Molecular Gradients of Bioinertness Reveal a Mechanistic Difference

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Langmuir 2009, 25, 1547-1553

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Molecular Gradients of Bioinertness Reveal a Mechanistic Difference between Mammalian Cell Adhesion and Bacterial Biofilm Formation Erik A. Burton,† Karen A. Simon,† Shuyu Hou,‡ Dacheng Ren,*,‡,§,| and Yan-Yeung Luk*,†,‡ Departments of Chemistry, Biomedical and Chemical Engineering, CiVil and EnVironmental Engineering, and Biology, Syracuse UniVersity, Syracuse, New York 13244 ReceiVed February 18, 2008. ReVised Manuscript ReceiVed NoVember 26, 2008 Chemical gradients play an important role in guiding the activities of both eukaryotic and prokaryotic cells. Here, we used molecularly well-defined chemical gradients formed by self-assembled monolayers (SAMs) on gold films to reveal that mammalian cell adhesion and bacterial biofilm formation respond differently to a gradient of surface chemistry that resists cell attachment. Gradient self-assembled monolayers (SAMs) consisting of two mixed alkanethiols were fabricated by differential exposure of the gold film to one alkanethiol, followed by soaking in another alkanethiol solution. A gradient in bioinertness that resisted cell attachment was created on SAMs from a gradient in the surface density of HS(CH2)11(OCH2CH2)3OH, backfilled with either HS(CH2)11OH or HS(CH2)11CH3. Measurements of the amounts of mammalian cells and bacterial biofilms on these gradient surfaces reveal that, for mammalian cells, a critical density of adhesion ligands from absorbed proteins on surfaces exists for supporting maximum adhesion and proliferation, whereas for the bacterium Escherichia coli, the amount of biofilm formed on surfaces increased linearly with the surface density of adhesive groups (methyl or hydroxyl groups) in different media. These results are consistent with mammalian cell adhesion requiring an anchorage via specific molecular recognitions and suggest that biofilms can form by immobilization of bacteria via nonspecific interaction between bacteria and surfaces.

Introduction Both eukaryotic and prokaryotic cells respond to physical gradients such as stiffness1 or topography2 of a surface and chemical gradients, either diffusible3-6 or surface-bound, as guiding cues for their activities.7,8 We are interested in using man-made materials and systems9,10 to study and control biological processes, including heterogeneous biocatalysis, mammalian cell adhesion,2 and bacterial biofilm formation.11 Here, we use molecularly well-defined and surface-bound chemical gradients to unravel important differences in the dependence of mammalian cell adhesion and bacterial biofilm formation on surface gradients that can help elucidate the mechanisms of these two processes. Most mammalian cells are anchorage dependent; denying their adhesion to a surface often results in programmed cell death.12 Adhesion of mammalian cells is an important process for many * To whom correspondence should be addressed. (Y.-Y.L.) E-mail: [email protected]. Phone: (315) 443-7440. Fax: (315) 443-4070. (D.R.) E-mail: [email protected]. Phone: (315) 443-4409. Fax: (315) 443-9175. † Department of Chemistry. ‡ Department of Biomedical and Chemical Engineering. § Department of Civil and Environmental Engineering. | Department of Biology. (1) Lo, C.-M.; Wang, H.-B.; Dembo, M.; Wang, Y.-L. Biophys. J. 2000, 79, 144–152. (2) Simon, K. A.; Burton, E. A.; Han, Y.; Li, J.; Huang, A.; Luk, Y.-Y. J. Am. Chem. Soc. 2007, 129, 4892–4893. (3) Hu, H. Neuron 1999, 23, 703–711. (4) Hu, H. Nat. Neurosci. 2001, 4, 695–701. (5) Zou, Y. Trends Neurosci. 2004, 27, 528–532. (6) Lyuksyutova, A. I.; Lu, C.-C.; Milanesio, N.; King, L. A.; Guo, N.; Wang, Y.; Nathans, J.; Tessier-Lavigne, M.; Zou, Y. Science 2003, 302, 1984–1988. (7) Norbeck, B. A.; Feng, Y.; Denburg, J. L. DeVelopment 1992, 116, 467–79. (8) Baier, H.; Bonhoeffer, F. Science 1992, 255, 472–5. (9) Simon, K. A.; Sejwal, P.; Gerecht, R. B.; Luk, Y.-Y. Langmuir 2007, 23, 1453–1458. (10) Han, Y.; Cheng, K.; Simon, K. A.; Lan, Y.; Sejwal, P.; Luk, Y.-Y. J. Am. Chem. Soc. 2006, 128, 13913–13920. (11) Hou, S.; Burton, E. A.; Simon, K. A.; Blodgett, D.; Luk, Y.-Y.; Ren, D. Appl. EnViron. Microbiol. 2007, 73, 4300–4307. (12) Mrksich, M. Chem. Soc. ReV. 2000, 29, 267–273.

