Atomic Force Microscopic Analysis of Highly Defined Protein Patterns

This paper describes the formation of highly defined two-dimensional protein arrays onto a polymeric substrate expressing biotin functionalities at it...
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Atomic Force Microscopic Analysis of Highly Defined Protein Patterns Formed by Microfluidic Networks Nikin Patel,*,† Giles H. W. Sanders,† Kevin M. Shakesheff,† Scott M. Cannizzaro,‡ Martyn C. Davies,† Robert Langer,‡ Clive J. Roberts,† Saul J. B. Tendler,† and Philip M. Williams† Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K., and Department of Chemical Engineering, Massachusetts Institute of Technology, E25-342, 45 Carleton Street, Cambridge, Massachusetts 02139 Received September 16, 1998. In Final Form: May 27, 1999 This paper describes the formation of highly defined two-dimensional protein arrays onto a polymeric substrate expressing biotin functionalities at its surface. Micron-scale arrays of avidin were created by exploiting its interaction with biotin, thereby creating a versatile patterned surface onto which any biotinylated species can be subsequently immobilized. The patterning process utilized the technique of microfluidic networks (µFN) to spatially confine the flow of protein solution on a substrate. We describe methods developed to control protein deposition with nanometer-scale precision. Utilizing atomic force microscopy (AFM), these patterned surfaces were analyzed to the molecular level. In addition, the applicability of this technique to a wider range of substrates has been investigated. In particular, developments within the µFN technique have enabled the successful formation of protein micropatterns onto a highly hydrophilic surface, a substrate which is generally not suited to the technique of µFN.

1. Introduction The advancement of patterning techniques in recent years has significantly enhanced our ability to spatially control surface chemistry at the micron level.1,2 The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors,3 combinatorial library screening,4 and the formation of tissue engineering templates.5 Biomolecular patterning has been realized utilizing a variety of techniques, the most common involving lithography-based methods. Photolithography-based methods have been utilized to create patterns of self-assembled monolayers (SAMs) of compounds such as alkanethiols6 and aminosilanes.7 By exploiting differential adsorption or variations in surface free energy, biomolecule adhesion can be mediated onto these surfaces. In addition, photolithography techniques have been utilized to selectively photoactivate molecules attached to a surface.4,8,9 For example, Hengsakul and Cass generated patterns of immobilized biotin groups onto polystyrene and nitrocellulose surfaces * Corresponding author. Present address: Molecular Profiles Limited, University Park, Nottingham NG7 2RD, U.K. E-mail: [email protected]. † The University of Nottingham. ‡ Massachusetts Institute of Technology. (1) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55. (2) Qin, D.; Xia, Y.; Rogers, J. A.; Jackman, R. J.; Zhao, X.-M.; Whitesides, G. M. Top. Curr. Chem. 1998, 194, 1. (3) Mrksich, M.; Whitesides, G. M. Trends Biotechnol. 1995, 13, 228. (4) Fodor, S. P. A.; Reed, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (5) Patel, N.; Padera, R.; Sanders, G. H. W.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M. FASEB J. 1998, 112, 1447. (6) Huang, J.; Hemminger, J. C. J. Am. Chem. Soc. 1993, 115, 3342. (7) Hickman, J. J.; Bhatia, S. K.; Quong, J. N.; Shoen, P.; Stenger, D. A.; Pike, C. J.; Cotman, C. W. J. Vac. Sci. Technol. A 1994, 12, 607. (8) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2272. (9) Hengsakul, M.; Cass, A. E. G. Bioconjugate Chem. 1996, 7, 249.

