Facile Immobilization of Biomolecules onto Various Surfaces Using

Feb 6, 2012 - The surface modifications of plastic or glass substrate and the subsequent immobilization of biomolecules onto the surfaces has been a c...
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Facile Immobilization of Biomolecules onto Various Surfaces Using Epoxide-Containing Antibiofouling Polymers Daekyung Sung,† Sangjin Park,‡ and Sangyong Jon*,†,‡ †

Department of Medical System Engineering (DMSE), ‡Cell Dynamics Research Center, School of Life Sciences, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea S Supporting Information *

ABSTRACT: The surface modifications of plastic or glass substrate and the subsequent immobilization of biomolecules onto the surfaces has been a central feature of the fabrication of biochips. To this end, we designed and synthesized new epoxide-containing random copolymers that form stable polymer adlayers on plastic or glass surface and subsequently react with amine or sulfhydryl functional groups of biomolecules under aqueous conditions. Epoxide-containing random copolymers were synthesized by radical polymerization of three functional monomers: a monomer acting as an anchor to the surfaces, a PEG group for preventing nonspecific protein adsorption, and an epoxide group for conjugating to biomolecules. Polymer coating layers were facilely formed on cyclic olefin copolymer (COC) or glass substrate by simply dipping each substrate into a solution of each copolymer. The polymer-coated surfaces characterized by a contact angle analyzer and Xray photoelectron spectroscopy (XPS) showed very low levels of nonspecific immunoglobulin G (IgG) adsorption compared to the uncoated bare surface (control). Using a microcontact printing (μCP) method, antibodies as representative biomolecules could be selectively attached onto the copolymers-coated glass or COC surface with high signal-to-noise ratios.

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INTRODUCTION Facile methods for immobilization of bioactive molecules, including antibodies, enzymes, peptides, polysaccharides, and nucleic acids, on surface materials have been attracting considerable attention in the field of biosensors and biochips.1 The most widely practiced chemistry for the conjugation of biomolecules involves coupling an N-hydroxysuccinimide (NHS) ester with amine groups of biomolecules.2 However, the NHS esters typically suffer from rapid hydrolysis under aqueous conditions and thus the functional activity of NHS ester-modified surfaces becomes compromised over time.3 Epoxy chemistry, by contrast, presents another alternative coupling system for biomolecule immobilization, due to its stability under aqueous conditions and its reactivity toward several nucleophilic groups such as amine and sulfhydryl groups.4 In one study, epoxide groups were linked to a substrate surface through the self-assembly of epoxysilanes.5 The hydrolysis of trialkoxysilane moieties induced conjugation of organosilanes to the glass or silicon oxide substrate, resulting in siloxane bonds on the surface. However, excessive polymerization or incomplete monolayer formation often occurs, resulting in poor reproducibility. Another method involves the use of glycidyl methacrylate (GMA) photopolymers grafted onto polyethylene terephthalate (PET) surfaces to introduce high-density epoxide groups for efficient protein immobilization.6 This approach involves multiple time-consuming steps and unwanted high nonspecific adsorption of proteins presumably due to the absence of antibiofouling functionality. © 2012 American Chemical Society

Therefore, there is a need for development of a new platform that permits the specific immobilization of biomolecules on glass or plastic substrates with reduced biofouling in a reproducible manner. Recently, a novel method based on polymeric self-assembled monolayers (pSAMs) was developed by us for the selective immobilization of proteins or cells on silicon oxide,7 indium tin oxide,8 or plastic substrates,9 yielding the advantages of reproducible immobilization and reduced nonspecific adsorption relative to conventional SAMs. In the previous studies, we demonstrated that rationally designed random copolymers containing a surface anchoring moiety (dodecyl, benzyl, or trimethoxysilanyl), an antibiofouling polyethylene glycol (PEG) component, and an NHS ester functional group for conjugation could be used to modify the surfaces of plastic or Si/SiO2 wafer surfaces, forming stable polymer adlayers at low pH on which biomolecules could be selectively immobilized.7,9b However, the storage lifetime of NHS ester-modified surfaces is short because the functional group is easily hydrolyzed under aqueous conditions, limiting their uses as a general platform for biochip construction. With those considerations in mind, we have designed and synthesized new epoxide-containing random copolymers to overcome the limitations of the previous NHS ester-containing copolymers. Here, we report the synthesis of the copolymers, Received: December 13, 2011 Revised: January 10, 2012 Published: February 6, 2012 4507

