Functionalized self-assembled monolayers on ultraflat gold as platforms for single molecule force spectroscopy and...

Area of Science:

• Biophysics
• Materials Science
• Biochemistry

Background:

• Single molecule force spectroscopy (SMFS) is crucial for protein unfolding and nanomechanical property analysis.
• Current methods often lack clarity on whether proteins are truly single and isolated, leading to potential artifacts.
• Undesirable protein/surface interactions can obscure critical data in force curves.

Purpose of the Study:

• To develop a controlled method for isolating single proteins for SMFS.
• To optimize protein immobilization techniques for enhanced data quality and reproducibility.
• To reduce nonspecific interactions in SMFS experiments.

Main Methods:

• Utilized mixed self-assembled monolayers (SAMs) with N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on gold surfaces.
• Covalently immobilized proteins via lysine residues to NHS groups within an OEG protein-resistant layer.
• Employed atomic force microscopy (AFM) for imaging and verifying single protein distribution and reducing tip-surface interactions.

Main Results:

• Achieved controlled isolation of single proteins through optimized lysine-NHS linkages on OEG SAMs.
• AFM imaging confirmed uniform single protein distribution on the surface.
• Significantly reduced nonspecific tip-surface interactions using the OEG layer.
• Generated high-quality, reproducible force-extension curves from stretched single proteins.

Conclusions:

• The developed experimental strategy provides a robust platform for studying single protein structure, interactions, and nanomechanical properties.
• Optimized surface chemistry enables reliable SMFS measurements by ensuring single, isolated protein analytes.
• This approach enhances the accuracy and interpretability of SMFS data for protein research.