by adsorbing photobiotin and then selectively activating it by exposure to white light through a patterned mask.9 However, despite its success in creating micron-scale patterns, lithographic techniques suffer a number of disadvantages. These methods are applicable to only a small set of materials with chemical modification of the surface often resulting from a highly specialized reaction. In addition, patterning often requires several stages, some of which involve exposure of the surface to harsh solvents. The relatively high costs involved can also make this technique not easily accessible to chemists and biologists. These limitations can be avoided, in part, with the use of microcontact printing (µCP).10,11 This is a relatively convenient and inexpensive technique whereby an elastomeric mold is utilized for generating patterns of SAMs with submicron precision. By exploiting the terminal functionality of these patterned SAMs, biomolecules have been adsorbed chemically or physically. However, µCP and lithography techniques which rely on the generation of patterned SAMs are restricted to substrates on which these ordered arrays can form, typically coinage metals (Au, Ag, Cu) and hydroxyl-terminated surfaces (Si/SiO2, glass). Microfluidic networks (µFN), an extension to µCP, provide an approach for biomolecular patterning onto a variety of different substrates. The technique employs an elastomeric block with a continuous series of recessed channels in its surface. When brought into intimate contact with a solid substrate, a network of capillaries is formed enabling the flow of fluids along confined areas. This technique, pioneered by Whitesides and co-workers, was initially utilized to form microstructures of polymers by molding them within the capillaries.12 Delamarche and co-workers have applied µFNs for the attachment of protein arrays onto typically hydrophobic surfaces for (10) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498. (11) Lo´pez, G. P.; Biebuyck, H. A.; Ha¨rter, R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10774. (12) Kim, E.; Xia, Y.; Whitesides, G. M. Nature 1995, 376, 581.

10.1021/la981268v CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999

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bioassay development.13,14 We have previously described the use of µFNs for the design of biodegradable tissue engineering templates by micron-scale patterning of cell adhesive ligands, thereby enabling control over the spatial attachment and growth of specific cell types.5 The success of this procedure relied on the ability to create ligand patterns onto a biodegradable surface with nanometerscale definition and minimal nonspecific adsorption. In this paper, we focus on the µFN technique itself, describing methods we have employed first to generate highly defined two-dimensional protein arrays and second to create a versatile patterned surface onto which ligands can be specifically immobilized. Utilizing atomic force microscopy (AFM), these surfaces have been characterized to molecular resolution. Further, the application of the µFN technique to a wider range of substrates, in particular a hydrophilic surface, is described. It should be noted that in the past decade µFN systems have been incorporated into biosensor instruments such as the BIACORE instruments. Although proteins and other biomolecules can be successfully deposited within confined regions of a substrate, the diversity of the patterns is inflexible and restricted to the manufacturer’s design. Polymeric µFN systems are relatively easier to construct and can be tailored to incorporate custom designs. 2. Experimental Section Synthesis of Polymeric Substrate. R-amine ω-hydroxy poly(ethylene glycol) (Shearwater Polymers, Inc., average mol wt 3.8K) was stirred overnight with NHS-biotin (Fluka) and triethylamine in methylene chloride and acetonitrile at room temperature under argon. The biotinylated poly(ethylene glycol) was then isolated by vacuum filtration, and dried azeotropically from toluene. Then, recrystallized l-lactide was polymerized from the ω-hydroxy end of poly(ethylene glycol)-biotin by refluxing in toluene. Polylactide-poly(ethylene glycol)-biotin was precipitated from a methylene chloride solution by the addition of cold ether. The molecular weight of the polymer was determined by 1H NMR using the poly(ethylene glycol) signal as a reference. Gel permeation chromatography revealed one peak indicative of pure material. The polylactide molecular weight for this study was 21.4K. Preparation of µFNs. A patterned poly(dimethylsiloxane) mold was formed by curing its prepolymer (Dow Corning Sylgard Silicone Elastomer-184, ISL, West Midlands, UK) on a patterned master prepared photolithographically by exposing and developing a photoresist pattern on gallium arsenide wafers. The elastomeric mold bearing the negative pattern of the master was peeled off and washed repeatedly with ethanol, hexane, and finally deionized water. Following drying under argon, the mold was placed onto an epitaxially grown gold surface (see Figure 4).15 The capillaries were rendered hydrophilic by plasma treatment with O2 (load coil power ≈ 200 W) for 1 s (Bio-Rad RF Plasma Barrel Etcher). Avidin Pattern Formation. Within 1 min of plasma treatment the mold was placed onto a film of the polylactide-poly(ethylene glycol)-biotin. The film was formed from a 1 mg/mL solution of the polymer in trifluoroethanol, drop cast onto a polystyrene substrate, and dried under vacuum. A 500 µg/mL solution of rhodamine-labeled avidin (av-R) (Sigma, Dorset, U.K.) was prepared using distilled water and passed through a 20 µm Satorius filter (Satorius AG, Goettingen, Germany). Approximately 1 mL of this av-R solution was dropped onto the polymeric substrate. The drop was positioned so that the av-R solution wetted an end of the mold at which the capillaries were open. After 1 h of contact time the av-R solution was removed by blotting and replaced with approximately 20 mL of distilled water. After (13) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779. (14) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500. (15) DeRose, J. A.; Thundat, T.; Nagahara, L. A.; Lindsay, S. M. Surf. Sci. 1991, 256, 102.

