Research and Application of Different Biomembranes

Author:Xu Fan

Supervisor:ma yu qiang

Database:Doctor

Degree Year:2015

Download:133

Pages:112

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As parts of cells,biomembranes are important for keeping organisms functioning well.They control entry and egress of molecules for the corresponding membrane bounded compartments,signalling and communication of cells,and provide a distinct biochemical environment from the outside world.A single or several kinds of phospholipids are often used to establish artificial model system as substitutes of real biofilm.Such brief simplifications containing key elements lead to deep comprehension of self-assembled lipid membrane.In this thesis,we firstly explore how Ca2+ could influence formation of lipid membrane.Then we try to use similar lipid bilayers as decoration of nanoconfined Bombyx Mori silk fibroin film and combine phospholipid with inorganic nanoparticles to build bionanoparticle composite film.These works highly expand lipid membranes’ biological use.Furthermore,based on nanofabrication technology,we are able to rebuild and apply mechanical stimulation to 3D micro cell tissue in vitro which could certainly give guides to design of artificial organs and slowing down vascular aging.In the first chapter,we introduce basic structure and function of biomembrane as well as phospholipids.Depending on geometrical properties,different molecules can assemble into different structures.Various methods have been used to create artificial lipid membranes from free suspended membranes to membranes supported on a solid surface.We also introduce usage of biomolecules for fabrication of nanodevices and studies about organizational structure,mechanical behavior;and biological function of engineered tissue.In the second chapter,we present main instruments used in this thesis including Quartz Crystal Microbalance with Dissipation monitoring(QCM-D),Atomic Force Microscopy(AFM),Laser Scanning Confocal Microscope(Confocal),Langmuir-Blodgett instrument(LB film).Besides that,a review of applications of commonly used microfabrication technologies in biological systems is given.In the third chapter,the simplest models,supported lipid bilayers are formed under different conditions.Calcium cation(Ca2+)is a key element to the cell membrane functions.Its effects on liquid crystal vesicle deposition have already been learnt.Here,it is found that Ca2+ can also influence the gel vesicle deposition by controlling the vesicle rupture and fusion on Si02 substrate at temperatures lower than the main transition temperature.Particular analyses are given to the vesicle-SiO2 and inter-vesicle attractions that are originated from the Ca2+ bridging effect.It is concluded that the aggregate condition of vesicles should be taken into consideration when dealing with vesicle deposition on a solid substrate.In the fourth chapter,lipid membranes are used to decorate regenerated Bombyx mori silk fibroin which is widely used as biomaterial.The β-sheet structure of silk fibroin after methanol treatment provides water-insolubility and mechanical stability while on the other side leads to a hydrophobic surface which is less preferred by biological systems.In this work we prepare a novel type ofnanoconfined silk fibroin film with a thickness beneath 100 nm.The film has a flat while hydrophobic surface because of the β-sheet structure of it due to the z-direction confinement during formation.Different types of lipid monolayers,DOPC,DPPC and MO,are assembled on the silk film surface.The lipid coating,especially the DPPC membrane,provides a much smoother and more hydrophilic surface due to the gel phase tails of lipids,in comparison with the DOPC and MO ones which are in a liquid phase and have a much stronger interfacial association between silk film surface and lipid tails.Such lipid coating significantly reduced the protein adsorption effect of the silk film without disturbing the biocompatibility to cells of it,which promises potential applications as surface coating for materials for biological use.Further more,new kinds of biomolecule-based electrical bistable device composed of phospholipid-CdTe nanoparticle multilayered films were widely used.The composite film was fabricated by a facile solution-cast method.X-ray reflectivity and transmission electron microscopy measurements showed the homogeneous distribution of nanoparticles within the lamellar lipid matrix with long-range ordering.Current-voltage scans on the Al/(lipid-nanoparticle composite film)/ITO/glass structures at room temperature exhibited an obvious current bistable phenomenon.Further investigation of such bionanoparticle composite film promises to show its importance for applications in future memory nanodevices with tailored performance.In the fifth chapter,we describe the realization of an approach to magnetically actuate arrays of microtissue constructs for biomechanical measurements and long-term mechanical stimulation.Microtissue strain gauge devices,consisting of pairs of flexible poly(dimethylsiloxane)(PDMS)cantilevers around which cell/matrix constructs can be induced to self-assemble,have been shown to provide effective readouts of tissue contractility,and magnetic stretching of individual microtissues via magnetic microspheres mounted on the cantilevers has been used to elucidate the tissues’ elastic modulus and response to varying mechanical boundary conditions.The fabrication of arrays of micromagnetic structures that can transduce an externally applied uniform magnetic field to simultaneously actuate multiple microtissues is presented here.The arrays are fabricated on silicon-nitride coated Si wafers and contain electrodeposited Ni bars;together with through-etched holes that provide optical and culture media access when the devices are mounted on the PDMS microtissue scaffold devices.Both static forces and AC forces at physiological frequencies of magnitudes up to 20 μN on each microtissue are readily generated in external fields of 40 mT.Operation of the magnetic arrays was demonstrated via measurements of elastic modulus and dynamic stiffening in response to AC actuation of fibroblast populated collagen microtissues.In the seventh chapter,there is a summary of this thesis and we present outlooks for future work.