The Preparation Of Supported Lipid Bilayers And Its Functional Study

Cell membranes maintain order of each part of the structure in cells and play an important role in the process of the material exchange,information transmission and biosynthesis of large molecules between cells and the external environment.Artificial lipid membranes have been widely used to study the structure,property and function of biomembranes because of the structural complexity of the cell membrane makes it difficult to separate.Supported lipid bilayers(SLBs)are popular as model systems for cell membranes and are ideal material for fixing biological active proteins.In addition,SLBs can not only maintain the biological activity of proteins,but also inhibit other non-specific adsorption of biological molecules.SLBs are promising for future applications in studying the interaction between receptors and ligands on cell membrane surface,the formation of the immune synapse.However,there are still many problems to be solved in the preparation and functionalization of the SLBs,many scientists endeavor in the research of preparing excellent SLBs.Nano sizer and zeta-potential tester was used to measure the distribution of particle size and potential of muti-lamellar vesicle(MLV)and small unilamellar vesicles(SUV),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 hydrophilic glass slide,and the effects of the DOPC-to-DGS-NTA ratio,SUV producing method,sizes of bleaching areas and concentrations of loading proteins(P-selectin and intercellular cell adhesion molecule-1,ICAM-1)on the SLBs fluidity and diffusion coefficient were studied systematically in this paper.The results demonstrated that: 1)SUV made by probe sonication were more stable and exhibited more uniform and smaller size compared with that made by film extrusion;2)The whole process of SLBs formation must not be exposed to air,or otherwise the SLBs showed no fludity;3)With the proportion of DGS-NTA mole ratio inceasing from 2% to 20%,the fluidity and fluorescence recovery degree decreased gradually,and the SLBs would lose its fluidity if the ratio reached to 82?18;4)The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size.Although SLBs of different bleaching area sizes showed different recovery rates,all of their fluorescence intensity could recover to above 90% after being bleached in 250 s;5)The average fluorescence intensity of SLBs increased linearly with the loading protein concentration(10~40 nmol/L),and the protein showed good mobility on the SLBs.P-selectin and ICAM-1 are important signal molecules during inflammatory immune response and tumor metastasis.So,the study would not only provide a new perspective for preparing SLBs,but also build a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.