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Selective detection of the antigenic polar heads of model lipid membranes supported on metals from their vibrational nonlinear optical response
Chemical Physics Letters 489 (2010) 12–15
Contents lists available at ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Selective detection of the antigenic polar heads of model lipid membranes supported on metals from their vibrational nonlinear optical response Dan Lis a, Julien Guthmuller b,1, Benoît Champagne b, Christophe Humbert c, Bertrand Busson c, Abderrahmane Tadjeddine c, André Peremans a, Francesca Cecchet a,* a b c
Research Centre in Physics of Matter and Radiation (PMR), FUNDP-University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium Laboratory of Theoretical Chemistry (LCT), FUNDP-University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium Laboratoire de Chimie Physique, Université Paris-Sud, CNRS, Bâtiment 201 Porte 2, 91405 Orsay, France
a r t i c l e
i n f o
Article history: Received 2 February 2010 In final form 23 February 2010 Available online 26 February 2010
a b s t r a c t We have investigated a metal-supported lipid film by using a sum frequency generation (SFG) spectrometer coupled to a free electron laser (FEL). DFT calculations on a molecular model have enabled to assign the vibrational signatures. The SFG vibrations detected in the backbone region, between 1375 cm1 and 1000 cm1, originate from the vibrational motions of the 2,4-dinitrophenyl polar head of the lipid, which holds antigenic properties, and can therefore be used to selectively study the biological interactions with complementary antibodies. Theses results strengthen the role of SFG spectroscopy as a highly sensitive tool for biological detection. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Investigating the structure and the physico-chemical properties of biomimetic lipid membranes on surfaces is a fundamental step towards the detailed comprehension of biological processes and their selective detection. This also represents the platform to build up reliable bio-devices mimicking the membrane behaviour. The analysis of the physico-chemical properties of bio-interfaces can be achieved by probing the vibrational response of the chemical functions involved in the biological processes. However, this requires highly surface-sensitive techniques that are able to discriminate interfacial responses from those of the surrounding bulk phase. In this respect, sum frequency generation spectroscopy (SFG), which is intrinsically selective to interfaces [1–4] retrieves physical–chemical information by probing the nonlinear vibrational response of the bio-interface. Up to now, SFG spectroscopy has remarkably contributed to the investigation of the interfacial properties of lipid layers. However, most SFG studies on lipid layers (i) have focused on the analysis of the vibrational signatures of aliphatic CH stretching modes from the hydrophobic tails and from the choline head groups [5–25], (ii) have detected carbonyl stretchings of the lipid chains [26] and amide I vibrations originating from membrane proteins within
* Corresponding author. E-mail address: [email protected] (F. Cecchet). 1 Present address: Institut für Physikalische Chemie, Friedrich Schiller Universität Jena, Helmholtzweg 4, 07743 Jena, Germany. 0009-2614/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2010.02.061
the lipid layer [27–30], or (iii) have probed interfacial water layers interacting with the lipid film [9,16,31–34]. Only few reports have investigated the vibrational signatures of other chemical fingerprints of lipids [19,35–37]. For example, detecting backbone vibrations of interfacial bio-films allows to selectively investigate the fingerprints of chemical groups directly involved in biological processes. However, this requires the scanning of the entire vibrational frequency window, which is still not regularly achieved with SFG due to physical or technical limitations. Moreover, while lipid films have been regularly investigated with SFG at liquid/air, liquid/liquid or liquid/solid insulating interfaces, there is no example of SFG studies about lipid films at metal interfaces. The relevance in building-up metal-supported bio-membranes is related to the possibility to develop bio-devices compatible with a wide range of electrical or optical detection systems. In this work, we probe the SFG fingerprint of the backbone vibrations of 2,4-dinitrophenyl antigenic lipid films (DNP, Fig. 1a) with a table top spectrometer coupled to a free electron laser (FEL) infrared source [38]. The advantages of using the FEL source are related to the possibility of exploring the low frequency spectral region, where most of the backbone vibrations show up, and to the benefit of the high FEL power density allowing the detection of weak mid/far infrared nonlinear susceptibilities. Interest in DNP lipid stays in the antigenic properties of its polar head, which is representative of a synthetic chemical agents acting as a cellular metabolic poison. Studies on DNP membranes are performed with the purpose to deeper understand the mechanism of cell membrane damages, and to investigate the biological recognition of DNP with their complementary anti-DNP antibody.