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the polymer solutions for 1 h at ambient temperature, followed by five washes with deionized water. Polymer-coated COC surfaces were characterized by measuring the static water contact angle to confirm the success of the polymer coating. Poly(TMSMA-r-mPEGMA-rGMA) was used to modify the glass surfaces. The glass surfaces were cleaned with a concentrated “piranha” solution (H2SO4/H2O2 = 3:1), then were thoroughly rinsed with ethanol and deionized water prior to polymer treatment. Polymer-coated glass surfaces were prepared by immersing a glass surface in the polymer solution (20 mg/mL in deionized water) for 1 h at ambient temperature, followed by five washes with solvent. The polymer-coated glass surfaces were then cured at 100 °C for 5 min, and polymer-coated glass surfaces were characterized by measuring the static water contact angles and XPS spectra. Antibiofouling Test. Polymer-coated COC and glass surfaces were immersed in PBS (pH 7.4) for 1 h to reduce the reactivity of the epoxide group, followed by washing with distilled water and air-drying. A solution of rhodamine-labeled antirabbit IgG in PBS (40 μg/mL, pH 7.4) was introduced to the polymer-coated COC and glass surfaces at room temperature for 2 h, followed by several washes with PBS and distilled water and subsequent air-drying. The degree of rhodaminelabeled antirabbit IgG adsorption onto the COC and glass surface was measured with a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with a 100× objective and rhodamineoptimized filter sets (Omega Optical Inc., Brattleboro, VT, USA). Antibody Immobilization Using Microcontact Printing (μCP). A poly(dimethylsiloxane) (PDMS) stamp was cleaned with detergent, washed with ethanol and deionized water several times, and treated with O2 plasma (Expanded Plasma Cleaner; Harrick Plasma Corp., Ithaca, NY) for 30 s to remove dust and generate hydroxyl groups on the surface. After inking the PDMS stamp with a 50 × 50 μm2 circular pattern using a rabbit IgG solution (200 μg/mL in borate buffer, pH 9.0), the inked stamp was brought into contact with the already prepared polymer-coated COC and glass surface and incubated for 2 h at 37 °C in a humid atmosphere, allowing the rabbit IgG molecules to transfer to the substrate surface. After removing the stamp, the COC and glass substrate were dipped immediately in PBS (pH 7.4) and incubated for 1 h to minimize the reactivity of the remaining epoxide functional groups as well as to remove the nonspecifically bound rabbit IgG, followed by washing with distilled water and air-drying. A solution of rhodamine-labeled antirabbit IgG (40 μg/mL) in PBS (pH 7.4) was added to the rabbit IgG-patterned polymeric COC and glass surfaces for 1 h at ambient temperatures, then washed several times with PBS and distilled water. The patterns of rhodamine-labeled antirabbit IgG were visualized with use of a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with 200× objectives and rhodamine-optimized filter sets (Omega Optical Inc., Brattleboro, VT, USA). Antibody Immobilization through Immobilized Protein G. Protein G (100 μg/mL in borate buffer, pH 9.0) was contact printed onto polymer-modified COC surfaces over 2 h at 37 °C in a humidity chamber using the μCP protocols described above. The substrates were then immediately immersed in PBS (pH 7.4) and incubated for 1 h, followed by washing with distilled water and air-drying. Rhodaminelabeled antirabbit IgG (40 μg/mL in PBS) was treated to the protein G-patterned COC surface and incubated for 2 h at ambient temperatures. The patterns of antirabbit IgG were visualized with use of a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with 200× objectives and rhodamine-optimized filter sets (Omega Optical Inc., Brattleboro, VT, USA).

formation and antibiofouling properties of the polymer adlayers formed on plastic or glass substrates, and the subsequent selective immobilization of proteins (i.e., antibodies) onto the functionalized surfaces.

