Microfluidic Permeation Printing of Self-Assembled Monolayer

Jun 29, 2010 - Developing strategies to pattern well-defined molecular gradients on surfaces is difficult ... We introduce a new strategy, microfluidi...
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Microfluidic Permeation Printing of Self-Assembled Monolayer Gradients on Surfaces for Chemoselective Ligand Immobilization Applied to Cell Adhesion and Polarization Brian M. Lamb, Sungjin Park, and Muhammad N. Yousaf* Department of Chemistry and the Carolina Center for Genome Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 Received June 4, 2010. Revised Manuscript Received June 16, 2010 To study complex cell behavior on model surfaces requires biospecific interactions between the interfacing cell and material. Developing strategies to pattern well-defined molecular gradients on surfaces is difficult but critical for studying cell adhesion, polarization, and directed cell migration. We introduce a new strategy, microfluidic SPREAD (Solute PeRmeation Enhancement And Diffusion) for inking poly(dimethylsiloxane) (PDMS) microfluidic cassettes with a gradient of alkanethiol. Using SPREAD, an oxyamine-terminated alkanethiol is able to permeate into a PDMS microfluidic cassette, creating a chemical gradient, which can subsequently be transfer printed onto a gold surface to form the corresponding chemoselective gradient of oxyamine-alkanethiol self-assembled monolayer (SAM). By first patterning regions of the gold surface with a protective SAM using microfluidic lithography, directional gradients can be stamped exclusively onto unprotected bare gold regions to form single cell gradient microarrays. The microfluidic SPREAD strategy can also be extended to print micrometer-sized islands of radial SAM gradients with excellent geometric resolution. The immobilization of a cell adhesive Arg-Gly-Asp (RGD)-ketone peptide to the SPREAD stamped oxyamine-alkanethiol SAMs provides a stable interfacial oxime linkage for biospecific studies of cell adhesion, polarity, and migration.

Introduction Directed cell and tissue migration in space and time is crucial for many fundamental biological processes including wound healing, metastasis, inflammation, and development.1,2 For these phenomena, the precise timing and movement of cells and tissue are guided by a complex interplay of soluble molecules, immobilized ligands to extracellular matrix, hydrodynamic and physicomechanical forces.3,4 The ability for cells to compute, interpret, and then decide cellular function based on information from the dynamic microenvironment underlies the very nature of cellular systems biology. For cell polarity and cell migration, gradients of soluble factors (chemotaxis) and immobilized gradients (haptotaxis) of ligands on extracellular matrix are necessary to provide directional information. To study these complex processes a number of model substrates presenting patterns of ligands and gradients have been developed.5,6 Many of these studies have employed self-assembled monolayers of alkanethiolates on gold *To whom correspondence should be addressed. E-mail: mnyousaf@ email.unc.edu. (1) Wong, K.; Park, H. T.; Wu, J. Y.; Rao, Y. Curr. Opin. Genet. Dev. 2002, 12, 583–591. (2) Schmid, R. S.; Shelton, S.; Stanco, A.; Yokota, Y.; Kreidberg, J. A.; Anton, E. S. Development 2004, 131, 6023–6031. (3) Ridley, A. J.; Schwartz, M. A.; Burridge, K.; Firtel, R. A.; Ginsberg, M. H.; Borisy, G.; Parsons, J. T.; Horwitz, A. R. Science 2003, 302, 1704–1709. (4) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Cell 2006, 126, 677–689. (5) Yu, X.; Wang, Z.; Jiang, Y.; Zhang, X. Langmuir 2006, 22, 4483–4486. (6) Goldberg, M.; Langer, R.; Jia, X. J. Biomater. Sci., Polym. Ed. 2007, 18, 241–268. (7) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992–5996. (8) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (9) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3–30. (10) (a) Hoover, D. K.; Yousaf, M. N. Langmuir 2009, 25, 2563–2566. (b) Hodgson, L.; Chan, E. W. L.; Hahn, K. M.; Yousaf, M. N. J. Am. Chem. Soc. 2007, 129, 9264–9265. (c) Lamb, B. M.; Westcott, N. P.; Yousaf, M. N. ChemBioChem 2008, 9, 2220–2224.

