Measurement of the kinetics of biomolecular interactions using the IAsys resonant mirror biosensor

In this unit, the resonant mirror biosensor or IAsys, produced by Affinity Sensors, is discussed for the measurement of the kinetics of biomolecular interactions. All biosensors operate using a common principle--that binding events occurring on the sensing surface of the biosensor are converted by a transducer into an electronic signal which can then be processed by a computer to yield information on the amount of binding. Where biosensors vary is in their configuration and the nature of the transducer. Both the IAsys and BIACORE series of machines use optical techniques to detect the binding event, but the IAsys uses a resonant mirror device as the transducer, while the BIACORE uses surface plasmon resonance. When using biosensors the experiment can be divided into various phases, which are separately in this unit. The phases involve (1) the immobilization of the ligand onto the sensing surface; (2) addition of the ligate to the sensing surface, and following its association to the ligand, including the kinetics of binding; (3) washing of free ligate from chamber and following the dissociation of the ligand-ligate complex; (4) regeneration of the surface; repetition of phases 2 and 3; and (5) data analysis. A protocol is also included for optimization of ligand pH for immobilization to the carboxymethylated dextran surfaces described in this unit.