Dense Passivating Poly(ethylene glycol) Films on Indium Tin Oxide

Langmuir , 2007, 23 (20), pp 10244–10253. DOI: 10.1021/la7011414. Publication Date (Web): August 23, 2007. Copyright ... Generic Top-Functionalizati...
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10244

Langmuir 2007, 23, 10244-10253

Dense Passivating Poly(ethylene glycol) Films on Indium Tin Oxide Substrates Robert Schlapak,† David Armitage,⊥ Nadia Saucedo-Zeni,‡ Michael Hohage,‡ and Stefan Howorka*,†,# Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, A-4020 Linz, Austria, Department of Engineering, UniVersity of Leicester, LE1 7RH, U.K., Institute of Experimental Physics, Johannes Kepler UniVersity, A-4040 Linz, Austria, and Department of Chemistry, UniVersity College London, London WC1H 0AJ, U.K. ReceiVed April 18, 2007. In Final Form: May 30, 2007 We describe the formation and characterization of surface-passivating poly(ethylene glycol) (PEG) films on indium tin oxide (ITO) glass substrates. PEG chains with a molecular weight of 2000 and 5000 D were covalently attached to the substrates in a systematic approach using different coupling schemes. The coupling strategies included the direct grafting with PEG-silane, PEG-methacrylate, and PEG-bis(amine), as well as the two-step functionalization with aldehyde-bearing silane films and subsequent coupling with PEG-bis(amine). Elemental analysis by X-ray photoelectron spectroscopy (XPS) confirmed the successful surface modification, and XPS and ellipsometry provided values for film thicknesses. XPS and ellipsometry thickness values were almost identical for PEG-silane films but differed by up to 400% for the other PEG layers, suggesting a homogeneous layer for PEG-silane but an inhomogeneous distribution for other PEG coatings on the molecularly rough ITO substrates. Atomic force microscopy (AFM) and water contact angle goniometry confirmed the different degrees of surface homogeneity of the polymer films, with PEG-silane reducing the AFM rms surface roughness by 50% and the water contact angle hysteresis by 75% compared to uncoated ITO. The ability of the PEG layers to passivate the substrate against the nonspecific adsorption of biopolymers was tested using fluorescence-labeled immunoglobulin G and DNA oligonucleotides in combination with fluorescence microscopy. The results indicate a positive relationship between film density and homogeneity on one hand and the ability to passivate against biopolymer adhesion on the other hand. The most homogeneous layers prepared with PEG-silane reduced the nonspecific adsorption of fluorescence-labeled DNA by a factor of 300 compared to uncoated ITO. In addition, the study finds that the ratio of film thicknesses derived by ellipsometry and XPS is a useful parameter to quantify the structural integrity of PEG layers on molecularly rough ITO surfaces. The findings may be applied to characterize PEG or other polymeric films on similarly coarse substrates.

Introduction The coating of solid substrates with poly(ethylene glycol) (PEG) is an important strategy to avert the nonspecific adsorption of biopolymers or cells onto surfaces. This strategy has been used in biomedical materials, microfluidic devices, microarrays, and biosensors.1 Passivating PEG films can, for example, help improve the performance of implants by retarding the fouling process, aid cell biological research by generating localized patterns of cells on solid substrates, or increase the signal specificity in analyte detection schemes by reducing the background level of nonspecific binding. Several mechanisms have been proposed to explain the passivating behavior of PEG films.2 Probable molecular reasons are the entropic cost caused by the binding of a biopolymer to the flexible PEG chains,3 the electrostatic repulsion induced by the adsorption of hydroxide * Author for correspondence: E-mail: [email protected]. Telephone: 0044 20 7679 4702. Fax: 0044 20 7679 7463. † Center for Biomedical Nanotechnology. ⊥ University of Leicester. ‡ Johannes Kepler University. # Department of Chemistry, University College London. (1) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176186; Delamarche, E.; Donzel, C.; Kamounah, F. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749-8758; Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Langmuir 2001, 17, 4090-4095; http://proteinslides.com/ product.html; Germanier, Y.; Tosatti, S.; Broggini, N.; Textor, G.; Buser, D. Clin. Oral Implants Res. 2006, 17, 251-257; Pike, D. B.; Cai, S.; Pomraning, K. R.; Firpo, M. A.; Fisher, R. J.; Shu, X. Z.; Prestwich, G. D.; Peattie, R. A. Biomaterials 2006, 27, 5242-5251. (2) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359-9366.

ions onto the polymer surface,4,5 and/or the presence of a localized, high-viscosity water layer at the PEG/solvent interface.6 In all three cases, the PEG film is most effective when it is dense and has few structural defects.2 Reflecting the widespread use of PEGylated surfaces for basic science and biomedical applications,1 many methods have been developed to graft PEG layers onto solid substrates such as silicates, silicon, gold, and metal oxides. For silicate and glass, these methods include the coupling of aldehyde7 or epoxideterminated PEG8 onto aminopropyl trialkoxysilane-derived layers, the binding of PEG-bis(amine) onto aldehyde-bearing silanized surfaces,9 and the direct grafting of methoxy-PEG derivatives with terminal trialkoxysilane10 or methacrylate.11 Protocols for the modification (3) Jeon, S. I.; Andrade, J. D. J. Colloid. Interface Sci. 1991, 142, 159-166; Jeon, S. I.; Lee, J. H.; Andrade, J. D.; Degennes, P. G. J. Colloid. Interface Sci. 1991, 142, 149-158. (4) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141. (5) Chan, Y. H. M.; Schweiss, R.; Werner, C.; Grunze, M. Langmuir 2003, 19, 7380-7385. (6) Vogler, E. A. AdV. Colloid. Interface Sci. 1998, 74, 69-117; Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767-9773; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (7) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 20432056. (8) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid. Interface Sci. 1998, 202, 507-517. (9) Schlapak, R.; Pammer, P.; Armitage, D.; Zhu, R.; Hinterdorfer, P.; Vaupel, M.; Fruhwirth, T.; Howorka, S. Langmuir 2006, 22, 277-285. (10) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 1457-1460. (11) Roosjen, A.; van der Mei, H. C.; Busscher, H. J.; Norde, W. Langmuir 2004, 20, 10949-10955.

