Immunosensing with photo-immobilized immunoreagents on planar optical wave guides

Immunocomplexation at wave guiding TiO2/SiO2 surfaces was investigated using an integrated optical grating coupler. For extended application of this label-free monitoring system, F(ab')2 fragments of monoclonal antibodies were photo-immobilized by photolinker polymer-mediated procedures that do not require functionalization of either the immunoreagent or the TiO2/SiO2 surface. Covalent, light-dependent binding of photolinker polymer and F(ab')2 fragments was achieved using a single-step photo-reaction. Bovine serum albumin derivatized with aryldiazirines (T-BSA) served as a photolinker polymer. T-BSA suppressed the non-specific adsorption of analytes to wave guide surfaces. Immunoreagent binding and immunological activity were analyzed and modified surfaces were investigated by scanning force microscopy. Apparent immunoreagent surface densities were 16.7 fmol F(ab')2 per mm2 sensor surface. Optical analyses revealed linear, dose-dependent antigen binding with label free analytes. Immunocompetent surfaces were regenerable by treatment at pH 2.3, rendering the immunosensing system applicable for repetitive use.