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Notes Topographic SU-8 Substrates for Immobilization of Three-Dimensional Nanotube-Vesicle Networks Johan Hurtig, Mattias Karlsson, and Owe Orwar* Department of Chemistry and Bioscience and Microtechnology Centre at Chalmers, Chalmers University of Technology, SE 412 96 Go¨ teborg, Sweden Received January 21, 2004
Introduction There has been a strong development in the miniaturization of fluidic devices in the micrometer range resulting in highly complex and fully integrated chip-based systems with important applications in separation and analysis of biomolecules.1,2 Following such a reduction in size, for example, wall properties will have pronounced effects on solvent and solute behavior, and mixing by diffusion will be much more efficient than on the micrometer scale. Operations in these devices might be based on the handling of single molecules to unravel compositional information, for example, analysis of small-scale biological samples, single-polymer sequencing, synthesis, and the like. Today, devices made by methods of microfabrication with channels of nanometer scale, at least in one dimension, have been utilized in biochemical analysis. DNA separations have been performed in devices with channels down to 45 nm (ref 3) and by the use of topographic substrates comprised of arrayed pillars with 125-nm spacing serving as entropic barriers.4-6 In addition, capillary electrophoresis has been performed in 430-nm inner-diameter fused silica capillaries by Woods and coworkers to separate catecholamines.7 Nanoscale fluidic devices might also give unique insight and understanding of kinetics and mechanisms of chemical reactions that take place in confined media (For a general review, see Khairutdinov and Serpone).8 This capability will be particularly relevant for studies of reactions, electron, and energy transport that naturally take place in nanoscale compartments, such as biological cells and in nanoscale devices, including molecular electronics systems.9 * To whom the correspondence should be addressed. E-mail:
[email protected]. Phone: + 46-(0)31-772 3060. Fax: + 46-(0)31-772 3858. (1) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (2) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (3) Cao, H.; Yu, Z.; Wang, J.; Tegenfeldt, J. O.; Austin, R. H.; Chen, E.; Wu, W.; Chou, S. Y. Appl. Phys. Lett. 2002, 81, 174. (4) Han, J.; Craighead, H. G. Science 2000, 288, 1026-1029. (5) Turner, S. W.; Perez, A. M.; Lopez, A.; Craighead, H. G. J. Vac. Sci. Technol., B 1998, 16, 3835-3840. (6) Turner, S. W. P.; Cabodi, M.; Craighead, H. G. Phys. Rev. Lett. 2002, 88, 128103. (7) Woods, L. A.; Roddy, T. P.; Paxon, T. L.; Ewing, A. G. Anal. Chem. 2001, 73, 3687-3690. (8) Khairutdinov, R. F.; Serpone, N. Prog. React. Kinet. 1996, 21, 1-68. (9) Kubatkin, S.; Danilov, A.; Hjort, M.; Cornil, J.; Bre´das, J.-L.; Stuhr-Hansen, N.; Hedegård, P.; Bjørnholm, T. Nature 2003, 425, 698701.
Mainly solid-state materials have been utilized in the development of both nanochannels and nanocontainers using, for instance, electron beam lithography, sacrificial layers of carbon nanotubes (CNTs), colloidal lithography, and nano-imprinting.10,11 CNTs have been used for construction of directional/ordered structures12 and networks.13 Although Gogotsi and co-workers showed the trapping of water in a multiwalled enclosed CNT,14 this type of structure has still not been used for controlled nanofluidics. We have taken another route using unconventional fabrication procedures with great flexibility to produce nanofluidic soft matter devices of different functions and geometries.15-19 These systems are based on self-assembly, self-organization, and forced shape transformations in thin-shell surfactant20 bilayer systems.15 Using novel micromanipulation methods, circuits consisting of surfaceimmobilized unilamellar vesicles (∼5-25 µm in diameter) conjugated with suspended nanotubes 50-150 nm in radius can be produced with controlled geometry and topology.16,18 The membrane composition (e.g., lipids, transporters, receptors, and catalytic sites) and container contents (e.g., catalytic particles, organelles, and reactants) can be controlled on the single-container