D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
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Fig. 1. Schematic representation of (a) DNP, (b) a LB film of DNP on Pt, (c) a LS film of DNP on a DDT/Pt surface, and (d) the DNP model used for DFT calculations. The yellow balls represent the polar head of DNP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods 2.1. Preparation of the supported lipid films Lipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N(2,4-dinitrophenyl) (DNP) was supplied from AvantiÒ Polar Lipids, and dodecanethiol (P98%, DDT) was from Sigma Aldrich. Pt substrates (ArrandeeÒ) were thin metal layers deposited on Si. Langmuir–Blodgett monolayers of DNP have been adsorbed on a Pt surface (head-down), while Langmuir–Schaefer films have been deposited on top of a dodecanethiol self-assembled monolayer (DDT/SAM) on Pt (head-up). Langmuir films of DNP were prepared by spreading a 1 mg/mL chloroform solution of lipid onto the water surface in a Langmuir trough (MiniMicro System from KSV Instruments). The compression rate was of 5 mm/min and the transfer surface pressure was of 46 mN/m. LB films of DNP on Pt were obtained by a vertical lift up of the substrate at a rate of 3 mm/min. The LS films were obtained by a horizontal transfer of the DNP molecules onto the DDT/SAM, which was previously obtained by immersion of the Pt substrate in a 1 mM DDT ethanol solution for 24 h. In order to verify that the film adsorption occurred with the head-down and the head-up configurations, respectively, we have measured the water contact angle of the films. It results that headdown LB monolayers showed a contact angle of 90°, in agreement with hydrophobic tails pointing out from the surface. DDT/SAM, however, exhibits a contact angle of 108°, which is the characteristic value of highly ordered SAMs of alknethiols on metals. When the DNP lipid has been adsorbed on top of the DDT/SAM by the Langmuir–Schaefer method, the water contact angle of the surface decreased to 45°, testifying for the presence of the DNP polar head pointing out from the surface.
2.2. Sum frequency generation spectroscopy (SFG) coupled to the free electron laser (FEL) The backbone vibrations of DNP films have been preliminary investigated with a laboratory table top spectrometer [39], but
these measurements did not detect any resonant SFG signal of DNP, probably because of the low infrared power of regular OPO laboratory systems (working power: 2–10 mW, 15 ps) together with the weak SFG activity of mid-infrared vibrations. Then, the SFG spectra have been recorded by tuning the FEL (CLIO, Orsay, France) infrared beam (working power: 20–60 mW, 1 ps) [26], between 1375 cm1 and 1000 cm1, while the visible wavelength was kept constant at 523.5 nm. The spectra have been recorded with ppp and ssp (in the order SFG, Vis, IR) sets of polarizations. 2.3. DFT calculations The theoretical calculations were carried out at the density functional theory (DFT) level of approximation by using the B3LYP exchange–correlation functional and the 6-311++G(d,p) basis set. First, the geometries were optimized, then the vibrational frequencies and normal modes were calculated. In order to evaluate the IR and Raman intensities, the IR vectors were obtained analytically, whereas a two-point numerical differentiation procedure was employed to determine the static Raman tensors. More information about the computational procedure can be found in Ref. [40]. All the calculations were performed using the GAUSSIAN 03 program [41]. 3. Results and discussion Fig. 2 shows the SFG spectra of a LB (a) and a LS (b) film of DNP recorded between 1375 cm1 and 1275 cm1, in the ppp (top) and ssp (bottom) configurations of polarizations. In the ppp spectra (top), the high frequency peak is centred at 1344 cm1 in both films, while the low frequency peak shows-up at 1305 cm1 in the LB film, and at 1312 cm1 in the LS film. The ssp spectra (Fig. 2a and b, bottom) exhibit quite different features compared to ppp spectra, especially regarding the peak intensity ratios. Indeed, in the ssp spectrum of the LB film (Fig. 2a, bottom) the intensity of the higher frequency peak has decreased compared to the lower frequency peak, while in the ssp spectrum of the LS film (Fig. 2b, bottom) the former has almost disappeared. Fig. 2c shows
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D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
Fig. 2. SFG spectra of a LB film of DNP on Pt (a) and of a LS film of DNP onto a DDT/Pt surface (b), recorded with the ppp (top) and ssp (bottom) sets of polarizations between 1375 cm1 and 1275 cm1; (c) SFG spectrum (ppp) of a LB film of DNP on Pt recorded between 1100 cm1 and 1000 cm1. The numbers of the assigned normal modes are given in brackets.
