Evaluation of Taylor dispersion injections: Determining kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing

In label-free biomolecular interaction analysis, a standard injection provides an injection of uniform analyte concentration. An alternative approach exploiting Taylor dispersion produces a continuous analyte titration allowing a full analyte dose response to be recorded in a single injection. The enhanced biophysical characterization that is possible with this new technique is demonstrated using a commercially available surface plasmon resonance-based biosensor. A kinetic interaction model was fitted locally to Taylor dispersion curves for estimation of the analyte diffusion coefficient in addition to affinity/kinetic constants. Statistical confidence in the measured parameters from a single Taylor dispersion injection was comparable to that obtained for global analysis of multiple standard injections. The affinity constants for multisite interactions were resolved with acceptable confidence limits. Importantly, a single analyte injection could be treated as a high-resolution real-time affinity isotherm and was demonstrated using the complex two-site interaction of warfarin with human serum albumin. In all three model interactions tested, the kinetic/affinity constants compared favorably with those obtained from standard kinetic analysis and the estimates of analyte diffusion coefficients were in good agreement with the expected values. (C) 2011 Elsevier Inc. All rights reserved.