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Surface Modification of SU-8 for Enhanced Biofunctionality and Nonfouling Properties Sarah L. Tao,†,‡,§ Ketul C. Popat,†,‡,| James J. Norman,⊥ and Tejal A. Desai*,‡ Department of Physiology, DiVision of Bioengineering, UniVersity of CaliforniasSan Francisco, San Francisco, California 94158, The Charles Stark Draper Laboratory, Cambridge, Massachusetts 02139, Department of Mechanical Engineering/School of Biomedical Engineering, Colorado State UniVersity, Fort Collins, Colorado 80523, and Department of Mechanical Engineering, Stanford UniVersity, Stanford, California 94305 ReceiVed October 3, 2007. In Final Form: December 4, 2007 SU-8 is a chemically amplified, epoxy-based negative photoresist typically used for producing ultrathick resist layers during device manufacturing in the semiconductor industry. As a simple resist, SU-8 has garnered attention as a possible material for a variety of biomedical applications, including tissue engineering, drug delivery, as well as cell-based screening and sensing. However, as a hydrophobic material, the use of SU-8 is limited due to a high degree of nonspecific adsorption of biomolecules, as well as limited cell attachment. In this work, surface chemistry is utilized to modify the SU-8 surface by covalently attaching poly(ethylene glycol) (PEG) to increase biofunctionality and improve its nonfouling properties. Different molecular weights and concentrations of PEG were used to form films of various grafting densities on SU-8 surfaces. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the SU-8 surface. High-resolution C1s spectra show that, with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of the PEG films formed. The effect of PEG-modified SU-8 was examined in terms of protein adsorption on the surface and fibroblast-surface interactions.
Introduction Microfabrication techniques and the ability to control surface microarchitecture, topography, and feature size may potentially be used to develop novel biomedical devices with capabilities not possible with current systems. Research on microfabricated devices for biomedical applications (BioMEMS) has led to a diverse range of microsystems for diagnostic and therapeutic applications. Although materials such as silicon and silicon oxide are the materials of choice for electronic and mechanical devices, patterning of these materials requires a stepwise process of methods, including sputter-coating, photolithography, and etching. The transition from semiconductor materials to polymers, however, allows for simple processing, shorter fabrication times, and potential large-scale fabrication of complex devices. SU-8 (glycidyl ether of bisphenol A) is a chemically amplified, epoxy-based negative photoresist. SU-8 is a common structural component in device manufacturing, because of its chemical and thermal resistance, as well as its ability to produce a wide range of thicknesses (from 200 µm with single spin-coat processes). Patterning SU-8 with the simple process of photolithography has the capability to produce not only structures with high aspect ratio1-3 but with multilevel processing to create complex three-dimensional structures.4-8 SU-8 has been employed as a structural material in numerous bioanalytical * Corresponding author. E-mail:
[email protected]. † These authors contributed equally. ‡ University of CaliforniasSan Francisco. § The Charles Stark Draper Laboratory. || Colorado State University. ⊥ Stanford University. (1) del Campo, A.; Greiner, C. J. Micromech. Microeng. 2007, 17, R81. (2) Lee, K. Y.; LaBianca, N.; Rishton, S. A.; Zolgharnain, S.; Gelorme, J. D.; Shaw, J.; Chang, T. H. P. J. Vac. Sci. Technol. B 1995, 13, 3012. (3) Vora, K. D.; Peele, A. G.; Shew, B. Y.; Harvey, E. C.; Hayes, J. P. Microsyst. Technol. 2007, 13, 487. (4) Mata, A.; Fleischman, A. J.; Roy, S. J. Micromech. Microeng. 2006, 16, 276.
