The preparation of supported lipid bilayers and its functional study_Journal of Biomedical Engineering

Authors：

ZOU Hui , WU Jianhua ,  FANG Ying

School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, P.R.China;

Corresponding author：

FANG Ying, Email: yfang@scut.edu.cn

Keywords：

supported lipid bilayers; DOPC and DGS-NTA; fluorescence recovery after photobleaching; diffusion coefficient; fluidity

DOI：

10.7507/1001-5515.201801028

Video：

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Supported lipid bilayers (SLBs) have been widely used in biomedical and bioengineering research in vitro because its structure and function are similar to natural cell membrane. A fluorescence recovery after photobleaching (FRAP) technique was used to measure the lateral diffusion of the SLBs composed of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-［(N-(5-amino-1-carboxyp-entyl) iminodiacetic acid)］ (DGS-NTA) on the glass slide, and the effects of the DOPC-to-DGS-NTA ratio, small unilamellar vesicles (SUV) producing method, sizes of bleaching areas and concentrations of loading proteins on the SLBs fluidity and diffusion coefficient were studied systematically in this paper. The results demonstrated that: (1) SUV made by probe sonication exhibited more uniform and smaller size compared with that made by film extrusion, but the whole process of SLBs formation must not be exposed to air. (2) The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size. With the mole ratio of DOPC to DGS-NTA decreasing from 98∶2 to 84∶16, the fluidity and fluorescence recovery degree decreased gradually, and the SLBs would lose its fluidity if the ratio reached to 82∶18. (3) The average fluorescence intensity of SLBs increased linearly with the loading protein concentration (10–40 nmol·L^–1), and the protein showed good mobility on the SLBs. The study would provide a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.

Citation： ZOU Hui, WU Jianhua, FANG Ying. The preparation of supported lipid bilayers and its functional study. Journal of Biomedical Engineering, 2019, 36(1): 85-93. doi: 10.7507/1001-5515.201801028 Copy

