Langmuir 2008, 24, 503-510
503
Settlement of UlWa Zoospores on Patterned Fluorinated and PEGylated Monolayer Surfaces John A. Finlay,†,# Sitaraman Krishnan,‡,#,& Maureen E. Callow,† James A. Callow,† Rong Dong,‡,§ Nicola Asgill,‡,| Kaiming Wong,‡ Edward J. Kramer,⊥ and Christopher K. Ober*,‡ School of Biosciences, The UniVersity of Birmingham, Birmingham, B15 2TT, UK, Department of Materials Science and Engineering, Cornell UniVersity, Ithaca, New York 14853, Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14853, Department of Biomedical Engineering, Vanderbilt UniVersity, NashVille, Tennessee 37235, and Departments of Materials and Chemical Engineering, UniVersity of California at Santa Barbara, Santa Barbara, California 93106 ReceiVed July 27, 2007. In Final Form: October 5, 2007 Various designs for coatings that resist the attachment of marine organisms are based on the concept of “ambiguous” surfaces that present both hydrophobic and hydrophilic functionalities as surface domains. In order to facilitate the optimal design of such surfaces, information is needed on the scale of the domains that the settling stages of marine organisms are able to distinguish. Previous experiments showed that UlVa zoospores settle (attach) in high numbers onto fluorinated monolayers compared to PEGylated monolayers. The main aim of the present study was to determine, when zoospores of the green alga UlVa are presented with a choice of fluorinated or PEGylated surfaces, what the minimum dimensions of the two types of surface are that zoospores can detect and consequently settle on. Silicon wafers were chemically modified to produce a pattern of squares containing alternating fluorinated and PEGylated stripes of different widths on either a uniform fluorinated or PEGylated background. Each 1 cm × 1 cm square contained stripes with widths of 500, 200, 100, 50, 20, 5, or 2 µm as well as an unpatterned square with a chemistry opposite that of the background. Spores were selective in choosing where to settle, settling at higher densities on fluorinated stripes compared to PEGylated stripes. However, the magnitude of response, and the consequences for settlement on patterned areas overall, was dependent on both the width of the stripes and the chemistry of the background. The data are discussed in relation to the ability of spores to “choose” favorable sites for settlement and the implications for the development of novel antifouling coatings.
Introduction The fouling of ship hulls and other man-made marine structures causes high operational and maintenance costs to industry and increased carbon emissions across the world.1 Traditional antifouling paints have relied upon the inclusion of biocides, many of which are now considered to be environmentally undesirable. In recent years, research has focused on the development of environmentally benign fouling-release coatings, especially low modulus, low surface energy silicone elastomers.2 Although these materials readily release most macrofouling organisms,3-6 they do not deter settlement or colonization, i.e., †
The University of Birmingham. Department of Materials Science and Engineering, Cornell University. § Department of Chemistry and Chemical Biology, Cornell University. | Vanderbilt University. ⊥ Departments of Materials and Chemical Engineering, University of California at Santa Barbara. # These authors contributed equally. & Present address: Department of Chemical and Biomolecular Engineering, Clarkson University, Potsdam, New York 13699. ‡
(1) Townsin, R. L. The ship hull fouling penalty. Biofouling 2003, 19 (supplement), 9-15. (2) Brady, R. F.; Singer, I. L. Mechanical factors favoring release from fouling release coatings. Biofouling 2000, 15, 73-81. (3) Swain, G. E. Redefining antifouling coatings. ProtectiVe Coatings Europe; July 1999; pp 18-25. (4) Chaudhury, M. K.; Finlay, J. A.; Chung, J. Y.; Callow, M. E.; Callow, J. A. The influence of elastic modulus and thickness on the release of the softfouling green alga UlVa linza (syn. Enteromorpha linza) from poly(dimethylsiloxane) (PDMS) model networks. Biofouling 2005, 21, 41-48. (5) Kavanagh, C. J.; Quinn, R. D.; Swain, G. W. Observations of barnacle detachment from silicones using high-speed video. J. Adhes. 2005, 81, 843-868. (6) Holm, E. R.; Kavanagh, C. J.; Meyer, A. E.; Wiebe, D.; Nedved, B. T.; Wendt, D.; Smith, C. M.; Hadfield, M. G.; Swain, G.; Darkangelo, C.; Truby, K.; Stein, J.; Montemarano, J. Interspecific variation in patterns of adhesion of marine fouling to silicone elastomers. Biofouling 2006, 22, 233-243.