microdevices, sensors, bioassays, and drug delivery vehicles.7-15 Although it is not currently FDA approved, initial studies have shown that SU-8 may be classified as a nonirritant and biocompatible.16,17 However, the hydrophobicity of SU-8 presents a limitation to many biological applications. These limitations include challenges in surface wetting,18 biofouling,13 and limited cell attachment.19,20 Surface modification of SU-8 can alleviate many of these problems. A number of methods have been employed, including adsorption of biomolecules,21 surface coating,13 plasma treatment,22 graft polymerization,20,23 and (5) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461. (6) Taff, J.; Kashte, Y.; Spinella-Marno, V.; Paranjape, M. J. Vac. Sci. Technol. A 2006, 24, 742. (7) Tao, S. L.; Desai, T. A. AdV. Mater. 2005, 17, 1625. (8) Tao, S. L.; Popat, K.; Desai, T. A. Nat. Protoc. 2006, 1, 3153. (9) Evans, M.; Sewter, C.; Hill, E. Assay Drug DeV. Technol. 2003, 1, 199. (10) Jenke, M. G.; Schreiter, C.; Kim, G. M.; Vogel, H.; Brugger, J. Microfluid Nanofluid 2007, 3, 189. (11) Marie, R.; Schmid, S.; Johansson, A.; Ejsing, L. E.; Nordstrom, M.; Hafliger, D.; Christensen, C. B. V.; Boisen, A.; Dufva, M. Biosens. Bioelectron. 2006, 21, 1327. (12) Noble, P. F.; Cayre, O. J.; Alargova, R. G.; Velev, O. D.; Paunov, V. N. J. Am. Chem. Soc. 2004, 126, 8092. (13) Xu, B. J.; Jin, Q. H.; Zhao, J. L. Sens. Actuators, A 2007, 135, 292. (14) Chang-Yen, D. A.; Gale, B. K. Lab Chip 2003, 3, 297. (15) Cheng, M. C.; Gadre, A. P.; Garra, J. A.; Nijdam, A. J.; Luo, C.; Schneider, T. W.; White, R. C.; Currie, J. F.; Paranjape, M. J. Vac. Sci. Technol. A 2004, 22, 837. (16) Kotzar, G.; Freas, M.; Abel, P.; Fleischman, A.; Roy, S.; Zorman, C.; Moran, J. M.; Melzak, J. Biomaterials 2002, 23, 2737. (17) Voskerician, G.; Shive, M. S.; Shawgo, R. S.;von Recum, H.; Anderson, J. M.; Cima, M. J.; Langer, R. Biomaterials 2003, 24, 1959. (18) Nordstrom, M.; Marie, R.; Calleja, M.; Boisen, A. J. Micromech. Microeng. 2004, 14, 1614. (19) Li, M. G.; J. D.; Green, H.; Mills, D. K.; McShane, M. J.; Gale, B. K. 1st Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine & Biology; 2000, p 531. (20) Wang, Y. L.; P., J. H.; Lai, H. H.; Sims, C. E.; Bachman, M.; Li, G. P.; Allbritton, N. L. J. Micromech. Microeng. 2007, 17, 1371. (21) Wu, Z. Z.; Zhao, Y.; Kisaalita, W. S. Colloid Surf. B 2006, 52, 14. (22) Ge, J.; Kivilahti, J. K. J. Appl. Phys. 2002, 92, 3007.
10.1021/la703066z CCC: $40.75 © 2008 American Chemical Society Published on Web 02/15/2008
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chemical modification.8,11,18,24,25 In this work, the covalent linkage of poly(ethylene glycol) (PEG) to the surface of SU-8 is used to increase its biofunctionality and nonfouling properties. PEG has been widely used to form nonfouling thin films on silicon surfaces.26-28 It is a relatively simple molecule and has the following structure that is characterized by hydroxyl groups at either end of the molecule: HO(CH2CH2O)nCH2CH2OH It is a linear or branched, neutral polyether available in a variety of molecular weights and soluble in water and most organic solvents. It also behaves uniquely in an aqueous environment. The PEG chains are constantly in motion in aqueous environment. It functions as a molecular windshield wiper, inhibiting