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Microfluidic competition assay via equilibrium binding

Authors
Journal
Sensors and Actuators B Chemical
0925-4005
Publisher
Elsevier
Publication Date
Volume
139
Issue
2
Identifiers
DOI: 10.1016/j.snb.2009.03.077
Keywords
  • Receptor–Ligand
  • Competition Binding
  • Microfluidic
  • Equilibrium Constant
  • Transport Model
Disciplines
  • Mathematics

Abstract

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 ( K d) 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 ( K d = 0.17 μM) than the labeled insulin ( K d = 0.76 μM). The potential advantages of the method and further improvements in the model are discussed.

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