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FT-SERS Studies on Molecular Recognition Capabilities of Monolayers of Novel Nucleolipid Amphiphiles Jianguo Huang, Chun Li, and Yingqiu Liang* State Key Laboratory of Coordination Chemistry and Institute of Mesoscopic Solid State Chemistry, Nanjing University, Nanjing 210093, People’s Republic of China Received July 16, 1999. In Final Form: December 13, 1999 The molecular recognition effect in nucleic acids was simulated in the monolayers formed by six novel nucleolipid amphiphiles. Fourier transform surface-enhanced Raman scattering (FT-SERS) technique was introduced into the research area of molecular recognition occurring in an interface system. Highquality FT-SERS spectra of a single Langmuir-Blodgett (LB) monolayer of the nucleolipid amphiphiles were obtained. Characteristic vibrational modes of the corresponding complementary nucleic acid bases, which transferred along with the monolayers of nucleolipid amphiphiles into the LB films, were clearly seen. The mechanism of molecular recognition through multiple hydrogen bonds between complementary bases was described. It was proved that this technique can be used as a powerful tool for studying molecular recognition in interface systems because of its high sensitivity.
Introduction The mutual recognition of complementary bases in nucleic acids by means of multiple hydrogen bonding is known to proceed spontaneously and with high selectivity, and has proved to be one of the most efficient mechanisms of accumulating, storing, reproducing, and evolving genetic information.1 In recent years, studies to simulate the mutual recognition of nucleic acid bases through mutual complementary hydrogen bonding in the replication process of DNA occurring in cells in a surface monolayer system have been of interest. Organized surface molecular monolayers assembled by Langmuir-Blodgett (LB) technique provide unique environments for molecular interactions and consequently for molecular recognition.2-9 Until now, only those amphiphiles containing analogous structures of nucleic acid bases were used to set up the simulated system,10-14 and there was large disparity compared with the recognition between complementary bases in biological molecular systems. In our previous work, six novel nucleic base-containing nucleolipid amphiphiles have been synthesized successfully,15 and the molecular recognition effect between the monolayers of these lipids and the complementary base substrates in subphases was investigated. The influence of recognition effect on the monolayer behavior was revealed via surface pressure-molecular area (π-A) isotherm measure* To whom correspondence should be addressed. (1) Lehn, J. M. Supramolecular Chemistry; VCH: Weinheim, 1995. (2) Shimomura, M.; Nakamura, F.; Ijiro, K. Thin Solid Films 1996, 284/285, 691. (3) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (4) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371. (5) Kitano, H.; Ringsdorf, H. Bull. Chem. Soc. Jpn. 1985, 58, 2826. (6) Moy, V.; Florn, E.; Gaub, H. Science 1994, 266, 257. (7) Shimomura, M. Prog. Polym. Sci. 1993, 18, 295. (8) Kunitake, T. Supramol. Sci. 1996, 3, 45. (9) Bohanon, T.; Denzinger, S.; Fink, R. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033. (10) Kurihara, K.; Ohoto, K.; Honda, Y.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 5077. (11) Honda, Y.; Kurihara, K.; Kunitake, T. Chem. Lett. 1991, 681. (12) Bemdt, P.; Kurihara, K.; Kunitake, K. Langmuir 1995, 11, 3083. (13) Kurihara, K.; Abe, T.; Nakashima, N. Langmuir 1996, 12, 4053. (14) Martin, S.; Ringsdorf, H. J. Chem. Soc., Chem. Commun. 1996, 10, 1193. (15) Huang, J.; Liang, Y. Synth. Commun. 1997, 27, 681.
