Altman Lauren E, Grier David G
Department of Physics and Center for Soft Matter Research, New York University, New York, NY 10003, USA.
Biomed Opt Express. 2020 Aug 24;11(9):5225-5236. doi: 10.1364/BOE.401103. eCollection 2020 Sep 1.
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 a population of 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 effective-sphere analysis is rapid and numerically robust and so is useful for practical implementations of label-free immunoassays. Here, we assess how measured effective-sphere properties reflect the actual properties of molecular-scale coatings by modeling coated spheres with the discrete-dipole approximation and analyzing their holograms with the effective-sphere model.
全息分子结合测定法利用全息视频显微镜，通过监测微米级胶体微珠表面光散射特性的相关变化，直接检测与微珠表面结合的分子。通过基于光散射的洛伦兹 - 米氏理论拟合生成模型，对单个球体的全息图进行分析。每次拟合都能以足够的精度得出探针微珠的直径和折射率估计值，以便观察随着分子结合微珠群体的增长情况。然而，这些拟合并非对分子尺度的涂层进行建模，而是使用有效介质理论，将涂覆球体视为均匀球体来处理。这种有效球体分析快速且数值稳健，因此对于无标记免疫测定的实际应用很有用。在此，我们通过用离散偶极近似法对涂覆球体进行建模，并使用有效球体模型分析其全息图，来评估所测量的有效球体特性如何反映分子尺度涂层的实际特性。
