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Selective adsorption of protein on micropatterned flexible poly(ethylene terephthalate) surfaces modified by vacuum ultraviolet lithography
Applied Surface Science 258 (2012) 4222–4227
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Selective adsorption of protein on micropatterned flexible poly(ethylene terephthalate) surfaces modified by vacuum ultraviolet lithography Shaoying Li, Zhongkui Wu ∗ , Hongxiao Tang, Jun Yang School of Materials Science and Engineering, Wuhan University of Technology, 122 Luoshi Rd, Wuhan 430070, China
a r t i c l e
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Article history: Received 21 September 2011 Received in revised form 5 December 2011 Accepted 5 December 2011 Available online 9 December 2011 Keywords: Self-assembled monolayer Micropatterning Selective protein adsorption VUV PET
a b s t r a c t Protein micropattern was fabricated on the flexible poly(ethylene terephthalate) (PET) surfaces modified by vacuum ultraviolet lithography (VUV). Chemical composition and topographies changes of the modified PET surfaces were characterized and analyzed by X-ray photoelectron spectroscopy (XPS), atomic force microscope (AFM) and static water contact angle. As demonstrated in fluorescence microscope, the protein patterns were surrounded by a protein-repellant layer of poly(ethylene glycol) (PEG) that were faithful reproductions of the copper mesh. These results suggested that this technique can be extended to other polymeric materials and will be useful in fields where arrays of protein patterns are desired. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The fabrication of micro and nanoscale patterns of proteins has applications in a number of fields, such as biosensors, biochips, micro electro-mechanical systems (MEMS), neuronal cell arrangement, construction of biomimetic and so on [1–4]. There have been many reports about fabricating protein patterns on rigid substrates [2,5,6] such as silicon wafer, glass and gold, because their surface properties are easily modified by forming self-assembled monolayers (SAMs) with high degree organization and physical robustness. But there are still limited utility as biomaterials due to their characteristics of expensive fabrication cost, inflexibility and opacity. Therefore, an intense attention in exploiting organic materials as substrates for protein patterning has been achieved thanks to their superiorities [7,8] of flexibility, low cost, light weight, and roll-to-roll processing. Polymeric materials such as poly(methylmethacrylate) (PMMA), polyurethane (PU), poly(dimethylsiloxane) (PDMS) and poly(ethylene terephthalate) (PET) have been widely used as the substrate materials for protein adsorption, while researches about fabricating protein patterns on organic materials have rarely been reported [9–12]. Among these common organic materials, PET exhibits many unique properties [13–15] such as its biocompatibility, transparency, flexibility, chemical resistance, light weight, and low cost. All these superiorities make PET suitable for most characterization techniques and
∗ Corresponding author. Tel.: +86 27 87653405; fax: +86 27 87651779. E-mail address: [email protected] (Z. Wu). 0169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2011.12.027
ideal for the microelectronics, cell culturing, protein adsorption and other biomedical materials applications. However, conventional patterning methods such as microcontact printing (-CP), electron beam lithography and dip-pen nanolithography (DPN) limit practical applications of organic substrates [16] because of their chemical compatibility and thermal stability during the fabrication process. Meanwhile, contamination problems, geometrical constraints on the achievable patterns, low pattern homogeneity, and alignment difficulties can constantly be met [1]. The surface modification of polymeric materials through photoinduced chemical processes, which can provide hydroxyl (OH)-bearing polymer surfaces, has attracted much attention [17,18]. Among the numerous photochemical approaches toward polymer surface processing, the use of an incoherent VUV excimer lamp is one of the most promising, because it can treat a relatively large area on a polymer substrate at a single time with a moderate light intensity. Accordingly, the characteristic penetration depth of VUV light into polymers is only several hundreds of nanometers because of the high absorption coefficients (104 ∼105 /cm) [19]. Finally, the VUV excimer lamp radiates no infrared rays and, accordingly, does not heat the sample. Therefore, thermal damage to polymer surfaces is negligibly small. In addition, polymer surfaces can be arbitrarily modified at relatively low temperatures while retaining their bulk properties intact [18]. SAMs, spontaneously formed on the modified polymer surfaces through chemisorption of organosilane molecules to OH sites on the surfaces, are widely applied to control physical and chemical properties of their surfaces [20]. Micropatterning such organosilane SAMs is a key technology to utilize the SAMs to a
S. Li et al. / Applied Surface Science 258 (2012) 4222–4227
wide variety of advanced applications, e.g., spatial arrangement of biomolecules, templates for area-selective depositions of metals, etc. [21,22]. Among various patterning methods, photolithography is most practical, since it can transfer an entire pattern on a photomask to a SAM at a single time [20]. However, these photochemistries are not applicable to photopatterning of alkylsilane SAMs; in spite that these SAMs are widely used for surface modification since more hydrophobic surfaces than those with the aromatic and mercapto SAMs can be prepared. There have been a few reports on deep-UV photopatterning of alkylsilane SAMs based on the C C bond cleavage [23]. However, this process is relatively slow. An effective method in order to photopattern alkyl SAMs has been persistently demanded. A promising alternative way is the use of VUV light whose wavelength is much shorter than 200 nm [24]. Herein, the surface modification of PET employing a VUV light 172 nm in wavelength radiated from a Xe∗2 lamp ( = 172 nm, Intensity = 10 mW/cm2 ) has provided OH bearing polymer surfaces, and 3-Aminopropyltriethoxysilane (APTES) SAMs are fabricated on the modified polymer surface through chemisorption of APTES molecules to OH sites on the surfaces. Secondly a PEG layer was grafted onto APTES SAMs via chemical modification. Thirdly the PEG layer was patterned by VUV lithography, and finally the patterned surface would serve as a template for selective protein adsorption. Bovine serum albumin (BSA) molecules would be adsorbed to designated micro areas on the surface of PET, and uniform biological patterns would be obtained. 2. Experimental 2.1. Materials APTES (NH2 (CH2 )3 Si(OC2 H5 )3 ) and 4,4 -Methylene-bis(phenyl-isocyanate) (MDI) were purchased from Aldrich Chemical Co. PEG (HO (CH2 CH2 O)n CH2 CH2 OH, Mn = 1000) was obtained from Sinopharm Chemical Reagent Co., Ltd. and was dried by azeotropic distillation with anhydrous toluene before use. MilliQ-deionized water was used for all the experiments in this study. The PET sheets were subjected to a solution (a mixture of Sodium hydroxide, Sodium carbonate anhydrous, washing powder and deionized water) at 65 ◦ C for 5 min. After being flushed with plenty of deionized water, all the sheets were extracted with acetone for 24 h in a Soxhlet extractor. Afterwards the sheets were transferred into ethanol for an ultrasonic cleaning, and finally were blown dry by nitrogen gas before use. As mentioned earlier, in order to reduce nonspecific protein adsorption, the PET substrates were designed to be grafted with a PEG layer. The whole process includes surface modification of PET (hydroxylation, self-assembled monolayers and PEG grafting), micropatterning of the PET–PEG surfaces and selective protein adsorption. The protein patterning procedure was described in Scheme 1. The clean PET sheets were exposed to UV irradiation, and uniform hydroxyl (OH)-bearing PET surfaces were obtained, followed by ammonification with APTES. Then the PEG layers were grafted onto the APTES SAMs via MDI, which binds to hydroxyl groups of PEG between amino groups of the APTES SAMs.
