Room Temperature, Aqueous Post-Polymerization Modification of

Nov 9, 2010 - Room Temperature, Aqueous Post-Polymerization Modification of Glycidyl Methacrylate-Containing Polymer Brushes Prepared via Surface-Init...
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Room Temperature, Aqueous Post-Polymerization Modification of Glycidyl Methacrylate-Containing Polymer Brushes Prepared via Surface-Initiated Atom Transfer Radical Polymerization Raphael Barbey and Harm-Anton Klok*  Ecole Polytechnique F ed erale de Lausanne (EPFL), Institut des Mat eriaux and Institut des Sciences et Ing enierie Chimiques, Laboratoire des Polym eres, B^ atiment MXD, Station 12, CH-1015 Lausanne, Switzerland Received June 12, 2010. Revised Manuscript Received September 12, 2010 This manuscript reports on the post-polymerization modification of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMAx-co-PDEAEMAy) (co)polymer brushes prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). The aim of this study was to evaluate the ability of tertiary amine groups incorporated in the polymer brush to accelerate the ring-opening of the epoxide groups by primary amines and to facilitate the aqueous, room temperature post-polymerization modification of the brushes. Using Fourier transform infrared (FTIR) spectroscopy to monitor the ring-opening reaction of the epoxide groups, it was found that the incorporation of 2-(diethylamino)ethyl methacrylate (DEAEMA) groups in the PGMA brushes significantly accelerated the rate of the post-polymerization modification reaction with several model amines. The rate enhancement was dependent on the fraction of DEAEMA units incorporated in the copolymer brush. For example, whereas 24 h was necessary to obtain a conversion of approximately 40% for PGMA brushes immersed in a 1 M propylamine solution in water, the same conversion was reached, in identical reaction conditions, after 8 and 2 h with copolymer brushes containing 10 mol % and 25 mol % of DEAEMA along the copolymer chains, respectively. In a final series of proof-ofconcept experiments, the feasibility of the glycidyl methacrylate containing brushes to act as substrates for protein immobilization was studied. Using FTIR spectroscopy and quartz crystal microbalance with dissipation (QCM-D) experiments, it could be demonstrated that the incorporation of DEAEMA units not only enhanced the rate of the protein immobilization reaction, but also resulted in higher protein binding capacities as compared to a PGMA homopolymer brush. These features make PGMAx-co-PDEAEMAy brushes very attractive candidates for the development of protein microarrays, among others.

Introduction Polymer brushes, which are defined as ultrathin polymer coatings consisting of polymer chains that are tethered with one chain end to a substrate, have been extensively studied over the past two decades and have attracted interest for a wide variety of (bio)applications.1 Among other methods reported for the preparation of polymer brushes, surface-initiated controlled radical polymerization techniques, such as surface-initiated atom transfer radical polymerization (SI-ATRP), surface-initiated nitroxidemediated polymerization (SI-NMP), or surface-initiated reversible addition fragmentation chain transfer (SI-RAFT) polymerization, have been proven particularly attractive, as they allow precise control over brush thickness, composition, and architecture.1-4 These radical-based synthetic strategies are relatively tolerant to a wide range of functional groups and therefore offer the possibility to directly prepare polymer brushes bearing reactive pendant groups, such as hydroxyl, carboxylic acid, or epoxide groups, without the need for protective group chemistry. These different pendant groups can be further utilized as handles for specific post*E-mail: [email protected]. Fax: þ41 21 693 5650. Tel: þ41 21 693 4866. (1) Barbey, R.; Lavanant, L.; Paripovic, D.; Sch€uwer, N.; Sugnaux, C.; Tugulu, S.; Klok, H.-A. Chem. Rev. 2009, 109, 5437. (2) Pyun, J.; Matyjaszewski, K. Chem. Mater. 2001, 13, 3436. (3) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33, 14. (4) Tsujii, Y.; Ohno, K.; Yamamoto, S.; Goto, A.; Fukuda, T. Adv. Polym. Sci. 2006, 197, 1. (5) Brantley, E. L.; Holmes, T. C.; Jennings, G. K. J. Phys. Chem. B 2004, 108, 16077.

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polymerization modification reactions. Hydroxyl,5-12 carboxylic acid,13-16 and epoxide17-23 pendant groups, for example, can either be transformed into functional moieties that cannot be introduced via direct surface-initiated polymerization of the corresponding monomer or used to immobilize (bio)molecules.1 (6) Sun, L.; Baker, G. L.; Bruening, M. L. Macromolecules 2005, 38, 2307. (7) Xu, D.; Yu, W. H.; Kang, E. T.; Neoh, K. G. J. Colloid Interface Sci. 2004, 279, 78. (8) Zhai, G. Q.; Cao, Y.; Gao, J. J. Appl. Polym. Sci. 2006, 102, 2590. (9) Xu, F. J.; Li, Y. L.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2005, 6, 1759. (10) Tugulu, S.; Silacci, P.; Stergiopulos, N.; Klok, H.-A. Biomaterials 2007, 28, 2536. (11) Tugulu, S.; Arnold, A.; Sielaff, I.; Johnsson, K.; Klok, H.-A. Biomacromolecules 2005, 6, 1602. (12) Jain, P.; Sun, L.; Dai, J. H.; Baker, G. L.; Bruening, M. L. Biomacromolecules 2007, 8, 3102. (13) Dai, J.; Bao, Z.; Sun, L.; Hong, S. U.; Baker, G. L.; Bruening, M. L. Langmuir 2006, 22, 4274. (14) Harris, B. P.; Kutty, J. K.; Fritz, E. W.; Webb, C. K.; Burg, K. J. L.; Metters, A. T. Langmuir 2006, 22, 4467. (15) Navarro, M.; Benetti, E. M.; Zapotoczny, S.; Planell, J. A.; Vancso, G. J. Langmuir 2008, 24, 10996. (16) Jain, P.; Dai, J. H.; Baker, G. L.; Bruening, M. L. Macromolecules 2008, 41, 8413. (17) Edmondson, S.; Huck, W. T. S. J. Mater. Chem. 2004, 14, 730. (18) Edmondson, S.; Huck, W. T. S. Adv. Mater. 2004, 16, 1327. (19) Yu, W. H.; Kang, E. T.; Neoh, K. G. Langmuir 2005, 21, 450. (20) Xu, F. J.; Zhong, S. P.; Yung, L. Y. L.; Tong, Y. W.; Kang, E.-T.; Neoh, K. G. Biomaterials 2006, 27, 1236. (21) Xu, F. J.; Cai, Q. J.; Li, Y. L.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2005, 6, 1012. (22) Iwasaki, Y.; Omichi, Y.; Iwata, R. Langmuir 2008, 24, 8427. (23) Huang, J. S.; Li, X. T.; Zheng, Y. H.; Zhang, Y.; Zhao, R. Y.; Gao, X. C.; Yan, H. S. Macromol. Biosci. 2008, 8, 508.

