberg, W. D.; Hedrick, J. L.; Hawker, C. J. J. Am. Chem. Soc. 2000, 122, 1844. (5) Patten, T. E.; Xia, J.; Abernathy, T.; Matyjaszewski, K. Science 1996, 272, 866. (6) Jordan, R.; Ulman, A. J. Am. Chem. Soc. 1998, 120, 243. (7) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677. (8) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550. (9) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (10) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. ReV. 2004, 33, 14-22. (11) Advincula, R. C.; Brittain, W. J.; Caster, K. C.; Ru¨he, J. Polymer Brushes: Synthesis, Characterization, Applications; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2004. (12) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551.
available fabrication techniques, a simple and rapid technique to obtain nanosized patterns is still an important objective in nanotechnology. Recently, chemical lithography has been developed by utilizing specific chemical reactions between 4′-nitro-1,1′-biphenyl-4thiol (NBT) SAM and electron beams.13-15 Electron irradiation of NBT SAM causes reduction of the terminal nitro groups into amino groups, while the underlying aromatic biphenyl monolayer is dehydrogenated and cross-linked. The amino moiety in the monolayer can subsequently be used for selective binding of functional entities (chemical lithography).16,17 The combination of chemical lithography and consecutive SI-ATRP is anticipated to allow a superior control of pattern formation and amplification of the patterns with a wide choice of monomers and chemical functionalities. Poly(N-isopropylacrylamide) (PNIPAM) is a well-known thermal-sensitive polymer which possesses a lower critical solution temperature (LCST) of around 32 °C in pure water.18-20 Recently, these environmentally sensitive patterned PNIPAM films have attracted considerable attention as they can be exploited in fluidic devices, drug delivery systems, in protein affinity separation, and as cell culture substrates.21-28 Herein, we describe (13) Eck, W.; Stadler, V.; Geyer, W.; Zharnikov, M.; Go¨lzha¨user, A.; Grunze, M. AdV. Mater. 2000, 12, 805. (14) Ku¨ller, A.; Eck, W.; Stadler, V.; Geyer, W.; Go¨lzha¨user, A. Appl. Phys. Lett. 2003, 82, 3776. (15) Zharnikov, M.; Grunze, M. J. Vac. Sci. Technol., B 2002, 20, 1793. (16) Schmelmer, U.; Jordan, R.; Geyer, W.; Go¨lzha¨user, A.; Grunze, M. Angew. Chem., Int. Ed. 2003, 42, 559. (17) Go¨lzha¨user, A.; Eck, W.; Geyer, W.; Stadler, V.; Weimann, T.; Grunze, M. AdV. Mater. 2001, 13, 806. (18) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (19) Wischerhoff, E.; Zacher, T.; Laschewsky, A.; Rekai, E. Angew. Chem., Int. Ed. 2000, 39, 4602. (20) Pelton, R. AdV. Colloid Interface Sci. 2000, 85, 1. (21) Jones, D. M.; Smith, J. R.; Huck, W. T. S.; Alexander, C. AdV. Mater. 2002, 14, 1130. (22) Huber, L.; Manginell, R. P.; Samara, M. A.; Kim, B.; Bunker, B. C. Science 2003, 301, 352. (23) Kaholek, M.; Lee, W. K.; LaMattina, B.; Caster, K. C.; Zauscher, S. Nano Lett. 2004, 4, 373. (24) Tu, H. L.; Heitzman, C. E.; Braun, P. V. Langmuir 2004, 20, 8313. (25) Kaholek, M.; Lee, W. K.; Ahn, S. J.; Ma, H. W.; Caster, K. C.; LaMattina, B.; Zauscher, S. Chem. Mater. 2004, 16, 3688.
