Electron-Beam Surface-Patterned Poly(ethylene glycol

We analyzed the digital image data quantitatively using the Digital Micrograph software .... An alternate cross-linking mechanism was proposed by Merr...
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Electron-Beam Surface-Patterned Poly(ethylene glycol) Microhydrogels P. Krsko, S. Sukhishvili, M. Mansfield, R. Clancy,† and M. Libera* Stevens Institute of Technology, Hoboken, New Jersey 07030 Received January 29, 2003. In Final Form: April 28, 2003 This paper describes research addressing the question of whether microscopic hydrogels can be created from poly(ethylene glycol) [PEG 6800] and poly(ethylene oxide) [PEO 200K] using spatially resolved radiation from a scanning electron microscope with an approach similar to that used in the electron-beam patterning of polymeric photoresists. We demonstrate that, indeed, PEG hydrogels with micrometer and submicrometer feature sizes can be created by this approach, and we call these microhydrogels. Using solvent-free PEG 6800 and PEO 200K films ∼50-100-nm thick, we have identified sets of irradiation conditions where sufficient cross-linking occurs so that the exposed patterned polymer remains while the unexposed polymer dissolves during a post-irradiation solvent rinse. Arbitrary spatial patterns can be made. We have generated patterned dots with diameters below 200 nm. Using atomic force microscopy, in air and water, to study ∼5 × 5 µm PEG and PEO pads on silicon, we show that the patterned features generated by electron-beam cross-linking swell when exposed to water. The extent of swelling depends on the incident electron dose. Maximum swelling ratios of 14-16 have been observed. The swelling ratio decreases with increasing dose toward a limit of unity at the highest doses studied. Because of the significance of PEG in biomaterials applications, we examined the adsorption of fibronectin fragments onto the PEG microhydrogels using immunofluoresence optical microscopy. Undetectable Fn levels are observed on microhydrogels subjected to the lowest radiative exposure conditions where maximum swelling occurs. Fn adsorption increases with increasing dose and reaches a maximum at the highest doses where swelling ratios of unity are observed. This approach opens a new means for arbitrarily patterning the spatial distribution of proteins on surfaces and may be useful for controlling surface bioactivity.

Introduction Surface patterning at appropriate length scales is of significance to many emerging applications areas, particularly those involving proteins and cells.1,2 In addition to well-established technologies based on photolithography, surface patterning has been achieved by soft lithography,3-6 microfluidic patterning,7-9 three-dimensional printing,10 and dip-pen nanolithography,11 among other traditional and hybrid approaches. Patterning using electron beams has been practiced for several decades and has the advantage of being able to generate surfacepatterned structures with both arbitrary shapes and feature sizes as small as a few tens of nanometers. In the context of modifying surfaces for biorelevant applications, these properties are important because the control of * Author to whom correspondence should be addressed. † Hospital for Joint Disease, New York University School of Medicine, New York, NY 10003. (1) Ito, Y. Biomaterials 1999, 20, 2333-2342. (2) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (3) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (4) Hyun, J.; Ma, H.; Banerjee, P.; Cole, J.; Gonsalves, K.; Chilkoti, A. Langmuir 2000, 18, 2975-2979. (5) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741-744. (6) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264 (Apr 29), 696-698. (7) Chiu, D.; Jeon, N. L.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (6), 2408-2413. (8) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500-508. (9) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. (10) Park, A.; Wu, B.; Griffith, L. G. J. Biomater. Sci., Polym. Ed. 1998, 9 (2), 89-110. (11) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295 (Mar 1), 1702-1705.

protein and cell behavior on synthetic surfaces requires the control of surface structure and chemistry at length scales ranging across both the nano- and microscales. Because of their usefulness in biological systems due to their unique interactions with water, hydrogels have been and continue to be extensively studied in the context of biomedical device12,13 and drug-delivery technologies14 and, more recently, for microfluidic applications.15 Among the various synthetic water-soluble polymers developed and studied as hydrogels, poly(ethylene glycol) (PEG) has received considerable attention.16 PEG is an amphiphilic polymer17 with FDA approval for in vivo application. Its antifouling properties in biomaterials applications are well known, and it has been used extensively to resist protein and cell adhesion.18-24 Cross-linking has been achieved in PEG, and its higher-molecular-weight relative poly(12) Nguyen, K. T.; West, J. L. Biomaterials 2002, 23, 4307-4314. (13) Peppas, N. A.; Langer, R. Science 1994, 263 (Mar 25), 17151720. (14) Langer, R. Nature 1998, 392 (Apr 30), 5-10. (15) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B.-H. Nature 2000, 404 (Apr 6), 588-590. (16) Harris, J. M. In Poly(ethylene glycol) Chemistry; Harris, J. M., Ed.; Plenum: New York, 1992; pp 1-14. (17) Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 83788379. (18) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. J. Biomed. Mater. Res. 2000, 51, 343-351. (19) Andrade, J. D.; Hlady, V. Advances in Polymer Science; SpringerVerlag: Berlin, 1986; Vol. 79, pp 1-63. (20) Gombotz, W. R.; Wang, G.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547-1562. (21) Graham, N. B. In Poly(Ethylene Glycol) ChemistrysBiotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum: New York, 1992; pp 263-281. (22) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (23) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (24) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059-5070.

