PhD Defense of Sheniqua Brown

Glucose and glutamine are essential nutrients that act as important sources of energy in living organisms. Their presence or lack thereof can be important indicators in biomedical and bioprocessing applications; therefore, monitoring of these analytes are a key component for both successful and effective medical care and bioprocess production. This research focuses on the development of the optical biosensors, glucose (GBP) and glutamine (QBP) binding protein, capable of detecting micromolar levels of their target analyte. A fluorescence tag is attached at a site responsive to analyte binding and fluorescence changes are measured with respect to known analyte concentrations. Previous studies successfully track blood glucose (BG) noninvasively through transdermal glucose (TG) in neonates, adult subjects and animal models; however, many of the studies use the soluble form of the protein for assay analysis. For consideration as an actual sensor the proteins must be immobilized. In the first aim of this work a series of expression, purification and characterization studies verified that the L255C GBP and S179C QBP E. coli mutants allowed the proteins to maintain their activity and micromolar sensitivity with the addition of a poly-histidine tag that facilitates immobilization. To farther confirm the feasibility of the his-tagged L255C GBP as a sensor for TG sensing, preliminary noninvasive TG studies were conducted on both healthy adult subjects and pediatric diabetic ketoacidosis (DKA) patients. In these patients, there was a trend and a positive linear correlation (R2 = 0.95) between the TG measured with GBP and the corresponding BG. This set of studies helped to characterize the his-tag GBP sensor for its intended use in biomedical applications. The final study of the first aim explored and successfully optimized a cell-free expression system for automatic expression and purification of the selected optimal his-tagged binding protein. This automated system allows for the protein to be produced in a consistent manner and can eventually lead to complete automated fabrication of the biosensors. The second aim of this project implemented automation and minimally invasive techniques into the TG sampling protocol to improve diffusion through skin. Microneedles (MN) were used for the collection of TG on Yucatan miniature pig skin mounted to a static diffusion cell, as well has different skin sites of healthy human subjects. Collected TG samples were measured with L255C GBP. The binding constant (Kd == 0.67pM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the anima! and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN treated human skin a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In the third aim of this work, the L255C GBP and S179C QBP underwent bioaffinity immobilization on nickel-nitriiotriacetic acid (Ni-NTA) agarose beads via the poly-histidine tag for sensor consideration. Two portable analyte sensing setups integrating the immobilization protein with an in-house manufactured portable fluorometer were explored. Ultimately, the collected experimental data supports the feasibility of GBP and QBP being used as portable glucose and giutamine sensing devices in biomedical and bioprocessing applications.