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 104particles 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.
Holographic immunoassays: direct detection of antibodies binding to colloidal spheres
The size of a probe bead reported by holographic particle characterization depends on the proportion of the surface area covered by bound target molecules and so can be used as an assay for molecular binding. We validate this technique by measuring the kinetics of irreversible binding for the antibodies immunoglobulin G (IgG) and immunoglobulin M (IgM) as they attach to micrometer-diameter colloidal beads coated with protein A. These measurements yield the antibodies’ binding rates and can be inverted to obtain the concentration of antibodies in solution. Holographic molecular binding assays therefore can be used to perform fast quantitative immunoassays that are complementary to conventional serological tests.
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- Award ID(s):
- 2027013
- NSF-PAR ID:
- 10219740
- Date Published:
- Journal Name:
- Soft Matter
- Volume:
- 16
- Issue:
- 44
- ISSN:
- 1744-683X
- Page Range / eLocation ID:
- 10180 to 10186
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0SM01351J
