# Real-Time Probing of Molecular Affinity Using Optical Tweezers

## TL;DR

This paper introduces a new optical tweezer method to study molecular binding in real time, using a model system to demonstrate its effectiveness and advantages over existing techniques.

## Contribution

A novel optical tweezer-based method for real-time, label-free monitoring of molecular affinity with high sensitivity and adaptability.

## Key findings

- The method successfully detected nanoscale changes in particle size during streptavidin–biotin binding.
- The association rate constant (kon≈10⁶ M⁻¹ s⁻¹) matched literature values, validating the technique's accuracy.
- The approach offers real-time monitoring, adaptability, and compatibility with miniaturized systems compared to existing methods.

## Abstract

The ability to assess molecular binding kinetics in real time is critical for advancing our understanding of molecular interactions in biochemical and biotechnological systems. This work presents a novel optical tweezer (OT)-based method to monitor molecular affinity in real time, focusing on the high-affinity streptavidin–biotin system as a model. Transparent poly(methyl methacrylate) (PMMA) microparticles functionalized with streptavidin were trapped before, during, and after binding with biotinylated bovine serum albumin (biotin–BSA), enabling the analysis of forward-scattered signals to detect nanoscale changes in particle size. By applying the Power Spectral Density method, the friction coefficient of individual particles was calculated, allowing for real-time tracking of binding dynamics and the estimation of the association rate constant (kon≈106M−1s−1). These results are consistent with literature values and demonstrate the potential of this OT-based approach for non-invasive, label-free detection of molecular interactions. Compared to existing techniques, such as atomic force microscopy and cantilever-based sensors, this method offers significant advantages, including real-time monitoring, adaptability to different bioaffinity systems, and compatibility with miniaturized setups. This work establishes a foundation for using OT-based tools to monitor high-affinity molecular interactions in real time. While demonstrated here using biotinylated BSA as a model ligand, future studies will explore the method’s applicability to smaller ligands and more subtle surface modifications.