Published on Web 11/09/2010

DOI: 10.1021/la102400z

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Poly(glycidyl methacrylate) (PGMA) represents a very attractive platform for the introduction of complex functional groups and/or the immobilization of biomolecules via post-polymerization modification. Post-polymerization modification of PGMA brushes involves nucleophilic ring-opening of the oxirane side chain functional groups and has been performed both in organic solvents and in aqueous media. In organic media and/or at elevated temperatures, this reaction has been shown to be a relatively fast and efficient process. For instance, cross-linked polymer brushes have been prepared either by reacting linear PGMA brushes with 4 M ethylenediamine in DMF for 10 h at room temperature,24 0.5 M ethanolic 1,4-phenylenediamine,25 or octylamine17 solutions at 60 °C or by treatment with 2 M methanolic NaOH solutions at 60 °C for 25 min.18,26 Kang and co-workers used PGMA brushes as a platform for the construction of architecturally more complex brushes. Their strategy involved the introduction of ATRP initiating groups by reaction of the pendant epoxide moieties of the brush with halogenated propionic acid derivatives in organic media at elevated temperature (60-70 °C) followed by subsequent SI-ATRP, allowing, as a result, the preparation of comb-shaped polymer brushes.19,20 In contrast to the examples discussed in the previous paragraph, biomolecules such as proteins and nucleotides are typically immobilized onto PGMA brushes using aqueous-based protocols. Xu et al., for example, exposed 50-nm-thick PGMA brushes prepared via SI-ATRP for a period of 5 h to a 4 mg 3 mL-1 solution of glucose oxidase (GOD) in phosphate buffered saline (PBS) at room temperature.21 Another report describes the overnight coupling of amino-modified fluorescent oligonucleotides, in PBS at room temperature, on poly(N,N-dimethylacrylamide)-bPGMA block copolymer brush-coated microscope glass slides, which were prepared via SI-RAFT polymerization.27 The authors showed, by fluorescence experiments, the higher binding capacity of the block copolymer brushes compared to a self-assembled monolayer (SAM) made of (3-glycidyloxypropyl)trimethoxysilane. SI-ATRP has also been used to coat magnetic polymer microspheres with epoxide-containing polymer brushes.23,28 Glycidyl methacrylate (GMA) and 2,3-dihydroxypropyl methacrylate were copolymerized to obtain water-dispersible microspheres, which were subsequently used to immobilize bovine serum albumin (BSA) and penicillin G acylase from potassium phosphate buffer at room temperature for 12 and 24 h, respectively. Compared to their post-polymerization modification in organic media, generally relatively long reaction times (12-24 h) are used to immobilize biomolecules on PGMA brushes from aqueous media. Although, to the best of our knowledge, no studies have been reported that describe the kinetics of the PGMA modification in aqueous media, this seems to suggest that this reaction is relatively slow, which of course limits the attractiveness and feasibility of PGMA as a platform for biomolecule immobilization. In a number of reports, however, the ability of tertiary amines to act as catalysts for the nucleophilic ring-opening of epoxide groups has been documented.29-31 The objective of this manuscript is to elaborate synthetic strategies for the preparation (24) Yu, W. H.; Kang, E. T.; Neoh, K. G. Langmuir 2004, 20, 8294. (25) Comrie, J. E.; Huck, W. T. S. Langmuir 2007, 23, 1569. (26) Edmondson, S.; Frieda, K.; Comrie, J. E.; Onck, P. R.; Huck, W. T. S. Adv. Mater. 2006, 18, 724. (27) Pirri, G.; Chiari, M.; Damin, F.; Meo, A. Anal. Chem. 2006, 78, 3118. (28) Huang, J.; Han, B.; Yue, W.; Yan, H. J. Mater. Chem. 2007, 17, 3812. (29) Wu, J.; Xia, H.-G. Green Chem. 2005, 7, 708. (30) Li, M.-S.; Ma, C.-C. M.; Chen, J.-L.; Lin, M.-L.; Chang, F.-C. Macromolecules 1996, 29, 499. (31) Jiang, P.; Shi, Y.; Liu, P.; Cai, Y. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 2947.

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of tertiary amine-containing PGMA brushes and to evaluate the feasibility of these copolymer brushes as platforms for aqueous, room temperature post-polymerization modification, and protein immobilization. To this end, a series of (co)polymer brushes was prepared via surface-initiated atom transfer radical (co)polymerization of GMA and 2-(diethylamino)ethyl methacrylate (DEAEMA), which was selected as the tertiary amine containing comonomer. Using a number of model primary amines, the kinetics of the post-polymerization modification of the PGMAco-poly(2-(diethylamino)ethyl methacrylate) (PGMA x -coPDEAEMAy) brushes were investigated and compared with PGMA. Finally, first proof-of-concept experiments have been performed to investigate the feasibility of these PGMAx-coPDEAEMAy brushes to act as a platform for the immobilization of proteins. The model kinetic studies and first proof-of-concept experiments indicate that the incorporation of the tertiary amine containing comonomer both increases the rate of the post-polymerization modification reaction and enhances protein immobilization.

Experimental Section Materials. All chemicals were obtained from commercial suppliers and used as received unless stated otherwise. Ultrahigh-quality water with a resistance of 18.2 MΩ 3 cm (at 25 °C) was obtained from a Millipore Milli-Q gradient machine fitted with a 0.22 μm filter. Phosphate buffered saline (PBS) tablets were purchased from Sigma for the preparation of PBS (pH = 7.4), whereas borate buffer (pH = 8.2) was prepared from boric acid (>99.5%, Fluka) and sodium tetraborate decahydrate (>99.5%, SigmaAldrich). The inhibitor in glycidyl methacrylate (4-methoxyphenol) was removed by passing the monomer through a column of activated, basic aluminum oxide, whereas 2-(diethylamino)ethyl methacrylate was freed from its inhibitor (phenothiazine) via distillation under reduced pressure. Surface-initiated atom transfer radical polymerization (SI-ATRP) was performed on silicon wafers cut in pieces of 2 cm  0.8 cm or on silicon oxide-covered quartz crystal microbalance (QCM) sensors. Analytical Methods. Brush thicknesses were determined by means of a Philips Plasmos SD 2300 ellipsometer working with a He-Ne laser (λ = 632.8 nm) at an angle of incidence of 70°. The calculation method was based on a three-layer silicon/polymer brush/ambient model, assuming the polymer brush to be isotropic and homogeneous. A fixed refractive index value of 1.45 was used for the polymer layer. All reported ellipsometric film thicknesses represent an average over 5 data points taken from the same substrate and are corrected for the approximately 2-nm-thick native oxide layer on the silicon substrates. XPS data were recorded on an Axis Ultra instrument from Kratos Analytical. These measurements were carried out with a conventional hemispheric analyzer. The X-ray source employed was a monochromatic Al KR (1486.6 eV) source operated at 150 W and 10-9 mbar. The analysis area was 700  350 μm, and the experiments performed at an angle of 90° relative to the substrate surface (takeoff angle). The pass energies were 80 and 20 eV for survey scans and high-resolution elemental scans, respectively. The operating software used was Vision 2, which corrects for the transmission function. Charge compensation was performed with a self-compensating device (Kratos patent) using field-emitted low-energy electrons (0.1 eV). All XPS spectra were calibrated on the aliphatic carbon signal at 285.0 eV. Relative sensitivity factors (RSF) of 0.278 (C1s) and 0.477 (N1s) were used to correct area ratios that are necessary for the calculation of Table 1.32 Fourier transform reflectance infrared spectra were acquired using a nitrogen-purged Nicolet Magna-IR 560 spectrometer equipped with a Micro Specular Reflectance accessory (Specac) and processed by means of the software (32) Wagner, C. D. J. Electron Spectrosc. Relat. Phenom. 1983, 32, 99. The RSFs used in this study (and that are provided by the software CasaXPS) are based on those by Wagner but modified to account for the geometry of the Kratos Axis Ultra system.

