Interpreting Holographic Molecular Binding Assays with Effective Medium Theory

Abstract

Holographic molecular binding assays use holographic video microscopy to directly detect molecules binding to the surfaces of micrometer-scale colloidal beads by monitoring associated changes in the beads' light-scattering properties. Holograms of individual spheres are analyzed by fitting to a generative model based on the Lorenz-Mie theory of light scattering. Each fit yields an estimate of a probe bead's diameter and refractive index with sufficient precision to watch the beads grow as molecules bind. Rather than modeling the molecular-scale coating, however, these fits use effective medium theory, treating the coated sphere as if it were homogeneous. This effectivesphere analysis is rapid and numerically robust and so is useful for practical implementations of label-free immunoassays. Here, we assess how effective-sphere properties reflect the properties of molecular-scale coatings by modeling coated spheres with the discrete-dipole approximation and analyzing their holograms with the effective-sphere model.