Site-Selective Surface Modification Using Enzymatic Soft Lithography

May 25, 2011 - The strategy was applied to two applications: the first is dedicated to the creation of amino-silane patterned surfaces, and the second...
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Site-Selective Surface Modification Using Enzymatic Soft Lithography Aurelie Guyomard-Lack,† Nicolas Delorme,‡ Celine Moreau,† Jean-Franc- ois Bardeau,‡ and Bernard Cathala*,† † ‡

INRA, UR1268 Biopolymeres Interactions Assemblages, F-44316 Nantes, France Laboratoire de Physique de l’Etat Condense, UMR CNRS 6087, Universite du Maine

bS Supporting Information ABSTRACT:

Surface modification with functional polymers or molecules offers great promise for the development of smart materials and applications. Here, we describe a versatile and easy-to-use method of site-selective surface modification based on the ease of microcontact printing and the exquisite selectivity of enzymatic degradation. A micropatterned poly-L-lysine (PLL) layer on solid substrates was prepared by enzymatic degradation using trypsin enzyme immobilized on a prestructured poly(dimethlylsiloxane) (PDMS) stamp. After the enzymatic degradation of PLL and the removal of the degradation products, very well defined patterning was revealed over a large scale by fluorescence microscopy and atomic force microscopy (AFM). We investigate the advantage of our method by comparison with traditional microcontact printing and found that lateral diffusion was reduced, yielding a more accurate reproduction of the master. We also demonstrate that the stamp can be reused without reinking. The patterned surface was used for site-selective modification. The strategy was applied to two applications: the first is dedicated to the creation of amino-silane patterned surfaces, and the second illustrates the possibility of patterning polyelectrolyte multilayered thin films.

’ INTRODUCTION Surfaces patterned with micro- and nanoscale functional polymers or molecules that offer various physicochemical properties have attracting considerable interest1 for their potential applications in cell or tissue engineering,2 4 biomimetic approaches,5 and fundamental surface science research.6 Soft lithography, which includes microcontact printing (μCP), has undergone a spectacular evolution since its discovery and now can achieve very fine patterning that can be applied to a wide variety of substrates.7 10 μCP is simple enough to be used in a typical laboratory setting and does not require prohibitively expensive equipment. Moreover, μCP can also be used for the selective patterning of a surface when reactive or catalytic species are linked to the stamp. In the first case, reactive species are used as ink and transferred to the stamp. For instance, sulfonic acidfunctionalized monolayer-protected gold nanoparticles were used as an ink to catalyze the hydrolysis of trimetylsilyl ether self-assembly monolayers (SAM) locally after their transfer onto the surface by microcontact printing.11 However, despite the size r 2011 American Chemical Society

of the nanoparticles that limits the diffusion, the granular nature of the nanoparticles causes an inhomogeneity in inking. To overcome this problem, the same authors report a catalytic strategy by creating functional groups on the printing stamp surface that are not transferred onto the SAM but can chemically modify the SAM surface during stamping.12 This inkless strategy was used later by several groups to pattern surfaces by using either a chemical reaction13 15 or a biocatalytic reaction.16 In all cases, the catalytic microcontact improves the quality of the mold duplication by the limitation of lateral diffusion. The method reported here belongs to the catalytic strategy and is based on the deposition of a biopolymer layer on a surface and the hydrolysis of this layer by microcontact printing with an immobilized hydrolytic enzyme. Covalent attachment together with the exquisite selective degradation of enzyme provides high-quality Received: December 3, 2010 Revised: April 20, 2011 Published: May 25, 2011 7629

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Langmuir resolution of the final pattern. Thus, our approach offers versatile strategies for the controlled juxtaposition of chemically distinct and active areas, providing opportunities for further modification by masking/unmasking reactive groups. Here we present the proof of concept for this original strategy with two model systems. The first is focused on the specific functionalization of surfaces subjected to enzymatic lithography by an amino-silane. The second illustrates selective polymer adsorption and offers an original strategy for patterning a polyelectrolyte multilayer. In both cases, highly defined patterns over large areas are obtained by rapid, cheap, and easy-touse processes that are ideal for biological applications.