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Figure 1. Schematic representation of the µFN patterning technique. Following curing of the poly(dimethylsiloxane), the mold is cut to reveal opened channels on the underside. When placed onto a suitable substrate, conformal contact between the mold and surface results in the formation of capillaries through which fluids can flow. a further 5 min the water was removed. This washing procedure was repeated five times. Then, the sample was immersed in water and the mold removed by peeling it away from the polymeric substrate. The sample was then washed with a further 100 mL of water. Catalase Pattern Formation. An epitaxially grown gold film (∼50-100 nm thick) was immersed into a 1 mM ethanolic solution of 3-mercaptopropanoic acid (Aldrich, Dorset, U.K.) for 24 h before being rinsed in ethanol and dried under a stream of high-purity argon. The elastomeric mold following plasma treatment was placed onto this SAM. A 500 µg/mL solution of bovine liver catalase (EC 1.11.1.6, Sigma) was prepared using sodium phosphate buffer (10 mM, pH 5.0) and passed through a 20 µm Satorius filter. The protein solution was applied to the mold for patterning as detailed above. Atomic Force Microscopy. Images were obtained using a Digital Instruments (Santa Barbara, CA) Multimode scanning probe microscope with a Digital Instruments Nanoscope IIIa controller. Topographic images were acquired under ambient conditions with tapping mode16 using sharpened silicon nitride tips with cantilever resonance frequencies in the range of 307375 kHz and a scan rate of 1 Hz. Force modulation AFM17 was undertaken employing a Topometrix (Saffron Walden, U.K.) Lumina system with a 100 µm Accurex scanner. Topometrix premounted pyramidal contact mode tips were employed. A scan rate of 1 Hz and a modulation amplitude of 2 nm was used. Contact Angle Measurements. Contact angles were measured in air on static drops using a goniometer (Ealing ElectroOptics plc, Watford, U.K.). A 2 µL drop of sodium phosphate buffer (10 mM, pH 5.0), was applied to the surface, and contact angle measurements were performed on opposite edges of the drop. Measurements were repeated on at least four drops.

3. Results and Discussion Patterning Method. The basic µFN method for patterning involves creating networks of continuous capillaries by the conformal contact between a patterned elastomeric mold and a substrate (Figure 1). A solution containing molecules that will form the pattern is then flowed through the capillaries, thereby confining their immobilization to particular parts of the surface. In this study, the high-affinity avidin-biotin interaction was exploited for creating a two-dimensional array of proteins on the biotinylated biodegradable block copolymer, polylactide-poly(ethylene glycol), termed PLA-PEG-biotin18 (Figure 2A). As shown schematically in Figure 2B, when films of PLA-PEG-biotin are exposed to an aqueous environment, biotin moieties are presented at the surface by flexible PEG chains. Contact angle analysis shows that (16) Zhong, Q.; Innes, D.; Kjoller, K.; Elings, V. B. Surf. Sci. 1993, 290, 688. (17) Maivald, P.; Butt, H. J.; Gould, S. A. C.; Prater, C. B.; Drake, B.; Gurley, J. A.; Elings, V. B.; Hansma, P. K. Nanotechnology 1991, 2, 103. (18) Cannizzaro, S. M.; Padera, R.; Langer, R.; Rogers, R.; Black, F. E.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M. Biotechnol. Bioeng. 1998, 58, 529.

