Patterning of Magnetic Nanobeads on Surfaces by Poly

Jan 12, 2008 - Poly(dimethylsiloxane) (PDMS) stamps are widely used in soft lithographic methods. They are powerful tools for obtaining structures of ...
1 downloads 0 Views 952KB Size
Download PDF
888

Langmuir 2008, 24, 888-893

Patterning of Magnetic Nanobeads on Surfaces by Poly(dimethylsiloxane) Stamps Juliane Issle,*,† Mateu Pla-Roca,‡ Elena Martı´nez,‡ and Uwe Hartmann† UniVersity of Saarland, Institute of Experimental Physics, P.O. Box 151 150, 66041 Saarbruecken, Germany, and Nanobioengineering Laboratory, Institut de Bioenginyeria de Catalunya, Barcelona Science Park, C/Josep Samitier 1-5, 08028 Barcelona, Spain ReceiVed June 26, 2007. In Final Form: August 21, 2007 Poly(dimethylsiloxane) (PDMS) stamps are widely used in soft lithographic methods. They are powerful tools for obtaining structures of soft material in the micrometer to nanometer range by printing techniques. In this contribution, a new application of h-PDMS stamps for nanobead deposition is introduced. Magnetite-polysaccharide particles of an average diameter of 150 nm are used. They can be biologically functionalized by attaching various molecular groups. Deposition of these particles on a carrier substrate results in well-reproducible structures. This is achieved by means of PDMS stamps with different patterns using a microfluidic approach on one hand and a printing approach on the other hand. Furthermore, magnetic substrates with particular domain structures have been used. The beads can then be arranged in rather complicated but well-defined geometrical structures along the domain walls. The magnetic interaction considerably increases the adhesion of the beads to the carrier substrate. All involved materials are biocompatible. Thus the setup can be used in cell culture experiments in order to investigate influences of different particle-bound proteins and particle patterns on cell growth and vitality.

Introduction From basic as well as applied reasons, there is a strong interest in cell-surface interactions. In this context, new methods for structuring biocompatible substrates chemically and topographically are of interest. In cell cultures, various substrates are used, whereas the most common ones are glass and polystyrol (culture flasks). Various experiments have shown that cells can be affected by lithographically produced nano- and microstructures.1-9 A new approach based on utilizing magnetic thin layers with particular magnetic structures is presented in the following. However, most ferromagnetic materials are toxic to cells because they contain heavy metals such as Ni, Co, or Fe, which are released into the culturing medium.10 We determined by various experiments that yttrium iron garnet (YIG) layers are biocompatible and stable under culturing conditions. They show particular domain structures,11-14 which can be easily modified by external * Corresponding author. Tel.: +49 681 302 2957; fax: +49 681 302 3790; e-mail: [email protected]. † University of Saarland. ‡ Institut de Bioenginyeria de Catalunya. (1) Kane, S. K.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (2) Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. J. Cell Sci. 2004, 117, 41-52. (3) Rozkiewicz, D.; Kraan, Y.; Werten, M. W. T.; de Wolf, F. A.; Subramaniam, V.; Ravoo, B. J.; Reinhoudt, D. N. Chem.sEur. J. 2006, 12, 6290-6297. (4) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (5) Curtis, A.; Riehle, M. Phys. Med. Biol. 2001, 46, 47-65. (6) Quist, A. P.; Pavlovic, E.; Oscarsson, S. Anal. Bioanal. Chem. 2005, 381, 591-600. (7) The´ry, M.; Racine, V.; Pe´pin, A.; Piel, M.; Chen, Y.; Sibarita, J. B.; Bornens, M. Nat. Cell Biol. 2005, 7, 947-953. (8) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573-1583. (9) Clark, P.; Connolly, P.; Curtis, A. S. G.; Dow, J. A. T.; Wilkinson, W. J. Cell Sci. 1991, 99, 73-77. (10) Grimsdottir, M. R.; Hensten-Pettersen, A.; Kullmann, A. Eur. J. Orthod. 1992, 14, 47-53. (11) Hubert, A.; Schaefer, R. Magnetic Domains; Springer: Berlin, 1998. (12) Kooy, C.; Enz, U. Philips Res. Rep. 1960, 15, 7-29. (13) Molho, P.; Porteseil, J. L.; Souche, Y.; Gouzerh, J.; Levy, J. C. S. J. Appl. Phys. 1987, 61, 4188-4193. (14) Hubert, A.; Malzemoff, A. P.; DeLuca, J. C. J. Appl. Phys. 1974, 45, 3562-3571.

