# Simultaneous Holographic Molecular Binding Assays with Internal Calibration Standards

## TL;DR

This paper introduces a new holographic method to detect molecules using probe beads and reference standards for accurate and simultaneous measurements.

## Contribution

The novel contribution is a multicomponent holographic assay with internal calibration standards for label-free molecule detection.

## Key findings

- Simultaneous measurements using two functionalized probe beads and inert reference beads yield consistent analyte concentration results.
- Negative controls using alcohol dehydrogenase confirm the specificity of the binding assay.
- A new all-optical method measures polymer brush grafting density and determines the specific volume of poly(ethylene oxide).

## Abstract

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.