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Molecular recognition of Langmuir–Blodgett polymer films containing uracil groups
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Colloids and Surfaces A: Physicochem. Eng. Aspects 321 (2008) 60–64
Molecular recognition of Langmuir–Blodgett polymer films containing uracil groups N. Sugiyama ∗ , M. Hirakawa, H. Zhu, Y. Takeoka, M. Rikukawa Department of Chemistry, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan Received 16 July 2007; received in revised form 5 January 2008; accepted 15 January 2008 Available online 19 January 2008
Abstract Novel copolymers, poly(acryroyloxymethyluracil-co-hexylacrylamide)s (poly(AU-co-HAAm)s), have been synthesized by radical copolymerization with different monomer ratios. The poly(AU-co-HAAm)s formed stable monolayers at the air–water interface and could be deposited on solid substrates as Y-type films by the vertical dipping method. The molecular structure of poly(AU-co-HAAm) LB films was determined by reflection absorption and transmission FT-IR, and X-ray diffraction measurements. Evidence for preferential orientation of both the AU and HAAm units was found. The LB films deposited on quartz crystal microbalance substrates adsorbed adenosine molecules, and the adsorbed amounts increased with higher contents of uracil groups in poly(AU-co-HAAm)s. Some morphology changes of LB films were also observed by atomic force microscopy (AFM) after the molecular recognition. © 2008 Elsevier B.V. All rights reserved. Keywords: Uracil; Molecular recognition; Langmuir–Blodgett; Molecular orientation
1. Introduction The Langmuir–Blodgett (LB) technique provides many possibilities for the control of film thickness, dimensions, and molecular structures on the nanometer scale, and the air–water interfaces are an attractive area to investigate the chemical and physical interactions of functional molecules and polymers. As a result of sophisticated nano-technology, well-ordered thin films can be fabricated for the application of various electrical and optical devices [1–4]. The numbers of researches on LB films based on functional polymers have increased because of feasible applications in many fields such as biosensors [5,6], microlithography for data storage [7], and drug delivery systems [8,9]. The stereo-complexes and the hydrolysis of biodegradable polymers have also been investigated at the air–water interfaces of LB systems [10,11]. Nucleic acid bases and their derivatives have been widely studied as moieties that can participate in hydrogen-bonding in organic solvents [12], and at the air–water interface [13]. In particular, the molecular recognition of nucleic acid base
∗
Corresponding author. Tel.: +81 3 3238 4312; fax: +81 3 3238 3361. E-mail address: [email protected] (N. Sugiyama).
0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.01.020
derivatives in water has attracted much attention [14] because there are competitive interactions between the nucleic acid components and water molecules through the hydrogen-bonded bridges. Here, uracil has the strongest hydrogen-bonding ability since N–H and C O groups in the uracil moiety act as both the hydrogen-bonding donors and acceptors. Introduction of uracil units on polymers is an attractive strategy in order to investigate the presence of intramolecular and intermolecular interactions of functional polymers and biological materials through the hydrogen bonds of the uracil units [15]. For example, Aoki et al. reported that the uracil groups of poly(6-(acryroyloxymethyl)uracil) (poly(AU)) affected the phase transition temperature in response to species of the additive materials [16]. In this manner, LB polymer films with uracil groups are expected to be available for highly sensitive biosensors because of their specific molecular recognition ability for physiologically active substances. However, there are few reports about LB polymer films having uracil groups for such purposes. In this paper, novel amphiphilic copolymers, poly(acryroyloxymethyluracilco-hexylacrylamide)s (poly(AU-co-HAAm)s), with various amounts of uracil groups were synthesized to study the molecular recognition of the uracil groups on the LB films. These LB films were characterized by FT-IR, XRD, QCM, and AFM mea-
N. Sugiyama et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 321 (2008) 60–64
