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ABSTRACT The purpose of this study is to demonstrate that stable lipid bilayers can be set up on an array of silicon micropores and can be used as sites for self-inserting ion-channel proteins which can be studied independently of each other. In course of this study an acrylic based holder was designed and machined to ensure leak-free fluidic access to the silicon micropores and physical isolation of the individual array channels. To measure the ion-channel currents, we simulated, designed and manufactured low-noise transimpedance amplifiers and support circuits based on published patch clamp amplifier designs, using currently available surface-mount components. This was done in order to achieve a reduction in size and costs as well as isolation of individual channels without the need for multiplexing of the input. During the experiments performed, stable bilayers were formed across an array of four vertically mounted 30 µm silicon micropores and OmpF porins were added for self insertion in each of the bilayers. To further demonstrate the independence of these bilayer recording sites, the antibiotic Ampicillin (2.5 mM) was added to one of the fluidic wells. The ionic current in each of the wells was recorded simultaneously. Sub-conductance states of Ompf porin were observed in two of the measurement sites. In addition, the conductance steps in the site containing the antibiotic could be clearly seen to be larger compared to those of the unmodified site. This is due to the transient blocking of ion flow through the porin due to translocation of the antibiotic. Based on this demonstration, ion-channel array reconstitution is a potential method for efficient electrophysiological characterization of different types of ion-channels simultaneously as well as for studying membrane permeation processes.
ContributorsRamakrishnan, Shankar (Author) / Goryll, Michael (Thesis advisor) / Thornton, Trevor J (Committee member) / Blain Christen, Jennifer M (Committee member) / Arizona State University (Publisher)
Created2011