Article pubs.acs.org/Langmuir
Surfactant-Induced Polymer Segregation To Produce Antifouling Surfaces via Dip-Coating with an Amphiphilic Polymer Shunsuke Yamamoto, Shigeru Kitahata, Ayane Shimomura, Kaya Tokuda, Takashi Nishino, and Tatsuo Maruyama* Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan S Supporting Information *
ABSTRACT: We propose a rational strategy to control the surface segregation of an amphiphilic copolymer in its dip-coating with a low-molecular-weight surfactant. We synthesized a water-insoluble methacrylate-based copolymer containing oligo(ethylene glycol) (OEG) (copolymer 1) and a perfluoroalkylated surfactant (surfactant 1) containing OEG. The dip-coating of copolymer 1 with surfactant 1 resulted in the segregation of surfactant 1 on the top surface of the dip-coated layer due to the high hydrophobicity of its perfluoroalkyl group. OEG moieties of surfactant 1 were accompanied by those of copolymer 1 in its segregation, allowing the OEG moieties of copolymer 1 to be located just below the top surface of the dip-coated layer. The removal of surfactant 1 produced the surface covered by the OEG moieties of the copolymer that exhibited antifouling properties. Using this strategy, we also succeeded in the introduction of carboxy groups on the dip-coated surface and demonstrated that the carboxy groups were available for the immobilization of functional molecules on the surface. properties of solid polymeric materials,22,23 molecular design of a polymer only can neither anticipate nor control the surface properties of the solid polymeric material. Thus, control over the segregation of a polymer during evaporation of a polymer solution, while displaying hydrophilic functional groups on the surface via dip-coating with a copolymer, remains challenging.24,25 In this study, we propose a rational strategy to control the polymer segregation in the dip-coating of an amphiphilic copolymer using a low-molecular-weight surfactant that can introduce hydrophilic moieties on the top surface of a polymeric substrate. Surfactant 1 and a water-insoluble methacrylate-based copolymer containing oligo(ethylene glycol) (OEG) moieties (copolymer 1, Scheme 1) were prepared. Surfactant 1 contained a perfluoroalkyl group and an OEG moiety. We hypothesized that dip-coating with a solution mixture of copolymer 1 and surfactant 1 would result in the segregation of surfactant 1 on the top surface of the resulting dip-coated layer owing to the high hydrophobicity of the perfluoroalkyl group (Scheme 1). OEG moieties of surfactant 1 are also involved in the segregation process. As a result, the OEG moieties of copolymer 1 would be located just beneath the top surface of the dip-coated layer. Removal of surfactant 1 would afford a surface comprising OEG moieties of copolymer 1.
1. INTRODUCTION Antifouling properties of solid surfaces are of great importance in biomaterials, biomedical devices, and analytical instruments. For example, formation of blood clots and nonspecific protein adsorption in such devices are problematic.1 Preparation of hydrophilic surfaces is one of the most popular methods employed to impart antifouling properties to a substrate. Poly(ethylene glycol) (PEG), 2 oligo(ethylene glycol) (OEG),3−6 and zwitterionic groups (e.g., phosphorylcholine)7,8 provide protein-repellent properties on a wide variety of surfaces. Traditional approaches to introducing such antifouling moieties into material surfaces include plasma treatment,9 silane coupling treatment,10 graft polymerization,11 self-assembled monolayer formation,12 chemical vapor deposition,13−15 and physical adsorption of an appropriate substance onto the material surfaces.16−18 However, these methods except for chemical vapor deposition rely on the surface-specific chemistry of the substrate material, and some of them are not suitable for high throughput processing. Thus, many of these methods have limited applications. Physical adsorption involves desorption to some extent. In contrast, dip-coating is a simple method that can be used for altering surface properties and for functionalizing material surfaces and does not require any specific apparatus. However, dip-coating with a water-insoluble copolymer containing PEG or zwitterionic moieties followed by drying generates a hydrophobic surface. This occurs because the hydrophobic moieties of the copolymer are preferentially segregated at the air−polymer interface to minimize the surface energy.19−21 Owing to differences between the surface and bulk © XXXX American Chemical Society
Received: April 10, 2014