magnetic fields. This allows the creation of various and rather complicated magnetic domain patterns. A widely used method for producing micro- and nanoscale patterns on substrates is soft lithography.15 Biochemical structuring can be achieved by patterning of proteins or other relevant biomolecules on carrier substrates by microcontact printing methods.3,6,7,16,17 These are based on establishing local contacts between structured poly(dimethylsiloxane) (PDMS) stamps and substrate surface. In microcontact printing, these contacts18 are used to transfer molecules (inks), such as thiols, silanes, or other reactive groups.1 These are then locally used to deposit proteins onto the surfaces. Functionalized areas allow adherence, while unfunctionalized areas are uncovered. Delamarche et al.16 suggested a direct patterned deposition of proteins by adsorbing them to the PDMS and then transferring them to the surfaces directly. In the present work, the proteins are chemically attached to magnetite-polysaccharide carrier particles. The particles consist of 12 nm magnetite grains embedded in a polysaccharide matrix and are of a diameter of 250 nm at maximum. They can be immobilized magnetically through their interaction with the garnet films. Different chemical surface groups are available, which allow covalent binding of various proteins without sophisticated chemical steps. It is in particular not necessary to develop chemical binding strategies for each protein-substrate combination.19 Another advantage is that it is possible to change the protein concentration and spatial distribution of the proteins by influencing the bead adsorption by externally applied magnetic fields.19 The magnetic interaction between beads and garnet film is much stronger than adhesion to nonmagnetic substrates. Therefore, the beads remain in predesigned patterns during subsequent treatment and cell cultivation. A maximum flexibility in bead (15) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550-575. (16) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. AdV. Mater. 2000, 12, 1067-1070. (17) Hoff, J. D.; Cheng, L. J.; Meyhoefer, E.; Guo, L. J.; Hunt, A. Nano Lett. 2004, 4, 853-857. (18) Bietsch, A.; Michel, B. J. Appl. Phys. 2000, 88, 4310-4318. (19) Issle, J.; Hartmann, U., patent pending.

10.1021/la7018956 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/12/2008

Patterning Nanobeads on Surfaces by PDMS Stamps

Langmuir, Vol. 24, No. 3, 2008 889

Figure 1. Faraday microscopic image of the yttrium iron garnet domain configurations of a (a) bubble lattice, (b) stripe domain pattern, (c) maze structure, and (d) mixed state.

Figure 3. Schematic of the microfluidic method: (a) magnetic layer is brought into close contact with the PDMS stamp. (b) Bead suspension flows under the magnetic substrate by means of capillary forces. (c) Removal of excess water and drying process. (d) Beads remain in areas of negative stamp structure. Figure 2. TEM image of a magnetic bead composed of several single superparamagnetic magnetite crystals. The polysaccharide matrix is not visible here.

