Consecutive Mechano-Immunophenotyping of Single Cells in a Photopatterned Microfluidic Channel

Area of Science:

• Biophysics
• Cell Biology
• Microfluidics

Background:

• Single-cell analysis often neglects the interplay between physical forces and biochemical signals.
• Existing assays rarely capture both mechanical and molecular phenotypes concurrently.

Purpose of the Study:

• To develop a novel microfluidic device for simultaneous extraction of mechanical and molecular phenotypes from single cells.
• To investigate the correlation between cell deformability and surface antigen expression.

Main Methods:

• A two-stage microfluidic device was designed to measure cell deformability (via transit time through constrictions) and surface antigen density (via antibody capture).
• The device utilizes a photopatterned hydrogel microchannel functionalized with antibodies to capture cells proportionally to antigen density.
• Five cancer cell lines were profiled using biotinylated anti-CD64 and anti-CD44v9 antibodies.

Main Results:

• The device successfully measured mechanical (transit time, Td) and molecular (antigen capture delay, Ta) phenotypes in a single pass.
• A mechanical-immunochemical correlation was observed, with R² = 0.41 for HeLa cells, indicating significant heterogeneity in other cell lines like HT29.
• The platform achieves high throughput (∼100 cells/min) and sensitivity (∼100 membrane proteins/μm²), while maintaining cell viability.

Conclusions:

• The developed platform enables multiplex, mechanomolecular phenotyping for applications in precision oncology, stem-cell quality control, and drug screening.
• This integrated approach offers a powerful tool for understanding cell behavior under combined physical and biochemical stimuli.
• The ability to host multiple ligands on the hydrogel allows for comprehensive profiling of cell surface markers.