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so that hydrophilic hydroxyl groups can be introduced onto the PET substrates. This step is very important to the formation of APTES SAMs. 2.2.2. SAMs formed by chemical vapor deposition (CVD) After hydroxylation, all the sheets were carefully placed into a Teflon container with 50 l APTES, which was put into a small jar. The whole process was operated in a glove box, which can offer a dry and spotless environment. And then the Teflon container was cautiously removed into an oven to be heated at 120 ◦ C for 3 h, so that APTES can interact strongly with hydroxyl groups introduced onto the PET substrates modified by UV light. After that, the PET-APTES sheets were immediately gone through with a series of ultrasonic rinsing respectively in toluene, acetone, ethanol, deionized water for 5 min. At last, the PET-APTES sheets were blown dry by nitrogen gas. 2.2.3. PEG grafting After the formation of APTES SAMs, PET surfaces were modified using the approach reported elsewhere [25]. In short, PET sheets were immersed in an anhydrous toluene solution containing MDI and triethylamine. The reaction was carried out at 50 ◦ C for 100 min. After being treated by MDI, PET sheets were rinsed with anhydrous toluene three times, then immersed in another anhydrous toluene solution containing PEG, and reacted at 50 ◦ C for 24 h. After the reaction, the PET sheets were exposed to an ultrosonic cleaning with ethanol for 5 min and dried under nitrogen gas. Then the PET–PEG surfaces were obtained finally. 2.3. Surface analysis In order to investigate properties of different modified surfaces prepared in this study, surface analyses by XPS and static water contact angles have been tested. Thereinto, XPS was performed using a VG Multilab 2000 X-ray photoelectron spectrometer. All measurements were conducted using a Al K␣ (100 eV) source at a power lever of 300 W with a fixed take-off angle of 90◦ . High-resolution C1s spectra were also collected and analyzed using Thermo Advantage V3.45 software. Static water contact angle test (Automatic Contact Angle Meter Model C20 Series (SONLON TECH. (SHANGHAI))) was conducted to evaluate the hydrophilic properties of the modified surfaces. After adsorption of BSA-FITC, the micropatterned PET sheets were observed by OLYMPUS (BX51) fluorescence microscope. In addition, AFM (DI Nanoscope TV, Veeco, USA) was also applied to characterize surface morphology of the PET substrates before and after irradiation. 2.4. Micropatterned the PET–PEG surfaces and selective protein adsorption 2.4.1. Micropatterned the PET–PEG surfaces Initially, the PET–PEG sheets covered with copper meshes (Details about the meshes were illustrated in Fig. 5a) were laid onto a piece of glass slide. Then CaF2 sheets with excellent VUV transparency were blanketed onto both the PET–PEG sheets and the copper meshes. Later on all the sheets mentioned above were fixed with clips and exposed under VUV light for 15 min at room temperature in a low-pressure atmosphere of about 800 Pa. Subsequently the copper meshes were carefully removed and the micropatterned PET–PEG surfaces were successfully formed.
2.2. Surface modification of PET 2.2.1. Hydroxylation The original PET sheets were totally exposed under UV light (an excimer lamp, Ushio Inc., UER20-172 V, = 172 nm, intensity = 10 mW/cm2 ) for 10 min in atmosphere at room temperature,
2.4.2. Selective adsorption of protein Fluorescently labeled protein (BSA) with fluorescein isothiocyanate (FITC): BSA was dissolved by magneton stirring in sodium bicarbonate buffer solution (SB, pH = 8.5) at the concentration of 10 mg/ml. FITC was dissolved in anhydrous dimethylsulfoxide
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Scheme 1. Schematic of the protein patterning procedure.
