Binding Kinetics of Immobilized Biomolecules by Microchannel Flow Displacement

Abstract

Binding kinetics of biomolecular interaction is essentially important for the understanding of biological processes. In this project, I have developed the theoretical and experimental framework for kinetic rate measurement of immobilized biomolecules in PDMS (poly(dimethylsiloxane)) microchannels employing the displacement technique, where a labeled ligand is displaced from the binding site by an excess of unlabeled ligand.Firstly, a mathematical model describing the transient convection, diffusion and dissociation of the labeled ligand was developed and solved analytically and numerically. Secondly, experiments were carried out to demonstrate 1) immobilization of the receptor via avidin-biotin interaction in microchannels, 2) receptor ligand binding and dissociation, 3) effectiveness of blockers in preventing non-specific binding. A fiber optic fluorescence probe was used to monitor the displaced protein at the channel outlet. The results suggest that larger molecular weight ligands like insulin are not displaced from the binding site by unlabeled ligand. This method is expected to be especially suitable for the study of low molecular weight biomolecules.