level, allowing complex chemical programming of networks.21,22 Thus, networks of nanotubes and vesicles serve as a platform to build nanofluidic devices operating on the single molecule and particle level and offer opportunities to study chemistry in confined biomimetic compartments. We here present a method to create three-dimensional compact liposome networks, adhered to topographic substrates fabricated in the epoxy polymer SU-8, which is demonstrated to be an excellent substrate material for liposome adhesion, with low lipid-film formation and spreading. Because network density is directly proportional to surface density, the increase in compactness of these devices is roughly proportional to 4wh, where w is the width and h is the height of a pillar. Thus, these devices (10) Chou, S. Y.; Krauss, P. R.; Renstrom, P. J. Science 1996, 272, 85-87. (11) Xia, Y.; Whitesides, G. M. Langmuir 1997, 13, 2059-2067. (12) Pan, Z. W.; Zhu, H. G.; Zhang, Z. T.; Im, H. J.; Dai, S.; Beach, D. B.; Lowndes, D. H. J. Phys. Chem. B 2003, 107, 1338-1344. (13) Jung, Y. J.; Homma, Y.; Ogino, T.; Kobayashi, Y.; Takagi, D.; Wei, B. Q.; Vajtai, R.; Ajayan, P. M. J. Phys. Chem. B 2003, 107, 68596864. (14) Gogotsi, Y.; Naguib, N.; Libera, J. A. Chem. Phys. Lett. 2002, 365, 354-360. (15) Karlsson, A.; Karlsson, R.; Karlsson, M.; Can, A.-S.; Stromberg, A.; Ryttsen, F.; Orwar, O. Nature 2001, 409, 150-152. (16) Karlsson, M.; Sott, K.; Cans, A.-S.; Karlsson, A.; Karlsson, R.; Orwar, O. Langmuir 2001, 17, 6754-6758. (17) Sott, K.; Karlsson, M.; Pihl, J.; Hurtig, J.; Lobovkina, T.; Orwar, O. Langmuir 2003, 19, 3904-3910. (18) Karlsson, M.; Sott, K.; Davidson, M.; Cans, A.-S.; Linderholm, P.; Chiu, D.; Orwar, O. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1157311578. (19) Karlsson, A.; Karlsson, M.; Karlsson, R.; Sott, K.; Lundqvist, A.; Tokarz, M.; Orwar, O. Anal. Chem. 2003, 75, 2529-2537. (20) Seifert, U. Adv. Phys. 1997, 46, 13-138. (21) Karlsson, R.; Karlsson, M.; Karlsson, A.; Cans, A.-S.; Bergenholtz, J.; Åkerman, B.; Ewing, A. G.; Voinova, M.; Orwar, O. Langmuir 2002, 18, 4186-4190. (22) Davidson, M.; Karlsson, M.; Sinclair, J.; Sott, K.; Orwar, O. J. Am. Chem. Soc. 2003, 125, 374-378.
10.1021/la0498051 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/26/2004
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might be used to improve the information density and processing capability of such networks in applications such as nanofluidics and computational devices as well as for nanoscale templating. SU-8 scaffolds might also be used to assist in demanding manipulation schemes to, for example, create crossing knots, trivial knots, and other complex surfactant topologies. Materials and Methods Chemicals. The chemicals used for buffer solutions were of analytical grade and purchased from Sigma (St. Louis, MO). Soybean polar lipid extract was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). FM 1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide] was from Molecular Probes (Eugene, OR). SU-8 25 negative photoresist was from MicroChem Corp. (Newton, MA), XP SU-8 developer was from Microresist Technology (GmbH, Berlin, Germany), and UV-5 positive photoresist and MF24A developer were from Shipley (Marlborough, MA). The SC-1 wash was of the composition 1:1:5 NH4 (25%)/H2O2 (30%)/deionized H2O with pro analysi chemicals from VWR International AB (Stockholm, Sweden). Materials. Four-inch soda lime wafers coated with 1000 Å of low reflective chrome were from Nanofilm (Westlake Village, CA). Borosilicate coverslips were from Menzel Gla¨ser (Braunschweig, Germany), with thicknesses 0.08-0.12 mm (No. 0), and borosilicate capillaries 1.0 mm o.d. × 0.78 mm i.d. were from Harvard Apparatus (Edenbridge, U.K.). Scaffold Fabrication. The patterns for the scaffold components were drawn in AutoCAD format and transferred to a chrome mask using electron beam (JBX-5DII, JEOL, Ltd., Tokyo, Japan) lithography and wet etching. Borosilicate substrates were SC-1 cleaned, oven-dehydrated, and spin-coated with the ultrathick negative photoresist (SU-8 25). The pattern was transferred to the negative resist and developed in XP SU-8, forming a positive relief with the UV-lithography technique.23 