they are not “antifouling”.7 Therefore, a considerable research effort is now focused on examining the potential of a range of antifouling surface designs that reduce the initial attachment of organisms.8-10 In the biomedical field, success in preventing biofouling has been achieved at the hydrophilic end of the wettability spectrum, for example, through the use of poly(ethylene glycol) (PEG) materials.11 When PEG polymers are grafted to a surface, a close association exists between water molecules and PEG arising from hydrogen bonding. Such formation of a hydration layer has been shown to hinder the nonspecific adsorption of proteins12 and has the potential to deter the adhesion of cells or microorganisms.13 The green seaweed UlVa is a prominent fouling alga. Dispersal is chiefly via microscopic zoospores, which are pear-shaped, (7) Swain, G. W. J.; Nelson, W. G.; Preedeekanit, S. Biofouling adhesion and biotic disturbance on the development of fouling communities on non-toxic surfaces. Biofouling 1998, 12, 257-269. (8) Tang, Y.; Finlay, J. A.; Kowalke, G. L.; Meyer, A. E.; Bright, F. V.; Callow, M. E.; Callow, J. A.; Wendt, D. E.; Detty, M. R. Hybrid xerogel films as novel coatings for antifouling and fouling release. Biofouling 2005, 21, 59-71. (9) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Kramer, E. J.; Sohn, K. E.; Fischer, D. A. Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: Attachment strength of the diatom NaVicula and the green alga UlVa. Biomacromolecules 2006, 7, 1449-1462. (10) Genzer, J.; Efimenko, K. Recent developments in superhydrophobic surfaces and their relevance to marine fouling: a review. Biofouling 2006, 22, 339-360. (11) Senaratne, W.; Andruzzi, L.; Ober, C. K. Self-assembled monolayers and polymer brushes in biotechnology: current applications and future perspectives. Biomacromolecules 2005, 6, 2427-2448. (12) Harder, P, Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption. J. Phys. Chem. B 1998, 102, 426-436.
10.1021/la702275g CCC: $40.75 © 2008 American Chemical Society Published on Web 12/15/2007
504 Langmuir, Vol. 24, No. 2, 2008
Finlay et al.
“naked” cells, i.e., without a cell wall, with four flagella at the anterior end, approximately 5 µm in diameter and 7-8 µm in length.14 To complete the life cycle, the liberated zoospores must attach to a solid surface through a complex process involving surface sensing, commitment to settlement, and release of a preformed adhesive.14 During settlement, zoospores exhibit selectivity, responding to a variety of surface-associated cues including topography,15,16 physicochemical attributes of the substrate,17,18 and the presence of biofilms.19 Several studies on the settlement of UlVa zoospores have indicated a preference for hydrophobic compared to hydrophilic surfaces.17,18,20 However, the strength of attachment of settled zoospores is typically stronger to hydrophilic compared to hydrophobic surfaces,21 although this was not the case for an mPEG-DOPA3 surface20 or oligo(ethylene glycol) SAMs22 to which spores could only adhere extremely weakly. These differences in settlement and adhesion behavior have inspired the development of “ambiguous” surfaces, which present both hydrophilic and hydrophobic domains to settling (attaching) cells and organisms. In recent years, a number of novel coatings in which the surface layers phase-segregate to produce a mosaic of hydrophilic and hydrophobic domains have shown promise as foul-release coatings.23,24 In the amphiphilic networks formed from hyperbranched fluoropolymer and linear poly(ethylene glycol), the dimensions of the surface-segregated components are in the micrometer range,25 while the amphiphilic block copolymers of Krishnan et al.24 present a much more homogeneous surface. The optimum design of “ambiguous” segregated surfaces requires information regarding the scale at which settling cells detect hydrophobic or hydrophilic domains. This can be explored through the use of patterned model surfaces. Patterned surfaces, with pattern dimensions comparable to those of cells, continue to be of interest in the study of cell-surface interactions.26-28 We present, herein, a simple and reliable procedure for creating micropatterns of alternating PEGylated and fluorinated monolayers on silicon surfaces. In contrast to non-photolithographic
techniques such as writing patterns using micropen29 and e-beam,30 or using methods such as microcontact printing and micromolding in capillaries,31,32 the reported procedure combines the advantages of photolithography in covering large areas of substrates with high-fidelity micropatterns and the convenience of vapor phase deposition of silanes to create multicomponent SAMs. Such patterned surfaces, which combine SAMs at opposite ends of the wettability spectrum, are ideal model surfaces for a variety of fundamental studies in surface science. In the present paper, we have used this technique to produce patterned surfaces with which to explore how UlVa zoospores respond to combinations of hydrophilic and hydrophobic domains presented at varying length scales in the micrometer range. Spores were presented with a choice of alternating fluorinated and PEGylated monolayer stripes of different widths, imprinted onto silicon wafers as 1 cm × 1 cm squares. The wettability of the region outside of the squared area, termed the background, was also controlled, being itself covered with either a fluorinated or PEGylated monolayer. The results provide the first evidence that marine organisms can discriminate between surface domains of different wettability and of a specific size.