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showed a F 1s peak at 700 eV (Figure 1a), indicating the presence of surfactant 1 on the dip-coated surface. Following rinsing with water, the dip-coated substrate showed a markedly reduced F 1s peak intensity at 700 eV (Figure 1b). The calculated F/C area ratio of the rinsed substrate was only 0.09, whereas that of the nonrinsed substrate was 2.1. The bare substrate and substrate dip-coated in the absence of surfactant 1 did not exhibit any F 1s peaks (Figure 1c,d). C 1s narrow spectra are summarized in Figure S1. Figure S1a shows small peaks at 293 and 291 eV that were derived from CF3 and CF2. After rinsing, these peaks disappeared or decreased (Figure S1b). These XPS results indicated that surfactant 1 was segregated on the top surface of the dip-coated layer due to its high hydrophobicity of the perfluoroalkyl group and was easily removed by rinsing the substrate with deionized water. Water Contact-Angle Measurements of the DipCoated Surfaces. The hydrophilicity of the bare and dipcoated substrates was evaluated by contact-angle measurements of water droplets (Figure 2). Three different types of
Scheme 1. Schematic Illustration of the Dip-Coating Process with Copolymer 1 and Surfactant 1 To Control the Surface Segregation of Copolymer 1
2. RESULTS AND DISCUSSION XPS Measurements of the Dip-Coated Surfaces. To demonstrate our hypothesis, an acrylic (poly(methyl methacrylate), PMMA) substrate (1 cm × 1 cm, 0.5 mm thick) was dip-coated in an ethyl acetate solution containing 4 wt % copolymer 1 and 1 wt % surfactant 1. The substrate was then dried under vacuum. The dip-coated substrate maintained its transparency. Weight measurements of the substrates before and after dip-coating revealed that the thickness of the coated layer was 1.4 ± 0.1 μm on average. The coated copolymer was resistant against desorption or dissolution in an aqueous buffer or water more than 24 h. Furthermore, no weight change was observed following immersion of the coated substrates in an aqueous solution and subsequent drying. X-ray photoelectron spectroscopy (XPS) analysis was conducted on the bare substrate and dip-coated substrate (Figure 1). The surface of the substrate dip-coated with copolymer 1 and surfactant 1 Figure 2. Water contact angles of the surfaces of the bare and dipcoated substrates. Substrates were dip-coated with 4 wt % copolymer 1 in the absence or presence of 0.2 wt % surfactant.
surfactants were investigated, i.e., surfactant 1, octaethylene glycol monododecyl ether (EG8C12), and diethylene glycol monododecyl ether (EG2C12). EG8C12 and EG2C12 featured a non-fluorinated alkyl chain and OEGs of different lengths. Substrates were dip-coated with copolymer 1 in the absence or presence of surfactants. Prior to the measurements, each substrate was rinsed with a copious amount of deionized water to remove the surfactants from the surface. The bare substrate exhibited a contact angle of 65 ± 1°, whereas the substrate coated with copolymer 1 in the absence of a surfactant exhibited a contact angle of 77 ± 2° (Figure 2). Dip-coating of the amphiphilic copolymer increased the contact angle by 12°, indicating that the hydrophobic moieties of copolymer 1 were preferentially segregated on a top surface despite the presence of the hydrophilic moieties. In contrast, the substrate coated with copolymer 1 and surfactant 1 exhibited a contact angle of 10 ± 3°, which was 55° and 67° lower respectively than those of the bare substrate and substrate dip-coated in the absence of surfactant 1. These results suggest that the presence of surfactant 1 significantly increased the hydrophilicity of the dip-coated surface, which indicated the presence of OEGs from copolymer 1 on the surface. The presence of EG8C12 in the
Figure 1. XPS spectra of the (a) substrate surface coated with copolymer 1 and surfactant 1 before rinsing with water and (b) dipcoated substrate after rinsing with water, (c) substrate surface coated with copolymer 1 only, and (d) bare PMMA substrate. B
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dip-coating process also generated a low contact angle (7 ± 1°) that was indicative of a hydrophilic surface. Dip-coating in the presence of EG2C12, which had a short OEG moiety, resulted in a contact angle of 60 ± 3°, which was only 5° lower than that of the bare substrate. The low interfacial activity and the short ethylene glycol moiety of EG2C12 did not afford surface segregation of the OEG moieties of copolymer 1. It should be noted that the high hydrophilicity of the dip-coated surface prepared in the presence of surfactant 1 was maintained even after immersing in water for 24 h at 25 °C. One of the featured characteristics of dip-coating is versatility. We then studied the dip-coating of other kinds of polymer substrates with copolymer 1. Figure 3 shows the effect
Figure 4. Protein (BSA) adsorption on the bare and dip-coated substrates. Substrates were dip-coated with 4 wt % copolymer 1 in the absence or presence of 0.2 wt % surfactant. Substrates, which were initially rinsed with water, were immersed in 1 mg/mL BSA solution for 1 h at 25 °C.