10.1021/la7011414 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/23/2007

PEG on ITO

of silicon proceed via the Cl2-activation of the surfaces and the subsequent coupling of hydroxyl-terminated PEG.12 The surface modification of gold, by comparison, utilizes the much milder chemisorption of thiol-functionalized PEG or oligo(ethylene glycol).4,5,13 For the anionic surfaces of metal oxides such as Ta2O5 or TiO2, copolymers such as poly(L-lysine)-g-poly(ethylene glycol) are electrostatically adsorbed onto the substrate surface.14,15 Indium tin oxide (ITO) is another solid substrate of increasing importance in the area of biosensing and biocompatible materials. ITO is a mixture of In2O3 and SnO2 and was initially developed as a conductive transparent electrode for materials science. It is now widely used in liquid crystal displays, and increasingly is applied in organic light-emitting diodes16 and solar cells.17 On the basis of its favorable combination of conductive and optical properties, ITO has also been utilized for cell biological and sensing applications. For example, ITO-coated glass slides have found use as heat-producing transparent microscopy supports for cell biological studies or as heating elements in biosensors for the amplification-based detection of bacterial DNA samples.18 Furthermore, the electronic properties of ITO have been exploited to electronically detect immunoanalytes using impedance measurements,19 to exert electrochemical control over the binding or release of protein20,21 and DNA analyte22 from the sensor surface, and to effect the redox state of electrochemically active sensor proteins.23 In several cases, the ITO substrates have been embedded into microfluidic chips,18 enabling the electrokinetic collection and concentration of cells and particular analytes.24 In addition, the high-refractive index of ITO has been utilized in optical waveguides for the surface plasmon-based sensing of bioanalytes25 with optional electrochemical control of analyte binding.20 In several of these applications it is important to avoid the nonspecific adsorption of protein or DNA onto ITO, for instance, to improve the signal specificity of the biosensor. Following this demand, passivating PEG coatings have been prepared using poly(L-lysine)-g-poly(ethylene glycol) copolymers which electrostatically adsorb onto negatively polarized metal oxide surfaces.14,15,26 These surface coatings resist the nonspecific adsorption of biopolymers but can also be locally desorbed from (12) 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, 7798-7803. (13) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (14) Ruiz-Taylor, L. A.; Martin, T. L.; Zaugg, F. G.; Witte, K.; Indermuhle, P.; Nock, S.; Wagner, P. Proc. Nat. Acad. Sci. U.S.A. 2001, 98, 852-857. (15) Huang, N. P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489-498. (16) Tang, C. W.; VanSlyke, S. A. Appl. Phys. Lett. 1987, 51, 913-915; Burroughes, J. H.; Bradley, D. D. C.; Brown, A. R.; Marks, R. N.; Mackay, K.; Friend, R. H.; Burn, P. L.; Holmes, A. B. Nature 1990, 347, 539-541. (17) Tang, C. W.; VanSlyke, S. A.; Chen, C. H. J. Appl. Phys. 1989, 65, 3610-3616. (18) Yeung, S. W.; Lee, T. M.; Cai, H.; Hsing, I. M. Nucleic Acids Res. 2006, 34, e118. (19) Yang, L. J.; Li, Y. B.; Erf, G. F. Anal. Chem. 2004, 76, 1107-1113. (20) Asanov, A. N.; Wilson, W. W.; Oldham, P. B. Anal. Chem. 1998, 70, 1156-1163. (21) Tang, C.; Feller, L.; Rossbach, P.; Keller, B.; Voros, J.; Tosatti, S.; Textor, M. Surf. Sci. 2006, 600, 1510-1517. (22) Yang, X. H.; Wang, Q.; Wang, K. M.; Tan, W. H.; Yao, J.; Li, H. M. Langmuir 2006, 22, 5654-5659; Moore, E. J.; Curtin, M.; Ionita, J.; Maguire, A. R.; Ceccone, G.; Galvin, P. Anal. Chem. 2007, 79, 2050-2057. (23) Hedges, D. H. P.; Richardson, D. J.; Russell, D. A. Langmuir 2004, 20, 1901-1908. (24) Bhatt, K. H.; Grego, S.; Velev, O. D. Langmuir 2005, 21, 6603-6612. (25) Homolka, J. In Optical sensors: Industrial EnVironmental and Diagnostic Application; Narayanaswamy, R., Wolfbeis, O. S., Eds.; Springer: Berlin, 2004; pp 145-172; Bearinger, J. P.; Voros, J.; Hubbell, J. A.; Textor, M. Biotechnol. Bioeng. 2003, 82, 465-473. (26) Tang, C. S.; Dusseiller, M.; Makohliso, S.; Heuschkel, M.; Sharma, S.; Keller, B.; Voros, J. Anal. Chem. 2006, 78, 711-717.

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ITO via electrostatic control, opening up the possibility to generate surface patterns for microarray applications.26 A similar approach on the adsorption and electrically stimulated desorption has also been described for the triblock copolymer poly(propylene sulfidebl-ethylene glycol) (PPS-PEG).21 While the tunable desorption of PEG-containing polymers from ITO can be of benefit, it is also advantageous to couple the PEG layer onto the ITO substrate via a covalent linkage. For instance, covalent coupling can help avoid the potential desorption of the PEG layer during the electrical switching of the ITO substrate required for analyte detection or sensor recycling.20-22 Many studies have been published on the covalent modification of ITO surfaces using small organic molecules,27-29 but there are few reports on the covalent grafting of PEG layers onto ITO. Here we describe the formation of covalently attached PEG films on ITO substrates. We systematically apply four different coupling schemes and examine the resulting films using ellipsometry, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle goniometry. The ability of the PEG layers to passivate the surface against nonspecific adsorption of fluorophore-labeled protein and DNA oligonucleotides is tested using fluorescence microscopy. The detailed surface analysis is pursued in order to thoroughly characterize dense films of PEG, and to compare their homogeneity with their ability to resist the nonspecific adsorption of biopolymers onto the ITO surface. As ITO is a molecularly rough surface, we also aimed to identify those surface analytical methods that best describe the structural integrity and homogeneity of the PEG films. Methods such as AFM and goniometry have been successfully applied in the past to examine the homogeneity of polymer films on atomically flat substrates such as gold, quartz, or silicon.4,9,10,12,30-32 However, there are few studies on how well these methods can be used to investigate PEG films on molecularly irregular substrates. Our report attempts to characterize the films’ homogeneity by means of AFM and goniometry, and also with ellipsometry and XPS which have not usually been applied to describe the integrity of PEG films. In particular, we investigate whether the ratio of thicknesses derived by ellipsometry and XPS is capable of accurately describing the quality of the films, and to predict the layers’ ability to avert the nonspecific adsorption of biopolymers. (27) Markovich, I.; Mandler, D. J. Electroanal. Chem. 2000, 484, 194-202; Markovich, I.; Mandler, D. J. Electroanal. Chem. 2001, 500, 453-460; Morgado, J.; Barbagallo, N.; Charas, A.; Matos, M.; Alcacer, L.; Cacialli, F. J. Phys. D: Appl. Phys. 2003, 36, 434-438; Appleyard, S. F. J.; Willis, M. R. Opt. Mater. 1998, 9, 120-124; Appleyard, S. F. J.; Day, S. R.; Pickford, R. D.; Willis, M. R. J. Mater. Chem. 2000, 10, 169-173; Hatton, R. A.; Willis, M. R.; Chesters, M. A.; Rutten, F. J. M.; Briggs, D. J. Mater. Chem. 2003, 13, 38-43; Hill, I. G.; Kahn, A. J. Appl. Phys. 1999, 86, 2116-2122; Zharnikov, M.; Golzhauser, A.; Grunze, M. Langmuir 2000, 16, 6208-6215; Span, A. R.; Bruner, E. L.; Bernasek, S. L.; Schwartz, J. Langmuir 2001, 17, 953-956; Bruner, E. L.; Span, A. R.; Bernasek, S. L.; Schwartz, J. Langmuir 2001, 17, 5696-5702; Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S. J. Am. Chem. Soc. 1995, 117, 6927-6933; Bermudez, V. M.; Berry, A. D.; Kim, H.; Pique, A. Langmuir 2006, 22, 1111311125; Nuesch, F.; Rotzinger, F.; Si-Ahmed, L.; Zuppiroli, L. Chem. Phys. Lett. 1998, 288, 861-867; Zuppiroli, L.; Si-Ahmed, L.; Kamaras, K.; Nuesch, F.; Bussac, M. N.; Ades, D.; Siove, A.; Moons, E.; Gratzel, M. Eur. Phys. J. B 1999, 11, 505-512; Yan, C.; Zharnikov, M.; Golzhauser, A.; Grunze, M. Langmuir 2000, 16, 6208-6215; Hillebrandt, H.; Tanaka, M. J. Phys. Chem. B 2001, 105, 4270-4276. (28) Koh, S. E.; McDonald, K. D.; Holt, D. H.; Dulcey, C. S.; Chaney, J. A.; Pehrsson, P. E. Langmuir 2006, 22, 6249-6255. (29) Karsi, N.; Lang, P.; Chehimi, M.; Delamar, M.; Horowitz, G. Langmuir 2006, 22, 3118-3124. (30) Ebner, A.; Kienberger, F.; Stroh, C. M.; Gruber, H. J.; Hinterdorfer, P. Microsc. Res. Tech. 2004, 65, 246-251. (31) Piehler, J.; Brecht, A.; Valiokas, R.; Liedberg, B.; Gauglitz, G. Biosens. Bioelectron. 2000, 15, 473-481. (32) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436.

