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Resonance Energy Transfer Nanobiosensors Based on Affinity Binding between Apo-Enzyme and Its Substrate

2004; American Chemical Society; Volume: 5; Issue: 5 Linguagem: Inglês

10.1021/bm0496662

1526-4602

Swetha Chinnayelka, Michael J. McShane,

Advanced biosensing and bioanalysis techniques

Systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding using Concanavalin A (Con A) are problematic as a result of problems of toxicity, aggregation, and irreversible binding. This paper presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). The basic principle for transduction is identical to that used in assays based on Con A−dextran: a reduction in RET from fluorescein isothiocyanate (FITC) to tetramethyl rhodamine isothiocyanate (TRITC) occurs when FITC−dextran (donor) is displaced from TRITC−apo-GOx (acceptor) as a result of the competition of glucose. Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, measured as an increase in the donor peak relative to acceptor due to stepwise addition of glucose. The solution-phase assay showed strong signals and excellent repeatability, with a sensitivity of 0.0163 (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.