’ EXPERIMENTAL SECTION Stamp Preparation. Stamps for the microcontact printing process were produced by casting a poly(dimethysiloxane) (PDMS) silicone elastomer against a microstructured silicon master provided by the CEALETI (Grenoble, France). The PDMS (Sylgard 184, Dow Corning) was used in a 10:1 mixture (w/w) of base elastomer/curing agent and hardened at 70 °C for 2 h. After the curing procedure, PDMS stamps were submitted to UV ozone treatment, which is known to harden the PDMS surface, in order to limit nonreacted siloxane leakage, which can lead to a decrease in enzyme activity. Grafting Procedure of Tryspsin on the Stamp Surface. The surfaces of PDMS were sonicated in ethanol for 5 min and activated by O2 plasma for 3 min, followed by silanization with (3-aminopropyl)triethoxysilane (APTES) at 70 °C for 1 h, sonication in ethanol for 3 min, and drying under a stream of N2. The stamps were placed in glutaraldehyde solution (2.5% v/v) for 2 h and then rinsed first with phosphate buffer (100 mM, pH 6) and then with water. The stamps were immersed in a solution of trypsin (1 g/L) in phosphate buffer for 1 day at 4 °C and then rinsed with phosphate buffer and NaCl solution (0.5 M) and sonicated in water for 3 min. Trypsin immobilized on microstamps (PDMS-g-trypsin) was stocked in borate buffer (100 mM, pH 8.4) at 4 °C before use and was stable under this condition for several months. PLL Substrate. The layer of PLL was deposited on silicon or quartz substrates by spin coating. The substrates were previously cleaned using freshly prepared piranha solution for 30 min, followed by rinsing with water. Poly-L-lysine (0.5 mL) labeled with fluorescein isothiocyante (PLLFITC) at 1 g/L in 100 mM NaCl at pH 6 was poured onto a stationary substrate for 10 min, which was then accelerated at 400 rpm and spun at 2000 rpm for 3 min. The film was then rinsed with 0.5 mL of water for 1 min and spun at 2000 rpm for 3 min. Microcontact Printing Procedure. After rapidly drying, the PDMS-g-trypsin microstamps were placed on a layer of PLLFITC and a weight of 50 g was put on top to achieve contact. This setup was maintained for 5 min at room temperature, after which the stamps were removed and the surfaces were immediately rinsed with water and dried under a stream of N2. Selective Adsorption of Biopolymer on Micropatterned Surfaces. To prepare the scaffold described in Figure 6, the micropatterned surfaces were multilayers of PLL-pectin-PLL or PLL-pectinPLLFITC (PLL or PLLFITC solutions at 1 g/L in 100 mM NaCl at pH 6 and pectin solution at 1 g/L in 100 mM phosphate buffer at pH 6). This multilayer was deposited by spin coating on a quartz or silicon substrate under the same conditions as for the deposition of the PLL layer. To absorb chitosanFITC selectively on the micropatterned surfaces, 0.5 mL of chitosanFITC at 1 g/L in 100 mM NaCl at pH 4 filtered through 5 μm was deposited on micropatterned PLL-pectin-PLL for 20 min. The surfaces were then immersed in rinse baths of water, sonicated in water for 1 min, and dried under a stream of N2.

Selective Grafting of Silane on Micropatterned Surfaces. The micropatterned surfaces of PLL were silanized with (3-aminopropyl)triethoxysilane (APTES) at 70 °C for 1 h. The surfaces were placed

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in trypsin solution (1 g/L) in phosphate buffer (100 mM, pH 6) for 30 min and then immediately rinsed with water and dried under a stream of N2. The surfaces were then immersed in fluorescein isothiocyanate (FITC) solution (0.6 g/L) in ethanol for 12 h by stirring. Next, the surfaces were rinsed with water, followed by sonication steps in water and then in 0.5 M NaOH for 3 min each before drying under a stream of N2. Micropatterned Surface Characterization. The micropatterned surfaces were observed by fluorescence microscopy using an Olympus microscope with a 40 objective and a mercury burner lamp with an excitation filter with a bandwidth of 460 at 490 nm. Atomic force microscopy measurements of surfaces were performed with an Agilent 5500 AFM. All of the topography images were obtained in tapping mode in air using the same PPP-NCHR-W tip (nanosensor, spring constant 40 N/m, frequency 297 kHz). Image processing was performed with Gwyddion freeware.

