Development of Three Alternative Strategies for the Binding of Cells to Functionalized DeepTip^TM AFM Probes

Area of Science:

• Biomaterials Science
• Cellular Biophysics
• Surface Chemistry

Background:

• Designing biohybrid materials necessitates precise control over cell-material interactions.
• Single Cell Force Spectroscopy (SCFS) is a key technique for characterizing these interactions, but requires stable cell attachment to the atomic force microscopy (AFM) probe.
• Cell detachment during SCFS experiments can lead to unreliable data.

Purpose of the Study:

• To develop and evaluate robust methods for attaching diverse cell types to AFM probes for SCFS.
• To compare the efficacy of different crosslinking strategies and probe functionalization schemes.
• To establish reliable protocols for advancing biohybrid material research.

Main Methods:

• Three crosslinking strategies were employed: streptavidin/biotin, sulfhydryl group-based crosslinkers, and click chemistry.
• Three probe decoration schemes were explored: specific antibody, concanavalin A, and direct binding via azide-derivatized proteins.
• These methods were tested on both non-adherent (CD4+ T-lymphocytes) and adherent (mesenchymal stem cells, MSC) cell types.

Main Results:

• All three presented methods successfully facilitated cell attachment to AFM probes for SCFS.
• The strength of cell-material interaction varied significantly based on the functionalization strategy.
• Interaction strength followed a decreasing order: specific antibody > concanavalin A > binding through azide-derivatized proteins.

Conclusions:

• The developed crosslinking and functionalization strategies offer versatile solutions for stable cell attachment in SCFS.
• The choice of probe decoration significantly impacts the measured cell-material interaction forces.
• These advancements provide a more reliable foundation for the design and characterization of biohybrid materials.