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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
Description
Engineered nanoporous substrates made using materials such as silicon nitride or silica have been demonstrated to work as particle counters or as hosts for nano-lipid bilayer membrane formation. These mechanically fabricated porous structures have thicknesses of several hundred nanometers up to several micrometers to ensure mechanical stability of the membrane. However, it is desirable to have a three-dimensional structure to ensure increased mechanical stability. In this study, circular silica shells used from Coscinodiscus wailesii, a species of diatoms (unicellular marine algae) were immobilized on a silicon chip with a micrometer-sized aperture using a UV curable polyurethane adhesive. The current conducted by a single nanopore of 40 nm diameter and 50 nm length, during the translocation of a 27 nm polystyrene sphere was simulated using COMSOL multiphysics and tested experimentally. The current conducted by a single 40 nm diameter nanopore of the diatom shell during the translocation of a 27 nm polystyrene sphere was simulated using COMSOL Multiphysics (28.36 pA) and was compared to the experimental measurement (28.69 pA) and Coulter Counting theory (29.95 pA).In addition, a mobility of 1.11 x 10-8 m2s-1V-1 for the 27 nm polystyrene spheres was used to convert the simulated current from spatial dependence to time dependence.
To achieve a sensing diameter of 1-2 nanometers, the diatom shells were used as substrates to perform ion-channel reconstitution experiments. The immobilized diatom shell was functionalized using silane chemistry and lipid bilayer membranes were formed. Functionalization of the diatom shell surface improves bilayer formation probability from 1 out of 10 to 10 out of 10 as monitored by impedance spectroscopy. Self-insertion of outer membrane protein OmpF of E.Coli into the lipid membranes could be confirmed using single channel recordings, indicating that nano-BLMs had formed which allow for fully functional porin activity. The results indicate that biogenic silica nanoporous substrates can be simulated using a simplified two dimensional geometry to predict the current when a nanoparticle translocates through a single aperture. With their tiered three-dimensional structure, diatom shells can be used in to form nano-lipid bilayer membranes and can be used in ion-channel reconstitution experiments similar to synthetic nanoporous membranes.
To achieve a sensing diameter of 1-2 nanometers, the diatom shells were used as substrates to perform ion-channel reconstitution experiments. The immobilized diatom shell was functionalized using silane chemistry and lipid bilayer membranes were formed. Functionalization of the diatom shell surface improves bilayer formation probability from 1 out of 10 to 10 out of 10 as monitored by impedance spectroscopy. Self-insertion of outer membrane protein OmpF of E.Coli into the lipid membranes could be confirmed using single channel recordings, indicating that nano-BLMs had formed which allow for fully functional porin activity. The results indicate that biogenic silica nanoporous substrates can be simulated using a simplified two dimensional geometry to predict the current when a nanoparticle translocates through a single aperture. With their tiered three-dimensional structure, diatom shells can be used in to form nano-lipid bilayer membranes and can be used in ion-channel reconstitution experiments similar to synthetic nanoporous membranes.
ContributorsRamakrishnan, Shankar (Author) / Goryll, Michael (Thesis advisor) / Blain Christen, Jennifer (Committee member) / Dey, Sandwip (Committee member) / Thornton, Trevor (Committee member) / Arizona State University (Publisher)
Created2015