Finally, the processed coverslips were rinsed in 2-propanol. The desired thickness is 50 µm, and the actual thickness of the SU-8 structures was examined with a surface profilometer (Tencor Alpha Step 500, KLA-Tencor, San Jose, CA). Scanning Electron Microscopy. A scanning electron microscope (JSM-6301F, JEOL; Tokyo, Japan) was used for imaging of the microfabricated structures. To eliminate the charge-up effects of the glass substrate, the SU-8 process protocol just described was used with a 3-in. unoxidized silicon wafer as the substrate. Contact Angle Measurements. Static contact angle measurements were performed on in-house built goniometric equipment on a sessile drop. Water droplets of equal volume were placed on the substrates, and the contact angle was measured after equilibrium had been reached. Liposome adhesion contact angles24 were measured utilizing the method developed by Karlsson et.al.19 Substrates were mounted parallel to the beam path of an inverted microscope (Leica DM IRB, Wetzler, Germany), resulting in two-dimensional images of surface-adhered objects from the side. Liposome adhesion was monitored using time-lapse microscopy with a charge-coupled device camera (C6157-01, Hamamatsu, Kista, Sweden), and data were recorded on a digital video (DVCAM, DSR-11, Sony, Japan). The video frames were digitized with Adobe Premier and analyzed in Adobe Photoshop. Micropipet-Assisted Formation of Unilamellar Networks. Fabrication of unilamellar networks of nanotubeinterconnected liposomes, using a micropipet-assisted technique on soybean lipid giant unilamellar-multilamellar vesicles (GUVMLV), was performed as described previously.16 High-resolution micromanipulators (MWH-3, Narishige, Tokyo, Japan) allowed precise control of vesicle immobilization in the x-y-z position. Borosilicate capillaries were pulled on a CO2-laser puller instrument (model P-2000, Sutter Instrument Co., Novato, CA). A microinjection system (Eppendorf, CellTram Vario) and a pulse (23) MicroChem Corp. Nano SU-8 Negative Tone Photoresists Formulation 2-25, 2002. http://www.microchem.com/products/pdf/ SU8_2-25.pdf (accessed Apr 2003). (24) Seifert, U.; Lipowsky, R. Phys. Rev. A 1990, 42, 4768-4771.
Figure 1. (a) Computer-aided design drawing of the scaffold dimensions utilized for three-dimensional vesicle nanonetwork fabrication. Lengths are given in micrometers. The pattern is set up in an array format with an even spacing of 300 µm and on some substrates with rotation in between. (b) Scanning electron micrograph of SU-8 pillars in the array utilized. Note well the pillar substrate is not glass but unoxidized silicon to reduce charge-up effects during scanning electron microscopy imaging. generator (Digitimer Stimulator DS9A, Welwyn Garden City, U.K.) were used to control the electroinjections.25 Confocal Imaging. A confocal laser scanning microscopy system (Leica TCS SP2 RS, Wetzlar, Germany), with a PL APO 63×/1.32 objective, was used for acquisition of confocal fluorescence images. The 488-nm line of the Ar laser was used for excitation of membrane lipid probe FM 1-43, and emission was collected by a photomultiplier tube with set sensitivity in the 550-650-nm spectrum. During acquisition, images were lineaveraged, and for the final computer-generated confocal images, morphological filter functions and three-dimensional visualization tools in the Leica software were utilized.
Results and Discussion Fabrication and Characterization of Topographic SU-8 Substrates. The polymeric photoresist SU-8 is a material for construction of micrometer-sized structures and has been utilized in a wide range of applications, for example, as moulds for soft lithography26 and optical waveguides.27 The polymer is well-suited for creation of high-aspect-ratio objects,28 and we here use SU-8 process(25) Karlsson, M.; Nolkrantz, K.; Davidson, M. J.; Stro¨mberg, A.; Ryttse´n, F.; Åkerman, B.; Orwar, O. Anal. Chem. 2000, 72, 5857-5862. (26) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551-575. (27) Lin, C.-H.; Lee, G.-B.; Chen, S.-H.; Chang, G.-L. Sens. Actuators, A 2003, 107, 125-131. (28) Lorenz, H.; Despont, M.; Fahrni, N.; LaBianca, N.; Renaud, P.; Vettiger, P. J. Micromech. Microeng. 1997, 7, 121-124.