biological processes, including cell growth and differentiation,13-15 fetal development,16 wound healing,16 immune response,16 and integration of prosthetic joints and dental implants.12,16 A reduction in this anchoring dependence is often associated with the spread of cancer cells.12,17-19 In contrast, bacteria are able to grow in planktonic (free floating) form, but also have a high propensity to attach to surfaces and to form colonies embedded in a secreted polysaccharide matrixs collectively named biofilms.20-23 Such biofilms usually are strongly attached to surfaces, and the embedded bacteria often become highly tolerant to antibiotics and disinfection treatments, making these biofilms the source of a wide range of persistent problems, including medical-device-associated infections, dental plaque, spread of waterborne pathogens, and corrosion in industrial settings.20,21,23 At a gross level, the mechanisms of adhesion of mammalian cells and the formation of biofilms both include complex, multistep processes. For example, specific protein binding to surfaces, nonspecific protein adsorption, and cell attachment are involved in the adhesion of mammalian cells, and reversible attachment, irreversible attachment, and maturation with an increase in biofilm thickness and biomass are involved in biofilm formation.23-25 (13) Thiery, J. P. Curr. Opin. Genet. DeV. 2003, 13, 365–371. (14) Chang, H.-H.; Kau, J.-H.; Lo, S. J.; Sun, D.-S. Cell Biol. Int. 2003, 27, 123–133. (15) Koller, M. R.; Papoutsakis, E. T. Bioprocess Technol. 1995, 20, 61–110. (16) Anselme, K. Biomaterials 2000, 21, 667–681. (17) Schwartz, M. A.; Assoian, R. K. J. Cell Sci. 2001, 114, 2553–2560. (18) Assoian, R. K. J. Cell Biol. 1997, 136, 1–4. (19) Chiarugi, P.; Giannoni, E. Antioxid. Redox Signaling 2005, 7, 578–592. (20) Palmer, R. J., Jr.; White, D. C. Trends Microbiol. 1997, 5, 435–40. (21) Costerton, J. W. Int. J. Antimicrob. Agents 1999, 11, 217–221. (22) O’Toole, G.; Kaplan, H. B.; Kolter, R. Annu. ReV. Microbiol. 2000, 54, 49–79. (23) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. ReV. Microbiol. 2004, 2, 95–108. (24) Pruss, B. M.; Besemann, C.; Denton, A.; Wolfe, A. J. J. Bacteriol. 2006, 188, 3731–3739. (25) Stoodley, P.; Sauer, K.; Davies, D. G.; Costerton, J. W. Annu. ReV. Microbiol. 2002, 56, 187–209.

10.1021/la803261b CCC: $40.75  2009 American Chemical Society Published on Web 01/09/2009

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In the molecular realm, these two processes are governed by vastly different molecular biology and cell signaling processes. Study of these two processes is particularly challenging because both processes are associated with surfaces that are often complex and not well-defined. In vivo, mammalian cell adhesion occurs on an extracellular matrix consisting of a complex mixture of varying compositions of proteins and carbohydrates.12,14,15,19,26,27 Outside a biological system and under conditions that permit proliferation, mammalian cells adhere on almost any surfaces mediated by the adsorbed proteins that occur ubiquitouslys except those with surface chemistry that is deliberately designed to resist cell adhesion.12 These designed surfaces are termed “bioinert”.28-30 Likewise, bacterial biofilms form on almost all kinds of surfaces either inside or outside a host organism and can form under conditions that are harsher than those permitting mammalian cell proliferation. In spite of the ubiquitous nature of mammalian cell adhesion and bacterial biofilm formation, changes in either surface chemistry or surface topography do influence the details of these processes.2,11,12,31 These variations in surface features, combined with nonspecific protein adsorption, are significant challenges for understanding and the control of mammalian cell adhesion and bacterial biofilm formation. While the response of mammalian cells to surface-associated gradients, including chemistry,8 topography,2 and rigidity,1 have been studied, a detailed understanding of biofilm formation on these gradient surfaces is still lacking. The understanding of how cells respond to a molecular gradient can form the basis for building new tools to study the mechanisms of these biological processes.32-34 In this work, we use two sets of surface-bound chemical gradients to study the response of mammalian cells and bacteria to surface chemistry that resists their adhesion. Many different types of surface-associated gradients have been created to address a wide variety of fundamental questions and application problems.2,8,35-51 Methods of producing these (26) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491–7. (27) Chiarugi, P.; Fiaschi, T. Cell. Signalling 2007, 19, 672–682. (28) Brown, C. L.; Whitehouse, M. W.; Tiekink, E. R. T.; Bushell, G. R. Inflammopharmacology 2008, 16, 133–137. (29) Hayashi, K.; Inadome, T.; Tsumura, H.; Mashima, T.; Sugioka, Y. Biomaterials 1993, 14, 1173–9. (30) Lee, D.; Yang, S. Y.; Cohen, R. E.; Rubner, M. F. PMSE Prepr. 2004, 90, 526–527. (31) Luk, Y.-Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604–9608. (32) Abhyankar, V. V.; Lokuta, M. A.; Huttenlocher, A.; Beebe, D. J. Lab Chip 2006, 6, 389–393. (33) Genson, K. L.; Fasolka, M. J. PMSE Prepr. 2007, 97, 826. (34) Kunzler, T. P.; Drobek, T.; Sprecher, C. M.; Schuler, M.; Spencer, N. D. Appl. Surf. Sci. 2006, 253, 2148–2153. (35) Ruardy, T. G.; Schakenraad, J. M.; van der Mei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 29, 1–30. (36) Liedberg, B.; Tengvall, P. Langmuir 1995, 11, 3821–7. (37) Wijesundara, M. B. J.; Fuoco, E.; Hanley, L. Langmuir 2001, 17, 5721– 5726. (38) Kraus, T.; Stutz, R.; Balmer, T. E.; Schmid, H.; Malaquin, L.; Spencer, N. D.; Wolf, H. Langmuir 2005, 21, 7796–7804. (39) Julthongpiput, D.; Fasolka, M. J.; Zhang, W.; Nguyen, T.; Amis, E. J. Nano Lett. 2005, 5, 1535–1540. (40) Balss, K. M.; Fried, G. A.; Bohn, P. W. J. Electrochem. Soc. 2002, 149, C450-C455. (41) Choi, S.-H.; Newby, B. Z. Langmuir 2003, 19, 7427–7435. (42) Dertinger, S. K. W.; Chiu, D. T.; Jeon, N. L.; Whitesides, G. M. Anal. Chem. 2001, 73, 1240–1246. (43) Efimenko, K.; Genzer, J. AdV. Mater. 2001, 13, 1560–1563. (44) Fuierer, R. R.; Carroll, R. L.; Feldheim, D. L.; Gorman, C. B. AdV. Mater. 2002, 14, 154–157. (45) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311–8316. (46) Lestelius, M.; Engquist, I.; Tengvall, P.; Chaudhury, M. K.; Liedberg, B. Colloids Surf., B 1999, 15, 57–70. (47) Liedberg, B.; Wirde, M.; Tao, Y.-T.; Tengvall, P.; Gelius, U. Langmuir 1997, 13, 5329–5334. (48) Spijker, H. T.; Bos, R.; van Oeveren, W.; de Vries, J.; Busscher, H. J. Colloids Surf., B 1999, 15, 89–97. (49) Terrill, R. H.; Balss, K. M.; Zhang, Y.; Bohn, P. W. J. Am. Chem. Soc. 2000, 122, 988–989.