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Experimental Section Reagents. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Indium tin oxide-coated glass slides (50 mm × 24 mm × 0.1 mm) with an ITO thickness of 17 ( 2 nm and a sheet resistance of 1200 ( 200 Ω/0 were obtained from Hans Tafelmaier Du¨nnschicht-Technik GmbH (Rosenheim, Germany). R,ω-Bis(2aminoethyl)poly(ethylene glycol) (PEG-bis(amine)) with a MW of 2 kD (Mw ) 2071 D, Mn ) 1674 D, PDI ) 1.24) and R-methoxyω-{N-[3-(triethoxysilyl)propyl]-ureido}-poly(ethylene glycol) (MeOPEG-NH-CO-NH-(CH2)3-Si(OEt)3) (PEG-silane) with a MW of 5 kD (PDI ) 1.27) were obtained from Rapp Polymere (Tu¨bingen, Germany). R-Methoxy-ω-methacryloyl-poly(ethylene glycol) (PEGmethacrylate) MW of 5 kD (PDI ) 1.03) was from Polymer Source (Dorval, Canada). DNA oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA), and IgG-Cy3 (PA43004), from GE/Amersham Pharmacia. Surface Cleaning and PEG Grafting. The ITO slides were cleaned in 10:90, 50:50, and 90:10 methanol/CHCl3 for 15 min each in an ultrasonic bath followed by washing in deionized water and drying in a stream of nitrogen. The slides were then subjected to an oxygen plasma treatment at 5 × 10-1 mbar for 5 min at 200 W in a Plasma System Junior (Europlasma NV, Oudenaarde, Belgium). Coupling of PEG-bis(amine) onto an aldehyde-bearing silanized surface was performed as described.9 Briefly, ITO slides were silanized with 3-glycidoxypropyl trimethoxysilane (GPS), followed by mild acid hydrolysis of the epoxide group and the oxidative cleavage of the resulting diol using NaIO4. PEG-bis(amine) was grafted onto the aldehyde-terminated slides using a procedure which involved coating the slides with a 10 mM solution of PEG in chloroform, evaporating the solvent, and incubating the slides for 40 h at 74 °C to leave behind a macroscopically homogeneous layer of molten PEG. After grafting, excess PEG was removed by extensive rinsing of the slides with DI water and drying in a stream of nitrogen. The same CHCl3-based procedure was used to graft PEG-bis(amine) directly onto blank ITO slides. For the formation of PEG films with PEG-methacrylate, a procedure by Roosjen et al.11 was used which is based on the thermal grafting of PEG from a 2 mM solution in chloroform and the incubation of the slides for 16 h at 145 °C. PEG-silane was coupled to ITO surfaces by incubating the slides with 2 mM PEG-silane in anhydrous toluene containing 1% triethylamine as catalyst.33 The coupling reaction was performed by incubating the slides for 18 h at 60 °C. Loosely bound PEG was removed from the surface by sonication in toluene and acetone for 5 min each, followed by rinsing with deionized water and drying with a nitrogen stream. Ellipsometry. The thickness of PEG layers was determined using a Woollham M2000 ellipsometer. Spectra were acquired with a polar angle between 45° and 75° in 5° steps. To fit the optical properties of the samples, a three-layer model (PEG/ITO/glass) was employed. The optical properties of the ITO/glass substrates were characterized separately by ellipsometry. PEG layers were modeled as a Lorenzian-oscillator since the absorption of PEG in the UV-vis range is dominated by a strong adsorption at 248 nm.34 The absorption properties of the PEG derivates used in this study were determined independently and found to be consistent with the published PEG data.34 The model did not include possible effects on the optical properties which may have occurred due to the formation of covalent bonds between PEG and the substrate. However, this effect on the optical properties of the system has to be very small since the formation of covalent bonds affects only a small fraction of the polymer monolayer on the ITO substrate. The oscillator strength and the full-width-at-half-maximum (fwhm) were adjusted to match the adsorption spectra of PEG polymer. Thus, the only variable in the fitting procedure was the nominal thickness of the film. See Supporting Information S-2 for the fit to the spectral dependence of the refractive index. X-ray Photoelectron Spectroscopy. Surface analysis of unmodified and chemically modified ITO surfaces was performed using an ESCALAB 200i-XL spectrometer operated using the large area-XL (33) Veiseh, M.; Zhang, M. J. Am. Chem. Soc. 2006, 128, 1197-1203. (34) Scherman, O. A.; Rutenberg, I. M.; Grubbs, R. H. J. Am. Chem. Soc. 2003, 125, 8515-8522.

Schlapak et al. magnetic lens mode and an Al KR monochromated X-ray source operated with a 1-mm spot size. The takeoff angle was maintained at 0° to the surface normal. Survey spectra were collected at a pass energy of 100 eV, and detailed spectra, at 20 eV pass energy. Removal of Shirley backgrounds and Gaussian peak fitting was performed using CasaXPS (Casa Software Ltd., U.K.). To obtain an estimate for the thickness of the organic layer on the glass surface, the intensities of the indium 3d peaks were measured. The attenuation of this peak by the addition of a surface film is given by t ) -λ cos (θ) ln (I/I0)

(1)

where t is thickness of the film, I0 and I are the In3d intensity before and after film addition, θ is the takeoff angle (from the surface normal), and λ is the mean free path of the In3d photoelectron in the film, taken to be 3.5 nm.35 Atomic Force Microscopy. AFM topographs were obtained with a Digital Instruments Dimension 3100 AFM (Veeco, Woodbury, NY) in the tapping mode. Silicon nitride (Si3N4) tips with a heightto-base ratio of 2:1 were employed. For each sample macroscopically different areas were examined to ensure that the observed topographs are representative of the analyzed samples. Contact Angle Goniometry. Contact angles were determined using a dataphysics OCA 20 goniometer in combination with the SCA-20 software package (dataphysics, Filderstadt, Germany) using deionized water. Advancing and receding contact angles were measured with a starting volume of 5 µL, an oscillating volume of 8 µL, and a velocity of 0.2 µL/s at 20 °C. The measurements were performed within two hours after finishing the cleaning and modification procedure of the slides. Assays on the Passivation of PEG-Films Using Fluorescence Microscopy. The ability of PEG-films to passivate the ITO surface against the nonspecific adsorption of biopolymers was tested with fluorescence-labeled DNA oligonucleotides and IgG protein. The assays were performed with Cy3-labeled oligonucleotide DNACy3 with the sequence 5′-Cy3-AGG TGC GTG TTT GT-3′, oligonucleotide thiol-DNA-Cy3 with the sequence 5′-Cy3-CTA GAC CGG TAC AGA TGC GTT CGA A-SH-3′ carrying a thiol modifier at the 3′ end, and goat anti-rabbit IgG-Cy3 with a labeling ratio of eight fluorophores per IgG molecule. The samples were diluted in 10 mM Tris HCl 7.5 to a final concentration ranging from 1 to 1000 nM and used for the incubation of PEGylated slides at room temperature for 180 min. Unbound DNA or protein was removed by washing with 10 mM Tris HCl 7.5 and deionized water, followed by drying in a stream of nitrogen. Fluorescence images were obtained with an in-house developed fluorescence scanning device,36 which is based on an inverted epifluorescence microscope (Axiovert 200, Zeiss, Oberkochen, Germany). For our measurements, a 100× objective (Zeiss, R PlanFLUAR 100x/1.45) was used. Samples were mounted on a scanning stage (Ma¨rzha¨user, Wetzlar-Steindorf, Germany) and illuminated with a diode-pumped solid-state laser with an emission line of 532 nm (Millennia Iis, Spectra Physics, Irvine, CA). Images were taken with a Photometrics CoolSnap HQ digital camera (Roper Scientific, Trenton, NJ) (1392 × 1040-element CCD; pixel pitch, 6.45 µm × 6.45 µm; 12-bit; QE, 0.6) using a time-delayed integration mode.36 Slides were optionally scanned with a filter of the optical density 1 or 2 for bright fluorescent spots exceeding the maximum detection limit of the CCD camera. Image processing and analysis of the images were performed with V++ (Roper Scientific) and Matlab (The MathWorks, Natick, MA). Plots of the concentration dependence of the unspecific binding of biopolymers to the modified surface (Figure 5) were fitted to the Langmuir isotherm of eq 2, (35) Laibinis, P. E.; Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017-7021. (36) Hesse, J.; Sonnleitner, M.; Sonnleitner, A.; Freudenthaler, G.; Jacak, J.; Ho¨glinger, O.; Schindler, H.; Schutz, G. J. Anal. Chem. 2004, 76, 5960-5964; Hesse, J.; Wechselberger, C.; Sonnleitner, M.; Schindler, H.; Schutz, G. J. J. Chromatogr., B 2002, 782, 127-135.

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Langmuir, Vol. 23, No. 20, 2007 10247 θ ) KeqC/(1 + KeqC)

(2)

where θ is the surface coverage, Keq the equilibrium binding constant, and C the concentration of the adsorbed species.