any biomolecules that approach the surface, hence preventing biofouling.29-31 However, PEG modification has not been applied to SU-8 surfaces. Thus, in this work, we have covalently coupled PEG to the surface of SU-8 to improve its biofunctionality and nonfouling properties. The PEG-grafted SU-8 surfaces were characterized using X-ray photoelectron spectroscopy (XPS) to determine the chemical composition of the surface. Various concentrations and molecular weights of PEG were used. Further, protein adsorption and cell attachment on unmodified and PEG modified surfaces were also investigated. Experimental Section Reagents. Silicon wafers were purchased from Addison Engineering (San Jose, CA). SU-8 2010 and SU-8 developer were purchased from Microchem (Newton, MA). Poly(ethylene glycol) (molecular weight ) 1K, 2K, 6K), aminopropyltriethoxysilane, silicon tetrachloride, triethylamine, toluene, acetone, isopropyl alcohol, sulfuric acid, toluidine blue, bovine serum albumin (BSA), 0.25% Trypsin-EDTA, fetal bovine serum, and penicillinstreptomycin were purchased from Sigma-Aldrich Chemical Co. (St. Louis MO). Ethanol was purchased from Electron Microscopy Sciences (Hatfield, PA). The BCA Protein Assay Kit was purchased from Pierce (Rockford, IL). IMR-90 human fetal lung fibroblasts were obtained from the UCSF Cell Culture Facility (San Francisco, CA). Eagle’s minimum essential medium was purchased from ATCC (Manassas, VA). CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) and Hoecsht were purchased from Invitrogen (Eugene, OR). Substrate Preparation. Silicon wafers were spin-coated with SU-8 2010 at a final spin speed of 1600 rpm for 30 s to produce a 15 µm resist layer. The wafers were prebaked on a 65 °C hot plate for 2 min followed by 4 min at 95 °C and then allowed to cool to room temperature. The SU-8 photoresist film was exposed to 365 nm UV light at 300 mJ/cm2 utilizing a Karl Suss MJB mask aligner. After exposure, the SU-8 was cross-linked by performing a two-step contact bake process on a 65 °C hot plate for 1 min followed by 1 min at 95 °C. The SU-8 was then developed in SU-8 Developer and dried with nitrogen. SU-8-coated wafers were then diced using a dicing saw (Dicing Blade Technology, Basic-Dice II) into 1 cm × 1 cm coupons for modification. Hydroxylating the SU-8 Surface. To render the SU-8 hydrophilic, the epoxy rings were opened using an acid-catalyzed reaction to (23) Wang, Y. L.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Langmuir 2006, 22, 2719. (24) Cavalli, G.; Banu, S.; Ranasinghe, R. T.; Broder, G. R.; Martins, H. F. P.; Neylon, C.; Morgan, H.; Bradley, M.; Roach, P. L. J. Comb. Chem. 2007, 9, 462. (25) Joshi, M.; Pinto, R.; Rao, V. R.; Mukherji, S. Appl. Surf. Sci. 2007, 253, 3127. (26) Popat, K. C.; Johnson, R. W.; Desai, T. A. J. Vac. Sci. Technol. B 2003, 21, 645. (27) Sharma, S.; Johnson, R. W.; Desai, T. A. Appl. Surf. Sci. 2003, 206, 218. (28) Zhu, X. Y.; Jun, Y.; Staarup, D. R.; Major, R. C.; Danielson, S.; Boiadjiev, V.; Gladfelter, W. L.; Bunker, B. C.; Guo, A. Langmuir 2001, 17, 7798. (29) Andrade, J. D.; Hlady, V. AdV. Polym. Sci. 1986, 79, 1. (30) Jeon, S. I.; Andrade, J. D. J Colloid Interface Sci. 1991, 142, 159. (31) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; Degennes, P. G. J Colloid Interface Sci. 1991, 142, 149.