(13) Andruzzi, L.; Senaratne, W.; Hexemer, A.; Sheets, E. D.; Ilic, B.; Holowka, D.; Kramer, E. J.; Baird, B.; Ober, C. K. Exploring the potential of oligoethylene glycol containing polymer brushes as bio-selective surfaces. Langmuir 2005, 21, 2495-2504. (14) Callow, M. E.; Callow, J. A.; Pickett-Heaps, J. D.; Wetherbee, R. Primary adhesion of Enteromorpha (Chlorophyta, Ulvales) propagules: quantitative settlement studies and video microscopy. J. Phycol. 1997, 33, 938-947. (15) Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Schumacher, J. F.; Wilkerson, W.; Wilson, L. H.; Callow, M. E.; Callow, J A.; Brennan, A. B. Engineered antifouling microtopographies-correlating wettability with cell attachment. Biofouling 2006, 22, 11-21. (16) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson, L. H.; Callow, M. E.; Callow, J. A. Finlay, J. A.; Brennan, A. B. Engineered antifouling microtopographies-effect of feature size, geometry, and roughness on settlement of zoospores of the green alga UlVa. Biofouling 2007, 23, 55-62. (17) Callow, M. E.; Callow, J. A.; Ista, L. K.; Coleman, S. E.; Nolasco, A. C.; Lo´pez, G. P. The use of self-assembled monolayers (SAMs) of different wettability to study surface selection and primary adhesion processes of zoospores of the green alga Enteromorpha. Appl. EnViron. Microbiol. 2000, 66, 32493254. (18) Ista, L. K.; Callow, M. E.; Finlay, J. A.; Coleman, S. E.; Nolasco, A. C.; Simons, R. H.; Callow, J. A.; Lopez, G. P. Effect of substratum chemistry and surface energy on attachment of marine bacteria and algal spores. Appl. EnViron. Microbiol. 2004, 70, 4151-4157. (19) Callow, J. A.; Callow, M. E. Biofilms. Progress in Molecular and Subcellular Biology 2006, 42 (Antifouling Compounds), 141-169. (20) Statz, A.; Finlay, J.; Dalsin, J.; Callow, M.; Callow, J. A.; Messersmith, P. B. Algal antifouling and fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels. Biofouling 2006, 22, 391-399. (21) Finlay, J. A.; Callow, M. E.; Ista, L. K.; Lopez, G. P.; Callow, J. A. Adhesion strength of settled spores of the green alga Enteromorpha and the diatom Amphora: the influence of surface wettability. Integr. Comp. Biol. 2002, 42, 1116-1122. (22) Schilp, S.; Kueller, A.; Rosenhahn, A.; Grunze, M.; Pettitt, M. E.; Callow, M. E.; Callow, J. A. Settlement and adhesion of algal cells to hexa(ethylene glycol)-containing self-assembled monolayers with systematically changed wetting properties. Biointerphases 2007, 2, 143-150.
(23) Gudipati, C. S.; Finlay, J. A.; Callow, J. A.; Callow, M. E.; Wooley, K. L. The antifouling and fouling-release performance of hyperbranched fluoropolymer (HBFP) - poly(ethylene glycol) (PEG) composite coatings evaluated by adsorption of biomacromolecules and the green fouling alga UlVa. Langmuir 2005, 21, 30443053. (24) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry, R.; Ober, C. K.; Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A. Anti-biofouling properties of comb-like block copolymer with amphiphilic sidechains. Langmuir 2006, 22, 5075-5086. (25) Gudipati, C. S.; Greenlief, C. M.; Johnson, J. A.; Prayongpan, P.; Wooley, K. L. Hyperbranched fluoropolymer and linear poly(ethylene glycol) based amphiphilic crosslinked netwotks as efficient antifouling coatings: an insight into the surface compositions, topographies and morphologies. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 6193-6208. (26) The´ry, M.; Racine, V.; Piel, M.; Pe´pin, A.; Dimitrov, A.; Chen, Y.; Sibarita, J-B.; Bornens, M. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19771-19776. (27) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044-3063. (28) Zhang, S.; Yan, L.; Altman, M.; La¨ssle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biological surface engineering: a simple system for cell pattern formation. Biomaterials 1999, 20, 12131220. (29) Lo´pez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. Convenient methods for patterning the adhesion of mammalian cells to surfaces using self-assembled monolayers of alkanethiolates on gold. J. Am. Chem. Soc. 1993, 115, 5877-5878. (30) Senaratne, W.; Sengupta, P.; Harnett, C.; Craighead, H.; Baird, B.; Ober, C. K. Molecular templates for bio-specific recognition by low energy electron beam lithography. Nanobiotechnology 2005, 1, 23-34. (31) Hyun, J.; Ma, H.; Banerjee, P.; Cole, J.; Gonsalves, K.; Chilkoti, A. Micropatterns of a cell-adhesive peptide on an amphiphilic comb polymer film. Langmuir 2002, 18, 2975-2979. (32) Jun, Y.; Cha, T.; Guo, A.; Zhu, X.-Y. Patterning protein molecules on poly(ethylene glycol) coated Si(111). Biomaterials 2004, 25, 3503-3509.
