Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Feb 6;10(1):1932.
doi: 10.1038/s41598-020-58833-7.

Holographic molecular binding assays

Yvonne Zagzag  1   2 , M Francesca Soddu  3 , Andrew D Hollingsworth  1 , David G Grier  4
Affiliations

Holographic molecular binding assays

Yvonne Zagzag et al. Sci Rep. .

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.

PubMed Disclaimer

Conflict of interest statement

D.G.G. is a founder of Spheryx, Inc., which manufactures the xSight instrument used for this study.

Figures

Figure 1
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, dp, and refractive index, np, of the associated sphere. This measurement is sufficiently precise to detect the change in diameter associated with molecular binding. (e) Joint probability distribution, ρ(dp, np), 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
Figure 2
(a) Projected distribution ρ(dp) of particle diameters for biotinylated polystyrene spheres before and after incubation with NeutrAvidin. The width of the distribution, σd, reflects the population polydispersity in diameter. The difference, Δρ(dp), between these distributions is consistent with a statistically significant increase of Δdp = (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 Δdp = (3.2 ± 0.4) nm.
Figure 3
Figure 3
Dependence of holographically measured diameter, dp, and refractive index, np, on particle position, zp, 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Perera VY, Creasy MT, Winter AJ. Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens. J. Clin. Microbiol. 1983;18:601–608. doi: 10.1128/JCM.18.3.601-608.1983. - DOI - PMC - PubMed
    1. McHugh TM, Viele MK, Chase ES, Recktenwald DJ. The sensitive detection and quantitation of antibody to HVC by using a microsphere-based immunoassay and flow cytometry. Cytom. 1997;29:106–112. doi: 10.1002/(SICI)1097-0320(19971001)29:2<106::AID-CYTO2>3.0.CO;2-B. - DOI - PubMed
    1. Brenner S, et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat. Biotech. 2000;18:630. doi: 10.1038/76469. - DOI - PubMed
    1. Lee S-H, et al. Characterizing and tracking single colloidal particles with video holographic microscopy. Opt. Express. 2007;15:18275–18282. doi: 10.1364/OE.15.018275. - DOI - PubMed
    1. Cheong FC, et al. Flow visualization and flow cytometry with holographic video microscopy. Opt. Express. 2009;17:13071–13079. doi: 10.1364/OE.17.013071. - DOI - PubMed

Publication types