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Scheme 1. Representation of the Preparation Process of PNIPAM Brushes by Chemical Lithography and SI-ATRP on Au(111)a
a (a) NBT SAM was irradiated by electron-flood gun through a suitable mask or was written with a LEO 150 SEM with a Raith pattern generator, which results in intralayer cross-linking and conversion of the terminal nitro groups into amino groups. (b) The binding of BIBB into the amino sites gives a SAM that bears surface initiator. (c) Exposure to an N-isopropylacrylamide (NIPAM) and the radical polymerization results in a polymer brush layer at the irradiated regions.
how to utilize the chemical lithography and SI-ATRP to obtain functionalized nanopatterns where one or more of the patterned components are responsive to external stimuli. Experimental Section Materials. 4′-Nitro-1,1′-biphenyl-4-thiol (NBT) was synthesized according to the previously reported method.13 One hundred nanometer gold films were prepared by thermal evaporation on 5-nm titanium films on silicon wafers. SAMs of NBT were produced by immersion of the gold substrates in a degassed solution of 15 mmol NBT in N,N-dimethylformamide (DMF) for 72 h under nitrogen, followed by rinsing with DMF and ethanol and drying in a nitrogen stream. N-Isopropylacrylamide (NIPAM), N,N,N′,N′′,N′′-pentamethyldiethylenetrimine (PMDETA), bromoisobutyryl bromide (BIBB), copper(I) bromide (CuBr), and methanol were purchased from SigmaAldrich. NIPAM was recrystallized in n-hexane to remove the inhibitors. CuBr was purified by stirring in glacial acetic acid for 5 h, was washed with absolute ethanol and anhydrous diethyl ether, was dried under vacuum for 12 h at room temperature, and was stored under dry nitrogen. Dichloromethane (CH2Cl2) and triethylamine (Et3N) were distilled from calcium hydride prior to use. 1-Mercaptoundecyl-11-polyethylenglykol (PEG 2000, Mw ) ca. 2000) was synthesized following the previously described procedures.29 Water was purified with a Millipore desktop system and reached a specific resistance above 18.2 MΩ/cm. All other chemicals were received from Sigma-Aldrich and were used without further purification. (26) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427. (27) Ahn, S. J.; Kaholek, M.; Lee, W. K.; LaMattina, B.; LaBean, T. H.; Zauscher, S. AdV. Mater. 2004, 16, 2141. (28) Sun, T. L.; Wang, G. J.; Feng, L.; Liu, B. Q.; Ma, Y. M.; Jiang, L.; Zhu, D. B. Angew. Chem., Int. Ed. 2004, 43, 357. (29) Tokumitsu, S.; Liebich, A.; Herrwerth, S.; Eck, W.; Himmelhaus, M.; Grunze, M. Langmuir 2002, 18, 8862.
Figure 1. (a) Reflected FTIR spectra of PNIPAM-grafted film. (b) XPS C1s spectra for cABT SAM, BIBB-attached monolayer, and PNIPAM-grafted film on flat surface.
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Figure 2. (a) and (b) SEM micrographs of PNIPAM brushes generated by chemical lithography and subsequent SI-ATRP on a substrate that was irradiated by a flood gun through a mask with a 55-µm periodicity (35-µm diameter, 20-µm distance). (c) Tapping-mode AFM image of PNIPAM brushes with a 2.5-µm periodicity (1.8-µm diameter and 0.7-µm distance) in air and corresponding height profile. Polymerization time ) 60 min. Chemical Lithography. Chemical lithography was performed by the following two methods: (1) a flood gun (50 eV, 40 mC/cm2) was used to expose the sample surface through a stencil mask with different sizes and shapes placed on the NBT SAM covered substrate. (2) To extend chemical patterning to higher electron energies and avoid the limitation of masks to obtain smaller structures, a LEO 1530 scanning electron microscope (SEM) with a Raith Elphy Plus Pattern Generator System (REPGS) software was used to directly write amino-terminated lines and dots with a nominal width from 1000 to 50 nm into NBT SAMs. The e-beam energy was chosen at 3 keV, vacuum pressure ∼ 5 × 10-6 mbar. Surface Initiator Monolayers. The irradiated substrates bearing SAMs of cross-linked 4′-amino-1,1′-biphenyl-4-thiol (cABT) were placed in dried CH2Cl2 that contained triethyamine (2%, w/v). Then, the surface initiator bromoisobutyryl bromide (BIBB) was added dropwise at 0 °C. The mixture was left for 1 h at this temperature and then at