Simultaneous Holographic Molecular Binding Assays with Internal Calibration Standards

Holographic molecular binding assays detect macromolecules binding to colloidal probe beads by monitoring nanometer-scale changes in the beads' diameters with holographic microscopy. Measured changes are interpreted with Maxwell Garnett effective-medium theory to infer the surface coverage of analyte molecules and therefore to measure the analyte concentration in solution. We demonstrate a multicomponent holographic binding assay that tests for immunoglobulin G (IgG) using two different types of functionalized probe beads and provides internal negative controls using inert reference beads. The three label-free measurements are performed simultaneously and yield consistent results for the concentration of the analyte. Negative controls are validated by performing the same test on a solution of alcohol dehydrogenase (ADH), which has a similar molecular weight to IgG but does not bind to the probe beads' binding sites. To assess and mitigate run-to-run variations that might affect the assay's accuracy and reproducibility, we introduce a class of inert reference beads whose diameter and refractive index serve as standards for quantitative holographic microscopy measurements and whose polymer brush coating resists macromolecular binding. We characterize the reference beads' coating by introducing a general all-optical method to measure the grafting density of the polymer brush. This measurement also yields a value of (1.308 ± 0.004) nm³ kDa^-1 for the specific volume of poly(ethylene oxide). This proof-of-concept demonstration of simultaneous independent holographic binding assays can be generalized into a platform for multiplexed testing.