Results Formation of PEG Films. ITO surfaces were chemically modified with PEG using four different coupling chemistries, which are summarized in Scheme 1. Reaction pathway (1) proceeded via the direct reaction of PEG-bis(amine) with the In2O3- and SnO2-bearing surface to form dative bonds from nitrogen to In or Sn. Nitrogen-containing organic molecules are known to be capable of forming stable adducts with indium(III)37 and tin(IV) oxides and other oxygen derivatives.38 In2O3 or SnO2 surfaces also display hydroxyl groups39 which can be used for silanization reactions.40 Following this lead, coupling strategy (2) involved silanization using 3-glycidoxypropyl trimethoxysilane (GPS), the conversion of epoxide into aldehyde groups, and the subsequent coupling of PEG-bis(amine) to form an imine linkage. In approach (3), PEG-methacrylate was reacted with the hydroxyl groups of ITO to form an ether bridge via oxo-Michael addition. Finally, chemical pathway (4) aimed at the direct coupling of trimethoxysilane-bearing PEG to the hydroxyl groups on the ITO surfaces. Ellipsometry. The thicknesses of the PEG films were determined via ellipsometry by measuring the spectral dependence of the refractive index of bare and PEG-modified ITO surfaces. The experimental data were fitted to a three-layer model for glass/ITO/PEG (Supporting Information, S-2). To minimize the uncertainties of the fitting procedure, the PEG layers were modeled as a Lorenzian oscillator because the absorption of PEG in the UV-vis range is dominated by a strong adsorption at 248 nm.34 The absorption properties of the PEG derivates were determined independently and found to be consistent with published data.34 While a Lorenzian oscillator can only approximate the real properties of the PEG layer, any possible discrepancy between model and experiment has to be small because the adsorption peak of PEG was congruent with the calculated spectrum for the Lorenzian-oscillator. Based on the absence of any additional experimental absorption bands, no other photon energies were expected to influence the optical properties of PEG. In the fitting process, the oscillator strength and the full-width-at-half-maximum (fwhm) were adjusted to match the adsorption spectra of PEG. This analysis yielded thickness values which ranged from 2.2 ( 0.2 nm for PEGbis(amine) over 3.3 ( 0.2 nm for GPS/PEG-bis(amine) and 2.2 ( 0.3 nm for PEG-methacrylate to 2.7 ( 0.3 nm for PEG-silane (Table 1; column: ellipsometry thickness). These data imply that all coupling approaches produced films with appreciable thickness. X-ray Photoelectron Spectroscopy. PEG-coated ITO slides were subjected to XPS analysis to confirm the presence of the organic polymer films and to obtain second, independent measurements of the film thickness. Figure 1A shows the survey spectrum of a cleaned, bare ITO surface. The major peaks for In3d (445 and 453 eV), Sn3d (487 and 496 eV), and O1s (530 eV) are characteristic for ITO.29 A small C1s peak at 286 eV (37) Carmalt, C. J. Coord. Chem. ReV. 2001, 223, 217-264; Carmalt, C. J.; King, S. J. Coord. Chem. ReV. 2006, 260, 682-709. (38) Suh, S.; Hoffmann, D. M. Inorg. Chem. 1996, 35, 6164-6169. (39) Donley, C.; Dunphy, D.; Paine, D.; Carter, C.; Nebesny, K.; Lee, P.; Alloway, D.; Armstrong, N. R. Langmuir 2002, 18, 450-457. (40) Nawrocki, J.; Dunlap, C.; Li, J.; Zhao, J.; McNeff, C. V.; McCormick, A.; Carr, P. W. J. Chromatogr., A 2004, 1028, 31-62; Palma, R.; Laureyn, W.; Frederix, F.; Bonroy, K.; Pireaux, J. J.; Borghs, G.; Maes, G. Langmuir 2007, 23, 443-451.

indicates minor organic contaminations which could not be removed during the cleaning procedure. Spectra B-E of Figure 1 display the survey spectra for PEG-modified surfaces generated via methods 1-4, respectively. Treatment with PEG increased the magnitude of the C1s signals at 286 eV in all spectra (Figure 1, B-E) which is in line with the presence of immobilized PEG. In addition, the In3d peaks (445 and 453 eV) and Sn3d peaks (487 and 496 eV) were reduced for all PEG-surfaces (e.g., PEGsilane, Figure 1E; compare to Figure 1A). This indicates that the PEG layers attenuated the indium and tin signals of the underlying ITO surfaces. In addition, the In3d and Sn3d signals at 445 and 487 eV, respectively, were slightly shifted to higher binding energies for the PEG-treated surfaces (Supporting Information, S-3), which is consistent with the chemical modification of the ITO substrates. The changes in the elemental composition of the ITO surfaces upon PEGylation are summarized in Table 2. As was already apparent from the visual inspection of the survey spectra (Figure 1), the elemental composition (Table 2) was found to be dependent on the modification pathway, suggesting different degrees of PEG coupling. The attenuation of In3d signal upon PEG treatment was used to infer the thickness of the PEG layers. The indium signal is the preferred choice for determining the film thickness because indium is present in greater concentrations than tin making the measurements for In more accurate than for Sn. The In3d signal for cleaned ITO was at 32.3 ( 0.6% and dropped to 15.1 ( 1.3% after surface modification with PEG-silane (Table 2). Inserting the corresponding peak areas into eq 1 yielded a layer thickness of 2.5 ( 0.3 nm (Table 1; column: XPS thickness). This compares very well to the thickness value of 2.7 ( 0.3 nm derived by ellipsometry (Table 1, ellipsometry thickness). The XPS thickness for the PEG-methacrylate film was found to be 1.7 ( 0.4 nm which is within experimental error of the ellipsometry-derived thickness of 2.2 ( 0.2 nm (Table 1, XPS thickness and ellipsometry thickness). By contrast, surfaces produced with PEGbis(amine) and GPS/PEG-bis(amine) had thickness values below 1 nm (Table 1, XPS thickness). These low values are in apparent contrast to the ellipsometry analysis which yielded film thicknesses of 2.2 ( 0.2 nm and 3.3 ( 0.3 nm (Table 1, ellipsometry thickness), respectively. The discrepancy can be reconciled by assuming that these PEG films contained appreciable amounts of PEG which were, however, distributed inhomogeneously over the ITO surfaces. Irregular layers give rise to lower XPS thickness values because the increased attenuation of the In3d signals by small but high carbon-rich PEG patches does not compensate for the lack of attenuation in the uncovered ITO areas. As the XPS signal decays exponentially from the surface, the attenuation will be greater for a uniform thin layer compared to the same amount of material distributed in a less uniform manner. The ratio between ellipsometry thickness and XPS thickness was calculated in an attempt to obtain a descriptor for the homogeneity of the PEG films. The ratio is assumed to describe how evenly the polymer mass is being spread on the substrate surface. When applied to the four different PEG films, PEGbis(amine) and GPS/PEG-bis(amine) had ratios of 3.7 ( 0.3 and 4.1 ( 0.4, whereas PEG-methacrylate and PEG-silane had values of 1.3 ( 0.1 and 1.1 ( 0.1 (Table 1; column: ellipsometry thickness/XPS thickness). The data imply less contiguous films for PEG-bis(amine) and GPS/PEG-bis(amine), and rather homogeneous layers for PEG-methacrylate and PEG-silane. The thickness ratio correlates well with two other XPS-based measures of film homogeneity. The first measure is the ratio of magnitude of the C1s signal and XPS film thickness. The ratio, termed C1s/XPS thickness, appears to be a useful indicator of surface homogeneity. For example, a high ratio (.1) suggests

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Schlapak et al.

Scheme 1. Scheme Illustrating the Different Approaches to Graft PEG Films onto ITO Substrates via (1) the Direct Coupling with PEG-bis(amine), (2) Silanization to Introduce an Aldehyde Layer Followed by Grafting with PEG-bis(amine), (3) the One-Step Coupling with PEG-methacrylate or (4) PEG-trimethoxysilane

Table 1. Characteristics of PEG-Grafted ITO Surfaces as Determined by Ellipsometry, XPS, AFM, Water Contact Angle Goniometry, and Fluorescence Microscopy

surface ITO cleaned ITO-PEGbis(amine) ITO/GPS/PEGbis(amine) ITO/PEGmethacrylate ITO/PEG-silane

ellipsometry XPS ellipsometry thickness thickness thickness/ C1s/XPS a b (nm) (nm) XPS thickness thickness

improvement in nonspecific binding of a,d

In/Sn XPS

AFM rms roughness (nm)a

(θa)a,c (deg)