Experimental Section Materials. 2-[Methoxy(polyethylenoxy)propyl]trimethoxysilane (PEG, CH3O(CH2CH2O)6-9(CH2)3Si(OCH3)3, FW 460-590) and tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane (commonly known as fluorooctatrichlorosilane, FOTS, F(CF2)6CH2CH2SiCl3, CAS no. 78560-45-9, FW 481.55) were obtained from Gelest. Acetic acid, pyridine, anhydrous toluene, 96% sulfuric acid, and 30 wt % hydrogen peroxide solution in water were obtained from Sigma-Aldrich. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC, g7 mol FITC per mol albumin, Sigma), and phosphate buffered saline (PBS, Sigma) tablets were used as received. The PBS buffer solution was prepared by dissolving the PBS tablet in water to yield 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, and a pH of 7.4 at 25 °C. Shipley S1818 photoresist and AZ 300 MIF Developer were used for photopatterning of silicon substrates. Single-side polished Si(100) wafers with thickness of 356-406 µm were purchased from Montco Silicon Technologies.
Zoospore Settlement on Chemically Patterned Surfaces.
Langmuir, Vol. 24, No. 2, 2008 505
Figure 2. Diagram showing layout of chemically patterned Si(100) wafer. Image (right) shows close-up of the patterned squares at the end of step 4, Figure 1. Each square is 1 cm2 in area. 0 refers to a uniformly covered square of either PEGylated or fluorinated SAM opposite to the background. The number denotes squares with stripes of fluorinated and PEGylated SAMs of widths 2, 5, 20, 50, 100, 200, and 500 µm. Background is either pure PEG or pure fluorinated monolayer.
Figure 1. Schematic diagram of the process used for creating alternating stripes of FOTS and PEG on silicon substrates using photolithography: (1) vapor deposition of FOTS; (2) coating with a layer of positive tone photoresist; (3) resist exposure through a chrome-patterned glass mask; (4) dissolution of exposed regions of the resist in aqueous base; (5) etching of exposed FOTS with air or oxygen plasma; (6) back-filling with PEGylated silane; and (7) stripping of photoresist. (Diagram not to scale.) Photolithographic Patterning of FOTS and PEG Self-Assembled Monolayers. Chrome-patterned soda-lime glass masks were prepared using L-edit mask layout editor (Tanner Research Inc.) and GCA/Mann 3600F Pattern Generator. The silicon wafers were immersed in a piranha solution (7:3 v/v conc H2SO4/30% aq H2O2) for 30 min, rinsed thoroughly with distilled water, and dried using ultrahigh-purity nitrogen gas. The wafers were exposed to oxygen plasma in a Harrick Plasma Cleaner. Vapor deposition of FOTS was performed using a molecular vapor deposition system MV-100 (Applied Microstructures). The vapor deposition process involved a 5 min precleaning with oxygen plasma (150 sccm O2 flow, and a radio frequency power of 200 W) prior to the formation of selfassembled monolayers. FOTS vapor (0.7 Torr) and water vapor (6 Torr) were introduced into the reaction chamber and allowed to react for 20 min before evacuation and purging with nitrogen. Positive-tone photoresist Shipley S1818 was spin-coated on the FOTS-covered silicon wafers at a rotational speed of 4000 rpm and baked at 115 °C for 1 min to remove residual solvent. Contact/ proximity printing of the grating patterns on the mask onto the photoresist was carried out using a Hybrid Technology Group contact/ proximity mask aligner (HTG III-HR). A 405 nm light source with an intensity of ∼17 mW/cm2 was used. The exposure time was set to 4.5 s. A Hamatech-Steag wafer processor (HMP900) was used for developing the patterns using a spray-puddle process.
