Control of Antigen Mass Transport via Capture Substrate Rotation: Binding Kinetics and Implications on Immunoassay Speed and Detection Limits

TitleControl of Antigen Mass Transport via Capture Substrate Rotation: Binding Kinetics and Implications on Immunoassay Speed and Detection Limits
Publication TypeJournal Article
Year of Publication2009
AuthorsWang GF, Driskell JD, Porter MD, Lipert RJ
Journal TitleAnalytical Chemistry
Volume81
Pages6175-6185
Date Published08/01
ISBN Number0003-2700
Accession NumberISI:000268455600029
Keywordsantibody-binding, biosensor, diffusion, force microscopy, gold surfaces, limitation, model, monolayers, protein, shear
Abstract

In conventional heterogeneous immunoassays, assay speed is usually limited by the rate of mass transport, i.e., diffusion of antigen to an antibody-coated surface. We previously demonstrated that assay speed can be significantly increased, without losing analytical sensitivity, by rapidly rotating the capture substrate, which decreases the thickness of the diffusion layer. In this work, we raised the rotation speed and observed that the capture of antigens deviates from the mass transport-limited assumption. To examine this issue, a general equation was derived for the rate of immuno-reaction on a rotating capture surface that takes into account both diffusion and the rate of reaction between antigen and antibody, which applies over a wide range of rotation rates. Results show that by vigorously rotating the substrate, the binding of antigens reaches a regime of intermediate binding kinetics, for which mass transport is comparable to the reaction rate. With this general solution, we are able to determine the two important binding kinetics parameters: the diffusion coefficient and the reaction rate constant. Then, using porcine parvovirus as an example, we use these parameters to investigate the limit of the assay speed and the limit of detection achievable on a practical time scale through numerical simulations of the kinetic binding curves for various assay conditions.

URL<Go to ISI>://000268455600029
DOI10.1021/Ac900704t