(DMSO) at the concentration of 1.0 mg/ml. The labeling reaction started from adding 150 l freshly-prepared FITC/DMSO solution to the BSA solution mentioned above, gradually and slowly. After that, the mixture was transferred into a refrigerator to react for 8 h at 4 ◦ C. Soon afterwards, added NH4 Cl to terminate the reaction, and then separated the unbound FITC from the conjugate, removed impurity ions and concentrated by means of ultrafiltration. Finally, conjugated stained protein was obtained and named as BSA-FITC solution. The micropatterned PET–PEG sheets were placed into wells of a 96-well cell culture cluster (Corning, USA) respectively and carefully. And then the BSA-FITC solution (250 l) was injected into each well. A shaker was used for good adsorption. After adsorption of 2.5 h, the above-mentioned PET sheets were rinsed three times every 10 min with PBS buffer solution (pH = 7.4) and finally dried under nitrogen gas. The whole operation was conducted in a light-proof area. 3. Results and discussion Irradiation of polymers by UV light leads to the formation of a microrelief and variations in the chemical and physical properties of the surface layer, in particular, hydrophilicity and hydrophobicity, which depend on the morphology and chemical composition of the surface [26]. 3.1. Surface modification of PET The surface morphology in 3D images of PET substrates before and after irradiation has been examined by AFM. As shown in Fig. 1, discrete, spheroidal grains appeared on both of the PET substrates, while the size of these grains changed significantly after
irradiation with UV light for 10 min. According to the analysis of AFM, the diameter of grains on the PET surfaces decreased from 98.282 to 26.848 nm (mean), and the area decreased from 16,867 to 2699.4 nm2 (mean). Meanwhile, the average root-mean-square roughness (Rrms ) changed from 4.314 nm to 11.286 nm during the irradiation. More specific details about these grains were listed in Table 1. These results indicated that the PET surface after irradiation became much rougher than the original PET substrate. Main chains of PET were broken by UV light at the wavelength of 172 nm, due to its high energy. During the irradiation, lots of volatile small molecules (e.g., CO2 ) and hydrophilic groups such as COOH or OH were produced [13]. Then it can be deduced that the microrelief mentioned above was caused when small molecules volatilized. Results presented here are similar to the report [26] describing morphology changes on the PMMA surface during VUV irradiation. Measurements of static water contact angles with the use of water as a test liquid enable one to evaluate the hydrophilic properties of the modified surfaces [26,27]. Fig. 2 shows water contact angle changes of different stages of the modified PET surfaces. The hydrophilicity of the surfaces changed meaningfully as the surface modification proceeded. After UV irradiation for 10 min (PET–UV 10 min), the PET substrate became hydrophilic and exhibited lower water contact angle (25◦ ) compared with the water contact angle of PET–Control (82◦ ). This decrease is attributed to the formation of hydrophilic moieties ( COOH or OH) during UV treatment [13]. After silanization with APTES, the water contact angle of the PET substrate increased again to 47◦ , in consistent with the water contact angle of APTES SAMs in others’ literature [28]. Those hydrophilic moieties ( COOH or OH) mentioned above, which were surface-anchored and high-surface-energy, can serve as attachment points for organosilane molecules (APTES). These
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Table 1 Specific details about grains on both PET surfaces before (PET–Control) and after (PET–UV 10 min) irradiation. PET–Control Mean Height (nm) Area (nm2 ) Diameter (nm) Length (nm) Width (nm)
2.603 16,867 98.282 160.97 62.768
PET–UV 10 min Minimum −0.150 3.815 2.204 2.762 2.762
Maximum 7.484 103,137 362.38 613.73 213.19
Mean
Minimum
2.213 2699.4 26.848 46.988 21.921
−0.128 3.815 2.204 2.762 2.762
Maximum 18.881 40,607 227.38 470.95 243.77
Fig. 3. XPS spectra of (a) PET–Control (b) PET–UV 10 min (c) PET–APTES and (d) PET–PEG surfaces.
Fig. 1. Surface morphology of the PET substrates (a) before irradiation (PET–Control) and (b) after irradiation (PET–UV 10 min). The surface measuring region is 1 m × 1 m. (Unit: m).
results of water contact angles suggested that the APTES monolayer was successfully deposited onto the surface of the UV-treated PET substrate. After treatment of the amino-terminated PET substrates with MDI and then PEG, the water contact angle was 37◦ , which was in agreement with the literature reported by Popat and Desai [29]. These results suggest that the PEG chains were grafted onto the PET surfaces. The chemical composition of the PET surfaces at various stages of surface modification was determined by XPS (Fig. 3). Fig. 3a shows a typical XPS survey spectrum for the original PET surface: the characteristic signals for carbon (C1s at 285.0 eV) and oxygen (O1s at 531.7 eV) were clearly detected. Similar XPS spectrum of the original PET was reported by Yang et al. [30] in their literature. Additional nitrogen signal (N1s at 398.8 eV) was also detected. The latter feature can be attributed to the nitrogenous additive used in mold of PET sheets. After irradiation of the PET surfaces with UV light for 10 min, the characteristic signals attributed to carbon, oxygen and nitrogen were still detected (Fig. 3b). However, the oxygen signals are noticeable stronger compared with the original PET surface, as a result of additional oxygen binding to the surface during breakage of the chains of PET. No Si signals were present
Fig. 2. Static water contact angles of (a) PET–Control (b) PET–UV 10 min (c) PET–APTES and (d) PET–PEG surface.
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Fig. 4. XPS spectra of C1s for (a) PET–Control and (b) PET–PEG surface.
on both the PET–Control and PET–UV 10 min (Fig. 3a and b). After silanization with APTES, the characteristic signals attributed to carbon, oxygen, nitrogen and additional signals assigned to Si2s and Si2p at 150.5 eV, 100.1 eV respectively were detected. Silicon was detected, which indicated successful APTES-SAM deposition, since this element is only contained in this film material. Gu and Cheng [31] obtained similar XPS spectra of APTES on glass substrates and drew a similar conclusion in their study. In addition, the experimentally observed C/N ratio of APTES SAMs estimated by XPS was 7.62, very close to the theoretical maximum C/N ratio of 7.71. This result indicated APTES molecules were mostly hydrolyzed and that the APTES SAM may be close packed and highly organized [28]. For the PEG modified PET sheet (Fig. 3d), the characteristic peaks attributed to carbon, oxygen, nitrogen and silicon were again detected. Additional chlorine signal (Cl2p at 199.8 eV) was also detected. The latter feature can be attributed to the interfaces of unavoidable contamination of PET sheets during treatment and analysis. C1s spectra of the original PET substrate and the PET substrate grafted with PEG are reflected in Fig. 3. Deconvolution divided the C1s spectra into three features, which were identified according to the reported chemical shifts [17]. Spectrum (a) shows a typical C1s spectrum of the original PET substrate, consisting of three components centered at BEs of 284.6, 286.4, and 288.8 eV, corresponding to C C/C H, C O/C O C and O C O groups, respectively. The intensity ratios of these components are in good agreement with the expected values [13]. C1s spectrum of the PET substrate grafted with PEG is shown in Fig. 4b. The relative intensities of the C C
Fig. 5. (a) Optical microscope photograph of the copper mesh used as a photomask in this study. (Width of the UV exposed domains (square) was 37 m (AB), and width of the non-exposed domains (stripe) was about 16 m (BC), respectively.) (b) Fluorescence microscope photograph of BSA-FITC adsorbed patterned PET–PEG surface. Bar represents 37 m.