’ RESULTS AND DISCUSSION Patterning of a Poly-L-lysine (PLL) Surface by a TrypsinImmobilized Microstructured Stamp. Microcontact printing

methods face several drawbacks, among which lateral diffusion is a major limitation for the accurate reproduction of the transferred pattern. Accordingly, previous work has addressed the development of nondiffusive methods using a catalytic modification of the surface. For instance, Shestopalov et al.13 15 have achieved the patterning of a protected functionalized SAM grafted to passivated silicon using a polyurethane-acrylate stamp functionalized with covalently bound sulfonic acid. Besides chemical reagents, enzymes are powerful catalysts with rapid turnover rates and high substrate specificity that makes them good candidates for simple and efficient surface modification. They have been previously used to pattern surfaces either by microcontact printing16 or more frequently by dip-pen nanolithography.17 23 Biocatalytic nanolithography with immobilized enzymes has been used to pattern surfaces through a controlled movement and positioning of the enzyme-modified AFM tip. For instance, this method was applied either to the polymerization of aniline nanostructures by a peroxidase-modified tip22 or by hydrolyzing biomolecules such as peptides to achieve surface patterns.17 This method yields high-resolution patterns but is restricted to rather limited surface areas because of the capacity of AFM devices. In our study, we immobilized the enzyme onto a solid support (i.e., μCP stamp) to enhance the quality of the replication of the mold by limiting the enzyme mobility as a result of its covalent attachment. Another major advantage of enzymes is their selective action that will modify only their substrats and allow neighboring nonsubstrate polymers to remain unchanged. To ensure that immobilization is due to a chemical link and not to physical interactions between the surface and the enzyme, the protocol was optimized on a planar PDMS substrate. The first step in the immobilization procedure is the activation of a substrate by oxygen plasma that converts the O Si(CH3)2 units of PDMS into silanol groups ( OH).24 This chemical modification of the surface is then followed by the reaction of the silanol groups with 3-aminopropyltrietoxysilane (APTES). The reaction is carried out in the vapor phase to avoid any polymerization of APTES that might increase the roughness of the surface and lower the accuracy of further surface modifications. An APTES-treated surface is subsequently exposed to glutaraldehyde to place reactive aldehydes on the surface, allowing free 7630

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Langmuir terminal aldehyde groups to be cross-linked by the amino residues of trypsin through Schiff base formation. The homogeneity of the grafting procedure is demonstrated by the AFM image of the functionalized surface that displays a very low roughness (2.3 nm, Figure 1). The proteolytic activity of trypsin after the immobilization process on the PDMS stamp was checked using the N-R-benzoyl-L-arginine ethyl ester assay (BAEE). When hydrolyzed, BAEE releases N-R-benzoyl-L-

Figure 1. (a) AFM topography image of trypsin immobilized on PDMS and (b) absorbance at 253 nm of (•) BAEE alone, (0) BAEE with the washing water of trypsin immobilized on a microstamp, and (9) BAEE with trypsin immobilized on the microstamp as a function of time.