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Table 1. Dry Ellipsometric Thicknesses and Composition of PGMA, PDEAEMA, and PGMAx-co-PDEAEMAy (Co)polymer Brushes feed composition [%] dry ellipsometric thicknessa [-]

GMA

DEAEMA

surface compositionb [%] GMA

DEAEMA

PGMA 130 100 0 100 0 227 90 10 90.9 9.1 PGMA90-co-PDEAEMA10c 190 75 25 77.2 22.8 PGMA75-co-PDEAEMA25c 230 50 50 53.9 46.1 PGMA50-co-PDEAEMA50c PDEAEMA 50 0 100 0 100 a After a polymerization time of 6 h with an error estimated to (10%. b Values determined based on the XPS [N1s]/[C1s] ratio. c These PGMAx-coPDEAEMAy copolymer brushes were prepared in a different batch than the one used for Figure 1.

OMNIC ESP 5.1. Water contact angles were determined using a DataPhysics OCA 35 contact angle measurement system. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE-400 Ultra Shield spectrometer. Chemical shift values (δ) are reported in parts per million (ppm). The residual proton signal or the carbon signal of the NMR solvent was used as internal standard (CDCl3: δH =7.25 ppm, δC =77.0 ppm). pH values of reaction solutions were measured using a precalibrated pH electrode from Mettler Toledo. The mass of bound proteins was determined with a quartz crystal microbalance with dissipation (QCM-D, Q-Sense E4) using SiO2-coated quartz crystals (AT-cut, 5 MHz) purchased from ICM (International Crystal Manufacturing, Inc.) and recording the third, fifth, and seventh harmonic of the resonance frequency.

Synthesis of Hex-5-enyl 2-bromo-2-methylpropanoate. This protocol is based on a procedure reported earlier by Husseman et al.33 Briefly, 2-bromo-2-methylpropionyl bromide (27.6 g, 15 mL, 120 mmol, 1.2 equiv) was added dropwise under nitrogen to a stirred solution of 5-hexen-1-ol (10.0 g, 10 mL, 100 mmol, 1 equiv) and triethylamine (12.1 g, 17 mL, 120 mmol, 1.2 equiv) in dichloromethane (70 mL). After stirring at 0 °C under nitrogen for 1 h, the reaction mixture was allowed to warm to room temperature where it was stirred overnight. The precipitated triethylamine hydrochloride was removed by filtration, and the solution was washed with saturated aqueous ammonium chloride (4  150 mL) and water (4  150 mL). The organic phase was then dried over MgSO4 and filtered. The dichloromethane was then removed, and the crude product was purified by vacuum distillation (∼10-1 mbar, Tbath = 80 °C) to give the product as a colorless oil (yield: 21.31 g, 85.6%). 1H NMR (400 MHz, CDCl3) δ = 5.80-5.70 (m, 1H, CHdCH2), 5.00-4.91 (m, 2H, CHdCH2), 4.15-4.12 (t, 2H, CH2-O), 2.08-2.02 (m, 2H, CHdCH2-CH2), 1.88 (s, 6H, (CH3)2), 1.69-1.62 (m, 2H, CH2-CH2), 1.49-1.42 (m, 2H, CH2-CH2) ppm. 13C NMR (100 MHz, CDCl3) δ = 171.69, 139.14, 114.11, 66.13, 55.96, 33.76, 30.77, 28.32, 25.75 ppm.

Synthesis of 6-(Chloro(dimethyl)silyl)hexyl 2-bromo-2methylpropanoate. The following synthesis represents a slight

modification of the protocol published by R€ uhe and co-workers.34 Hex-5-enyl 2-bromo-2-methylpropanoate (12.1 g, 10 mL, 49 mmol, 1 equiv) was mixed with 100 mL (85.2 g, 900 mmol, 18.5 equiv) of dimethylchlorosilane under nitrogen. 100 mg of Pt/C (10% Pt) was added and the mixture was refluxed for 18 h. The excess chlorosilane was removed under reduced pressure and was trapped in liquid nitrogen. The oily product was then quickly filtered over anhydrous sodium sulfate (Na2SO4) through a glass frit (pore size 4, with two filter papers 0.2 μm) to remove the residual catalyst. To avoid any product loss, the Na2SO4 cake was rinsed several times with dichloromethane (6  2 mL). The filtrate was then placed under reduced pressure to remove dichloromethane. Finally, the crude product was purified by vacuum distillation (∼10-1 mbar, Tbath = 150 °C) to give the desired product as a colorless oil (yield: 15.1 g, 90.4%). 1H NMR (400 MHz, CDCl3) δ = 4.16-4.11 (t, 2H, CH2-O), 1.91 (s, 6H, C-(CH3)2), 1.69-1.60 (m, 2H, (33) Husseman, M.; Malmstr€om, E. E.; McNamara, M.; Mate, M.; Mecerreyes, D.; Benoit, D. G.; Hedrick, J. L.; Mansky, P.; Huang, E.; Russell, T. P.; Hawker, C. J. Macromolecules 1999, 32, 1424. (34) Ramakrishnan, A.; Dhamodharan, R.; R€uhe, J. Macromol. Rapid Commun. 2002, 23, 612.