10.1021/la034157r CCC: $25.00 © 2003 American Chemical Society Published on Web 06/03/2003

Poly(ethylene glycol) Microhydrogels

(ethylene oxide) (PEO), both by modification of the polymer chemistry and by exposure to radiation such as ultraviolet (UV) light, X-rays, or electrons.25-28 Exposure to highenergy electron irradiation has been employed to promote cross-linking reactions in dilute aqueous PEG solutions using MeV energy electrons from an accelerator.24,29-33 This approach has been used to study the effects of incident dose, polymer concentration in solution, polymer molecular weight, polymer architecture, and biophysical response, among other possible variables. In contrast to previous work on PEG and PEO hydrogels, we are using focused electron beams to create arbitrary patterns of fine-scale hydrogels on surfaces. We show that microhydrogels can be formed by this method, and we explore the nature of microhydrogels produced using polymers with two different molecular weights: PEG 6800 and PEO 200K. In both cases, there is a threshold incident dose of electrons above which stable microhydrogels form, and we show that the extent of microhydrogel swelling can be controlled by the electron dose. The swelling is anisotropic and principally occurs perpendicular to the plane of the polymer film. Lateral swelling is constrained by the adhesive bonds between the microhydrogels and the silicon substrate on which they are patterned. Because of PEG’s significance in the control of protein adsorption on biomaterials surfaces, we ask whether the adsorption of protein, specifically fragments of fibronectin (Fn) that contain the RGD binding motif, on these microhydrogels can be controlled. We show that there is a dramatic dependence of Fn adsorption on the swelling properties of the microhydrogels, and we can control this adsorption lithographically. Experimental Procedures Materials. Our experiments used PEG of molecular weight (M) 6800 Da (PEG 6800) and 200 000 Da PEO (PEO 200K) purchased from Scientific Polymer Products and used as received. HPLC-grade tetrahydrofuran (Aldrich), anhydrous 200-proof ethyl alcohol (Aldrich), HPLC-grade acetone (Pharmco), and dichloromethane (Acros Organics) were all used as received. Single-crystal 3-in. wafers of boron-doped [100]-oriented silicon approximately 0.5-mm thick and polished on one side were obtained from Virginia Semiconductor. Water with a resistivity exceeding 15 MΩ cm was produced using a Millipore Direct Q system for combined reverse osmosis and deionization treatments. We purchased human plasma Fn cell binding fragments from Upstate Biotechnology, rabbit antihuman Fn primary antibody from Chemicon International, and flourescein (FITC) conjugated AffiniPure goat antirabbit IgG H+L secondary antibody from Jackson ImmunoResearch. Phosphate buffer saline with Ca (PBS-C) was used at pH 7.4. Silicon Substrate Preparation. Silicon substrates were prepared by cleaving single-crystal wafers into sections 1 × 1 cm in size. In a first cleaning process, each substrate was sonicated in acetone for 5 min and then in ethanol for 5 min. The surface was dried using a nitrogen gas stream. In a second cleaning (25) Andreopoulis, F. M.; Beckman, E. J.; Russell, A. J. Biomaterials 1998, 19, 1343-1352. (26) Cruise, G. M.; Scharp, D. S.; Hubbell, J. A. Biomaterials 1998, 19, 1287-1294. (27) Lu, S.; Anseth, K. Macromolecules 2000, 33 (7), 2509-2515. (28) Mellott, M. B.; Searcy, K.; Pishko, M. V. Biomaterials 2001, 22, 929-941. (29) King, P. A. Irradiated Poly(ethylene oxide) and Processes Therefor. U.S. Patent 3,470,078, 1969. (30) Kofinas, P.; Athanassiou, V.; Merrill, E. W. Biomaterials 1996, 17, 1547-1550. (31) Merrill, E. W. Immobilized poly(ethylene oxide) star molecules for bioapplications. U.S. Patent 5,275,838, 1994. (32) Rosiak, J. M.; Ulanski, P. Radiat. Phys. Chem. 1999, 55, 139151. (33) Sofia, S. J.; Merrill, E. W. J. Biomed. Mater. Res. 1998, 40, 153163.