surements, and their monolayer formation, orientation structure, and selectivity of molecular recognition were investigated. 2. Experiments 2.1. Synthesis and characterization of poly(AU-co-HAAm)s 6-(Acryroyloxymethyl)uracil (AU) was synthesized according to the procedure described by Brahme et al. [16,17]. 1 H NMR (DMSO-d6 ) δ: 4.83 (s, 2H, O–CH2 ); 5.54 (s, 1H, 5-CH); 6.08 (m, 1H, vinilic CH); 6.43–6.22 (m, 2H, vinilic CH2 ); 11.05 (broad, 2H, NH) ppm. EI-MS: m/e, 196(M), 153(M+ -HNCO), 141(M+ -CH2 CHCO), 55(CH2 CHCO). N-Hexylacrylamide monomer (HAAm) was synthesized by the reaction of acrylamide with 1-bromohexane in anhydrous dimethylformamide (DMF) in the presence of KOH [18]. 1 H NMR (CDCl3 ) δ: 0.94 (m, 3H, CH3 ); 1.31 (m, 4H, CH3 CH2 CH2 CH2 –); 1.52 (m, 2H, CH3 CH2 –); 1.74 (s, 2H, CH3 CH2 CH2 CH2 CH2 –); 3.32 (q, 2H, CH3 CH2 CH2 CH2 CH2 CH2 –); 5.73 (broad, 1H, NH); 6.27–5.62 (m, 3H, vinilic H) ppm. EI-MS: 155(M), 140(M+ -CH3 ), 126, 112, 98, 84, 70(140-nCH2 ), 55(CH2 CHCO). Amphiphilic poly(AU-co-HAAm)s with different AU:HAAm molar ratios were synthesized by radical polymerization. The synthetic route is illustrated in Scheme 1. The monomers and 2,2 -azobis-(isobutyronitrile) (AIBN), which was used as an initiator, were degassed and sealed in an ampoule, and then were polymerized by shaking for 40 h at 70 ◦ C. The resulting polymers were precipitated by pouring the reacted solution into acetone and were purified by reprecipitation with DMSO and acetone. The chemical structure and copolymer composition were characterized by 1 H NMR, FT-IR, and elemental analyses. 2.2. Fabrication and characterization of LB films Monolayers were spread from a mixed solution of DMF and chloroform (50 mg/L, DMF content: 10%) on a water subphase that was purified with a TORAYPURE LV10-T system (TORAY). The surface pressure–area (π–A) isotherms were measured on an LB-200 (Nippon Laser & Electronics) at 20 ◦ C
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with a compression speed of 15 mm min−1 . Multilayer films were transferred onto various substrates by the vertical dipping method at 20 ◦ C and 25 mN m−1 . A dipping speed of 13–15 mm min−1 was used. Waiting time at the top position was 30 min and that at the bottom position was 34 s for all subsequent dips. The films were dried for 1 h after depositing the third layer. FT-IR spectroscopy was performed on a Nicolet Magna-IR 750. The LB films were deposited on Pt-coated substrates for measuring reflection–absorption FT-IR spectra and were deposited on zinc selenide (ZnSe) substrates for measuring transmission FTIR spectra. X-ray diffraction patterns of the obtained LB films on glass slides were collected at r.t. using Ni-filtered Cu–K␣ radiation on a Rigaku RAD-IIA (RINT2000) diffractometer at 40 kV and 30 mA. A commercially available quartz crystal microbalance (QCM, SEIKO EG&G) was used with a polished 9 MHz AT-cut quartz crystal (area size: 0.189 cm2 ). The topology and microstructure of obtained LB films before and after the molecular recognition examinations were evaluated by AFM studies, which were performed with a Nanoscope IIIA (Digital Instruments Inc.) operated in the tapping mode.
3. Results and discussion Fig. 1 shows the FT-IR spectra of AU, HAAm, and poly(AU-co-HAAm) (uracil content: 21 mol%) in pressed KBr pellets. Poly(AU-co-HAAm) containing 21 mol% of AU units is expressed as poly(AU-co-HAAm) (21%). Two absorption bands at 1625 cm−1 in the AU and HAAm spectra were assigned to C C group (stretching vibration) and were not observed in the poly(AU-co-HAAm) spectrum. The N–H stretching broad band around 3300 cm−1 , the C O stretching peak at 1750 cm−1 , and the N–H deforming vibration peak at 1460 cm−1 of uracil group were observed in the AU and poly(AU-co-HAAm) spectra. The several peaks in the range of 3000–2800 cm−1 attributed to alkyl chains were also observed in HAAm and poly(AU-co-HAAm) spectra. The molecular weights of copolymers are summarized in Table 1. While the poly(HAAm) and poly(AU) had MW = 65,000 and 58,000, the molecular weights of poly(AUco-HAAm)s with various molar ratios were about 11,000, with a polydispersity of 1.2–1.3. 1 H NMR spectra of poly(AU-co-
Scheme 1. Synthesis of HAAm, AU, and poly(AU-co-HAAm)s.
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Fig. 1. Transmission FT-IR spectra of AU (a), HAAm (b), and poly(AU-coHAAm) (21%) (c). Fig. 3. Schematic view of Langmuir monolayer of poly(AU-co-HAAm) and CPK models of AU and HAAm.
Fig. 2. Surface pressure–area (π–A) isotherms of poly(HAAm), poly(AU), and poly(AU-co-HAAm)s at 20 ◦ C.