the SFG signal of a LB film of DNP in the 1100–1000 cm1 range which is characterized by a broad resonant peak centred at 1056 cm1. The peak assignment is however not trivial because of the complexity of the molecular structure. In order to associate the resonant signals to molecular vibrations, DFT calculations have been carried out on a model molecule, which holds the main chemical functions of DNP (Fig. 1d). The calculations have provided the vibrational modes and their infrared vectors and Raman tensors, which enable to predict the SFG active vibrations [28,42–45]. Those latter are depicted in the top of Fig. 3 and their characteristics (i.e. frequency, IR and Raman intensities) are summarized in the bottom of Fig. 3. In the spectral range covered by Fig. 2a and b (1375–1275 cm1), DFT calculations predict three SFG active vibrations, namely at 1349 cm1 (100), at 1319 cm1 (98) and at 1292 cm1 (95), all mainly localized on the polar head of the lipid molecule. The highest frequency peak in the SFG spectra is as-
signed to vibration 100. This latter is mostly due to a superposition of the para-NO2 symmetric stretching with CH2 bendings (waggings) and has a strong SFG activity, as testified by the strong IR and Raman intensities. The experimental peaks at 1305 cm1 for the LB film and at 1312 cm1 for the LS film are assigned to the superposition of vibrations 98 and 95, which correspond to bending modes of the phenyl and methylene units and show comparable SFG activities. The broadening, the asymmetric peak features, and the frequency shift between the LB (1305 cm1) and the LS (1312 cm1) films may be attributed to the coexistence of different stable molecular conformations, that can be expected for these long and flexible DNP polar heads. The presence of several stable molecular conformations has been confirmed by DFT calculations, strengthening the hypothesis that adsorption onto the surface has occurred with different molecular orientations of the polar head, which may hold a large orientational distribution, even if the aliphatic carbon skeleton is well-packed and highly organized. The
Fig. 3. Top: representation of the vibrational modes, the IR vectors (pink arrows) and the Raman tensors (red for negative, green for positive values) as obtained from DFT calculations for the DNP model molecule. Bottom: theoretical parameters of the vibrational modes (frequency, IR intensity, Raman intensity) as predicted from DFT calculations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
lowest vibrational frequency at 1056 cm1 is a superposition of CH bendings (81, 80, 77), which are rather delocalized on the entire molecular structure. The peak profiles, namely the peak intensity ratios and the peak frequency shifts, provide orientational information. In fact, each polarization set probes selected components of the nonlinear susceptibility (ZZZ, XXZ, XZX, and ZXX with ppp, and YYZ with ssp), and therefore the peak intensities depend on the orientation of ð2Þ the susceptibility tensor vijk of the vibrational modes with respect to the interface [46]. This implies that the different peak profiles of the LB and LS films (Fig. 1) in the ppp and ssp sets are ascribed to different orientations of the molecular groups involved in the SFG active vibrations. Even if the ppp spectra recorded for the LB and LS films (Fig. 2a and b, top) exhibit quite similar peak profiles, the ssp spectra (Fig. 2a and b, bottom) show rather different features. Therefore, this attests to a different orientational distribution of the polar head in the two film configurations. One can notice that this orientational difference can be highlighted only using polarization-dependent SFG measurements (with both ppp and ssp sets). However, this is challenging since detecting SFG signatures with other polarization than ppp is not regularly achieved on metals [32]. Here, this was achieved thanks to the higher FEL power density, regarding both the beam power and pulse. 4. Conclusions To our knowledge, this is the first study reporting on biological layers investigated with SFG spectroscopy coupled to a FEL radiation. This has allowed to probe backbone vibrations within metal-supported lipid films, and to measure selected components of the molecular nonlinear susceptibility in the 1375–1000 cm1 spectral window. Moreover, the approach – coupled to DFT calculations – has succeeded in identifying vibrations, which are purely localized on the polar head. This study has demonstrated that SFG can selectively follow the vibrational nonlinear response of chemical groups located on the DNP polar head. On this basis, the vibrational signatures will enable to carefully characterize the biological recognition of DNP lipid films with complementary anti-DNP antibodies, therefore strengthening the role of SFG spectroscopy as a highly sensitive tool for biological detection [47–49].