patterning is obtained if magnetically induced deposition is combined with microcontact printing techniques. Materials and Methods For cell cultivation, garnet films of the composition Y2.5Bi0.5Fe5-δGaδO12 (δ ) 0.5-1)20 have been found to be very suitable. The ferrimagnetic film is grown on a paramagnetic (111) gallium gadolinium garnet (GGG) substrate. The saturation magnetization determined by vibrating sample magnetometry was 15 kA/m. The films show metastable magnetic domain configurations,11-14,20,21 where each one represents a local minimum of magnetic energy. There are several possibilities to change from one structure to another.12-14 Figure 1 shows these magnetic configurations, obtained by Faraday microscopy. The latter utilizes the rotation of the polarization axis of linearly polarized light propagating through magnetized matter. The surface of the ferromagnetic layer is atomically flat, which is useful for bead deposition. The contact angle of the ferromagnetic garnet film is 52° for water. An angle of 62° is obtained for the paramagnetic substrate. For reference experiments, the substrate is used also in cell culturing experiments. Magnetic nanobeads composed of magnetite grains and a polysaccharide matrix (fluidMAG-ARA, Chemicell) have been used as mentioned before. The grains of approximately 12 nm diameter are shown in a transmission electron microscopy (TEM) image in Figure 2. The grains’ diameter is well below the critical diameter for single domain behavior, and room temperature is well above the blocking temperature.22 Thus, the polysaccharide-magnetite complex behaves largely superparamagnetically, as proven by superconducting (20) Helseth, L. E.; Backus, T.; Johansen, T. H.; Fischer, T. M. Langmuir 2005, 21, 7518-7523. (21) Issle, J.; Hartmann, U. J. Phys.: Conf. Ser. 2007, 61, 487-491. (22) Bean, C. P.; Livingston, J. D. J. Appl. Phys. 1959, 30, 120-129.

quantum interference device (SQUID) measurements. The average diameter of the composite particles is 150 nm. The volume fraction of magnetite is 80%. Terminal surface groups allow for covalent binding of different biomolecules (proteins, growth factors, etc.) to the magnetic beads. Patterning of the substrates with the beads was achieved by soft stamps as known from microcontact printing. h-PDMS and PDMS (Sylgard 184, Dow Corning) stamps were replicated from siliconbased molds, as previously described.23,24 Two bead transferring methods were used to generate patterns on the garnet films in a 1 cm2 area. In the microfluidic approach, the PDMS stamp was first treated in an O2 plasma to hydrophilize it. Subsequently, the substrate was put onto the stamp. An optical microscope was used to check the contact and the stability of the PDMS structures above the garnet films. After approximately 1 min, the h-PDMS stamp was in strong contact with the flat surface18 of the substrate as shown in Figure 3a. A droplet of bead suspension was delivered to the edge between carrier substrate and stamp. Because of capillary forces, the bead solution was sucked into the channels between stamp and substrate as shown in Figure 3b. After 30 s, the excess water was removed with a pipet as shown in Figure 3c. A 1 h drying and sterilization process in UV light at 60 °C followed. The PDMS stamp was removed carefully, while the beads remained on the substrate in their original pattern as shown in Figure 3d. For two-dimensional structures such as discs, a soft molding process is more favorable.25 A droplet of bead suspension was put onto the flat substrate as shown in Figure 4a. Immediately afterward, the stamp was brought into close contact with the surface by slightly being pressed, as shown in Figure 4b. The bead suspension was fully displaced underneath the exposed parts of the stamp. The remaining suspension in between the dry locations was dried at 60 °C for 1 h to prevent smearing of the pattern. The substrate was removed carefully by bending the PDMS stamp. (23) Odom, T. W.; Love, J. C.; Wolfe, D. B.; Paul, K. E.; Whitesides, G. M. Langmuir 2002, 18, 5314-5320. (24) Kang, H.; Lee, J.; Park, J.; Lee, H. H. Nanotechnology 2006, 17, 197200. (25) Kim, Y. S.; Suh, K. Y.; Lee, H. H. Appl. Phys. Lett. 2001, 79, 2285-2287.

890 Langmuir, Vol. 24, No. 3, 2008

Issle et al.

Figure 5. Schematic drawing of the maze domain structure. Magnetic nanobeads can be deposited onto domain walls between two antiparallel domains.

Figure 4. Schematic of the soft molding method: (a) a droplet of bead suspension is placed onto the magnetic layer. (b) PDMS stamp and magnetic film are brought into close contact. The positive structure of the stamp displaces the bead suspension. (c) After drying, the beads remain in areas of the negative structure. The medium for cell culture was composed of DMEM High Glucose, 10% FBS, 1% penicillin/streptomycin, and 100 mg/L sodium pyruvate (all PAA). MG 63 (ATCC CRL-1427) osteoblasts were used.