HAAm)s showed each chemical shift for uracil rings of AU and hexyl groups of HAAm. The copolymer compositions were calculated by the ratio between the CH of AU ring (5.5 ppm) and the CH3 of hexyl group (0.8 ppm), and were consistent with the results of elemental analysis. These results indicate the poly(AUco-HAAm) copolymers were successfully synthesized by our procedure. Fig. 2 shows π–A isotherms of the homopolymers and poly(AU-co-HAAm)s with various AU contents at 20 ◦ C. Although poly(AU) monolayers were unstable, poly(AU-co-
HAAm)s and poly(HAAm) exhibited stable condensed solid phases up to 20 mN m−1 , indicating that the long alkyl chains of HAAm units resulted in stable amphiphilic properties. The areas per unit of poly(AU-co-HAAm)s listed in Table 1 were esti˚ 2 , which were larger than that of poly(AU) mated to be 9.3–30 A 2 ˚ ). The areas of cross section of HAAm and AU molecules (8.9 A estimated from the π–A isotherms agreed with the areas calculated by the Corey–Pauling–Koltun (CPK) model as shown in Fig. 3. This result suggests that the plane of the uracil ring in poly(AU-co-HAAm)s is orientated perpendicular to the water surface. Consequently, the AU groups in poly(AU-co-HAAm)s provide enough hydrophilicity to form stable monolayers at the air–water interface. Monolayers of poly(AU-co-HAAm) (21%) and poly(AUco-HAAm) (45%) were transferred onto various substrates as Y-type LB films with nearly 100% transfer ratio. This may be due to the flexibility and excellent amphiphilic balance of poly(AUco-HAAm) monolayers. The strong intermolecular interaction of uracil groups may also have a positive effect on the excellent deposition. Poly(AU-co-HAAm) (67%) and poly(AU) monolayers, however, could not be transferred sufficiently. This suggests that excess uracil content perturbs the amphiphilic balance of copolymers and causes a molecular aggregation of uracil groups. Reflection absorption FT-IR (RAIR) and transmission FTIR (TIR) have been shown to provide direct information about
Table 1 Characteristics of poly(AU-co-HAAm)s Abbreviation
AU molar ratioa (%)
Mw b
PDIb
˚ 2) Area per unitc (A
Poly(HAAm) Poly(AU-co-HAAm) (21%) Poly(AU-co-HAAm) (45%) Poly(AU-co-HAAm) (67%) Poly(AU)
0 21.3 45.4 67.0 100
65,000 12,000 11,400 11,500 58,000
2.24 1.21 1.27 1.27 1.93
34 30 15 9.3 8.9
a b c
Calculated by 1 H NMR. Determined by GPC (DMF eluent). Estimated by π–A isotherms.
N. Sugiyama et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 321 (2008) 60–64
Fig. 4. Reflection–absorption (R) and transmission (T) FT-IR spectra of poly(AU-co-HAAm) (21%) LB films with ten layers.
the molecular orientation of the various molecules in LB films. Function groups oriented perpendicular to the film substrate will exhibit stronger vibrational bands in RAIR than in TIR. As can be seen in Fig. 4, the absorption bands at 1750 cm−1 and 1200 cm−1 due to the stretching vibration of two C O groups and stretching vibration of C–N of the uracil ring was noticeably stronger and more defined in the RAIR of a poly(AU-co-HAAm) (21%) LB film than those in the TIR. The asymmetric (2920 cm−1 ) and symmetric (2852 cm−1 ) CH2 stretching vibrations observed in the TIR exhibit significantly stronger intensities than the asymmetric (2957 cm−1 ) and symmetric (2879 cm−1 ) CH3 stretching vibrations, whereas in the RAIR, the asymmetric and symmetric CH2 stretching vibrations are more comparable in intensities to their CH3 counterparts. This type of polarization dependence is characteristic of highly ordered hydrocarbon tail groups oriented with their fully extended chains nearly perpendicular to the substrate. The absorption band around 3300 cm−1 attributed to the stretching vibration of N–H groups was observed stronger in RAIR than in TIR, indicating that N–H groups take a perpendicular direction to the substrate. These results suggest that the uracil rings and hydrocarbon tails in the poly(AU-co-HAAm) (21%) LB film are preferentially orientated normal to the surface. The poly(AU-co-HAAm) LB films showed only one order of diffraction, which implied a thickness per monolayer of about ˚ Assuming that the copolymer adopts the configuration 34.0 A. that is shown in Fig. 3, we can conclude that the thickness estimated from XRD data is compatible with the single layer spacing of copolymer calculated by the CPK model. Fig. 5 exhibits the molecular recognition behavior of poly(AU-co-HAAm) (21%) LB films deposited on QCM substrates. When a QCM substrate of poly(AU-co-HAAm) (21%) LB film with six layers was soaked into a 0.5 mM adenosine aqueous solution (pH 5.4), a linear increase in mass (−F) was observed and the mass reached a constant value after 30 min. The amount of adenosine adsorption is calculated to be 237 ng cm−2 from the F value. For the mixed aqueous solution of adenosine and thymidine (0.5 mM and
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Fig. 5. Time dependence of frequency shifts for poly(AU-co-HAAm) (21%) LB films (six layers) on QCM substrates soaked in various solutions at 20 ◦ C; adenosine aqueous solution (0.5 mM) (a), mixed aqueous solution of adenosine and thymidine (0.5 and 0.5 mM) (b), and thymidine aqueous solution (0.5 mM) (c).