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gradients include the formation of monolayers of alkanethiols on gold films with a gradient in surface topography,2 differential application of chemical components by migration of solution in a wedge-shaped capillary,8 diffusion of alkanethiols across a gold surface,36,46 differential polyatomic ion deposition on a polymer surface,37 microcontact printing of alkanethiols on gold surfaces with specially designed PDMS stamps,38 graded UVozonolysis of self-assembled monolayers of alkanethiols,39 patterning of alkanethiols by electrochemical methods,40,49,52 differential application of organosilanes on a silicon surface,41 application of solutions via microfluidic networks,42,45,50 diffusion of alkylchlorosilanes on silicon surfaces,43 replacement lithography of self-assembled monolayers,44 grafting of PEO moieties on a surface by differential corona discharge,51 and differential gas plasma treatment of polyethylene films.48 Spencer and co-workers reported an efficient differential exposure method for the fabrication of chemical gradients by slow immersion of a gold film into an alkanethiol solution to afford an incomplete self-assembled monolayer (SAM) with a gradient in the spacing of the alkanethiols, followed by backfilling the surface with another alkanethiol solution.53 This method results in a two-component self-assembled monolayer, with a gradient in surface composition, such that one end of the SAM is composed primarily of the first component, while the other end consists mainly of the complementary component, with a continuous change in the surface density between. Among other surfaces, SAMs on gold films are molecularly well-defined and provide a wide variety of surface chemistries, including those for inhibiting or promoting protein adsorption, mammalian cell adhesion,2,12,31,35,54-61 and biofilm formation.11,62 In this work, we used SAMs to create chemical gradients of bioinertness on surfaces (Figure 1). Monolayers formed by oligo(ethylene glycol)-terminated alkanethiol (e.g., HS(CH2)11(OCH2CH2)3OH) exhibit strong resistance to protein adsorption, mammalian cell adhesion,31,54,57 and bacterial biofilm formation,11 whereas monolayers formed by other alkanethiols, including HS(CH2)11CH3 and HS(CH2)11OH, exhibit different degrees of affinity for mammalian cell adhesion31 and biofilm formation.11 Using the differential exposure method,53 we first immersed a gold film, supported on a glass substrate, into a solution of either methyl- or hydroxyl-terminated alkanethiol (3 µM) solution at a controlled rate (40 µm/s), resulting in a continuously decreasing exposure time from the entering end of the glass substrate. Upon reaching full immersion, the slide was removed, rinsed with ethanol, and then soaked in HS(CH2)11(50) Fosser, K. A.; Nuzzo, R. G. Anal. Chem. 2003, 75, 5775–5782. (51) Jeong, B. J.; Lee, J. H.; Lee, H. B. J. Colloid Interface Sci. 1996, 178, 757–63. (52) Plummer, S. T.; Bohn, P. W. Langmuir 2002, 18, 4142–4149. (53) Morgenthaler, S.; Lee, S.; Zuercher, S.; Spencer, N. D. Langmuir 2003, 19, 10459–10462. (54) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877–8. (55) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164–7. (56) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55–78. (57) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388–2391. (58) Zhang, S.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213– 1220. (59) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu, W.-S. Chem. Biol. 1997, 4, 731–737. (60) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270–274. (61) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Angew. Chem., Int. Ed. 2001, 40, 1093–1096. (62) Ista, L. K.; Fan, H.; Baca, O.; Lopez, G. P. FEMS Microbiol. Lett. 1996, 142, 59–63.

