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Surface Plasmon Resonance Studies of Protein Binding on Plasma Polymerized Di(ethylene glycol) Monovinyl Ether Films Z. Zhang,† B. Menges,† R. B. Timmons,‡ W. Knoll,† and R. Fo¨rch*,† MPI fu¨ r Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany, and Department of Biochemistry and Chemistry, University of Texas at Arlington, Arlington, Texas 76019 Received December 11, 2002. In Final Form: March 4, 2003 The attachment of fibrinogen, bovine serum albumin, and immunoglobulin on continuous wave (CW) and pulsed plasma polymerized di(ethylene glycol) monovinyl ether was studied using surface plasmon resonance (SPR) spectroscopy and waveguide mode spectroscopy (WaMS). Structural analysis of the films was carried out using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and atomic force microscopy. Plasma conditions employed during depositions produced significant differences in the chemical and physical properties of the resultant polymer films. Films deposited under CW and higher plasma duty cycles showed relatively high refractive indices (n > 1.57) and essentially a constant thickness if immersed in aqueous buffer solutions and exhibited a high adsorption affinity for proteins. In contrast, films produced under lower plasma duty cycles were of lower refractive index (n < 1.4), exhibited significant swelling if immersed in aqueous buffer, and were extremely effective in preventing protein adsorption. The SPR and WaMS data suggest that the relatively non-cross-linked films produced at the lower duty cycles exhibit hydrogel-like behavior when immersed in aqueous solutions. It is believed that these hydrated films are responsible for the remarkably effective nonfouling properties of the films deposited at low duty cycles. The relationship between film structure, polymer stability in aqueous buffer, and protein binding affinities are discussed.
Introduction The controlled adsorption of proteins and other biomolecules onto surfaces in contact with an aqueous solution is a crucial step in the fabrication of sensors and medical implant devices. Molecules containing ethylene oxide (EO) units attached to surfaces exhibit a welldocumented tendency to reduce the adsorption of proteins.1 Methods employed to attach EO-containing molecules to surfaces have included simple physical adsorption processes,2-4 PEO grafting to surfaces,5-8 radiation and chemical cross-linking methods,9 self-assembled monolayer techniques,10 and plasma polymerization methods.11,12-16 Lopez et al. first demonstrated that plasma polymers of tetra(ethylene glycol) dimethyl ether lead to * To whom correspondence may be addressed: Tel: +49 6131 379 487. Fax: +49 6131 379 100. E-mail:
[email protected]. † MPI fu ¨ r Polymerforschung. ‡ University of Texan at Arlington. (1) Poly(ethylene glycol) Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Ed.; ACS Symposium Series 680; American Chemical Society: Washington, DC, 1997. (2) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed Mater. Res. 1989, 23, 351-368. (3) Maechling-Strasser, C.; Dejardin, P.; Galin, J. C.; Schmitt, A. J. Biomed Mater. Res. 1989, 23, 1385-1393. (4) Maechling-Strasser, C.; Dejardin, P.; Galin, J. C.; Schmitt, A. J. Biomed Mater. Res. 1989, 23, 1395-1410. (5) Sun, Y. H.; Gombotz, W. R.; Hoffman, A. S. J. Bioact. Compat. Polym. 1986, 1, 316-334. (6) Sun, Y. H.; Gombotz, W. R.; Hoffman, A. S. Polym. Prep. 1987, 28, 292-294. (7) Grainger, D. W.; Nojiri, C.; Okano, T.; Kim, S. W. J. Biomed Mater. Res. 1988, 22, 231-249. (8) Grainger, D. W.; Knutsen, K.; Kim, S. W.; Feijin, J. J. Biomed Mater. Res. 1990, 24, 403-431. (9) Sheu, M. S.; Hoffman, A. S.; Ratner, B. D.; Feijen, J.; Harris, J. M. in Plasma Surface Modification of Polymers: Relevance to Adhesion; Strobel, M., Lyons, C. S., Mittal, K. L., Eds.; VSP: The Netherlands 1994; pp 197-208. (10) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 714-721. (11) Grunkemeier, J. M.; Tsai, W. B.; Alexander, M. R.; Castner, D. G.; Horbett, T. A. J. Biomater. Mater. Res. 2000, 51, 669-679.
surfaces with a high resistance to proteins.13 Beyer et al. showed that the pulsed plasma assisted polymerization of tri(ethylene glycol) monoallyl ether provided highly efficient nonfouling surfaces.14,15 More recently, using radiolabeled albumin and fibrinogen, Wu et al. showed that a minimum of two ethylene oxide units in the monomer molecule are necessary to obtain outstanding nonfouling properties under low duty cycle pulsed-plasma conditions.16,17 The present work provides additional information on the adsorption of fibrinogen (Fbg), bovine serum albumin (BSA), and immunoglobulin (IgG) on plasma polymers of di(ethylene glycol) monovinyl ether (EO2) as a function of the polymer film structure. Surface plasmon resonance (SPR) spectrocopy and waveguide mode spectroscopy (WaMS) were employed to provide the first real time studies of protein attachment on plasmapolymerized EO2 films. These optical techniques allow for the determination of the thickness (d) and the refractive index (n) of the plasma polymer in solution, as well as real time measurements of reactions occurring at the interface. A combination of these two methods provides new insights into the dynamics of protein binding, plus important data on the behavior of the plasma polymer films when submersed in solution. Experimental Section Materials and Substrates. The monomer and proteins were purchased from Sigma, Germany. The monomer was freeze-dried three times to remove excess adsorbed gases but was not purified (12) Favia, P.; d’Agostino, R.; Palumbo, F. J. Phys IV 1997, 7, 199208. (13) Lopez, G. P.; Ratner, B. D.; Tidwell, C. D.; Haycox, L. L.; Rapoza, R. J.; Horbett, T. A. J. Biomol. Med. Res. 1992, 26, 415-439. (14) Beyer, D.; Knoll, W.; Ringsdorf, H.; Wang, J. H.; Timmons, R. B.; Sluka, P. J. Biomed. Mater. Res. 1997, 36, 181-189. (15) Beyer, D. Ph.D. Thesis, Universita¨t Mainz, Germany, 1996. (16) Wu, Y. J.; Timmons, R. B.; Jen, J. S.; Molock, F. E. Colloids Surf., B 2000, 18, 235-248. (17) Wu, Y. J. Ph.D. Thesis, University of Texas at Arlington, Arlington, TX, 2000.
10.1021/la026980d CCC: $25.00 © 2003 American Chemical Society Published on Web 05/02/2003
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further. High index (n ) 1.9) LaSFN9 glass required for SPR measurements was purchased from Hellma Optik, Jena, Germany. Gold (d ) 50 nm) was thermally evaporated onto LaSFN9 glass and used as substrates for the SPR measurements. For studies in aqueous solution, an adhesion layer is required to ensure adhesion of the plasma polymer on the gold SPR substrates. This was achieved by self-assembling short-chain thiols on the gold-coated glass slides and allowing the selfassembled monolayer (SAM) to participate in the subsequent plasma polymerization reaction. Details of the procedures involved have been described elsewhere.18,19 For Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements, samples of the plasma polymer films were prepared on 1 mm thick double-sided polished silicon wafers without an additional adhesion layer. The substrates for the WaMS measurements consisted of Ta2O5 film (df ≈ 163 nm, nf ≈ 2.22, Λ ) 745 nm) on a glass substrate. These substrates were cleaned in an Ar/O2 plasma, operated at 100 W for 10 min prior to use. Ta2O5 waveguides were chosen since these previously showed minimal swelling (
