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Functionalized Poly(ethylene glycol)-Grafted Polysiloxane Monolayers for Control of Protein Binding Nan Xia,† Yunhua Hu,§,| David W. Grainger,§ and David G. Castner*,†,‡ National ESCA and Surface Analysis Center for Biomedical Problems, Departments of Chemical Engineering and Bioengineering, University of Washington, Box 351750, Seattle, Washington 98195-1750, and Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 Received September 11, 2001. In Final Form: January 24, 2002 Two new grafted polysiloxane polymers, a copolysiloxane and a terpolysiloxane, have been synthesized. Both polysiloxane backbones are grafted with dialkyl disulfide chains and 600 MW methoxy-terminated poly(ethylene glycol) (PEG) chains, while the terpolysiloxane also has 3400 MW PEG side chains terminated with N-hydroxysuccinimide (NHS) reactive ester groups. The two polymers spontaneously form monolayers on gold surfaces with estimated thicknesses of 23 and 31 Å, respectively. Combined analysis with angledependent X-ray photoelectron spectroscopy and static time-of-flight secondary ion mass spectrometry showed that most grafted dialkyl disulfide side chains were bound to the gold surface, forming multiple molecular linkages at the monolayer/gold interface. The PEG-grafted chains were concentrated in the outermost exposed region of the monolayers. Protein adsorption on the two immobilized polymer monolayers was examined with surface plasmon resonance (SPR). The copolymer-covered surface resisted protein adsorption from buffer, while the terpolymer monolayer bound significant amounts of protein. The NHS headgroup in the terpolymer was the primary site for protein binding. These two polymers have potential applications for SPR biosensor surface modification as protein-resistant and protein-immobilizing surfaces, respectively.
1. Introduction In 1983, Nuzzo and Allara reported the first preparation of organothiol monolayer assemblies on a gold surface.1 Since then, the behavior of various sulfur-containing molecules adsorbing onto gold surfaces has been widely explored.2-8 It is now generally accepted that the Au-S bond formed between the adsorbates and gold surface plays a key role in governing the structure and stability of the monolayers. One hypothesis, derived from the above fact, is that by increasing the number of Au-S bonds per adsorbate molecule, the stability of the organic adlayers can be increased. Robust substrate anchoring of monolayers would benefit many interfacial applications. Consequently, a series of chemisorbing polymers have been synthesized.9-13 These polymers have a flexible polysiloxanebackbone,with grafted side chains containing * Corresponding author: Professor David G. Castner, Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195-1750. Phone: (206)543-8094. Fax: (206)543-3778. E-mail:
[email protected]. † Department of Chemical Engineering, University of Washington. ‡ Department of Bioengineering, University of Washington. § Department of Chemistry, Colorado State University. | Present address: Bone Tissue Engineering Center, Carnegie Mellon University, 5000 Forbes Ave., Pittsburgh, PA 15213-3890. (1) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (2) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365. (3) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (4) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155. (5) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (6) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164. (7) Keller, H.; Schrepp, W.; Fuchs, H. Thin Solid Films 1992, 210/ 211, 799. (8) Li, Y.; Huang, J.; McIver, R. T., Jr.; Hemminger, J. C. J. Am. Chem. Soc. 1992, 114, 2428. (9) Mao, G.; Castner, D. G.; Grainger, D. W. Chem. Mater. 1997, 9, 1741.
