(C-96) Thermal stabilization of glucose oxidase for continuous glucose monitoring

Introduction::

Across the United States, 34 million type 2 diabetic patients must regulate their glucose levels via glucose monitoring. This condition is characterized by insulin resistance, thus requiring glucose monitoring and insulin therapy to avoid hyperglycemia and hypoglycemia. A common approach includes continuous glucose monitors (CGM) employing a sensing enzyme, glucose oxidase (GOx), to detect glucose levels within dermal interstitial fluid [1]. Prolonged exposure to physiological temperatures causes irreversible enzymatic denaturation, thus requiring replacements of monitors after 10-14 days [1]. Enhancing enzyme stability would lengthen sensing lifetime and improve glucose reading accuracy allowing for less frequent CGM replacements. To stabilize GOx, copolymers can be mixed with the enzyme to create polymer-enzyme complexes (PEC) in which the copolymer is hypothesized to act as a stabilizing chaperone to the native enzyme [2]. The PEC stabilization approach transcends traditional techniques, such as screening for enzymes produced by extremophiles or sourcing a stable enzyme from a genetically modified organism. In this work, we investigate utilizing GOx-PECs for long-term stability at accelerated aging conditions compared to the native enzyme.

Materials and Methods::

Random copolymers are synthesized via Photo-induced Electron/energy Transfer-Reversible Addition-Fragmentation Chain Transfer (PET-RAFT) polymerization in an automated laboratory setup using a Hamilton liquid handling robot. Polymer B4 is composed of DEAEMA, 2-HPMA, MMA, and PEGMA monomers, while polymer B6 is composed of DEAEMA, BMA, and PEGMA monomers. The Gormley Lab takes a machine learning (ML) based approach to optimize retained enzymatic activity (REA) based on a Learn-Design-Build-Test loop to predict polymer designs best tailored to stabilize an enzyme based on previous iterations [2]. Synthesized copolymers are added to enzyme solutions to form PECs, whose REA is quantified via a thermostability assay. A colorimetric, double enzymatic assay was developed to study the kinetics of GOx and GOx-PEC by measuring optical density at a wavelength of 405 nm using UV-Vis spectroscopy. The assay employs 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the indicator, glucose as the substrate, horseradish peroxidase (HRP), and GOx. GOx-PEC accelerated aging screenings are performed above the native enzyme’s melting temperature to identify promising copolymer candidates for further evaluation for long term exposure to physiological conditions seen in CGM technology. Statistical analysis of GOx-PEC and GOx enzymatic activity was performed using OriginPro software.

Results, Conclusions, and Discussions::

The assay was designed in which a rate limiting step would ensure GOx and GOx-PEC enzymatic activity quantification, and not that of HRP, after exposure to heat stress. Native GOx activity was quantified at various concentrations to achieve target absorbance levels to show that there is a linear relationship between GOx concentration and change in activity through a calibration curve (Figure 1). Copolymers were mixed with the enzyme to create GOx-PECs, which prompted determination of optimal copolymer concentration to allow for improved enzymatic activity when combined. Preliminary accelerated aging results demonstrate that 30 minutes of exposure to 65ºC results in 21.22 ± 3.62% and 18.25 ± 3.74% retained activity for GOx-PEC-B4 and GOx-PEC-B6, respectively, as compared to native GOx retaining 7.75 ± 4.31% activity (Figure 2). Post-hoc analysis following a two-way ANOVA was used to analyze heat exposure time effect on enzymatic activity (α = 0.05, p = 0.004). A statistically significant difference in enzymatic activity with a copolymer, hypothesized to have favorable surface interactions with the enzyme to allow for conformational changes, shows promise in extending CGM lifetime via GOx-PEC implementation [3]. Different characteristics, such as charge, hydrophobicity, and hydrophilicity are exhibited by copolymers, which in turn affect the interactions with GOx when complexed into GOx-PECs. Further investigation into monomer composition and degree of polymerization effect on retained enzymatic activity during exposure to denaturing temperatures is necessary to better characterize GOx-PEC performance under thermal stress. Future work aims to analyze GOx using dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) to study how the addition of stabilizing copolymers aids in prevention of enzyme aggregation and causes any conformational changes in the enzyme. Support was provided by a grant from the National Science Foundation DMREF-2118860 and NSF-EEC-1950509, REU Site in Cellular Bioengineering: From Biomaterials to Stem Cells.

Acknowledgements (Optional): :

References (Optional): : [1] Harris, J., Journal of Diabetes, 2013 [2] Tamasi, M., Adv. Mat., 2022 [3] Waltmann, C., Proc. Natl. Acad., 2022