# Probing the Dynamic Strength of Biomolecular Interactions with Single-Cell Centrifugation

## TL;DR

This paper introduces a high-throughput method using a centrifuge force microscope to study how molecular interactions respond to mechanical stress, with applications in immunotherapy.

## Contribution

A next-generation centrifuge force microscope with dual-channel fluorescence imaging for real-time, high-throughput analysis of receptor–ligand binding mechanics.

## Key findings

- The CFM quantifies receptor–ligand avidity in thousands of single cells under mechanical stress.
- BiTE-mediated adhesion between T and cancer cells shows time-dependent increases in bond strength.
- Ligand concentration correlates with bond strength in T and B cell interactions with BiTE-modified surfaces.

## Abstract

Molecular interactions
between receptors and ligands
govern critical
biological processes, from immune surveillance and T-cell activation
to tissue development. However, current techniques for studying binding
avidity often sacrifice throughput or precision. We introduce a high-throughput
method for quantifying molecular and cellular binding kinetics using
a centrifuge force microscope (CFM)a compact imaging system
integrated into a benchtop centrifuge. The CFM performs real-time
force measurements on thousands of single cells in parallel, probing
receptor–ligand interactions under controlled mechanical stress.
To extend these capabilities, we developed a next-generation CFM with
dual-channel fluorescence imaging that enables tracking of individual
cell unbinding events. To demonstrate its utility, we profiled the
binding mechanics of Bispecific T-cell Engager (BiTE) molecules, immunotherapeutic
proteins that facilitate T-cell targeting of cancer cells. In cell–protein
assays, we quantified the avidity of T and B cells interacting with
BiTE-modified surfaces, revealing receptor-specific correlations between
ligand concentration and bond strength. In cell–cell assays,
we characterized BiTE-mediated adhesion between Jurkat and Nalm6 cells,
demonstrating a time-dependent increase in avidity. By integrating
force spectroscopy with fluorescence imaging, the CFM provides a high-throughput
approach for investigating the mechanochemical principles underlying
receptor-mediated interactions, with broad implications for biophysical
chemistry, molecular recognition, and therapeutic development.