(191bs) Engineering Glucose Binding Proteins with a Chemo-Enzymatic Tag for Glucose Detection in Exhaled Breath Condensates (EBC)

Despite recent advances in glucose biosensors and glucose detection, reliable measurement of glucose at the very low concentrations is highly desired. Glucose concentrations in non-invasive body fluids, such as saliva, sweat, and exhaled breath are only a fraction of those found in blood. In particular, we have found that exhaled breath condensate (EBC) contains a consistent fractional glucose concentration compared to blood glucose owing to the rapid glucose exchange between lung fluid and blood. However, this concentration is approximately 1/1500 of blood glucose concentration, as confirmed by our preliminary human trials conducted using a selective EBC condenser developed in our lab. While electrochemical sensors based on glucose oxidase (GOx) are the current gold standard, the lack of stability due to environmental interferences and the high dissociation constant of the enzyme prevent them from being able to resolve the minute changes in glucose at low concentrations [1]. The E. coli glucose binding protein (GBP) has a stronger and more specific affinity to glucose (K_D = 0.35 µM) compared to that of GOx (K_D = 20 mM) [2]. We propose to harness this protein to achieve a lower limit of detection and higher sensitivity via fluorescence-based competitive binding biosensor.

We take advantage of the competitive binding of glucose (K_D = 0.35 µM) and galactose (K_D = 1.4 µM) to GBP to develop a fluorescent biosensor that is quenched when bound to galactose-analog and fluorescent when bound to glucose. We engineer GBP to contain a biorthogonal azide tag which will allow conjugation to fluorophores, quantum dots, or another matrix of interest directly from cell lysate. This will enable the competitive detection of glucose via galactose-analog displacement upon interaction with EBC samples. Our chemoenzymatic tagging method [3] selectively and efficiently labels the N-terminus of the GBP with 12-azidodecanoic acid (12-ADA). We simultaneously develop a fluorescently conjugated galactose that quenches the fluorescence of the quantum dot when bound to GBP.

This presentation will discuss our initial results characterizing wt-GBP and 12-ADA-GBP binding to glucose and galactose. This work will also include assessing the binding capabilities of 12-ADA-GBP to TAMRA fluorophores via click chemistry directly from cell lysate. We find that our chemoenzymatic tagging method enables us to directly bind the 12-ADA-GBP from cell lysates containing overexpressed proteins to fluorophores of interest without any purification steps.

[1] Chen, C ., Q. Xie, D. Yang, H. Xiao, Y. Fu, Y. Tan, and S. Yao. “Recent advances in electrochemical glucose biosensors: a review.” RSC Adv. vol. 3, pp. 4473-4491, 2013

[2] L. Tolosa and G. Rao, “The Glucose Binding Protein as Glucose Sensor,” in Glucose Sensing, C. D. Geddes and J. R. Lakowicz, Eds. Boston, MA: Springer US, 2006, pp. 323–331.

[3] C. Kulkarni, M. Lo, J. G. Fraseur, D. A. Tirrell, and T. L. Kinzer-Ursem, “Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications,” Bioconjug. Chem., vol. 26, no. 10, pp. 2153–2160, Oct. 2015.