Langmuir, Vol. 19, No. 14, 2003 5619 Table 1. Average Dose of Electron Irradiation for the Exposure Pattern pattern

dwell time (ms)

dose (C/m2)

1 2 3 4 5 6 7 8

0.001 0.004 0.007 0.01 0.04 0.07 0.1 0.4

0.003 0.012 0.021 0.031 0.122 0.214 0.306 1.223

pattern

dwell time (ms)

dose (C/m2)

9 10 11 12 13 14 15

0.7 1 4 7 10 40 70

2.140 3.057 12.228 21.399 30.570 122.279 213.988

process, the substrates were exposed to UV irradiation from a mercury grid lamp with a maximum power of 450 W for 5 min. These substrates were then further exposed to 5% hydrofluoric acid in water for 5 min, rinsed in distilled water, and finally exposed to an RF oxygen plasma for 7 min. These two substratecleaning methods led to comparable results when later casting PEG and PEO thin films on them. Solvent Casting of PEG Thin Films. We cast thin polymer films by dropping approximately 50 µL of a 1 wt % solution of PEG or PEO in THF at 323 K onto the polished side of the cleaned silicon wafer spinning at approximately 4000 rpm. The silicon substrate was fixed to the spin-coating device using double-sided adhesive. After 20 min of spinning, the wafer was annealed at 320 K under a vacuum of approximately ∼50 mTorr for 2 h. In general, the PEO 200K films tended to be thicker than the PEG 6800 films under nominally identical conditions. This procedure produced films with thicknesses of approximately 40 and 120 nm for the PEG 6800 and PEO 200K films, respectively. Electron-Beam Patterning. Polymer films on silicon substrates were exposed to electron irradiation in a LEO 982 DSM field-emission scanning electron microscope (FEG-SEM). The vacuum in the specimen chamber during electron irradiation was approximately 10-6 hPa. The electron accelerating energy used was 10 keV, and a typical beam current was in the range from 20 to 100 pA. The electron-beam position and dwell time at each pixel position were controlled using an Emispec Vision data acquisition and control computer system (Emispec Systems). Exposure patterns ranging from individual points, to lines generated by a linear sequence of points, to square pads generated by a two-dimensional array of exposure points could all be created using the scripting capabilities of the Emispec Vision software. Square exposure areas were generated by digitally rastering an electron beam, approximately 10 nm in diameter, across the polymer surface in a square array of exposure points. An average dose, D, for such an exposure was determined by normalizing the total number of electrons to the total area, A, exposed to electron irradiation: D ) ItN/A, where I is the beam current, t is the dwell time per pixel, and N is the number of pixels in the array. Arrays of irradiated polymer were created in three rows of five square irradiation areas. Each square irradiation area was generated by a two-dimensional raster of 60 by 60 pixels over an area of 5.4 × 5.4 µm. The interpixel spacing was 90 nm. The average doses for each exposed area are summarized in Table 1. Removal of lower-solubility polymer after irradiation corresponds to the development of a resist. In the present experiments, irradiation led to cross-linking. Unirradiated or insufficiently cross-linked material was soluble in water, dichloromethane, and tetrahydrofuran (THF). We developed the irradiated specimens immediately after removal from the microscope vacuum environment. They were immersed and gently agitated for 5 min in 200 mL of THF and then rinsed by immersion in 200 mL of type I water from a Millipore Direct Q water purification system. The developed specimens were dried under flowing nitrogen gas. Sometimes micrometer-sized particulate was observed on the specimen surfaces after development. This particulate matter does not appear to affect the present experiments in a substantive manner. Exposure to Fn and Antibodies. Patterned surfaces generated by electron irradiation and subsequent development were exposed to human plasma Fn fragments at 37 °C for 2 h at a concentration of 0.1 mg/mL in PBS-C buffer. After rinsing in PBS-C for three iterations of 5 min each, the specimens were