Figure 1. Surface chemistry of SU-8. (A) Acid-catalyzed reaction to open SU-8 epoxy rings. (B) Reaction mechanism of PEG immobilization on SU-8 surfaces using a covalent coupling technique. provide hydroxyl groups at the surface.8 The SU-8 samples were treated in 95% sulfuric acid at 80 °C for 10 s followed by a wash in DI water for 5 min (Figure 1A). Covalent Coupling of Poly(ethylene glycol) on the SU-8 Surface. PEG immobilization on the SU-8 surface was achieved by modifying a covalent coupling technique described by Sharma et al. for silicon surfaces.27 This technique forms more stable films compared to physical adsorption on the surface. In this technique, a PEG-silane couple is formed by reacting PEG with silicon tetrachloride in the presence of triethylamine as a catalyst. The reaction results in the formation of PEG-OSiCl3, which then reacts with the trace level -OH groups on the surface to form a network of Si-O-Si bonds, resulting in the immobilization of PEG on the surface. The PEG-silane couple formation and its immobilization on the surface were performed in anhydrous conditions to prevent hydrolysis and undesired side reactions. In brief, PEG (molecular weight ) 1K, 2K, 6K) was dissolved in anhydrous toluene to form a PEG solution at a concentration of 10 mM. Then 0.975 mmol of triethylamine was added drop by drop to the PEG solution at 25 °C. The reaction mixture was gently shaken for 1 h. After this, 0.175 mmol of silicon tetrachloride was added and the reaction mixture was further shaken for 15 min at room temperature. The reaction mixture was then filtered through a sintered glass funnel and the filtrate was used directly to immobilize the surfaces without further purification, since an excess of unreacted PEG is not expected to have harmful effects on the silanization process. Immobilization of the PEG-silane couple with SU-8 surfaces was carried out for 1 h. To study the effect of PEG concentration, the surfaces were immobilized with various concentrations (10, 20, 40 and 80 mM) of PEG (MW ) 1K) for 1 h. After coupling, the surfaces were rinsed thoroughly with anhydrous toluene, acetone, and deionized water and were air-dried and stored under argon until further use. Figure 1B shows the schematic representation of PEG immobilization of SU-8 surfaces. X-ray Photoelectron Spectroscopy. To determine the surface composition of PEG-modified SU-8 surfaces, XPS analysis was carried out. The surfaces were mounted on an XPS stage. Three
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spots per sample were analyzed. The analysis was conducted on a SSI-S Probe X-ray photoelectron spectrometer with a monochromatic Al KR X-ray small spot source (1486.6 eV) and multichannel detector. The SSI XPS samples a surface area of about 400 µm by 700 µm. A concentric hemispherical analyzer (CHA) was operated in the constant analyzer transmission mode to measure the binding energies of emitted photoelectrons. The binding energy scale was calibrated by the Au4f7/2 peak at 83.9 eV, and the linearity was verified by the Cu3p1/2 and Cu2p3/2 peaks at 76.5 and 932.5 eV, respectively. Survey spectra were collected from 0 to 1100 eV with a pass energy of 160 eV. All spectra were referenced by setting the hydrocarbon C1s peak to 285.0 eV to compensate for residual charging effects. Data for percent elemental composition and elemental ratios were calculated using manufacturer supplied software with the XPS. Film Thickness from XPS Data. Film thickness can be determined from the attenuation of XPS signals from the substrates. The thickness can be obtained by using the standard uniform overlayer model, which is given by eq 1.32,33
L)
(
I ) I0
t -1 EL‚sin θ
)
(1)
where I0 is the intensity of carbon peaks before surface modification, Il is the intensity of carbon peaks from after modification with PEG, t is the thickness of the film, EL is the electron attenuation length for the carbon peak, and θ is the angle at which the X-ray hits the surface. In order to obtain film thickness from this equation, the electron attenuation lengths (L) for the carbon peak needs to be calculated. L was calculated using eq 2. L)
xE 49 + 0.11 F E2F
(2)
where E is the electron energy (X-ray core energy - core binding energy for carbon) ) (1486 - 285) ) 1201 eV, and F is the density of PEG (1.1 g/cm3). Calculation of PEG Surface Concentration and Grafting Density. The values of thickness obtained from the standard uniform overlayer model can be used to calculate the PEG grafting density. The PEG grafting σ density is given by eq 3.39 σ)
(La)
2
(3)
where a is size of a monomer unit (∼3 Å)40,41 and L is the average distance between PEG chains grafted to the surface. The average distance between PEG chains grafted to the surface L42 can be estimated by determining the surface concentration of PEG Γ,43 which is given by eq 4 (32) Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Langmuir 2004, 20, 429. (33) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059. (34) NIST Electron EffectiVe Attenuation Length Database, version 1.0 (SRD82); National Institute of Standards and Technology: Gaithersburg, MD, 2001. (35) Schweppe, J.; Deslattes, R. D.; Mooney, T.; Powell, C. J. J. Electron Spectrosc. 1994, 67, 463. (36) NIST X-ray Photoelectron Spectroscopy Database, version 3.1; National Institute of Standards and Technology: Gaithersburg, MD, 2001. (37) Band, I. M. K.; Fomichev, Y. I.; Trzhaskovska, M. B. J. Phys. B: At. Mol. Phys. 1979, 23, 443. (38) Stenberg, M.; N., H. J. J Phys-Paris. 1983, C-10, 83. (39) de Gennes, P. G. Macromolecules 1980, 13, 1069. (40) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (41) Szleifer, I. Biophys. J. 1997, 72, 595. (42) Harris, J. M. Poly(ethylene glycol)sBiotechnical and Biomedical Applications; Plenum Press: New York, 1992. (43) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. C. J. Biol. Chem. 1983, 258, 2426.