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arginine (BA), which can be monitored spectrophotometrically (maximum absorbance at 253 nm). Figure 1b shows the increase in absorbance at 253 nm as a function of time as PDMS-g-trypsin is immersed in BAEE solution (as compared to the BAEE solution alone). In addition, no evolution of absorbance is observed in the washing water of PDMS-g-trypsin, indicating that there is no further enzyme release. All of these results demonstrated that the tryspin is covalently cross-linked on the PDMS microstamp and that its enzymatic activity is preserved during the immobilization procedure. An accurate site-selective chemical modification of a surface is obtained by using silicon microstructures as masters to prepare the poly(dimethylsiloxane) (PDMS) stamps employed to pattern the surfaces. The use of high-resolution masters combined with the optimization of the PDMS stamp preparation yields precise surface modification over large areas with a suitably high, reproducible definition of the resulting pattern. Results arising from the patterning of fluorescently labeled PLL surfaces with trypsin are presented in Figure 2. Trypsin is a protease that cleaves protein chains when a basic amino acid (lysine or arginine) is present and is well known to hydrolyze PLL.25 However, the use of other enzymatic systems will be considered in future applications. Experimentally, a thin layer of PLLFITC adsorbed on a silicon substrate was patterned with PDMS-g-trypsin microstamps. The microstamp pattern consisted of 1 mm lines spaced 3 mm apart. After contact, the PLLFITC surface was examined by fluorescence microscopy and atomic force microscopy (Figure 2). The fluorescence microscopy image (Figure 2b) revealed uniform lines with good linearity over hundreds of micrometers due to the degradation of the PLLFITC layer by trypsin. In Figure 2c, the gap size resulting from degradation is 1 mm; such a result demonstrates the perfect replication of the template pattern on the surface. Control experiments with a PDMS stamp devoid of enzyme have also been performed to ensure that the patterning is not due to the etching of the PLL layer by the PDMS stamp (Supporting Information). These experiments reveal that the surfaces remain identical to those observed before the stamping. Thus, the patterning is not due to the etching of the PLL layer by

Figure 2. (a) Schematic representation of the enzymatic microlithography process. (b) Fluorescence microscopy image of the PLLFITC surface patterned by enzymatic microlithography. (c) AFM topography image of the patterned PLL surface previously created. (d) Line profile of the corresponding AFM image. (e) Enlargement of a single line of the previous profile. 7631

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Figure 3. Schematic representations (a) of the enzymatic microlithography process with an adsorbed enzyme on the stamp and (c) of the microcontact printing process of PLLFITC (b, d) with fluorescence microscopy images of the results for each process.

the PDMS stamp but to the effective degradation of the PLLFITC layer by the enzyme as observed by AFM. No residual material is apparent on the silicon surface (Figure 2d,e). After the enzyme action, the edge of the gap is very sharp. The resolution (here understood as the distance between the top of the PLL layer and the bottom of the degradation pattern) of the edge of the strip is about 100 nm. Moreover, this height is constant all along the patterned lines. The height difference between the top of the layer and the silicon surface that corresponds to the thickness of the PLL layer was found to be 0.4 nm on average (Figure 2), in agreement with previous results obtained on adsorbed PLL of bare silicon.26 Moreover, the same layer thickness was measured after mechanically scratching the surface with the AFM tip (Supporting Information). Therefore, the image of the silicon surface obtained either by enzymatic or mechanical removal displays similar surface roughness, suggesting that no materials remain on the degraded surface. Accordingly, the accessible silicon surface is suitable for further modification and the PLL-covered area can be protected from any reaction or interaction process. To evaluate the efficiency of our approach compared to standard microcontact printing approaches, we investigated the patterns obtained when trypsin and PLL are used as ink and directly transferred onto the surface. When trypsin is used as ink and printed onto the PLL layer (Figure 3a,b), lateral diffusion occurs in the PLL layer and definition of the strips is lower. When a PDMS stamp of 1 μm lines spaced by 3 μm was used, the PLL strips were almost all degraded, thus we present results obtained with a stamp consisting of 60 μm lines separated by 100 μm (Figure 3b). Obviously, the pattern obtained is not an accurate replica of the stamp and the degradation of the PLL layer is not limited to the stamp contact zone. Such a phenomenon is known to be one of the main limitations of microcontact printing, and it can be somehow controlled by environmental factors.27 The covalent attachment of the enzyme to the stamp appears to be a powerful solution to increase the fidelity of the stamped pattern. To go further in the comparison of our method to traditional ones, we investigated the direct transfer of PLL on a silicon surface (Figure 3c,d). The patterns obtained present a more ordered organization, but the frontiers between PLL and silicon