different anchoring and exposed functional groups. Thiol or disulfide anchoring side chains present in several polysiloxanes10-13 provide multipoint attachments to gold surfaces. Selection of the exposed side chains depends on the desired functionality for the modified surface: both perfluorinated and alkoxy side chains have been reported.9-13 In the work reported here, the focus was on developing polymeric monolayers for surface plasmon resonance (SPR) biosensors. This requires SPR surfaces (often gold) that resist nonspecific absorption of biomolecules from solution. SPR sensors are rapidly gaining recognition and application as powerful biotechnological tools.14-29 By de(10) Wang, W.; Castner, D. G.; Grainger, D. W. Supramol. Sci. 1997, 4, 83. (11) Sun, F.; Grainger, D. W.; Castner, D. G.; Leach-Scampavia, D. K. Macromolecules 1994, 27, 3053. (12) Sun, F.; Mao, G.; Grainger, D. W.; Castner, D. G. Thin Solid Films 1994, 242, 106. (13) Sun, F.; Castner, D. G.; Mao, G.; Wang, W.; McKeown, P.; Grainger, D. W. J. Am. Chem. Soc. 1996, 118, 1856. (14) Lundstrom, I. Biosens. Bioelectron. 1994, 9, 725. (15) Lukosz, W. Biosens. Bioelectron. 1997, 12, 175. (16) Melendez, J.; Carr, R.; Bartholomew, D.; Taneja, H. Development of a surface plasmon resonance sensor for commercial applications. Sixth International Conference on Chemical Sensors, Washington, DC, 1996. (17) Melendez, J.; Carr, R.; Bartholomew, D. U.; Kukanskis, K.; Elkind, J.; Yee, S.; Furlong, C.; Woodbury, R. Sens. Actuators, B 1996, 35, 212. (18) Kooyman, R. P. H.; Kolkman, H.; Gent, J. V.; Greve, J. Anal. Chim. Acta 1988, 213, 35. (19) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513. (20) O’Shannessy, D. J. Curr. Opin. Biotechnol. 1994, 5, 65. (21) Liedberg, B.; Lundstrom, I.; Stenberg, E. Sens. Actuators, B 1993, 11, 63. (22) Liedberg, B.; Nylander, C.; Lundstro¨m, I. Biosens. Bioelectron. 1995, 10, i. (23) Mrksich, M.; Grunwell, J. R.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 12009. (24) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383.
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tecting changes in the refractive index at the interface of the sensor surface and the analyte, these sensors can monitor in real time the adsorption/desorption process of biomolecules onto the sensor surface from liquid solution. The commonly used SPR sensor surface is composed of a thin gold layer deposited on glass or other optically transparent media. Modification or functionalization of the SPR sensor surface is consistently viewed as a key step to enhance the biocompatibility, biospecificity, and surface sensitivity of the sensor surface. Self-assembled monolayers (SAMs) are one method widely used to modify the surface properties of SPR sensors.30-43 However, one concern with using monomeric SAMs to functionalize SPR surfaces is possible inadequate stability when exposed to buffer with nonphysiological pH, oxidative chemicals, electrochemical environments, or large biomolecules (thermal desorption energy of the Au-S bond has been estimated to be 30 kcal/mol44). The stability of polymeric monolayers with multiple attachments to gold surfaces is significantly improved compared to monomeric SAMs.10,13,61 Therefore, polymeric monolayers are promising candidates for preparing functionalized SPR sensors. The two polymers synthesized for this study, PEG-Sil and NHS-PEG-Sil, are shown in Figure 1. PEG-Sil is the control polymer, which has dialkyl disulfide anchoring side chains and methoxy-terminated poly(ethylene glycol) (PEG) side chains grafted to a polysiloxane backbone. The plural disulfide side chains provide multipoint attachment to the gold surface, while the 600 MW PEG side chain should reduce nonspecific protein adsorption. The other polymer, NHS-PEG-Sil, is analogous