High-resolution and high-repetition-rate vibrational sum-frequency generation spectroscopy of one- and two-component phosphatidylcholine monolayers


We present broadband vibrational sum-frequency generation (VSFG) spectra of Langmuir-Blodgett monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and different mixtures of them as model systems of pulmonary surfactants. The systematic study explored the dependence of the vibrational spectra as a function of surface tension and mixture ratio in various polarization combinations. The extremely short acquisition time and the high spectral resolution of our recently developed spectrometer helped minimize sample degradation under ambient conditions throughout the duration of the measurement and allowed the detection of previously unseen vibrational bands with unprecedented signal-to-noise ratio. The dramatically improved capability to record reliable vibrational spectra together with the label-free nature of the VSFG method provides direct access to native lipid structure and dynamics directly in the monolayer. The resulting data deliver quantitative information for structural analysis of multi-component phospholipid monolayers and may aid in the development of new synthetic pulmonary surfactants.

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  1. 1.

    Goerke J. Pulmonary surfactant: functions and molecular composition. Biochim Biophys Acta (BBA) - Mol Basis Dis. 1998;1408(2):79–89. https://doi.org/10.1016/S0925-4439(98)00060-X.

    Article  CAS  Google Scholar 

  2. 2.

    Wüstneck R, Perez-Gil J, Wüstneck N, Cruz A, Fainerman VB, Pison U. Interfacial properties of pulmonary surfactant layers. Adv Colloid Interf Sci. 2005;117(1):33–58. https://doi.org/10.1016/j.cis.2005.05.001.

    Article  CAS  Google Scholar 

  3. 3.

    Yu S, Harding PGR, Smith N, Possmayer F. Bovine pulmonary surfactant: chemical composition and physical properties. Lipids. 1983;18(8):522–9. https://doi.org/10.1007/BF02535391.

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Ramanathan R. Animal-derived surfactants: where are we? The evidence from randomized, controlled clinical trials. J Perinatol. 2009;29:S38. https://doi.org/10.1038/jp.2009.31.

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Sardesai S, Biniwale M, Wertheimer F, Garingo A, Ramanathan R. Evolution of surfactant therapy for respiratory distress syndrome: past, present, and future. Pediatr Res. 2016;81:240. https://doi.org/10.1038/pr.2016.203.

    Article  PubMed  Google Scholar 

  6. 6.

    Pfister RH, Soll R, Wiswell TE. Cochrane review: Protein containing synthetic surfactant versus animal derived surfactant extract for the prevention and treatment of respiratory distress syndrome. Evid Based Child Health. 2010;5(1):17–51. https://doi.org/10.1002/ebch.517.

    Article  Google Scholar 

  7. 7.

    Halliday HL. Surfactants: past, present and future. J Perinatol. 2008;28:S47. https://doi.org/10.1038/jp.2008.50.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Olżyńska A, Zubek M, Roeselova M, Korchowiec J, Cwiklik L. Mixed DPPC/POPC monolayers: all-atom molecular dynamics simulations and Langmuir monolayer experiments. Biochim Biophys Acta Biomembr. 2016;1858(12):3120–30. https://doi.org/10.1016/j.bbamem.2016.09.015.

    Article  CAS  Google Scholar 

  9. 9.

    Wang H-F, Velarde L, Gan W, Fu L. Quantitative sum-frequency generation vibrational spectroscopy of molecular surfaces and interfaces: lineshape, polarization, and orientation. Annu Rev Phys Chem. 2015;66(1):189–216. https://doi.org/10.1146/annurev-physchem-040214-121322.

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Wang H-F, Gan W, Lu R, Rao Y, Wu B-H. Quantitative spectral and orientational analysis in surface sum frequency generation vibrational spectroscopy (SFG-VS). Int Rev Phys Chem. 2005;24(2):191–256. https://doi.org/10.1080/01442350500225894.

    Article  CAS  Google Scholar 

  11. 11.

    Baumler SM, Allen HC. Chapter 5. Vibrational spectroscopy of gas–liquid interfaces. In: Faust JA, House JE, editors. Physical chemistry of gas-liquid interfaces. Elsevier; 2018. p. 105–33.

  12. 12.

    Liu W, Wang Z, Fu L, Leblanc RM, Yan ECY. Lipid compositions modulate fluidity and stability of bilayers: characterization by surface pressure and sum frequency generation spectroscopy. Langmuir. 2013;29(48):15022–31. https://doi.org/10.1021/la4036453.

