Thomas E. Ryan

Refractive index-based detection of biomolecular interactions

A biosensor was constructed by modifying the optics of a critical angle refractometer to enable it to measure changes in surface plasmon resonance. Initially, the utility of the critical angle refractometry to measure biomolecular interactions was examined using the Leica Automatic Critical Angle Refractometer 600 (AR600). While initial results suggested that the biomolecular interactions could be measured by critical angle refractometry, these proved to be an artifact of protein buildup on the surface. At lower concentrations of target protein, no change in the refractive index was observed. The AR600 was reconfigured from a critical angle refractometer to surface plasmon resonance technology to study molecular interactions between biomolecules. The performance of the SPR biosensor was demonstrated by measuring protein-protein interactions; specifically, avidin:anti-avidin interactions and streptavidin:anti-streptavidin interactions. Equilibrium dissociation constants were obtained by data analysis according to pseudo-first order binding kinetics. Polyclonal anti-streptavidin and monoclonal anti-avidin demonstrated affinity constants of 9.7 x 10-5 M-1 and 4.3 x 10-5 M-1 for their respective ligands.