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as the model substrate.7-10 There are several advantages of using SAMs on gold as a platform for preparing model systems to study cell phenomena over other materials: (1) SAMs are synthetically flexible (routine organic synthesis to generate almost any alkanethiol tethered molecule); (2) several surface spectroscopies exist to characterize interfacial reactions or associations (for example, surface plasmon resonance spectroscopy, atomic force microscopy, scanning tunneling microscopy); (3) many soft-lithography approaches to pattern surfaces with different surface chemistries; (4) for biointerfacial studies, SAMs can become inert to nonspecific protein adsorption (incorporation of ethylene glycol-terminated alkanethiols renders the surface inert); and (5) the method is compatible with cell culture conditions (pH 7, 37 °C) and livecell high-resolution optical and fluorescence microscopy. Taken together, these features allow for the generation of well-defined surfaces for probing biospecific interactions between ligands presented on the SAM and cell surface receptors. Although patterning gold surfaces is routine, generating patterned molecular gradients with control of geometry and generating radial surface gradients for cell biological studies is much more difficult. Current methods use complex photochemical or microfluidic approaches to generate gradients on surfaces.11-15 Until now, there has been no strategy that allows for rapid and inexpensive printing of chemoselective gradients in patterns and radial geometric patterns. This capability would significantly enhance studies of complex cell behavior on model substrates. (11) Dillmore, W. S.; Yousaf, M. N.; Mrksich, M. Langmuir 2004, 20, 7224–7231. (12) Chan, E. W.; Yousaf, M. N. Mol. Biosyst. 2008, 4, 746–753. (13) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311–8316. (14) Genzer, J.; Bhat, R. R. Langmuir 2008, 24, 2294–2317. (15) Dertinger, S. K. W.; Chiu, D. T.; Jeon, N. L.; Whitesides, G. M. Anal. Chem. 2001, 73, 1240–1246.

Published on Web 06/29/2010

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Lamb et al. Scheme 1. Structures of Molecules Used for Surface Tailoring

Herein, we develop and demonstrate a new methodology, microfluidic Solute PeRmeation Enhancement And Diffusion (SPREAD), for inking PDMS stamps with a gradient of alkanethiol, which can then be stamped onto a gold surface to generate a patterned gradient self-assembled monolayer. We demonstrate that an oxyamine-alkanethiol (11-(Aminooxy)undecane-1-thiol) (1) can permeate and diffuse into a PDMS microfluidic cassette and then be printed directly onto a gold surface to form a gradient SAM (Scheme 1). By backfilling the remaining bare gold regions with a tetra(ethylene)glycol-undecanethiol (EG4) (2), an inert patterned gradient surface is produced for studies of cell behavior. The profile of the surface gradients generated by the SPREAD stamping technique relies on diffusion and the use of nonpolar solvents that promote permeation of the inking molecule into the PDMS. The extent of alkanethiol permeation in PDMS can be controlled by simply adjusting the parameters for the time of diffusion, the polarity of the solvent, and the extent of PDMS cross-linking. By combining these practical and adjustable experimental parameters with another patterning strategy, microfluidic lithography (μFL), we are able to generate a complex array of patterned linear gradient features.16 We further show the flexibility of the SPREAD methodology by producing geometric islands of radial gradients by performing SPREAD in a microfluidic chamber encompassing the island microarray features. Finally, we show direct application of these SPREAD gradient patterns by immobilizing a cell adhesive Arg-Gly-Asp (RGD) peptide to the oxyamine-alkanethiol SAMs for studies of cell polarity. This methodology allows for the rapid printing of chemoselective gradients in arrays with control of geometry that can immobilize a wide variety of ligands for a range of biotechnological cell based assays to fundamental studies of cell behavior (adhesion, polarization, migration, growth, differentiation).

Results and Discussion For many microfluidic and soft lithography applications of polydimethylsiloxane (PDMS), the permeability of solvents and small molecules into the polymer outside the intended channels is considered a severe limitation to its usefulness as a polymer material for microfluidic devices. This known limitation has accelerated (16) Lamb, B. M.; Barrett, D. G.; Westcott, N. P.; Yousaf, M. N. Langmuir 2008, 24, 8885–8889.