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MATERIALS AND METHODS

Materials. Dodecyl methacrylate (DMA), benzyl methacrylate (BMA), 3-(trimethoxysilyl)propyl methacrylate (TMSMA), poly(ethylene glycol) methyl ether methacrylate (mPEGMA, average Mn ≈ 475), glycidyl methacrylate (GMA), 2,2′-azobisisobutyronitrile (AIBN), and rabbit IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cyclic olefin copolymer (COC) was obtained from TOPAS Advanced Polymers (Florence, KY, USA). Glass slides were purchased from Marienfeld Laboratory Glassware (LaudaKö nigshofen, Germany). Antirabbit IgG and rhodamine-labeled antirabbit IgG was purchased from Invitrogen (Carlsbad, CA, USA). All organic solvents were used as received without further purification. Measurements. 1H NMR (400 MHz) spectra were recorded on a JEOL JNM-ECX400 spectrometer (Tokyo, Japan). Organic phase gel permeation chromatography (GPC) was performed with a Waters 1515 series isocratic pump and a Rheodyne model 7725 injector with a 100 L injection loop at a flow rate of 0.4 mL/min. A Phoenix300 contact angle and surface tension analyzer (Surface Electro Optics, Kyunggi-do, Korea) equipped with video camera and monitor was used to measure contact angles. X-ray photoelectron spectroscopy (XPS) spectra were acquired with use of a Kratos AXIS Ultra Imaging X-ray Photoelectron Spectrometer with a monochromatized Al K Xray source. Synthesis of Polymers. Prior to polymerization, neat mPEGMA was passed over an inhibitor-removal column (Sigma-Aldrich, Milwaukee, WI, USA). DMA, BMA, or TMSMA (3.5 mmol, 3.5 equiv), mPEGMA (3 mmol, 1.425 g, 3 equiv), and GMA (3.5 mmol, 0.592 g, 3.5 equiv) were placed in a vial and dissolved in 10 mL of tetrahydrofuran (anhydrous, 99.9%, inhibitor-free). This mixture was degassed for 10 min by bubbling with a stream of N2 gas. After adding 0.1 mmol of AIBN (16.5 mg, 0.1 equiv) as a radical initiator, the vial was sealed with a Teflon-lined screw cap. The polymerization reaction was carried out at 70 °C for 24 h. The final product solution was cooled to room temperature and stored at 4 °C until use. 1H NMR (400 MHz, CDCl3): poly(DMA-r-mPEGMA-r-GMA) δ 4.29 (br, 1H, CO2−CH2 of GMA), 4.1 (br, 2H, CO2−CH2 of mPEGMA), 3.92 (br, 2H, CO2−CH2 of DMA), 3.81 (br, 1H, CO2−CH2 of GMA), 3.79− 3.58 (br, 30H of mPEGMA), 3.38 (s, 3H, O−CH3 of mPEGMA), 3.21 (br, 1H of GMA), 2.84 (br, 1H of GMA), 2.63 (br, 1H of GMA), 1.89−1.82 (br, 10H), 1.61 (br, 2H), 1.38−1.22 (br, 20H of DMA), 1.04 (br, 3H of DMA), 0.92−0.86 (br, 8H); poly(BMA-r-mPEGMA-rGMA) δ 7.33 (br, 5H, benzene of BMA), 4.99 (br, 2H, CO2−CH2 of BMA), 4.29 (br, 1H, CO2−CH2 of GMA), 4.08 (br, 2H, CO2−CH2 of mPEGMA), 3.81 (br, 1H, CO2−CH2 of GMA), 3.78−3.56 (br, 30H of mPEGMA), 3.38 (s, 3H, O−CH3 of mPEGMA), 3.2 (br, 1H of GMA), 2.8 (br, 1H of GMA), 2.6 (br, 1H of GMA), 1.9−1.8 (br, 10H), 1.1−0.83 (br, 5H); poly(TMSMA-r-mPEGMA-r-GMA) δ 4.29 (br, 1H, CO2−CH2 of GMA), 4.1 (br, 2H, CO2−CH2 of mPEGMA), 3.9 (br, 2H, CO2−CH2 of TMSMA), 3.82 (br, 1H, CO2−CH2 of GMA), 3.77−3.61 (br, 30H of mPEGMA), 3.38 (s, 3H, O−CH3 of mPEGMA), 3.21 (br, 1H of GMA), 2.84 (br, 1H of GMA), 2.63 (br, 1H of GMA), 1.9−1.8 (br, 10H), 1.73 (br, 2H of TMSMA), 1.1−0.83 (br, 5H), 0.66 (br, 2H of TMSMA). GPC: poly(DMA-r-mPEGMA-rGMA), Mn = 28 997 with Mw/Mn = 1.89; poly(BMA-r-mPEGMA-rGMA), Mn = 25 261 with Mw/Mn = 1.78; poly(TMSMA-r-mPEGMAr-GMA), Mn = 25 711 with Mw/Mn = 1.83. Surface Modification. Modification of the COC surface was carried out by the following methods. First, COC substrates were cut into specific sizes and washed with ethanol and distilled water to remove contaminants. Poly(DMA-r-mPEGMA-r-GMA) and poly(BMA-r-mPEGMA-r-GMA) for COC surface modification were obtained as viscous liquids by evaporating away THF under vacuum for 10 min, and the polymers were dissolved in deionized water (40 mg/mL) for subsequent coating. COC substrates were immersed in