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Li B, Lu X, Han X, Wu F-G, Myers JN, Chen Z. Interfacial Fresnel coefficients and molecular structures of model cell membranes: from a lipid monolayer to a lipid bilayer. J Phys Chem C. 2014;118(49):28631–9. https://doi.org/10.1021/jp509272k.

    Article  CAS  Google Scholar 

  14. 14.

    Ma G, Allen HC. DPPC Langmuir monolayer at the air–water interface: probing the tail and head groups by vibrational sum frequency generation spectroscopy. Langmuir. 2006;22(12):5341–9. https://doi.org/10.1021/la0535227.

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Sung W, Seok S, Kim D, Tian CS, Shen YR. Sum-frequency spectroscopic study of Langmuir monolayers of lipids having oppositely charged headgroups. Langmuir. 2010;26(23):18266–72. https://doi.org/10.1021/la103129z.

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Nojima Y, Suzuki Y, Yamaguchi S. Weakly hydrogen-bonded water inside charged lipid monolayer observed with heterodyne-detected vibrational sum frequency generation spectroscopy. J Phys Chem C. 2017;121(4):2173–80. https://doi.org/10.1021/acs.jpcc.6b09229.

    Article  CAS  Google Scholar 

  17. 17.

    Liljeblad JFD, Bulone V, Rutland MW, Johnson CM. Supported phospholipid monolayers. The molecular structure investigated by vibrational sum frequency spectroscopy. J Phys Chem C. 2011;115(21):10617–29. https://doi.org/10.1021/jp111587e.

    Article  CAS  Google Scholar 

  18. 18.

    Li B, Li X, Ma Y-H, Han X, Wu F-G, Guo Z, et al. Sum frequency generation of interfacial lipid monolayers shows polarization dependence on experimental geometries. Langmuir. 2016;32(28):7086–95. https://doi.org/10.1021/acs.langmuir.6b01944.

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Feng R-J, Li X, Zhang Z, Lu Z, Guo Y. Spectral assignment and orientational analysis in a vibrational sum frequency generation study of DPPC monolayers at the air/water interface. J Chem Phys. 2016;145(24):244707. https://doi.org/10.1063/1.4972564.

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Smits M, Sovago M, Wurpel GWH, Kim D, Müller M, Bonn M. Polarization-resolved broad-bandwidth sum-frequency generation spectroscopy of monolayer relaxation. J Phys Chem C. 2007;111(25):8878–83. https://doi.org/10.1021/jp067453w.

    Article  CAS  Google Scholar 

  21. 21.

    Liljeblad JFD, Bulone V, Tyrode E, Rutland MW, Johnson CM. Phospholipid monolayers probed by vibrational sum frequency spectroscopy: instability of unsaturated phospholipids. Biophys J. 2010;98(10):L50–L2. https://doi.org/10.1016/j.bpj.2010.02.009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Velarde L, Wang H-F. Unified treatment and measurement of the spectral resolution and temporal effects in frequency-resolved sum-frequency generation vibrational spectroscopy (SFG-VS). Phys Chem Chem Phys. 2013;15(46):19970–84. https://doi.org/10.1039/C3CP52577E.

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Li Y, Feng R, Lin L, Liu M, Guo Y, Zhang Z. Ordering effects of cholesterol on sphingomyelin monolayers investigated by high-resolution broadband sum-frequency generation vibrational spectroscopy. Chin Chem Lett. 2018;29(3):357–60. https://doi.org/10.1016/j.cclet.2017.11.006.

    Article  CAS  Google Scholar 

  24. 24.

    Heiner Z, Petrov V, Mero M. Compact, high-repetition-rate source for broadband sum-frequency generation spectroscopy. APL Photonics. 2017;2(6):066102. https://doi.org/10.1063/1.4983691.

    Article  CAS  Google Scholar 

  25. 25.

    Yesudas F, Mero M, Kneipp J, Heiner Z. Vibrational sum-frequency generation spectroscopy of lipid bilayers at repetition rates up to 100 kHz. J Chem Phys. 2018;148(10):104702. https://doi.org/10.1063/1.5016629.

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Roberts G. Langmuir-Blodgett films. Boston: Springer; 1990.

    Google Scholar 

  27. 27.

    Motschmann H, Mohwald H. Langmuir–Blodgett films. In: Holmberg K, editor. Handbook of applied surface and colloid chemistry. Wiley; 2001.

  28. 28.

    Tredgold RH. The physics of Langmuir-Blodgett films. Rep Prog Phys. 1987;50(12):1609.

    Article  CAS  Google Scholar 

  29. 29.