1.	Mcconnell H M, Watts T H, Weis R M, et al. Supported planar membranes in studies of cell-cell recognition in the immune system. Biochim Biophys Acta, 1986, 864(1): 95-106.
2.	Attwood S J, Choi Y, Leonenko Z. Preparation of DOPC and DPPC supported planar lipid bilayers for atomic force microscopy and atomic force spectroscopy. Int J Mol Sci, 2013, 14(2): 3514-3539.
3.	Jass J, Tjärnhage T, Puu G. From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: An atomic force microscopy study. Biophys J, 2000, 79(6): 3153-3163.
4.	Nair P M, Salaita K, Petit R S, et al. Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. Nat Protoc, 2011, 6(4): 523-539.
5.	Wu Jianhua, Fang Ying, Zarnitsyna V I, et al. A coupled diffusion-kinetics model for analysis of contact-area FRAP experiment. Biophys J, 2008, 95(2): 910-919.
6.	Santos L C, Blair D A, Kumari S, et al. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability. Immunol Cell Biol, 2016, 94(10): 981-993.
7.	Zhou X, Cao H, Yang D, et al. Two-dimensional alignment of self-assembled organic nanotubes through Langmuir-Blodgett technique. Langmuir the Acs Journal of Surfaces & Colloids, 2016, 32(49): 13065-13072.
8.	Komolov A S, Lazneva E F, Zhukov Y M, et al. Atomic composition and stability of Langmuir-Blodgett monolayers based on siloxane dimer of quaterthiophene on the surface of polycrystalline gold. Physics of the Solid State, 2017, 59(12): 2491-2496.
9.	Iyer A, Schilderink N, Claessens M M, et al. Membrane-bound alpha synuclein clusters induce impaired lipid diffusion and increased lipid packing. Biophys J, 2016, 111(11): 2440-2449.
10.	Castellana E T, Cremer P S. Solid supported lipid bilayers: From biophysical studies to sensor design. Surf Sci Rep, 2006, 61(10): 429-444.
11.	Curran A R, Templer R H, Booth P J. Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer. Biochemistry, 1999, 38(29): 9328-9336.
12.	Nye J A, Groves J T. Kinetic control of histidine-tagged protein surface density on supported lipid bilayers. Langmuir, 2008, 24(8): 4145-4149.
13.	Yu Chenghan, Groves J T. Engineering supported membranes for cell biology. Med Biol Eng Comput, 2010, 48(10, SI): 955-963.
14.	Lin W C, Yu C H, Triffo S, et al. Supported membrane formation, characterization, functionalization, and patterning for application in biological science and technology. Curr Protoc Chem Biol, 2010, 2(4): 235-269.
15.	Jousma H, Talsma H, Spies F, et al. Characterization of liposomes. The influence of extrusion of multilamellar vesicles through polycarbonate membranes on particle size, particle size distribution and number of bilayers. Int J Pharm, 1987, 35(3): 263-274.
16.	Kunding A H, Mortensen M W, Christensen S M, et al. A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys J, 2008, 95(3): 1176-1188.
17.	Cho N J, Hwang L Y, Solandt J J R, et al. Comparison of extruded and sonicated vesicles for planar bilayer self-assembly. Materials (Basel), 2013, 6(8): 3294-3308.
18.	Traïkia M, Warschawski D E, Recouvreur M, et al. Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and ³¹P-nuclear magnetic resonance. Eur Biophys J, 2000, 29(3): 184-195.
19.	Zheng Peilin, Bertolet G, Chen Yuhui, et al. Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer. J Vis Exp, 2015(96): 52502.
20.	Zhang Shaosen, Xu Liling, Zhao Xingwang, et al. A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments. Plos One, 2013, 8(5): e63735.
21.	Fischer N O, Blanchette C D, Chromy B A, et al. Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles. Bioconjug Chem, 2009, 20(3): 460-465.
22.	Mcever R P. Selectins: initiators of leukocyte adhesion and signaling at the vascular wall. Cardiovasc Res, 2015, 107(3): 331-339.
23.	Tanaka M, Hermann J, Haase I, et al. Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes. Langmuir, 2007, 23(10): 5638-5644.
24.	Seu K J, Cambrea L R, Everly R M, et al. Influence of lipid chemistry on membrane fluidity: Tail and headgroup interactions. Biophys J, 2006, 91(10): 3727-3735.
25.	Tolentino T P, Wu Jianhua, Zarnitsyna V I, et al. Measuring diffusion and binding kinetics by contact area FRAP. Biophys J, 2008, 95(2): 920-930.
26.	Tian Ye, Schwieters C D, Opella S J, et al. High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH. J Biomol NMR, 2017, 67(1): 35-49.
27.	Tolentino T P. Measuring ligand diffusivity and receptor binding kinetics within a cell membrane contact area. Georgia Institute of Technology, 2001, 86(S421): 67-71.
28.	Dustin M L, Bromley S K, Davis M M, et al. Identification of self through two-dimensional chemistry and synapses. Annu Rev Cell Dev Biol, 2001, 17(1): 133-157.
29.	Dustin M L. Supported bilayers at the vanguard of immune cell activation studies. J Struct Biol, 2009, 168(1): 152-160.
30.	Kiessling V, Yang S T, Tamm L K. Supported lipid bilayers as models for studying membrane domains. Current Topics in Membranes, 2015, 75: 1-23.
31.	Liu W, Tobias M, Pavel T, et al. Antigen affinity discrimination is an intrinsic function of the B cell receptor. J Exp Med, 2010, 207(5): 1095-1111.