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Figure 1. The strategy for generating gradients of molecules in PDMS microfluidic cassettes via solute permeation and diffusion (SPREAD). (A) A PDMS microfluidic cassette is placed directly onto the surface of a glass substrate. An alkanethiol (R-SH) is flowed into the cassette and permeates and diffuses through the walls of the microfluidic channels with the aid of nonpolar solvents (e.g., methylene chloride). Evacuation of the microfluidic cassette, timed diffusion, and removal from the glass substrate provides a PDMS stamp with gradients of alkanethiol molecules embedded in the matrix originating from the sidewalls of the microfluidic channels. (B) The corresponding top view of the SPREAD technique.

the development of less solvent/reagent permeable polymers to retain the reagents within the proper channels in the microfluidic device.17,18 This phenomenon, diffusion of solutes through the walls of a microfluidic device, has been well studied. Because of its ubiquitous use in soft lithography applications, PDMS has been investigated for organic solvent permeation and diffusion for a (17) de Jong, J.; Lammertink, R. G.; Wessling, M. Lab Chip 2006, 6, 1125–1139. (18) Roman, G. T.; Hlaus, T.; Bass, K. J.; Seelhammer, T. G.; Culbertson, C. T. Anal. Chem. 2005, 77, 1414–1422.

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Figure 2. SPREAD stamping of a gradient of alkanethiol for chemoselective immobilization of ligands to inert surfaces. (A) A microfluidic cassette SPREAD inked with an oxyamine tethered alkanethiol is stamped onto a bare gold surface. The oxyaminealkanethiol rapidly forms a gradient self-assembled monolayer. (B) The remaining bare gold regions are backfilled with tetra(ethyleneglycol) undecanethiol (EG4) to render the surface inert to nonspecific protein adsorption or cell attachment. (C) Addition of a ketone tethered ligand to the surface allows for a rapid and stable interfacial reaction generating an oxime linkage. As an example, a ketone-functionalized rhodamine was immobilized and the resulting surface imaged with fluorescence microscopy. A clear gradient is visible emanating from the side-walls of the empty microfluidic channel. (scale bar = 200 μm).

number of applications.19-21 We have recently shown that certain organic solvents that permeate into PDMS microfluidic channels can also enhance its permeability to alkanethiols being flowed through a channel.16 To further understand this effect and attempt to utilize it for novel applications, a three-step experimental procedure was performed to ink the microfluidic device with a chemical gradient (Figure 1). Briefly flowing a solution of alkanethiol into the channel followed controlled diffusion time results in reproducible chemical gradients emanating from the channels of the microfluidic device. During this process, the alkanethiol is essentially extracted from the channels through the side-walls of the microfluidic cassette, which can be further enhanced diffusion rate due by the inclusion of a nonpolar solvent. To generate a patterned and chemoselective gradient SAM that can immobilize a wide range of ligands, we used the SPREAD strategy to print an oxyamine-terminated alkanethiol (Figure 2). It has recently been shown that the oxyamine-alkanethiol molecule has several features that allows it to be an ideal SAM system for interfacial reactions on gold surfaces: (1) the oxyamine group (19) Ismagilov, R. F.; Ng, J. M.; Kenis, P. J.; Whitesides, G. M. Anal. Chem. 2001, 73, 5207–5213. (20) Bennett, M.; Brisdon, B. J.; England, R.; Field, R. W. J. Membr. Sci. 1997, 137, 63–69. (21) Lee, N. J.; Park, C.; Whitesides, G. M. Anal. Chem. 2003, 75, 6544–6554.