room temperature for 12 h. Finally, the substrates were cleaned with acetone and toluene and were dried under a nitrogen flow. Fabrication of PNIPAM Brushes. Preparation of PNIPAM brushes was achieved by immersing the substrates with the initiator grafted onto the patterned surface under N2 atmosphere into a degassed solution of N-isopropylacrylamide (NIPAM) (25% w/v), CuBr (0.35 mmol), and N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA, 1.05 mmol) in a 1:1 (v/v) mixture of water and methanol (4 mL). The polymerization was allowed to proceed for 60 or 180 min at room temperature, after which the substrates were removed from the reaction flask and were quickly rinsed with water and methanol and were dried under N2. Cell Culture. The patterned PNIPAM samples were first placed into a Petri dish with an iodine atmosphere about 5 min. Thus, the un-cross-linked NBT was removed and the sample was rinsed with dimethylformamid (DMF) several times. Next, the sample was immersed into an ∼1 mM solution of PEG 2000 in DMF for 24 h and subsequently was rinsed several times with DMF and was blown in a nitrogen stream. REF52-fibroblasts were employed for the cell culture experiments. The patterned PNIPAM brushes surrounded with PEG 2000 were sterilized by treatment with 70% (v/v) ethanol and were washed two times with phosphate-buffered saline (PBS) at room temperature. The fibroblasts were cultured in DMEM media supplemented with 5% fetal bovine serum and 1% L-glutamine. Cells were seeded at a density of ∼104 cells/cm2 in wells containing the PNIPAM samples. They were kept in a thermostatic device
(37 °C in 5% CO2 atmosphere) for 18 h. Finally, cells were washed two times with 37 °C PBS, were fixed with 3.7% paraformaldehyde in PBS for 25 min, and were then observed by an SEM. Characterization. Fourier transform infrared spectra were recorded on a JASCO FT/IR-660 plus spectrophotometer in reflection mode. X-ray photoelectron spectroscopy (XPS) data were recorded with an ESCA Lab 220 I-XL spectrometer, using an Al KR source operated at 207 W. Advancing contact angles were measured with an OCA 20 machine (data Physics, Germany) at saturated humidity, and the temperature was controlled by a superthermostat (Julabo F 25, Germany). Atomic force microscopy (AFM) images were obtained in the tapping mode equipped with a liquid cell and a high-temperature facility (Nanoscope IIIa Digital Instruments, Santa Barbara). The brush widths were obtained by measuring their full width at half-maximum (fwhm). Scanning electron micrographs were obtained with a LEO 1530 scanning electron microscope with primary electron energy of 3 keV and an in-lens detector for secondaryelectron detections.
Results and Discussion Polymer brush patterns are fabricated on the basis of the procedures described in Scheme 1. To realize the SI-ATRP reaction on the SAM surface, we need to introduce initiators on the SAM. At first, a SAM of NBT was converted into crosslinked 4′-amino-1,1′-biphenyl-4-thiol (cABT) monolayer by irradiation with electron beams (50 eV, 40 mC/cm2) so that most of the nitro groups were reduced to the amino groups according to the previous XPS results.13 Then, the surface initiator, bromoisobutyryl bromide (BIBB), was added to create a surfacebound initiator monolayer. The substrates containing surface initiator monolayer were exposed to a degassed mixture of water and methanol (1:1, v/v) containing N-isopropylacrylamide (NIPAM) and catalyst for 60 min. After the SI-ATRP reaction, we carried out the measurements of the reflected Fourier transform infrared (FTIR) spectra with the obtained samples. The specific absorption peak at 3291 cm-1 demonstrates the stretch vibration of the secondary amide-NH group. The asymmetric stretching vibration of -CH3 group occurs at 2975 cm-1, the secondary amide CdO stretching gives a rise to a strong band at 1645 cm-1, and the asymmetric bending deformation of -CH3 occurs
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Figure 3. (a) AFM height images of different line-width PNIPAM patterns obtained at room temperature in air. (b) and (c) are the 3D magnification images and corresponding height profiles, respectively. Polymerization time ) 60 min.