the surfaces that were coated with copolymer 1 in the presence of surfactant 1, EG8C12, and EG2C12 were 0.66 ± 0.43, 0.17 ± 0.07, and 1.2 ± 0.25 μg/cm2, respectively. Dip-coating in the presence of surfactant 1 and EG8C12 largely reduced protein uptake, indicative of the presence of low-fouling surfaces. The low adsorption amounts were comparable to those achieved by low-fouling surfaces reported previously.27 Li et al. reported that self-assembled monolayer composed of oligoethylene glycol exhibited only 18 ng/cm2 of fibrinogen, which was determined by SPR. They described that the self-assembled monolayer on gold has a densely packed monolayer of oligoethylene glycol moieties, which would contribute to the remarkably low fouling. However, their measurement method and the adsorption conditions were much different from our present study.28 Since the presence of EG8C12 gave the lowest adsorption of BSA, the effect of the EG8C12 concentration on protein adsorption was investigated (Figure 5). The amount of
Figure 3. Water contact angles of different kinds of substrates. Substrates were dip-coated with 4 wt % copolymer 1 in the absence or presence of 0.2 wt % surfactant 1. PET represents poly(ethylene terephthalate).
of dip-coating on the contact angles of different kinds of substrates. Being similar to the results of acrylic substrates, we observed the remarkable decreases in water contact angles of nylon-6,10 and poly(ethylene terephthalate) (PET) substrates after dip-coating with copolymer 1 with surfactant 1. The dipcoating without surfactant 1 also resulted in slightly higher contact angles (around 80°) than bare nylon-6,10 and poly(ethylene terephthalate) substrates. These results demonstrate the broad applicability of the present strategy. Protein Adsorption onto the Dip-Coated Surfaces. To evaluate the antifouling property of the surfaces, protein adsorption studies using bovine serum albumin (BSA) as a model foulant were conducted. The bare substrate and dipcoated substrate were immersed in a 0.1 M phosphate buffer solution (pH 7.6) containing 1 mg/mL BSA for 1 h at 25 °C, followed by rinsing with phosphate buffer. The amount of BSA adsorbed onto the substrate surfaces was measured using micro-BCA assay.26 Substrates were immersed in a mixture of the micro-BCA working solution (1 mL) and a phosphate buffer (1 mL). After incubation, the absorbance of the mixture solution (at 562 nm) was measured to determine the protein amount on the substrates. Figure 4 shows the amount of BSA adsorbed onto the substrate surfaces. The bare substrate adsorbed a BSA content of 3.6 ± 0.77 μg/cm2, whereas the surface coated with copolymer 1 in the absence of a surfactant adsorbed a BSA content of 1.7 ± 0.14 μg/cm2. As expected from the watercontact angle measurements, coating with the copolymer reduced the nonspecific adsorption of BSA on the surface to some extent. In contrast, the amounts of BSA adsorbed onto
Figure 5. Effect of concentration of EG8C12 on the amount of protein (BSA) adsorbed onto the dip-coated surface. Substrates were dipcoated with 4 wt % copolymer 1 in the absence or presence of EG8C12. Substrates, which were initially rinsed with water, were immersed in 1 mg/mL BSA solution for 1 h at 25 °C.