Mammalian Cell Adhesion and Bacterial Biofilm Formation

Figure 1. Schematic representation of the gradient of bioinert chemistry by two-component SAMs prepared by controlled immersion of gold films into a solution of either hydroxyl- or methyl-terminated alkanethiol, followed by soaking in a solution of tri(ethylene glycol)-terminated alkanethiol.

(OCH2CH2)3OH solution (2 mM) overnight, resulting in a twocomponent molecular gradient SAM. On these molecular gradients (Figure 1), we studied the adhesion of Swiss 3T3 albino fibroblast cells and the biofilm formation of E. coli. Adherent mammalian cells were directly counted using an optical microscope, while biofilm formation was measured by quantifying the surface coverage using COMSTAT software.11,63 We also used X-ray photoelectron spectroscopy (XPS) to characterize the atomic composition of the gradients at several points along their surfaces.53,64-66 Because the SAMs presenting gradients in bioinert chemistry are in contact with culture media that contain a wide range of proteins, and because adsorbed proteins support cell adhesion, the characterization of protein density on the chemical gradient provides a direct measure of the adhesiveness of the surface for mammalian cells.67,68

Experimental Section Chemicals. Alkanethiols were purchased from Aldrich Chemicals (Milwaukee, WI). Ethanol (99.98%) was from Pharmco (Brookfield, CT) and was used as a solvent for all of the alkanethiol solutions and for washing the modified surfaces. HS(CH2)11(OCH2CH2)3OH was synthesized using previously reported methods.69 Glass Substrates. Substrates for the gold films were Fisher’s finest premium microscope slides (Fisher Scientific, Pittsburgh, PA). Prior to gold deposition, the slides were cleaned in piranha solution. Warning! Piranha solution is extremely corrosiVe and also has the potential for detonation if contaminated with a significant amount of oxidizable material. The slides were kept in piranha solution (7 parts concentrated sulfuric acid, 3 parts 35% hydrogen peroxide) at 70 °C for 45 min. After cooling, the piranha solution was poured off and the slides were rinsed 20 times with water having a resistivity of 18 MΩ cm (Millipore, Billerica, MA). This was followed by 10 rinses of ethanol and 10 of methanol. The slides were then dried individually with a stream of nitrogen and stored in an 80 °C oven overnight. Gold Films. Semitransparent gold films of approximately 18 nm thickness were deposited onto the glass substrates with an electron beam evaporation system (Thermionics, Port Townsend, WA). A (63) Heydorn, A.; Nielsen, A. T.; Hentzer, M.; Sternberg, C.; Givskov, M.; Ersboll, B. K.; Molin, S. Microbiology 2000, 146, 2395–2407. (64) Blondiaux, N.; Zuercher, S.; Liley, M.; Spencer, N. D. Langmuir 2007, 23, 3489–3494. (65) Zheng, Y. F.; Liu, X. L.; Zhang, H. F. Surf. Coat. Technol. 2008, 202, 3011–3016. (66) Clements, L. R.; Khung, Y.-L.; Thissen, H.; Voelcker, N. H. Proc. SPIEsInt. Soc. Opt. Eng. 2008, 6799, 67990W/1–67990W/9. (67) Stadler, V.; Kirmse, R.; Beyer, M.; Breitling, F.; Ludwig, T.; Bischoff, F. R. Langmuir 2008, 24, 8151–8157. (68) Thierry, B.; Zimmer, L.; McNiven, S.; Finnie, K.; Barbe, C.; Griesser, H. J. Langmuir 2008, 24, 8143–8150. (69) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12–20.