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immersed in rabbit antihuman primary antibody, at a concentration of 5 µL/mL in PBS-C, for 30 min at room temperature. These were then rinsed in PBS-C for three iterations of 5 min each and exposed to FITC-conjugated goat antirabbit IgG secondary antibody, at a concentration of 10 µL/mL in PBS-C, at room temperature for 30 min. The specimens were then washed in PBS-C for three iterations of 5 min each, rinsed in water for 30 s, and finally dried under flowing nitrogen gas. To confirm that the fluorescent signal observed was due to Fn adsorption rather than to the adsorption of either the primary or the secondary antibodies, we performed control experiments where patterned surfaces were not exposed to Fn but instead were exposed (1) to the primary antibody and then the secondary antibody as outlined above or (2) only to the secondary antibody. These control experiments indicated that undetectable fluorescent intensity derives from direct primary or secondary antibody adsorption on the patterned surfaces in the absence of Fn. Morphological Analysis of Micropatterned PEG. We used three methods to study the morphology of our specimens. SEM imaging was done with the same LEO 982 FEG SEM used to write the microhydrogel patterns. SEM imaging was either done at a dose much lower than that used to write the patterns or on specimens that were not subsequently used for swelling and protein-adsorption measurements. Quantitative measurements of the film height using a NanoScope IIIa scanning probe microscope in contact mode (Digital Instruments, Veeco Metrology Group) employed Veeco Nanoprobe tips (model NP-20). When making these height measurements, specimens were mounted in a Digital Instruments liquid cell. Pad heights were first determined from the pads in a dry state. The liquid cell was then filled with purified water, and measurements of the pad height in the hydrated state were made after equilibrating the specimen in water for approximately 10 min. The atomic force microscopy (AFM) imaging force was minimized to limit the deformation of the pads by the AFM tip. Fluorescence optical microscopy was done using a Nikon Eclipse E1000 microscope with 20×, 50×, and 100× LU Plan objective lenses. We analyzed the digital image data quantitatively using the Digital Micrograph software system from Gatan, Inc. Calculation of Average Molecular Weight between Cross-Links. For a polymer of molecular weight M, a key parameter characterizing the gel is the average molecular weight between cross-links Mc. In a good solvent, an unsolvated crosslinked polymer with a volume V0 will swell to an equilibrium volume V in a solvent where the interaction between the polymer and the solvent is quantified by the Flory-Huggins interaction parameter, χ. Because the swelling we observed is constrained laterally, we derived an expression (Appendix I) for onedimensional swelling. Like the classical expression first derived by Flory for the three-dimensional swelling of a cross-linked gel,34,35 the one-dimensional expression considers the free energy of polymer-solvent mixing and the elastic energy associated with stretching a cross-linked network and gives

-[ln(1 - v2m) + v2m + χ(v2m)2] )

[ ][

][

]

v1 Mc v2m 1-2 v2m1-2/d vj Mc M 2

(1)

where v2m is the volume fraction of polymer in the solvated gel and vj is the specific volume of polymer. At the swelling equilibrium, the swelling ratio q is equal to the ratio V/V0 which, in turn, is 1/v2m. For our calculations, we used values of 0.861 cm3/g for the specific volume of the polymer, vj ; 18.1 cm3/mol for the molar volume of the solvent, v1; and 0.42630,36 for the FloryHuggins interaction parameter, χ. (34) Bell, C. L.; Peppas, N. A. In Advances in Polymer Science: Biopolymers II; Peppas, N. A., Langer, R. S., Eds.; Springer: Berlin, 1995; Vol. 122, pp 127-175. (35) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (36) Merrill, E. W.; Dennison, K. A.; Sung, C. Biomaterials 1993, 14, 1117-1126.

Figure 1. (A) SEM image of electron-beam patterned PEG 6800 after unirradiated PEG has been removed by solvent washing; (B) binarized and skeletonized image derived from part A, illustrating a typical test pattern for dose-dependent irradiation.

Results and Discussion Figure 1A shows the results of the irradiation and development of a PEG 6800 film. As is outlined in the skeletonized image in Figure 1B, there are 15 areas subjected to exposure. A fiducial pattern with Roman numerals I-V was written by the electron beam to facilitate identification of the various exposure areas. This fiducial pattern was generated by writing lines at a dose of 70 C/m2. At the lowest doses, no polymer remained on the surface after washing with THF (at positions 1-5, Table 1), presumably due to insufficient cross-linking and attachment to the surface. At high doses, square pads remained at the surface after washing with THF (positions 6-15, Table 1). One can conclude that, within this higher dose range, electron exposure leads to a net cross-linking effect37 and attachment to the surface. PEO 200K shows a similar behavior. Under the conditions of these experiments, PEG and PEO behave like negative photoresists. Cross-linking reduces the solubility of the polymer in the PEG and PEO pads in a good solvent. Although the results presented here concentrate only on development using THF, other good solvents for PEG were successfully used to develop the irradiated films. The microhydrogel pad structures resulting from development by these different solvents were similar with some variations in the sharpness of the resulting pattern. Figure 1 shows that the pads increase in lateral size and surface uniformity with increasing dose. We suggest that both of these observations can be attributed to the proximity effect. The proximity effect38,39 refers to the regions of polymer not exposed by electron radiation directly from the incident beam but rather by electrons backscattered from the substrate. These backscattered (37) Zhang, L.; Zhang, W.; Zhang, Z.; Yu, L.; Zhang, H.; Qi, Y.; Chen, D. Radiat. Phys. Chem. 1992, 40 (6), 501-505. (38) Murata, K.; Kyser, D. F.; Ting, C. H. J. Appl. Phys. 1981, 52 (7), 4396-4405. (39) Rai-Choudary, P. Handbook of Microlithography, Micromachining, and Microfabrication; SPIE: Bellingham, 1997; Vol. I.

