Holographic molecular binding assays

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.

Figure 1

Holographic molecular binding assay. (a) Functionalized colloidal spheres are incubated with target molecules that bind to the beads’ surface groups. (b) Conventional analysis requires washing and incubation with fluorescent labels that also bind to the surface-bound target molecules. These labels’ fluorescence is read out in a flow cytometer. (c) Fluid-borne beads travel down a microfluidic channel where they are illuminated by a collimated laser beam. Scattered light interferes with the rest of the beam to form holograms that are recorded with a video camera. (d) Each hologram is fit pixel-by-pixel to predictions of the Lorenz-Mie theory of light scattering to measure the diameter, d_p, and refractive index, n_p, of the associated sphere. This measurement is sufficiently precise to detect the change in diameter associated with molecular binding. (e) Joint probability distribution, ρ(d_p, n_p), of particle diameter and refractive index measurements for biotinylated polystyrene spheres before and after incubation with NeutrAvidin. Each point represents the properties of one bead.

Figure 2

(a) Projected distribution ρ(d_p) of particle diameters for biotinylated polystyrene spheres before and after incubation with NeutrAvidin. The width of the distribution, σ_d, reflects the population polydispersity in diameter. The difference, Δρ(d_p), between these distributions is consistent with a statistically significant increase of Δd_p = (1.4 ± 0.1) nm in the mean particle diameter. (b) Analogous data for IgG binding to spheres coated with protein A shows an increase of Δd_p = (3.2 ± 0.4) nm.

Figure 3

Dependence of holographically measured diameter, d_p, and refractive index, n_p, on particle position, z_p, within the sample cell for (a) biotinylated spheres before (yellow squares) and after (red circles) binding by NeutrAvidin and (b) spheres coated with Protein A before and after binding by IgG. Population averages and standard deviations are calculated at each height using kernel density estimators.