The patterned wafers were rinsed with distilled water and subjected to oxygen plasma treatment for at least 10 min in a Harrick Plasma Cleaner. The wafers were then immersed in a solution of the PEGylated silane (parts by volume: 4 mL of PEG, 100 mL of anhydrous toluene, 25 µL of pyridine, and 10 µL of acetic acid) for ∼12 h in a glove box, after which they were rinsed with toluene to remove excess silane. The resist was stripped off by thoroughly rinsing with acetone. The wafers with patterned stripes of FOTS and PEG were finally baked in a vacuum oven at 100 °C for 10 min to ensure covalent attachment of FOTS and PEG to the silicon surface. The overall procedure is shown in Figure 1. Protein Adsorption. Chemically patterned wafer surfaces were incubated with a 50 µg/mL solution of fluorescein-tagged BSA protein in PBS buffer solution (pH ∼7.4) at room temperature, in the dark, for 2 h. The surfaces were then rinsed with PBS buffer solution and observed under an Olympus BX51 microscope with a 40× UPlan Fluorite 40× dry objective (N.A. 0.75). Images were acquired using a Roper CoolSnap HQ CCD camera and Image Pro image acquisition and processing software. The fluorescent patterns were observed with a 450 nm excitation and 550 nm emission filter sets. Fluorescence images reported here were analyzed using the Image J 1.36b software. Zoospore Settlement Assays. Plants of UlVa linza were collected from Wembury Beach, United Kingdom (50°18′N, 4°02′W). Zoospores were released from reproductive tips and prepared for assays as described in ref 14. Three replicates of each type of silicon wafer were placed in 9 cm diameter Petri dishes and equilibrated with 25 mL artificial seawater (Tropic Marine) for 10 min prior to the start of the experiment. The seawater was removed, and 25 mL of spore suspension was added containing 1.25 × 106 spores/mL. Spores were allowed to settle for 1 h in darkness, after which the wafers were gently washed in fresh seawater and fixed for 15 min in 2.5% glutaraldehyde in seawater and then washed as described previously.14 The density of settled spores was determined using a Zeiss epifluorescence microscope with video camera and image analysis software (Imaging Associates Ltd.) as described by Callow et al.33 Spores were visualized by the fluorescence signal from the chlorophyll they contain. Thirty fields of view in each square, on each of three replicate wafers, were captured as images for analysis (90 fields of view per treatment in total). Data are presented as the mean ( 95% confidence limits. The data were analyzed, where indicated, by a two-way ANOVA, and significant differences between treatments determined by a post-hoc Tukey test comparing the differences between the means with a variance value derived from the data set. (33) Callow, M. E.; Jennings, A. R.; Brennan, A. B.; Seegert, C. E.; Gibson, A.; Wilson, L.; Feinberg, A.; Baney, R.; Callow, J. A. Microtopographic cues for settlement of zoospores of the green fouling alga Enteromorpha. Biofouling 2002, 18, 237-245.
506 Langmuir, Vol. 24, No. 2, 2008
Finlay et al.
Figure 3. Images and intensity profiles showing fluorescence from BSA-FITC adsorbed on the hydrophobic fluorinated regions of the patterned squares: 2 µm stripes on FOTS background (left); 2 µm stripes on PEG background (center); and 5 µm stripes on PEG background (right). The left and right images were acquired at the boundaries of the striped regions and the corresponding backgrounds. The lines on each image denote the transect for intensity profiles.
Results Pattern Formation. Silicon surfaces with alternating stripes of FOTS and PEG were prepared on silicon wafers using a positive tone photoresist process exposed to 405 nm UV light. Figure 1 shows a schematic of the steps involved in the fabrication of hydrophilic PEGylated stripes in a hydrophobic fluorinated background. Silicon wafers were first functionalized with FOTS using vapor deposition,34 and then covered with a layer (∼1.5 µm thick) of a standard diazonaphthoquinone-novolac resist system. The resist-covered FOTS SAM was exposed through a glass mask that had transparent stripes of 2, 5, 20, 50, 100, 200, and 500 µm width in an opaque background (Figure 2).35 The lines and spaces in each 1 cm × 1 cm patterned region were of equal widths. The exposed regions of the resist were dissolved in an aqueous base solution, and the unprotected FOTS was etched using oxygen plasma. To ensure a high extent of surface hydroxylation for reaction with the PEGylated silane, the wafers were exposed to plasma longer than the etch time needed to completely remove FOTS from the exposed regions.36 The resulting surface silanol groups were reacted with the PEGylated silane in a toluene solution. The resist was both quite resistant to plasma etching, as evident from its high retained thickness after the etching step (5 in Figure 1), and it was also sufficiently resistant to toluene36 to enable PEG deposition in only the exposed regions. Surfaces consisting of FOTS stripes in a PEGylated background were similarly prepared starting from a silicon wafer that was covered with PEGylated SAM with additional oxygen plasma (30 min) etching to remove PEG SAMs in the exposed regions. (34) Mayer, T. M.; de Boer, M. P.; Shinn, N. D.; Clews, P. J. Chemical vapor deposition of fluoroalkylsilane monolayer films. J. Vac. Sci. Technol., B 2000, 18, 2433-2440. (35) Diffraction of light at the edge of an opaque stripe on the mask limits the resolution of patterns. For a periodic grating consisting of opaque and transparent stripes of equal width, w, the minimum theoretical resolution is given by wmin ) 1.5xλ(s+0.5t), where λ is the exposure wavelength, s is the width of the gap between mask and resist, and t is the resist thickness. For a 405 nm UV light, a 4 µm gap, and a resist thickness of 1.5 µm, the minimum feature size possible by the technique used is about 2 µm. Other patterning methods would enable smaller features to be produced. (36) Asgill, N.; Krishnan, S.; Ober, C. K. (2005) Fabrication of patterned hydrophobic and hydrophilic surfaces to study cell adhesion. Cornell University Nanobiotechnology Center Home Page. http://www.nbtc.cornell.edu/mainstreetscience/reu_2005/N_Asgill.ppt (accessed Aug 2005).