    Raoult F, Boscheron ACL, Husson D, Sauteret C, Modena A, Malka V, et al. Efficient generation of narrow-bandwidth picosecond pulses by frequency doubling of femtosecond chirped pulses. Opt Lett. 1998;23(14):1117–9. https://doi.org/10.1364/OL.23.001117.

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Nejbauer M, Radzewicz C. Efficient spectral shift and compression of femtosecond pulses by parametric amplification of chirped light. Opt Express. 2012;20(3):2136–42. https://doi.org/10.1364/OE.20.002136.

    Article  PubMed  Google Scholar 

  31. 31.

    Qiao L, Ge A, Liang Y, Ye S. Oxidative degradation of the monolayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in low-level ozone. J Phys Chem B. 2015;119(44):14188–99. https://doi.org/10.1021/acs.jpcb.5b08985.

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Liu J, Conboy JC. Structure of a gel phase lipid bilayer prepared by the Langmuir–Blodgett/Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy. Langmuir. 2005;21(20):9091–7. https://doi.org/10.1021/la051500e.

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Lu R, Gan W, Wu B-h, Chen H, Wang H-f. Vibrational polarization spectroscopy of CH stretching modes of the methylene group at the vapor/liquid interfaces with sum frequency generation. J Phys Chem B. 2004;108(22):7297–306. https://doi.org/10.1021/jp036674o.

    Article  CAS  Google Scholar 

  34. 34.

    Lu R, Gan W, Wu B-h, Zhang Z, Guo Y, Wang H-f. C–H stretching vibrations of methyl, methylene and methine groups at the vapor/alcohol (n = 1–8) interfaces. J Phys Chem B. 2005;109(29):14118–29. https://doi.org/10.1021/jp051565q.

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Velarde L, Wang H-f. Capturing inhomogeneous broadening of the –CN stretch vibration in a Langmuir monolayer with high-resolution spectra and ultrafast vibrational dynamics in sum-frequency generation vibrational spectroscopy (SFG-VS). J Chem Phys. 2013;139(8):084204. https://doi.org/10.1063/1.4818996.

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Velarde L, Zhang X-y, Lu Z, Joly AG, Wang Z, Wang H-f. Communication: Spectroscopic phase and lineshapes in high-resolution broadband sum frequency vibrational spectroscopy: resolving interfacial inhomogeneities of “identical” molecular groups. J Chem Phys. 2011;135(24):241102. https://doi.org/10.1063/1.3675629.

    Article  CAS  PubMed  Google Scholar 

  37. 37.

    Feng R-j, Lin L, Li Y-y, Liu M-h, Guo Y, Zhang Z. Effect of Ca2+ to sphingomyelin investigated by sum frequency generation vibrational spectroscopy. Biophys J. 2017;112(10):2173–83. https://doi.org/10.1016/j.bpj.2017.04.026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Fu L, Chen S-L, Wang H-F. Validation of spectra and phase in sub-1 cm−1 resolution sum-frequency generation vibrational spectroscopy through internal heterodyne phase-resolved measurement. J Phys Chem B. 2016;120(8):1579–89. https://doi.org/10.1021/acs.jpcb.5b07780.

    Article  CAS  PubMed  Google Scholar 

  39. 39.

    Mifflin AL, Velarde L, Ho J, Psciuk BT, Negre CFA, Ebben CJ, et al. Accurate line shapes from sub-1 cm−1 resolution sum frequency generation vibrational spectroscopy of α-pinene at room temperature. J Phys Chem A. 2015;119(8):1292–302. https://doi.org/10.1021/jp510700z.

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    MacPhail RA, Strauss HL, Snyder RG, Elliger CA. Carbon-hydrogen stretching modes and the structure of n-alkyl chains. 2. Long, all-trans chains. J Phys Chem. 1984;88(3):334–41. https://doi.org/10.1021/j150647a002.

    Article  CAS  Google Scholar 

  41. 41.

    Ward RN, Duffy DC, Davies PB, Bain CD. Sum-frequency spectroscopy of surfactants adsorbed at a flat hydrophobic surface. J Phys Chem. 1994;98(34):8536–42. https://doi.org/10.1021/j100085a037.

    Article  CAS  Google Scholar 

  42. 42.

    Conboy JC, Messmer MC, Richmond GL. Investigation of surfactant conformation and order at the liquid–liquid interface by total internal reflection sum-frequency vibrational spectroscopy. J Phys Chem. 1996;100(18):7617–22. https://doi.org/10.1021/jp953616x.

    Article  CAS  Google Scholar 

  43. 43.

    Miranda PB, Pflumio V, Saijo H, Shen YR. Chain–chain interaction between surfactant monolayers and alkanes or alcohols at solid/liquid interfaces. J Am Chem Soc. 1998;120(46):12092–9. https://doi.org/10.1021/ja9732441.