Langmuir, Vol. 25, No. 3, 2009 1549 layer of titanium (approximately 5 nm thick) was applied first for adhesion of the gold film. Films were deposited at an oblique angle of 45° to the normal of the substrate. Pressure was maintained at or below 2 × 10-6 Torr throughout the deposition. Molecular Gradients. Self-assembled monolayers of two alkanethiols with gradient density were prepared according to the method described by Spencer and co-workers,53 but differing in the types of alkanethiols used. Gold slides were clamped in position on a syringe pump (KD Scientific, Holliston, MA), which was programmed to lower the slide at 40 µm/s. The slides were immersed into 3 µM solutions of 12-dodecanethiol or 1-mercapto-11-undecanol. Upon reaching full immersion, the slides were removed, thoroughly rinsed with ethanol, and dried with a stream of nitrogen. The slides were then soaked in the complementary alkanethiol solution containing 2 mM HS(CH2)11(OCH2CH2)3OH in ethanol for 10 h. The slides were then removed, rinsed thoroughly with ethanol, and dried with a stream of nitrogen prior to use in mammalian cell and bacterial cultures. Mammalian Cell Culture. Swiss 3T3 albino fibroblast cells from the American Type Culture Collection were cultured according to standard methods.35 SAM-modified slides (∼1.13 cm × 4 cm) were placed in 25 mL Falcon tissue culture flasks (Becton Dickson, Franklin Lakes, NJ). The cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; pH 7.4) containing 10% fetal bovine serum, 10 µL/mL penicillin-streptomycin, and 24 µL/mL nystatin and added to the flasks. The cells were incubated at 37 °C and 5% carbon dioxide concentration. The culture medium was changed every four days. Optical micrographs of adhered cells were taken at 10 positions along the gradient at different time points over a 140 h period. The cells were counted within the field of view of each picture. At low cell density, all cells were directly counted. At higher cell density, the cells were counted in a defined region of the field of view, and the total number of cells was calculated by multiplying this value by the ratio of the total area of the field of view to the area in which the cells were counted. Biofilm Formation of E. coli. E. coli RP437 was labeled with a plasmid, pRSH103, that expresses red fluorescent proteins constitutively so that the biofilms can be visualized by confocal laser scanning microscopy (CLSM). The plasmid pRSH103 was constructed by replacing the ampicillin-resistant marker (AmpR) of the prokaryotic expression vector pDsRed-Express (Clontech Laboratories, Inc., Mountain View, CA) with a tetracycline-resistant marker (TetR). E. coli RP437/pRSH103 was cultured on the two different chemical gradient SAM surfaces. Overnight cultures grown in Luria-Bertani (LB) medium (containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride) supplemented with 10 µg/mL tetracycline were used to inoculate the biofilm cultures to an optical density at 600 nm (OD600) of 0.05 as measured by a Genesis 5 spectrophotometer (Spectronic Instruments, Rochester, NY). The chemical gradient slides were incubated in a plastic Petri dish (100 × 15 mm), containing E. coli in 20 mL of LB medium and 10 µg/mL tetracycline at 37 °C without shaking. To analyze the E. coli biofilms using CLSM, each gradient SAM with adherent biofilm was washed gently by being dipped vertically into 0.85% NaCl buffer three times (change to fresh buffer after each dipping). This washing procedure does not remove bacteria embedded in a biofilm, but only the unattached planktonic cells.11 Then the surface was put upside down on a microscope coverglass (24 × 60 mm, no. 2, VWR International, West Chester, PA) and analyzed with a Zeiss LSM 5 Pascal confocal microscope (Carl Zeiss, Inc., Berlin, Germany). The red florescence was visualized by excitation with a helium-neon laser at 543 nm. Emission of fluorescence was detected with a long-pass filter at 560 nm. At least five spots were examined for each sample. The experiments were performed in duplicate. X-ray Photoelectron Spectroscopic Analysis of the Gradients. Chemical gradients (HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH) of SAMs were placed in aqueous protein solutions and incubated at 37 °C for two days to simulate the cell culture conditions, followed by rinsing with water and drying with a stream of nitrogen. Protein solutions consisted of either complete mammalian cell culture growth medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine

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Figure 2. Micrographs of cultured cells at different times (shown to the left) on a 4 cm long gradient SAM prepared by first immersing the slide into 3 µM HS(CH2)11OH at 40 µm/s, followed by overnight soaking in 2 mM HS(CH2)11(OCH2CH2)3OH ethanol solution. The pictures, left to right, show cell adhesion on the highest to lowest surface density of HS(CH2)11(OCH2CH2)3OH on the chemical gradient. Scale bar ) 190 µm.

serum) or 3 mg/mL bovine serum albumin. XPS spectra were obtained on a Surface Science Instruments model SSX-100, using a monochromated Al KR X-ray source at the Center for Materials Research at Cornell University.

Results Response of Mammalian Cell Adhesion to a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH Molecular Gradient. Figure 2 shows the cultured Swiss 3T3 albino fibroblasts on SAMs composed of a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH molecular gradient. At the early stage of 24 h of cell culture, relatively few cells have attached over the entire surface of the HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradient, but the surface showed more cells in the region of lowest surface density of HS(CH2)11(OCH2CH2)3OH. At 86 h of cell culture, more cells appeared intheregionsofintermediateandlowsurfacedensityofHS(CH2)11(OCH2CH2)3OH without reaching confluency of cell adhesion. At 116 h the most bioinert region of the gradient still had minimal cell adhesion, but both the regions presenting intermediate and low surface density of HS(CH2)11(OCH2CH2)3OH of the molecular gradient were covered with a nearly confluent layer of mammalian cells. These results indicate that the molecular gradient of HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH affords a gradual decrease in the ability to resist cell adhesion as the surface density of HS(CH2)11(OCH2CH2)3OH decreases. We quantified the adhesion of cells on the molecular gradient by counting the number of adhered cells at five positions over the 4 cm long gradient, these positions being 0.0, 0.9, 1.8, 2.7, and 3.6 cm from the end of the gradient having the highest density of HS(CH2)11(OCH2CH2)3OH (Figure 3). Means and standard deviations of cell numbers in four regions within each position on the chemical gradient were obtained for each position on the chemical gradient. The experiments were conducted in duplicate. The trend in the resistance to cell adhesion was not linear over the chemical gradient. We observed a slope of decreasing bioinertness only in the region over low surface density of HS(CH2)11(OCH2CH2)3OH, which was followed by a plateau of minimal resistance over the remainder of the gradient. Cells adhered and became confluent in 116 h on the region of the gradient having the lowest surface density of HS(CH2)11(OCH2CH2)3OH. In contrast, the region of the surface

Figure 3. Surface density of cells at different times on a 4 cm long gradient SAM prepared by first immersing the slide into 3 µM HS(CH2)11OH at 40 µm/s, followed by overnight soaking in 2 mM HS(CH2)11(OCH2CH2)3OH in ethanol. Means and standard deviations of the cell numbers in four regions within each position were obtained for each position on the chemical gradient. The experiments were conducted in duplicate.