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Figure 3. Gradient length characterization of printed oxyaminealkanethiols on gold surfaces obtained using the SPREAD procedure in a microfluidic device. (A) Diffusion length dependence on permitted diffusion time of an ethanolic solution of 5 mM oxyamine alkanethiol. (B) Gradient length dependence on CH2Cl2 cosolvent inclusion in a 5 mM oxyamine alkanethiol solution with 600 s diffusion time. The intensity profile of all characterized chemical gradients is characteristically linear (Supporting Information Figure 1).

reacts rapidly and chemoselectively with ketone-containing ligands to generate a stable interfacial oxime conjugate. By using appropriate ligands, surfaces can present a range of molecules for biointerfacial and material applications; (2) the coupling reaction is biocompatible and can be performed in the presence of live cells without deleterious effects to the cell; (3) the reaction is synthetically flexible where the ketone functional group can be easily introduced into many biomolecules via routine solid-phase or solution chemistry.22 A SPREAD-inked oxyamine-alkanethiol PDMS microfluidic cassette was generated and stamped to a bare gold surface (Figure 2). Removal of the PDMS stamp and exposing the surface to a tetra(ethylene)glycol-undecanethiol (EG4) solution for 12 h results in a patterned inert SAM surface presenting a gradient of oxyamine-alkanethiol capable of chemoselective ligand immobilization (Figure 2). The EG4 is known to prevent nonspecific protein adsorption and cell attachment to surfaces.23 As an added feature, the SPREAD-inked PDMS stamp can be used repeatedly to print patterned gradient SAMs to many additional gold surfaces. However, similar to the classic PDMS microcontact printing strategy, each subsequent stamping (22) Park, S.; Yousaf, M. N. Langmuir 2008, 24, 6201–6207. (23) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714–10721.

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Figure 4. A protect and print strategy for creating chemoselective gradient arrays. (A) A PDMS microfluidic cassette is placed directly onto a bare gold surface. A μFL technique is used to flow a solution of tetra(ethylene)glycol alkanethiol EG4 (1 mM, EtOH, 30 s) through the channels to generate a patterned EG4 SAM. Removal of the cassette reveals a high density EG4 monolayer that prevents further alkanethiol surface patterning in these regions. (B) A SPREAD-inked PDMS cassette (oxyamine-alkanethiol) is then stamped onto the surface (15s) to create a oxyamine-alkanethiol SAM gradient on the bare gold surface. (C) Alkanethiol transfer does not occur on the blocked EG4 SAM regions. This allows for spatial separation of the gradient features. (D) The remainder of the surface is then backfilled with EG4. (E,F) Subsequent reaction with a ketone-functionalized rhodamine shows an ordered gradient array. (scale bar = 80 μm).

consumes a significant amount of the total ink (alkanethiol) remaining within the stamp. To characterize the fidelity of the SPREAD-stamping strategy, a ketone-functionalized rhodamine (3) was reacted to the oxyamine terminated gradient SAMs.22 Visualization of the oxime conjugated surface with fluorescence microscopy clearly shows a SAM gradient with a linear slope (Supporting Information Figure 1). As a control, the surface was exposed to nonfunctionalized rhodamine, which lacks the ketone group, and no fluorescence was observed. Furthermore, when the oxyamine SAM surface is first reacted with acetone then the ketone-rhodamine, no fluorescence is observed. To demonstrate the reproducibility and control that can be exerted with the SPREAD methodology, we characterized how the length of the gradient depends on diffusion time within the PDMS and the carrier solvent composition. By adjusting these parameters, a wide range of oxyamine alkanethiol SAM gradient lengths can be obtained (Figure 3). As shown in Figure 3A, by controlling the oxyamine alkanethiol diffusion durations, varying gradient lengths can be produced for subsequent patterning. Figure 3B shows the role of solvent composition on diffusion within the PDMS for generating different length gradients. These data clearly show that a wide range of gradient lengths and therefore slopes can be readily produced with a single PDMS channel using the SPREAD strategy by using easily controlled parameters, such as cosolvent composition and set time of diffusion. To extend this strategy to other materials, we have successfully created chemical gradients of polar alkylphosphonates SAMs on transparent indium tin oxide (ITO) surfaces 12820 DOI: 10.1021/la1022642