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Figure 2. Monte Carlo simulation of the trajectories of 2500 20-keV electrons incident over a 30-nm-diameter region of a 100-nm film of amorphous polymer on top of a silicon substrate. The inset details the incident and backscattered (bs) electron trajectories in the polymer film.

electrons can emerge from the specimen many tens or hundreds of nanometers from their point of incidence, and they transfer energy to the polymer film more efficiently during their second pass through the polymer film because of their lower energy. We used Monte Carlo simulations of electron trajectories to illustrate the origin of the proximity effect. One of our results is shown in Figure 2 for 2500 electron trajectories. The upper part of the inset shows that there is some broadening of the electron beam, but most electrons pass through the specimen into the substrate. The lower part of the inset shows that backscattered electrons emerge from the substrate and expose the polymer thin film at distances as high as 1 µm or more from the point of incidence. The experimental results of Figure 1 indicate that at higher doses the extent of this exposure is sufficient to cross-link PEG 6800 in the spaces between adjacent exposure points. In addition, we can attribute the increase in lateral size of the high-dose pads to this same effect. To understand the mechanism of cross-linking in the present experiments, where dry, solid PEG and PEO films are exposed to electron irradiation under a vacuum on the order of 10-6 hPa, we can consider the results obtained by other groups in related experiments. In particular, King29 suggested that PEO cross-linking tends to occur in environments such as a vacuum where free oxygen is present at low levels and radicals on the polymer chain can survive until a cross-linking reaction occurs. This mechanism is consistent with that believed to occur in the radiation-induced cross-linking of ultrahigh molecular weight polyethylene used in load-bearing surfaces of many medical implants.40 Using electron energy-loss spectroscopy (EELS) in the transmission electron microscope, Ditchfield et al.41 have shown that the irradiation of polyethylene by 80 keV electrons in a vacuum produces unsaturated carbon bonds and attribute this to the radiation-induced loss of hydrogen. We believe that a similar cross-linking mechanism may be occurring in our experiments, and we are currently pursuing EELS studies to experimentally elaborate on the effects of electron irradiation on PEO and PEG in a vacuum. An alternate cross-linking mechanism was proposed by Merrill and coworkers42,43 for hydrated systems, where water radiolysis produces hydroxyl radicals that react with PEO/PEG hydrogen atoms to create active PEG/PEO sites that can, in turn, react with each other to form cross-links. This (40) Kurtz, S. M.; Meuratoglu, O. K.; Evans, M.; Edidin, A. A. Biomaterials 1999, 20, 1659-1688. (41) Ditchfield, R. W.; Grubb, D. T.; Whelan, M. J. Philos. Mag. 1973, 27 (6), 1267-1280. (42) Merrill, E. W.; Salzman, E. W. J. Am. Soc. Artificial Internal Organs 1982, 6, 60-64.

Figure 3. AFM images of PEG 6800 irradiated to a dose of 0.31 C/m2 imaged while (A) dry (average height ) 31.5 nm) and (B) immersed in water (average height ) 238.2 nm). The bottom plot shows the height profiles from each of these images.

mechanism is not likely to occur in our experiments because of the absence of water during electron irradiation. Figure 3 illustrates the basic swelling phenomenon observed in the cross-linked PEG/PEO pads. The upper image shows an AFM image of a pad (pad 7, Figure 1) of PEG 6800 in its dry state after development. The lower image was collected in situ and shows the same pad in its hydrated state. The pad height increased from an average of 31.5 nm in the dry state to an average of 238.2 nm in the wet state. Also striking from Figure 3 is that the swelling is highly anisotropic with little change in the lateral pad dimensions presumably as a result of the constraints imposed by the binding of the pad to the silicon surface. Figure 4 shows height profiles characteristic of 10 PEG 6800 pads created at different exposure doses (pads 6-15, Table 1). These profiles were generated from AFM image data on dry pads and in situ AFM measurements in water. Interestingly, the edges of the height profiles characterizing the high-dose pads in the wet state (Figure 4) are significantly higher than those characterizing the central portion of these same pads. We attribute this to the proximity effect, because the radiative exposure at the pad edges is less than that in the pad centers. The edge swelling is less apparent in the range of lower doses, for example, less than 1-10 C/m2, in part because it is masked in this range by the significant swelling of the central portion of the pad. For larger doses, the swelling of the pad centers is relatively small, so the edge swelling is more visible. More significant from Figure 4 is that the swelling of the central area of each pad depends strongly on the radiative dose. The pad-swelling behavior as a function of the incident electron dose is presented in Figure 5 for both PEG 6800 and PEO 200K. Each point in these figures represents an average determined from either five (PEG (43) Merrill, E. W.; Wright, K. A.; Sagar, A.; Dennison, K. A.; Tay, S.-W.; Sung, C.; Chaikoff, E.; Rempp, P.; Lutz, P.; Callow, A. D.; Connolly, R.; Ramberg, K.; Verdon, S. In Polymers in Medicine: Biomedical and Pharmaceutical Applications; Ottenbrite, R. M., Chiellini, E., Eds.; Technomic: Lancaster, PA, 1992; pp 39-56.