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RESULTS AND DISCUSSION Synthesis of Epoxide-Containing Copolymers. In previous our studies, we showed that NHS ester-containing random copolymers composed of surface anchoring, antibiofouling, and conjugating moieties enabled facile formation of the preactivated polymer adlayers and the subsequent immobilization of biomolecules onto plastic or Si/SiO2 substrates.7,9b However, a major drawback associated with the 4508

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Scheme 1. (a) Chemical Structures of the Three Epoxide-Activated Antibiofouling Polymers Described in This Studya and (b) Schematic Representation of the Procedure for Modifying a Plastic or Glass Surface, the Antibiofouling Properties, and Attachment of the Antibodies, Using Epoxide-Containing Copolymers

a

The dodecyl and benzyl groups were used to modify plastic surfaces. Trimethoxysilane groups provided components for anchoring to the glass surface.

copolymer (40 mg/mL in deionized water) at ambient temperature. In the case of previous NHS ester-containing copolymers, we were not able to use neutral pH buffer solution for the polymer coating process as well as for the subsequent immobilization step of biomolecules due to the rapid hydrolysis of the NHS ester, limiting their uses. For glass surface modifications, the glass substrate was immersed in the poly(TMSMA-r-mPEGMA-r-GMA) solution (20 mg/mL in deionized water) for 1 h at ambient temperature, followed by curing at 100 °C for 5 min to further ensure covalent bond formation via dehydration between the silane groups in the copolymer and the hydroxyl groups of the substrate.7,10 Each polymer-coated COC and glass surface was characterized by measuring the static water contact angles, which were compared with those of the pristine surfaces. The water contact angle of the bare COC substrate decreased moderately from 94.5 ± 0.6° to 76.1 ± 1.5° and 75.7 ± 0.4° after surface modification with poly(DMA-r-mPEGMA-r-GMA) and poly(BMA-r-mPEGMA-r-GMA), respectively, presumably because the hydrophilic PEG component were exposed on COC surfaces. On the other hand, the water contact angle of the bare glass substrate (25.8 ± 1.2°) increased substantially to 65.6 ± 1.5° after surface modification with poly(TMSMA-r-mPEGMAr-GMA), indicating that the polymer layers were formed on the surfaces. Further, the glass surfaces coated with poly(TMSMAr-mPEGMA-r-GMA) were characterized by XPS. The C (1s) XPS intensity of the bare glass substrate (8.4%) increased considerably to 48.2%, however, the O (1s) (64.0%) and Si