15
The authors acknowledge the support of C. Six and A. Gayral for the SFG measurements at the CLIO facility. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39]
Acknowledgments
[40] [41]
F.C., Y.C., and A.P., are postdoctoral researcher, research associate, and research director of the Fund for the Scientific Research F.R.S.-FNRS. D.L. thanks the FRIA for his Ph.D. student fellowship. J.G. thanks the F.R.S.-FNRS for his postdoctoral grant under the Conventions No. 2.4.509.04.F. The calculations were performed on the Interuniversity Scientific Computing Facility (ISCF, University of Namur, Belgium) for which the authors gratefully acknowledge the financial support of the F.R.S.-FRFC and the ‘Loterie Nationale’ under Contract No. 2.4.617.07.F, and of the FUNDP.
[42] [43] [44] [45] [46] [47] [48] [49]
Y.R. Shen, Nature 337 (1989) 519. C. Bain, J. Chem. Soc. Faraday Trans. 91 (1995) 1281. Y.R. Shen, Pure Appl. Chem. 10 (2001) 1589. P. Guyot-Sionnest, Surf. Sci. 585 (2005) 1. W. Pohle, M. Saß, C. Selle, K. Wolfrum, J. Löbau, Vib. Spectrosc. 19 (1999) 321. S. Roke, J. Schins, M. Müller, M. Bonn, Phys. Rev. Lett. 90 (2003) 128101. G. Kim, M.C. Gurau, S.-M. Lim, P.S. Cremer, J. Phys. Chem. B 107 (2003) 1403. J. Liu, J.C. Conboy, J. Am. Chem. Soc. 126 (2004) 8894. J. Liu, J.C. Conboy, J. Am. Chem. Soc. 126 (2004) 8376. M. Bonn, S. Roke, O. Berg, L.B.F. Juurlink, A. Stamouli, M. Muller, J. Phys. Chem. B 108 (2004) 19083. S. Ye, H. Noda, T. Nishida, S. Morita, M. Osawa, Langmuir 20 (2004) 357. A.W. Doyle et al., Langmuir 20 (2004) 8961. Z.S. Nickolov, D.W. Britt, J.D. Miller, J. Phys. Chem. B 110 (2006) 15506. T. Johansson, G.W. Leach, J. Phys. Chem. B 110 (2006) 16567. T. Nishida, C.M. Johnson, J. Holman, M. Osawa, P.B. Davies, S. Ye, Phys. Rev. Lett. 96 (2006) 077402. G. Ma, H.C. Allen, Langmuir 22 (2006) 5341. J. Liu, J.C. Conboy, J. Phys. Chem. C 111 (2007) 8988. N.A. Anderson, L.J. Richter, J.C. Stephenson, K.A. Briggman, J. Am. Chem. Soc. 129 (2007) 2094. G. Ma, X. Chen, H.C. Allen, J. Am. Chem. Soc. 129 (2007) 14053. M. Sovago, G.W.H. Wurpel, M. Smits, M. Müller, M. Bonn, J. Am. Chem. Soc. 129 (2007) 11079. K.L. Harper, H.C. Allen, Langmuir 23 (2007) 8925. X. Chen, J.E. Wang, C.B. Kristalyn, Z. Chen, Biophys. J. 93 (2007) 866. F.J. Pavinatto et al., Langmuir 25 (2009) 10051. H.D. Jayathilake, J.A. Driscoll, A.N. Bordenyuk, L. Wu, S.R.P. da Rocha, C.N. Verani, A.V. Benderskii, Langmuir 25 (2009) 6880. S. Ye, M. Osawa, Chem. Lett. 38 (2009) 386. P. Viswanath, A. Aroti, H. Motschmann, E. Leontidis, J. Phys. Chem. B 113 (2009) 14816. X. Chen, J. Wang, A.P. Boughton, C.B. Kristalyn, Z. Chen, J. Am. Chem. Soc. 129 (2007) 1420. X. Chen, A.P. Boughton, J.J.G. Tesmer, Z. Chen, J. Am. Chem. Soc. 129 (2007) 12658. K.T. Nguyen, S.V. Le Clair, S. Ye, Z. Chen, J. Phys. Chem. B 113 (2009) 12358. C.W. Avery, A. Som, Y. Xu, G.N. Tew, Z. Chen, Anal. Chem. 81 (2009) 8365. J. Kim, G. Kim, P.S. Cremer, Langmuir 17 (2001) 7255. M.R. Watry, T.L. Tarbuck, G.L. Richmond, J. Phys. Chem. B 107 (2003) 512. G.W.H. Wurpel, M. Sovago, M. Bonn, J. Am. Chem. Soc. 129 (2007) 8420. M. Sovago, R.K. Campen, H.J. Bakker, M. Bonn, Chem. Phys. Lett. 470 (2009) 7. D.K. Hore, D.K. Beaman, G.L. Richmond, J. Am. Chem. Soc. 127 (2005) 9356. T.M. Nobre, J. Phys. Chem. B 113 (2009) 7491. G. Ma, J. Liu, L. Fu, E.C.Y. Yan, Appl. Spectrosc. 63 (2009) 528. C. Humbert, B. Busson, C. Six, A. Gayral, M. Gruselle, F. Villain, A. Tadjeddine, J. Electroanal. Chem. 621 (2008) 314. A.A. Mani, L. Dreesen, C. Humbert, P. Hollander, Y. Caudano, P.A. Thiry, A. Peremans, Appl. Surf. Sci. 502–503 (2002) 261. J. Guthmuller et al., ChemPhysChem 10 (2009) 2132. M.J. Frisch et al., GAUSSIAN 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004. L.J. Richter et al., J. Phys. Chem. B 108 (2004) 12547. D. Lis et al., J. Phys. Chem. C 113 (2009) 9857. F. Cecchet et al., ChemPhysChem 11 (2010) 607. F. Cecchet et al., J. Phys. Chem. C 114 (2010) 4106. H.-F. Wang, W. Gan, R. Lu, Y. Rao, B.-H. Wu, Int. Rev Phys. Chem. 24 (2005) 191. L. Dreesen et al., ChemPhysChem 5 (2004) 1719. Y. Sartenaer, G. Tourillon, L. Dreesen, D. Lis, A.A. Mani, P. Thiry, A. Peremans, Biosens. Bioelectron. 22 (2007) 2179. C. Howell, R. Schmidt, V. Kurz, P. Koelsch, Biointerphases 3 (2008) FC47.