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Figure 6. Vertical swell ratio as a function of the incident electron dose for PEG 6800 and PEO 200K. Figure 4. AFM images (60 × 60 µm) of the PEG 6800 exposure pattern shown in the (A) dry and (B) wet conditions. The height profiles in the plot below were generated by averaging over approximately 50 pixels, as is indicated by the dotted box in the upper left AFM image.

Figure 5. Height of (solid) wet and (dashed) dry pads of (A) PEG 6800 and (B) PEO 200K, each normalized to their maximum swelling height (193 and 1250 nm, respectively). Data were not collected from PEO 200K for doses exceeding 3 C/m2 because the extensive swelling of proximity-effect cross-linked polymer at the pad edges obscured the central pad area. The lines are sketched to facilitate interpretation of the data.

6800) or three (PEO 200K) different experiments. On the basis of the dry thickness measurements, we can identify three different regions of pad stability marked on Figure 5: unstable pads, partially stable pads, and stable pads. There is a lower limiting dose below which no pads remained at the surface. These pads are unstable, and, as is discussed in the following, we attribute their

instability to insufficient cross-linking and adhesion to the substrate. In the regime of partially stable pads, the dry pad height varies weakly with incident dose. We attribute this behavior to partial cross-linking and adhesion such that a portion of the polymer molecules are insufficiently bound to the pad or substrate and are washed away during the post-irradiation development step. Finally, at higher doses, the dry pad height is constant. The pads fabricated at these higher doses are stable. An important observation from Figure 5 is that, except for the lower dose portions of the partially stable regions, the hydrated pad height decreases with increasing dose. At the highest doses, the PEG 6800 dry and wet pad heights are essentially identical. This observation is consistent with the expected decrease of swelling with an increase of cross-linking density, assuming that the number of cross-links between PEG or PEO molecules increases with the radiation dose. These same swelling trends as a function of the radiative dose are manifested by the swelling ratios plotted in Figure 6. The swelling ratio, q, was determined from the vertical pad heights: q ) hwet/hdry, where hwet and hdry are the wet and dry pad heights, respectively. Figure 6 shows that the PEG 6800 and PEO 200K pads achieve maximum swelling ratios of about 14 and 16, respectively. Beyond these maxima, the microhydrogels are stable and the swelling ratios fall monotonically with increasing dose toward a limit of unity. We presume that this decrease in the swell ratio with dose is due to an increase in the crosslink density or, in other words, to a decrease in the average molecular weight of the polymer chains between crosslinks, Mc. As is detailed in the Experimental Section, we have derived eq 1 to describe one-dimensional swelling, and, using this equation together with our experimentally determined swell ratios, we calculated Mc. The results are presented in Figure 7. The curves both have maxima corresponding to approximately half of the nominal molecular weight of each polymer, 90 000 for PEO 200K and 3400 for PEG 6800, suggesting that, on average, each PEO or PEG molecule is composed of two segments separated by a single cross-link. For both polymers, Mc decreases with increasing dose indicating that, on average, higher electron doses generate multiple cross-links that partition the parent molecules into more and smaller segments. Figure 8 shows the average number of cross-links per molecule, as was determined from the Mc values plotted in Figure 7A,B for both PEO 200K and PEG 6800. For the range of doses where stable pads are generated, above about 0.03 C/m2 for PEO 200K and 1 C/m2 for PEG 6800, the number of cross-links per molecule increases linearly

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Figure 9. Inset: fluorescence optical micrograph of pads 6-15 in the exposure array shown in Figure 1. The Fn concentration scales with the fluorescent intensity. The fluorescent intensity averaged over the central portion of each pad is plotted as a function of the pad swell ratio. The vertical dotted line indicates a swelling ratio of 1. The horizontal dashed line indicates the level of nonspecific adsorption onto the exposed silicon substrate.

Figure 7. Average molecular weight between cross-links as a function of the incident electron dose for (A) PEO 200K and (B) PEG 6800.