groups in spectra (b) have increased markedly; in contrast, the relative intensities of the C O and O C O groups have decreased. This indicates that the C C groups on the PET wafer surfaces were enhanced for the graft of PEG chains. Relative changes in the percentage of C C, C O, and C O peaks listed in Table 2 can also deduce that PEG has been grafted onto the PET substrates. Combined the water contact angles with the C1s spectra results, a conclusion can be draw that a PEG layer was successfully grafted onto the PET substrate. 3.2. Micropatterned PET–PEG surfaces and selective protein adsorption In this study, BSA was used as a model protein to characterize the effectiveness of selective protein adsorption. FITC emitted green fluorescence at 520 nm when excited at 488 nm using a laser Table 2 The relative changes in the percentage of C C, C O, and C O peaks for the different films. Composition
PET-Control
PET–PEG
C C C C
62.40% 38.14% 6.97% 8.95:5.47:1
79.75% 21.38% 2.52% 31.65:8.48:1
C O O C/C O/C O
S. Li et al. / Applied Surface Science 258 (2012) 4222–4227
(excitation wavelength of FITC is 495 nm). As mentioned in Section 2, the PET–PEG sheet was micropatterned by VUV lithography, which served as a template for selective protein adsorption. After incubation in BSA-FITC solution (1.0 mg/ml) for 2.5 h, the micropatterned PET–PEG surface was characterized by fluorescence microscope, which was used to provide qualitative information about the spatial distribution of protein on the patterned PET–PEG surface and the results were shown in Fig. 5. As shown in Fig. 5b, the fluorescence intensity in UV exposed domains (square) is stronger than that in non-exposed domains (stripe). It indicated that the protein was adsorbed on the exposed domains, where the PEG layer was ablated by the high-powered VUV excimer source; while the protein was repelled [11,32] on the non-exposed regions, in which the PEG layer still remained. Results presented here are similar to Gan et al.’s report [11]. The possible mechanism by which BSA absorbs to the exposed domains of PET surface has been explained briefly as follows. The PEG layer in the exposed domains was ablated by the high-powered VUV excimer source and resulted in the formation of hydrophilic moieties ( COOH or OH) [20,33,34]. Protein adsorption may be realized by the covalent bonding between COOH on the substrate and NH2 in the protein [35,36]. In addition, hydrophilic moieties can not only serve as attachment points for protein, but also can supply hydrophilic environment to maintain bioactivity for the protein; Hydroxyl and carboxyl groups are typical polar groups, which may form hydrogen bonds with electric negative atomic nitrogen in the protein. Furthermore, hydrogen bonds play an important role in maintaining the space structure of proteins. The fluorescence micropatterns with clear, sharp, and defined edges that are faithful reproductions of the copper mesh were obtained on the patterned PET–PEG surface, for the green fluorescence intensity is proportional to the amount of absorbed BSA-FITC. By this measure BSA was adsorbed on the designated domains and selective protein adsorption was realized finally. 4. Conclusions In summary, a simple and convenient method has been reported for realizing protein patterning on the flexible PET substrates by VUV lithography. Fluorescence microscopic observation verified that selective protein adsorption was realized and uniform protein patterns with good fidelity were obtained. By tailoring surface properties of organic substrates and using a photomask with the desired pattern, micro-and nanostructures with complex patterns can be fabricated. Results obtained in this study can be regarded as exploratory work for the research of flexible biosensors. Acknowledgments We thank Prof. Ming SUN of State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University for help
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in fluorescence microscope photograph. This work is financially supported by the National Natural Science Foundation of China (50973087/E0310). References [1] S. Lenci, L. Tedeschi, C. Domenici, C. Lande, A. Nannini, G. Pennelli, F. Pieri, S. Severi, Mater. Sci. Eng. C 30 (2010) 1221. [2] H. Sorribas, C. Padeste, L. Tiefenauer, Biomaterials 23 (2002) 893. [3] M. Yamaguchi, O. Nishimura, S.H. Lim, K. Shimokawa, T. Tamura, M. Suzuki, Colloids Surf. A: Physicochem. Eng. Aspects 284–285 (2006) 532. [4] M. Mrksich, G.M. Whitesides, Trends Biotechnol. 13 (1995) 228. [5] Q. Tang, C.H. Xu, S.Q. Shi, L.M. Zhou, Synth. Met. 147 (2004) 247. [6] S.H. Lim, M. Yamaguchi, O. Nishimura, Y. Mie, T. Tamura, B.W. Kim, M. Suzuki, J. Nanosci. Nanotechnol. 9 (2009) 7318. [7] H. Lee, S. Hong, K. Yang, K. Choi, Microelectron. Eng. 83 (2006) 323. [8] M.C. Choi, Y. Kim, C.S. Ha, Prog. Polym. Sci. 33 (2008) 581. [9] K.E. Schmalenberg, H.M. Buettner, K.E. Uhrich, Biomaterials 25 (2004) 1851. [10] H. Chen, L. Yuan, W. Song, Z.K. Wu, D. Li, Prog. Polym. Sci. 33 (2008) 1059. [11] J.B. Gan, H. Chen, F. Zhou, H. Huang, J. Zheng, W. Song, L. Yuan, Z.K. Wu, Colloids Surf. B: Biointerfaces 76 (2010) 381. [12] Z.P. Yang, A.M. Belu, A.L. Vinson, H. Sugg, A. Chilkoti, Langmuir 16 (2000) 7482. [13] A.E. Ozcam, K. Efimenko, C. Jaye, R.J. Spontak, D.A. Fischer, J. Genzer, J. Electron Spectrosc. Relat. Phenom. 172 (2009) 95. [14] C. Chollet, S. Lazare, F. Guillemot, M.C. Durrieu, Impact of RGD micro-patterns on cell adhesion, Colloids Surf. B: Biointerfaces 75 (2010) 107–114. [15] S.H. Hong, J.Y. Hwang, H. Lee, H.C. Lee, K.W. Choi, Microelectron. Eng. 86 (2009) 295. [16] J. Kretz, L. Dreeskornfeld, J. Hartwich, W. Rösner, Microelectron. Eng. 67–68 (2003) 763. [17] J.Y. Zhang, H. Esrom, U. Kogelschatz, G. Emig, Appl. Surf. Sci. 69 (1993) 299. [18] A. Hozumi, T. Masuda, K. Hayashi, H. Sugimura, O. Takai, T. Kameyama, Langmuir 18 (2002) 9022. [19] A. Höllander, J.E. Klemberg-Sapieha, M.R. Wertheimer, J. Polym. Sci. Part A: Polym. Chem. 33 (1995) 2013. [20] H. Sugimura, K. Ushiyama, A. Hozumi, O. Takai, Langmuir 16 (2000) 885. [21] S. Sawada, Y. Masuda, P.X. Zhu, K. Koumoto, Langmuir 22 (2006) 332. [22] Y. Miura, H. Sato, T. Ikeda, H. Sugimura, O. Takai, K. Kobayashi, Biomacromolecules 5 (2004) 1708. [23] B. Lee, N.A. Clark, Langmuir 14 (1998) 5498. [24] T.M. Bloomstein, M.W. Horn, M. Rothschild, R.R. Kunz, S.T. Palmacci, R.B. Goodman, J. Vac. Sci. Technol. B 15 (1997) 2112. [25] Z.K. Wu, L.B. Ding, H. Chen, L. Yuan, H. Huang, W. Song, Colloids Surf. B: Biointerfaces 69 (2009) 72. [26] A.V. Mitrofanov, O.V. Karban, A. Sugonyako, M. Lubomska, X-ray, synchrotron and neutron techniques, J. Surf. Invest. 