THE RESONANT MIRROR - A NOVEL OPTICAL SENSOR FOR DIRECT SENSING OF BIOMOLECULAR INTERACTIONS .2. APPLICATIONS

The application of a novel optical evanescent wave sensor, the Resonant Mirror (RM), to the direct study of biological recognition studies is described. In particular, measurement of antigen/antibody and enzyme/substrate/inhibitor interactions in real-time without labelling is detailed. The RM, described in the preceding paper (Cush et al., 1992), detects changes in refractive index and/or thickness occuring within a few hundred nanometers of the sensor surface. Several methods for immobilisation of biomolecules at the sensor surface have been investigated using antibodies and radioactively labelled antigens as model systems. One of the immobilisation techniques involves deposition of carboxymethyl-dextran on the sensor device and subsequent covalent attachment of biomolecules to the gel via their amino groups. With an anti-theophylline antibody, this method resulted in at least 212 femtomoles of active antibody per mm2 of the surface, a surface density equivalent to at least 9 monolayers of antibody. Immunoassays performed on the RM with immobilised antibodies resulted in sensitivities in the nM range for the complementary antigens. The specificity and sensitivity of the system was further improved by the use of sandwich or multi-site immunoassays and, by the use of enhancer particles, such as colloidal gold particles. The broader applicability of the RM to studies on molecular interaction studies was demonstrated in an assay for the proteolytic enzyme trypsin and the specific inhibition of enzyme activity by alpha1-anti-trypsin.