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Figure 2. (A) Chemical structure of the PLA-PEG-biotin polymer. (B) Schematic representation for the immobilization of biomolecules onto PLA-PEG-biotin. In an aqueous environment, the polymer surface presents biotin on the end of flexible PEG chains (I). Biotin moieties are used to immobilize tetrameric avidin molecules (II). Free biotin binding sites on the avidin molecules can then be used to anchor any biotinylated ligand (III).

the polymer surface is initially hydrophobic (contact angle 79° ( 4°); however, within a few minutes the surface becomes more hydrophilic (contact angle decreases to 45° ( 5°) due to surface expression of PEG units in an aqueous environment. The addition of the protein avidin to this environment results in its immobilization to biotin via one of its four binding sites. The other free binding sites are then available for the attachment of a biotinylated ligand. The analysis of specific interactions between avidin and PLA-PEG-biotin has been previously described by Black et al.19 To create patterns of avidin, the elastomeric mold was placed onto the PLA-PEG-biotin film and a solution of avidin fluorescently tagged with rhodamine was flowed through the capillaries. The transparent nature of the mold and substrate enabled pattern formation to be monitored by both optical and fluorescence microscopy. After 1 h of flow, the capillaries were flushed with water followed by removal of the mold by peeling. Figure 3 displays resulting patterns of av-R generated on the PLA-PEG-biotin. These patterns were formed either from a mold bearing 12 µm wide, 2 µm deep channels separated by 250 µm (Figure 3A) or from a mold bearing a series of channels of 30, 50, and 70 µm wide, 20 µm deep separated by 250 µm (Figure 3B). In both cases the channels were 16 mm long. The volume of protein solution required to fill the channels is small, with quantities of liquid as small as 10 µL sufficient to fill a 20 mm wide section of the above molds. Indeed, Delamarche and coworkers have demonstrated that a 1 µL quantity of protein solution was sufficient to fill 100 channels which were 3 mm long, 3 µm wide, and 1.5 µm deep.13 An important aspect for the successful implementation of this patterning procedure involves an intimate contact between the mold and substrate which needs sustaining to preserve the precise dimensions of the capillaries and prevent any fluid leakage. The elastomeric mold in its native state is hydrophobic (contact angle of 105° with water) which enables it to form a tight reversible seal (19) Black, F. E.; Hartshorne, M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M.; Cannizzaro, S. M.; Kim, I.; Langer, R. Langmuir 1999, 15, 3157-3161.

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Figure 3. Fluorescence microscopy images displaying patterns of avidin-rhodamine generated on PLA-PEG-biotin: (A) 12 µm wide avidin stripes (light gray regions) separated by 250 µm; (B) series of avidin stripes 30, 50, 70, and 70 µm wide, separated by 250 µm.

with flat substrates. However, this hydrophobicity inhibits the flow of aqueous solutions through capillaries. One method for promoting capillary flow involves exposing the whole elastomeric mold to an oxygen plasma to render it hydrophilic (contact angle of 0° with water).13 A problem often encountered in these studies when using this technique is the hydrophilic nature of the mold base compromises its ability to maintain a seal with the PLAPEG-biotin film for long periods of time. Within minutes of introducing the av-R solution to the mold edge, the seal fully deteriorates resulting in seepage of fluid between capillaries. Therefore, a selective plasma etching technique has been developed to render the capillary walls hydrophilic while maintaining the hydrophobicity of the mold base (Figure 4). By placing the mold onto a clean, flat, preferably hydrophobic surface, the resultant conformal contact can shield base regions from the activity of the oxygen plasma. Contact angle analysis confirms the differential hydrophobicity/hydrophilicity of shielded and nonshielded regions. Transfer of the mold to the PLAPEG-biotin surface results in a seal which can last for many hours. Subsequent patterning of av-R (Figure 3) can be achieved with no seepage of protein solution between capillaries. Another important factor for successful patterning by µFN is the generation of a continuous flow of solution through the capillaries. This is required to ensure an even distribution of immobilized molecules along the whole length of the capillaries. When protein solution is flowed into a channel, the diffusion of protein molecules and their interaction with channel walls can cause their rapid depletion from solution with immobilization only occurring along a very short (micron scale) length of the capillary.14 This effect is promoted when using smaller capillaries which have a higher surface-to-volume ratio and protein solutions of lower concentration. Therefore, to achieve immobilization along the entire length of the capillaries, the flow must be sufficient to provide an adequate delivery