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Figure 4. Fluorescence microscopy images of a fluorescently labeled PLL surface patterned after (a) the first, (b) the second, and (c) the third contact of a trypsin-functionalized stamp successively on thin layers of fluorescently labeled PLL adsorbed on a silicon surface.

are not as well-defined as when the PLL layer is degraded by the covalently linked enzyme stamp. The last advantage offered by our method is that the stamp can be used for the printing of several surfaces without reinking, contrary to the standard approaches. We used the same stamp to pattern three different surfaces successively (Figure 4). The same patterns were obtained for all of the samples, indicating that enzymes remain active and are still efficient after several contacts. From all of these results, the enzymatic microcontact printing approach has proved to be an alternative to overcoming some of the limitation of microcontact printing. To take advantage of these results, we investigated two examples of the site-selective modification of surfaces. The first one is related to surface modification by silane molecules, and the second is related to the specific adsorption of polymers on modified multilayered polyelectrolyte thin films. Selective Silane Reaction on a Patterned PLL Surface. Silanes are widely used for the production of self-assembled monolayers (SAMs); however, highly defined patterning by soft lithography is seldom achieved.28,29 Here, silane patterning was obtained by subjecting the PLL-patterned surface to a (3-aminopropyl)triethoxysilane (APTES) atmosphere (Figure 5). An evaluation of the selective reactivity of the exposed silicon surface with APTES was tested by dipping the patterned surface in trypsin solution to degrade the remaining PLL on the surface. Finally, the APTES patterning was revealed by FITC, which reacts with the amino groups of APTES. The process is summarized in Figure 5, and the fluorescence micrographs in Figure 5a,b show, respectively, the fluorescently trypsin-degraded PLL surface and the pattern obtained after reaction with the FTIC. Lines of 3 μm in width corresponding to the nondegraded PLL lines were clearly observed in Figure 5a, and Figure 5b displays the exact inverse organization. Fluorescent lines of 1 μm in width are inserted between the lines of 3 μm corresponding to the substrate (dark zones). This latter well-define pattern provides direct proof that (i) APTES reacts directly with the silicon dioxide of the substrate that is not covered by PLL, (ii) PLL is fully removed by the trypsin treatment, and (iii) we can easily form 2D arrays of ordered guest molecules on the surface using enzymatic soft lithography. Surface Modification of Polyelectrolyte Multilayer Thin Films for Site-Specific Polymer Adsorption. The layer-by-layer technique pioneered by Decher in the early 1990s30 is based on an 7632

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Figure 5. Schematic diagram of the selective modification of surfaces by (3-aminopropyl)triethoxysilane (APTES). The patterned PLL surface was subjected to an APTES atmosphere. The remaining PLL was then degraded by dipping the surface into a trypsin solution, and the APTES pattern was revealed by reaction with FITC. (a) Starting surface displaying fluorescent 3 μm PLL lines. (b) Resulting 1 μm lines observed on the surface after the FTIC reaction.