3 (2009) 519. [27] Z. Zhu, M.J. Kelley, Appl. Surf. Sci. 236 (2004) 416. [28] X.X. Zeng, G.H. Xu, Y. Gao, Y. An, J. Phys. Chem. B 115 (2011) 450. [29] K.C. Popat, T.A. Desai, Biosens. Bioelectron. 19 (2004) 1040. [30] P. Yang, X.X. Zhang, B. Yang, H.C. Zhao, J.C. Chen, W.T. Yang, Adv. Funct. Mater. 15 (2005) 1418. [31] Q.L. Gu, X.H. Cheng, Appl. Surf. Sci. 253 (2007) 6802. [32] H. Chen, X.Y. Hu, Y.X. Zhang, D. Li, Z.K. Wu, T. Zhang, Colloids Surf. B: Biointerfaces 61 (2008) 237–243. [33] T. Koga, M. Morita, H. Ishida, H. Yakabe, S. Sasaki, O. Sakata, H. Otsuka, A. Takahara, Langmuir 21 (2005) 905–910. [34] L. Hong, H. Sugimura, T. Furukawa, O. Takai, Langmuir 19 (2003) 1966–1969. [35] N. Patel, M.C. Davies, M. Hartshorne, R.J. Heaton, C.J. Roberts, Langmuir 13 (1997) 6485. [36] A.J. Guiomar, J.T. Guthrie, S.D. Evans, Langmuir 15 (1999) 1198.
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Selective adsorption of protein on micropatterned flexible poly(ethylene terephthalate) surfaces modified by vacuum ultraviolet lithography Shaoying Li, Zhongkui Wu ∗ , Hongxiao Tang, Jun Yang School of Materials Science and Engineering, Wuhan University of Technology, 122 Luoshi Rd, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 21 September 2011 Received in revised form 5 December 2011 Accepted 5 December 2011 Available online 9 December 2011 Keywords: Self-assembled monolayer Micropatterning Selective protein adsorption VUV PET
a b s t r a c t Protein micropattern was fabricated on the flexible poly(ethylene terephthalate) (PET) surfaces modified by vacuum ultraviolet lithography (VUV). Chemical composition and topographies changes of the modified PET surfaces were characterized and analyzed by X-ray photoelectron spectroscopy (XPS), atomic force microscope (AFM) and static water contact angle. As demonstrated in fluorescence microscope, the protein patterns were surrounded by a protein-repellant layer of poly(ethylene glycol) (PEG) that were faithful reproductions of the copper mesh. These results suggested that this technique can be extended to other polymeric materials and will be useful in fields where arrays of protein patterns are desired. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The fabrication of micro and nanoscale patterns of proteins has applications in a number of fields, such as biosensors, biochips, micro electro-mechanical systems (MEMS), neuronal cell arrangement, construction of biomimetic and so on [1–4]. There have been many reports about fabricating protein patterns on rigid substrates [2,5,6] such as silicon wafer, glass and gold, because their surface properties are easily modified by forming self-assembled monolayers (SAMs) with high degree organization and physical robustness. But there are still limited utility as biomaterials due to their characteristics of expensive fabrication cost, inflexibility and opacity. Therefore, an intense attention in exploiting organic materials as substrates for protein patterning has been achieved thanks to their superiorities [7,8] of flexibility, low cost, light weight, and roll-to-roll processing. Polymeric materials such as poly(methylmethacrylate) (PMMA), polyurethane (PU), poly(dimethylsiloxane) (PDMS) and poly(ethylene terephthalate) (PET) have been widely used as the substrate materials for protein adsorption, while researches about fabricating protein patterns on organic materials have rarely been reported [9–12]. Among these common organic materials, PET exhibits many unique properties [13–15] such as its biocompatibility, transparency, flexibility, chemical resistance, light weight, and low cost. All these superiorities make PET suitable for most characterization techniques and
∗ Corresponding author. Tel.: +86 27 87653405; fax: +86 27 87651779. E-mail address: [email protected] (Z. Wu). 0169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2011.12.027
ideal for the microelectronics, cell culturing, protein adsorption and other biomedical materials applications. However, conventional patterning methods such as microcontact printing (-CP), electron beam lithography and dip-pen nanolithography (DPN) limit practical applications of organic substrates [16] because of their chemical compatibility and thermal stability during the fabrication process. Meanwhile, contamination problems, geometrical constraints on the achievable patterns, low pattern homogeneity, and alignment difficulties can constantly be met [1]. The surface modification of polymeric materials through photoinduced chemical processes, which can provide hydroxyl (OH)-bearing polymer surfaces, has attracted much attention [17,18]. Among the numerous photochemical approaches toward polymer surface processing, the use of an incoherent VUV excimer lamp is one of the most promising, because it can treat a relatively large area on a polymer substrate at a single time with a moderate light intensity. Accordingly, the characteristic penetration depth of VUV light into polymers is only several hundreds of nanometers because of the high absorption coefficients (104 ∼105 /cm) [19]. Finally, the VUV excimer lamp radiates no infrared rays and, accordingly, does not heat the sample. Therefore, thermal damage to polymer surfaces is negligibly small. In addition, polymer surfaces can be arbitrarily modified at relatively low temperatures while retaining their bulk properties intact [18]. SAMs, spontaneously formed on the modified polymer surfaces through chemisorption of organosilane molecules to OH sites on the surfaces, are widely applied to control physical and chemical properties of their surfaces [20]. Micropatterning such organosilane SAMs is a key technology to utilize the SAMs to a