to PEG-Sil but has a fraction of longer 3400 MW PEG side chains terminated with the reactive ester N-hydroxysuccinimide (NHS). The ester-NHS linkage readily reacts with amine groups to form amide bonds. Thus, any ligand with nucleophilic NH2 groups, such as proteins with surface exposed lysine amino acids, can be covalently attached to NHS-PEG-Sil monolayer surfaces via formation of amide bonds. (25) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55. (26) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731. (27) Kunz, U.; Katerkamp, A.; Renneberg, R.; Spener, F.; Cammann, K. Sens. Actuators, B 1996, 32, 149. (28) Yeatman, E. M. Biosens. Bioelectron. 1996, 11, 635. (29) BIAcore System Manual; Pharmacia Biosensor AB, Scientific Information: Uppsala, Sweden, 1991. (30) Wink, T.; vanZuilen, S. J.; Bult, A.; vanBennekom, W. P. Analyst 1997, 122, R43. (31) Lo¨fås, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 21, 1526. (32) Lo¨fås, S.; Johnsson, B.; Edstro¨m, Å.; Hansson, A.; Mu¨ller Hillgren, R.-M.; Stigh, L. Biosens. Bioelectron. 1995, 10, 813. (33) Ha¨ussling, L.; Ringsdorf, H.; Schmitt, F.-J.; Knoll, W. Langmuir 1991, 7, 1837. (34) Schmitt, F.-J.; Ha¨ussling, L.; Ringsdorf, H.; Knoll, W. Thin Solid Films 1992, 210/211, 815. (35) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012. (36) Mittler-Neher, S.; Spinke, J.; Liley, M.; Nelles, G.; Weisser, M.; Back, R.; Wenz, G.; Knoll, W. Biosens. Bioelectron. 1995, 10, 903. (37) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361. (38) Van den Heuvel, D. J.; Kooyman, R. P. H.; Drijfhout, J. W.; Welling, G. W. Anal. Biochem. 1993, 215, 223. (39) Kooyman, R. P. H.; Van den Heuvel, D. J.; Drijfhout, J. W.; Welling, G. W. Thin Solid Films 1994, 244, 913. (40) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (41) Duschl, C.; Se´vin-Landais, A.-F.; Vogel, H. Biophys. J. 1996, 70, 1985. (42) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (43) Piscevic, D.; Lawall, R.; Veith, M.; Liley, M.; Okahata, Y.; Knoll, W. Appl. Surf. Sci. 1995, 90, 425. (44) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437.
Xia et al.
Figure 1. The chemical structures of PEG-Sil and NHSPEG-Sil.
Adlayers formed by adsorbing the PEG-Sil and NHSPEG-Sil polymers onto gold surfaces were characterized by angle-dependent X-ray photoelectron spectroscopy (XPS), static time-of-flight secondary ion mass spectrometry (ToF-SIMS), and SPR. 2. Materials and Methods 2.1. Materials. The copolysiloxane and terpolysiloxane were synthesized (see section 3). Reagents for the synthesis, including 5-bromo-1-penten (Lancaster), thiolacetic acid (Aldrich), lithium tetrahydroaluminum (Aldrich), trifluoromethanesulfonic anhydride (Aldrich), ethanethiol (Aldrich), allyl bromide (Aldrich), R-allyl-ω-methyl poly(ethylene glycol) (ZALP 0600, MW 600, a gift from Goldschmidt Chemical, Essen, Germany), R-hydroxyω-propionic acid-poly(ethylene glycol) (MW 3400, Shearwater Polymers, Inc.), poly(hydrogen methylsiloxane) (PHMS, MW 4500, Hu¨ls), platinum 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex (3% solution in toluene, Hu¨ls), N-hydroxysuccinimide (Aldrich), N,N′-dicyclohexylcarbodiimide (DCC, Sigma), 4-(dimethylamino)pyridine (DMAP, Sigma), toluene (HPLC grade, Fisher Scientific), ethanol (reagent grade, VWR), and ethanolamine (Aldrich), were used as received. Bovine albumin, bovine fibrinogen, and bovine IgG were purchased from Sigma Chemical Co. All water used was purified by treating in a reverse osmosis unit followed by a Millipore unit (18 mΩ resistivity). 