0.5 mM, pH 5.5), the amount of molecular adsorption slightly decreased, and little molecular adsorption was observed for a 0.5 mM thymidine aqueous solution (pH 4.6). These phenomena reveal that uracil groups can recognize only adenosine moieties through the complementary hydrogen bonding interaction. Poly(AU-co-HAAm) (45%) showed greater adsorption capacity than that of poly(AU-co-HAAm) (21%), and the amount of adenosine adsorption was 382 ng cm−2 . In order to estimate the adenosine adsorption level per uracil unit, the number of uracil molecules on a QCM substrate was calculated from the data of
Fig. 6. 3D height images of poly(AU-co-HAAm) (45%) LB films with fifty layers before (a) and after (b) soaking in adenosine aqueous solution (0.5 mM).
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π–A isotherms. The molar ratio of uracil group and adsorbed adenosine was found to be about 1:0.41. Fig. 6 shows the height AFM images of a poly(AUco-HAAm) (45%) LB film before and after the adenosine recognition. After the film was soaked in an adenosine solution, the surface morphology was remarkably changed. The formation of grain structure and an increase in surface roughness were observed after the soaking in an adenosine solution. On the contrary, after soaking in pure water, the AFM images of the polymer films did not showed obvious changes. Therefore, the complementary adsorption of adenosine on poly(AU-co-HAAm) LB films could also be confirmed by the morphology observations with AFM. Detailed studies on the adsorption mechanism are in progress. 4. Conclusion We have demonstrated that LB films of poly(AU-co-HAAm)s can be fabricated without the use of traditional surface-active molecules. By controlling the AU content in the copolymer, it is possible to formed well-ordered Y-type LB films. Most notably, the combination of AU and HAAm promotes a high amphiphilic balance and good molecular orientation. The uracil group and alkyl chains in the LB films are easily oriented perpendicular to the substrate. The poly(AU-co-HAAm) LB films preferentially adsorb adenosine, probably due to the complementary hydrogen bonding interaction. Biomimetic behavior of nucleic acids is reproduced in our system. The use of poly(AU-co-HAAm) and LB technique opens up the possibility for high resolution biosensing on solid devices. Acknowledgements This work was supported by a Grant-in-Aid for Science Research in the Priority Area of “Super-Hierarchical Structures”
from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. A part of the research is supported by an “Academic Frontier” Project for Private Universities: matching fund subsidy from MEXT. References [1] R. Ravindranath, P.K. Ajikumar, R.C. Advincula, W. Knoll, S. Valiyaveettil, Langmuir 22 (2006) 9002. [2] M. Rikukawa, Y. Tabuchi, K. Ochiai, K. Sanui, N. Ogata, Thin Solid Films 327 (1998) 469. [3] K. Ochiai, M. Rikukawa, K. Sanui, N. Ogata, Y. Ueno, K. Ema, Synth. Met. 101 (1999) 84. [4] (a) T. Nakanishi, K. Ariga, M. Morita, H. Kozai, N. Taniguchi, H. Murakami, T. Sagara, N. Nakashima, Colloids Surf. A 284 (2006) 607; (b) T. Nakanishi, H. Kozai, M. Morita, H. Murakami, T. Sagara, K. Ariga, N. Nakashima, J. Nanosci. Nanotechnol. 6 (2006) 1779. [5] F. Yin, H. Shin, Y. Kwon, Talanta 67 (2005) 221. [6] K. Ariga, T. Nakanishi, T. Michinobu, J. Nanosci. Nanotechnol. 6 (2006) 2278. [7] X. Chen, N. Lu, H. Zhang, M. Hirtz, L. Wu, H. Fuchs, L. Chi, J. Phys. Chem. B 110 (2006) 8039. [8] M. Li, M. Xin, Des. Monomers Polym. 