Figure 4. The mean surface density of UlVa spores (spores mm-2) settled on patterned squares with either (a) a fluorinated or (b) a PEGylated background. The total surface area of each type of stripe covered exactly 50% of each square. Also shown (horizontal lines) are the densities of spores settled on the fluorinated background and the pure (100%) PEG square (a) and on the PEGylated background and the pure (100%) fluorinated square (b). Each point is the mean from 90 counts on 3 replicate patterns. Bars show 95% confidence limits.
Protein Adsorption and Visualization of Chemical Patterns by Fluorescence Microscopy. Adsorption of FITC-labeled bovine serum albumin was used to confirm the fidelity of the patterning process. The resistance of PEG to protein adsorption and cell adhesion is well-documented in the biofouling literature.11 The hydrophilic PEGylated regions of all patterns showed lower adsorption of fluorescent BSA compared to the hydrophobic
Zoospore Settlement on Chemically Patterned Surfaces.
Langmuir, Vol. 24, No. 2, 2008 507
Figure 5. Images of spores settled on wafers with a fluorinated background. Images were taken under a low-power dissecting microscope using incident illumination. Images are shown at 10× microscope magnification except the 5 µm and 2 µm images, which are shown at 20× magnification for clarity.
fluorinated regions: Figure 3 shows representative fluorescence microscopy images and intensity profiles. Comparison Between Zoospore Settlement on PEGylated and Fluorinated Backgrounds. Visual inspection of the background areas of wafers after the settlement assay showed a green covering on the wafers with a fluorinated background compared to those with a PEGylated background indicating a higher settlement density of spores on the nonpolar surface. This visual impression was confirmed by the spore counts (Figure 4). The mean spore densities were 484 ( 37 (2 × standard error) and 136 ( 10 spores mm-2 for the fluorinated and PEGylated backgrounds, respectively. Two-way analysis of variance with Tukey tests showed that these differences were statistically significant (F 1, 1602 ) 143; P < 0.05). Comparison Between Zoospore Settlement on Fluorinated and PEGylated Stripes. The patterns were so designed that the total area of fluorinated and PEGylated surface within each 1 cm × 1 cm striped square, irrespective of the composition of the background, was identical (i.e., a 50/50 distribution of PEGylated and fluorinated areas). Therefore, the null hypothesis tested in these experiments was that the total number of spores settled on
each patterned 1 cm × 1 cm square will be the same. Departures from this would show that the spores were influenced by the patterning, thus rejecting the null hypothesis. The mean density of settled spores on each square is shown in Figure 4. On the wafers with a fluorinated background, there was no detectable trend to suggest that the patterns were influencing settlement; all showed a level of settlement per unit area approaching or exceeding that of the fluorinated background (the lowest level of settlement on the 2 µm striped squares was only 23% less than the pure FOTS background). The level of settlement on the 100% PEGylated (i.e., unpatterned) square on the fluorinated background was, as anticipated, the lowest level recorded on the fluorinated wafer (Figure 4a). In contrast, spore density on the striped squares of wafers with a PEGylated background showed a marked influence of feature size. There was a high density of settled spores on squares with stripes ranging from 20 to 500 µm, approaching that of the density of the 100% fluorinated (i.e., unpatterned) square (Figure 4b). However, on squares with stripes that were 2 and 5 µm wide, the spore density was low and similar to that on the PEGylated background itself. Two-way analysis of variance and Tukey tests
508 Langmuir, Vol. 24, No. 2, 2008
Finlay et al.
