ABSTRACT
In a typical experiment, a capture molecule is first immobilized covalently on the sensor surface. Subsequently, the residual binding sites are blocked. After a wash with a buffer of low ionic strength, the interaction sample is passed over the sensor, followed again by buffer. Figure 2 illustrates a typical SPR sensogram. Response is measured with time and is proportional to the mass of the adsorbed molecule [6,11]. As ligand adsorption on the immobilized capture molecule occurs, the adsorption profile allows the determination of the association rate constant, kon. After passing of the sample plug, dissociation of the complex follows as buffer passes over the sensor. The logarithmic decay of the dissociation reaction then allows the calculation of the dissociation rate constant koff. Earlier studies exploring the potential of SPR in the field of biomolecular interactions employed simple adsorption to the metal surface for the immobilization of the capture component. This strategy comes with certain limitations. Some proteins may denature when absorbed directly to the metal; hapten-type ligands are very difficult to immobilize on metal. Furthermore, the performance of the sensor depends on the even and reproducible distribution as well as correct orientation of the adsorbed molecules. With a directly immobilized ligand there can be undesired interaction