2.2. Polymer Monolayer Film Assembly. Silicon wafers were cut into 1.0 × 1.0 cm pieces and cleaned with piranha solution (7:3 (v/v) H2SO4 and H2O2). (Caution: piranha solution reacts violently with organic solvents and should be handled with extreme care.) Cleaned wafers were first coated by electron beam evaporation with about 100 Å of Ti as an adhesive underlayer and then with about 1000 Å of gold at pressures below 1 × 10-6 Torr. For SPR measurements, clean glass microscope slides were first coated with 20 Å of Ti and then 500 Å of Au using the same evaporation system. For monolayer assembly, gold substrates were placed in a 1 mM solution of PEG-Sil or a saturated (100 Da) ejected from the surface are electrically unstable and tend to be neutralized or fragmented during the desorption process.52 When the polymer monolayers were dosed with sodium ions, a dramatic enhancement in the intensity of large mass fragment peaks was observed in the positive spectra of both polymer monolayers. This cationization phenomenon has been detected on various PEG-modified surfaces, and some systematic studies on it have been published.52-54 Consistent with previous studies9 of ultrathin polysiloxane polymer films, the following characteristic fragments from the polysiloxane backbone were observed in (52) Michel, R.; Luginbuhl, R.; Graham, D. J.; Ratner, B. D. Langmuir 2000, 16, 6503. (53) Gusev, A. I.; Choi, B. K.; Hercules, D. M. J. Mass Spectrum. 1998, 33, 480-485. (54) Grade, H.; Winograd, N.; Cooks, R. G. J. Am. Chem. Soc. 1977, 99, 7725-7726.
Figure 4. Low mass range (0-100 m/z) positive ToF-SIMS spectra for (a) PEG-Sil and (b) NHS-PEG-Sil.
Figure 5. Low mass range (200-475 m/z) negative ToF-SIMS spectrum for PEG-Sil.
the positive and negative spectra: CH3SiO+ (m/z ) 58), SiC3H9+ (m/z ) 73), SiOHC2H6+ (m/z ) 75), CH3Si- (m/z ) 43), CH3SiO- (m/z ) 59), CH3SiO2- (m/z ) 75). Evidence for the chemical bonding between polymer dialkyl disulfide chains and the gold substrate appears in the negative spectra (Figure 5). Peaks due to the ions with a universal formula of AunSm- are the most prominent ion clusters in the high mass range. They are always present in the negative SIMS spectra for thiol SAMs on gold.52,55,56 Peaks corresponding to small fragments from gold-dialkyl disulfide chain complexes are also observed, such as AuSC2H5-, AuS2C2H5-, and Au(SC2H5)2-. The
Functionalized PEG-Grafted Polysiloxane Monolayers
negative spectra for short-chain thiol SAMs exhibit analogous significant peaks as gold-molecular fragments, such as Au(M - H)- and AuS(M - H)-, where M represents the adsorbate molecule.52 In our case, however, since the dialkyl disulfide chain in PEG-Sil is grafted to the long, flexible polysiloxane main chain, peaks due to fragments of the dialkyl disulfide chain are observed instead of the intact molecular ion. Since PEG-Sil and NHS-PEG-Sil have an analogous chemical structure, it is anticipated that the NHS-PEGSil spectra would exhibit many features observed in the PEG-Sil spectra. However, several major differences were detected. First, a lack of significant peaks from Au-S fragments was noticed in the negative spectra for NHSPEG-Sil monolayers. This is probably due to the surface sensitivity of ToF-SIMS (less than 20 Å) and the thickness difference between the two monolayers, which was confirmed by both the XPS and the ellipsometry measurements. Second, the positive NHS-PEG-Sil spectra (Figure 4b) exhibit a large enhancement of the NH4+ fragment peak (m/z ) 18) and several other nitrogencontaining peaks in the low mass range. This is due to the presence of NHS groups in the NHS-PEG-Sil polymer. A difference was also observed in the PEG fingerprint peaks. C2H5O+ and C3H7O+ ions are the two strongest peaks in the PEG-Sil spectra; however, in the NHSPEG-Sil spectra, C3H7O+ exhibits a dramatic decrease in intensity, while the C2H5O+ peak remains unchanged. Molecular fragmentation in ToF-SIMS is sensitive to both polymer chain molecular weight and end groups.57,58 Because the C3H7O+ fragment originates primarily