Elucidating the Mechanism of Electrochemical Inactivation of Direct Electron Transfer Type Glucose Dehydrogenase Enzyme-Employing Biosensor
Authors: Sode Koji , Kerrigan Joseph 
Published: December 2024

Introduction: Diabetes management has evolved through the introduction of self-monitoring blood glucose (SMBG) and continuous glucose monitoring (CGM) systems, enabling real-time patient feedback.[1] Of these, CGM systems, capable of prolonged, continuous monitoring, hinge upon the dependable performance of the enzymes to maintain precise measurements and long-term sensing capabilities. Current CGM systems utilize fungi-derived glucose oxidase (GOx) to catalyze the reaction with glucose. This enzyme is able to record glucose values within the physiological range with high accuracy; however, this enzyme also creates hydrogen peroxide during continuous operation leading to self-inactivation via radical oxygen species(ROS) formation. Our group has been involved in the characterization and functional engineering of a FAD-dependent glucose dehydrogenase enzyme derived from Burkholderia cepacia (BcGDH) which is capable of direct electron transfer (DET) with an electrode.[2, 3] By eliminating the need for hydrogen peroxide or toxic external mediators—as are common with 1st and 2nd generation CGM sensors, respectively—we are able to simplify our sensing platform and create the ideal sensing modality. Additionally, we have improved the thermal stability of BcGDH by introducing inter-subunit disulfide bonds which has greatly improved the in vitro half-life of our enzyme.[4] Although we have been able to engineer our BcGDH to have a much greater half-life in vitro than the wild type BcGDH or GOx, we observe uncharacteristic inactivation during continuous applied potential electrochemistry experiments. This study focuses on the elucidation of the electrochemical inactivation mechanism of a DET-type BcGDH enzymatic sensor.

Materials & Methods: BcGDH engineered enzyme, from the introduction of inter-subunit disulfide bonds was recombinantly produced and purified. A ϕ=2mm gold disk electrode was polished and electrochemically cleaned before functionalized with dithiobis(succinimidyl hexanoate) to form a self-assembled monolayer(SAM). The purified BcGDH mutant was then immobilized to the SAM. Variations in potentials (none, 100mV or 400mV vs Ag/AgCl), glucose concentration (none or 20mM), and oxygen (argon purged or ambient oxygen) were used for the incubation conditions. Every three hours (0 to 9 hours) calibration curves were investigated unilaterally at 45°C, 400mV vs Ag/AgCl applied potential, ambient oxygen, and varying concentrations from 0 to 20mM glucose.

Results & Discussion: The disulfide bond stabilized BcGDH engineered enzyme was stable at more than 80°C confirming its thermal stability and extended half-life at supraphysiological temperatures. However, when utilized in a sensor format at 45°C, engineered enzyme was inactivated much more rapidly than expected. The largest rate of inactivation was revealed to be due to application of a high over-potential (+400mV vs Ag/AgCl) coupled with hyper-physiological glucose levels with ambient oxygen showing a sensor half-life of 2.89±0.74 hours at 45°C. The impact of oxygen-purging the system by utilizing argon gas was shown to improve the sensor half-life to 35.04±6.17 hours at 45°C. Additionally, without the high over-potential, the hyper-physiological glucose concentrations had no detriment to the sensor and no sensor inactivation was observed. . The high over-potential combined with hyper-physiological glucose and ambient oxygen were assumed to produce radical oxygen species (ROS) which are responsible for the inactivation of the enzyme.

Conclusions: We demonstrate the ability to use this DET-type GDH electrochemical sensor to investigate the factors which accelerate its inactivation. While hyper-physiological glucose levels do not significantly inactivate the enzyme-sensor, the high over-potential incubation inactivates the sensor much faster—likely due to the formation of ROS from generation of H2O2 at the cathode. By removing oxygen from the system via an Argon purge, the conversion of oxygen to hydrogen peroxide is thwarted. In the future, studies should be done to mitigate the ROS generation as glucose levels cannot be controlled. This strategy may be done by applying a lower over-potential to the sensor would minimize the formation of ROS even in the presence of hyper-physiological glucose.

References:

[1] I. Lee et al., “Continuous glucose monitoring systems - Current status and future perspectives of the flagship technologies in biosensor research -,” Biosensors and Bioelectronics, vol. 181, p. 113054, Jun. 2021, doi: 10.1016/j.bios.2021.113054.
[2] K.Sowa et al., “Direct Electron Transfer–Type Oxidoreductases for Biomedical Applications”, Annual Reviews of Biomedical Engineering, 2024, 26
[3] J. O-Shimazaki et al., Bioelectrochemistry, “FAD dependent glucose dehydrogenases – Discovery and engineering of representative glucose sensing enzymes-,” Volume 132, April 2020, 107414 https://doi.org/10.1016/j.bioelechem.2019.107414
[4] J. O-Shimazaki et al., “Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase,” Commun Biol, vol. 5, no. 1, p. 1334, Dec. 2022, doi: 10.1038/s42003-022-04286-9.