adsorbed protein decreased with increasing EG8C12 concentrations. Above 0.5 wt % EG8C12, the amount of adsorbed BSA reached the lowest plateau. The adsorption results were in accordance with the water-contact angle measurements, suggesting the presence of OEG moieties on the surface. C
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The above XPS, water-contact angle measurements, and protein adsorption studies strongly support our hypothesis: (1) dip-coating of a substrate with a water-insoluble copolymer containing OEG moieties followed by drying generated a relatively hydrophobic surface; (2) dip-coating with a copolymer and a low-molecular-weight surfactant resulted in the segregation of surfactant 1 on the top surface of the dipcoated layer and OEG moieties just beneath the top surface of the dip-coated layer; (3) a low-molecular-weight surfactant can be easily removed from the top surface by rinsing the substrate surface, resulting in the display of a hydrophilic surface. Immobilization of Functional Molecules on the DipCoated Surfaces. The present methodology can be used to immobilize functional molecules with a controlled surface density on a substrate, while imparting antifouling properties.18,29,30 To this effect, we synthesized another copolymer (copolymer 2, Figure 6). Copolymer 2 was composed of
presence of EG8C12. Copolymer 2 on the surface was deprotected with a HCl aqueous solution to expose the carboxy group on the surface. A cleavable fluorescent compound as a functional molecule was used for examining the immobilization process on the dip-coated surface (Scheme 2).25 The fluorescent compound has a reactive amino group and a cleavable disulfide bond, which allows the release of the fluorescent compound in the presence of a reducing agent. Thus, the amount of immobilized fluorescent compound in solution can be quantitatively determined. The cleavable fluorescent compound was conjugated with the carboxy groups on the surface using water-soluble carbodiimide. Upon reaction with a reducing agent, release of the fluorescent moiety occurred. Figure 6 shows the amount of immobilized fluorescent compound on the different substrates that were dip-coated with varying amounts of copolymer 2. As the content ratio of copolymer 2 in the copolymer mixture increased, the amount of fluorescence increased up to ∼25 pmol/cm2. The results indicated that the density of the carboxy group can be controlled by varying the mixing ratio of the two copolymers. It should be noted that the amount of fluorescent compound adsorbed onto the surface coated with copolymer 1 only (i.e., 0% copolymer 2) was 0.71 ± 0.21 pmol/cm2, whereas that on the bare substrate was 7.0 ± 4.4 pmol/cm2. This also indicates that the surface coated with copolymer 1 exhibits an antifouling property against the fluorescent compound.
Figure 6. Control over the amount of carboxy groups on the surface by dip-coating with a mixture of two different types of copolymers. Substrates were dip-coated with 2 wt % copolymer (copolymers 1 + 2) in the presence of 0.2 wt % EG8C12. The cleavable fluorescent compound was conjugated with the carboxy groups using watersoluble carbodiimide. The immobilized cleavable fluorescent compound was reduced using tris(2-carboxyethyl)phosphine hydrochloride to release the fluorescent moiety in solution.
3. CONCLUSIONS We successfully controlled polymer segregation using lowmolecular-weight surfactants during dip-coating with a polymer solution. The present strategy produced a hydrophilic and lowfouling surface via simple dip-coating with an amphiphilic copolymer. Furthermore, the use of a combined amphiphilic copolymer and copolymer containing reactive groups in dipcoating allowed the immobilization of functional molecules on the low-fouling surface. The current strategy presents great potential for the functionalization of solid-polymer surfaces and also for the preparation of biomedical and bioanalytical materials.