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wide variety of advanced applications, e.g., spatial arrangement of biomolecules, templates for area-selective depositions of metals, etc. [21,22]. Among various patterning methods, photolithography is most practical, since it can transfer an entire pattern on a photomask to a SAM at a single time [20]. However, these photochemistries are not applicable to photopatterning of alkylsilane SAMs; in spite that these SAMs are widely used for surface modification since more hydrophobic surfaces than those with the aromatic and mercapto SAMs can be prepared. There have been a few reports on deep-UV photopatterning of alkylsilane SAMs based on the C C bond cleavage [23]. However, this process is relatively slow. An effective method in order to photopattern alkyl SAMs has been persistently demanded. A promising alternative way is the use of VUV light whose wavelength is much shorter than 200 nm [24]. Herein, the surface modification of PET employing a VUV light 172 nm in wavelength radiated from a Xe∗2 lamp ( = 172 nm, Intensity = 10 mW/cm2 ) has provided OH bearing polymer surfaces, and 3-Aminopropyltriethoxysilane (APTES) SAMs are fabricated on the modified polymer surface through chemisorption of APTES molecules to OH sites on the surfaces. Secondly a PEG layer was grafted onto APTES SAMs via chemical modification. Thirdly the PEG layer was patterned by VUV lithography, and finally the patterned surface would serve as a template for selective protein adsorption. Bovine serum albumin (BSA) molecules would be adsorbed to designated micro areas on the surface of PET, and uniform biological patterns would be obtained. 2. Experimental 2.1. Materials APTES (NH2 (CH2 )3 Si(OC2 H5 )3 ) and 4,4 -Methylene-bis(phenyl-isocyanate) (MDI) were purchased from Aldrich Chemical Co. PEG (HO (CH2 CH2 O)n CH2 CH2 OH, Mn = 1000) was obtained from Sinopharm Chemical Reagent Co., Ltd. and was dried by azeotropic distillation with anhydrous toluene before use. MilliQ-deionized water was used for all the experiments in this study. The PET sheets were subjected to a solution (a mixture of Sodium hydroxide, Sodium carbonate anhydrous, washing powder and deionized water) at 65 ◦ C for 5 min. After being flushed with plenty of deionized water, all the sheets were extracted with acetone for 24 h in a Soxhlet extractor. Afterwards the sheets were transferred into ethanol for an ultrasonic cleaning, and finally were blown dry by nitrogen gas before use. As mentioned earlier, in order to reduce nonspecific protein adsorption, the PET substrates were designed to be grafted with a PEG layer. The whole process includes surface modification of PET (hydroxylation, self-assembled monolayers and PEG grafting), micropatterning of the PET–PEG surfaces and selective protein adsorption. The protein patterning procedure was described in Scheme 1. The clean PET sheets were exposed to UV irradiation, and uniform hydroxyl (OH)-bearing PET surfaces were obtained, followed by ammonification with APTES. Then the PEG layers were grafted onto the APTES SAMs via MDI, which binds to hydroxyl groups of PEG between amino groups of the APTES SAMs.
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so that hydrophilic hydroxyl groups can be introduced onto the PET substrates. This step is very important to the formation of APTES SAMs. 2.2.2. SAMs formed by chemical vapor deposition (CVD) After hydroxylation, all the sheets were carefully placed into a Teflon container with 50 l APTES, which was put into a small jar. The whole process was operated in a glove box, which can offer a dry and spotless environment. And then the Teflon container was cautiously removed into an oven to be heated at 120 ◦ C for 3 h, so that APTES can interact strongly with hydroxyl groups introduced onto the PET substrates modified by UV light. After that, the PET-APTES sheets were immediately gone through with a series of ultrasonic rinsing respectively in toluene, acetone, ethanol, deionized water for 5 min. At last, the PET-APTES sheets were blown dry by nitrogen gas. 2.2.3. PEG grafting After the formation of APTES SAMs, PET surfaces were modified using the approach reported elsewhere [25]. In short, PET sheets were immersed in an anhydrous toluene solution containing MDI and triethylamine. The reaction was carried out at 50 ◦ C for 100 min. After being treated by MDI, PET sheets were rinsed with anhydrous toluene three times, then immersed in another anhydrous toluene solution containing PEG, and reacted at 50 ◦ C for 24 h. After the reaction, the PET sheets were exposed to an ultrosonic cleaning with ethanol for 5 min and dried under nitrogen gas. Then the PET–PEG surfaces were obtained finally. 2.3. Surface analysis In order to investigate properties of different modified surfaces prepared in this study, surface analyses by XPS and static water contact angles have been tested. Thereinto, XPS was performed using a VG Multilab 2000 X-ray photoelectron spectrometer. All measurements were conducted using a Al K␣ (100 eV) source at a power lever of 300 W with a fixed take-off angle of 90◦ . High-resolution C1s spectra were also collected and analyzed using Thermo Advantage V3.45 software. Static water contact angle test (Automatic Contact Angle Meter Model C20 Series (SONLON TECH. (SHANGHAI))) was conducted to evaluate the hydrophilic properties of the modified surfaces. After adsorption of BSA-FITC, the micropatterned PET sheets were observed by OLYMPUS (BX51) fluorescence microscope. In addition, AFM (DI Nanoscope TV, Veeco, USA) was also applied to characterize surface morphology of the PET substrates before and after irradiation. 2.4. Micropatterned the PET–PEG surfaces and selective protein adsorption 2.4.1. Micropatterned the PET–PEG surfaces Initially, the PET–PEG sheets covered with copper meshes (Details about the meshes were illustrated in Fig. 5a) were laid onto a piece of glass slide. Then CaF2 sheets with excellent VUV transparency were blanketed onto both the PET–PEG sheets and the copper meshes. Later on all the sheets mentioned above were fixed with clips and exposed under VUV light for 15 min at room temperature in a low-pressure atmosphere of about 800 Pa. Subsequently the copper meshes were carefully removed and the micropatterned PET–PEG surfaces were successfully formed.