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Figure 5. Schematic for SPREAD stamping of chemoselective radial gradient islands. (A) A PDMS microfluidic chamber containing patterned microfeatures is reversibly sealed onto a glass substrate. (B) Oxyamine-alkanethiol solution (5 mM, in 1:1 EtOH/ CH2Cl2) is flowed through the microfluidic chamber and slowly permeates and diffuses into the PDMS features. The microfluidic chamber is then solvent evacuated by negative pressure, and the PDMS stamp is removed. (C) The SPREAD-inked stamp is then placed directly onto a bare gold surface to transfer print the oxyamine alkanethiol to generate SAM islands of radial gradients to the surface. (D) The surface is backfilled with EG4 for 12 h then exposed to a ketone-functionalized rhodamine and imaged using fluorescence microscopy to characterize the printed surface features. Altering the polarity of the flowed solvent and the duration of alkanethiol diffusion during SPREAD correlates with the degree of permeation into the PDMS and subsequent steepness and length of the printed gradient features. (scale bar = 30 μm).

(Supporting Information Figure 2). The gradient profiles imparted to the ITO surface are characteristically linear and the shape of the gradients can potentially be controlled by modifying flow durations and cosolvent conditions (data not shown). To illustrate the flexibility of the SPREAD strategy to generate high-throughput and multiplex surfaces for biotechnological and cell based assays, we generated an array of SAM gradients using a protect and print method (Figure 4). This strategy relies on first patterning a protective inert monolayer onto a gold surface. This pattern inhibits the subsequent printing of a SPREAD alkanethiol gradient onto the protected features but not on the bare gold regions. Using a microfluidic lithography (μFL) strategy, a high-density alkanethiol monolayer can be patterned directly onto a gold surface.10,16 For the SPREAD microarray, we used μFL to pattern a high density EG4 monolayer. This monolayer can be formed in as little as 30 s when a solution of 1 mM EG4 in ethanol is flowed through the microfluidic cassette. The EG4 alkanethiol in ethanol does not significantly permeate the PDMS cassette over the short time interval of μFL. After selective surface Langmuir 2010, 26(15), 12817–12823

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Figure 6. Cell adhesion and polarization on biospecific gradient features created with SPREAD inking and stamping. (A) A fluorescent gradient array with the corresponding (B) RGD-peptide array patterned with cells. The cells adhere and polarize selectively on the patterned RGD presenting gradient array (see text for details, scale bar = 100 μm). (C) Cartoon of the polarity vector defining the direction between the nucleus center, centrosome center, and golgi center of a cell. (D) Image of a representative cell on a single SPREAD gradient. The vector was used to define internal polarity on the SPREAD array gradients for the cells (scale bar = 5 μm). (E) An oval radial gradient with the corresponding RGD-peptide presenting gradient (scale bar = 30 μm). (F) Cells adhered to the high peptide density region and polarized to the edge. (G) The net cell polarity vector consistently pointed outward (>80%) from the center of the pattern for all radial gradient shapes.

protection, a preformed SPREAD-inked PDMS stamp of oxyamine-alkanethiol is printed onto the surface in a bisected orientation, which immediately generates a SAM gradient exclusively onto the unprotected bare gold surface regions. After backfilling the remaining surface with EG4, a patterned gradient array of oxyamine-alkanethiol is formed. To visualize the array, a fluorescent ketone-functionalized rhodamine was immobilized. Characterization by fluorescence microscopy revealed the high fidelity of the protection process (Supporting Information Figure 1 and Figure 4). Using this simple general method, it is possible to make microarrays of various shapes and sizes. The level of control provided by the SPREAD protect and print strategy to generate inert and chemoselective gradient arrays rapidly and inexpensively will find wide use in many biological and material applications. To print radial SAM gradients of nearly any geometry, we integrated the microfluidic SPREAD procedure with a modified PDMS microfluidic microcontact stamp chamber containing protruding geometric features. A schematic for microfluidic SPREAD inking of the microcontact printing stamps is shown in Figure 5. By using a slight variation of classic microcontact printing, a PDMS stamp was designed with protruding islands of various shapes encased within a single microfluidic chamber (Figure 5A). By flowing oxyamine-alkanethiol into the chamber with a permeating nonpolar solvent (CH2Cl2), a SPREAD-inked PDMS stamp (Figure 5) was generated containing radial gradients of oxyamine-alkanethiol within the isolated features. The stamp was then removed from the glass surface and printed directly onto a bare gold surface followed by backfilling with EG4. Since only the PDMS protruding features contact the gold surface during the stamping procedure, only the array of geometrical shapes with radial gradients of oxyamine-alkanethiol are imparted onto the surface. The fidelity of the patterning technique was characterized by immobilizing a ketone-functionalized rhodamine to the oxyamine-terminated SAM gradients and visualizing Langmuir 2010, 26(15), 12817–12823

