支撑脂双层膜的制备及其功能化研究
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摘要
支撑脂双层膜(SLBs)因其结构与功能均类似天然细胞膜而广泛应用于生物医学及生物工程研究。本文以玻璃载玻片为基底,以1, 2-油-锡-磷脂-3-卵磷脂(DOPC)与{亚氨基二乙酸丁二酰(镍盐)}(DGS-NTA)为原料,采用荧光漂白恢复技术(FRAP)研究原料配比、制备方法、漂白区域大小及蛋白加载浓度对SLBs流动性及扩散系数的影响。结果表明:①声波破碎法优于薄膜挤压法,预处理脂质体得到的小囊泡粒径更小、更均匀,并且SLBs形成过程中须避免接触空气;②膜的荧光恢复速度和扩散系数随漂白区域的增大而减小,随着DOPC与DGS-NTA摩尔质量比由98∶2变为84∶16,SLBs流动性及荧光恢复率逐渐减小,当两者配比为82∶18时,膜已失去流动性;③加载蛋白浓度在10～40 nmol·L~(–1)范围时,SLBs的荧光强度随蛋白浓度线性增加,蛋白在SLBs上具有很好的流动性。该研究为细胞膜分子之间的相互作用及后续信号响应的研究搭建了一个良好的生物膜平台。
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)](DGSNTA) 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.
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)](DGSNTA) 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.
引文
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19 Zheng Peilin,Bertolet G,Chen Yuhui,et al.Super-resolution imaging of the natural killer cell immunological synapse on a glasssupported 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.
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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.
2 Attwood S J,Choi Y,Leonenko Z.Preparation of DOPC andDPPC supported planar lipid bilayers for atomic force microscopy and atomic force spectroscopy.Int J Mol Sci,2013,14(2):3514-3539.
3 Jass J,Tjarnhage 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 diffusionkinetics 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 polymerizationdependent 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 selfassembled 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 fromsingle-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 Trai'kia 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 glasssupported 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.