In this work a number of instrumental aspects related to different optical configurations of Total Internal Reflection Fluoresence (TIRF) systems are introduced, and the influence of some biological reaction parameters on the performance of optical TIRF immunosensors are discussed. The manner in which fundamental quantities, such as the dissociation constant of the immunological reaction in the adsorbed phase, affect the measurement range of a TIRF immunosensor is described. The effects of light scattering at the interface and non-specific binding are also examined. Using a simple equilibrium model, in which experimental values of the surface density of active antibodies and of the dissociation constants in the immobilized phase are used, these parameters are directly related to the range of concentrations in which a competitive immunosensor gives a useful response. It is shown that amongst the most critical parameters affecting sensor performance are the dissociation constant of the antibody-antigen and antibody-labelled antigen reactions in the immobilized phase. Methods of controlling the effects of these parameters are considered. © 1995.

cited By 14