methyl methacrylate and methacrylate containing OEG with a carboxy group at its terminus. The carboxy group was first protected with a tert-butyl alcohol. A substrate was dip-coated in a solution mixture of copolymer 1 and copolymer 2 in the
Scheme 2. Schematic Illustration of the Measurement of Carboxy Groups on a Solid Surface Using Cleavable Fluorescent Compound
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monomer was identified by 1H NMR and ESI-MS. 1H NMR (300 MHz, CDCl3, δ): 6.58 (s, H, −NH), 5.75 (s, H, −CHCH), 5.3 (s, H, −CHCH), 3.62 (t, 28H −COCH2CH2CO), 1.42 (s, 9H, −CCH3). Synthesis of Methacrylate-Based Copolymer Containing Carboxy Groups. A random copolymer composed of methyl methacrylate (MMA, 90 mol %) and methacrylate-based monomer containing OEG-CO-t-butyl ester (10 mol %) was synthesized via free radical polymerization. MMA (1.5 mmol), the monomer (0.16 mmol), and AIBN (3.7 μmol) were dissolved in 2.2 mL of toluene in a glass vial, followed by purging with nitrogen gas. Polymerization was performed under a nitrogen atmosphere at 60 °C overnight. The copolymer was precipitated by adding the solution to 200 mL of hexane and vacuum-dried. The copolymer synthesized was identified by 1H NMR and SEC. SEC suggested that the synthesized poly(MMA-r-PEGMA) had a Mn of 1.6 × 105 g/mol and a Mw/Mn of 1.6. 1 H NMR (300 MHz, CDCl 3 , δ): 3.66 (t, 49H, −COCH2CH2CO), 1.52−2.10 (m, 20H, −CCH2), 1.42 (s, 10H, −CCH3), 0.79−1.16 (s, 14H, −CCH3). Anal. Calcd for C72H122O29: C, 58.7; H, 8.40; N, 0.964. Found: C, 58.7; H, 8.4; N, 0.96. Synthesis of Cleavable Fluorescent Compound. Cystamine (4.0 mmol) and triethylamine (1.2 mL) were dissolved in 11.5 mL of THF/water (1:2 v/v). A fluorescein isothiocyanate (FITC) (0.1 M) solution in THF (3.8 mL) was added dropwise to the solution. The solution was stirred at room temperature overnight. A HCl (1 M) aqueous solution was added to adjust the solution pH to 1−2. The resulting solid was collected by centrifugation, washed with HCl aqueous solution (1 mM, 10 mL) thrice, followed by washing with deionized water (10 mL) twice, and freeze-dried overnight. The cleavable fluorescent compound was characterized by 1H NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) (UltrafleXtreme, Bruker, Billerica, MA). 1 H NMR (300 MHz, CDCl3, δ): 6.99−6.54 (m, 9H, fluorescence), 3.16−2.94 (m, 8H, −CH2). MALDI TOF-MS (m/z): [M + Br]+ calcd for C25H23N3O5S3, 541.7; found, 542.4. Dip-Coating of Poly(MMA-r-PEGMA) on an Acrylic Substrate. Dip-coating on an acrylic substrate was carried out as follows. Poly(MMA-r-PEGMA) and surfactant 1 were dissolved in ethyl acetate to give a copolymer concentration of 4 wt % and a surfactant concentration of 0.2 wt %. A PMMA substrate (1 cm × 1 cm, 0.5 mm thick) was immersed in the copolymer solution (dip-coating) for a few seconds and dried in a vacuum desiccator overnight at 25 °C. The dipcoated substrate was rinsed with ∼10 mL of water to remove the surfactant from the surface of the dip-coated substrate. The substrate was subjected to contact angle measurements, protein adsorption experiments, and XPS analysis. X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS measurements were performed with an ESCA-850 (Shimadzu, Kyoto, Japan) equipped with a Mg Kα source (powered at 30 mA and 8 kV) at a constant dwelling time of 200 ms. The pressure in the analysis chamber was about 10−15 Pa. The spectra were calibrated relative to the C 1s binding energy (284.6 eV). Contact Angle Measurements. Contact angles of water droplets (5 μL) were measured using a digital automated contact angle goniometer (Kyowa Interface Science Co., Ltd.) at 25 °C. Twenty seconds after placing a droplet (5 μL), images of the droplets