electrostatic self-assembly process and is a powerful tool for the creation of thin multilayer films with a nanostructuration perpendicular to the substrate. This method is based on the successive adsorption of oppositely charged polymers, which leads to the formation of multilayered thin films. Despite the occurrence of diffusion phenomena,31 these films exhibit internal organization within their thicknesses.30 However, this method cannot be used to create a planar structuration of the polymers. Few recent studies investigated the possibility of creating lateral organization at the surface of multilayered films by using polyelectrolytes as ink in microcontact printing.32,33 With the aim of opening up new opportunities for the investigation and creation of polyelectrolyte thin films, we applied our patterning strategy using trypsin enzymatic lithography to a pectin/PLL assembly. A PLL/pectin/PLL thin film was produced by the alternating deposition of both polymers by a spin-coating procedure. First, the PLL/pectin/ PLLFITC film was subjected to trypsin action as shown in Figure 6a. The presence of dark lines indicates that fluorescently labeled PLL was degraded by the stamp as described above (Figure 6b); however, the AFM image (Figure 6c) is different than the previous one (Figure 2c). Indeed, surface patterning was hardly visible on the AFM images. In the previous example, the bottom of the patterning was silicon, which is a hard material with very low roughness. In the case of the PLL/pectin/PLLFITC assembly, the patterned surface consists of soft materials formed by an alternation of PLL/pectin strips with a roughness close to the thickness of the PLL layer (0.4 nm). Because the roughness of the pectin layer is in the range of the height of the PLL layer, the visualization of the height difference between PLL and the pectin strips is hindered. Thus, AFM cannot distinguish any step at the frontier between PLL and the pectin layers. This suggests that only the last PLL layer that is exposed to the stamp is degraded by the enzyme; otherwise, a gap would be evidenced by AFM as shown in the previous degradation of a single layer of PLL. This implies also that the PLL layer within the film remains intact and is protected by the pectin layer. This point is not really surprising by taking into account the high specificity of enzymes for their substrates. In other

Figure 6. (a) Schematic representation of the process of selective patterning of PLL/pectin/PLLFITC films by trypsin stamps. (b) Fluorescence microscopy and (c) AFM topographic images of PLL/pectin/ PLLFITC after enzymatic microlithography. (d) Schematic representation of the process of selective patterning of PLL/pectin/PLL films by trypsin stamps, followed by the adsorption of fluorescently labeled chitosan. Fluorescence microscopy images (e) before and (f) after the adsorption of fluorescently labeled chitosan on PLL/pectin/PLL patterned by enzymatic microlithography.

words, trypsin is unable to degrade the glycosidic linkages of the pectin. The patterning of PLL at the surface of the film was confirmed by the selective adsorption of fluorescently labeled chitosan on the unlabeled patterned PLL/pectin surface (Figure 6e,f). At the pH used here, the chitosan is positively charged and therefore interacts with pectin but is repulsed by positively charged PLL. After rinsing to remove any loosely bound polymers, the fluorescence images show the expected pattern (Figure 6f). Fluorescent 1 μm chitosan lines are clearly visible, separated by 3 μm dark zones corresponding to unlabeled PLL. Figure 6f is the perfect negative of Figure 6b that was obtained after the degradation of PLL by trypsin. Thus, the final surface is positive but alternates chitosan and PLL. To our knowledge, such 3D patterning of multilayered thin films has never before been reported. Thus, because of the reliability and versatility of the technique, this strategy opens up opportunities for basic investigations of polyelectrolyte film construction and the design of microscale patterned surfaces for new applications requiring the localization of molecules or polymer of interest. The use of μCP combined with enzymes offers new opportunities to complete the panel of the method already available8,9,34 by offering a low-cost, versatile method for site-selective chemical surface patterning with high definition. A comparison with the standard procedure of microcontact printing in which PLL or trypsin is used as an ink demonstrates that the accuracy of the replica is greatly enhanced by our method because the lateral diffusion of ink is avoided. Moreover, the covalent attachment of 7633

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Langmuir the enzyme on the surface provides the possibility to reuse the stamp several times without reinking because trypsin is still active after printing. This versatile strategy offers a wide range of possibility to create various chemical functions on the surface by selective modification of the surface. Finally, we foresee this method finding application in a variety of contexts, such as biomedical surfaces for cells or objects to be selectively captured or released or versatile coatings for smart packaging in food or environmental industries. Efforts are underway to extend the range of application by using other classes of enzymes that can create smart, new physicochemical properties of surfaces.

’ ASSOCIATED CONTENT

bS

Supporting Information. AFM topographic image of a PLL surface and depth profile of a surface scratch. Fluorescence microscopy images of PLL surfaces after the patterned PDMS stamp was removed from the surface. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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