1. Mcconnell H M, Watts T H, Weis R M, et al. Supported planar membranes in studies of cell-cell recognition in the immune system. Biochim Biophys Acta, 1986, 864(1): 95-106.
2. Attwood S J, Choi Y, Leonenko Z. Preparation of DOPC and DPPC supported planar lipid bilayers for atomic force microscopy and atomic force spectroscopy. Int J Mol Sci, 2013, 14(2): 3514-3539.
3. Jass J, Tjärnhage T, Puu G. From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: An atomic force microscopy study. Biophys J, 2000, 79(6): 3153-3163.
4. Nair P M, Salaita K, Petit R S, et al. Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. Nat Protoc, 2011, 6(4): 523-539.
5. Wu Jianhua, Fang Ying, Zarnitsyna V I, et al. A coupled diffusion-kinetics model for analysis of contact-area FRAP experiment. Biophys J, 2008, 95(2): 910-919.
6. Santos L C, Blair D A, Kumari S, et al. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability. Immunol Cell Biol, 2016, 94(10): 981-993.
7. Zhou X, Cao H, Yang D, et al. Two-dimensional alignment of self-assembled organic nanotubes through Langmuir-Blodgett technique. Langmuir the Acs Journal of Surfaces & Colloids, 2016, 32(49): 13065-13072.
8. Komolov A S, Lazneva E F, Zhukov Y M, et al. Atomic composition and stability of Langmuir-Blodgett monolayers based on siloxane dimer of quaterthiophene on the surface of polycrystalline gold. Physics of the Solid State, 2017, 59(12): 2491-2496.
9. Iyer A, Schilderink N, Claessens M M, et al. Membrane-bound alpha synuclein clusters induce impaired lipid diffusion and increased lipid packing. Biophys J, 2016, 111(11): 2440-2449.
10. Castellana E T, Cremer P S. Solid supported lipid bilayers: From biophysical studies to sensor design. Surf Sci Rep, 2006, 61(10): 429-444.
11. Curran A R, Templer R H, Booth P J. Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer. Biochemistry, 1999, 38(29): 9328-9336.
12. Nye J A, Groves J T. Kinetic control of histidine-tagged protein surface density on supported lipid bilayers. Langmuir, 2008, 24(8): 4145-4149.
13. Yu Chenghan, Groves J T. Engineering supported membranes for cell biology. Med Biol Eng Comput, 2010, 48(10, SI): 955-963.
14. Lin W C, Yu C H, Triffo S, et al. Supported membrane formation, characterization, functionalization, and patterning for application in biological science and technology. Curr Protoc Chem Biol, 2010, 2(4): 235-269.
15. Jousma H, Talsma H, Spies F, et al. Characterization of liposomes. The influence of extrusion of multilamellar vesicles through polycarbonate membranes on particle size, particle size distribution and number of bilayers. Int J Pharm, 1987, 35(3): 263-274.
16. Kunding A H, Mortensen M W, Christensen S M, et al. A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys J, 2008, 95(3): 1176-1188.
17. Cho N J, Hwang L Y, Solandt J J R, et al. Comparison of extruded and sonicated vesicles for planar bilayer self-assembly. Materials (Basel), 2013, 6(8): 3294-3308.
18. Traïkia M, Warschawski D E, Recouvreur M, et al. Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and ³¹P-nuclear magnetic resonance. Eur Biophys J, 2000, 29(3): 184-195.
19. Zheng Peilin, Bertolet G, Chen Yuhui, et al. Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer. J Vis Exp, 2015(96): 52502.
20. Zhang Shaosen, Xu Liling, Zhao Xingwang, et al. A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments. Plos One, 2013, 8(5): e63735.
21. Fischer N O, Blanchette C D, Chromy B A, et al. Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles. Bioconjug Chem, 2009, 20(3): 460-465.
22. Mcever R P. Selectins: initiators of leukocyte adhesion and signaling at the vascular wall. Cardiovasc Res, 2015, 107(3): 331-339.
23. Tanaka M, Hermann J, Haase I, et al. Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes. Langmuir, 2007, 23(10): 5638-5644.
24. Seu K J, Cambrea L R, Everly R M, et al. Influence of lipid chemistry on membrane fluidity: Tail and headgroup interactions. Biophys J, 2006, 91(10): 3727-3735.
25. Tolentino T P, Wu Jianhua, Zarnitsyna V I, et al. Measuring diffusion and binding kinetics by contact area FRAP. Biophys J, 2008, 95(2): 920-930.
26. Tian Ye, Schwieters C D, Opella S J, et al. High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH. J Biomol NMR, 2017, 67(1): 35-49.
27. Tolentino T P. Measuring ligand diffusivity and receptor binding kinetics within a cell membrane contact area. Georgia Institute of Technology, 2001, 86(S421): 67-71.
28. Dustin M L, Bromley S K, Davis M M, et al. Identification of self through two-dimensional chemistry and synapses. Annu Rev Cell Dev Biol, 2001, 17(1): 133-157.
29. Dustin M L. Supported bilayers at the vanguard of immune cell activation studies. J Struct Biol, 2009, 168(1): 152-160.
30. Kiessling V, Yang S T, Tamm L K. Supported lipid bilayers as models for studying membrane domains. Current Topics in Membranes, 2015, 75: 1-23.
31. Liu W, Tobias M, Pavel T, et al. Antigen affinity discrimination is an intrinsic function of the B cell receptor. J Exp Med, 2010, 207(5): 1095-1111.

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The preparation of supported lipid bilayers and its functional study

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