were captured, from which the contact angles were determined. The measurements were performed at three different locations on each substrate. Error bars represent standard deviations. Protein Adsorption and Assay of Protein Adsorbed on Surfaces. Bovine serum albumin (BSA) was used as a model protein for the adsorption studies. BSA was dissolved in a phosphate buffer (pH 7, 100 mM) to give a BSA concentration of 1 mg/mL. Substrates were initially rinsed with water, then immersed in the BSA solution for 1 h at 25 °C, and then rinsed with excess amounts of water. The amount of protein adsorbed onto the surfaces was measured using a micro-BCA protein assay kit (ThermoScientific, Waltham, MA). Briefly, a substrate was immersed in a mixture of a micro-BCA working reagent (1 mL) and a phosphate buffer (1 mL), followed by incubation
4. EXPERIMENTAL SECTION Materials. Methyl methacrylate (MMA), 2,2′-azobis(isobutyronitrile) (AIBN), triethylamine, N,N-dimethyl-4-aminopyridine (DMAP), N,N-dimethylformamide (DMF), chloroform, and Nhydroxysuccinimide (NHS) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Poly(ethylene glycol) methyl ether methacrylate (average Mn 300) was purchased from Sigma (St. Louis, MO). Tridecafluoroheptanoyl chloride was purchased from Aldrich (Milwaukee, WI). NH2-octaethylene glycolCOO-C(CH3) (amino-dPEG8-t-butyl ester 8-amine) was purchased from Quanta Biodesign, Ltd. (Powell, OH). Octaethylene glycol monododecyl ether (EG8C12) and diethylene glycol monododecyl ether (EG2C12) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was purchased from Dojindo Molecular Technologies Inc. (Kumamoto, Japan). Diethylene glycol monododecyl ether and tris(2carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Tokyo Chemical Industry (Tokyo, Japan). MMA was distilled at reduced pressure prior to use, and other reagents were used without further purification. The water used was high-quality deionized water (DI water, >15 MΩ·cm) produced by an Elix-5 system (Millipore, Molsheim, France). An acrylic (PMMA) substrate (1 mm thick) was obtained from Nitto Jushi Kogyo Co., Ltd. (Tokyo, Japan). Nylon-6,10 and poly(ethylene terephthalate) (PET) were obtained from Toray (Tokyo, Japan) and Hitachi Maxell, Ltd. (Tokyo, Japan), respectively. Substrates were cleaned with a detergent solution and cut into 1 cm × 1 cm pieces prior to use. Synthesis of Methacrylate-Based Copolymers Containing Oligo(ethylene glycol) (OEG). A random copolymer composed of methyl methacrylate (MMA, 90 mol %) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, average Mn 300, 10 mol %) was prepared via free radical polymerization. Inhibitor was removed from PEGMA using activated alumina. MMA (90 mmol), PEGMA (10 mmol), and AIBN (0.20 mmol) were dissolved in 50 mL of toluene in a glass vial, followed by purging with nitrogen gas. Polymerization was performed under a nitrogen atmosphere at 90 °C overnight. The copolymer was precipitated by adding the solution to 200 mL hexane and vacuum-dried. The copolymer synthesized was identified by 1H NMR and size-exclusion chromatography (SEC). The monomer compositions were determined by elemental analysis. The copolymer poly(MMA-r-PEGMA) consisted of adhesive moieties (MMA) and antifouling moieties (PEGMA). The MMA moieties served as an adhesive matrix in some polymeric substrates and are insoluble in water. Size-exclusion chromatography suggested that the synthesized poly(MMA-r-PEGMA) had a Mn of 2.5 × 105 g/mol and a Mw/Mn of 1.6. 