| As the most fundamental physiological barrier of living cells,cell membrane（or organelle membrane）plays an important role in controlling material transport,and intercellular information exchange,etc.These physiological functions cannot be performed without membrane proteins closely related to biological membranes.Studying the effects of biofilm-membrane protein interactions on the structure,function,and kinetics of both is essential to deepen the understanding of relevant physiological processes to guide the development of novel drugs and the treatment of diseases.Therefore,developing membrane-protein interaction characterization techniques to gain a deeper understanding of the interaction has been the direction of researchers’efforts.Surface/interface-sensitive sensing technology is a powerful tool for membrane and membrane-protein interaction studies because of its respond to molecular adsorption and desorption at the surface interface,structural changes,and other events in real time and without additional labeling,based on small changes in relevant measurement parameters caused by alterations in the surface environment.The first step to transferring a membrane-protein research system to a surface interface analysis platform is to construct a complete phospholipid bilayer on the surface of the sensing substrate.The technology of constructing amphiphilic phospholipid membranes with almost no net charge is well established and can meet the needs of some membrane-protein systems,such as transmembrane protein insertion.However,the function of some membrane proteins depends on functional negatively charged phospholipid molecules,and it is difficult for negatively charged liposomes to spontaneously rupture on negatively charged surfaces to form two-dimensional support membranes due to factors such as electrostatic repulsion.Hence the assembly of the supported lipid bilayer（SLB）on similarly charged solid surfaces has long been a challenge.Based on these questions,the main research aspects of this thesis include the following:（1）A new method for constructing the negatively charged intact phospholipid bilayers on the widely used negatively charged Si O2surface is proposed,and provides a novel idea for the preparation of surface-supported phospholipid membranes.The core of the method is to use PBS buffer solution to overcome electrostatic repulsion,mediating the rupture and fusion of bicelle on the Si O2surface.The formed phospholipid membrane has no non-negligible defects and can be used in surface optical and acoustical analysis techniques.In addition,the kinetic processes of the film formation process were monitored using quartz crystal microbalance with dissipation（QCM-D）,and reasonable speculations and explanations were provided for the possible mechanisms behind the different kinetic stages of the film formation process.This method does not rely on the pretreatment of liposomes and the assistance of organic solvents,which makes it low-cost,easy to operate,and biocompatible.（2）Phospholipid bilayers containing 30%functional negatively charged lipids（dioleoyl phosphatidylserine and cardiolipin）were assembled on the Si O2surface using the proposed support membrane preparation method,and the interaction of the positively charged membrane protein cytochrome c（Cytc）with the negatively charged support membrane was dynamically monitored in situ using the QCM-D.After Cytc adsorbed onto the negatively charged SLB,hydrogen peroxide and calcium ions were introduced.QCM-D monitored their effects on the membrane-protein complex to simulate the effect of the increase of pro-apoptotic factors in mitochondria on the interaction of Cytc with the membrane during the initiation of apoptosis.Based on the experimental results,the understanding of the key question of whether Cytc departs from the inner mitochondrial membrane at the initiation of apoptosis was given.（3）In order to be able to monitor the interfacial adsorption molecular kinetic processes while having a more precise insight into the structural changes at the molecular level,the idea of combining plasmon waveguide resonance（PWR）,a sensitive sensing technique,with enhanced Raman spectroscopy is proposed for the study of membrane-protein interactions.PWR.Thanks to its high high-quality factor,it can be used for the sensitive sensing of molecular events at the surface interface and to obtain molecular adsorption kinetic parameters and anisotropic information of the film.Meanwhile,the PWR chip structure can provide interfacial evanescent fields with enhanced electric field amplitudes compared to incident light,which can be used for spectral enhancement of Raman spectra with inherently low sensitivity.The parameters of resonance angular displacement,the full width of half maximum of resonance peak,and electric field amplitude distribution of the sensing substrate are obtained by using the finite difference time domain（FDTD）method,which provides theoretical guidance for chip processing and characterization technique implementation. |
