Microfluidic competition assay via equilibrium binding

Abstract

Competitive binding of analytes in solution, to an immobilized receptor, is a preferred method for quantifying molecules in complex mixtures. We have developed a theoretical and experimental framework for the microfluidic competition assay. A mathematical model describes the transient, convection-dispersion of solutes, undergoing equilibrium binding to immobilized receptors, while entrained in a low Reynolds number incompressible fluid flowing through a microchannel. The proposed method involves monitoring of the elution profile of a reference molecule and ligand in the presence of a competitor. The time difference between the two breakthrough curves provides a measure of the unknown concentration of the competitor. Theoretical results illustrate the general method for determining the equilibrium dissociation constant (Kd) of the ligand and competitor, as well as the competitor concentration. Experimental data is presented for the binding of fluorescein labeled insulin and unlabeled insulin to a monoclonal antibody. It is found that the unlabeled insulin binds with higher affinity (Kd = 0.17 μM) than the labeled insulin (Kd = 0.76 μM). The potential advantages of the method and further improvements in the model are discussed. © 2009 Elsevier B.V. All rights reserved.