the surface with fluorescence microscopy (Supporting Information Figure 1) (Figure 5). Characterization of the gradient revealed that inwardly fading radial SAM gradients were directly imparted onto the surface. As shown in the bottom left of figure 5, the degree of alkanethiol permeation into the microisland features can be controlled by simply limiting or extending diffusion time (30 s to 4 min) to result in defined length radial gradients. By manipulating diffusion time and solvent content, combined with existing soft lithography fabrication methods, the SPREAD strategy may be used to print SAM gradient features ranging in size from 20 μm to several millimeters. It should be noted that the alkanethiol permeation occurs in all dimensions of the PDMS microfluidic stamp chamber exposed to fluid during the SPREAD procedure, but alkanethiol permeation into features other than the PDMS protruding islands or the borders of the microfluidic chamber are not printed onto the gold surface. To demonstrate an application of the gradients manufactured with the SPREAD technique, we examined the ability of gradient substrates to induce cell polarization. The ability for a cell to polarize and therefore generate asymmetry within itself is a fundamental cellular process strongly influenced by environmental factors and is critical for a range of cell biological processes.24,25 The role of the underlying adhesive environment is critical to establish cell polarity, but is not well understood.26 To study how biospecific ligand-receptor interactions can affect cell polarity, we immobilized the cell adhesive Arg-Gly-Asp (RGD) ketone peptide (4). The RGD peptide sequence is found in the extracellular matrix proteins, including fibronectin, and is known to facilitate cell adhesion via the cells integrin receptors.27 For cells to migrate, cells must first evaluate their environment and polarize, establishing (24) Moissoglu, K.; Schwartz, M. A. Biol. Cell. 2006, 98, 547–555. (25) Nelson, W. J.; Ridley, A. J.; Schwartz, M. A. Nature 2003, 422, 766–774. (26) Thery, M.; Racine, V.; Piel, M.; Pepin, A.; Dimitrov, A.; Chen, Y.; Sibarita, J. B.; Bornens, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19771–19776. (27) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309, 30–33.

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intracellular asymmetry within the cell. To observe the underlying surface chemistry effects of RGD on cell polarity, Swiss 3T3 fibroblast cells were confined to small patterns (50 μm width  150 μm gradient length) where the cells could attach and become polarized. The most conclusive method to determine cell polarity is to measure the vector between the cell nucleus, concentrated golgi, and centrosome. The polarity of a cell can be experimentally observed and measured through the systematic reorientation and alignment of these organelles, which can be visualized using fluorescent dyes to map the direction of polarity.28 As can be seen in the series of representative fluorescent micrographs in Figure 6, the adherent cells adopt a morphology in which the cells polarize toward the high-density RGD region. No consistent directional polarity on nongradient patterns were observed, however cells on gradient arrays and radial patterns were cells consistently polarized toward the higher density RGD regions based on the nucleus and the concentrated golgi/centrosome vectors. As controls, addition of soluble RGD peptide (1 mM, 20 min) detaches cells adhered to RGD immobilized gradients. Furthermore, when no RGD peptide is immobilized, almost no cells adhered to the surface, indicating the surface is inert to nonspecific cell attachment. These results clearly show that cell polarity can be directly influenced gradients produced with the SPREAD methodology .