( ) M ΓNA
1/2
(4)
where M is the molecular weight of PEG (1000 in this case), Γ ()Ft) is the surface concentration (g/nm2), NA is Avogadro’s number (6.023 × 1023 mol-1), F is the density of dry PEG layer (assumed to be constant, 10-21 g/nm3),44 and t is the thickness of PEG layer (nm) Therefore, by combining eqs 2 and 3, a simplified relationship for σ can be obtained, which is given by eq 5. σ)
( ) a2ΓNA M
(5)
Using eq 5, the grafting density of PEG modified surfaces can be calculated. Contact Angle Measurement. Contact angle measurements of deionized water droplets on substrate surfaces were used to characterize wettability and the hydrophilic/phobic nature of the surface. The contact angle is defined as the angle between the substrate support surface and the tangent line at the point of contact of the liquid droplet with the substrate. Contact angles were measured using the Cam-Micro contact angle meter (Tantec, Inc., Schaumburg, IL). Determining -OH Group Density. SU-8 samples were stained with a 0.1% w/v solution of toluidine blue for 5 min, rinsed with water, and then dried with nitrogen. The toluidine blue was then desorbed from the sample surface by immersion in a 10% acetic acid solution for 1 h. The optical absorption of the toluidine blue released was measured at a wavelength of 633 nm using a 96-well plate reader. The amount of toluidine blue originally bound to the SU-8 was then back-calculated from the optical density assuming a 1:1 ratio between hydroxyl groups and bound toluidine blue. Protein Adsorption. SU-8 samples were incubated in 1 mg/mL BSA solution for 1 h at room temperature. The concentrations of protein prior and subsequent to incubation with SU-8 were determined colormetrically by a BCA protein assay. Changes in color were measured utilizing a 96-well plate reader at a wavelength of 562 nm. Fibroblast Cell Culture. IMR-90 cells were grown in culture medium consisting of Eagle’s minimum essential media supplemented with Earle’s balanced salt solution, 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, 10% fetal bovine serum, and 100 units/mL penicillin and 100 µg/mL streptomycin antibiotic. Cells were maintained in a humidified 5% CO2/95% air atmosphere at 37 °C while renewing media every second day. Cells were subcultured by trypsinization with 0.25% trypsin-EDTA. Cell Attachment and Growth. All SU-8 surfaces were prepared for cell seeding by UV sterilization for 30 min and subsequent incubation in 70% ethanol for 30 min. The surfaces were then washed twice with PBS and incubated in growth media at 37 °C for 30 min. After replacing with fresh media, cells were seeded at 5 × 104 cells/cm2. The number of adherent cells on substrates was quantified after either 16 or 72 h. Briefly, substrates were transferred to new tissue culture plates, and live cells were stained with a 10 µM solution of CMFDA and their nuclei with a 2.5 µg/mL solution of Hoecsht dye. The substrates were imaged randomly using a fluorescence microscope, with 3-5 fields of view per sample. Images were analyzed using Image Pro Plus Analysis Software. Statistical Analysis. Multiple samples were collected for each measurement (at least n ) 3) and expressed as a mean ( standard error mean. One-way ANOVA was used to assess the statistical significance of the results. A post-hoc Dunnett’s test was used to perform a pairwise comparison of means between the SU-8 surface and the experimental groups.
Results and Discussion SU-8 may be chemically modified in order to form appropriate biological interactions for a variety of tissue engineering and (44) Vogel, A. C.; W.; Leicester, J. J. Phys. Chem. 1954, 58, 174.