    Article  CAS  Google Scholar 

  44. 44.

    Watry MR, Tarbuck TL, Richmond GL. Vibrational sum-frequency studies of a series of phospholipid monolayers and the associated water structure at the vapor/water interface. J Phys Chem B. 2003;107(2):512–8. https://doi.org/10.1021/jp0216878.

    Article  CAS  Google Scholar 

  45. 45.

    Backus EHG, Bonn D, Cantin S, Roke S, Bonn M. Laser-heating-induced displacement of surfactants on the water surface. J Phys Chem B. 2012;116(9):2703–12. https://doi.org/10.1021/jp2074545.

    Article  CAS  PubMed  Google Scholar 

  46. 46.

    Lambert AG, Davies PB, Neivandt DJ. Implementing the theory of sum frequency generation vibrational spectroscopy: a tutorial review. Appl Spectrosc Rev. 2005;40(2):103–45. https://doi.org/10.1081/ASR-200038326.

    Article  CAS  Google Scholar 

  47. 47.

    Kim J, Kim G, Cremer PS. Investigations of water structure at the solid/liquid interface in the presence of supported lipid bilayers by vibrational sum frequency spectroscopy. Langmuir. 2001;17(23):7255–60. https://doi.org/10.1021/la0017274.

    Article  CAS  Google Scholar 

  48. 48.

    Chen X, Hua W, Huang Z, Allen HC. Interfacial water structure associated with phospholipid membranes studied by phase-sensitive vibrational sum frequency generation spectroscopy. J Am Chem Soc. 2010;132(32):11336–42. https://doi.org/10.1021/ja1048237.

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Mondal JA, Nihonyanagi S, Yamaguchi S, Tahara T. Structure and orientation of water at charged lipid monolayer/water interfaces probed by heterodyne-detected vibrational sum frequency generation spectroscopy. J Am Chem Soc. 2010;132(31):10656–7. https://doi.org/10.1021/ja104327t.

    Article  CAS  PubMed  Google Scholar 

  50. 50.

    Doǧangün M, Ohno PE, Liang D, McGeachy AC, Bé AG, Dalchand N, et al. Hydrogen-bond networks near supported lipid bilayers from vibrational sum frequency generation experiments and atomistic simulations. J Phys Chem B. 2018;122(18):4870–9. https://doi.org/10.1021/acs.jpcb.8b02138.

    Article  CAS  PubMed  Google Scholar 

  51. 51.

    Mondal JA, Nihonyanagi S, Yamaguchi S, Tahara T. Three distinct water structures at a zwitterionic lipid/water interface revealed by heterodyne-detected vibrational sum frequency generation. J Am Chem Soc. 2012;134(18):7842–50. https://doi.org/10.1021/ja300658h.

    Article  CAS  PubMed  Google Scholar 

  52. 52.

    Notter RH, Tabak SA, Holcomb S, Mavis RD. Postcollapse dynamic surface pressure relaxation in binary surface films containing dipalmitoyl phosphatidylcholine. J Colloid Interface Sci. 1980;74(2):370–7. https://doi.org/10.1016/0021-9797(80)90206-4.

    Article  CAS  Google Scholar 

  53. 53.

    Ma G, Liu J, Fu L, Yan ECY. Probing water and biomolecules at the air–water interface with a broad bandwidth vibrational sum frequency generation spectrometer from 3800 to 900 cm−1. Appl Spectrosc. 2009;63(5):528–37.

    Article  CAS  PubMed  Google Scholar 

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We thank K. Balasubramanian (SALSA, Humboldt-Universität zu Berlin) and R. M. Iost for providing support in the sample cleaning processes.


This project is financed by the Deutsche Forschungsgemeinschaft (DFG) project GSC 1013 SALSA. F.Y. is grateful for the support by a fellowship in SALSA. Z. H. acknowledges the funding of her Julia Lermontova fellowship by GSC 1013 SALSA.

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry with guest editors Erin Baker, Kerstin Leopold, Francesco Ricci, and Wei Wang.

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Yesudas, F., Mero, M., Kneipp, J. et al. High-resolution and high-repetition-rate vibrational sum-frequency generation spectroscopy of one- and two-component phosphatidylcholine monolayers. Anal Bioanal Chem 411, 4861–4871 (2019). https://doi.org/10.1007/s00216-019-01690-9

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  • Lung surfactants
  • 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
  • 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)
  • Broadband vibrational sum-frequency generation spectroscopy
  • Two-component phosphatidylcholine
  • Langmuir-Blodgett monolayer