Holographic molecular binding assays

M. Francesca Soddu,Andrew D. Hollingsworth,David G. Grier,Yvonne Zagzag

doi:10.1038/s41598-020-58833-7

M. Francesca Soddu, Andrew D. Hollingsworth + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-58833-7

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Journal: Scientific Reports	Publication Date: Feb 6, 2020
Citations: 11	License type: open-access

Affiliation: New York University, University of Pennsylvania

Abstract

We demonstrate that holographic particle characterization can directly detect binding of proteins to functionalized colloidal probe particles by monitoring the associated change in the particles’ size. This label-free molecular binding assay uses in-line holographic video microscopy to measure the diameter and refractive index of individual probe spheres as they flow down a microfluidic channel. Pooling measurements on 104 particles yields the population-average diameter with an uncertainty smaller than 0.5 nm, which is sufficient to detect sub-monolayer coverage by bound proteins. We demonstrate this method by monitoring binding of NeutrAvidin to biotinylated spheres and binding of immunoglobulin G to spheres functionalized with protein A.

Highlights

We demonstrate that holographic particle characterization can directly detect binding of proteins to functionalized colloidal probe particles by monitoring the associated change in the particles’ size
High-speed automated fitting of in-line holographic microscopy images such as the example in Fig. 1(d) yields precise measurements of the probe beads’ diameters before and after they are incubated with the target molecule
Interpreting holographic characterization data with the effective-sphere model[6,7,8] and rigorous statistical methods[9] yields a label-free assay that is fast, cost-effective and sensitive. We demonstrate this method through measurements on two model systems: avidin binding to biotinylated polystyrene spheres, and immunoglobulin G (IgG) binding to polystyrene spheres functionalized with protein A

Summary

Introduction

We demonstrate that holographic particle characterization can directly detect binding of proteins to functionalized colloidal probe particles by monitoring the associated change in the particles’ size. Bead-based molecular binding assays use micrometer-scale colloidal spheres as solid substrates for functional sites to which target molecules can bind. We demonstrate that holographic particle characterization[4,5] can eliminate the extra steps associated with fluorescence detection by directly measuring the increase in the beads’ diameters caused by target molecules binding to their surfaces.

Results

Conclusion