RET nanobiosensors using affinity of an apo-enzyme toward its substrate

Abstract

Fluorescent biosensors can be highly specific and sensitive, and may be engineered as implantable devices for metabolic monitoring. Commonly-used systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding involve Concanavalin A (Con A), which is known to be toxic, and has problems of aggregation and irreversible binding. This work presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, observed as an increase in the donor peak relative to acceptor with the stepwise addition of glucose solution. The assay showed strong signals and excellent repeatability, with a sensitivity of 9x10/sup -4/ (ratio units)/mM over the range of 0-90 mM glucose. If properly encapsulated, these sensors can potentially be used for in vivo sensing without the concern of toxicity associated with Con A.