(θr)a,c (deg)

hysteresisa

IgG-Cy3

thiolDNA-Cy3 DNA-Cy3

N.A. 2.2 ( 0.2

N.A 0.6 ( 0.1

N.A 3.7 ( 0.3

N.A 32 ( 5

4.3 ( 0.2 5.3 ( 0.3

1.7 ( 0.2 1.2 ( 0.2

56 ( 2 37 ( 2 19 ( 2 62 ( 2 41 ( 2 21 ( 2

N.A. 0.8e

N.A. 0.0e

N.A. 0.2e

3.3 ( 0.3

0.8 ( 0.1

4.1 ( 0.4

26 ( 3

3.9 ( 0.2

3.1 ( 0.3

60 ( 2 33 ( 1 27 ( 2

2.3 ( 0.4

0.3e

0.3e

2.2 ( 0.2

1.7 ( 0.4

1.3 ( 0.1

16 ( 2

4.4 ( 0.2

1.9 ( 0.2

58 ( 3 37 ( 2 21 ( 2

14 ( 2

5.2 ( 1.7

7.5 ( 1.7

2.7 ( 0.3

2.5 ( 0.3

1.1 ( 0.1

15 ( 1

4.4 ( 0.2

0.8 ( 0.1

38 ( 1 33 ( 1

81 ( 18

300 ( 50

5 ( 1 150 ( 20

The values represent the arithmetic mean ( S.D. of three independent measurements from separately prepared samples. The average and the standard deviation for measurements of three different places within a given sample surface were similar to the average and standard deviation for three separately prepared samples. b The values represent the arithmetic mean ( S.D. of three independent measurements from two sets of samples for ITO/PEG-silane and ITO, one set of samples for the remaining surfaces. c Advancing (θa) and receding (θr) contact angles of water. d The factors on the improvement on the nonspecific binding were derived as defined in eq 3. e The improvement factors are too small to give a reasonable standard deviation of the average. NA, not applicable. a

an unusually high carbon content for a nominally thin, and hence inhomogeneous, film. On the other hand, a low ratio (∼1) implies that the carbon-containing polymer is spread as a thick layer, which is a characteristic feature of a homogeneous film. When the C1s/XPS thickness ratios were calculated for the four PEG layers, PEG-bis(amine) and GPS/PEG-bis(amine) were highest, whereas PEG-methacrylate and PEG-silane had lower values (Table 1; column: C1s/XPS thickness). This order is similar to the sequence found for the ellipsometry thickness/XPS thickness ratio except that the C1s/thickness ratio was lower for GPS/ PEG-bis(amine) than for PEG-bis(amine) (Table 1, compare C1s/ XPS thickness and ellipsometry thickness/XPS thickness). It was also found that the ellipsometry/XPS thickness ratio correlated with the XPS-derived In/Sn ratio. This value is 4.3 ( 0.2 for the bare ITO surface used in this study (Table 1, In/Sn XPS). Likely defective PEG-bis(amine) and GPS/bis(amine) surfaces had divergent In/Sn ratios of 5.3 ( 0.3 and 3.8 ( 0.2, respectively (Table 1, In/Sn XPS). Diverging ratios have also been reported for other surface modifications.28,29,41 Following these literature studies, our results could be explained by assuming that the largely homogeneous ITO substrate42 exhibited some minor

variations in the chemical composition at the nanoscale level such as between or within grains.43 On the basis of this assumption, the higher In/Sn ratio would indicate that PEG-bis(amine) modification occurred with a slightly higher preference at SnO2rich regions. By contrast, a lower In/Sn ratio would imply that GPS/bis(amine) reacted with a somewhat higher probability with In2O3-rich regions of the ITO surface. By comparison, assumed homogeneous PEG-methacrylate and PEG-silane surfaces had an In/Sn ratio of 4.4 ( 0.2 which is very similar to the value of 4.3 ( 0.2 for bare ITO (Table 1, In/Sn XPS). Following our assumption of a slightly inhomogeneous ITO surface, these values suggest that PEG was in these cases coupled with equal probability onto indium- and tin oxide-rich grains of the substrate. Our ellipsometry and XPS analysis yielded thickness values of 2.7 and 2.5 nm, respectively, for the PEG-silane layer. The consensus value of 2.6 nm compares favorably to literature values for PEG films which vary between 1 and 5 nm depending on the (41) Brewer, S. H.; Brown, D. A.; Franzen, S. Langmuir 2002, 18, 68576865. (42) Hartnagel, H. L.; Dawar, A. L.; Jain, A. K.; Jagadish, C. Semiconducting Transpartent Thin Films; IOP Publishing: Bristol, 1995. (43) Vink, T. J.; Walrave, W.; Daams, J. L. C.; Baarslag, P. C.; Vandenmerrakker, J. Thin Solid Films 1995, 266, 145-151.

PEG on ITO

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Figure 1. XPS survey spectra illustrating the elemental changes upon surface modification. (A) Cleaned bare ITO surface, and ITO surfaces modified with (B) PEG-bis(amine), (C) GPS/PEG-bis(amine), (D) PEG-methacrylate, and (E) PEG-silane. Table 2. Full Chemical Quantification of ITO Surfaces Carrying PEG Layers as Determined by XPSa

ITO ITO/PEG-bis(amine) ITO/GPS/PEG-bis(amine) ITO/PEG-methacrylate ITO/PEG-silane

C1s (%)

F1sb (%)

In3d (%)

N1s (%)

O1s (%)

Si2pb (%)

Sn3d (%)

In/Sn

8.9 ( 1.0 19.1 ( 3.4 21.2 ( 1.5 26.7 ( 4.2 38.9 ( 1.5

0.4 ( 0.3 0.7 ( 0.4 0.8 ( 0.7 0.9 ( 0.6 0.5 ( 0.3

32.3 ( 0.6 28.1 ( 0.3 24.4 ( 0.3 22.2 ( 2.6 15.1 ( 1.3

0.3 ( 0.1 0.5 ( 0.4 0.4 ( 0.1 0.7 ( 0.2 0.9 ( 0.3

49.3 ( 0.1 44.0 ( 2.9 43.7 ( 0.9 41.2 ( 1.5 36.0 ( 0.7

1.5 ( 0.5 2.5 ( 1.6 3.3 ( 0.7 3.2 ( 1.6 5.0 ( 1.0

7.5 ( 0.3 5.3 ( 0.3 6.3 ( 0.3 5.1 ( 0.4 3.5 ( 0.2

4.3 ( 0.2 5.3 ( 0.3 3.9 ( 0.2 4.4 ( 0.2 4.4 ( 0.2

The values represent the arithmetic mean of at least three independent measurements ( the standard deviation from two sets of samples for ITO/PEG-silane and ITO, and one set of samples for the other surfaces. b The peak positions of Si2p and F1s are 100-104 eV and 685-689 eV, respectively. a

extent of surface coverage and the size of the PEG.12,44 In another report, for example, grafting of PEG with a MW of 2 kD yielded a dense film of 3.5 nm whichsvia the known polymer densitys corresponds to a surface coverage of 195 pmol/cm2.9 Analogous calculations for our layer of PEG-silane with a MW of 5 kD and a thickness of 2.6 nm yielded a surface coverage of 62 pmol/cm2. This surface coverage is equivalent to an average molecular cross section of 2.7 nm2 per polymer chain. This value compares well with the molecular cross section of an idealized helical PEG chain32 of 0.21 nm2 because longer PEG chains with MW 5 kD, as used in our experiments, cannot be approximated as an extended helical chain but rather as a compressible and dynamically changing polymer ball.45 Atomic Force Microscopy. AFM was applied to study the molecular structure of the PEG films. Topographic images were obtained in the tapping mode. A representative micrograph of cleaned, unmodified ITO is shown in Figure 2A. The image and the height profile display several peaks of up to 10 nm, in line with the known coarse surface structure of the metal oxide.46 ITO surfaces modified with PEG using the four methods were also analyzed with AFM. The topographic images and the (44) Andruzzi, L.; Senaratne, W.; Hexemer, A.; Sheets, E. D.; Ilic, B.; Kramer, E. J.; Baird, B.; Ober, C. K. Langmuir 2005, 21, 2495-2504. (45) Doi, M. Introduction to Polymer physics; Clarendon Press: Oxford, 1996; Kenworthy, A. K.; Hristova, K.; Needham, D.; McIntosh, T. J. Biophys. J. 1995, 68, 1921-1936; Rex, S.; Zuckermann, M. J.; Lafleur, M.; Silvius, J. R. Biophys. J. 1998, 75, 2900-2914. (46) Amaral, A.; Brogueria, P.; Nunes de Carvalho, C.; Lavareda, C. Surf. Coat. Technol. 2000, 125, 151-156.