having the highest surface density of HS(CH2)11(OCH2CH2)3OH remained highly resistant to cell adhesion, with less than 50 cells/mm2 even after six days of cell culture. It is important to note that when the transition of cell population switched from a linear increase to a plateau at 86 h at the position 1.8 cm from the highest surface density of HS(CH2)11(OCH2CH2)3OH), the cell adhesion on the surface did not become confluent. The number of cells continued to increase 2-fold on the plateau region of cell adhesion (low surface density of HS(CH2)11(OCH2CH2)3OH) by 140 h of cell culture. We believe that this transition in the cell adhesion behavior (from a linear increase to a plateau in cell population) represents a critical value of surface adhesiveness for mammalian cell adhesion beyond which the lower surface density of HS(CH2)11(OCH2CH2)3OH) no longer resists cell adhesion. This critical adhesiveness is not caused by the area covered by the spread of the adhered cells, as cell adhesion was not confluent and the cells continued to proliferate. Response of Mammalian Cell Adhesion to a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 Molecular Gradient. On a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 gradient, bioin-

Mammalian Cell Adhesion and Bacterial Biofilm Formation

Figure 4. Surface density of cultured cells at different times on a 4 cm long gradient SAM prepared by first immersing the slide into 3 µM HS(CH2)11CH3 in ethanol solution at 40 µm/s, followed by overnight soaking in 2 mM HS(CH2)11(OCH2CH2)3OH in ethanol. Means and standard deviations of the cell numbers in four regions within each position were obtained for each position on the chemical gradient. The experiments were conducted in duplicate.

ertness of resisting cell adhesion was not apparent. The amount of mammalian cells adhered on the surface did not correlate with the surface density of HS(CH2)11(OCH2CH2)3OH; similar amounts of cell adhesion at the high and low surface density regions of this alkanethiol were observed (Figure 4). We note that, in general, the rate of SAM formation is much faster for alkanethiols in ethanol presenting methyl groups than for those terminated with other polar functional groups. Thus, even though the same low concentration (3 µM) was used for both HS(CH2)11CH3 and HS(CH2)11OH for coating the gold films, the surface density of HS(CH2)11CH3 was higher than that of HS(CH2)11OH. The increase in adhesiveness afforded by the methyl-terminated alkanethiols may be great enough to override the effect of the bioinertness from the tris(ethylene glycol)terminated alkanethiols for mammalian cell adhesion. E. coli. Biofilm Formation on HS(CH2)11(OCH2CH2)3OH/ HS(CH2)11CH3 and HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH Molecular Gradients. To measure the amount of biofilm formed on molecular gradients, E. coli RP437 carrying plasmid pRSH103, which expresses red fluorescent protein constitutively, was cultured on SAMs presenting the molecular gradients. The amount of biofilm formed on a SAM over time was monitored by measuring the fluorescence signal from E. coli cells embedded in the biofilm (Figure 5). This measurement provides a method to quantify the amount of biofilm formed along the molecular gradient. Figure 5 shows the fluorescence signal from E. coli biofilms on the two molecular gradients after 24 h of incubation. For both of the molecular gradients at the region having the highest surface density of HS(CH2)11(OCH2CH2)3OH (region farthest from the entering end dipped in the solution of HS(CH2)11CH3 or HS(CH2)11OH), weak fluorescence was observed, indicating few bacteria attached. For both gradients, the trend in bioinertness is visible, with the surface coverage of biofilms increasing with distance from the end of the gradient having the highest density of HS(CH2)11(OCH2CH2)3OH. Similar to mammalian cell adhesion, the bacteria density in the biofilm was also much higher on the HS(CH2)11CH3 gradient than it was on the HS(CH2)11OH gradient (Figure 5). In contrast to the nonlinear response of mammalian cell adhesion to the chemical gradient, the surface coverage by E. coli biofilm formation increased linearly along the entire molecular gradient as the bioinertness decreased (Figure 6). At four equally spaced positions along the gradient, the surface coverage resulting from 24 h of E. coli biofilm formation was 6%, 16%, 25%, and 33% on the HS(CH2)11(OCH2CH2)3OH/

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Figure 5. Confocal images of E. coli biofilm formation on a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 gradient (I) and on a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradient (II) 24 h after inoculation. The pictures, left to right, show the biofilm at 0.7 cm (a), 1.4 cm (b), 2.1 cm (c), and 2.8 cm (d) from the end of the gradient having the highest surface density of HS(CH2)11(OCH2CH2)3OH. Scale bar ) 40 µm.

Figure 6. Surface coverage of the E. coli biofilm after 24 h of incubation at four positions on a 4 cm long chemical gradient of HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 in LB medium (A) and in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (B) and on a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradient in LB medium (C). The positions showing surface coverage are 0.7, 1.4, 2.1, and 2.8 cm from the region of the highest surface density of HS(CH2)11(OCH2CH2)3OH. Each datum shows the mean and standard deviation of surface coverage at each position on the basis of duplicate samples with five confocal images taken for each position.

HS(CH2)11CH3 gradient. The linear relationship was also observed for the HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradient, although only 2% of the most bioinert region of this gradient was covered by the biofilm, and only 7% of the surface was covered on the least bioinert region (Figure 6). Because the standard LB medium for culturing E. coli has a composition (e.g., yeast extract and tryptone) different from that of fetal bovine serum used in the mammalian cell culture medium, surfaces exposed to LB medium may not have the same chemistry and adhesiveness as those exposed to the mammalian cell culture medium. To compare with mammalian cell adhesion, E. coli cells were also cultured in the growth medium used for mammalian cell culture (Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum) and were presented to a HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 gradient. This experiment provided the same surface condition and chemical environment for biofilm formation as those for mammalian cell adhesion.