previous copolymer systems is that the NHS ester is rapidly hydrolyzed under aqueous conditions at neutral or basic pH.3 To overcome this problem, in the present work, we synthesized a set of next generation random copolymers containing an epoxide as a functional group. The chemical structures of the three epoxide-containing copolymers are shown in Scheme 1a: poly(DMA-r-mPEGMA-r-GMA), poly(BMA-r-mPEGMA-rGMA), and poly(TMSMA-r-mPEGMA-r-GMA). As illustrated in Scheme 1b, dodecyl or benzyl and trimethoxysilyl moieties are used for anchoring the corresponding polymers onto COC and glass surfaces, respectively, PEG is able to block the nonspecific binding of proteins, and the epoxide moiety acts as a functional group for protein attachment. The copolymers were synthesized from three kinds of monomers by radical polymerization using a molar feed ratio of 3.5:3:3.5. 1H NMR analysis of the copolymers revealed that the actual composition of each monomer unit was 3.4:2.8:3.8 for poly(TMSMA-rmPEGMA-r-GMA), 3.3:3.4:3.3 for poly(DMA-r-mPEGMA-rGMA), and 3.7:3:3.3 for poly(BMA-r-mPEGMA-r-GMA) (Supporting Information, Figure S1). Formation of the Copolymer Adlayers on COC and Glass Surfaces. We examined whether the copolymers are able to coat hydrophobic plastic or hydrophilic glass substrates. Poly(DMA-r-mPEGMA-r-GMA) or poly(BMA-r-mPEGMA-rGMA) was used for COC surface coating, whereas poly(TMSMA-r-mPEGMA-r-GMA) was tested for coating the glass surface. COC surface coating was achieved by simply immersing the substrate into an aqueous solution of the 4509

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(2p) (27.6%) intensities decreased to 40.5% and 11.3%, respectively, after polymer coating (Table 1). These results Table 1. Elemental Composition of the Poly(TMSMA-rmPEGMA-r-GMA)-Coated Glass Surface Measured by XPS elemental composition (%) substrate

C1s

O1s

Si2p

bare glass modified glass

8.39 48.21

64.04 40.53

27.57 11.26

indicated that significant fractions of intrinsic Si and O atoms in the glass surface became buried under a more hydrophobic polymer layers containing C atoms corresponding to an alkyl chain of the copolymer after surface modification. An Evaluation of Nonspecific Protein Adsorption on the Polymer-Coated COC and Glass Surfaces. The resistance of the polymer-coated COC and glass surfaces to protein adsorption was estimated by using rhodamine-labeled antirabbit IgG as a model protein. The coated substrates were incubated in a solution containing rhodamine-labeled antirabbit IgG (40 μg/mL, PBS) at room temperature for 2 h to induce nonspecific adsorption of the antibody. The surfaces were subsequently washed and air-dried. The fluorescence intensities on the control (bare surface) and polymer-coated COC or glass surfaces were measured to quantify nonspecific IgG adsorption. Compared to the uncoated COC surface (control), the relative fluorescence intensities of poly(DMA-r-mPEGMA-r-GMA)and poly(BMA-r-mPEGMA-r-GMA)-coated surfaces were 20.4% and 21.1%, respectively, indicating that both polymerscoated COC surfaces are highly resistant to nonspecific antibody adsorption (Figure 1). In the case of the poly(TMSMA-r-mPEGMA-r-GMA)-coated glass surface, the relative fluorescence intensity was 32.7% of the uncoated glass surface (control) (Figure 2). Although the ability to block protein adsorption on the polymer-coated glass surface is lower than the case of the COC surface, these results suggest that all three epoxide-containing copolymers were capable of blocking unwanted nonspecific protein adsorption compared to bare surfaces probably due to the exposure of the multiple PEG groups. Antibody Immobilization on the Polymer-Coated COC Surfaces. The suitability of the polymer-coated COC and glass surfaces for biomolecule immobilization was examined by preparing a micropattern of antibodies, using a μCP method, a soft lithographic technique.11 Figure 3a shows a schematic representation of the entire surface modification and immobilization of rabbit IgG as a model protein on the poly(BMA-r-mPEGMA-r-GMA)-coated COC surfaces. Because the amine groups of the antibody can react with the epoxide groups of the polymer-coated surface under moderate alkaline pH conditions (borate buffer, pH 9.0),12 we expected that rabbit IgG could be immobilized onto the surface via covalent bond formation. In the first step, the rabbit IgG was contact-printed onto the reactive polymer layers by using a positive PDMS stamp with 50 × 50 μm2 circular patterns. Subsequently, rabbit IgG-patterned COC surfaces were immersed in PBS (pH 7.4) to remove nonspecifically bound rabbit IgG. The substrates were then incubated in a solution containing rhodamine-labeled antirabbit IgG to probe the rabbit IgG attachment on the stamped area at pH 7.4. Figure 3b shows fluorescence microscopy images of the rhodaminelabeled antirabbit IgG pattern in which a high apparent signal-