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CH2-CH2-O), 1.44-1.27 (m, 6H, (CH2)3), 0.84-0.74 (m, 2H, Si-CH2), 0.39-0.35 (m, 6H, Si-(CH3)2) ppm. 13C NMR (100 MHz, CDCl3) δ = 171.63, 65.97, 55.91, 32.36, 30.73, 28.16, 25.35, 22.80, 18.81, 1.61 ppm. ATRP Initiator Functionalized Substrates. The silicon wafers were sonicated in acetone, ethanol, and Milli-Q water for 5 min each, and the QCM sensors were stirred for 10 min in acetone, then both substrates were rinsed with ethanol and dried under vacuum. Subsequently, the QCM sensors were placed in an oxygen plasma generator (200 W) for 15 min, whereas the silicon wafers were immersed in piranha solution (H2SO4/H2O2 = 7:3, v/v) for 1 h at 140 °C (Caution! This mixture reacts violently with organic materials.) The silicon slides were extensively rinsed with deionized water and ethanol, then dried. Next, the substrates were exposed to a 2 mM solution of initiator in toluene for 2 h at room temperature. After that, the substrates were removed from the reaction mixture and rinsed extensively with toluene and acetone, sonicated 15 s in acetone (silicon wafer only), rinsed with ethanol, sonicated 15 s in ethanol (silicon wafer only), and then rinsed with Milli-Q water and ethanol. Finally, the initiator-functionalized substrates were dried under vacuum and stored until needed for a polymerization.

Preparation of Poly(glycidyl methacrylate) (PGMA) and Poly(glycidyl methacrylate)-co-poly(2-(diethylamino)ethyl methacrylate) (PGMAx-co-PDEAEMAy) (Co)polymer Brushes.

This procedure is based on a paper by Edmondson et al.17 Monomers, glycidyl methacrylate (GMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA), were mixed in predefined molar ratios (i.e., 100:0, 99:1, 90:10, 75:25, 50:50, 0:100; GMA/DEAEMA) and then added to a methanol/water solution (monomer(s)/methanol/ water = 5:4:1; v/v/v) under stirring. After two freeze-pump-thaw cycles, 2,20 -bipyridyl (bpy), CuICl, and CuIIBr2 were added in quantities so as to obtain a molar ratio of monomer(s)/CuICl/ CuIIBr2/bpy of 2000/20/1/50. After that, the mixture was subjected to two additional freeze-pump-thaw cycles. Once at room temperature, the reaction mixture was transferred via canula to nitrogen-purged reaction vessels containing the initiator-functionalized substrates. After a defined time, the substrates were removed from the ATRP solution, extensively rinsed with methanol and then successively washed in methanol (1 h), dichloromethane, and acetone (30 min each). Finally, the (co)polymer brush-coated substrates were rinsed with ethanol, dried under vacuum, and characterized by ellipsometry as well as by FTIR spectroscopy and XPS. Throughout the text, copolymer brushes are referred to as PGMAx-co-PDEAEMAy, where the subscripts x and y refer to the mole percentage of the monomer in the polymerization solution.

Post-Polymerization Modification of the (Co)polymer Brushes. After recording an FTIR spectrum of the pristine sample, the (co)polymer brushes were incubated in aqueous solutions, which contained either a model primary amine or a protein. The amines used in the present study were propylamine (1 M), amino-2propanol (1 M), taurine (0.5 M), and (2-aminoethyl)trimethylammonium chloride hydrochloride (0.5 M), and the proteins used were BSA (100 mg 3 mL-1) and lysozyme (100 mg 3 mL-1). The post-polymerization modification reactions with the different model primary amines were carried out in water, whereas the reactions DOI: 10.1021/la102400z

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Barbey and Klok Scheme 1. Preparation of PGMAx-co-PDEAEMAy Brushes via SI-ATRP

with BSA and lysozyme were carried out in PBS. After defined times, the substrates were taken out from the reaction solution, extensively rinsed with Milli-Q water, and dried under a stream of compressed air. After acquisition of the reflectance spectra, the substrates were placed back into the reaction solution until another predefined time was reached. To monitor the kinetics of the postpolymerization modification reactions, this process was repeated several times over a 48 h period. Then, the dry ellipsometric thickness of the post-modified (co)polymer brushes was determined by ellipsometry. Long-Term Stability of the (Co)polymer Brushes. The longterm (up to one month) stability of the prepared (co)polymer brushes toward hydrolysis in air, water, PBS, 0.01 M NaOH, and 2-(diethylamino)ethyl methacrylate, has also been studied by FTIR spectroscopy following the procedure reported in the previous paragraph. Quantification of Immobilized Proteins. The amount of immobilized proteins, BSA and lysozyme, has been determined using QCM-D by measuring the shift in resonance frequency of the (co)polymer-coated QCM-D sensors after incubation in protein-containing solutions for 6, 24, and 48 h. Briefly, the (co)polymer-coated sensors were measured in their dry state, then taken out and incubated in 100 mg 3 mL-1 protein (BSA or lysozyme) solutions in PBS. After predefined times, the substrates were extensively rinsed with water and ethanol and finally dried under vacuum. Then, the resonance frequency of the sensors was measured, and the amount of immobilized protein determined by means of the Sauerbrey relation assuming the film to be thin and rigid in its dry state.35 Δmass ¼ - C 3

fn, t - fn, 0 Δfn ¼ -C3 n n

ð1Þ

where Δmass represents the area averaged mass (ng 3 cm-2), C is a constant equal to 17.7 ng 3 cm-2 3 Hz-1 (mass sensitivity for ATcut, 5 MHz quartz crystals), n (= 1, 3, 5, 7, etc.) is the overtone number, fn,0 and fn,t are the resonance frequencies before incubation and after exposure to the protein solution for a predefined time, respectively.

Results and Discussion Brush Synthesis and Characterization. As depicted in Scheme 1, PGMA and PGMAx-co-PDEAEMAy brushes were grown at room temperature from silicon substrates via SI-ATRP in a methanol/water solvent mixture using CuICl as activator, CuIIBr2 as deactivator, and 2,20 -bipyridyl (bpy) as ligand. The first step for the preparation of the (co)polymer brushes consisted of the modification of the silicon substrates with the ATRP initiator (6-(chloro(dimethyl)silyl)hexyl 2-bromo-2-methylpropanoate). The deposition of the ATRP initiator was accomplished by (35) Sauerbrey, G. Z. Phys. 1959, 155, 206.

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Figure 1. Evolution of the dry ellipsometric thickness of PGMA (9), PGMA99-co-PDEAEMA1 ((), PGMA90-co-PDEAEMA10 (2), PGMA75-co-PDEAEMA25 (O), PGMA50-co-PDEAEMA50 (b), and PDEAEMA (0) brushes as a function of polymerization time.

placing cleaned, activated silicon substrates in a 2 mM solution of the initiator in toluene for 2 h under gentle stirring at room temperature. The successful grafting of the ATRP initiator was evidenced by ellipsometry and water contact angle measurements, which revealed a homogeneous film thickness of ∼0.8 nm as well as an increase in the advancing water contact angle to approximately 75°, suggesting that the ATRP initiator functionalized monochlorosilane used in this study forms well-defined selfassembled monolayers (SAMs) on silicon substrates. Although less reactive than their trifunctional analogues, monofunctional organosilanes offer the advantage that they only contain one hydrolyzable group that can react, which prevents the formation of multilayer films.36 It is worth mentioning that, compared to other published procedures,17,34,37 the reaction time for the immobilization of ATRP initiator was decreased to 2 h and the reaction was not carried out under any special conditions, such as the use of dried solvents, the exclusion of light, or the addition of triethylamine as a weak base. The 2-bromoisobutyrate-functionalized surfaces were then used for the preparation of PGMAx-co-PDEAEMAy (co)polymer brushes via SI-ATRP in methanol/water solvent mixtures at room temperature. For all experiments described below, the molar ratios of monomer(s)/CuICl/CuIIBr2/bpy were kept constant as 2000/20/ 1/50. Figure 1 shows the evolution of the dry ellipsometric film (36) Fadeev, A. Y.; McCarthy, T. J. Langmuir 2000, 16, 7268. (37) Riachi, C.; Sch€uwer, N.; Klok, H.-A. Macromolecules 2009, 42, 8076.