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Figure 7. XPS of the HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradient incubated in Dulbecco’s modified Eagle’s medium containing 10 wt % fetal bovine serum at 37 °C for two days. Scans were taken at 0.5 cm intervals across the surface of the gradient.

The biofilm formed by E. coli cultured in the medium for mammalian cells also responded linearly to the surface gradient (Figure 6), but the slope of increase in the biofilm surface coverage along the chemical gradient was much smaller than in the bacterial culture medium (LB medium). Overall, there was significantly less biofilm mass formed when Dulbecco’s modified Eagle’s medium was used instead of the LB medium, a result probably due to the difference in nutrient composition and potential inhibitory compounds in serum. Protein Adsorption on a HS(CH2)11(OCH2CH2)3OH/ HS(CH2)11OH Gradient. In Dulbecco’s modified Eagle’s medium, the chemical gradients in the self-assembled monolayers are in contact with proteins from serum. These proteins will adsorb on the surface depending on the alkanethiol composition in the chemical gradient. To better correlate mammalian cell adhesion and biofilm formation to the surface properties of the chemical gradients, we directly characterized the amount of protein adsorbed on surfaces by using XPS, which has been shown in previous studies to be an effective means of analyzing adsorbed proteins.67,68 Because XPS measures the elemental composition of a surface by analyzing the emitted photoelectrons which are characteristic of the emitting atoms,70 and because the chemical gradient of mixed alkanethiols on gold films does not contain any nitrogen atoms, the XPS signals from nitrogen atoms can be solely attributed to the protein adsorbed on the positions along the chemical gradient, providing a direct measurement of the amount of protein on the chemical gradient. In contrast, the carbon and oxygen signals from XPS are the result of a mixture of alkanthiols and adsorbed proteins on the gradient. We incubated SAMs presenting HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH gradients in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37 °C for two days to simulate the condition of the mammalian cell culture. Figure 7 shows the carbon, oxygen, and nitrogen signals of XPS from the chemical gradient treated by this medium. Because the chemical composition changed continuously in the SAM itself, we measured the absolute intensity of the XPS signal from each element at different positions along the chemical gradient. The XPS signal from nitrogen increased linearly from the bioinert end (high surface density of HS(CH2)11(OCH2CH2)OH) to the more adhesive end (high surface density of HS(CH2)11OH), indicating that there was a continuous (70) Alov, N. V. J. Anal. Chem. 2005, 60, 297–300.

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increase in the surface density of adsorbed proteins. Notably, the XPS signals from oxygen and carbon also increase monotonically as the surface adhesiveness increases. Because the carbon and oxygen contents decrease in the SAM as the surface density of HS(CH2)11(OCH2CH2)OH decreases, this result suggests that the amount of protein adsorbed on the surface affords more elements, which override the decreasing content of carbon and oxygen in the SAM. Furthermore, the relative excess of carbon over oxygen and the excess of oxygen over nitrogen are proportional to the average abundance of carbon, oxygen, and nitrogen elements from a protein. Collectively, these results suggest that the content of adsorbed proteins increased linearly on the chemical gradients and that the transition of a linear increase to a plateau of mammalian cell adhesion is due to the adhesion mechanism of mammalian cells and not an unexpected pattern in protein adsorption or surface inhomogeneity. Because the protein content of fetal bovine serum is undefined and varies among manufacturers and lots,71,72 we also examined, as a control, the XPS results of the same chemical gradient (HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH) treated with bovine serum albumin (3 mg/mL) and observed an increase in absorbed protein on the surface with decreasing surface density of HS(CH2)11(OCH2CH2)3OH (see the Supporting Information).

Discussion Our work shows that mammalian cell adhesion and bacterial biofilm formation exhibited a drastic difference on the molecular gradients. Although mammalian cell adhesion and biofilm formation share some similarities, including cell attachment and nonspecific and specific binding events, the molecular biologies governing the mechanism of each process are different. For mammalian cells, adhesion on surfaces is orchestrated by a collection of temporally and spatially resolved events, which include a specific molecular binding between the membrane protein integrin and the peptide sequence arginine-glycine-aspartic acid (RGD) that is common to fibronectin, fibrinogen, laminin, and other extracellular proteins.15,26 This binding event further induces the assembly of a wide collection of specific proteins at the intracellular domain of integrin and the development of actin filaments, which are necessary for vital cell activity and proliferation. In addition to the specific molecular recognition for the initial ligand receptor binding, the binding sites must be close enough in proximity for the receptor proteins (integrin) on cell membranes to form clusters such that focal adhesion can be initiated.12,14-16,26,73 If any of the above steps are disrupted, the cell activity will be impaired and could lead to eventual programmed cell deathsapoptosis. Such complex and well-orchestrated events imply a highly nonlinear dependence of adhesion on surface properties. For instance, a study using well-resolved ligands on surfaces reported that a critical distance shorter than about 57-70 nm between the ligands presented on surfaces is required to support full focal adhesion and spreading of the mammalian cells; if the specific binding sites are separated by more than 70 nm, then the cell adhesion is weak, and less spreading and more cell migration are observed.74 Our results of mammalian cell adhesion on the molecular gradient of HS(CH2)11(OCH2CH2)3OH/HS(CH2)11CH3 (71) Boone, C. W.; Mantel, N.; Caruso, T. D., Jr.; Kazam, E.; Stevenson, R. E. In Vitro 1971, 7, 174–89. (72) Zheng, X.; Baker, H.; Hancock, W. S.; Fawaz, F.; McCaman, M.; Pungor, E., Jr. Biotechnol. Prog. 2006, 22, 1294–1300. (73) Lim, J. Y.; Dreiss, A. D.; Zhou, Z.; Hansen, J. C.; Siedlecki, C. A.; Hengstebeck, R. W.; Cheng, J.; Winograd, N.; Donahue, H. J. Biomaterials 2007, 28, 1787–1797. (74) Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Biophys. J. 2007, 92, 2964–2974.

