Inactivated Enzyme-Nanotube Based Nanosensors for Continuous Glucose Monitoring in Biological Fluids

Abstract

In-blood continuous glucose monitoring is important for diabetes management but remains a technical challenge owing to the dearth of tissue-transparent glucose sensors. In this study, we present the development of nanosensors capable of immediate and reversible responses upon exposure to glucose in serum and in translational biological applications. Here, we present on the development of near-infrared fluorescent single-walled carbon nanotube (SWCNT) based sensors, directly functionalized with glucose oxidase (GOx), to catalyze and thus fluorescently detect the oxidation of glucose.We prepared GOx-SWCNT nanosensors by facile direct sonication of SWCNT with GOx in a manner that – surprisingly – does not compromise the ability of GOx to detect glucose. We next studied the mechanism of glucose detection by GOx-SWCNT through an increase in SWCNT near-infrared fluorescence. Specifically, we prepared the nanosensors using an irreversibly deactivated GOx, to show that the fluorescence modulation of our nanosensors does not require enzymatic activity and confirming the fluorescence modulation of our nanosensors is purely affinity-based. These results are important, because consumption of the analyte typically compromises nanosensor reversibility, whereas our results show using a deactivated version of an enzyme could enable generation of reversible glucose sensors. Next, based on our observations, we developed sensitive and reversible glucose nanosensors using apo-GOx, where the cofactor was removed from the glucose enzyme, rendering it inactive but otherwise conformationally similar to native GOx. Because the apo-form of GOx preserves its native structure, these nanosensors showed superior sensitivity and stability to that of the native nanosensors with a change in fluorescence upon glucose exposure of up to dF/F=40 % within 2 seconds. We further show that apo-GOx-SWCNTs enable glucose imaging reversibly, and in serum, motivating the use of these nanosensors for continuous in-tissue monitoring of glucose. Separately, we show that sonication-based synthesis of enzyme-SWCNT conjugates enables nanosensor generation of a range of additional targets other than glucose, potentially opening the door to rapid nanosensor generation through direct enzyme conjugation. Our work herein both generates a tissue-translatable glucose nanosensor and motivates direct sonication and enzyme-SWCNT physisorption as a generalizable method for nanosensor generation.