2.2. Surface modification of PET 2.2.1. Hydroxylation The original PET sheets were totally exposed under UV light (an excimer lamp, Ushio Inc., UER20-172 V, = 172 nm, intensity = 10 mW/cm2 ) for 10 min in atmosphere at room temperature,
2.4.2. Selective adsorption of protein Fluorescently labeled protein (BSA) with fluorescein isothiocyanate (FITC): BSA was dissolved by magneton stirring in sodium bicarbonate buffer solution (SB, pH = 8.5) at the concentration of 10 mg/ml. FITC was dissolved in anhydrous dimethylsulfoxide
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Scheme 1. Schematic of the protein patterning procedure.
(DMSO) at the concentration of 1.0 mg/ml. The labeling reaction started from adding 150 l freshly-prepared FITC/DMSO solution to the BSA solution mentioned above, gradually and slowly. After that, the mixture was transferred into a refrigerator to react for 8 h at 4 ◦ C. Soon afterwards, added NH4 Cl to terminate the reaction, and then separated the unbound FITC from the conjugate, removed impurity ions and concentrated by means of ultrafiltration. Finally, conjugated stained protein was obtained and named as BSA-FITC solution. The micropatterned PET–PEG sheets were placed into wells of a 96-well cell culture cluster (Corning, USA) respectively and carefully. And then the BSA-FITC solution (250 l) was injected into each well. A shaker was used for good adsorption. After adsorption of 2.5 h, the above-mentioned PET sheets were rinsed three times every 10 min with PBS buffer solution (pH = 7.4) and finally dried under nitrogen gas. The whole operation was conducted in a light-proof area. 3. Results and discussion Irradiation of polymers by UV light leads to the formation of a microrelief and variations in the chemical and physical properties of the surface layer, in particular, hydrophilicity and hydrophobicity, which depend on the morphology and chemical composition of the surface [26]. 3.1. Surface modification of PET The surface morphology in 3D images of PET substrates before and after irradiation has been examined by AFM. As shown in Fig. 1, discrete, spheroidal grains appeared on both of the PET substrates, while the size of these grains changed significantly after
irradiation with UV light for 10 min. According to the analysis of AFM, the diameter of grains on the PET surfaces decreased from 98.282 to 26.848 nm (mean), and the area decreased from 16,867 to 2699.4 nm2 (mean). Meanwhile, the average root-mean-square roughness (Rrms ) changed from 4.314 nm to 11.286 nm during the irradiation. More specific details about these grains were listed in Table 1. These results indicated that the PET surface after irradiation became much rougher than the original PET substrate. Main chains of PET were broken by UV light at the wavelength of 172 nm, due to its high energy. During the irradiation, lots of volatile small molecules (e.g., CO2 ) and hydrophilic groups such as COOH or OH were produced [13]. Then it can be deduced that the microrelief mentioned above was caused when small molecules volatilized. Results presented here are similar to the report [26] describing morphology changes on the PMMA surface during VUV irradiation. Measurements of static water contact angles with the use of water as a test liquid enable one to evaluate the hydrophilic properties of the modified surfaces [26,27]. Fig. 2 shows water contact angle changes of different stages of the modified PET surfaces. The hydrophilicity of the surfaces changed meaningfully as the surface modification proceeded. After UV irradiation for 10 min (PET–UV 10 min), the PET substrate became hydrophilic and exhibited lower water contact angle (25◦ ) compared with the water contact angle of PET–Control (82◦ ). This decrease is attributed to the formation of hydrophilic moieties ( COOH or OH) during UV treatment [13]. After silanization with APTES, the water contact angle of the PET substrate increased again to 47◦ , in consistent with the water contact angle of APTES SAMs in others’ literature [28]. Those hydrophilic moieties ( COOH or OH) mentioned above, which were surface-anchored and high-surface-energy, can serve as attachment points for organosilane molecules (APTES). These
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Table 1 Specific details about grains on both PET surfaces before (PET–Control) and after (PET–UV 10 min) irradiation. PET–Control Mean Height (nm) Area (nm2 ) Diameter (nm) Length (nm) Width (nm)
2.603 16,867 98.282 160.97 62.768
PET–UV 10 min Minimum −0.150 3.815 2.204 2.762 2.762
Maximum 7.484 103,137 362.38 613.73 213.19
Mean
Minimum
2.213 2699.4 26.848 46.988 21.921
−0.128 3.815 2.204 2.762 2.762
Maximum 18.881 40,607 227.38 470.95 243.77
Fig. 3. XPS spectra of (a) PET–Control (b) PET–UV 10 min (c) PET–APTES and (d) PET–PEG surfaces.
Fig. 1. Surface morphology of the PET substrates (a) before irradiation (PET–Control) and (b) after irradiation (PET–UV 10 min). The surface measuring region is 1 m × 1 m. (Unit: m).
results of water contact angles suggested that the APTES monolayer was successfully deposited onto the surface of the UV-treated PET substrate. After treatment of the amino-terminated PET substrates with MDI and then PEG, the water contact angle was 37◦ , which was in agreement with the literature reported by Popat and Desai [29]. These results suggest that the PEG chains were grafted onto the PET surfaces. The chemical composition of the PET surfaces at various stages of surface modification was determined by XPS (Fig. 3). Fig. 3a shows a typical XPS survey spectrum for the original PET surface: the characteristic signals for carbon (C1s at 285.0 eV) and oxygen (O1s at 531.7 eV) were clearly detected. Similar XPS spectrum of the original PET was reported by Yang et al. [30] in their literature. Additional nitrogen signal (N1s at 398.8 eV) was also detected. The latter feature can be attributed to the nitrogenous additive used in mold of PET sheets. After irradiation of the PET surfaces with UV light for 10 min, the characteristic signals attributed to carbon, oxygen and nitrogen were still detected (Fig. 3b). However, the oxygen signals are noticeable stronger compared with the original PET surface, as a result of additional oxygen binding to the surface during breakage of the chains of PET. No Si signals were present
Fig. 2. Static water contact angles of (a) PET–Control (b) PET–UV 10 min (c) PET–APTES and (d) PET–PEG surface.