Mammalian Cell Adhesion and Bacterial Biofilm Formation

indicate that there is a critical density of adhesion ligands, below which the cell resistance is linearly dependent on the surface density of tris(ethylene glycol)-terminated alkanethiol and above which cell adhesion readily occurs and is insensitive to the amount of tri(ethylene glycol) on the surface. In contrast to mammalian cells, the details at the molecular level for the initial attachment of bacteria on a surface are not as clear and certain. Bacteria attachment can occur directly on a surface or can be mediated by the protein of a host.20,21 Bacterial cells attach to surfaces using different structures (e.g., flagella, fimbriae, or curli) that are known to involve reversible (flagella) and irreversible (fimbriae and curli) attachment.24 Whether a specific molecular recognition is necessary for the initial stage of biofilm formation on different surfaces is not clear. van der Mei and co-workers suggested that the molecular interactions responsible for specific interaction by adhesion molecules may also be responsible for conducting nonspecific adhesion onto surfaces.75 Bacteria in general can attach to a wide range of materials and are well-known to migrate on surfaces through twitching and swarming.76 Our results, together with other observations,75,76 suggest that biofilm formation on certain surfaces does not require a specific molecular recognition event between the bacteria and the substrate. It is important to note that these results do not imply that bacteria cannot or do not have specific molecular interactions with certain ligands on a surface or in a host, but that in the absence of such a specific molecular interaction biofilms can still form if immobilization is permitted via certain nonspecific molecular interactions. One major difference between bacterial and mammalian cell adhesion is that, if bacteria are only weakly attached or are prevented from attaching to a surface, they can still survive and proliferate, whereas mammalian cells will not have full vital activity if the attachment is impaired by insufficient contact points or is denied altogether.74 Our results of a linear increase in the biofilm mass as the bioinertness of the surface decreases (as a result of the decrease in the surface density of HS(CH2)11(OCH2CH2)3OH) indicate that, unlike mammalian cells, no critical density of adhesiveness exists in our system that supports maximum adhesion of E. coli. Together, these results suggest that, at least for biofilm formation on the surfaces in our study, bacteria can be immobilized in space merely via some nonspecific interactions such as ionic interactions, van der Waals interactions, and/or hydrogen bonding. Such an immobilization (75) Busscher, H. J.; Cowan, M. M.; van der Mei, H. C. FEMS Microbiol. ReV. 1992, 8, 199–209. (76) Allison, C.; Hughes, C. Sci. Prog. 1991, 75, 403–22.

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dependence is fundamentally different from the anchorage dependence of mammalian cell adhesion, which requires specific molecular recognition between the integrin membrane protein and ligands in the exctracellular matrix.

Conclusions This work shows that surfaces presenting a gradient in bioinert chemistry are useful tools for studying the mechanism of both mammalian cell adhesion and bacterial biofilm formation on surfaces. Our results suggest that biofilms formed on chemical gradients increase linearly with an increase in the surface adhesiveness for protein adsorption, but there is a critical value of adhesiveness for mammalian cell adhesion on surfaces, beyond which point the resistance to protein adsorption no longer affects the adhesion of mammalian cells. These results are consistent with the anchorage dependence of mammalian cell adhesion and support a mere immobilization dependence for biofilm formation on the surfaces that does not require specific molecular interactions. We note that, in addition to creating gradients in bioinertness, other chemical gradients that present increasing surface density of desired ligands can be prepared. We are currently studying the response of mammalian cells and bacteria to such gradients of specific ligands. These chemical gradients can be prepared on gold films having a gradient in nanometerscale surface topography that are known to enhance surface chemistry.2 Overall, these methods have potential for studying unexplored areas such as directed bacterial biofilm formation and for exploring the use of three-dimensional gradients in biofilm mass. Acknowledgment. We thank Syracuse University, NSFCMMI (Grant Nos. 0727491 and 0826288), and the Syracuse Center of Excellence CARTI award supported by the U.S. Environmental Protection Agency (Grant X-83232501-0) for partial financial support. E.A.B. also thanks the Syracuse Center of Excellence for a fellowship under the same EPA grant. S.H. was supported by a fellowship from the Syracuse Biomaterials Institute. We are grateful to Jonathan Shu (Cornell University) for help with the XPS analysis. Supporting Information Available: XPS analysis of the HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH chemical gradient treated with bovine serum albumin and duplicate of the mammalian cell culture on HS(CH2)11(OCH2CH2)3OH/HS(CH2)11OH molecular gradients. This information is available free of charge via the Internet at http://pubs.acs.org. LA803261B