at 1462 cm-1. The two bands at 1369 and 1389 cm-1 of almost equal intensity are assigned to the two -CH3 groups in the isopropyl functionality. It is deduced that a covalent bond from the PNIPAM film is formed (Figure 1a). Furthermore, XPS spectra obtained by the original cABT, BIBB-bound, and polymer-grafted surfaces show that the C1s peak intensity of BIBB-bound surface at 284.6 eV is obviously larger than that of the original cABT SAM surface (Figure 1b). The polymerization of NIPAM monomer leads to the further increase of the intensity of C1s peak at 284.6 eV and meanwhile produces a new C1s peak at 287.2 eV, which is ascribed to the existence of -CON- group. Overall, analysis of the substrate with reflected FTIR and XPS confirms the formation of the surface initiator monolayer as well as the existence of PNIPAM brush. Figure 2a shows a typical SEM micrograph of a structured SAM onto which a PNIPAM brush has been grown by chemical lithography and subsequent SI-ATRP. The difference in the secondary electron between the original SAM/gold substrate and PNIPAM brush creates the contrast of the black and white. The dark dots represent areas where the nitro groups in the NBT SAM were converted into amino groups by electron irradiation, through a stencil mask, followed by PNIPAM brush formation by SI-ATRP. The surface covered by PNIPAM brush is rougher
than that uncovered as shown in Figure 2b. Obviously, chemical lithography is not limited to a particular sample size since the use of electron-flood guns with a suitable mask can produce large area patterns. AFM image in Figure 2c reveals that the PNIPAM brush dots have the form of “chairs”. The inner and the circumference height of the polymer films are about 59 and 68 nm, respectively. The variation in height is attributed to the higher density of surface initiators near the outer rim created by an effectively higher electron dose because of scattering of electrons by the holes in the mask. It results in the reduction of more nitro groups and hence the higher surface initiator concentration at the perimeter. Brush height is a function of initiator density, where low initiator densities produce low brush heights. This was recently shown by Jones et al.21 The AFM images in Figure 3a shows PNIPAM brush patterns with different line widths from micro- to nanometer size. The left figures (1000, 500, 250, 100, and 50 nm) stand for the nominal projected line widths. AFM measurements of PNIPAM brushes show that the matching lateral features (full width at halfmaximum or fwhm) are approximately 1003, 506, 258, 119, and 70 nm, respectively. Compared with the grafting region, the widths of polymer brushes measured by AFM have an obviously
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Figure 4. (a) The curve of contact angle of PNIPAM film on flat substrate as a function of temperature. (b) and (c) AFM images of 70-nm line-width PNIPAM patterns obtained in water and the corresponding height profile at room temperature and 40 °C, respectively. Polymerization time ) 60 min.
expansive behavior, but the polymerization still exhibits linear growth. Figure 3b and c shows the 3D magnification images and corresponding height profiles, respectively. The corresponding height profiles illustrate that the heights of 1003, 506, 119, and 70 nm line-width polymer brushes are 89, 86, 81, and 76 nm, respectively. Although the polymerization conditions and the used electron doses are identical, the brush height is higher than those of the PNIPAM brushes of micrometer size shown above (Figure 2) obtained with the flood gun at lower energies. The differences in brush height may be due to an energy-dependent cross section for reduction of the nitro groups or to the higher yield of secondary electrons. In contrast to the polymer brushes in Figure 2, also the root-mean-square roughness value of the nanostructures in Figure 3 decreases approximately by 1.7 nm from 2.7 to 1.0 nm. To test the external responsivity of the PNIPAM patterns, thermosensitivity of the patterns has been detected. First, we measured the change of contact angle of PNIPAM film on flat substrate with the temperature (Figure 4a). It indicates that the PNIPAM swells in water to create a hydrophilic surface with a contact angle of 62.5 ( 2.4 ° at room temperature (below the LCST). Above the LCST at 40 °C, the measured contact angle is rapidly rising and reaches to ca. 90 °C, indicating that water in the PNIPAM film was expelled and that the polymer collapsed, which leads to the more hydrophobic PNIPAM-grafted surface. Furthermore, AFM equipped with a fluid cell was used to characterize the thermosensitive property of patterned PNIPAM brush (Figure 4b and c). The AFM images show that the height of 70-nm line-width PNIPAM pattern is significantly influenced by the water temperature. When exposed to water at room
