Nonspecific interactions in AFM force spectroscopy measurements

Area of Science:

• Biophysics
• Surface Science

Background:

• Atomic force microscopy (AFM) force spectroscopy is vital for studying ligand-receptor interactions.
• Sample-probe contact duration (dwell time) and loading force significantly influence measurement accuracy.
• Prolonged contact times may be necessary for slow binding kinetics but can increase nonspecific interactions.

Purpose of the Study:

• To investigate methods for reducing nonspecific interactions in AFM force measurements requiring extended substrate-probe contact.
• To evaluate the impact of loading force and dwell time on nonspecific adhesion during AFM measurements.

Main Methods:

• Utilized AFM force spectroscopy to measure interactions between bovine serum albumin (BSA)-functionalized cantilevers and various surfaces (BSA-coated glass, PEG-functionalized glass, Pluronic-treated dishes, agarose beads).
• Systematically varied loading force and contact dwell time to assess their effect on nonspecific adhesion.
• Investigated the role of forced unfolding of BSA in mediating nonspecific interactions.

Main Results:

• Nonspecific interactions between the BSA-functionalized cantilever and tested samples increased with both loading force and dwell time.
• Forced unfolding of BSA was identified as a contributing factor to increased nonspecific adhesion.
• Reducing loading force enabled extended contact durations (minutes) with agarose beads without significant nonspecific adhesion.

Conclusions:

• Optimizing loading force is critical for minimizing nonspecific interactions in AFM force spectroscopy.
• Lowering loading force allows for extended dwell times, facilitating the study of slow binding kinetics without compromising specific interaction detection.
• This approach enhances the reliability of AFM for investigating biological interactions under conditions requiring prolonged sample-probe contact.