Results and Discussion Magnetic Structure. Figure 1a is an example for the socalled bubble configuration of the magnetic garnet film, Figure 1b shows a stripe domain pattern, Figure 1c shows a maze structure, and finally, Figure 1d shows a mixed state of bubbles and mazes. The configurations presented here result from different preparations under different magnetic environments: first, the samples were heated above their Curie temperature and then cooled down in either zero or inhomogeneous magnetic field.12,13 Each domain configuration is stable at room temperature and in nonmagnetic environments. The maze domain structure, schematically shown in Figure 5, is the most stable configuration being suitable for the experiments described in the following discussion. Each domain is magnetized perpendicular to the surface, with alternating magnetization from domain to domain. The shape of the domains varies in external magnetic fields.11,12,14,21 The average width of each domain in remanence is 2-5 µm at a film thickness range of 4-6 µm. Between two adjacent antiparallel domains, the magnetization rotates continuously, forming a domain wall of Bloch-type.11,26 Above the walls, the near-surface stray field has its maximum gradient. As the garnet layer is in its most stable state with maze domains, the experiments have been performed preferably at such a domain configuration as shown in Figures 1c and 6a,b. The garnet could be used without further treatment. It is, however, also possible (26) Kittel, C. ReV. Mod. Phys. 1949, 21, 541-583.

to achieve the patterning on other domain configurations. In Figure 6c, a domain configuration of bubbles has been used. Therefore, the garnet layer was first heated above the Curie temperature of approximately 425 K. During cooling down, a magnetic field nearly parallel to the surface (within 1-2°) was applied. The magnetic particles can be adsorbed to the magnetic garnet film via magnetostatic interaction. The locally acting force caused by the inhomogeneous surface stray field B, which is acting on the induced dipole moment m of a particle is given by F ) -grad(B‚m). The beads thus gather at the domain walls where the gradient is highest, as schematically shown in Figure 5.11,20 The beads can follow slow wall motions caused by changes of the domain configuration by application of external magnetic fields.20 Patterning Methods. The employed stamps constitute different profiles, for example, line profiles (20 µm width, 1.2 µm height, 40 µm distance from center to center), discs (20 µm diameter, 0.8 µm height, 20 µm distance), or octagons (50 µm width, 0.8 µm height, 54 µm distance from center to center). All stamps had an additional h-PDMS layer to prevent roof collapse. It is possible to obtain very accurate stripes with and without beads by using the microfluidic method as shown in Figure 6. The beads are deposited in areas of 20 µm width, which exactly corresponds to the microfluidic channels during the deposition process. The stamp stays in close contact with the substrate until the water is completely evaporated. The substructure, which is clearly visible, results from the domain structure of the garnet layer as shown in Figures 6 and 7. The domains exhibit the maze pattern (Figures 1c and 5). The AFM image obtained in contact mode, with cantilever resonant frequency 70 kHz, in Figure 7 shows that the beads are partly well-ordered in the substructure. They do not pile up but arrange in structures with a maximum height of 150 nm. This is important for cell culture as some cell types react strongly to topographical patterns. With this information, it is possible to estimate the amount of proteins offered to the cells if functionalized beads are used. The dark lines in Figure 6 are along the flow direction of the suspension and result from bead agglomeration at the edge between stamp and garnet layer. This is a consequence of a fairly high bead concentration chosen to enhance contrast in the light microscope. High concentration in this context implies longer deposition times as discussed below. If a nonmagnetic substrate is used, the beads are also restricted to the microfluidic channels as shown in Figure 8. However, no substructure does occur. Again, a high bead concentration was used. During the drying process, the excess particles adhered to the stamp and were thus removed. Apart from line structures, the microfluidic method could be applied to a structure of positive

Patterning Nanobeads on Surfaces by PDMS Stamps

Langmuir, Vol. 24, No. 3, 2008 891

Figure 7. AFM investigation on magnetic beads, deposited in a line structure. It is clearly visible that the beads do not pile up and that an accurate structure is obtained by means of the fluidic method.

Figure 8. AFM image of a linear bead structure obtained in the absence of magnetic interaction between substrate and beads (50 µm × 50 µm).