ments,16-18 whereas in the high-frequency region of Fourier transform infrared (FTIR) spectra, the broad band of the hydrogen bond was observed.17,19 Moreover, the results of UV spectra measurements indicated that the photodimerization of thymine or uracil moieties was facilitated in LB film matrix at room temperature.16,18 However, no scattering band could be observed in the FTRaman spectra of the LB films of the lipids. In the present paper, the monolayers of the nucleolipid amphiphiles were transferred onto silver island film substrates that were prepared through chemical procedure, and high-quality Fourier transform surface-enhanced Raman scattering (FT-SERS) spectra for a single complementary basecontaining monolayer LB film were obtained. The scattering intensity of vibrational modes of a moiety in direct contact with the metal surface is preferentially enhanced. The vibrational bands of the related groups in the middle frequency region were clearly identifiable, whereas in FTIR spectra, they could not be distinguished. FT-SERS spectroscopy is a powerful tool with high selectivity for studying molecular recognition in a monolayer system. FT-SERS can be used to investigate the molecular recognition mechanism between the complementary bases in simulated systems on the molecular level. This is important not only for understanding molecular interactions on biological cell surfaces but also for designing novel biosensors as well as antivirus and antitumor drugs.20-23 Experimental Section Preparation of SERS-Active Silver Substrates. Fresh 5% NaOH solution (2.5 mL) was added to 50 mL of 3% AgNO3 solution whereupon a dark-brown AgOH precipitate was formed; then concentrated NH3‚H2O was added to the mixture dropwise to (16) Ding, D.; Zhang, Z.; Shi, B.; Luo, X.; Liang, Y. Colloids Surf. 1996, 112, 25. (17) Huang, J.; Liang, Y. Spectrosc. Lett. 1997, 30, 1441. (18) Huang, J.; Liang, Y. Thin Solid Films 1998, 326, 217. (19) Huang, J.; Liang, Y. Thin Solid Films 1998, 325, 210. (20) Helgeson, R.; Paek, K.; Knobler C. J. Am. Chem. Soc. 1996, 118, 5590. (21) Maverick, E.; Cram, D.; Donald, J. Compr. Supramol. Chem. 1996, 1, 213. (22) Maverick, E.; Cram, D. Compr. Supramol. Chem. 1996, 2, 367. (23) Kubo, I.; Sugawara, T.; Arikawa, Y. Anal. Lett. 1991, 24, 1711.
10.1021/la990947i CCC: $19.00 © 2000 American Chemical Society Published on Web 03/11/2000
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Figure 1. Chemical structures of the six novel nucleolipid amphiphiles. the point at which the precipitate redissolves. D-Glucose (10%, 15 mL) was added to the prepared Tollen’s reagent with swift and careful agitation to ensure mixing. Immediately the solution was then poured into a culture dish in which the frosted glass slides (dimensions 2 × 2 cm, thickness 5 mm) that had been cleaned with nitric acid and distilled water were placed horizontally. After about 5 min, the solution was poured out and the silver-coated slides were rinsed several times with distilled water and sonicated in distilled water for 1 min. The slides were then stored in distilled water before the LB film fabrication. Preparation of LB Film. The instruments and methods for LB film fabrications used here were the same as those reported previously.15-17 FT-Raman Spectra Measurements. A Bruker RFS 100 Fourier transform Raman spectrophotometer with a Nd/YAG laser emitting at 1064 nm as the near-infrared excitation source and a liquid nitrogen-cooled germanium detector were used to acquire the FT-Raman spectra of the LB film deposited on glass substrates and FT-SERS spectra of the LB film deposited on silver island film substrates. Spectra of the samples were collected at 4 cm-1 resolution by accumulating 500 scans (measurement time 17 min) with a laser power at the samples of 75 mW.
Results and Discussion The synthesis and purification of the six novel nucleolipid amphiphiles have been reported previously15 and the chemical structures of them are shown in Figure 1. Each amphiphile can form a stable monolayer at the air/ water interface.24 The nucleic bases in the headgroups of these amphiphiles, together with the corresponding complementary bases in subphases, are summarized in Table 1. The FT-SERS spectra of these molecular recognition systems show that, for the amphiphiles containing the same base in the headgroup, the FT-SERS spectra of their single-layer LB films are similar, indicating that the base moieties in the headgroups were absorbed on the silver island substrate. Therefore, FT-SERS spectra of the molecular recognition systems can be classified into two types: one contains lipids 1, 2, 5, 6 (for lipid 5, the base in the headgroup is uracil, in which the methyl group (24) Huang, J. Ph.D. Thesis, Nanjing University, 1998.
