Microfluidic Homogeneous Mobility Shift Assays at the Bench and the Bedside

Binding interactions underpin all biological processes. As a result, understanding binding interactions has implications in the development of novel diagnostics as well as advancing our understanding of biological processes and the molecular mechanisms of human disease./nElectrophoresis is a widely used chemical separation technique that is used to study molecular binding interactions. In this technique, molecules are separated based on differences in their electrophoretic mobilities. By harnessing the molecular binding interactions that are prevalent throughout biology and medicine, electrophoretic mobility shift assays rely on the evolutionarily-derived binding affinity and specificity of biomolecules to induce a change in electrophoretic mobility between molecular populations. These assays use a molecular probe that binds with a target molecule, causing a change in weight or size, shape, and/or charge that leads to a detectable change in electrophoretic mobility./nElectrophoretic mobility shift assays benefit from operation on the microscale. Microfluidic assays have emerged as powerful tools that can be used to study and detect binding interactions between many of the major classes of biological macromolecules. Performing electrophoresis on the microscale enables the application of higher electric fields which in turn speeds up separations and enables increased resolution of analyte fractions./n/nThis dissertation reports the development of microfluidic electrophoretic mobility shift assays and their application to relevant research questions affecting both clinical medicine and basic research. The unifying theme of the assays presented here is the use of induced mobility differences in order to probe for the functionality of a target biomolecule of interest. All the applications presented here take advantage of a specific benefit imparted by adapting electrophoresis to the microscale including the small sample volume requirements which are appropriate for analysis of volume-limited tear fluid from dry eye patients and the enhanced mobility resolution enabled by lower diffusion timescales as is appropriate for detecting the small conformational changes of functional riboswitch aptamers./n/nIn this work, I first demonstrate and characterize a first-in-kind microfluidic homogeneous immunoassay that is able to probe for protein biomarkers in human tear fluid. This format capitalizes on the binding affinity and specificity of antibodies to impart a mobility shift between the bound and unbound forms of a target protein biomarker. I optimize the assay conditions including gel pore-size and pH to minimize the nonspecific binding interactions that complicate measurement of tear proteins. As a result, I demonstrate detection of tear protein biomarkers with a specificity and speed that outperforms conventional tools such as ELISA or slab gel electrophoresis. With the target-user and the applicability of the assay to the broader community in mind, gaps in the literature that are relevant to translation of the assay presented here are also considered including the impact of upstream biospecimen sample processing on the assay read-out and the integration of additional ophthalmological tests for dry eye severity assessment into a single device. This work has the potential to revolutionize our understanding of ocular disease pathology, enable non-invasive diagnosis of systemic disease (where biomarkers are available) using tears, and may be used to stratify Sjögren's syndrome patients from those with other forms of dry eye. Taken in sum, this work has broad implications in the proteomic analysis of tear fluid and can be used in the advancement of both basic science and clinical medicine./n/nSecondly, I demonstrate and characterize a first-in-kind microfluidic mobility shift assay for riboswitch screening applications. Riboswitches are RNA sequences that undergo a conformational change in response to binding by a regulatory small molecule ligand, resulting in modulation of gene expression. I optimize assay conditions in order to demonstrate the ability to utilize a ligand binding-induced conformational change to impart a mobility shift to the bound and unbound riboswitch populations. As a result, I utilize the assay to screen five never-before characterized riboswitch candidates for functionality. I also demonstrate the ability to utilize the fine mobility resolution of microfluidic formats to be able to study the binding affinity of both fast and slow interconverting riboswitch pairs./n/nIn sum, this work makes important contributions toward creating robust analytical tools capable of probing the binding interactions that underpin all of biology. By harnessing the speed, resolution, and portability of electrophoresis on the microscale, this work has the potential to enable these powerful analytical techniques to increasingly be used both at the bench and the bedside.

Advisor: Herr, Amy E