Fluorescently Labeled Periplasmic Binding Proteins as Optical Biosensors for Detecting Micromolar Levels of Glucose and Glutamine in Biomedical & Bioprocess Applications

Abstract

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 a working sensor the proteins were 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 further 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. 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. 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. In the third aim of this work, the L255C GBP and S179C QBP underwent bioaffinity immobilization on nickel-nitrilotriacetic 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. In the final portable setup, immobilized GBP and QBP were trapped in a microfluidic chromatography column. In this setup, both sensors had a response time of approximately 10 seconds. In addition, flushing the column with PBS reversed the analyte-sensor reaction permitting reusability of the sensor. Ultimately, the collected experimental data supports the feasibility of GBP and QBP being used as a noninvasive to minimally invasive portable glucose and glutamine sensing devices in biomedical and bioprocessing applications.