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Table 1. Effect of Poly(ethylene glycol) Molecular Weight on Atomic Surface Concentration and Surface Properties of SU-8
SU-8 OH 1K PEG 2K PEG 6K PEG a
O/C
C1s intensity (eV)
0.22 0.34 0.31 0.24 0.19
2111 2595 2732 3485 4562
high-resolution C1s thickness (nm)
C-H
C-O
CA (deg)
L (nm)a
σb
3.62 4.48 5.41
0.714 0.696 0.652 0.517 0.675
0.286 0.304 0.348 0.483 0.625
80 26 40 17 20
0.677 0.861 1.356
0.189 0.121 0.048
Distance between PEG chains. b PEG grafting density. Table 2. Effect of Poly(ethylene glycol) Concentration on Atomic Surface Concentration and Surface Properties of SU-8
SU-8 OH 10 mM 20 mM 60 mM 80 mM 100 mM a
O/C
C1s intensity (eV)
0.22 0.34 0.31 0.28 0.23 0.17 0.13
2111 2595 2732 2885 2950 3398 3794
high-resolution C1s thickness (nm)
C-H
C-O
CA (deg)
L (nm)a
σb
3.62 3.82 3.93 4.39 4.78
0.714 0.696 0.652 0.624 0.575 0.521 0.403
0.286 0.304 0.348 0.376 0.425 0.479 0.597
80 26 40 35 28 25 28
0.677 0.659 0.649 0.614 0.589
0.189 0.207 0.213 0.238 0.259
Distance between PEG chains. b PEG grafting density.
Figure 2. XPS high-resolution C1s scan of (A) PEGylated SU-8 with varying PEG molecular weight and (B) PEG-modified SU-8 with varying PEG concentration.
drug delivery applications. In this work, XPS analysis was performed to ensure the presence of PEG moieties on the SU-8 surface. Survey scans were taken to determine the elemental surface composition of various elements present, and highresolution C1s scans were taken to study the carbon chemistries. A survey scan of the unmodified SU-8 surface showed distinct peaks for O1s (528 eV) and C1s (285 eV) with an oxygen to carbon peak ratio of approximately 0.22 (Table 1 and 2). Acidcatalyzed opening of the epoxy ring produced an augmented oxidation state in the SU-8 surface, transforming it from a hydrophobic to a hydrophilic surface. The increase in the peak area ratio of oxygen to carbon from 0.22 to 0.34 reflects an augmented oxidation as compared with the elemental composition of the original epoxy surface (Table 1 and 2). After modification with PEG, there is an increase of Si2p (100 eV) peaks, due to the silicon tetrachloride that is coupled with PEG, and an increase in C1s (285 eV) peaks, which results in a decrease in the peak area ratio of oxygen to carbon. This trend is followed when either the molecular weight (Table 1) or the concentration of PEG (Table 2) is increased. Since XPS is a depth-sensitive technique, these results confirm the presence of PEG on the surface. To further support the presence of PEG moieties on the surface, analysis of functional groups on the surface was performed using
high-resolution C1s spectra. The high-resolution C1s peak for unmodified SU-8 consisted of two well-defined peaks, a hydrocarbon (C-H or C-C) peak at 285 eV and an C-O peak at 286.5 eV. The intensity of the C-O in the C1s peak increased with both increasing molecular weight and concentration of PEG (Figure 2). Using the peak fit analysis software provided with the XPS instrument, the relative percentages of C-C and C-O in the C1s peak for PEG-modified surfaces were determined (Table 1 and 2). The relative percentage of C-O in the overall C1s peak is an indirect measure of PEG grafting on the surface. A convolution of Gaussian components was assumed for all peak shapes. Further, a standard overlayer model was used along with the XPS intensities to determine the PEG film thickness on SU-8 surfaces. As expected, the PEG film thickness increased with an increase in PEG molecular weight (Table 1) and concentration (Table 2). The values of thickness obtained from the standard uniform overlayer model were then used to calculate the PEG grafting density. Grafting density is an important parameter, since it is the measure of formation of PEG chains on the surface for various conditions. As the molecular weight of PEG was increased, the average distance between grafted PEG chains also increased, thus resulting in a decrease in grafting density (Table 1). However, by increasing the concentration of PEG (for 1K
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Figure 3. The density of OH groups on the SU-8 surfaces with varying (A) PEG molecular weight and (B) PEG concentration. The amount of protein adsorbed to the SU-8 surface with varying (C) PEG molecular weight and (D) PEG concentration. *p < 0.01, †p
