Monitoring Biological Processes and Interactions at Lipid Membranes using Ion Channel-Based Sensors and Membrane Microarrays.

Supported Lipid Membrane;Planar Lipid Bilayer;Ion Channel;Gramicidin;Membrane Array;Membrane Interactions;Biomedical Engineering;Engineering;Biomedical Engineering

Majd Zarringhalam Araghy, SheereenTakayama, Shuichi ;

University of Michigan

关键词: Supported Lipid Membrane;    Planar Lipid Bilayer;    Ion Channel;    Gramicidin;    Membrane Array;    Membrane Interactions;    Biomedical Engineering;    Engineering;    Biomedical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/64664/sheereen_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Many cellular processes involve molecular interactions at the cell membrane. Due to the complexity of living cells, these interactions are usually studied on model membranes. This thesis introduces two platforms based on model membranes for studying biological interactions and processes on cell membranes. In the first part of this thesis, we employed planar lipid bilayers to develop a novel, label-free, and sensitive assay for monitoring the activity of phospholipases D and C that are critical for cell signaling. The activities of these enzymes typically change the surface charge of the membrane. The present assay employs the ion channel-forming peptide gramicidin A to probe these changes and to monitor the activity of these phospholipases in situ and in real-time. Quantitative results from this assay, allowed us to investigate the kinetics of the heterogeneous catalysis of these enzymes. In addition we applied this gramicidin-based sensor to monitor the binding of two therapeutic drugs to various bilayers. Quinine, an anti-malaria agent, and imipramine, an anti-depressant, are positively-charged under physiological conditions and, once bound to a membrane, alter the membrane surface charge. The present assay probes these changes and makes it possible to quantify these binding events. In the second part of this work, we developed a technique that employs topographically-patterned hydrogel stamps to fabricate arrays of membranes and membrane proteins for screening of membrane interactions. This method takes advantage of the porous, hydrated, and biocompatible nature of hydrogels to print spatially-addressable arrays of membranes in a rapid and parallel fashion. We employed this method for two distinct approaches; one approach takes advantage of the storage capability of agarose stamps and minimizes the required time and amount of membrane preparations by generating multiple copies of a membrane array. The other approach takes advantage of on-stamp preconcentration of cellular membrane fragments to generate arrays of multilayered-membranes with high contents of proteins and enhances detection sensitivity. We used these arrays for screening the interactions of a protein (annexin V) and an anti-inflammatory drug (nimesulide) with various bilayers. We also carried out ligand-binding assays on these arrays and showed that the stamped proteins retained their binding activity.