from the terminus of methoxy-PEG chains, both increasing the PEG chain length and substituting NHS for some of the methoxy terminal groups reduce the fragmentation yield of C3H7O+. The C2H5O+ ion fragment primarily originates from the middle of the PEG chain, so the end group and PEG chain length will have less effect on its yield compared to the C3H7O+ ion yield. Finally, the most important difference between the two monolayer spectra is shown in Figure 6. Two peaks observed in the NHS-PEG-Sil spectra, C4H4NO2+ (m/z ) 98, Figure 6a) and C4H4NO3- (m/z ) 114, Figure 6b), were not present in the PEG-Sil spectra. The two fragments are attributed to (succinimide - H)+ and (hydroxysuccinimide - H)- species, respectively, both which originate from the NHS group in NHS-PEG-Sil. The chemical bonds connecting each of these two fragments with the PEG chain (the carboximide bond for succinimide and the ester bond for N-hydroxysuccinimide) are the two most reactive sites in the NHS-PEG structure. Moreover, succinimide is an electrophilic group, while N-hydroxysuccinimide is more nucleophilic. As a result, the two fragments are detected as positive and negative ions, respectively. XPS results in the previous section have revealed the presence of nitrogen in the NHS-PEG-Sil monolayer. ToF-SIMS has further shown that this nitrogen is due to the NHS group, which remains in the NHS-PEG-Sil monolayer after chemisorbing from solution onto the gold surface. 4.4. Surface Plasmon Resonance Analyses. The first SPR experiment demonstrated the ability of the PEGSil monolayer to resist nonspecific protein adsorption. For (55) Offord, D. A.; John, C. M.; Linford, M. R.; Griffin, J. H. Langmuir 1994, 10, 883. (56) Offord, D. A.; John, C. M.; Griffin, J. H. Langmuir 1994, 10, 761. (57) Shard, A. G.; Davies, M. C.; Schacht, E. Surf. Interface Anal. 1996, 24, 787. (58) Vanden Eynde, X.; Reihs, K.; Bertrand, P. Macromolecules 1999, 32, 2925.
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Figure 6. Comparison of the (a) positive and (b) negative ToFSIMS spectra for PEG-Sil and NHS-PEG-Sil, showing fragment peaks from the NHS group in the latter monolayers. The spectra have been offset from zero for clarity.
comparison, adsorption from a 1 mg/mL solution of bovine serum albumin (BSA) onto a bare gold surface is shown in Figure 7a. Curves b and c in Figure 7 are the SPR sensorgrams obtained by successively introducing proteins BSA, fibrinogen, and IgG (1 mg/mL each in PBS) over the PEG-Sil monolayer surface. Each protein injection was followed by an 8-10 min incubation, and then the monolayer surface was rinsed with pure PBS buffer. In curves b and c, the SPR wavelength shift observed after each protein injection was roughly the same (∼0.6 nm). This corresponds to a local refractive index (RI) increase of 1.8 × 10-4 RIU, close to the RI increase of the bulk solution for an addition of 1 mg of protein per mL of buffer.59 Moreover, after each injection of the rinse buffer, the wavelength in the sensorgram returns to the baseline of the monolayer in pure PBS buffer prior to exposure to the protein solution. (Note: The upward drift of the baseline in curve b is a commonly observed problem with singlechannel SPR biosensing systems. It is generally believed that this drift is caused by the instability of the experimental conditions, for example, temperature. A dualchannel system, with the addition of a reference channel, can avoid this problem.) Therefore, we conclude that the SPR wavelength differences caused by injecting protein solutions over the PEG-Sil monolayer surface are primarily due to the bulk RI changes between protein solutions and pure buffer. Amounts of protein adsorbed onto the PEG-Sil monolayer are consistently below the detection limit of the SPR instrument (
