Mobile phospholipid bilayers on a polyion/alkylthiol layer pair as a biosensor model for selective peptide detection.

摘要

Solid supported phospholipid bilayers, as model biomembranes, draw great attraction in biosensor applications as well as the biophysics and biochemistry fields. To mimic the natural cell membranes, it becomes very important to minimize the effects of the close proximity of solid substrates, which may hinder the lateral diffusivity of the model membranes and limit the space for transmembrane protrusions of proteins and peptides. This dissertation describes the use of a polyelectrolyte/alkylthiol layer pair that lifts the model membranes away from the solid substrates, creating aqueous environments on both sides of the membranes and retaining the mobility of the membranes. Lipid mixtures of major components of natural cell membranes, phosphatidylcholine (PC), phosphatidylserine (PS) and cholesterol, are used to build bilayers on the polyion/alkylthiol layer pair by electrostatic attractions. The kinetics of the bilayer formation and the thicknesses of the bilayers are examined by surface plasmon resonance (SPR). The lateral mobility of the bilayers is studied by fluorescence recovery after photobleaching (FRAP).; A second major part of this dissertation revolves around the insertion of pore forming peptides and the insulating properties of the supported membranes, which are important for biosensor fabrications. If the supported membranes have enough structural integrity and thus charge blockage, the effect of the ion-channels formed by the inserted peptides can be easily detected due to facilitated charge transfer. Electrochemical experiments by cyclic voltammetry (CV) are performed with SPR in a combined set-up to study the insulating properties of the supported bilayers with different lipid compositions. Ion-channel forming peptides and antimicrobial peptides are introduced to the supported membranes. The association of the peptides with the membranes is detected by the SPR and the channel-forming activities of the peptides are measured by the CV experiments in an electrochemical cell.; The third part of the dissertation focused on the use of atomic force microscopy (AFM) to monitor the surface morphology change with the layer-by-layer deposition in situ. The adsorption process is presented in correlation to the electrochemical (CV) and SPR studies in the second part of the dissertation. The soft-contact mode used in this study is able to detect the nanoscopic architecture of the biomembranes and the effect of the peptides associated with the membranes.