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Figure 4. Scheme for the selective O2 plasma treatment of the elastomeric mold. This treatment increases the hydrophilicity of any poly(dimethylsiloxane) surface that is not protected by the gold surface. Transferring the treated mold to the PLAPEG-biotin surfaces produces capillaries with hydrophilic walls. Avidin solution flow across the PLA-PEG-biotin surface is restricted to the capillary regions due to an intimate contact between the hydrophobic mold base and substrate.

Figure 6. Optical and fluorescence images showing the difference in flow of rhodamine-labeled avidin molecules compared to the advancement of the liquid front through the capillaries. (A) Optical image showing a partly filled capillary alongside two capillaries which are fully filled with protein solution. (B) The corresponding fluorescence image shows that although the latter two capillaries are filled with liquid, there is a lag in the flow of avidin molecules through the capillaries.

Figure 5. Fluorescence microscopy image showing the flow of avidin-rhodamine solution out of two 70 µm wide capillaries. Accumulation of solution along the hydrophilic edge of the mold maintains a flow through the microcapillaries.

of protein molecules. The creation of highly hydrophilic capillary walls by selective plasma treatment results in an initial surge of protein solution into the capillaries. Within a few seconds, the entire length of the capillaries is filled with liquid. A flow is maintained through the capillaries by liquid exiting and accumulating along the hydrophilic outer edge of the elastomeric mold (Figure 5). On using the elastomeric mold for the first time, a significant protein depletion effect is observed whereby the flow of fluorescent avidin molecules was significantly retarded compared to the advancement of the liquid front through the capillary (Figure 6). Filling of the 16 mm long capillaries with fluorescent avidin molecules typically occurred within 1 h. However, on repeat use of the same elastomeric mold, filling of capillaries with avidin molecules was significantly reduced to approximately 10 min. This suggests that the deposition of avidin molecules onto the walls of the capillaries during initial use significantly reduces the effects of protein depletion on subsequent use.20 Although a natural flow of liquid through capillaries has been generated in this patterning technique, a

relatively concentrated protein solution (500 µg/mL) was utilized to ensure that the initial capillary filling occurred relatively quickly (∼1 h). Sjo¨lander and Urbaniczky have shown that a biosensor with incorporated miniaturized flow cells with mechanical liquid flow-through devices was capable of flowing and analyzing a solution of both low concentration (picomolar range) and volume (microliter quantity) relatively quickly (10 min). Therefore, future advances in elastomeric patterning technology could incorporate mechanical-flow through devices.21 AFM Analysis of Patterns. AFM analysis was utilized to determine both the sharpness of avidin patterns and the level of avidin immobilization within in the patterns. Figure 7 displays a tapping-mode AFM image of a boundary between a 12 µm wide avidin stripe and bare PLA-PEG-biotin. Tapping-modeAFM was utilized due to its relatively high resolution and nondestructive nature compared with the traditional technique of contact-mode AFM.16 Within the stripe, individual avidin molecules are observed with a diameter of approximately 5-10 nm, which correlates reasonably with X-ray crystallography data of 5.6 nm × 5.0 nm × 4.0 nm.22 The small difference between observed and actual dimensions of avidin molecules could be explained by the phenomenon of tip (20) Selective plasma treatment was employed prior to each use of the elastomeric mold. (21) Sjo¨lander, J.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345. (22) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993, 231, 698.