Conclusion We have developed a simple and powerful new strategy for creating biospecific and chemoselective surface gradients. The microfluidic SPREAD technique utilizes solvent permeation and enhanced alkanethiol diffusion into the walls of a PDMS microfluidic cassette to subsequently pattern SAM gradients onto gold surfaces. By manipulating the permeation duration and the carrier cosolvent conditions, various gradient lengths can be produced for subsequent chemoselective tailoring on a range of surfaces. By first blocking features on the gold surface with a protective monolayer using microfluidic lithography, we demonstrate a protect and print technique for creating gradient microarrays. By using SPREAD in combination with microfluidic stamp chambers, we further demonstrate the ability to pattern geometric islands of radial gradients. The surface gradients were characterized via fluorescence microscopy and, by immobilizing the cell adhesive peptide RGD, demonstrated how the underlying surface chemistry can polarize cells and promote selective cell attachment. SPREAD is a rapid and inexpensive methodology to generate well-defined surface gradients and is straightforward to apply to a range of cell adhesion, polarization, and migration studies based on extracellular matrix adhesion. Future studies will expand the strategy to diffuse multiple alkanethiols within the PDMS stamp to generate multicomponent alkanethiol gradient arrays on gold. The methodology may also be used to pattern molecules on other important materials (e.g., indium tin oxide (ITO) and glass, Supporting Information Figure 2) for a range of optoelectronic and material applications.29,30 Furthermore, by adjusting the amount of cross-linking within the PDMS, the size and nature of the diffusing molecule (molecular weight, molecule type, e.g., small molecule, DNA, RNA, peptides) will also provide increased utility and flexibility for the SPREAD methodology for a range of biotechnological applications. Other polymer compositions will (28) Ueda, M.; Graf, R.; MacWilliams, H. K.; Schliwa, M.; Euteneuer, U. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9674–9678. (29) Pulsipher, A.; Westcott, N. P.; Luo, W.; Yousaf, M. N. J. Am. Chem. Soc. 2009, 131, 7626–7632. (30) Luo, W.; Westcott, N. P.; Pulsipher, A.; Yousaf, M. N. Langmuir 2008, 24, 13096–13101.

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also be explored and may be used to generate three-dimensional gradients of molecules within the material for tissue engineering applications.31-33

Methods Synthesis of Alkanethiols and Rhodamine Ketone. Tetra(ethylene)glycol-undecane thiol (EG4) was synthesized as reported previously.23 11-Amino-oxyundecanethiol (oxyamine-alkanethiol) and ketone functionalized rhodamine were synthesized according to Park et al.22 Solid-Phase Peptide Synthesis of GRGDS-ketone. Ketone functionalized GRGDS peptide was synthesized using a peptide synthesizer (CS Bio) at 0.1 mmol scale. 4-Acetyl butyric acid was used without protection of the ketone group. The peptide was obtained from the resin after treating with 10 mL of TFA containing 5% water and 5% methylene chloride for 2 h under stirring and filtration. The filtrate was mixed with ethyl ether (40 mL) and the mixture was centrifuged at 3000 rpm for 15 min to obtain a white precipitate. The precipitate was lyophilized overnight to obtain a white solid. ESI mass H2O calcd 714.4; found 714.3. Microfabrication. The microfluidic cassettes were fabricated using soft lithography.34 Patterns were designed using masks drawn in Adobe Illustrator CS3 and photoplotted by Pageworks (NH, U.S.A.) onto transparencies. These masks were then used to pattern SU-8 50 (Microchem) using the manufacturer’s directions to obtain 50 μm channel depth using these masks. Sylgard 184 (Dow Corning) was cast onto the mold in a 1:10 curing agent to elastomer w/w. The prepolymer was degassed for 15 min and then poured over the mold. The prepolymer was cured for 1 h at 75 °C. The PDMS was removed from the master and microfluidic holes were punched into the PDMS to allow fluid flow. Preparation of Gold Substrates. Gold substrates were prepared by electron beam deposition of first titanium (6 nm) and then gold (12 nm) on 24 mm  100 mm glass microscope slides. The slides were cut into 1  2 cm2 pieces and washed with absolute ethanol before use. Microfluidic Lithography (μFL). A 1 mM ethanolic solution of EG4 was flowed into the channels for 30 s twice (once through each inlet to ensure a high density monolayer) to install a protective EG4 SAM on the gold surface.16 The microfluidic cassette was then rinsed with ethanol for 10 s, and the PDMS cassette removed. PDMS Cleansing. Prior to use, all newly fabricated PDMS microfluidic cassettes were Soxhlet extracted with ethyl acetate for 6 h.35 After each use, the stamps were immersed in CH2Cl2 for 30 min to extract remaining thiol absorbed in the PDMS, then dried in a vacuum chamber for 2 h. This procedure could be performed over 30 times without any degradation of the stamps. The stamps could be stored and reused indefinitely after drying. SPREAD Inking and Printing Procedure. The clean PDMS cassettes were sealed onto the surface of a glass microscope slide, then 5 mM oxyamine-alkanethiol solutions (mixtures of EtOH and CH2Cl2) were pulsed into the microfluidic stamp rapidly and then kept static for 60 s. The cassette was then evacuated with the aid of negative pressure, and the alkanethiol was allowed to diffuse into the PDMS (to achieve variable but controlled gradient lengths; see Figure 3). After diffusion, the PDMS cassette was removed from the glass surface and stamped onto a gold-coated microscope slide for 5 s. After stamping, the surfaces were backfilled with EG4 to make the surface inert to nonspecific cell attachment. (31) Barrett, D. G.; Yousaf, M. N. Macromolecules 2008, 41, 6347–6352. (32) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2480–2487. (33) Luo, W.; Yousaf, M. N. Chem. Commun. 2009, No.10, 1237–1239. (34) Deng, T.; Tien, J.; Whitesides, G. M. Langmuir 1999, 15, 10706–10714. (35) Thibault, C.; Severac, C.; Mingotaud, A. F.; Vieu, C.; Mauzac, M. Langmuir 2007, 23, 10706–10714.