Figure 8. The number of cross-links per molecule for PEO 200K and PEG 6800 for a subset of the incident electron doses studied.

with the dose on this log-log plot. In this regime, the number of cross-links per molecule, X, as a function of the dose, D, can be modeled by a power law as X ) ADn, where A and n are constants. Assuming that cross-linking occurs as a consequence of ionization events caused by inelastic scattering between the incident energetic electrons and atomic/molecular electrons in the polymer films, one would expect that the exponent n should be unity because the number of inelastic scattering events scales directly with the number of incident electrons. The exponents determined from the slopes of the two straight lines in Figure 8 are n200K ) 0.82 and n6800 ) 0.88. The fact that these are similar to each other suggests that, over the range of doses leading to approximately 1-20 cross-links per molecule, PEO 200K and PEG 6800 respond similarly to incident

electron irradiation to form stable gels. The fact that these exponents are close to unity suggests that cross-linking is a consequence of the random ionization of polymer chains due to inelastic electron scattering. Above these doses, the efficiency of cross-linking per unit of incident dose increases in a nonlinear manner with power-law exponents exceeding unity. For clarity, these data are not shown in Figure 8. In this high-dose regime, however, the microhydrogels are characterized by swelling ratios close to unity, suggesting that segment mobility and confinement may influence the cross-linking efficiency. The Mc data of Figure 8 also suggest an explanation for the partially stable and unstable gels observed at lower doses for both PEO 200K and PEG 6800. A critical dose can be defined for these polymers by extrapolating the power-law model to the dose where only one cross-link per molecule is generated. The condition of one cross-link per molecule is generally required for the onset of gelation. The critical doses for PEO 200K and PEG 6800 are 0.015 C/m2 and 0.7 C/m2, respectively. These values correspond closely to the doses indicated in Figure 5A,B, separating the partially stable microhydrogels from the stable ones. Partially stable microhydrogels form when there is an insufficient electron dose to cross-link all of the molecules in a pad. Uncross-linked molecules are removed during development, leaving behind a pad of reduced dry height with, on average, one cross-link per molecule. In the limit of even lower dose, an insufficient fraction of molecules in the film are cross-linked, and all are washed away during the development step together with any unexposed polymer. While we can at present only speculate about the mechanism of radiation-induced binding between the polymers and the silicon substrate that is responsible for pad adhesion, the fact that partially stable pads can be formed that remain well adhered to the substrate suggests that the efficiency of radiation-induced adhesion to the substrate is at least as high as that for polymer-polymer cross-linking. Finally, we address the issue of Fn adsorption onto the PEG microhydrogels. Typical results are summarized by Figure 9. The inset shows a 256-bit gray scale fluorescence optical micrograph of the pad array described in the SEM image of Figure 1. The fluorescence is due to the

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localization of FITC-labeled secondary antibody with adsorbed fragments of human Fn. The brighter contrast corresponds to higher concentrations of adsorbed Fn. The background surrounding the electron-beam written pad array has nonzero intensity. This corresponds to the nonspecific adsorption of Fn onto the silicon substrate after washing away unexposed PEG. Clear from the inset image is that the extent of Fn adsorption is a function of the electron dose used to create a particular pad. Fn adsorption is hindered relative to the background when pads are created with low incident doses (pads 6-10). Fn adsorption is enhanced, however, for pads created with high doses (pads 11-15). The fact that one can regulate the extent of Fn adsorption simply by varying the incident electron dose is remarkable. To create Figure 9, we averaged the fluorescent intensity over the central portion of each pad and plotted these average values as a function of the swell ratio. The curve represents the average of two different sets of such data. The background in the Figure 9 inset due to nonspecific adsorption on the exposed silicon substrate was modeled and subtracted from the image. Consequently, Figure 9 plots the relative intensities where 0 corresponds to nonspecific adsorption. Microhydrogels that are only lightly cross-linked, and thus swell substantially, hinder the adsorption of Fn. Indeed, Figure 9 shows that swell ratios exceeding approximately 2 are sufficient to enhance the protein adsorption resistance relative to the background. At the highest swell ratio observed in this particular experiment, the fluorescent intensity was below the detectability limit of our microscope system. This observation is consistent with the often-reported finding that PEG surfaces tend to resist the adsorption of most proteins. In contrast, the adsorption of Fn is enhanced relative to the background when the swell ratio falls below about 1.5. The amount of Fn adsorption increases dramatically as the swell ratios approach unity. We have not yet pursued experiments to separate the possible effects on Fn adsorption of radiation-induced chemical changes that do not lead to cross-linking. These may become particularly significant at high doses. Figure 10 demonstrates that arbitrary patterns of Fnadsorbing PEG can be generated using the electron-beam patterning approach. This figure was created using a binarized logo for the Stevens Institute of Technology (lower left) with an interpixel spacing of 100 nm and dose conditions like those of pad 11 in Figure 1. Figure 10A is a reflected-light optical micrograph, where the dark contrast corresponds to regions where cross-linked PEG 6800 remains after development. Figure 10B is a fluorescence optical micrograph that shows the spatial distribution of adsorbed Fn on the patterned PEG. The bright contrast corresponds to a higher concentration of adsorbed Fn and is localized most strongly in areas where the polymer received the highest exposure from the direct electron beam. Perhaps most striking from Figure 10 is the fact that continuous PEG surface pads can be generated where the affinity for Fn adsorption can be modulated at a submicrometer length scale. This can be seen most clearly by observing that the words “Stevens Institute of Technology” are visible in the fluorescence image but are not apparent in the reflected-light image, the latter showing that polymer is present and the former showing that Fn does not adsorb there. The linear profile of the fluorescent intensity shown in Figure 10C was collected across the “l” in the word Technology in Figure 10B and was averaged over 25 pixels vertically. The full width at half-maximum of the decreased intensity associated with this feature is

Krsko et al.