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Figure 2. XPS survey and high-resolution C1s and O1s spectra of (A) 80-nm-thick PGMA, (B) 230-nm-thick PGMA50-co-PDEAEMA50, and (C) 25-nm-thick PDEAEMA brushes.

thickness as a function of polymerization time for reaction mixtures containing different amounts of the two comonomers. The dry thickness of both homopolymer brushes increased linearly with polymerization time. After 6 h, approximately 130- and 50-nmthick PGMA and PDEAEMA brushes were obtained. Analysis of the surface-initiated GMA/DEAEMA copolymerizations revealed markedly different kinetics. Whereas the copolymerization of a monomer feed containing 1 mol % DEAEMA was still relatively similar to the GMA and DEAEMA homopolymerizations, copolymerizations carried out with higher DEAEMA content in the feed were extremely fast during the first 30-60 min and then leveled off, indicative of the loss of the “living” character of the copolymerization. Moreover, compared to the PGMA and PDEAEMA homopolymer brushes, the PGMAx-co-PDEAEMAy copolymer brushes were thicker. Qualitatively similar results were reported by other groups, who described the surface-initiated atom transfer radical copolymerization of n-butyl38 and tert-butyl methacrylate39 with 2-(dimethylamino)ethyl methacrylate. The reasons for this rate enhancement during copolymerization are not fully understood at the moment and are the subject of ongoing investigations. Most likely, however, the increased rate of polymerization may be attributed to the presence of the tertiary amine group of DEAEMA. As observed by Tang et al., the rate of (38) Xu, C.; Barnes, S. E.; Wu, T.; Fischer, D. A.; DeLongchamp, D. M.; Batteas, J. D.; Beers, K. L. Adv. Mater. 2006, 18, 1427. (39) Sanjuan, S.; Tran, Y. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 4305.

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polymerization can be significantly increased in the presence of tertiary amine compounds (i.e., triethylamine or tributylamine) in the ATRP solution.40,41 The different (co)polymer brushes were characterized by XPS. Figure 2 shows the XPS survey and high-resolution C1s and O1s scans of a PGMA (Figure 2A), a PGMA50-co-PDEAEMA50 (Figure 2B), and a PDEAEMA (Figure 2C) brush. In agreement with the elemental composition of the (co)polymer brushes, the survey spectra reveal the presence of C1s, O1s and, for (co)polymer brushes containing DEAEMA units, N1s signals. For the PGMA brush, the high-resolution C1s signals can be fitted with the expected peak area ratios using five model Gaussian/Lorentzian curves, which correspond to the aliphatic carbon atoms of the polymer backbone (C-C/C-H, 285.0 eV), the secondary carbons of the ester groups (C-CdO, 285.5 eV), the C-O moieties (286.5 eV), the carbon atoms of the oxirane rings (C-O-C, 287.0 eV), and the carbon atoms of the ester groups (O-CdO, 289.1 eV). The C1s high-resolution scan of the PGMA50-co-PDEAEMA50 brush can be fitted in the same way, but with peak areas different from those reported for the PGMA brush. As expected from the chemical structure of PGMA50-co-PDEAEMA50, after being normalized by the area of the backbone aliphatic carbons the contribution of (40) Tang, H. D.; Arulsamy, N.; Radosz, M.; Shen, Y. Q.; Tsarevsky, N. V.; Braunecker, W. A.; Tang, W.; Matyjaszewski, K. J. Am. Chem. Soc. 2006, 128, 16277. (41) Tang, H. D.; Shen, Y. Q.; Li, B.-G.; Radosz, M. Macromol. Rapid Commun. 2008, 29, 1834.

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Figure 3. FTIR reflectance spectra of (A) PGMA (150 nm), (B) PGMA90-co-PDEAEMA10 (220 nm), (C) PGMA75-co-PDEAEMA25 (210 nm), (D) PGMA50-co-PDEAEMA50 (200 nm), and (E) PDEAEMA (50 nm) brushes. All spectra were normalized with respect to the carbonyl band at 1730 cm-1. Scheme 2. Post-Polymerization Modification of PGMAx-co-PDEAEMAy (Co)polymer Brushes

the oxirane ring carbons was decreased by a factor 3 compared to PGMA homopolymer brushes, while the peak area of C-O signals, together with a contribution to the same peak area from the carbons adjacent to amino moieties (C-N) was multiplied by 5/3. In agreement with the elemental composition of PDEAEMA, the contribution from the oxirane ring carbons was absent in the C1s high-resolution scan of the PDEAEMA brush, whereas the other signals remain. The O1s high-resolution scans can also be fitted with Gaussian/Lorentzian curves according to the chemical structures of the (co)polymer brushes and with the correct peak area ratios. The O1s signals of the PGMA and PGMA50-coPDEAEMA50 scans can be fitted with three curves corresponding to the oxygen atoms from O-CdO (533.9 eV), C-O-C (533.2 eV), and O-CdO (532.3 eV). Compared to PGMA, where the areas underneath the different O1s signals are equivalent, the peak area from the C-O-C signal in PGMA50-co-PDEAEMA50 is two times smaller than the two other contributions. As expected, the signal from the oxirane ring oxygens is absent in the O1s highresolution scan of the PDEAEMA brush, while the contributions from oxygen atoms of the ester groups remain in the predicted peak area ratios. XPS analysis was also used to compare the monomer feed ratio with the composition of the copolymer brushes. The composition of the copolymer brushes was estimated 18224 DOI: 10.1021/la102400z

from the [N1s]/[C1s] ratio. The results, which are included in Table 1, indicate that there is good agreement between the composition of the monomer feed and that of the copolymers. The (co)polymer brushes were further analyzed with FTIR spectroscopy (Figure 3). In the spectrum of the PGMA brush (Figure 3A), the peaks at 3060 cm-1, 2997 cm-1, and 2949 cm-1 can be assigned to C-H stretching, whereas the intense peak at 1730 cm-1 represents the contribution from the carbonyl groups of the ester moieties. The peak at 908 cm-1 represents the antisymmetric ring deformation band of the epoxide groups42 and will be used in the remainder of this contribution to study the post-polymerization modification of PGMA and PGMAxco-PDEAEMAy (co)polymer brushes. For PDEAEMA brushes (Figure 3E), the C-H and N-H stretching peaks were localized at 2967 cm-1, 2935 cm-1, and 2807 cm-1, while the contribution from the carbonyl groups remain at 1730 cm-1. The FTIR spectra of the copolymer brushes (Figure 3B-D) show features of both homopolymer in ratios that reflect their compositions. (42) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteritic Frequencies of Organic Molecules; Academic Press, Inc.: San Diego, 1991.

