Recognition kinetics of biomolecules at the surface of different-sized spheres

Bead-based assay is widely used in many bioanalytical applications involving the attachment of proteins and other biomolecules to the surface. For further understanding of the formation of a sphere-biomolecule complex and easily optimizing the use of spheres in targeted biological applications, it is necessary to know the kinetics of the binding reaction at sphere/solution interface. In our presented work, a simple fluorescence analysis method was employed to measure the kinetics for the binding of biotin to sphere surface-bound FITC-SA, based on the fact that the fluorescence intensity of FITC was proportionally enhanced by increasing the binding amount of biotin. By monitoring the time-dependent changes of FITC fluorescence, it was found that the binding rate constant of biotin to sphere surface-immobilized FITC-SA was much smaller than that of biotin to freely diffusing FITC-SA. This can be attributed to the decreased encounter frequency of the reaction pair, restricted motion of the attached biomolecule, and the weakened steric accessibility of the binding site. These factors would become more obvious when increasing the size of the sphere upon which the FITC-SA was immobilized. Additionally, the effect of nanoparticles on the diffusion-controlled bimolecular binding reaction was more evident than that on the chemical recognition-controlled binding reaction.