temperature (below the LCST), the height of PNIPAM brush pattern is noticeably increased up to 140 nm (Figure 4b). Above the LCST, at 40 °C, the PNIPAM height reduces to ca. 60 nm (Figure 4c) because of the water expelled. It indicates that the obtained NIPAM nanopatterns have the obviously thermosensitive feature. The reversibility of such thermal-responsive nanopatterns is consistent with those obtained by other techniques.23-28 Furthermore, we find that the height of PNIPAM brush can reach to around 370 nm when the polymerization time extends to 180 min (Figure 5a). It indicates that the height of PNIPAM brushes prepared by ATRP can be definitely controlled by varying the polymerization time as reported by others.5,7,25 Such patterns are detected by AFM in water at 20 °C (below the LCST). It shows in Figure 5b that the height of the patterns is dramatically increased to about 890 nm, indicating the expanding of the polymer brush. Figure 6 shows the representative SEM micrographs of fibroblast adhered to the different-shaped PNIPAM patterns but inhibited by the surrounding PEG 2000. Here, the dark regions stand for PNIPAM brushes and the surrounding bright regions mean the backfill of PEG 2000. Obviously, PEG 2000 layer is very effective in preventing cells from attaching and spreading owing to increased hydrophilicity (Figure 6a). Furthermore, the high-surface mobility and steric stabilization effects of the PEG chains in water also lower the extent of cell attachment and growth.30,31 Figure 6b shows the single cell adhered on the multiple (30) Gotoh, Y.; Tsukada, M.; Minoura, N.; Imai, Y. Biomaterials 1997, 18, 267. (31) Wang, P.; Tan, K. L.; Kang, E. T.; Neoh, K. G. J. Mater. Chem. 2001, 11, 2951.
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Figure 5. AFM images of PNIPAM brushes with a polymerization time of 180 min in air (a) and in water (b) corresponding to their height profiles, respectively. Pattern periodicity: 2.5 µm.
Figure 6. SEM images of fibroblasts spread on multiple PNIPAM circle patterns surrounded with PEG 2000; (a) circle patterns (diameter: 15 µm; space: 30 µm) and (c) mixed patterns; (b) and (d) are magnification image of a and c, respectively.
PNIPAM circle patterns. In other words, cell body can spread across the interspacing nonadhesive areas (i.e., PEG 2000 layer) of the substrate and elongate from one small adhesive dot to another. These results prove that cells can adhere, spread, and proliferate on the patterned PNIPAM film at 37 °C (above (32) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomateriols 1995, 16, 297.
LCST).32-35 Furthermore, the SEM image in Figure 6c displays that fibroblasts spread on the mixed PNIPAM patterns and (33) Ista, L. K.; Perez-luna, V. H.; Lopez, G. P. Appl. EnViron. Microbiol. 1999, 65, 1603. (34) Hirose, M.; Kwon, O. H.; Yamato, M.; Kikuchi, A.; Okano, T. Biomacromalecules 2000, 1, 377. (35) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425.
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flattened against the surface were inhibited by the surrounding PEG 2000. The mixed polymer patterns were made up of different diameter circular patterns. Similarly, the enlarged image (Figure 6d) further demonstrates that single cell can adhere to the multiple, closely spaced PNIPAM dot patterns but cannot spread across two dot stripes when the interspaces between two dot stripes (i.e., nonadhesive areas or PEG 2000 layer) are too large. With spatial control over where a cell can adhere and where it cannot, we can separate changes in a cell’s fate because of the changes of the adhered cell shapes.35,36 The physically extended degree of a cell can conveniently determine the proliferation or death of a cell.
Conclusions We have demonstrated that the combination of chemical lithography and SI-ATRP can be applied to prepare micro- to (36) Sniadecki, N. J.; Desai, R. A.; Ruiz, S. A.; Chen, C. S. Ann. Biomed. Eng. 2006, 34, 59.
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nanopatterns of stimulus-responsive (“smart”) polymer brush on gold substrate. The smallest size can reach to 70-nm line width and dots. We also demonstrate the reversibility of thermal responsiveness of the nanopatterns and the ability to selectively attach cells to patterned PNIPAM brushes. At 37 °C (above LCST), cells can adhere, spread, and proliferate on patterned PNIPAM brushes. This fabrication approach allows creating spatially defined polymer patterns and provides a simple and versatile method to construct complex micro- and nanopatterned polymer brushes with spatial and topographic control in a single step. Acknowledgment. This work was financially supported by the National Nature Science Foundation of China (No. 20471063, 20403022, 90206035) and the European Union through the IP “Ambio” and Germany-China DAAD (PPP) collaboration grant. A. Ku¨ller thanks B. Holzapfel for help with sample preparation. LA062793U