9 (2006) 89. [9] S. Ni, W. Yin, M.K. Ferguson-McPherson, S.K. Satija, J.R. Morris, A.R. Esker, Langmuir 22 (2006) 5969. [10] W.K. Lee, T. Iwata, J.A. Gardella Jr., Langmuir 21 (2005) 11180. [11] Y. Duan, J. Liu, H. Sato, J. Zhang, H. Tsuji, Y. Ozaki, S. Yan, Biomacromolecules 7 (2006) 2728. [12] L. Zendlova, P. Hobza, M. Kabelac, J. Phys. Chem. B 111 (2007) 2591. [13] C. Li, J. Huang, Y. Liang, Langmuir 16 (2000) 7701. [14] H. Asanuma, T. Ban, S. Gotoh, T. Hishiya, M. Komiyama, Macromolecules 31 (1998) 371. [15] T. Sugiki, Y. Inaki, J. Photopolym. Sci. Technol. 16 (2003) 691. [16] T. Aoki, K. Nakamura, K. Sanui, A. Kikuchi, T. Okano, Y. Sakurai, N. Ogata, Polym. J. 31 (1999) 1185. [17] N.M. Brahme, W.T. Smith Jr., J. Polym. Sci. Polym. Chem. 22 (1984) 813. [18] T. Miyashita, H. Yoshida, T. Murakata, M. Matsuda, Polymer 28 (1987) 311.
Colloids and Surfaces A: Physicochem. Eng. Aspects 321 (2008) 60–64
Molecular recognition of Langmuir–Blodgett polymer films containing uracil groups N. Sugiyama ∗ , M. Hirakawa, H. Zhu, Y. Takeoka, M. Rikukawa Department of Chemistry, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan Received 16 July 2007; received in revised form 5 January 2008; accepted 15 January 2008 Available online 19 January 2008
Abstract Novel copolymers, poly(acryroyloxymethyluracil-co-hexylacrylamide)s (poly(AU-co-HAAm)s), have been synthesized by radical copolymerization with different monomer ratios. The poly(AU-co-HAAm)s formed stable monolayers at the air–water interface and could be deposited on solid substrates as Y-type films by the vertical dipping method. The molecular structure of poly(AU-co-HAAm) LB films was determined by reflection absorption and transmission FT-IR, and X-ray diffraction measurements. Evidence for preferential orientation of both the AU and HAAm units was found. The LB films deposited on quartz crystal microbalance substrates adsorbed adenosine molecules, and the adsorbed amounts increased with higher contents of uracil groups in poly(AU-co-HAAm)s. Some morphology changes of LB films were also observed by atomic force microscopy (AFM) after the molecular recognition. © 2008 Elsevier B.V. All rights reserved. Keywords: Uracil; Molecular recognition; Langmuir–Blodgett; Molecular orientation
1. Introduction The Langmuir–Blodgett (LB) technique provides many possibilities for the control of film thickness, dimensions, and molecular structures on the nanometer scale, and the air–water interfaces are an attractive area to investigate the chemical and physical interactions of functional molecules and polymers. As a result of sophisticated nano-technology, well-ordered thin films can be fabricated for the application of various electrical and optical devices [1–4]. The numbers of researches on LB films based on functional polymers have increased because of feasible applications in many fields such as biosensors [5,6], microlithography for data storage [7], and drug delivery systems [8,9]. The stereo-complexes and the hydrolysis of biodegradable polymers have also been investigated at the air–water interfaces of LB systems [10,11]. Nucleic acid bases and their derivatives have been widely studied as moieties that can participate in hydrogen-bonding in organic solvents [12], and at the air–water interface [13]. In particular, the molecular recognition of nucleic acid base
∗
Corresponding author. Tel.: +81 3 3238 4312; fax: +81 3 3238 3361. E-mail address: [email protected] (N. Sugiyama).