representative height profiles are shown in B-E of Figure 2 (please note the two different z-scales). The visual inspection revealed that modification by PEG-bis(amine) (Figure 2B) minimally affected the surface, whereas treatment with GPS/ PEG-bis(amine) (Figure 2C) increased the number and dimensions of the elevated features with a height of up to 20 nm. The additional peaks could represent aggregates of PEG molecules. PEGmethacrylate surfaces (Figure 2D) showed a smoother surface background which was, however, decorated with large elevated features up to 20 nm in height. A smooth background surface with fewer peaks of a maximum height of 5 nm was observed for PEG-silane samples (Figure 2E). It is likely that the smooth surfaces of PEG-methacrylate and PEG-silane represent the dense PEG films, which were also detected with XPS and ellipsometry thickness measurements. Changes in the surface morphology upon PEGylation were also apparent when the densities of the topographic peaks and their dimensions were compared. The results of the analysis (Supporting Information, S-4) indicate lower peak densities for PEG-methacrylate and PEG-silane, but larger peak widths for PEG-methacrylate and higher peaks for GPS/PEG-bis(amine) and PEG-methacrylate. The root-mean-square (rms) roughness of the topographic images was used as a quantitative measure to compare the changes in the surface of ITO upon PEGylation. The rms roughness of bare ITO had a value of 1.7 ( 0.2 nm (Table 1, AFM rms roughness). By comparison, the rms value of GPS/PEG-bis(amine) was 3.2 ( 0.2 nm (Table 1, AFM rms roughness) which

10250 Langmuir, Vol. 23, No. 20, 2007

Figure 2. AFM micrographs and height profiles along the beige lines of (A) bare ITO surfaces and surfaces modified with PEG films via (B) PEG-bis(amine), (C) GPS/PEG-bis(amine), (D) PEGmethacrylate, (E) PEG-silane. Note the different scale bars for (A), (B), (E), and (C), (D). Two different z-scales were chosen to represent the full range of the height differences in highly inhomogeneous surfaces compared to the structural details of smoother surfaces. Each micrograph displays an area of 1.7 µm2. Bars, 400 nm.

is twice the value of bare ITO. This implies that GPS/PEGbis(amine) actually increased the surface roughness, most likely due to the uneven distribution of the PEG film. In comparison, the rms values of PEG-bis(amine) and PEG-methacrylate surfaces were 1.3 ( 0.2 nm and 1.9 ( 0.2 nm which are slightly smaller than and equal to the blank ITO, respectively (Table 1, AFM rms roughness). The lower value of PEG-bis(amine) suggests that holes and/or topographically lower areas of the ITO were filled with PEG. By contrast, PEG layers obtained via the attachment of PEG-silane resulted in an rms value of 0.8 ( 0.1 (Table 1, AFM rms roughness) which is about half the value of bare ITO and suggests the smoothening of the uneven ITO substrate surface by a PEG layer. Water Contact Angle Goniometry. Goniometry was used as complementary method to assess the homogeneity of the polymeric films. Goniometry is a sensitive surface analytical tool and can detect subtle changes in the structure of organic interfaces as shown by several studies.9,31,47 The advancing (θa) and receding (θr) contact angles of water were measured. For cleaned ITO, θa and θr were 56 ( 2° and 37 ( 2°, respectively (Table 1; columns: (θa) and (θr)). The high hysteresis of 19° suggests surface inhomogeneities which are in agreement with the coarse surface structure of ITO (Table 1; column: hysteresis). The contact angles for PEG-treated surfaces obtained by methods 1-3 (Scheme 1) were of similar value, ranging between 58 ( 3° and 62 ( 2° for θa, and 33 ( 1° and 41 ( 2° for θr (Table 1, θa and θr). The corresponding hysteresis values were between 21 ( 2 and 27 ( 2° (Table 1, hysteresis), implying that these (47) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96; Folkers, J. P.; Gorman, C. B.; Laibinis, P. E.; Buchholz, S.; Whitesides, G. M.; Nuzzo, R. G. Langmuir 1995, 11, 813-824; Robbins, M. O.; Joanny, J. F. Europhys. Lett. 1987, 3, 729-735; Good, R. J. J. Am. Chem. Soc. 1952, 74, 5041-5042.

Schlapak et al.

Figure 3. Fluorescence micrographs of bare ITO (A) and PEGfunctionalized surfaces (B-F) treated with 1 µM IgG-Cy3 (A-E). The micrographs show an area of 78 µm × 78 µm.

PEG films did not reduce the surface inhomogeneities of the underlying ITO surface. The data compare well with the low film thicknesses derived by XPS for PEG-bis(amine) and GPS/ PEG-bis(amine) (Table 1, XPS thickness). It is possible that the high hysteresis values are due to the numerous, irregular, elevated nanoscale features seen in AFM micrographs of GPS/PEG-bis(amine) and PEG-methacrylate surfaces (Figure 2, C and D). By contrast, PEG films generated via direct modification with PEGsilane had very low advancing and receding contact angles of 38 ( 1° and 33 ( 1° (Table 1, θa and θr), characteristic for hydrophilic PEG surfaces.9,31 Importantly, the low hysteresis of 5 ( 1° strongly indicates that the PEG-silane film had few structural defects.31 The high homogeneity of PEG-silane as detected by goniometry is in very good agreement with the XPS/ ellipsometry analysis on a dense polymer film and the AFM data on the smoothened PEG-ITO surface. Fluorescence Microscopy. The ability of PEG films to avert the nonspecific adsorption of biopolymers was determined by using fluorescence-based assays in combination with direct and sensitive fluorescence microscopy readout. Assays on the nonspecific adsorption were performed with Cy3-labeled immunoglobulin (IgG-Cy3) or with Cy3-tagged DNA. Dilute solutions of the fluorescence-labeled biopolymers were added to bare ITO or PEG-modified ITO surfaces, incubated for 180 min, and removed, followed by a detergent-free washing step. Biopolymers which had nonspecifically bound onto the ITO surface were detected and quantified using epifluorescence microscopy. Figure 3A displays a representative fluorescence micrograph of bare ITO after incubation with 1 µM IgG-Cy3. The surface is much brighter (36000 ( 5000 counts) after protein incubation than it is before (140 ( 15 counts) (Figure 3F), implying that IgG nonspecifically adsorbed onto the surface. Nonspecific adsorption was also found for ITO surfaces coated with PEG, albeit to a lower extent. For example, ITO modified with PEGbis(amine) (Figure 3B) and GPS/PEG-bis(amine) (Figure 3C) displayed a lower fluorescence brightness of 21000 ( 3000 counts

PEG on ITO

Figure 4. Amount of IgG-Cy3, Thiol-DNA-Cy3, and DNA-Cy3 nonspecifically adsorbed onto ITO surfaces depends on the type of PEG-coating. The numbers are the average counts obtained from fluorescence micrographs, n ) 3.

and 12000 ( 1700 counts, respectively. PEGylated surfaces obtained with PEG-methacrylate had even less adsorption (Figure 3D; 2600 ( 300 counts). The bright features visible on micrographs in Figure 3 B-D were on average 20% brighter than the surrounding area and most likely represent spots with multiple biomolecules. By comparison, the fluorescence brightness of PEG-silane surfaces after protein challenge (Figure 3E; 430 ( 80 counts) were very close to the PEG-silane surfaces without protein incubation (Figure 3F; 140 ( 15 counts). A bar chart in Figure 4 (black bars) graphically summarizes the quantitative results of the average fluorescence brightness of different PEG surfaces after IgG-Cy3 challenge. The background fluorescence of PEGylated ITO surfaces without IgG-Cy3 is not included in the bar chart as almost all PEG films did not increase the background fluorescence compared to ITO; only the autofluorescence of ITO/PEG-methacrylate was 2 times higher than that of ITO (Supporting Information, S-5 and S-6). To compare the changes in nonspecific binding achieved by the four PEG coatings, an improvement factor, defined in eq 3, was introduced,

improvement factor ) (BITO/biopolymer - BITO)/ (BITO-PEG/biopolymer - BITO-PEG) - 1 (3) where BITO/biopolymer and BITO are the average fluorescence brightness of bare ITO after and before incubation with the labeled biopolymer, respectively, and BITO-PEG/biopolymer and BITO-PEG are the average fluorescence brightness of PEG-coated ITO after and before incubation with the labeled biopolymer, respectively. An improvement factor of zero implies that the PEG coating does not lead to a reduction in the nonspecific binding. When quantified using this measure, PEG-silane films were the best in passivating the ITO surface because the nonspecific adsorption was reduced by a factor of 150 ( 20. PEG-methacrylate followed with a factor of 14 ( 2, and PEG-bis(amine) and GPS/ PEG-bis(amine) were poorest with factors of 0.8 and 2.3 ( 0.4, respectively (Table 1; column: improvement in nonspecific binding of IgG-Cy3). The ability of PEG films to avoid the adsorption of biopolymers was also tested with 1 µM solutions of Cy3-labeled oligonucleotides DNA-Cy3 with 14 nucleotides and thiol-DNA-Cy3 of 25 bases with a terminal disulfide group. As disulfide- and thiolterminated compounds are known to bind to ITO surfaces,29 thiol-DNA-Cy3 represented a potentially difficult biopolymer. The fluorescence micrographs of the incubated surfaces are shown in Supporting Information (S-5 and S-6), while the quantitative results of the assays on the nonspecific adsorption of DNA oligos are summarized in the bar chart in Figure 4. Thiol-DNA-Cy3 (Figure 4, gray bars) and DNA-Cy3 (Figure 4, white bars)

