Article pubs.acs.org/Langmuir
High-Resolution Micropatterned Teflon AF Substrates for Biocompatible Nanofluidic Devices Ilja Czolkos,† Bodil Hakonen, Owe Orwar, and Aldo Jesorka* Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden ABSTRACT: We describe a general photolithography-based process for the microfabrication of surface-supported Teflon AF structures. Teflon AF patterns primarily benefit from superior optical properties such as very low autofluorescence and a low refractive index. The process ensures that the Teflon AF patterns remain strongly hydrophobic in order to allow rapid lipid monolayer spreading and generates a characteristic edge morphology which assists directed cell growth along the structured surfaces. We provide application examples, demonstrating the well-controlled mixing of lipid films on Teflon AF structures and showing how the patterned surfaces can be used as biocompatible growth-directing substrates for cell culture. Chinese hamster ovary (CHO) cells develop in a guided fashion along the sides of the microstructures, selectively avoiding to grow over the patterned areas.
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INTRODUCTION Surfaces with fabricated small-scale patterns can be exploited to facilitate adhesion and interfacing of biological structures, such as cells, tissues, and surface-supported lipid films. Such surfaces are integral parts of technological platforms which mimic certain biological features. Surface-supported phospholipid biand monolayers, which share fundamental characteristics with biological membranes, are an active research topic with one focus on biomedical studies and sensing applications and another on the choice of substrate materials and their structural design.1 Tissue engineering requires three-dimensional matrices with specific surface requirements toward cell adhesion and viability.2,3 The technology builds upon the concept of cell contact guidance, which is the behavior of cells to grow preferably along surface structural features in the micrometer and nanometer range, as well as on chemically engineered surfaces.4,5 Suitable patterning techniques encompass positive and negative patterning. In the former, distinctive areas of a surface are promoted to be more adhesive for cells than untreated areas, whereas in the latter, surfaces are partially coated with a cytophobic material to stimulate cell growth in the uncoated areas only.6 Commonly used cytophobic materials are fluorocarbons such as PTFE, Teflon AF (amorphous fluoropolymer), and other types. For site-specific cell adhesion, Valle et al. used negative patterning of Teflon AF with laminin,7 whereas Varma et al. created a fluorocarbon film with voids (glass) in which cells grow (positive patterning).8 Apart from its cytophobicity, Teflon AF is a very interesting material for various applications due to its hydrophobicity, low refractive index,9 high gas permeability,10 and low level of fluorescence. Recent research by Zhang et al. highlights the unique transport properties of Teflon AF and reviews applications where Teflon AF plays a role, underlining the importance of the material in a wide range of fields.11,12 For instance, fluorocarbons are © 2011 American Chemical Society
exploited in conjunction with aperture-spanning black lipid membranes (BLM) for ionophore investigations. Nowadays, micro- and nanofabrication techniques allow for small-scale apertures with superior electrical seals as well as high stability of the phospholipid films.13,14 Because of the high affinity to hydrophobic lipid tails, phospholipids spread exclusively as monolayers on Teflon AF. We observed that the spreading rates and lateral lipid diffusion on Teflon AF is significantly increased compared to previously studied epoxy resists.15,16 Here, we report a straightforward technique to micropattern Teflon AF on solid substrates. Our approach is based on widely applied microfabrication processes to create surface-adhering Teflon AF structures. The feature size of such structures can be as low as 1.8 μm, while the gaps between Teflon AF features can be smaller than 1 μm. Only methods involving expensive mask-less irradiation are known to give better pattern resolution of Teflon AF or other fluorocarbon films.17−19 Furthermore, our fabrication technique allows the patterned Teflon AF to be used in small-scale biologically inspired applications. We show that the distinct topographical edge feature of the Teflon AF structures we produce is especially suitable to guide cell growth. Chinese hamster ovary (CHO) cells grow along this feature, surrounding the Teflon AF surfaces while they avoid covering them. We also explored Teflon AF as substrates for self-spreading and mixing of molecular surface-supported lipid films. We have thus created a method to embody a biocompatible platform, which has high potential for interfacing live cells with supported biomimetic membranes. Received: November 14, 2011 Revised: December 26, 2011 Published: December 28, 2011 3200
dx.doi.org/10.1021/la2044784 | Langmuir 2012, 28, 3200−3205
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Article
zeocin and blasticidin in the same quantities as above. CHO cells were kept at 37 °C and 5% CO2 and were inspected daily for 10 days. Confocal Microscopy. Experimental details regarding confocal microscopy, buffers, and the micromanipulation technique for multilamellar lipid vesicles and their fabrication can be found in ref 16.