Contents lists available at ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Selective detection of the antigenic polar heads of model lipid membranes supported on metals from their vibrational nonlinear optical response Dan Lis a, Julien Guthmuller b,1, Benoît Champagne b, Christophe Humbert c, Bertrand Busson c, Abderrahmane Tadjeddine c, André Peremans a, Francesca Cecchet a,* a b c
Research Centre in Physics of Matter and Radiation (PMR), FUNDP-University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium Laboratory of Theoretical Chemistry (LCT), FUNDP-University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium Laboratoire de Chimie Physique, Université Paris-Sud, CNRS, Bâtiment 201 Porte 2, 91405 Orsay, France
a r t i c l e
i n f o
Article history: Received 2 February 2010 In final form 23 February 2010 Available online 26 February 2010
a b s t r a c t We have investigated a metal-supported lipid film by using a sum frequency generation (SFG) spectrometer coupled to a free electron laser (FEL). DFT calculations on a molecular model have enabled to assign the vibrational signatures. The SFG vibrations detected in the backbone region, between 1375 cm1 and 1000 cm1, originate from the vibrational motions of the 2,4-dinitrophenyl polar head of the lipid, which holds antigenic properties, and can therefore be used to selectively study the biological interactions with complementary antibodies. Theses results strengthen the role of SFG spectroscopy as a highly sensitive tool for biological detection. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Investigating the structure and the physico-chemical properties of biomimetic lipid membranes on surfaces is a fundamental step towards the detailed comprehension of biological processes and their selective detection. This also represents the platform to build up reliable bio-devices mimicking the membrane behaviour. The analysis of the physico-chemical properties of bio-interfaces can be achieved by probing the vibrational response of the chemical functions involved in the biological processes. However, this requires highly surface-sensitive techniques that are able to discriminate interfacial responses from those of the surrounding bulk phase. In this respect, sum frequency generation spectroscopy (SFG), which is intrinsically selective to interfaces [1–4] retrieves physical–chemical information by probing the nonlinear vibrational response of the bio-interface. Up to now, SFG spectroscopy has remarkably contributed to the investigation of the interfacial properties of lipid layers. However, most SFG studies on lipid layers (i) have focused on the analysis of the vibrational signatures of aliphatic CH stretching modes from the hydrophobic tails and from the choline head groups [5–25], (ii) have detected carbonyl stretchings of the lipid chains [26] and amide I vibrations originating from membrane proteins within
* Corresponding author. E-mail address: [email protected] (F. Cecchet). 1 Present address: Institut für Physikalische Chemie, Friedrich Schiller Universität Jena, Helmholtzweg 4, 07743 Jena, Germany. 0009-2614/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2010.02.061