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Figure 2. Schematic drawing of the principles of three-dimensional-network construction. (a) A tapered borosilicate-glass micropipet with an outer-tip diameter of 0.5-1 µm, back-filled with aqueous medium and mounted onto an electroinjection system,25 was pressed against the membrane of a surface-immobilized vesicle. By applying direct current voltage pulses of field strengths between 10 and 40 V/cm and duration of 1-4 ms over the micropipet, the lipid membrane was penetrated. The micropipet is pulled out and away from the mother vesicle, forming a lipid nanotube connection between the mother liposome and the pipet tip. Aqueous medium is injected into the nanotube using a pressurized-air-driven microinjector, thus, forming a small satellite vesicle at the outlet of the micropipet tip. (b) This newly created vesicle can then be immobilized onto the substrate surface at the desired coordinates. The manipulators are graded to a resolution of 0.2 µm, allowing for precise control of the x-y-z position. (c) After the attachment of a vesicle, repeating the retraction and injection steps, further vesicles can be connected, with flow of lipid material from the mother vesicle over the vesicle and tube network to the new vesicle, to expand the network, resulting in complex threedimensional structures (d).
ing technology for construction of topographic substrates consisting of scaffolds for immobilization of fluid-state lipid membrane vesicles. The design of the scaffold structures consists of double-L-shaped pillars; dimensions are according to Figure 1a, with pillars arranged in an array format (Figure 1b). Because high numerical aperture optics was used for liposome network construction, the scaffold height was set to ∼50 µm. The main benefit of L-shaped pillars is that a large surface area is generated that is easily accessible for the micropipet tools. The fabrication process results in a topographic substrate consisting of two types of materials, polymeric SU-8 pillars located on a borosilicate surface. Both materials are transparent for wavelengths above 360 nm. To evaluate the surface properties of the microfabricated compound substrate, pure SU-8 and borosilicate surfaces were analyzed separately with static contact angle experiments. Directly after the scaffold microfabrication process,23 planar SU-8 polymer substrates displayed static contact angles of 77° ( 1 and borosilicate coverslips subjected to the scaffold microfabrication process displayed contact angles of 73° ( 2. Thus, the compound substrates will display similar surface properties with respect to water contact angles. Typically, unprocessed borosilicate coverslips display contact angles of ∼45°.19 Thus, it is likely
that SU-8 residues from the microfabrication process are present on the processed borosilicate substrate. Over time, however, the contact angle on the processed borosilicate substrate slowly decreased, and after two months the processed borosilicate substrate exhibited similar contact angles as observed on unprocessed borosilicate. Accordingly, the topographic substrates were used within 2 weeks of fabrication to ensure similar surface properties on the SU-8 scaffolds and the borosilicate surface. Vesicle Adhesion Investigations. Substrate surface properties are of paramount importance when designing substrates for liposome network construction. The contact area of an adhering liposome will, because of the Brownian motion of the membrane surface, grow progressively until it reaches mechanical equilibrium. The surface free energy of a bound vesicle will then be given by the balance between the adhesive forces, lateral tension, and bending rigidity:
E)
Ka Kc (A - A0)2 + (c1 + c2 - c0)2 dA - φA* 2A0 2
∫
where the first term represents stretching energy and the second bending energy, φ is the effective contact potential, and A* is the vesicle contact area.24 Conse-
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Figure 3. Confocal microscopy images of three-dimensional nanotube-vesicle networks stained by FM 1-43 on SU-8 scaffolds (color coded in orange) in three different conformations. Panel A: three-dimensional, two-liposome network displaying the use of SU-8 as scaffold material. Panel B: three-liposome network. Panel C: seven-liposome network with tight tube angles and off-equatorial tube extraction. The images are contrast-enhanced and pillars are color coded (Adobe Photoshop) to aid the visualization of the three-dimensional networks because SU-8 is auto-fluorescent for the excitation and emission wavelength of the dye.
quently, the shape of a bound vesicle reflects the strength of adhesion in the system. At low contact potentials (weak adhesion regime), a bound vesicle is largely spherical with a very small contact area and undefinable contact angle. As the contact potential increases (strong adhesion regime), a vesicle will interact more strongly with the substrate surface and appear as truncated spheres with well-defined contact angles. If the contact potential becomes excessively large, adhesive forces will lead to tension-induced lysis, and the vesicle will rupture and spread onto the substrate.29 For lipid nanotube-vesicle network construction, it is desirable to have a substrate that mediates intermediate vesicle adhesion, enabling secure anchoring of vesicle containers without compromising network integrity and stability. The adhesion of a giant unilamellar lipid vesicle was investigated using microscopy as described in Materials and Methods. Giant unilamellar vesicles adhered rapidly to both SU-8 and glass substrates and displayed strong adhesion behavior with similar contact angles. After the initial adhesion event, vesicles slowly increased their (29) Bernard, A.-L.; Guedeau-Boudeville, M.-A.; Jullien, L.; Meglio, J.-M. d. Langmuir 2000, 16, 6809-6820.
