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 Resonan t Mirror (RM), to the direct study of biological recognition studies i s described. In particular, measurement of antigen/antibody and enzyme /substrate/inhibitor interactions in real-time without labelling is de tailed. 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 imm obilisation of biomolecules at the sensor surface have been investigat ed using antibodies and radioactively labelled antigens as model syste ms. One of the immobilisation techniques involves deposition of carbox ymethyl-dextran on the sensor device and subsequent covalent attachmen t of biomolecules to the gel via their amino groups. With an anti-theo phylline antibody, this method resulted in at least 212 femtomoles of active antibody per mm2 of the surface, a surface density equivalent t o 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 immu noassays and, by the use of enhancer particles, such as colloidal gold particles. The broader applicability of the RM to studies on molecula r interaction studies was demonstrated in an assay for the proteolytic enzyme trypsin and the specific inhibition of enzyme activity by alph a1-anti-trypsin.