Figure 1. An evaluation of nonspecific protein adsorption on the polymer-coated COC surfaces. The fluorescence intensities of the control (bare COC) and polymer-coated COC surfaces were measured after incubation of rhodamine-labeled antirabbit IgG at room temperature for 2 h. The relative amounts of protein adsorbed onto each surface were calculated as the percentage of protein adsorption onto unmodified, bare COC surfaces. Signals of five spots were averaged.

to-noise ratio was seen, indicating that rabbit IgG was selectively immobilized on the polymer-modified COC surface

Figure 2. An evaluation of nonspecific protein adsorption on the polymer-coated glass surfaces. The fluorescence intensities of the control (bare glass) and polymer-coated glass surfaces were measured after incubation of rhodamine-labeled antirabbit IgG at room temperature for 2 h. The relative amounts of protein adsorbed onto each surface were calculated as the percentage of protein adsorption onto unmodified, bare glass surfaces. Signals of five spots were averaged. 4510

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Figure 3. (a) Schematic representation of the procedure for surface modification and immobilization of antibodies onto the polymer-coated COC surfaces. Poly(DMA-r-mPEGMA-r-GMA) and poly(BMA-r-mPEGMA-r-GMA) were chosen selected for COC surface modification. (b) Fluorescence microscopy images of the rhodamine-labeled antirabbit IgG patterns prepared following μCP of rabbit-IgG on the polymer-coated COC surfaces using a 50 × 50 μm circularly patterned PDMS stamp. (c) The mean fluorescence intensity of the attached rhodamine antirabbit IgG patterns obtained as in part b.

Figure 4. (a) Schematic representation of the glass surface modification process and antibody immobilization with use of poly(TMSMA-rmPEGMA-r-GMA). (b) Fluorescence microscopy images of the rhodamine-labeled anti rabbit-IgG patterns prepared following μCP of rabbit-IgG on the polymer-coated glass surfaces, using a 50 × 50 μm circular patterned stamp. (c) Mean fluorescence intensities of the immobilized rhodamine antirabbit IgG patterns obtained as in panel b.

along with low nonspecific adsorption in the background area. A comparison of the relative signal intensity in the circular areas revealed that poly(DMA-r-mPEGMA-r-GMA)-coated surface had slightly better ability to immobilize the antibody than poly(BMA-r-mPEGMA-r-GMA)-coated surface (184 ± 26 versus 158 ± 24 au, respectively) (Figure 3c). Notably, despite no treatment of blocking agents such as BSA, gelatin, or milk

proteins, the background signal in the nonpatterned areas was very low. In the conventional approaches either backprinting with PEG or precoating with a blocking reagent is necessary to minimize the nonspecific adsorption of proteins on biochip surfaces.13 However, the presence of intrinsic PEG groups in the copolymers removed the need for such extra steps in preparing protein immobilization onto the polymer-coated 4511

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Figure 5. (a) Schematic representation of the surface-modification process, orientation of the protein G, and immobilization of antibodies onto the polymer-coated COC surfaces. (b) Fluorescence microscopy images of the rhodamine-labeled antirabbit IgG patterns on the polymer-coated COC surfaces patterned with 50 × 50 μm circular patterns of protein G imprinted by μCP. (c) Mean fluorescent intensities of the immobilized rhodamine antirabbit IgG patterns obtained as in panel b.