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Figure 4. FTIR reflectance spectra of a PGMA75-co-PDEAEMA25 brush (initial thickness: 190 nm) exposed to a 1 M aqueous solution of propylamine for different reaction times. All spectra were normalized with respect to the carbonyl band at 1730 cm-1.

An important question in view of the primary amine and protein post-polymerization modifications, which will be discussed in the next sections (vide infra), is to demonstrate that no side reactions, such as cross-linking due to unexpected oxirane ring-opening, take place during the preparation of the copolymer brushes. The perfect assignments and peak area ratios obtained from the XPS C1s and O1s high-resolution scans, the expected surface composition determined from the XPS [N1s]/[C1s] ratios, as well as the absence of a broad band in the hydroxyl region (∼3200-3700 cm-1) in the FTIR spectra are good indicators to ascertain the structure of the prepared (co)polymer brushes. Furthermore, at room temperature, PGMA brushes were found to be stabile upon exposure to DEAEMA monomer (Figure S1, Supporting Information), which is another indication of the stability of the oxirane rings during the copolymerization. After 14 days of incubation in DEAEMA, FTIR spectroscopic analysis of the PGMA brushes did not reveal a decrease in intensity of the characteristic oxirane signal at 908 cm-1. These observations, however, do not necessarily apply to all tertiary amine-containing methacrylates. Attempts, for example, to produce PGMA-co-poly(2-(dimethylamino)ethyl methacrylate (PGMA-co-PDMAEMA) brushes failed after 1 h due to the formation of an insoluble gel in the polymerization solution. This is in agreement with the observation reported by Jiang et al. that the addition of DMAEMA to a solution of PGMA resulted in the formation of a viscous gel.31 The different reactivities of DMAEMA and DEAEMA have been attributed to the difference in nucleophilicity between the dimethylamino- and the diethylaminocontaining monomers. Post-Polymerization Modification. In the next series of experiments, the post-polymerization modification of the epoxide-containing (co)polymer brushes with various primary amines was investigated. To this end, PGMA, PGMA90-co-PDEAEMA10 and PGMA75-co-PDEAEMA25 brushes were incubated in aqueous solutions of various primary amines at room temperature (Scheme 2). FTIR spectroscopy was used to measure the conversion of epoxide groups as a function of reaction time. As an example, Figure 4 shows FTIR spectra of a PGMA75-co-PDEAEMA25 brush that was incubated in a 1 M propylamine solution in water Langmuir 2010, 26(23), 18219–18230

for different reaction times. Epoxide group conversions were determined by monitoring the peak area at 908 cm-1, which is characteristic for the epoxide group, and calculating the ratio of this area relative to that of the ester carbonyl peak at 1730 cm-1. As the reaction time increases, the relative area ratio of the epoxide signal at 908 cm-1 decreases, which is good evidence for the ring-opening of the oxirane rings and successful aminolysis of the (co)polymer brush. The successful ring-opening is further evidenced by the appearance of a broad band in the hydroxyl region (∼3200-3700 cm-1) of the FTIR spectrum. From the FTIR spectra, the conversion of epoxide groups was determined using the following equation: A908, t A1730, t0 Conversion ¼ 1 A908, t0 3 A1730, t

! ð2Þ

Four amine-containing aqueous solutions were investigated, namely, propylamine (1 M, nonpolar, pH = 12.8), amino-2propanol (1 M, polar, pH = 12.1), taurine (0.5 M, negatively charged, pH = 4.7), and (2-aminoethyl)trimethylammonium chloride hydrochloride (0.5 M, positively charged, pH=3.7). Figure 5 represents the epoxide group conversion of PGMA, PGMA90-coPDEAEMA10, and PGMA75-co-PDEAEMA25 brushes as a function of post-polymerization reaction time. For all investigated amines, the results in Figure 5 clearly show that the incorporation of the tertiary amine comonomer accelerates the rate of the postpolymerization modification reaction and leads to increased epoxide group conversion, which reflects the ability of the tertiary amine group to catalyze the epoxide ring-opening reaction. In addition to the catalytic activity of the tertiary amine groups, another factor that could contribute to the observed differences among the PGMA, PGMA90-co-PDEAEMA10, and PGMA75-coPDEAEMA25 brushes could be variations in the swelling behavior of the different coatings. However, since the advancing water contact angles of the PGMA (∼66°) and PGMA75-co-PDEAEMA25 (∼60°) brushes are relatively close, differences in initial swelling behavior are likely to be small. It is important to realize, DOI: 10.1021/la102400z

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Figure 5. Epoxide conversion of homo- and copolymer brushes as a function of post-polymerization modification reaction time at room temperature for aqueous solutions of (A) propylamine (1 M), (B) amino-2-propanol (1 M), (C) taurine (0.5 M), and (D) (2-aminoethyl)trimethylammonium chloride hydrochloride (0.5 M). PGMA (9, initial thickness: 110 nm), PGMA90-co-PDEAEMA10 (2, initial thickness: 240 nm), and PGMA75-co-PDEAEMA25 (b, initial thickness: 210 nm). Table 2. Epoxide Group Conversion and Variation in Dry Film Thickness after 48 h Post-Polymerization Modification of PGMA, PGMA90-coPDEAEMA10, and PGMA75-co-PDEAEMA25 Brushes with Different Primary Amines PGMA a

conversion [%]

PGMA90-co-PDEAEMA10

Δ thickness [nm]

b

a

conversion [%]

PGMA75-co-PDEAEMA25

Δ thickness [nm]

b

conversiona [%]

Δ thicknessb [nm]

propylamine 61.1 þ32.8 69.5 þ51.4 78.7 þ60.4 amino-2-propanol 8.9 þ5.7 28.4 þ17.9 44.6 þ26.7 taurine 0.6 þ1.5 9.7 þ8.1 34.8 þ22.8 (2-aminoethyl) trimethylammonium 0.4 -0.9 21.5 þ12.3 47.6 þ23.2 chloride hydrochloride a Epoxide group conversion determined by FTIR after 48 h of post-polymerization modification. b Variation in dry ellipsometric thickness for (co)polymer brushes after 48 h of incubation in primary amines solutions.

