Performance enhancement of fluorescence energy transfer-based biosensors by site-directed mutagenesis of the transducer

Abstract

We have recently described a biosensor for zinc and other metals which is based upon fluorescence energy transfer from a site or sites on the enzyme carbonic anhydrase to an inhibitor whose binding to the active site is largely metal-dependent. The concentration of the metal ion analyte is a simple function of the occupancy of the active site by the colored inhibitor, which is transduced as a change in intensity or lifetime of the fluorescent donor. We and others have demonstrated assays for several analytes based on energy transfer with moderate success. In this work we demonstrate by simulation and experiment that precise control of the donor:acceptor distance is valuable in optimizing the response of the system.