Langmuir, Vol. 23, No. 20, 2007 10251

Figure 5. Concentration dependence of nonspecifically adsorbed IgG-Cy3 onto bare ITO surfaces (empty circles) and surfaces modified with PEG-silane (filled circles). The numbers represent the average fluorescence counts measured for micrographs from two independent experiments.

adsorbed onto ITO with average fluorescence brightness values of 12000 ( 2000 counts and 8100 ( 1800 counts, respectively. These values are lower than the brightness of ITO surfaces challenged with IgG-Cy3 of 36000 ( 5000 counts (Figure 4, black bar). The lower values do not mean that less DNA adsorbed onto the surface because the fluorophore-labeling ratios of DNACy3 and IgG-Cy3 were different. DNA oligos were labeled at a stoichiometry of 1:1 whereas IgG protein was tagged with an average of 8 fluorophores. After correcting for the higher labeling ratio, the relative amount of adsorbed protein was approx 50% lower than that for DNA oligonucleotides. When the nonspecific adsorption of thiol-DNA-Cy3 and DNA-Cy3 onto the four PEGylated surfaces was compared (Figure 4, gray and white bars), PEG-bis(amine) and GPS/PEG-bis(amine) films did not appear to significantly passivate ITO while PEG-methacrylate andsto an even greater extentsPEG-silane reduced the amount of nonspecifically bound DNA. Expressed as factors, PEG-bis(amine) and GPS/PEG-bis(amine) films led to a reduction by zero and 0.3, and 0.2 and 0.3 in the binding of thiol-DNA-Cy3 and DNA-Cy3, respectively (Table 1; columns: improvement in nonspecific binding of thiol-DNA-Cy3 and improvement in nonspecific binding of DNA-Cy3). By contrast, in the case of PEG-methacrylate the factors for thiol-DNA-Cy3 and DNACy3 were 5.2 ( 1.7 and 7.5 ( 1.7 (Table 1). PEG-silane surfaces fared best in the assays, leading to reduction by a factor of 81 ( 18 and 300 ( 50 for thiol-DNA-Cy3 and DNA-Cy3, respectively (Table 1). The ability of PEG-silane films to avert the nonspecific adsorption of IgG-Cy3 was tested in dependence of the concentration of the protein. The concentration of IgG-Cy3 ranged from 1 µM to 1 nM. Figure 5 shows the results of the fluorescence analysis of bare and PEG-silane-coated ITO. A semilogarithmic plot was chosen to display the strong binding to bare ITO and weak binding to the PEG-surface within one graph. IgG-Cy3 showed concentration-dependent binding onto both surfaces. The data points were fitted to the Langmuir isotherm of eq 2 and yielded apparent equilibrium binding constants, Keq. The Keq values for ITO and PEG-ITO were (3.3 ( 1.2) × 107 M-1 and (2.0 ( 0.6) × 107 M-1. The similar values may reflect that IgG protein bound via similar molecular interactions to both bare and PEG-coated substrate surfaces.

Discussion This report describes the covalent grafting of PEG films on ITO substrates for biopassivation purposes. Different coupling chemistries were tested to identify those conditions which lead to a dense PEG layer. The polymer coatings were examined with five different surface analytical tools to probe the films’ properties and, in particular, their homogeneity. While ellipsometry suggested that all layers had a thickness of at least 2 nm, XPS provided a more nuanced picture on the formation of PEG films.

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The thickness values of two PEGylated surfaces were in agreement with the ellipsometry results, whereas the XPS thickness values of two other PEG films were below 0.8 nm, most likely because PEG molecules were aggregated in small patches also seen in AFM images leading to inhomogeneous films. Despite lacking nanoscale-lateral resolution, XPS in combination with ellipsometry was clearly capable of inferring molecular differences in the films’ structural integrity. AFM and goniometry, which were used as additional analytical tools, supported the finding of different degrees of the films’ quality. When all surface analytical data were combined, it was possible (i) to draw a coherent picture of the quality of a given PEG layer and (ii) to predict the quality to the film’s ability to avert the nonspecific biopolymer adsorption. For example, all surface analytical results indicated that the PEG-silane film was dense as both ellipsometry thickness (2.5 ( 0.3 nm) and XPS thickness (2.7 ( 0.3 nm) were high and very similar in value (Table 1, ellipsometry thickness and XPS thickness), and the thickness ratio was close to unity (Table 1, ellipsometry thickness/XPS thickness). The presence of a polymer film was also apparent in AFM measurements because the rms roughness of ITO was reduced by 50% to a value of 0.8 ( 0.1 nm (Table 1, AFM rms roughness). In addition, the PEG coating led to a drop of the water contact angle hysteresis by a factor of almost four to a final value of 5 ( 1° (Table 1, hysteresis). Taken together, these data are all consistent with a molecular, dense film with few structural defects. In line with expectations, the PEG-silane layer was very effective in repelling fluorescence-labeled biopolymers from the substrate surface with an improvement factor of 150 ( 20 for IgG-Cy3 and 300 ( 50 for DNA-Cy3, compared to unprotected ITO surfaces (Table 1; columns: improvement in nonspecific binding of IgG-Cy3 and improvement in nonspecific binding of DNA-Cy3). The combined analysis yielded a slightly different picture for PEG-methacrylate, which was characterized by a thinner and less homogeneous polymer film. Ellipsometry thickness (2.2 ( 0.2 nm) and XPS thickness (1.7 ( 0.4 nm) were lower than those of PEG-silane (Table 1, ellipsometry thickness and XPS thickness). In addition, the higher thickness ratio of 1.3 ( 0.1 (Table 1, ellipsometry thickness/XPS thickness) indicated some molecular inhomogeneities. Elevated features were observed in AFM topographs, and the rms roughness values for the PEGylated surface (1.9 ( 0.2 nm) and blank ITO (1.7 ( 0.2 nm) were very close (Table 1, AFM rms roughness). Furthermore, the high water contact angle hysteresis for PEG-methacrylate (21 ( 2°) and blank ITO (19 ( 2°) were similar (Table 1, hysteresis). As would be expected for a thinner and less homogeneous PEG layer, the ability to avert the nonspecific adsorption was improved only by a factor of 14 ( 2 for IgG-Cy3 and 7.5 ( 1.7 for DNACy3 (Table 1, improvement in nonspecific binding of IgG-Cy3 and improvement in nonspecific binding of DNA-Cy3), which are about 11 and 40 times smaller than for the thicker and more homogeneous PEG-silane film, respectively. The surface analytical examination of surfaces treated with PEG-bis(amine) and GPS/PEG-bis(amine) pointed at very inhomogeneous films. There was a large discrepancy between ellipsometry thickness values (2.2 ( 0.2 nm and 3.3 ( 0.3 nm) and XPS thickness values (0.6 ( 0.1 nm and 0.8 ( 0.1 nm, thickness ratios of 3.7 ( 0.3 and 4.1 ( 0.4, respectively) implying an uneven distribution of PEG on the substrate surfaces (Table 1, ellipsometry thickness and XPS thickness). The AFM rms roughness of PEG-bis(amine) was lower (1.2 ( 0.2 nm) while the rms value of GPS/PEG-bis(amine) was higher (3.1 ( 0.3 nm) than the bare ITO surface (1.7 ( 0.2 nm) (Table 1, AFM