however, that every epoxide ring-opening generates one new secondary amine and one new hydroxyl group. These new functional groups may enhance the swelling of the post-modified brushes as compared to the original PGMA or PGMAx-coPDEAEMAy brushes. As a consequence, as post-polymerization modification proceeds, swelling of the polymer brushes may increase, which may add to the catalytic activity of the DEAEMA tertiary amine groups and confer an autocatalytic character to the post-polymerization modification process. To substantiate these hypotheses and differentiate between the influence of the catalytic activity of the DEAEMA tertiary amine groups and the change in swelling properties during post-polymerization modification, detailed in situ swelling studies would be needed, which would go beyond the scope of the present contribution. For any given primary amine, the post-polymerization modification reaction rates and degrees of modification increase from the PGMA to the PGMA90-co-PDEAEMA10 to the PGMA75-co-PDEAEMA25 (co)polymer brush. Although the reaction kinetics are easy to compare in a series of substrates for a given primary amine, a 18226 DOI: 10.1021/la102400z

comparison of the post-polymerization modification kinetics of different amines is more complex as the nucleophilicity, polarity, and charge of the different amines vary, as well as the pH and wettability of the different reaction solutions. Another technique which (at least semiquantitatively) allows the monitoring of the post-polymerization modification of the PGMA and PGMAx-co-PDEAEMAy brushes is ellipsometry. Table 2 summarizes the variations in the dry film thickness of the different brushes upon post-polymerization modification. These results are in good agreement with the data presented in Figure 5 and confirm the ability of the tertiary amine groups to accelerate the post-polymerization modification reaction. To further confirm that the conversion of the epoxide groups as evidenced by the FTIR experiments is indeed due to the postpolymerization modification with the primary amines and not due to, for example, hydrolysis, the stability of the (co)polymer brushes toward hydrolysis was studied (Figure 6). To evaluate the stability of the different brushes, changes in the epoxide signal were monitored as a function of time and expressed as apparent Langmuir 2010, 26(23), 18219–18230

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Figure 6. Stability of epoxide groups of PGMA (9, initial thickness: 110 nm), PGMA90-co-PDEAEMA10 (2, initial thickness: 240 nm), and PGMA75-co-PDEAEMA25 (b, initial thickness: 210 nm) brushes toward hydrolysis in (A) air, (B) water, (C) PBS, and (D) NaOH 0.01 M.

Figure 7. Epoxide conversion as a function of reaction time for PGMA brushes (initial thickness: 130 nm) immersed in 1 M propylamine solutions which contained no triethylamine (9), 1 mol % (b), 10 mol % (2), or 25 mol % (right-facing triangle) triethylamine (with respect to propylamine), as well as for a PGMA75-co-PDEAEMA25 (O, initial thickness: 210 nm) brush in propylamine without addition of triethylamine.

conversions as was also done in Figure 5. In air, equivalent to shelf storage, all (co)polymer brushes are stable for at least 1 month. In water and PBS (pH=7.4), PGMA brushes are virtually unreactive, whereas copolymer brushes were hydrolyzed to a certain extent. The susceptibility of the brushes toward hydrolysis increased upon incorporation of DEAEMA and increased with increasing DEAEMA content. It is important to note, however, that the rates of the hydrolysis reaction are much slower, and Langmuir 2010, 26(23), 18219–18230

the extent to which hydrolysis takes place is much smaller, as compared to post-polymerization modification with a primary amine. (Note that the x-axis in Figure 5 is in hours and the x-axis in Figure 6 in days.) Figure 6D does not contain any data points beyond 14 days. This is due to the fact that exposing PGMA and PGMAx-co-PDEAEMAy (co)polymer brushes to 0.01 M NaOH (pH = 12.2) for periods longer than 14 days resulted in detachment of the brushes as evidenced by FTIR and ellipsometry. Instead of copolymerization of a tertiary amine-containing monomer such as DEAEMA, another possible strategy to enhance the rate and degree of post-polymerization modification of PGMA brushes with primary amines would be to add a tertiary amine, such as triethylamine, to the reaction mixture. To evaluate this strategy and its feasibility compared to the copolymerization of GMA and DEAEMA, a final series of experiments was carried out. In these experiments, the effect of adding increasing amounts of triethylamine to a reaction mixture containing propylamine on the post-polymerization modification of a PGMA brush was studied by monitoring the epoxide group conversion as a function of time with FTIR spectroscopy. The results of these experiments, which are summarized in Figure 7, show that the addition of triethylamine to the propylamine solution also accelerates the rate of epoxide group conversion. However, relatively large amounts, up to 25 mol % (with respect to propylamine), are required to obtain epoxide post-polymerization modification reaction profiles that are comparable to, for instance, a PGMA75-co-PDEAEMA25 brush. With the substrate dimensions and reaction volumes used for the post-polymerization modification experiments in this work, the absolute amount of triethylamine molecules that need to be added to the reaction mixture in order to achieve an identical acceleration of the reaction can be DOI: 10.1021/la102400z

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Figure 8. FTIR reflectance spectra of PGMA75-co-PDEAEMA25 brushes (initial thickness: 210 nm) recorded at different reaction times upon exposure to (A) BSA or (B) lysozyme solutions. All spectra were normalized with respect to the carbonyl band at 1730 cm-1. Inserts represent details of the 1750-700 cm-1 region.

Figure 9. Epoxide group conversion as a function of reaction time upon exposure of different brushes to (A) BSA and (B) lysozyme in PBS. (9) PGMA (initial thickness: 110 nm); (2) PGMA90-co-PDEAEMA10 (initial thickness: 240 nm); (b) PGMA75-co-PDEAEMA25 (initial thickness: 210 nm).

estimated to be more than 6 orders of magnitude larger than the amount of DEAEMA residues in the copolymer brushes. Protein Immobilization. The experiments discussed in the previous section clearly demonstrate that the incorporation of DEAEMA units in the polymer brush accelerates the postpolymerization modification of the GMA epoxide groups with primary amines. The enhanced reactivity of the PGMAx-coPDEAEMAy brushes compared to the PGMA homopolymer brush makes these copolymer brushes an interesting platform for the immobilization of biomolecules, such as proteins, which typically contain several surface-exposed amine groups. As a first proof-of-concept, protein immobilization experiments were carried out on PGMA, PGMA90-co-PDEAEMA10, and PGMA75co-PDEAEMA25 brushes. To this end, the different brushes were exposed to solutions of BSA and lysozyme (100 mg 3 mL-1 in PBS, pH = 7.4) and the epoxide group conversion studied via FTIR spectroscopy. Figure 8 shows the FTIR spectra of PGMA75-coPDEAEMA25 brushes as a function of incubation time in BSA (Figure 8A) and lysozyme (Figure 8B) solutions. As expected, the 18228 DOI: 10.1021/la102400z

intensity of the characteristic epoxide peak at 908 cm-1 decreases, whereas a broad peak appears in the hydroxyl region (∼32003700 cm-1). Moreover, small peaks appear at 1650 cm-1 and 1540-1550 cm-1, which can be assigned to the amide I and amide II bands of bound proteins, respectively. Figure 9 plots the epoxide group conversion as determined from FTIR spectroscopy as a function of time for the immobilization of BSA and lysozyme on PGMA, PGMA90-co-PDEAEMA10, and PGMA75co-PDEAEMA25 brushes. Interestingly, under the investigated conditions, no epoxide ring-opening was observed for the PGMA homopolymer brushes, whereas the extent of modification of the copolymer brushes increased with increasing DEAEMA content. It is important to note here that the epoxide conversions reported in Figure 9 are higher than those shown in Figure 6, which were due to hydrolysis (note again that the x-axis in Figure 6 is in days and that of Figure 9 in hours). Comparison of Figure 6 and Figure 9, however, also clearly indicates that the epoxide conversions reported in Figure 9 are not only due to protein immobilization but are also the result of concurrent hydrolysis, which in some Langmuir 2010, 26(23), 18219–18230