Figure 6. Images of spores settled on wafers with a PEGylated background. Images were taken under a low-power dissecting microscope using incident illumination. Images are shown at 10× microscope magnification except the 5 µm and 2 µm images, which are shown at 20× magnification for clarity.
showed that the density of settled spores on the 2 and 5 µm stripes and the PEG background) were not significantly different from each other (F 8, 1602 ) 77; P < 0.05). The level of spores settled on the pure PEG square on the FOTS background (Figure 4a) was approximately 35% higher than the level of spores settling on the pure PEG background (Figure 4b). A possible reason for this lies in the ability of settled spores to influence the settlement of swimming spores, possibly through the release of chemical signals (ref 14 and unpublished data). Spores settling on the FOTS background immediately surrounding the PEG square could stimulate attraction of spores to that region of the wafer, leading to a higher level of settlement on the PEG square, compared with a uniformly PEGylated background, i.e., we are witnessing the outcome of competing signalssthose that attract spores to the vicinity balanced by the uncongenial surface properties of PEG. The distribution of settled spores within the patterns was explored by microscopy. Low-magnification images (Figure 5) clearly showed differences in spore settlement density between adjacent stripes. On the fluorinated background, a striped distribution of spores could clearly be seen on the 5, 20, 50, 100, 200, and 500 µm patterns, but no striping could be seen on the
2 µm patterns, which is anticipated, since a spore itself is approximately 5 µm in diameter. On the wafers with a PEGylated background, a difference in the density of settled spores could be seen on the 20, 50, 100, 200, and 500 µm stripes, but not on either the 5 or 2 µm patterns (Figure 6). Higher-magnification images of selected regions of the backgrounds and the boundaries between stripes are shown in Figure 8. The differences in spore density between nonpatterned fluorinated and PEGylated regions can clearly be seen, and relatively discrete differences in spore distribution can be observed at the boundaries between stripes in the patterned areas. It can also be observed that spores settled gregariously on the fluorinated surfaces, forming large clumps of spores, in contrast to the single spores that predominated on the PEGylated surfaces. In order to verify that the density of settled spores on patterned squares determined by random sampling truly reflected the density on the squares overall, more detailed counts were made on PEGylated and fluorinated stripes of the 500 µm patterns (the chemistry of the counted stripes was determined by reference to the background). From this, a “calculated mean” coverage was determined for the whole square, being the sum of 50% of the spore density of fluorinated stripes and 50% of the spore density
Zoospore Settlement on Chemically Patterned Surfaces.
Figure 7. Spore settlement on squares with 500 µm stripes. Black columns from wafer with fluorinated background; white columns from wafer with PEG background. “500 F stripe” is the spore density on 500-µm-wide fluorinated stripes; “500 P stripe” is the spore density on 500-µm-wide PEG stripes. “Sum stripes” is the calculated mean for spore distribution on a square based on the sum of 50% spore density on fluorinated stripes and 50% spore density on PEG stripes. “F 500” and “P 500” are the mean counts of spore settlement on 500 µm patterns on fluorinated and PEGylated backgrounds, respectively, as determined by random sampling of the square, without regard to the pattern (i.e., the same data as shown in Figure 4). Error bars show 95% confidence limits from 90 counts made on each section of one wafer.
of the PEGylated stripes. Results (Figure 7) showed that the “calculated mean” values were very similar to the mean values determined by sampling the whole area of the patterned square, thus validating the counts shown in Figure 4.
Discussion Settlement of zoospores on a surface results in irreversible adhesion to that surface, achieved by the rapid discharge of a glycoprotein adhesive37 and withdrawal of the flagella (motility organelles) inside the cell. Prior to settlement, a number of patterns of zoospore exploratory behavior can be observed.14,38 Some
Langmuir, Vol. 24, No. 2, 2008 509
spores commit within seconds to minutes to permanent adhesion, while others continue to explore close to the surface, or if the surface is “inhospitable” for settlement, spores move away from the surface back into the water column. The rate of settlement is determined by the response of spores to a number of surface cues, including topography15,16 and physicochemical properties such as wettability.17,18 In the latter case, higher numbers of spores settle on uniformly hydrophobic surfaces compared to uniformly hydrophilic surfaces. The purpose of the experiments reported in the present paper was to explore how spores respond when presented with a choice of surfaces of contrasting wettability. The null hypothesis tested was that spores would not be influenced by the patterns and that the overall density of settled spores on each patterned square would be the same, since all patterned squares contained a 1:1 ratio of PEGylated and fluorinated areas. Departures from this would show that the spores were influenced by the patterning, thus disproving the null hypothesis. The results reported here for patterns created on the wafers with a fluorinated background appear to support the null hypothesis, since there appeared to be little influence of feature size on settlement. Furthermore, the overall level of settlement appeared to reflect that of the fluorinated background. This suggests that spores regarded the patterns created on the fluorinated background as uniformly fluorinated, i.e., there was little influence of the less favorable PEGylated regions. However, on the wafers with the PEGylated background, the null hypothesis is clearly not supported, since there was a clear effect of feature size on settlement. For stripe widths of 2 and 5 µm, the level of settlement on the patterned squares was low, and similar to the PEGylated background itself. This implies that the surface domains of fluorinated graft were of insufficient size to attract spores. On the other hand, squares containing stripes above 5 µm had high levels of spore settlement, similar to that on the uniformly fluorinated square. This implies that the spores were being attracted to the squares with stripe widths above 5 µm in the same density as they were to the uniformly fluorinated square. This result can only be explained if more spores per unit area settled on the fluorinated stripes than on the uniformly
Figure 8. Autofluorescence images of settled spores on regions of a wafer with a PEGylated background. Images: Top row, pure PEG background (left), FOTS square (right). Bottom row, border region between 500 µm PEG and 500 µm FOTS stripe (left), 200 µm striped square centered on FOTS region (right). The dotted lines indicating the boundaries between PEGylated and fluorinated areas are provided to assist interpretation. Magnification factor 900×.