the lipid layer [27–30], or (iii) have probed interfacial water layers interacting with the lipid film [9,16,31–34]. Only few reports have investigated the vibrational signatures of other chemical fingerprints of lipids [19,35–37]. For example, detecting backbone vibrations of interfacial bio-films allows to selectively investigate the fingerprints of chemical groups directly involved in biological processes. However, this requires the scanning of the entire vibrational frequency window, which is still not regularly achieved with SFG due to physical or technical limitations. Moreover, while lipid films have been regularly investigated with SFG at liquid/air, liquid/liquid or liquid/solid insulating interfaces, there is no example of SFG studies about lipid films at metal interfaces. The relevance in building-up metal-supported bio-membranes is related to the possibility to develop bio-devices compatible with a wide range of electrical or optical detection systems. In this work, we probe the SFG fingerprint of the backbone vibrations of 2,4-dinitrophenyl antigenic lipid films (DNP, Fig. 1a) with a table top spectrometer coupled to a free electron laser (FEL) infrared source [38]. The advantages of using the FEL source are related to the possibility of exploring the low frequency spectral region, where most of the backbone vibrations show up, and to the benefit of the high FEL power density allowing the detection of weak mid/far infrared nonlinear susceptibilities. Interest in DNP lipid stays in the antigenic properties of its polar head, which is representative of a synthetic chemical agents acting as a cellular metabolic poison. Studies on DNP membranes are performed with the purpose to deeper understand the mechanism of cell membrane damages, and to investigate the biological recognition of DNP with their complementary anti-DNP antibody.
D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
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Fig. 1. Schematic representation of (a) DNP, (b) a LB film of DNP on Pt, (c) a LS film of DNP on a DDT/Pt surface, and (d) the DNP model used for DFT calculations. The yellow balls represent the polar head of DNP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods 2.1. Preparation of the supported lipid films Lipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N(2,4-dinitrophenyl) (DNP) was supplied from AvantiÒ Polar Lipids, and dodecanethiol (P98%, DDT) was from Sigma Aldrich. Pt substrates (ArrandeeÒ) were thin metal layers deposited on Si. Langmuir–Blodgett monolayers of DNP have been adsorbed on a Pt surface (head-down), while Langmuir–Schaefer films have been deposited on top of a dodecanethiol self-assembled monolayer (DDT/SAM) on Pt (head-up). Langmuir films of DNP were prepared by spreading a 1 mg/mL chloroform solution of lipid onto the water surface in a Langmuir trough (MiniMicro System from KSV Instruments). The compression rate was of 5 mm/min and the transfer surface pressure was of 46 mN/m. LB films of DNP on Pt were obtained by a vertical lift up of the substrate at a rate of 3 mm/min. The LS films were obtained by a horizontal transfer of the DNP molecules onto the DDT/SAM, which was previously obtained by immersion of the Pt substrate in a 1 mM DDT ethanol solution for 24 h. In order to verify that the film adsorption occurred with the head-down and the head-up configurations, respectively, we have measured the water contact angle of the films. It results that headdown LB monolayers showed a contact angle of 90°, in agreement with hydrophobic tails pointing out from the surface. DDT/SAM, however, exhibits a contact angle of 108°, which is the characteristic value of highly ordered SAMs of alknethiols on metals. When the DNP lipid has been adsorbed on top of the DDT/SAM by the Langmuir–Schaefer method, the water contact angle of the surface decreased to 45°, testifying for the presence of the DNP polar head pointing out from the surface.