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Fluorescence Visualization. For visualization, the surfaces were reacted with a fluorescent ketone-functionalized rhodamine22 (10 mM, 3hrs) and characterized by fluorescence microscopy. RGDS-Ketone Functionalization of SPREAD Surfaces. Surfaces were exposed to a 10 mM solution of RGDS-ketone for 3 h then rinsed thoroughly with water. Cell Culture. Swiss Albino 3T3 fibroblasts (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (Gibco) containing 10% calf bovine serum and 1% penicillin/streptomycin. Cells were removed with a solution of 0.05% trypsin in 0.53 mM EDTA, resuspended in serum-free medium (100 000 cells/mL) for cell seeding, and allowed 2 h to attach to the surface prior to the addition of serum-containing media. For passage, cells were resuspended in the same 10 mL of medium that they were growing in, and 3 mL was transferred to 7 mL of fresh medium in a new flask. Cell Staining and Microscopy.36. Cells were fixed with 3.2% formaldehyde in Dulbecco’s PBS buffer (Sigma, St. Louis, MO). The cells were then permeated with Dulbecco’s PBS buffer containing 0.1% Triton X - 100, a combination of fluorescent dyes were used to visualize the fibroblasts: DAPI (40 ,6-diamidino2-phenylindole dihydrochloride for the nucleus, Sigma, St. Louis, (36) Hoover, D. K.; Chan, E. W.; Yousaf, M. N. J. Am. Chem. Soc. 2008, 130, 3280–3281.

Langmuir 2010, 26(15), 12817–12823

Article MO), phalloidin-tetramethylrhodamine B isothiocyanate (Sigma, St. Louis, MO) for F-actin cytoskeleton, antigiantin (Covance Research Products, Berkeley, CA) with a fluorescent tagged secondary antibody (fluorescein-conjugated goat antirabbit IgG, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) targeting the golgi apparatus, and mouse monoclonal antigamma tubulin (Sigma) to track centrosome position. Fluorescence images were taken using a Nikon Eclipse TE2000-E inverted microscope (Nikon USA, Inc., Melville, NY) and data analyzed by metamorph software.

Acknowledgment. This work was supported by the Carolina Center for Cancer Nanotechnology Excellence (NCI) and grants from the Burroughs Wellcome Foundation (Interface Career Award) and National Science Foundation (Career Award). We also thank Professors Michael Ramsey, Joe Desimone, Rudy Juliano, and Klaus Hahn for their comments and suggestions. Supporting Information Available: Figures showing line scans of fluorescent gradients on gold surfaces and SPREAD printing of hydroxy-alkane phosphonates to transparent indium tin oxide (ITO) surfaces characterized by scanning electron microscopy. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la1022642

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