Figure 10. (A) Reflected-light optical micrograph of the Stevens logo (lower left) showing patterned regions of PEG 6800 (dark contrast); (B) fluorescent image mapping the spatial distribution of Fn (bright contrast) adsorbed onto the pattern of part A; (C) profile of fluorescent intensity across the “l” in Technology.

260 nm. The width of the “l” corresponds to three electronbeam exposure points, and we have made no accommodation to mitigate the proximity effects that further degrade the spatial resolution. We have achieved feature sizes of 100 nm in single-pixel exposures with a different polymer system by using a thin-film substrate rather than a bulk silicon substrate and will report on this in a subsequent publication. We see no fundamental limit to achieving feature sizes on the order of a few tens of nanometers, as has been done with polymeric resists for device technologies.44 Conclusion and Outlook Microhydrogels can be synthesized from solvent-free thin films of PEG and PEO using focused electron-beam irradiation in a FEG-SEM. Such electron exposure crosslinks these polymers. With no special precaution toward enhancing the spatial resolution, submicrometer-sized and micrometer-sized microhydrogels can be generated. These microhydrogels swell when immersed in water, and the extent of swelling can be controlled via the incident dose. These properties alone suggest technological opportunities associated with microfiltering, valving, and small-molecule delivery. We have, furthermore, demonstrated that the extent of Fn adsorption can be controlled by the degree of swelling in such a way that we have the equivalent of a writable gray scale of adsorbed protein. The ability to create continuous surface films with modulated affinity for Fn opens the possibility of generating polymer pads not only to which cells can adhere but also whose adhesiveness for cells can also be modulated at subcellular length scales relevant to the focal adhesions and focal contacts that can influence cell behavior and expression.3,45,46 (44) Goodberlet, J. G.; Hastings, J. T.; Smith, H. I. J. Vac. Sci. Technol., B 2001, 19 (6), 2499-2503. (45) Folkman, J.; Moscona, A. Nature 1978, 273, 345-349. (46) Ingber, D. E. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3579-3584.

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Acknowledgment. The authors thank Dr. Manoj Misra and Dennis Palatini of Unilever Research and Mr. Chris Stucchio of Stevens for their help on this research. We are grateful to the National Science Foundation for supporting the Stevens facilities for scanning electron microscopy. This research has been partially supported by the Army Research Office (Grant DAAD19-00-1-0481) and the NIH-National Institute for Biomedical Imaging and Bioengineering (Grant P41 EB 000922-01). Appendix I: Swelling Equilibrium The classical treatment of swelling equilibrium35 uses the standard Flory-Huggins model to treat the polymersolvent interaction, and it uses the standard theory of Gaussian chain stretching to account for the elasticity of the network. The equilibrium dimensions of the gel are taken as those that minimize the free energy. The resulting formula provides a direct relationship between the extent of cross-linking and the equilibrium dimensions of the swollen gel and permits us to predict the former from the latter. However, because we consider gels attached to a surface that are only free to swell in one dimension, the classical treatment of isotropic expansion in three dimensions is not appropriate in the present case. The general expression for the stretching free energy of a Gaussian chain is

kT 2 R + R2y + R2z - 3 - ln(RxRyRz)] 2[ x

(A1)

where Rx, Ry, and Rz represent the stretching ratios in each of three orthogonal directions.35 The isotropic case

of free expansion in all three directions (d ) 3) is treated by setting Rx ) Ry ) Rz ) R to yield

kT 2 3R - 3 - ln R3] 2[

(A2)

However, with expansion constrained to occur in only one direction (d ) 1), we set Rx ) Ry ) 1 and Rz ) R. And, although the result is not needed here, the d ) 2 case may be obtained by setting Rx ) 1 and Ry ) Rz ) R. In general, we set d of the Rj’s equal to R and 3 - d of them equal to 1. Therefore, all three cases can be represented by a single formula:

kT dR2 - d - ln Rd] 2[

(A3)

Note also that for arbitrary d the swelling ratio obeys

q ) Rd

(A4)

We now repeat Flory’s derivation35 using eq A3 in place of eq A2 to obtain the following as a condition for the swelling equilibrium:

-[ln(1 - v2m) + v2m + χ(v2m)2] )

[ ][

][

]

Mc v2m v1 1-2 v2m1-2/d (A5) vj Mc M 2

Setting d ) 3 yields the expression found in ref 35, whereas setting d ) 1 yields eq 1 above. LA034157R