Figure 6. Light microscopic image of (a) a line structure, obtained by using a PDMS stamp with 20 µm wide lines for depositing magnetic beads. The substructures arise from the domain walls of the magnetic garnet layer. (b) The black lines occur because of bead agglomeration at the edges of the stamp and are an artifact due to high bead concentration. (c) Magnetic pretreatment of the garnet formed bubble domains before beads were deposited in lines.

discs of a diameter of 20 µm as shown in Figure 9. The exposed parts of the stamp pattern prevented the beads from being deposited onto the substrate. Deposition is restricted to areas defined by the cavities between stamp and substrate. The flow direction indicated in Figure 9 causes an additional anisotropy. To obtain circular structures, the soft molding method is more favorable. A droplet of bead suspension is put onto the ferromagnetic garnet, and the stamp is pressed onto the droplet. Figure 10 shows the resulting pattern. The circular structures are

Figure 9. PDMS stamp with 20 µm positive holes has been used as a microfluidic device. The arrow indicates the flow direction of the bead suspension. Light microscopic image.

formed in between the positive discs of the stamp, where the solution remains. The microfluidic method is suitable for all stamp topographies where flow through percolating channels is possible along a millimeter range. It is very important to ensure that wider structures with low aspect ratios do not collapse or deform in a way that they prevent continuous flow of the suspension.27 An important aspect is the atomic flatness of the garnet surface, which enables precise contact between stamp and substrate. (27) Sharp, K. G.; Blackman, G. S.; Glassmaker, N. J.; Jagota, A.; Hui, C. Y. Langmuir 2004, 20, 6430-6438.

892 Langmuir, Vol. 24, No. 3, 2008

Issle et al.

Figure 10. Circles are obtained by a stamping process of aqueous bead suspension with a PDMS stamp of positive 20 µm circles. The beads remain in between the positive structure, which is indicated by the dashed circle. Light microscopic image.

Figure 11. Light microscopic image of MG 63 osteoblasts grown on magnetic garnet. The dark lines represent deposited nanobeads.

The bead concentration of the original solution as provided by the manufacturer was 35 mg/mL and 6.5 × 1013/g. In the experiments described previously, a fairly high particle concentration of 1:10 with respect to the original solution was used. Since per experiment, 5 µL was needed, this led to 1.1 × 108 beads in total. However, the final bead concentration on the substrate or domain wall, respectively, depends predominantly on the time that is given between deposition and removal of the excess suspension. Comparison of Figures 6 and 7 shows this effect. In Figure 6b, the incubation time was nearly 1 min, whereas in Figure 7, the excess was removed after approximately 30 s. Shorter periods prevent bead agglomeration at the edges between stamp and substrate such as appears in Figure 6b (black lines). In addition to this, the number of deposited particles depends on the channel height or cavity dimensions. In the described experiments, PDMS structure heights of 0.8 and 1.2 µm were used. Lower structures around 400 nm led to clogging and inadequate patterning. Both described approaches allow fairly accurate reproduction and a high throughput. The covered area of about 1 cm2 makes the methods suitable for cell culture experiments. The topographical, magnetic, and biochemical properties of the generated patterns can be altered without much effort. The topography is determined by the stamps but additionally by the substrate domain structure. The latter can be changed by applying external fields

Figure 12. Light microscopic images of MG 63 osteoblasts grown on patterned surfaces. (a) Linear structure, after 24 h. (b) Same line structure as in panel a, after 48 h. (c) Octagon structure.

eventually combined with heat treatments. However, also bead size and concentration have an influence on the obtained pattern. A wealth of biochemical functionalizations is obtained through binding of functional molecules to the beads. Cell Culture. Garnet films turned out to be biocompatible.19,21 MG 63 osteoblasts grow until confluency within several days. Figure 11 shows some osteoblasts grown on a garnet layer with a large amount of deposited beads 24 h after seeding. An additional video shows osteoblasts grown over a period of 2 h on the

