Simultaneous Holographic Molecular Binding Assays with Internal Calibration Standards

Area of Science:

• Biophysics
• Analytical Chemistry
• Nanotechnology

Background:

• Holographic microscopy offers label-free detection of nanoscale changes.
• Quantifying molecular binding requires accurate measurement of analyte concentration.

Purpose of the Study:

• To develop a multicomponent holographic binding assay for simultaneous, label-free detection of analytes.
• To introduce inert reference beads for improved accuracy and reproducibility in holographic assays.
• To establish a general method for characterizing polymer brush coatings on reference beads.

Main Methods:

• Utilizing holographic microscopy to monitor nanometer-scale diameter changes in colloidal probe beads upon macromolecular binding.
• Applying Maxwell Garnett effective-medium theory to interpret bead diameter changes and infer analyte concentration.
• Employing functionalized probe beads and inert reference beads for simultaneous, multicomponent, and label-free binding assays.

Main Results:

• Demonstrated a multicomponent assay for immunoglobulin G (IgG) with consistent results using internal negative controls.
• Validated negative controls using alcohol dehydrogenase (ADH) to confirm specificity.
• Introduced inert reference beads for quantitative holographic microscopy and characterized their polymer brush coating, yielding specific volume for poly(ethylene oxide).

Conclusions:

• Simultaneous independent holographic binding assays provide a robust platform for multiplexed testing.
• The developed method enhances accuracy and reproducibility in molecular binding detection.
• This approach can be generalized for various analyte detection and characterization applications.