510 Langmuir, Vol. 24, No. 2, 2008
fluorinated square. The data in Figures 5 and 8 showed this was the case and suggests that spores that approached the PEGylated stripes on the patterned squares moved over to the adjacent, more “hospitable” fluorinated area on which they settled. A similar type of behavior has previously been recorded for spores settling on surfaces fabricated in polydimethylsiloxane (PDMS) that had 1 cm × 1 cm squares with alternating channels and ridges, each 5 µm wide and deep.33 Spores were attracted to settle in the channels, and an area depleted in settled spores was seen surrounding the patterned area. The mean density of settled spores in all patterned squares thus depended on whether the background was attractive or unattractive for settlement. Why should the chemistry of the background have such a strong influence? It is important to realize that the wafer backgrounds represent a large proportion of the total surface area of each wafer (approximately 82% of a 45 cm2 wafer), and therefore, a large proportion of spores can escape making the choice between a chemically more complex area presenting both attractive and unattractive domains. However, where the background is in itself unattractive for settlement (i.e., PEGylated), spores are more likely to explore the squares containing the more attractive fluorinated regions. Consistent with this, it was noted that the spore density on the patterns with a PEGylated background was markedly higher for all squares, except where the pattern spacing was 2 or 5 µm, than the equivalent squares on the wafer with the fluorinated background. The “inhospitable” PEG background in effect encourages more spores to search for more hospitable areassand thus there are high levels of settlement on pure fluorinated squares, and the fluorinated regions of squares with stripe widths of 5 µm above. (37) Callow, J. A.; Callow, M. E. The UlVa Spore Adhesive System. In Biological AdhesiVes; Smith, A. M., Callow. J. A., Eds. Springer: Heidelberg, 2006; pp 63-78. (38) Heydt, M.; Rosenhahn, A.; Grunze, M.; Pettitt, M.; Callow, M. E.; Callow, J. A. Digital in-line holography as three dimensional tool to study motile marine organisms during their exploration of surfaces. J. Adhes. 2007, 83, 417-430.
Finlay et al.
However, at some critical dimension below 20 µm, presumably the contrasting chemistries presented by the 2 and 5 µm patterns could not be distinguished. The spores regarded these patterns as pure PEG and avoided settlement on them, in preference to the larger stripes patterns where the spores could more clearly locate the favorable fluorinated areas. The aim of the present study was to use patterned model surfaces to explore spore settlement behavior, and we do not wish to imply that these specific patterns or chemistries are the basis of future practical, antifouling coatings. Nevertheless, the demonstration here that spore settlement is reduced on patterns of contrasting wettability below a certain size, compared with a purely hydrophobic surface, might provide conceptual guidance to those developing practical antifouling coatings based on mosaics of hydrophilic and hydrophobic domains. Such coatings could include, for example, phase-segregating polymer blends or amphiphilic polymers with copolymer additives capable of producing chemically heterogeneous surfaces. In future work, we will explore more complex patterns including randomized domains or domains of different shapes to investigate whether these give different patterns of spore settlement compared to the linear patterns investigated here. Acknowledgment. The authors are grateful to the Office of Naval Research for support through grants N00014-02-1-0170 (C.K.O. and E.J.K.) and N00014-05-1-0134 (J.A.C. and M.E.C.). Photolithographic patterning was performed at the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS 03-35765). We also benefited from the facilities at the Nanobiotechnology Center (NBTC) and Cornell Center for Materials Research (CCMR), both of which are supported by the NSF. N.A. acknowledges NBTC support through the Research Experience for Undergraduates program. LA702275G