EXPERIMENTAL SECTION
Fabrication of Microstructured Teflon AF. Microscope coverslides #1 from Menzel Gläser were thoroughly cleaned and plasmatreated in a Tepla Plasma Batch System 300, a microwave plasma system of AMO GmbH with oxygen plasma at 250 W for 2 min. A titanium adhesion layer (thickness 2 nm) followed by a gold layer (thickness 8 nm) was deposited onto the cleaned coverslides with dc magnetron sputtering at a deposition rate of 5 and 20 Å/s, respectively. Where only titanium was used, that layer was 8 nm thin, and it was deposited at 5 Å/s. HMDS (Micro Resist Technology GmbH) primer was applied to the substrate for 30 s and spun off at 2000 min−1 for 30 s, followed by spin-coating of lift-off resist LOR 1A (Microchem Corp.) at 2000 min−1 for 1 min and a subsequent bake at 200 °C for 10 min. Positive photoresist S1813 (Rohm and Haas Co.) was spin-coated at 400 min−1 for 60 s and baked at 110 °C for 2 min. After cooling down, the substrates were exposed to 400 nm UV light at 6 W/cm2 through a dark-field mask with a Karl Süss MA6 mask aligner for 20 s. S1813 is developed with MF-319 (Rohm and Haas Co.) for 40−45 s, rinsed with water, blow-dried, and treated with oxygen plasma in a 30 s ash step in a Batchtop m/95. Teflon AF solution grade 601S1-100-6 (Du Pont, 6% (w/w) solids contents, based on Teflon AF1600, glass transition temperature Tg = 160 °C) was diluted to 1.2% (w/w) solids contents with a perfluorinated solvent (CAS 86508-42-1, Larodan Fine Chemicals AB). The 1.2% (w/w) Teflon AF solution was spin-coated twice onto the S1813 layer (2000 min−1, 1 min) and then baked for 5 min at 180 °C to surpass Teflon AF’s glass transition temperature. After cooling down, the substrates were subjected to Shipley Remover 1165 (Rohm and Haas Co.) (60−70 °C). Immediately after submerging the substrates, the reddish S1813 forms holes and starts to lift off the Teflon AF film within minutes where S1813 had not been exposed. The remover solution turns red during this process. After the major part of the Teflon AF film has been lifted off, the substrate is swivelled in a bath of isopropanol and then water and is blow-dried thereafter. Spraying of the substrates and excessive transfer across liquid/air interfaces should be avoided since the capillary forces may jeopardize the adhesion of the Teflon AF structures. The substrate is then placed in a fresh Shipley Remover 1165 bath in order to extract S1813 residue. This is important since it significantly decreases autofluorescence of the substrate. The substrates are then cleaned in isopropanol and water as before and are blow-dried. The lift-off process may be repeated if development of the Teflon AF structures is incomplete at first. The dark-field photomask for the S1813 process was prepared on a JEOL JBX-9300FS electron-beam lithography system. A UV-5/0.6 resist (Shipley Co.) coated Cr/soda-lime mask was exposed, developed, and etched using a common process for micrometer resolution.20 Pattern files were prepared on the AutoCAD 2006 platform (Autodesk). All fabrication procedures were executed under cleanroom atmosphere (class 3−6 according to ISO 14644-1). Surface Characterization. The structured surfaces were characterized with a scanning probe microscope (Veeco Dimension 3000 SPM) in tapping mode with a NSG01 DLC probe (NT-MDT Europe BV, The Netherlands). Cell Culture. Substrates with Teflon AF patterns for directed cell growth studies were fabricated on 47 mm diameter #1.5 coverslides from Menzel Gläser (Germany, supplied by WillCo Wells BV, The Netherlands) as described above. They were assembled into cell culture dishes with glass bottom dishes (WillCo Wells BV, The Netherlands) under cleanroom conditions and were packaged sterile. T-REx CHO cells were cultured in 5 mL of DMEM/F12 medium with 10% FBS (from PAA) and 350 μg/mL zeocin (Invitrogen) and 5 μg/mL blasticidin (Sigma) in T25 polystyrene flasks (Nunc) until they reached confluence. The medium was removed, and 2 mL of accutase solution (from PPA) were added to desorb cells from the bottom of the flask. Then, 2 mL of DMEM/F12 culturing medium with FBS and zeocin and blasticidin in the same quantities as above were added. 10 μL of this suspension, which contains ≈8 × 105 cells/mL, was pipetted onto the glass bottom dishes with the Teflon AF patterns and was diluted with 2 mL of DMEM/F12 culturing medium with FBS and
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RESULTS AND DISCUSSION Teflon AF Patterning. The principal problem in surfacepatterning of Teflon AF or similar materials has always been the weak adhesion of fluoropolymers to other materials. Despite this obstacle, we achieve to pattern Teflon AF on the small micrometer length scale in a straightforward process involving thin-layer deposition and UV photolithography. In short, a gold- or titanium-coated borosilicate coverslide is coated with the positive resist S1813, exposed to UV light through a mask, and is developed. From there on, the process is illustrated in more detail in Figure 1. The fabrication is based on spin-coating
Figure 1. Illustration of the fabrication process. (i) A patterned S1813 surface is fabricated by conventional UV lithography. A layer of LOR1A was spin-coated prior to S1813 but is not shown here. (ii) Deposition of a thin Teflon AF film on the patterned S1813 surface. The red arrows indicate the weakest point of the Teflon AF film at the edge of S1813 structures. (iii) After a baking step, the substrate is immersed in lift-off solution (Shipley R1165 at ∼65 °C). (iv) S1813 is swelling and dissolving beneath the thin polymer film. (v) Detachment of the bulk polymer film at the predetermined breaking points (red arrows). (vi) After rinsing and drying, the patterned Teflon AF surface remains.
a continuous Teflon AF film onto predetermined resist structures (Figure 1ii) which are swelling in the lift-off solvent (Figure 1iv), tearing the Teflon film at the pattern edges, and thereby selectively lifting off the overhead Teflon AF film 3201
dx.doi.org/10.1021/la2044784 | Langmuir 2012, 28, 3200−3205
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Article
Figure 2. Topography of Teflon AF patterns. (a) Bright-field image of patterned Teflon AF. (b) Bright-field image of a lane