Patterning Nanobeads on Surfaces by PDMS Stamps

described substrate (Supporting Information). Temporal changes of the bead patterns and thus topographical changes can even be generated in cell culturing experiments by means of varying external magnetic fields. Initial results in cell culturing experiments were obtained. MG 63 osteoblasts were grown on structures as discussed here. The beads used for deposition were not coated or functionalized. Their matrix consisted of polysaccharide with embedded magnetite particles. The structures in Figure 12 are not as clearly visible as in Figure 7b, because a shorter period of deposition was chosen. Beads that agglomerate at the edges between stamp and substrate should be avoided. Figure 12a shows two cells on a linear bead structure as described before, 12 h after seeding. Figure 12b is the same sample 48 h after seeding. Figure 12c shows cells grown on an octagonal structure, where beads are located in the octagon area and spaces in between are without beads. In comparison to Figure 11, where the cells are randomly distributed and elongated, some changes occur. In Figure 12a, the cells align with the structure. It seems that they try to avoid direct contact with the beads. When the osteoblasts have sufficient cell-cell contacts, which happens after a certain incubation time, they reorient and do not remain in the elongated state as shown in Figure 12b. Once they can establish contact with each other they can overcome the alignment given by the substrate structure. Further detailed experiments showed more precisely that it is not the contact to the beads that is avoided. Rather, cells cannot easily build up focal adhesions on areas with deposited beads as in Figure 12c. It is clearly visible that the lamellipodia of the osteoblasts are restricted to the spaces in between the octagons. They have elongated or triangular shapes, which is in contrast to Figure 11. But obviously, the cells do not avoid contact with the beads completely. Most parts of the membrane of the triangular cells cover areas with deposited beads, as also shown in Figure 12b. It is thus a possible strategy to address cells via transmembrane pathways utilizing functionalized beads. Further experiments with different structures and immunochemical investigations will clarify the reasons for the unequal cell growth. First explanations consider the fact that areas with beads exhibit elastic behavior when the cells try to build up some force via their focal adhesions to the surface. Therefore, they are not optimal from an energetic point of view.28 Another explanation could be based on the changing topography between areas with and without beads, which loses influence if the cells try to build up cell-cell contacts. Nevertheless, it is possible for cells to (28) Bischofs, I. B.; Schwarz, U. S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9274-9279.

Langmuir, Vol. 24, No. 3, 2008 893

adhere and grow nondirectionally on the bead structure as shown in Figure 11, if they do not have the choice between coated and uncoated areas or if they have enough time to establish cell-cell contacts.

Conclusion The presented microfluidic and soft molding methods enable the structuring of substrates in the micro- to nanometer range in a straightforward and effective way. The patterns obtained were geometrically fairly accurate. The patterned substrates can be used in cell cultures with additional biofunctionalization of the employed magnetic beads. Thus, a large variety of topographical, magnetic, and biochemical substrate features is easily accessible.19 Furthermore, it is possible to investigate patterns of different geometric properties, as it is easily possible to influence the shape of the patterns by means of external magnetic fields. Biocompatibility of the setup could be proven. The nanobeads deposited onto garnet films are not subject to endocytosis. Further experiments will clarify the response of the cells to several bead structures, varying in width and shape. Bead types of different size and composition of the biocompatible shell will be tested as well. Automatic domain variations by driven external fields open the opportunity of influencing cells over long periods in a controlled manner. Also, it is possible to lead cells with certain patterns to directional growth. It is evident that this possibility is of considerable importance to high-throughput applications. Acknowledgment. The authors thank Prof. Tom H. Johansen, Department of Physics, University of Oslo, Norway for kindly providing the garnet films and AMO, Gesellschaft fu¨r Angewandte Mikro- und Optoelektronik GmbH in Aachen, Germany, for kindly providing the silicon masters. Also, assistance in cell culture and substrate preparation by Susanne Kirsch is acknowledged. This paper and the work it concerns were generated in the context of the CellPROM project, funded by the European Community as Contract NMP4-CT-2004-500039 under the Sixth Framework Program for Research and Technological Development. Supporting Information Available: Movie with osteoblasts grown on beads deposited onto garnet films shows the biocompatibility of the setup. This material is available free of charge via the Internet at http://pubs.acs.org. LA7018956