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rms roughness). A possible explanation for the smoothing of the surface for PEG-bis(amine) might be the preferential linkage to Sn-rich grains. If these happened to form the lowest parts of the ITO surface, it could result in an apparent smoothing of the surface with alternating patches of PEG and metal oxide. Further analysis, for example with pulse force mode AFM,28 would be required to clarify this point. Nevertheless, the high hysteresis values from goniometry analysis (21 ( 2° and 27 ( 2°, Table 1) supported the notion that these surfaces had structural defects. The poor homogeneity of the films was clearly related to their low ability to biopassivate the surface. IgG-Cy3 binding on PEGbis(amine) and GPS/PEG-bis(amine) was reduced by only 0.8 and 2.3 ( 0.4, and DNA-Cy3 binding was also reduced by a similarly small factor of 0.2 and 0.3, respectively (Table 1, improvement in nonspecific binding of IgG-Cy3 and improvement in nonspecific binding of DNA-Cy3). It cannot be ruled out that the positively charged amine group at the free end of the PEG chain affected the nonspecific binding of negatively charged DNA strands or IgG protein. However, any possible influence would have to be very small as the properties of one terminal nitrogen would be masked by the remaining 135 carbon and oxygen atoms in a PEG chain of MW 2000. Furthermore, in a hydrated PEG chain the free end is not permanently located at the polymer surface butson a time-based averagesdistributed within a polymer ball,45,48 thereby minimizing the influence of the amine group on the adsorption of biopolymers. The ellipsometry/XPS thickness ratio along with water contact angle goniometry and AFM rms roughness successfully identified the most homogeneous polymer film. All three measures (Table 1, ellipsometry thickness/XPS thickness, hysteresis, AFM rms roughness) were lowest for the PEG-silane film, which correlates well with the big improvement factors observed in the biopassivation assays (Table 1, improvement in nonspecific binding). The three surface parameters were, however, not quantitatively correlated to the improvement factor for the three other PEG films. Water contact angle hysteresis was the same for PEGmethacrylate and GPS/PEG-bis(amine) (Table 1, hysteresis) even though the former performed better in the biopassivation assays (Table 1, improvement in nonspecific binding). The second surface parameter, AFM roughness, also failed to provide a clear link to the performance in the tests on the absorption of biopolymers. For example, the rms roughness of PEG-bis(amine) with a value of 1.2 ( 0.2 was lower than the value of PEGmethacrylate (1.9 ( 0.2) (Table 1, AFM rms roughness) even though the latter was better in averting nonspecific adsorption. The thickness ratio (Table 1, ellipsometry thickness/XPS thickness) was qualitatively related to the results of the biopassivation assays. The relation was, however, not quantitative as a small increase in the thickness ratio from 1.1 ( 0.1 (PEGsilane) to 1.3 ( 0.1 (PEG-methacrylate) was accompanied by a 10-fold drop in the improvement factor for IgG-Cy3, while a much bigger step in the thickness ratio to 4.1 ( 0.4 (GPS/PEGbis(amine)) was associated with a smaller decrease in the nonspecific binding to a factor of 2.3 ( 0.4 (Table 1). While PEG film homogeneities have been described in the past by water contact angle goniometry,32 roughness measurements in AFM topographic images,4,10,12 compressibility studies using AFM phase modulation, and scratching experiments with cantilevers,12 we contend that the thickness ratio is a valuable, new, additional parameter for the quality of PEG layers, which has not been described in any previous study. The thickness ratio can, in combination with water contact angle hysteresis, paint (48) Kong, C. Y.; Muthukumar, M. J. Am. Chem. Soc. 2005, 127, 1825218261.

PEG on ITO

a nuanced picture of the structural integrity of PEG films. This measure may be used to characterize other polymer coatings which are uniformly spread over substrate surfaces. It might also be adapted to describe nonuniform polymer coatings featuring regular patterns under certain conditions. This is the case when the distance between the polymer-coated areas is smaller than the wavelength used for the ellipsometry because in this range the optical signal depends only weakly on topography. For polymer separations larger than the wavelength, the thickness ratio cannot be applied. The four different PEG-grafting approaches produced PEG films of different quality on ITO surfaces. What are the chemical reasons that PEG-silane produced the most homogeneous and effective films, followed by PEG-methacrylate, with GPS/PEGbis(amine) and PEG-bis(amine) faring worst? One cannot give the exact reason because ITO is a mixture of two metal oxides whose exact surface composition depends on the details of the cleaning treatment.39 Nevertheless, a discussion of the possible influence of various factors is warranted. Both PEG-trimethoxysilane and PEG-methacrylate share similarities which together may be the key for successful coupling reactions. (i) Both PEG reagents only need one simple coupling step, thus avoiding some side reactions which can occur in multistep reactions. In addition, (ii) the two PEG reagents were applied as a water-free molten film (see Experimental Section) which ensures a high concentration of reactive groups close to the surface and might also limit the deactivation of the reactive groups by hydrolysis. Finally, (iii) both PEG reagents couple to the hydroxyl groups of the ITO surface. The chemistries of PEG-trimethoxysilane and PEGmethacrylate differ with respect to their ability to form networks with other PEG chains. Only trimethoxysilane can potentially form silane-oxygen bonds to other reactive heads of the PEG chains. The extent to which these networks actually form is, however, likely limited as the molecular cross section of the PEG chain restricts contact between the reactive head groups. Hydroxyl moieties of ITO were the anchoring points not only for PEG-silane and PEG-methacrylate but also for the poorperforming GPS/PEG-bis(amine). The quality of the GPS/PEGbis(amine) film was low even though a similar procedure has been used successfully in a previous study on glass-displaying silanol groups.9 Possibly, this could reflect the different surface chemistries of the ITO and glass substrates. Finally, PEG-bis(amine) did not couple effectively to ITO. Apparently dative bonds between the nitrogen and the metal oxides did not form throughout the ITO surface even though coupling conditions were chosen to avoid interference with water which is known to be an important parameter in the formation of bonds to indium alkoxides.37 The dense films of PEG obtained in this report are limited not only to help avoid the nonspecific binding of biopolymers to the ITO surface. In the case of biosensor and microarray substrates, PEG chains with suitable terminal functional groups can also be used as flexible linkers to immobilize antibodies or DNA strands onto the solid substrate. Flexible PEG and OEG linkers have been used in the past to improve the biomolecular recognition between the immobilized receptors and the free analyte molecules.9,14,49-51 Compared to the direct linker-less immobilization, the flexible PEG chains are thought to enhance binding due to the greater steric freedom of the receptors and

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favorable binding kinetics. The best PEG-coupling chemistries used in this reportssilanization with trimethoxysilane-terminated PEG chains, and oxo-Michael addition with methacrylateterminated PEG chainssare compatible with the linker-mediated immobilization of biomolecular receptors. For instance, the other terminus of the PEG chain could be functionalized with biotin to enable extravidin binding followed by coupling to biotinylated antibodies.51 Alternatively, it may also be feasible to use maleimide,9,52 Diels-Alder,50 or 1,3-dipolar addition-based53 immobilization schemes.

Conclusions In this report, we have described the formation of dense films of PEG on ITO substrates. To the best of our knowledge, this is the first time covalently coupled PEG layers on ITO have been described. The study is also of interest because it demonstrates that the homogeneity of polymer films can be accurately determined on molecularly coarse surfaces. By using PEG and ITO as a model system, measurement on AFM roughness and contact angle hysteresis were shown to successfully identify the most homogeneous PEG film even though both surface parameters did not explain the different degrees of nonspecific adsorption seen for the three other PEG films. In addition, the ratio of the film thicknesses derived by ellipsometry and XPS was introduced as a useful descriptor for the quality of polymer films with the most homogeneous and dense coatings having a thickness ratio close to unity. This measure may be applied to other uniform polymer coatings. Finally, the study highlights that the molecular density and homogeneity of the PEG films correlate very well with their ability to passivate the surface against nonspecific adsorption against biopolymers. In summary, the report identifies the critical film parameters which can help rationalize and predict the capacity of the PEG layers for biopassivation. The findings might be applicable to other coarse substrates and other polymeric films. Acknowledgment. This work has been supported by the Austrian Science Foundation (Project N00104-NAN) and the state of Upper Austria. We thank Hermann Gruber and Helen Kinns for critically reading the manuscript and Peter Hinterdorfer, Ferry Kienberger, and Franco Cacialli for helpful suggestions. Supporting Information Available: Ellipsometric analysis of polymer film thickness, detailed In and Sn XPS spectra, the quantitative analysis of the AFM topographs, and fluorescence micrographs of bare ITO and PEG-functionalized surfaces treated with 1 µM thiol-DNACy3 and DNA-Cy3. This material is available free of charge via the Internet at http://pubs.acs.org. LA7011414 (49) Nivens, D. A.; Conrad, D. W.; Esker, A. R.; Brode, P. F.; Rubingh, D. N.; Rauch, D. S.; Yu, H.; Gast, A. P.; Robertson, C. R.; Trigiante, G. Langmuir 2000, 16, 2198-2206; Stadler, B.; Falconnet, D.; Pfeiffer, I.; Hook, F.; Voros, J. Langmuir 2004, 20, 11348-11354. (50) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270-274. (51) Huang, N. P.; Voros, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220-230. (52) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031-2039. (53) Sun, X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L. Bioconjug. Chem. 2006, 17, 52-57.