2.2. Sum frequency generation spectroscopy (SFG) coupled to the free electron laser (FEL) The backbone vibrations of DNP films have been preliminary investigated with a laboratory table top spectrometer [39], but
these measurements did not detect any resonant SFG signal of DNP, probably because of the low infrared power of regular OPO laboratory systems (working power: 2–10 mW, 15 ps) together with the weak SFG activity of mid-infrared vibrations. Then, the SFG spectra have been recorded by tuning the FEL (CLIO, Orsay, France) infrared beam (working power: 20–60 mW, 1 ps) [26], between 1375 cm1 and 1000 cm1, while the visible wavelength was kept constant at 523.5 nm. The spectra have been recorded with ppp and ssp (in the order SFG, Vis, IR) sets of polarizations. 2.3. DFT calculations The theoretical calculations were carried out at the density functional theory (DFT) level of approximation by using the B3LYP exchange–correlation functional and the 6-311++G(d,p) basis set. First, the geometries were optimized, then the vibrational frequencies and normal modes were calculated. In order to evaluate the IR and Raman intensities, the IR vectors were obtained analytically, whereas a two-point numerical differentiation procedure was employed to determine the static Raman tensors. More information about the computational procedure can be found in Ref. [40]. All the calculations were performed using the GAUSSIAN 03 program [41]. 3. Results and discussion Fig. 2 shows the SFG spectra of a LB (a) and a LS (b) film of DNP recorded between 1375 cm1 and 1275 cm1, in the ppp (top) and ssp (bottom) configurations of polarizations. In the ppp spectra (top), the high frequency peak is centred at 1344 cm1 in both films, while the low frequency peak shows-up at 1305 cm1 in the LB film, and at 1312 cm1 in the LS film. The ssp spectra (Fig. 2a and b, bottom) exhibit quite different features compared to ppp spectra, especially regarding the peak intensity ratios. Indeed, in the ssp spectrum of the LB film (Fig. 2a, bottom) the intensity of the higher frequency peak has decreased compared to the lower frequency peak, while in the ssp spectrum of the LS film (Fig. 2b, bottom) the former has almost disappeared. Fig. 2c shows
14
D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
Fig. 2. SFG spectra of a LB film of DNP on Pt (a) and of a LS film of DNP onto a DDT/Pt surface (b), recorded with the ppp (top) and ssp (bottom) sets of polarizations between 1375 cm1 and 1275 cm1; (c) SFG spectrum (ppp) of a LB film of DNP on Pt recorded between 1100 cm1 and 1000 cm1. The numbers of the assigned normal modes are given in brackets.
