Holographic molecular binding assays.

Area of Science:

• Biophysics
• Nanotechnology
• Analytical Chemistry

Background:

• Accurate detection of molecular binding is crucial for diagnostics and drug development.
• Existing methods often require labels or complex procedures.
• There is a need for sensitive, label-free assays to quantify protein-particle interactions.

Purpose of the Study:

• To develop and demonstrate a label-free method for detecting protein binding to functionalized particles.
• To utilize holographic particle characterization for precise size and refractive index measurements.
• To establish the sensitivity of this method for detecting sub-monolayer protein coverage.

Main Methods:

• In-line holographic video microscopy was employed to track individual colloidal probe particles in a microfluidic channel.
• The diameter and refractive index of each particle were measured to detect changes associated with molecular binding.
• Population-average measurements from thousands of particles were used to achieve high precision.

Main Results:

• Holographic particle characterization directly detected protein binding by monitoring changes in particle size.
• The method achieved a population-average diameter uncertainty below 0.5 nm, enabling detection of sub-monolayer protein coverage.
• Successful demonstration of binding detection for NeutrAvidin to biotinylated spheres and immunoglobulin G to protein A-functionalized spheres.

Conclusions:

• Holographic particle characterization provides a sensitive, label-free approach for studying molecular binding events.
• This technique can be applied to various protein-functionalized particle systems.
• The method offers a powerful tool for quantitative analysis in biophysics and nanotechnology.