0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.01.020
derivatives in water has attracted much attention [14] because there are competitive interactions between the nucleic acid components and water molecules through the hydrogen-bonded bridges. Here, uracil has the strongest hydrogen-bonding ability since N–H and C O groups in the uracil moiety act as both the hydrogen-bonding donors and acceptors. Introduction of uracil units on polymers is an attractive strategy in order to investigate the presence of intramolecular and intermolecular interactions of functional polymers and biological materials through the hydrogen bonds of the uracil units [15]. For example, Aoki et al. reported that the uracil groups of poly(6-(acryroyloxymethyl)uracil) (poly(AU)) affected the phase transition temperature in response to species of the additive materials [16]. In this manner, LB polymer films with uracil groups are expected to be available for highly sensitive biosensors because of their specific molecular recognition ability for physiologically active substances. However, there are few reports about LB polymer films having uracil groups for such purposes. In this paper, novel amphiphilic copolymers, poly(acryroyloxymethyluracilco-hexylacrylamide)s (poly(AU-co-HAAm)s), with various amounts of uracil groups were synthesized to study the molecular recognition of the uracil groups on the LB films. These LB films were characterized by FT-IR, XRD, QCM, and AFM mea-
N. Sugiyama et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 321 (2008) 60–64
surements, and their monolayer formation, orientation structure, and selectivity of molecular recognition were investigated. 2. Experiments 2.1. Synthesis and characterization of poly(AU-co-HAAm)s 6-(Acryroyloxymethyl)uracil (AU) was synthesized according to the procedure described by Brahme et al. [16,17]. 1 H NMR (DMSO-d6 ) δ: 4.83 (s, 2H, O–CH2 ); 5.54 (s, 1H, 5-CH); 6.08 (m, 1H, vinilic CH); 6.43–6.22 (m, 2H, vinilic CH2 ); 11.05 (broad, 2H, NH) ppm. EI-MS: m/e, 196(M), 153(M+ -HNCO), 141(M+ -CH2 CHCO), 55(CH2 CHCO). N-Hexylacrylamide monomer (HAAm) was synthesized by the reaction of acrylamide with 1-bromohexane in anhydrous dimethylformamide (DMF) in the presence of KOH [18]. 1 H NMR (CDCl3 ) δ: 0.94 (m, 3H, CH3 ); 1.31 (m, 4H, CH3 CH2 CH2 CH2 –); 1.52 (m, 2H, CH3 CH2 –); 1.74 (s, 2H, CH3 CH2 CH2 CH2 CH2 –); 3.32 (q, 2H, CH3 CH2 CH2 CH2 CH2 CH2 –); 5.73 (broad, 1H, NH); 6.27–5.62 (m, 3H, vinilic H) ppm. EI-MS: 155(M), 140(M+ -CH3 ), 126, 112, 98, 84, 70(140-nCH2 ), 55(CH2 CHCO). Amphiphilic poly(AU-co-HAAm)s with different AU:HAAm molar ratios were synthesized by radical polymerization. The synthetic route is illustrated in Scheme 1. The monomers and 2,2 -azobis-(isobutyronitrile) (AIBN), which was used as an initiator, were degassed and sealed in an ampoule, and then were polymerized by shaking for 40 h at 70 ◦ C. The resulting polymers were precipitated by pouring the reacted solution into acetone and were purified by reprecipitation with DMSO and acetone. The chemical structure and copolymer composition were characterized by 1 H NMR, FT-IR, and elemental analyses. 2.2. Fabrication and characterization of LB films Monolayers were spread from a mixed solution of DMF and chloroform (50 mg/L, DMF content: 10%) on a water subphase that was purified with a TORAYPURE LV10-T system (TORAY). The surface pressure–area (π–A) isotherms were measured on an LB-200 (Nippon Laser & Electronics) at 20 ◦ C
61
with a compression speed of 15 mm min−1 . Multilayer films were transferred onto various substrates by the vertical dipping method at 20 ◦ C and 25 mN m−1 . A dipping speed of 13–15 mm min−1 was used. Waiting time at the top position was 30 min and that at the bottom position was 34 s for all subsequent dips. The films were dried for 1 h after depositing the third layer. FT-IR spectroscopy was performed on a Nicolet Magna-IR 750. The LB films were deposited on Pt-coated substrates for measuring reflection–absorption FT-IR spectra and were deposited on zinc selenide (ZnSe) substrates for measuring transmission FTIR spectra. X-ray diffraction patterns of the obtained LB films on glass slides were collected at r.t. using Ni-filtered Cu–K␣ radiation on a Rigaku RAD-IIA (RINT2000) diffractometer at 40 kV and 30 mA. A commercially available quartz crystal microbalance (QCM, SEIKO EG&G) was used with a polished 9 MHz AT-cut quartz crystal (area size: 0.189 cm2 ). The topology and microstructure of obtained LB films before and after the molecular recognition examinations were evaluated by AFM studies, which were performed with a Nanoscope IIIA (Digital Instruments Inc.) operated in the tapping mode.
