AdvisorAspinwall, Craig A.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
EmbargoRelease after 08/27/2019
AbstractCellular secretion regulates cell communication and function. The ability to detect and quantify the release of hormones and neurotransmitters provides deeper understanding of cell signaling pathways. Ion channel sensors demonstrate a high potential for detecting cellular secretion with high sensitivity and selectivity, as well as adequate spatial and temporal resolution for real-time subcellular detection. Ion channel sensors utilize ligand-gated ion channels (LGICs) as recognition elements, enabling detection of ligand-receptor binding with high specificity. LGICs serve as signal transducers that transduce ligand binding events into highly sensitive current measurements, allowing label-free detection of hormone and neurotransmitters that are neither optically, nor electrochemically active. The work within this dissertation describes three new approaches to further advance ion channel sensor development. First, in vitro expression of eGFP-Kir6.2 was explored and verified using fluorescence microscopy, SDS-PAGE and dot blot. Electrophysiological measurement confirmed the successful expression of functional ion channels with expected pore conductance and antagonist sensitivity. The new expression method allowed fast and purification-free protein production, greatly reducing the time and technical barrier for ion channel sensor fabrication. Second, a dual-barrel ion channel probe was described to provide precise positioning of sniffer sensor using access resistance as feedback signal. Selective formation of polymer scaffold stabilized black lipid membrane across one barrel was confirmed and enabled membrane protein insertions. Precise positioning of the sensor will increase sensor reproducibility, thus providing accurate measurements of cellular release. Finally, a surface modified microfluidic valve was fabricated with > 70 fold enhancement in electrical resistance, enabling the isolation of ion channel signals in pA regime. The microfluidic valve provides a simple but cost-effective alternative for high throughput parallel electrophysiology. The efforts to advance the development of ion channel sensors will greatly improve our understanding of the biological system, benefiting disease diagnosis and treatment.
Degree ProgramGraduate College