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Figure 7. Tapping-mode AFM image displaying a sharp boundary between avidin molecules within a 12 µm wide stripe and bare PLA-PEG-biotin. Individual molecules of avidin within a monolayer stripe are observed. No features with dimensions similar to avidin are observed between stripes. Image z-scale: 0-10 nm (dark to light color).

broadening which arises when the surface feature being imaged by AFM is much sharper than the apex of the probe, resulting in the sample feature imaging the probe. Under these circumstances, the feature appears larger than its actual size.23,24 Due to the nondestructive nature of tapping-mode AFM and the high-affinity avidin-biotin interaction, no evidence of sample destruction by the AFM probe was observed. Cross-sectional analysis of the boundary recorded a step height of approximately 5 nm, which is indicative of a monolayer coverage. On some areas of the avidin stripe, large protein aggregates are observed which are resistant to washing with water. In addition, this image demonstrates the exceptional control of avidin distribution along the boundary edge. Lateral deviations along the edge presented in Figure 7 are less than 30 nm, equivalent to only approximately six avidin molecules. This high level of pattern definition results from the selective plasma treatment which generates and maintains an intimate contact between the elastomeric mold and polymeric substrate, preventing any leakage of protein solution underneath the mold. As shown in Figure 7, no avidin molecules are observed in areas contacted by the elastomeric mold. In the above example, patterns were generated utilizing an elastomeric mold which had previously been used several times resulting in stripes composed of a monolayer of avidin with even distribution along the entire length of the pattern. However, when using a mold for the first time, a significantly lower coverage of avidin is observed due to protein depletion effects. Figure 8 demonstrates this differential coverage. Here, a new mold was used to generate a pattern of 12 µm wide stripes of avidin. The same mold is then used to repeat the patterning procedure at approximately 90° to the initial pattern resulting in a crisscross formation. Fluorescence microscopic imaging (Figure 8A) displays the initial pattern having a significantly lower fluorescence intensity compared with the repeat pattern. AFM imaging correlates lower fluorescence intensity with lower protein coverage. These differences (23) Allen, M. J.; Hud, N. V.; Balooch, M.; Tench, R. J.; Siekhaus, W. J.; Balhorn, R. Ultramicroscopy 1992, 42, 1095. (24) Williams, P. M.; Shakesheff, K. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B. J. Vac. Sci. Technol. B 1996, 14, 1557.

Figure 8. (A) Fluorescence microscopy image of a crisscross pattern. Protein depletion effects during initial use of elastomeric mold (horizontal 12 µm wide stripes) result in the formation of lower fluorescence intensity stripes compared with those formed during repeat patterning (vertical stripes). Forcemodulation (B) and tapping-mode (C) AFM images of the crisscross pattern highlight differences in avidin coverage between the two sets of lines. Image z-scales: (B) 0-1.5 nA/ nm, (C) 0-15 nm (dark to light color).

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Figure 9. Tapping-mode AFM image displaying a pattern of catalase molecules adsorbed onto a carboxylic acid terminated SAM on gold. The image displays a boundary region at the edge of a 12 µm wide catalase stripe. Individual molecules of catalase are observed within a monolayer (inset: 100 nm × 100 nm). No features with dimensions similar to catalase are observed between stripes. Image z-scale: 0-20 nm (dark to light color).