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Figure 10. Area averaged mass of proteins immobilized on different (co)polymer brushes upon exposure to 100 mg 3 mL-1 solutions of (A)

BSA and (B) lysozyme in PBS for different reaction times (6, 24, and 48 h): PGMA (initial thickness: 100 nm); PGMA90-co-PDEAEMA10 (initial thickness: 100 nm); PGMA75-co-PDEAEMA25 (initial thickness: 70 nm). (C) Area averaged mass of bound protein on PGMA brushes of two different thicknesses (100 and 25 nm) upon exposure to a 50 mg 3 mL-1 OVA solution in borate buffer for 6, 24, and 48 h.

cases even accounts for the major part of epoxide conversion. Nevertheless, the data in Figure 8 and Figure 9 support the successful immobilization of BSA and lysozyme on the copolymer brushes. Moreover, an increase in the dry ellipsometric thickness (Table S1, Supporting Information) of the (co)polymer brushes after immersion in protein solutions was measured, which is also indicative of a successful protein immobilization. The FTIR spectroscopy and ellipsometry experiments were complemented with quartz crystal microbalance with dissipation (QCM-D) studies in an attempt to more quantitatively assess and compare the protein binding capacity of the different (co)polymer brushes. In these experiments, the amount of protein immobilized on (co)polymer brush-coated QCM-D sensors was determined from the differences in resonance frequencies before and after exposure to a 100 mg 3 mL-1 solution of BSA or lysozyme in PBS. Using QCM-D chips coated with a PGMA, PGMA90-coPDEAEMA10, or PGMA50-co-PDEAEMA50 brush, three series of experiments were performed with incubation times of 6, 24, and 48 h, respectively. The results of these experiments are summarized in Table S2 in the Supporting Information and are also presented in Figure 10. Figure 10A,B clearly shows that the amount of protein that can be immobilized on the (co)polymer brushes increases with increasing DEAEMA content. This confirms that the incorporation of DEAEMA not only enhances the rate of protein immobilization compared to a PGMA homopolymer brush, but also increases the protein binding capacity. For example, whereas the area averaged mass of bound proteins levels off to approximately 1 μg 3 cm-2 on PGMA brushes, values of up to approximately 3.5 and 5 μg 3 cm-2 are reached for PGMA75-coPDEAEMA25 brushes incubated for 48 h in BSA and lysozyme solutions, respectively. It is interesting to note that this effect is observed even though the thickness of the PGMA75-co-PDEAEMA25 brush (70 nm) is smaller than that of PGMA or PGMA90co-PDEAEMA10 brushes (100 nm). The reason for the difference in the amount of bound protein between BSA and lysozyme is mainly attributed to the difference in mass (i.e., size) between the two proteins (approximately 66 kDa for BSA and 16 kDa for lysozyme). The charge of the proteins at pH = 7.4, i.e., negative for BSA (pI = 5.8) and positive for lysozyme (pI = 9.4), or the Langmuir 2010, 26(23), 18219–18230

number of lysine amino acids that compose BSA (60) and lysozyme (6) do not seem to influence the protein binding capacity of our (co)polymer brushes, but the smaller-sized lysozyme seems to be more readily immobilized as compared to the larger BSA. Finally, to demonstrate the influence of the 3D nature of the polymer brush coatings on the protein binding capacity, two QCM-D chips coated with PGMA brushes of 100 and 25 nm thickness were exposed to a 50 mg 3 mL-1 ovalbumin (OVA) solution in borate buffer for 6, 24, and 48 h. The data in Figure 10C and Table S3 (Supporting Information) demonstrate that the protein binding capacity of PGMA brushes increases with increasing brush thickness, which illustrates the 3D nature of the polymer brush layer.

Conclusions This paper has investigated the reactivity of poly(glycidyl methacrylate) (PGMA) brushes toward post-polymerization modification with primary amines and has elaborated strategies that allow a facile, room temperature, and aqueous post-polymerization modification of these brushes with biomolecules such as proteins. The proposed strategy involves the introduction of tertiary amine groups, which can catalyze the epoxide ring-opening reaction, by surface-initiated atom transfer radical copolymerization of glycidyl methacrylate (GMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA). PGMAx-co-PDEAEMAy copolymer brushes with DEAEMA contents between 1 and 50 mol % and thicknesses ranging from 190 and 240 nm could be prepared via SIATRP in aqueous methanol at room temperature using a mixed CuICl/CuIIBr2/2,20 -bipyridyl (bpy) catalyst system. Model experiments, which investigated the room temperature, aqueous postpolymerization modification of these (co)polymer brushes with several primary amines revealed that the incorporation of DEAEMA in the brushes significantly increased both the rate and the degree of post-polymerization modification. Post-polymerization modification of the PGMA and PGMAx-co-PDEAEMAy copolymer brushes is proposed to be an autocatalytic process in which the catalytic activity of the DEAEMA tertiary amine groups is amplified by changes in the swelling properties of the polymer DOI: 10.1021/la102400z

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brush coatings as the reaction proceeds. The rate and degree of post-polymerization modification were found to increase with increasing DEAEMA content. Finally, first proof-of-concept experiments were carried out to evaluate the feasibility of the DEAEMA-containing brushes to act as platforms for the immobilization of proteins. These experiments were carried out by exposing the different brushes to solutions of lysozyme or bovine serum albumin (BSA) in phosphate buffered saline (PBS) at room temperature. Evaluation of the protein immobilization kinetics using FTIR spectroscopy revealed that the incorporation of DEAEMA enhanced the rate of the reaction. These studies were complemented by quartz crystal microbalance with dissipation (QCM-D) experiments, which demonstrated that the incorporation of the DEAEMA comonomer not only increases the rate

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of the protein immobilization reaction, but also leads to an enhanced protein binding capacity. The ability of the PGMAx-coPDEAEMAy brushes to facilitate the covalent immobilization of proteins at room temperature from aqueous solution makes them a very attractive platform for the development of protein microarrays, for example. Acknowledgment. This research was supported by CTI/KTI. Supporting Information Available: Stability of PGMA brushes in DEAEMA followed by FTIR spectroscopy, as well as details on the evaluation of protein immobilization by ellipsometry and QCM-D. This material is available free of charge via the Internet at http://pubs.acs.org.

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