Reversible fiber-optic immunosensor measurements☆

Abstract Reversible binding of analyte to antibody is necessary for analytical sensor systems that can continuously measure increases and decreases in analyte concentration. Reversible response occurs when the dissociation rate is sufficiently fast that the analyte will dissociate in a short enough time to permit a rapid re-equilibration to be established in response to a change in analyte concentration. The previously reported phenytoin sensor is based on a homogeneous fluorescence energy-transfer immunoassay and has a reversible response to increases and decreases in phenytoin concentration with a response time of approximately 15 min. The first-order dissociation rate constant for the labeled analyte-antibody complex, phenytoin-B-phycoerythrin (BPE), is 4 × 10 −3 s −1 . Whole antibody and Fab give equivalent sensor performance, which agrees closely with that predicted from a model for the simultaneous equilibrium reactions in the sensor. These results suggest that the antibody binds monovalently to the phenytoin-BPE, and that each antibody binding site acts independently. Optimization of the labeled reactants and reaction conditions results in a sensor which has a dynamic range in the clinically important 1–20 μM concentration range for free phenytoin.