Design, manufacture and characterization of an optical fiber glucose affinity sensor based on an homogeneous fluorescence energy transfer assay system

Abstract

Optical fiber biosensors based on fluorescence assays have several distinct advantages when measuring biological analytes such as metabolites, cofactors, toxins, etc. Not only are optical signals immune to electronic interferences, but the polychromatic nature of most fluorochemical assays provides more potentially useful data about the system being studied. One of the most common difficulties normally encountered with optical biosensors is the inability to routinely recalibrate the optical and electronic components of the system throughout the life of the sensor. With this in mind, an optical biosensor system for glucose has been constructed along with the peripheral electronic instrumentation. The biochemical assay is based on an homogeneous singlet/singlet energy transfer affinity assay. The sensor probe indirectly measures glucose concentrations from the level of fluorescence quenching caused by the homogeneous competition assay between TRITC labeled concanavalin A (receptor) and FITC labeled Dextran (ligand). The FITC signal is used as an indicator for glucose concentrations and the TRITC signal is used for internal calibration. Chemical derivatization procedures using succinic anhydride were developed to prevent aggregation of the receptor protein in solution, and the TRITC/ConA ratios were optimized to achieve the best sensor performance. Using this sensor system, the FITC-Dextran detection limit was 0.05 μg/ml and glucose concentrations up to 1600 mg/dl could be detected with a time response of approximately 10 min.