the SFG signal of a LB film of DNP in the 1100–1000 cm1 range which is characterized by a broad resonant peak centred at 1056 cm1. The peak assignment is however not trivial because of the complexity of the molecular structure. In order to associate the resonant signals to molecular vibrations, DFT calculations have been carried out on a model molecule, which holds the main chemical functions of DNP (Fig. 1d). The calculations have provided the vibrational modes and their infrared vectors and Raman tensors, which enable to predict the SFG active vibrations [28,42–45]. Those latter are depicted in the top of Fig. 3 and their characteristics (i.e. frequency, IR and Raman intensities) are summarized in the bottom of Fig. 3. In the spectral range covered by Fig. 2a and b (1375–1275 cm1), DFT calculations predict three SFG active vibrations, namely at 1349 cm1 (100), at 1319 cm1 (98) and at 1292 cm1 (95), all mainly localized on the polar head of the lipid molecule. The highest frequency peak in the SFG spectra is as-
signed to vibration 100. This latter is mostly due to a superposition of the para-NO2 symmetric stretching with CH2 bendings (waggings) and has a strong SFG activity, as testified by the strong IR and Raman intensities. The experimental peaks at 1305 cm1 for the LB film and at 1312 cm1 for the LS film are assigned to the superposition of vibrations 98 and 95, which correspond to bending modes of the phenyl and methylene units and show comparable SFG activities. The broadening, the asymmetric peak features, and the frequency shift between the LB (1305 cm1) and the LS (1312 cm1) films may be attributed to the coexistence of different stable molecular conformations, that can be expected for these long and flexible DNP polar heads. The presence of several stable molecular conformations has been confirmed by DFT calculations, strengthening the hypothesis that adsorption onto the surface has occurred with different molecular orientations of the polar head, which may hold a large orientational distribution, even if the aliphatic carbon skeleton is well-packed and highly organized. The
Fig. 3. Top: representation of the vibrational modes, the IR vectors (pink arrows) and the Raman tensors (red for negative, green for positive values) as obtained from DFT calculations for the DNP model molecule. Bottom: theoretical parameters of the vibrational modes (frequency, IR intensity, Raman intensity) as predicted from DFT calculations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
D. Lis et al. / Chemical Physics Letters 489 (2010) 12–15
lowest vibrational frequency at 1056 cm1 is a superposition of CH bendings (81, 80, 77), which are rather delocalized on the entire molecular structure. The peak profiles, namely the peak intensity ratios and the peak frequency shifts, provide orientational information. In fact, each polarization set probes selected components of the nonlinear susceptibility (ZZZ, XXZ, XZX, and ZXX with ppp, and YYZ with ssp), and therefore the peak intensities depend on the orientation of ð2Þ the susceptibility tensor vijk of the vibrational modes with respect to the interface [46]. This implies that the different peak profiles of the LB and LS films (Fig. 1) in the ppp and ssp sets are ascribed to different orientations of the molecular groups involved in the SFG active vibrations. Even if the ppp spectra recorded for the LB and LS films (Fig. 2a and b, top) exhibit quite similar peak profiles, the ssp spectra (Fig. 2a and b, bottom) show rather different features. Therefore, this attests to a different orientational distribution of the polar head in the two film configurations. One can notice that this orientational difference can be highlighted only using polarization-dependent SFG measurements (with both ppp and ssp sets). However, this is challenging since detecting SFG signatures with other polarization than ppp is not regularly achieved on metals [32]. Here, this was achieved thanks to the higher FEL power density, regarding both the beam power and pulse. 4. Conclusions To our knowledge, this is the first study reporting on biological layers investigated with SFG spectroscopy coupled to a FEL radiation. This has allowed to probe backbone vibrations within metal-supported lipid films, and to measure selected components of the molecular nonlinear susceptibility in the 1375–1000 cm1 spectral window. Moreover, the approach – coupled to DFT calculations – has succeeded in identifying vibrations, which are purely localized on the polar head. This study has demonstrated that SFG can selectively follow the vibrational nonlinear response of chemical groups located on the DNP polar head. On this basis, the vibrational signatures will enable to carefully characterize the biological recognition of DNP lipid films with complementary anti-DNP antibodies, therefore strengthening the role of SFG spectroscopy as a highly sensitive tool for biological detection [47–49].
15
The authors acknowledge the support of C. Six and A. Gayral for the SFG measurements at the CLIO facility. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39]
Acknowledgments
[40] [41]
F.C., Y.C., and A.P., are postdoctoral researcher, research associate, and research director of the Fund for the Scientific Research F.R.S.-FNRS. D.L. thanks the FRIA for his Ph.D. student fellowship. J.G. thanks the F.R.S.-FNRS for his postdoctoral grant under the Conventions No. 2.4.509.04.F. The calculations were performed on the Interuniversity Scientific Computing Facility (ISCF, University of Namur, Belgium) for which the authors gratefully acknowledge the financial support of the F.R.S.-FRFC and the ‘Loterie Nationale’ under Contract No. 2.4.617.07.F, and of the FUNDP.
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