Related Experiment Video
Updated: May 16, 2026
10:37
Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy
Published on: March 16, 2020
A simple bioconjugate attachment protocol for use in single molecule force spectroscopy experiments based on mixed
Simon J Attwood1, Anna M C Simpson, Rachael Stone
1Nanoscience Centre, Department of Engineering, Cambridge University, Cambridge, CB3 0FF, UK. mew10@eng.cam.ac.uk.
International Journal of Molecular Sciences
|December 4, 2012
Summary
This study introduces a new method for single molecule force spectroscopy using mixed self-assembled monolayers and a crosslinker. The technique successfully probed biotin-avidin interactions, confirming its effectiveness for biomolecular force measurements.
Area of Science:
- Biophysics
- Surface Chemistry
- Biomolecular Interactions
Background:
- Single molecule force spectroscopy (SMFS) is vital for studying biomolecular interactions at the single-molecule level.
- The tip-sample coupling chemistry in SMFS experiments is critical for reliable data acquisition.
- Existing methods for surface functionalization can be complex and may affect experimental outcomes.
Purpose of the Study:
- To develop and validate a novel coupling chemistry for SMFS experiments.
- To improve the reliability and specificity of biomolecular force measurements using SMFS.
- To demonstrate the efficacy of the new protocol using a well-characterized biomolecular interaction.
Main Methods:
- Utilized a novel approach combining mixed self-assembled monolayers of alkanethiols with a heterobifunctional crosslinker for tip-sample functionalization.
- Employed single molecule force spectroscopy to probe the biotin-avidin interaction.
- Performed specificity tests by blocking with free avidin.
Main Results:
- Successfully measured unbinding forces for the biotin-avidin interaction.
- Obtained unbinding force values comparable to previously reported data at similar loading rates.
- Demonstrated significant reduction in avidin binding after blocking, confirming assay specificity.
Conclusions:
- The novel mixed self-assembled monolayer and crosslinker approach provides an effective and specific method for biomolecular force measurements in SMFS.
- This protocol enhances the reliability of SMFS by optimizing tip-sample coupling chemistry.
- The demonstrated biotin-avidin interaction validates the utility of this new technique for probing biomolecular recognition events.