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on the ring is absent compared with the thymine moiety in the headgroups of the other three lipids, but the spectra were not affected.); the other contains lipids 3 and 4. In this paper, the molecular recognition capabilities of the lipids in Table 1 were discussed through the FT-SERS spectroscopic investigation of molecular recognition systems of lipids 1 and 3. Figure 2a shows the FT-Raman spectrum of a singlelayer LB film of nucleolipid amphiphile 1 [(3′,5′-distearoyl)2′-deoxythymidine] deposited on glass substrate; no scattering band can be observed. Figure 2b and 2c shows FT-SERS spectra of 1 as a single-layer LB film deposited on chemical-procedured silver island film substrates from the surface of pure water and aqueous 5 mM adenosine solution, respectively. In the region of 1750-500 cm-1, high-quality FT-SERS spectra were obtained, and the enhanced bands of the related groups were clearly seen. For an LB film of 1 deposited from pure water subphase (Figure 2b), the enhanced bands of thymine moiety near 1642 cm-1 [C(4)dO stretching], 1601 cm-1 [a combination of C(4)dO and C(5)dC(6) stretching], 1575 cm-1 (C-N stretching), 1165 cm-1 [a combination of CH deformation and C(2)N(3) stretching], 827 cm-1 (C-N symmetric stretching), and 615 cm-1 (C-NdO deformation) are found.25,26 By comparing Figure 2c (aqueous 5 mM adenosine subphase) and Figure 2b (pure water subphase), four new pronounced enhanced peaks attributed to adenine moiety appear at 734 cm-1 (adenine ring breathing mode),27 1333 cm-1 (adenine ring skeletal vibration), 1370 cm-1 [C(2)-H bending],28 and 1451 cm-1 [a combination of N(1)-C(2) and N(3)-C(2) stretching].25 These changes indicate that amphiphile 1 monolayer at the air-water interface is capable of interacting with dissolved adenosine in the subphase, and the adenosine is also transferred onto solid substrate along with the surface monolayer, which is consistent with the results of molecular recognition system of lipid 2 through π-A isotherm measurements.16,17 Moreover, the bands near 1601 cm-1, 927 cm-1, and 615 cm-1, attributed to the thymine moiety, are almost absent in Figure 2c, which corresponds to the FTIRattenuated total reflection (ATR) results in the NHstretching high-frequency regions (the appearance of a broad absorption band in the NH stretching regions indicates the formation of an adenine-thymine complex through multiple complementary hydrogen bonds,17 whereas no distinguished bands can be observed in the middle frequency region). Furthermore, the NH2 asymmetric and symmetric deformation vibration of the adenine ring was reported to be observed near 1644 and 1632 cm-1, respectively,29 whereas in Figure 2c, both could not be observed clearly, suggesting that the NH2 group of the adenine ring is hydrogen-bonded with the thymine moiety of the nucleolipid amphiphile. According to Raman selective rule,28 it can be deduced from Figure 2c that the molecular recognition species (complementary base pairs) are close to the silver surface, whereas no enhanced information detected in the high-frequency region indicates that the alkyl chains remain far from the silver surface. The above spectral features indicate that the molecular recognition occurred between the monolayer of (25) Otto, C.; van den Tweel, T. J. J.; de Mul, F. F. M.; Greve, J. J. Raman Spectrosc. 1986, 17, 289. (26) Aamouche, A.; Ghomi, M.; Couombeau, C.; Grajcar, L.; Baron, M. H.; Jobic, H.; Berthier, G. J. Phys. Chem. A 1997, 101, 1808. (27) Ervin, K. M.; Koglin, E.; Sequaris, T. M.; Valenta, P.; Nurnberg, H. W. J. Electroanal. Chem. 1980, 114, 179. (28) Watanade, T.; Kawanami, O.; Kaoh, H.; Honda, K.; Nishimura, Y.; Tsuboi, M. Surf. Sci. 1985, 158, 341. (29) Stepanian, S. G.; Sheina, G. G.; Radchenko, E. D.; Blagoi, Y. P. J. Mol. Struct. 1985, 131, 333.
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Table 1. Novel Nucleolipid Amphiphiles and Complementary Bases nucleolipid amphiphiles
bases in headgroup
complementary bases
3′,5′-distearoyl-2′-deoxythymidine (1) octadecanoyl ester of 1-(2-carboxyethyl) thymine (2) octadecanoyl ester of 1-(2-carboxyethyl) adenine (3) 3′,5′-distearoyl-2′-deoxyadenosine (4) 5′-stearoyl-uridine (5) double headgroup surface-active derivative of thymine (6)
thymine thymine adenosine adenosine uracil thymine
adenosine adenosine thymine, uracil thymine, uracil adenosine adenosine
Figure 2. FT-Raman spectrum of nucleolipid amphiphile (3′,5′distearoyl)-2′-deoxythymidine (1): FT-Raman spectrum of a single-layer LB film deposited on glass substrate (a); FT-SERS spectrum of a single-layer LB film deposited on silver island film substrate from the surface of pure water (b) and aqueous 5 mM adenosine solution (c).