used to immobilize different types of antibodies for a variety of applications because it can specifically bind a Fc region of an antibody and allows it to keep biologically active orientation.14 To achieve antibody immobilization, protein G was first microcontact-printed onto poly(DMA-r-mPEGMA-r-GMA)or poly(BMA-r-mPEGMA-r-GMA)-coated COC surface and subsequently rhodamine-labeled antirabbit IgG was treated (Figure 5a). As shown in Figure 5b, the rhodamine-labeled antirabbit IgG was bound exclusively to the protein Gimmobilized areas. Again, very low nonspecific binding of rhodamine-labeled IgG was observed on the background areas, which lacked protein G immobilization. In addition, relative fluorescence intensity measurements on the patterned areas revealed that the poly(DMA-r-mPEGMA-r-GMA)-coated surface showed better antibody immobilization than the poly(BMA-r-mPEGMA-r-GMA)-coated surface. (Figure 5c) These results indicate that the protein G-immobilized COC surface with the epoxide-containing copolymer coating layers may be useful in constructing antibody-based biosensors and biochips.

surfaces. To further demonstrate the usefulness of epoxide polymer compared to previous NHS ester polymer, we carried out a stability test of the poly(BMA-r-mPEGMA-r-GMA)coated COC surface under aqueous conditions. We found that the polymer-coated surface after immersion in aqueous solution for 24 h still enabled efficient immobilization of proteins (Supporting Information, Figure S2). Antibody Immobilization on the Polymer-Coated Glass Surfaces. Figure 4a shows a schematic representation of the entire antibody immobilization and detection process on the poly(TMSMA-r-mPEGMA-r-GMA)-coated glass surface. Rabbit IgG was contact-printed onto the polymer-coated glass surfaces with use of a μCP method. Rhodamine-labeled antirabbit IgG was then applied to the rabbit IgG micropatterned surface. Nonspecific adsorption of rhodamine-labeled antirabbit IgG was also very low in the areas lacking rabbit IgG immobilization probably due to the presence of multiple PEGs on the surface, resulting in a high signal-to-noise ratio (Figure 4b). A mean fluorescence intensity of the rabbit IgG immobilized circular areas was also measured by Image J (213 ± 5 au) (Figure 4c). These results demonstrate that antibodies can be easily immobilized onto the epoxidecontaining copolymer-coated glass surfaces along with a low background signal. In a stability test, we found that the poly(TMSMA-r-mPEGMA-r-GMA)-coated glass surface after immersion in aqueous solution for 24 h still enabled efficient immobilization of proteins (Supporting Information, Figure S3). In a specificity test, rhodamine-labeled antirabbit IgG was bound exclusively to the rabbit IgG-immobilized areas, whereas there was little binding of nonrelevant mouse IgG (Supporting Information, Figure S4), indicating that rabbit IgG was specifically bound to those patterned areas. Protein G-Mediated Antibody Immobilization on the Polymer-Coated COC Surfaces. Protein G has been widely

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CONCLUSION In this work, we demonstrated that epoxide-containing copolymers enabled selective immobilization of antibodies on COC plastic and glass surfaces with a high signal-to-noise ratio. The epoxide-containing copolymer coating layers on COC or glass substrate, unlike our previous NHS ester-containing copolymers, are stable enough for further biomolecule conjugation under aqueous conditions at ambient temperature. There have been a variety of applications requiring immobilization of biomolecules onto various materials’ surfaces, including biochips, biosensors, drug delivery, nanoparticle-based imaging, etc. Because the copolymers may be able to coat a variety of substrates, including other plastics, Si/SiO2, metal oxides, 4512

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carbon nanotubes, graphenes, etc., the epoxide-containing copolymers described herein may warrant applications to the fields of biochips.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of poly(TMSMA-r-mPEGMA-r-GMA), poly(DMA-r-mPEGMA-r-GMA), and poly(BMA-r-mPEGMAr-GMA) and stability and specificity test of the polymers-coated surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+82) 62-715-2504, Fax: (+82) 62-715-2484, E-mail: [email protected].

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ACKNOWLEDGMENTS We are very grateful to Dr. Yongwon Jung at Korea Research Institute of Bioscience and Biotechnology for supplying protein G. This work was supported by the R&D Program of MKE/ KEIT [10031930, Technology for future aptamer-based IVD] and by a grant from Cell Dynamics Research Center, NRF (2010-0001625, 2011-0001163).

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REFERENCES

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