Development of a regenerable fiber-optic biosensor

Fiber-optic chemical sensors have been developed that employ immunochemicals to perform fluoroimmunoassays. We have developed a regenerable fiber-optic sensor with a capillary delivery system to perform repetitive assays using antibodies immobilized to beads, "immunobeads." The sensitivity of these sensors is directly proportional to the amount of antibody present. For the immunobeads, the amount immobilized (loaded) and the ability of the immobilized antibody to maintain antibody recognition (activity) are two criteria which affect the sensitivity of the sensors. This study evaluates four reagents [1,1' carbonyldiimidazole (GDI), Protein A, glycidoxypropyltrimethoxysilane (GOPS), and 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP)] using these two criteria. Two antibody/antigen systems were employed to investigate the four procedures. The first system is a polyclonal rabbit anti-human IgG with a F(ab)'₂ fragment human IgG as the antigen. This represents a polyvalent, large-antigen case whereas the second system is a monoclonal mouse anti-benzo(a)pyrene tetraol (BPT) IgG with BPT as the antigen. This represents a monovalent, small hapten (small antigens with M.W. < 5000 are usually called haptens) case. The regenerable sensor, termed a "microscale regenerable biosensor" (MRB) is evaluated using immunobeads for the direct assays of BPT. Both a diffusion-based and an aspiration-based MRB are evaluated based on their precision, dynamic range, limit of detection, and selectivity. The main emphasis of the study is to show that a direct assay of BPT is feasible and not to optimize all operational parameters.