in avidin coverage are highlighted by force-modulation AFM (Figure 8B), which generates image contrast from variations in sample compliance. This image displays the initial stripe with lower force-modulation contrast compared with the repeat pattern. This suggests that the initial stripes have a lower protein coverage. Threshold analysis of a tapping-mode AFM image (Figure 8C) shows the initial stripe as having approximately 25% coverage of avidin compared with 88% coverage for the repeat pattern. Crisscross patterns whereby both sets of stripes have similar coverage of immobilized avidin can be created by utilizing a mold which has been previously used several times before (data not shown). AFM analysis of these crisscross patterns has confirmed multiple patterning steps can be performed without destruction of existing patterns. The intimate contact between elastomeric mold and patterned substrate does not alter the coverage of the immobilized avidin molecules. The ability to employ multiple patterning steps has the potential to greatly increase the complexity of patterns formed by µFN. Patterning onto a Hydrophilic Surface. As detailed above, the selective plasma treatment of the elastomeric mold is essential for maintaining a tight seal between the mold and PLA-PEG-biotin substrate. In addition to enabling the formation of sharp avidin micropatterns on this relatively hydrophobic substrate, the selective plasma treatment procedure has created the opportunity of protein patterning onto more hydrophilic substrates. For hydrophilic substrates, plasma treatment of the whole mold results in a seal which typically lasts for only a few seconds. However, on selective plasma treatment, a seal can be generated which lasts for several hours. The applicability of this technique to a highly hydrophilic substrate is demonstrated here by the micropatterning of the enzyme catalase onto a carboxylate-terminated SAM on gold (contact angle of 22° ( 2° with water). Catalase solution of pH 5.0 was flowed through an elastomeric mold to form 12 µm wide catalase stripes separated by 250 µm gaps. At this pH, catalase has a net positive charge and can be adsorbed to the negatively charged SAM via an electrostatic interaction. To ensure that a monolayer coverage

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of catalase was obtained along the entire length of the mold capillaries, the patterning method involved the use of a mold which had previously been filled with catalase. On subsequent use, filling of capillaries for approximately 10 min resulted in monolayer coverage of catalase. Figure 9 displays a boundary region at the edge of a catalase stripe. Within the stripe, individual catalase molecules are observed with a diameter of approximately 10-12 nm and height of 4 nm, which correlate well with X-ray crystallography data.25 The largest lateral deviations along the boundary are approximately 100 nm. This increase in lateral deviation compared with patterns formed on PLAPEG-biotin may result from an increase in roughness of the underlying gold surface. However, the intimate seal generated by the hydrophobic mold base prevents any leakage of catalase molecules. Despite electrostatic interactions between protein and substrate being weaker compared with covalent immobilization, the nondestructive nature of tapping-mode AFM enables these adsorbed protein monolayers to be imaged without any destructive effects arising from interactions between AFM probe and sample. 4. Conclusions This paper has demonstrated that the µFN technique can be utilized to generate precisely defined twodimensional protein arrays onto a polymeric surface. A fundamental step for the generation of these patterns has been the selective plasma treatment of the elastomeric mold. This has proved important for maintaining an intimate seal between the mold and PLA-PEG-biotin film while simultaneously generating and maintaining flow of solution through microcapillaries. This procedure can also be applied to highly hydrophilic surfaces which are generally more difficult to pattern by µFN due to insufficient sealing between elastomeric mold and substrate. Surface analysis by AFM has played a key role in determining the quality of the patterns generated at the molecular level. AFM has confirmed that patterns with monolayer quantities of protein can be produced with exceptional sharpness, at the nanometer scale, with no leakage of protein molecules from microcapillaries. In addition AFM has demonstrated that multiple steps to form complex arrays can be performed successfully without damage to initial patterns. Finally, the ability to form micropatterns of avidin molecules using PLA-PEG-biotin has created a generic technique by which any biotinylated species can be immobilized into defined patterns. Exposure of an avidin pattern to an aqueous solution of a biotinylated species would limit specific binding to patterned regions only with the presence of PEG at the surface of nonpatterned regions preventing any nonspecific binding. Acknowledgment. The authors thank G. Laws and J. R. Middleton for their assistance in the preparation of the patterned master. M.C.D., S.J.B.T., and C.J.R. acknowledge Eli Lilly for the funding of a studentship to N.P. G.H.W.S. acknowledges support from a BBSRC postdoctoral fellowship, and S.M.C. acknowledges support from a NIH postdoctoral fellowship. K.M.S. is an EPSRC Advanced Research Fellow. R.S.L. acknowledges the National Science Foundation for support. LA981268V (25) Murthy, M. R. N.; Reid, T. J., III.; Sicignano, A.; Tanaka, N.; Rossmann, M. G. J. Mol. Biol. 1981, 152, 465.