1H NMR (300 MHz, CDCl3, δ): 3.52 (t, 68H, −COCH2CH2CO), 1.79−2.10 (m, 20H, −CCH2), 0.81−1.19 (m, 36H, −CCH3). Anal. Calcd for C60H100O25: C, 59.0; H, 8.20. Found: C, 58.7; H, 8.09. Synthesis of Surfactant 1. Octaethylene glycol monomethyl ether (1.5 mmol) and triethylamine (2.2 mmol) were dissolved in 2.5 mL of dichloromethane. Tridecafluoroheptanoyl chloride (1.45 mmol) was added dropwise to the solution. The solution was stirred at room temperature overnight. Water was added to the solution to remove octaethylene glycol monomethyl ether and triethylamine. The extraction with water was repeated three times. The dichloromethane phase was collected and evaporated. The residue was dried under vacuum overnight. The product (surfactant 1) was identified by ESIMS and 1H NMR. 1H NMR (300 MHz, CDCl3, δ): 4.60 (t, 2H, −CO2 CH 2), 3.52 (t, 26H, −COCH 2CH 2 CO), 3.40 (s, 3H, −COCH3). Synthesis of Methacrylate Monomer Containing OEG-CO-tbutyl Ester. Amino-dPEG8-t-butyl ester 8-amine (0.20 mmol) and triethylamine (0.30 mmol) were dissolved in 1 mL of dichloromethane. Methacryloyl chloride (0.21 mmol) was added dropwise to the solution. The solution was stirred at room temperature overnight. Water was added to the solution. The dichloromethane phase was collected. The extraction using an aqueous solution was repeated thrice. The dichloromethane phase was evaporated. The synthesized E
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at 60 °C for 1 h. The absorbance of the solution was measured at 562 nm. Immobilization of a Fluorescent Compound on a DipCoated Surface. The copolymer containing carboxy groups, poly(MMA-r-PEGMA), and surfactant 1 were dissolved in ethyl acetate. The total copolymer concentration was 2 wt %, and surfactant concentration was 0.2 wt %. The content of the copolymer containing carboxy groups was varied from 0 to 0.75. Dip-coating was carried out using these solutions. The copolymer-coated substrates were first immersed in deionized water to remove surfactant 1 from the surface and then immersed in 1 mL of HCl (4 M) for 1 h at 25 °C to remove the protecting tert-butyl groups. The substrates were immersed in 2 mL of phosphate buffer (0.1 M, pH 7.6) containing 5 vol % DMSO, 0.1 mM cleavable fluorescent compound, 0.2 mM NHS, and 1 mM EDC for 2 h at 25 °C (Scheme 2). The substrates were washed with phosphate buffer (5 mL) thrice and immersed in 15 mL of NaOH (1 mM) for 1.5 h at 40 °C. The NaOH aqueous solution was replaced with a fresh NaOH aqueous solution, and the solution-containing substrate was agitated for 2 h at 40 °C to wash out the nonreacted physically absorbed fluorescent compound. The substrates were finally rinsed with the phosphate buffer (5 mL) thrice and immersed in 2 mL of phosphate buffer (0.1 M, pH 7.6) containing 2 mM TCEP for 1 h at 40 °C to cleave the disulfide bonds in the fluorescent compound. The fluorescence intensity of the solution was measured using a fluorescence spectrophotometer (FP-8200, Jasco, Tokyo, Japan). The excitation and emission wavelengths were 495 and 515 nm, respectively. The excitation and emission bandwidths were 5 nm. An intermediate sensitivity setting was used. Typically, dip-coating and the quantification were carried out in triplicate unless otherwise stated.
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ASSOCIATED CONTENT
S Supporting Information *
XPS C 1s narrow spectra of bare and dip-coated substrates (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax +81-78-803-6070; e-mail
[email protected] (T.M.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported financially by a Grant-in-Aid for Challenging Exploratory Research 25630380, by The Ogasawara Foundation for the Promotion of Science & Engineering, and by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan. The authors thank Professors A. Mori and H. Matsuyama (Kobe University) for their technical help and advice.
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