3. Results and discussion Fig. 1 shows the FT-IR spectra of AU, HAAm, and poly(AU-co-HAAm) (uracil content: 21 mol%) in pressed KBr pellets. Poly(AU-co-HAAm) containing 21 mol% of AU units is expressed as poly(AU-co-HAAm) (21%). Two absorption bands at 1625 cm−1 in the AU and HAAm spectra were assigned to C C group (stretching vibration) and were not observed in the poly(AU-co-HAAm) spectrum. The N–H stretching broad band around 3300 cm−1 , the C O stretching peak at 1750 cm−1 , and the N–H deforming vibration peak at 1460 cm−1 of uracil group were observed in the AU and poly(AU-co-HAAm) spectra. The several peaks in the range of 3000–2800 cm−1 attributed to alkyl chains were also observed in HAAm and poly(AU-co-HAAm) spectra. The molecular weights of copolymers are summarized in Table 1. While the poly(HAAm) and poly(AU) had MW = 65,000 and 58,000, the molecular weights of poly(AUco-HAAm)s with various molar ratios were about 11,000, with a polydispersity of 1.2–1.3. 1 H NMR spectra of poly(AU-co-
Scheme 1. Synthesis of HAAm, AU, and poly(AU-co-HAAm)s.
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Fig. 1. Transmission FT-IR spectra of AU (a), HAAm (b), and poly(AU-coHAAm) (21%) (c). Fig. 3. Schematic view of Langmuir monolayer of poly(AU-co-HAAm) and CPK models of AU and HAAm.
Fig. 2. Surface pressure–area (π–A) isotherms of poly(HAAm), poly(AU), and poly(AU-co-HAAm)s at 20 ◦ C.
HAAm)s showed each chemical shift for uracil rings of AU and hexyl groups of HAAm. The copolymer compositions were calculated by the ratio between the CH of AU ring (5.5 ppm) and the CH3 of hexyl group (0.8 ppm), and were consistent with the results of elemental analysis. These results indicate the poly(AUco-HAAm) copolymers were successfully synthesized by our procedure. Fig. 2 shows π–A isotherms of the homopolymers and poly(AU-co-HAAm)s with various AU contents at 20 ◦ C. Although poly(AU) monolayers were unstable, poly(AU-co-
HAAm)s and poly(HAAm) exhibited stable condensed solid phases up to 20 mN m−1 , indicating that the long alkyl chains of HAAm units resulted in stable amphiphilic properties. The areas per unit of poly(AU-co-HAAm)s listed in Table 1 were esti˚ 2 , which were larger than that of poly(AU) mated to be 9.3–30 A 2 ˚ ). The areas of cross section of HAAm and AU molecules (8.9 A estimated from the π–A isotherms agreed with the areas calculated by the Corey–Pauling–Koltun (CPK) model as shown in Fig. 3. This result suggests that the plane of the uracil ring in poly(AU-co-HAAm)s is orientated perpendicular to the water surface. Consequently, the AU groups in poly(AU-co-HAAm)s provide enough hydrophilicity to form stable monolayers at the air–water interface. Monolayers of poly(AU-co-HAAm) (21%) and poly(AUco-HAAm) (45%) were transferred onto various substrates as Y-type LB films with nearly 100% transfer ratio. This may be due to the flexibility and excellent amphiphilic balance of poly(AUco-HAAm) monolayers. The strong intermolecular interaction of uracil groups may also have a positive effect on the excellent deposition. Poly(AU-co-HAAm) (67%) and poly(AU) monolayers, however, could not be transferred sufficiently. This suggests that excess uracil content perturbs the amphiphilic balance of copolymers and causes a molecular aggregation of uracil groups. Reflection absorption FT-IR (RAIR) and transmission FTIR (TIR) have been shown to provide direct information about
Table 1 Characteristics of poly(AU-co-HAAm)s Abbreviation
AU molar ratioa (%)
Mw b
PDIb
˚ 2) Area per unitc (A
Poly(HAAm) Poly(AU-co-HAAm) (21%) Poly(AU-co-HAAm) (45%) Poly(AU-co-HAAm) (67%) Poly(AU)
0 21.3 45.4 67.0 100
65,000 12,000 11,400 11,500 58,000
2.24 1.21 1.27 1.27 1.93
34 30 15 9.3 8.9
a b c
Calculated by 1 H NMR. Determined by GPC (DMF eluent). Estimated by π–A isotherms.