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Fig. 4. XPS spectra of C1s for (a) PET–Control and (b) PET–PEG surface.
on both the PET–Control and PET–UV 10 min (Fig. 3a and b). After silanization with APTES, the characteristic signals attributed to carbon, oxygen, nitrogen and additional signals assigned to Si2s and Si2p at 150.5 eV, 100.1 eV respectively were detected. Silicon was detected, which indicated successful APTES-SAM deposition, since this element is only contained in this film material. Gu and Cheng [31] obtained similar XPS spectra of APTES on glass substrates and drew a similar conclusion in their study. In addition, the experimentally observed C/N ratio of APTES SAMs estimated by XPS was 7.62, very close to the theoretical maximum C/N ratio of 7.71. This result indicated APTES molecules were mostly hydrolyzed and that the APTES SAM may be close packed and highly organized [28]. For the PEG modified PET sheet (Fig. 3d), the characteristic peaks attributed to carbon, oxygen, nitrogen and silicon were again detected. Additional chlorine signal (Cl2p at 199.8 eV) was also detected. The latter feature can be attributed to the interfaces of unavoidable contamination of PET sheets during treatment and analysis. C1s spectra of the original PET substrate and the PET substrate grafted with PEG are reflected in Fig. 3. Deconvolution divided the C1s spectra into three features, which were identified according to the reported chemical shifts [17]. Spectrum (a) shows a typical C1s spectrum of the original PET substrate, consisting of three components centered at BEs of 284.6, 286.4, and 288.8 eV, corresponding to C C/C H, C O/C O C and O C O groups, respectively. The intensity ratios of these components are in good agreement with the expected values [13]. C1s spectrum of the PET substrate grafted with PEG is shown in Fig. 4b. The relative intensities of the C C
Fig. 5. (a) Optical microscope photograph of the copper mesh used as a photomask in this study. (Width of the UV exposed domains (square) was 37 m (AB), and width of the non-exposed domains (stripe) was about 16 m (BC), respectively.) (b) Fluorescence microscope photograph of BSA-FITC adsorbed patterned PET–PEG surface. Bar represents 37 m.
groups in spectra (b) have increased markedly; in contrast, the relative intensities of the C O and O C O groups have decreased. This indicates that the C C groups on the PET wafer surfaces were enhanced for the graft of PEG chains. Relative changes in the percentage of C C, C O, and C O peaks listed in Table 2 can also deduce that PEG has been grafted onto the PET substrates. Combined the water contact angles with the C1s spectra results, a conclusion can be draw that a PEG layer was successfully grafted onto the PET substrate. 3.2. Micropatterned PET–PEG surfaces and selective protein adsorption In this study, BSA was used as a model protein to characterize the effectiveness of selective protein adsorption. FITC emitted green fluorescence at 520 nm when excited at 488 nm using a laser Table 2 The relative changes in the percentage of C C, C O, and C O peaks for the different films. Composition
PET-Control
PET–PEG
C C C C
62.40% 38.14% 6.97% 8.95:5.47:1
79.75% 21.38% 2.52% 31.65:8.48:1
C O O C/C O/C O
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(excitation wavelength of FITC is 495 nm). As mentioned in Section 2, the PET–PEG sheet was micropatterned by VUV lithography, which served as a template for selective protein adsorption. After incubation in BSA-FITC solution (1.0 mg/ml) for 2.5 h, the micropatterned PET–PEG surface was characterized by fluorescence microscope, which was used to provide qualitative information about the spatial distribution of protein on the patterned PET–PEG surface and the results were shown in Fig. 5. As shown in Fig. 5b, the fluorescence intensity in UV exposed domains (square) is stronger than that in non-exposed domains (stripe). It indicated that the protein was adsorbed on the exposed domains, where the PEG layer was ablated by the high-powered VUV excimer source; while the protein was repelled [11,32] on the non-exposed regions, in which the PEG layer still remained. Results presented here are similar to Gan et al.’s report [11]. The possible mechanism by which BSA absorbs to the exposed domains of PET surface has been explained briefly as follows. The PEG layer in the exposed domains was ablated by the high-powered VUV excimer source and resulted in the formation of hydrophilic moieties ( COOH or OH) [20,33,34]. Protein adsorption may be realized by the covalent bonding between COOH on the substrate and NH2 in the protein [35,36]. In addition, hydrophilic moieties can not only serve as attachment points for protein, but also can supply hydrophilic environment to maintain bioactivity for the protein; Hydroxyl and carboxyl groups are typical polar groups, which may form hydrogen bonds with electric negative atomic nitrogen in the protein. Furthermore, hydrogen bonds play an important role in maintaining the space structure of proteins. The fluorescence micropatterns with clear, sharp, and defined edges that are faithful reproductions of the copper mesh were obtained on the patterned PET–PEG surface, for the green fluorescence intensity is proportional to the amount of absorbed BSA-FITC. By this measure BSA was adsorbed on the designated domains and selective protein adsorption was realized finally. 4. Conclusions In summary, a simple and convenient method has been reported for realizing protein patterning on the flexible PET substrates by VUV lithography. Fluorescence microscopic observation verified that selective protein adsorption was realized and uniform protein patterns with good fidelity were obtained. By tailoring surface properties of organic substrates and using a photomask with the desired pattern, micro-and nanostructures with complex patterns can be fabricated. Results obtained in this study can be regarded as exploratory work for the research of flexible biosensors. Acknowledgments We thank Prof. Ming SUN of State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University for help
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