contact areas for ∼10 min before equilibrium was reached. Vesicles immobilized on SU-8 stabilized at contact angles of ∼130°, whereas contact angles for vesicles on borosilicate continued to decrease and then later stabilized around ∼122°; that is, there is a weaker adhesion to SU-8 than to glass. Furthermore, we never observed lipid-film spreading on SU-8 substrates, also indicating a weaker adhesion to this type of substrate. Vesicles immobilized on SU-8 substrates could be maintained for long periods of time (several hours). Consequently, SU-8 provides a highly suitable substrate for secure immobilization of vesicles and for maintaining network integrity over long periods of time. Three-Dimensional Network Construction. Fluidstate lipid nanotube-vesicle networks were produced on topographic substrates on inverted microscopes using a micropipet manipulation technique.16 As the starting material for production of networks, we used giant vesicles made from soybean polar extracts by a dehydration/ rehydration technique.25 A droplet (∼300 µL) of liposome suspension was placed on the topographic substrates, where vesicles where allowed to adhere randomly over the surface. A unilamellar-multilamellar vesicle complex
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(GUV-MLV) located close to a three-dimensional structure was chosen as a membrane source. The use of graded micromanipulators gave precise control (resolution 0.2 µm) of vesicle positioning in x-y-z directions. The surface properties of the microfabricated substrate enabled secure immobilization of vesicles, which snapped readily to the SU-8 pillars as well as the borosilicate substrate, allowing pipet retraction and release of the liposomes without any visible sign of leakage. The procedure of formation of threedimensional networks using scaffold structures is schematically illustrated in Figure 2a-d. Topographic substrates allow formation of very dense networks with small footprint areas because liposomes can be positioned anywhere on the polymer scaffolds as well as on the planar substrate surface. Confocal fluorescence images of various three-dimensional networks are shown in Figure 3. In panel A, a three-dimensional two-vesicle system is shown, where one liposome is attached on the borosilicate surface and one is adhered to an elevated position on the vertical wall of the polymer pillar. In panel B is a three-vesicle network disconnected from the multi/unilamellar complex where also a fourth vesicle is present and still connected to the mother liposome off-image (Figure 2, panel B). In panel C, the top, side, and perspective views depict seven connected vesicles. The vesicle on the left pillar is connected to three other vesicles at a tight angle, were the possibility of three-dimensional placement gives access to off-equatorial sites for nanotube extraction. Conclusion The polymeric photoresist SU-8 is a highly suitable material for soybean lipid vesicle immobilization displaying sufficient contact potential for secure anchoring of liposomes and minimal lipid spreading. Thus, this material allows fluid-state lipid membrane structures to retain their
structural integrity for long time periods (several hours). The properties of SU-8 can be utilized for lipid nanotube positioning or guidance while still maintaining nanotube properties as well as localized (patterned) adhesion of vesicles facilitating construction of complex liposome networks. To the best of our knowledge, this is the first demonstration of SU-8 as a vesicle adhesion substrate. We have further demonstrated a fabrication principle of three-dimensional nanotube networks on topographically patterned substrates, imaged by confocal microscopy. The use of three-dimensional construction techniques increases compactness of lipid nanotube networks as the tube density from a single vesicle is increased when access to the off-equatorial area is provided. The capability of building complex nanoscale surfactant networks in three dimensions is important for increasing the information density and processing capability of such networks in applications such as nanofluidics and computational devices as well as for nanoscale templating. Acknowledgment. We thank Leica Microsystems AB for lending the confocal microscopy system, Cellectricon AB for use of their laboratory facilities, and Polymer Technology, Chalmers, for use of the contact angle instrument. The authors wish to thank the Swedish Research Council (VR), the Swedish Foundation for Strategic Research (SSF), and the Chalmers Bioinitiative for financial support. Supporting Information Available: Measured changes in the contact angles of liposomes adhered to glass and SU-8 substrates during a 40-min time-lapse experiment, representation of a vesicle adhering to a substrate, and definition of the contact angle. This material is available free of charge via the Internet at http://pubs.acs.org. LA0498051