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Fig. 4. Reflection–absorption (R) and transmission (T) FT-IR spectra of poly(AU-co-HAAm) (21%) LB films with ten layers.
the molecular orientation of the various molecules in LB films. Function groups oriented perpendicular to the film substrate will exhibit stronger vibrational bands in RAIR than in TIR. As can be seen in Fig. 4, the absorption bands at 1750 cm−1 and 1200 cm−1 due to the stretching vibration of two C O groups and stretching vibration of C–N of the uracil ring was noticeably stronger and more defined in the RAIR of a poly(AU-co-HAAm) (21%) LB film than those in the TIR. The asymmetric (2920 cm−1 ) and symmetric (2852 cm−1 ) CH2 stretching vibrations observed in the TIR exhibit significantly stronger intensities than the asymmetric (2957 cm−1 ) and symmetric (2879 cm−1 ) CH3 stretching vibrations, whereas in the RAIR, the asymmetric and symmetric CH2 stretching vibrations are more comparable in intensities to their CH3 counterparts. This type of polarization dependence is characteristic of highly ordered hydrocarbon tail groups oriented with their fully extended chains nearly perpendicular to the substrate. The absorption band around 3300 cm−1 attributed to the stretching vibration of N–H groups was observed stronger in RAIR than in TIR, indicating that N–H groups take a perpendicular direction to the substrate. These results suggest that the uracil rings and hydrocarbon tails in the poly(AU-co-HAAm) (21%) LB film are preferentially orientated normal to the surface. The poly(AU-co-HAAm) LB films showed only one order of diffraction, which implied a thickness per monolayer of about ˚ Assuming that the copolymer adopts the configuration 34.0 A. that is shown in Fig. 3, we can conclude that the thickness estimated from XRD data is compatible with the single layer spacing of copolymer calculated by the CPK model. Fig. 5 exhibits the molecular recognition behavior of poly(AU-co-HAAm) (21%) LB films deposited on QCM substrates. When a QCM substrate of poly(AU-co-HAAm) (21%) LB film with six layers was soaked into a 0.5 mM adenosine aqueous solution (pH 5.4), a linear increase in mass (−F) was observed and the mass reached a constant value after 30 min. The amount of adenosine adsorption is calculated to be 237 ng cm−2 from the F value. For the mixed aqueous solution of adenosine and thymidine (0.5 mM and
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Fig. 5. Time dependence of frequency shifts for poly(AU-co-HAAm) (21%) LB films (six layers) on QCM substrates soaked in various solutions at 20 ◦ C; adenosine aqueous solution (0.5 mM) (a), mixed aqueous solution of adenosine and thymidine (0.5 and 0.5 mM) (b), and thymidine aqueous solution (0.5 mM) (c).
0.5 mM, pH 5.5), the amount of molecular adsorption slightly decreased, and little molecular adsorption was observed for a 0.5 mM thymidine aqueous solution (pH 4.6). These phenomena reveal that uracil groups can recognize only adenosine moieties through the complementary hydrogen bonding interaction. Poly(AU-co-HAAm) (45%) showed greater adsorption capacity than that of poly(AU-co-HAAm) (21%), and the amount of adenosine adsorption was 382 ng cm−2 . In order to estimate the adenosine adsorption level per uracil unit, the number of uracil molecules on a QCM substrate was calculated from the data of
Fig. 6. 3D height images of poly(AU-co-HAAm) (45%) LB films with fifty layers before (a) and after (b) soaking in adenosine aqueous solution (0.5 mM).
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π–A isotherms. The molar ratio of uracil group and adsorbed adenosine was found to be about 1:0.41. Fig. 6 shows the height AFM images of a poly(AUco-HAAm) (45%) LB film before and after the adenosine recognition. After the film was soaked in an adenosine solution, the surface morphology was remarkably changed. The formation of grain structure and an increase in surface roughness were observed after the soaking in an adenosine solution. On the contrary, after soaking in pure water, the AFM images of the polymer films did not showed obvious changes. Therefore, the complementary adsorption of adenosine on poly(AU-co-HAAm) LB films could also be confirmed by the morphology observations with AFM. Detailed studies on the adsorption mechanism are in progress. 4. Conclusion We have demonstrated that LB films of poly(AU-co-HAAm)s can be fabricated without the use of traditional surface-active molecules. By controlling the AU content in the copolymer, it is possible to formed well-ordered Y-type LB films. Most notably, the combination of AU and HAAm promotes a high amphiphilic balance and good molecular orientation. The uracil group and alkyl chains in the LB films are easily oriented perpendicular to the substrate. The poly(AU-co-HAAm) LB films preferentially adsorb adenosine, probably due to the complementary hydrogen bonding interaction. Biomimetic behavior of nucleic acids is reproduced in our system. The use of poly(AU-co-HAAm) and LB technique opens up the possibility for high resolution biosensing on solid devices. Acknowledgements This work was supported by a Grant-in-Aid for Science Research in the Priority Area of “Super-Hierarchical Structures”
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