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

Area of Science:

• Biophysical Chemistry
• Molecular Recognition
• Immunology

Background:

• Receptor-ligand interactions are crucial for biological processes like immune surveillance and tissue development.
• Existing methods for studying binding avidity lack either high throughput or precision.
• Understanding molecular binding kinetics is essential for therapeutic development.

Purpose of the Study:

• To develop a high-throughput method for quantifying molecular and cellular binding kinetics.
• To enhance existing centrifuge force microscopy (CFM) with dual-channel fluorescence imaging.
• To profile the binding mechanics of Bispecific T-cell Engager (BiTE) molecules.

Main Methods:

• Utilized a centrifuge force microscope (CFM) for real-time, parallel force measurements on thousands of single cells.
• Developed a next-generation CFM with dual-channel fluorescence imaging to track individual cell unbinding events.
• Performed cell-protein and cell-cell assays to quantify avidity and characterize BiTE-mediated adhesion.

Main Results:

• Quantified the avidity of T and B cells interacting with BiTE-modified surfaces, showing receptor-specific correlations between ligand concentration and bond strength.
• Characterized BiTE-mediated adhesion between Jurkat and Nalm6 cells, demonstrating a time-dependent increase in avidity.
• Demonstrated the CFM's capability to investigate mechanochemical principles of receptor-mediated interactions.

Conclusions:

• The integrated CFM provides a high-throughput approach for studying receptor-ligand interactions and binding avidity.
• This method has broad implications for biophysical chemistry, molecular recognition, and the development of immunotherapies.
• The CFM facilitates a deeper understanding of the mechanochemical underpinnings of cellular adhesion and immune responses.