Figure 3. Schematic illustration of the complementary multiple hydrogen bonds between the adenine group and thymidine (a), and between the adenine group and uridine (b).
1 and the adenosine substrate in the subphase through multiple complementary hydrogen bonds, together with the FTIR-ATR results. A schematic illustration is shown in Figure 3a. For the molecular recognition systems of nucleolipid amphiphiles 2, 5, and 6, similar results were obtained. Figure 4a shows the FT-Raman spectrum of a singlelayer LB film of nucleolipid amphiphile 3 [octadecanoyl ester of 1-(2-carboxyethyl) adenine] deposited on glass
Figure 4. FT-Raman spectrum of nucleolipid amphiphile octadecanoyl ester of 1-(2-carboxyethyl) adenine (3): FT-Raman spectrum of a single-layer LB film deposited on glass substrate (a); FT-SERS spectrum of a single-layer LB film deposited on silver island film substrate from the surface of pure water (b), aqueous 5 mM thymidine solution (c), and aqueous 5 mM uridine solution (d).
substrate; no scattering band can be observed. Figure 4b, 4c, and 4d shows FT-SERS spectra of 3 as a single-layer LB film deposited on chemical-procedured silver island film substrates from the surface of pure water, aqueous 5 mM thymidine solution, and aqueous 5 mM uridine solution, respectively. The large changes of the spectra before and after the molecular recognition effect occurred between the surface monolayer and the complementary bases in the subphase could be clearly seen. These results are consistent with those of π-A isotherm and FTIR-ATR measurements of the same molecular recognition system.18,19 In the spectrum of a single-layer LB film of 3 before molecular recognition occurred (Figure 4b), the enhanced bands of adenine caused by the certain orientation of the moiety on the silver surface could be observed:25,30 1639 cm-1 (NH2 deformation); 1601 cm-1 [C(4)dC(5) stretching]; 1574 cm-1 (C-N stretching); 1165 cm-1 [C(6)-N(1)-C(2) stretching]; 1038 cm-1 [C(8)-H outof-plane blending], and 827 cm-1 (C-N symmetric stretching). All these bands decreased in intensity or disappeared after the molecular recognition effect occurred (Figure 4c and 4d). Three characteristic enhanced bands of uracil, which combined into the single-layer LB film of 3 through multiple complementary hydrogen bonds, could be observed25 (Figure 4d): 1630 cm-1 [C(4)dO stretching]; 1388 cm-1 (uracil ring stretching); 798 cm-1 (uracil ring breathing). The spectral feature of Figure 4c is similar, but the enhanced effect of the thymine moiety is much weaker than the uracil moiety, which results from the (30) Itoch, K.; Minami, K.; Tsujino, T.; Kim, M. J. Phys. Chem. 1991, 95, 1339.
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steric effect caused by the methyl group in the thymine ring; in the uracil-containing LB film the uracil ring remains nearer to the silver surface and the amount of uracil moieties is larger. 18,19 However, in 1750-1500 cm-1 region, the enhanced bands of the adenine moiety near 1639, 1601, and 1575 cm-1 are absent in Figure 4c and 4d. This should be due to the fact that the molecular recognition effect occurred between adenine and thymine or uracil moieties through multiple complementary hydrogen bonds, and the molecular orientation is different from that of the moieties in the LB film deposited from pure water. The multiple hydrogen bonds between uracil and adenine moieties is shown in Figure 3b. Similar results were obtained for the molecular recognition system of 4. Conclusion FT-SERS technique was used for the first time to investigate the molecular recognition capabilities between monolayers of six novel nucleolipid amphiphiles and the
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complementary base substrates in the subphase. Highquality FT-SERS spectra were obtained for a single-layer LB film; abundant spectral information was obtained in the middle frequency region. By combining the FTIR-ATR results, the molecular recognition mechanism between the complementary bases was discussed. It is shown that FT-SERS can be used as a powerful tool of high sensitivity in the area of interface recognition. The molecular recognition of nucleolipid amphiphiles to the complementary nucleobases should be important bearing on the related process occurring at the surface of the biological molecular system. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (no. 29873022) & State Science and Technology Commission of China. LA990947I
