Going Vertical To Improve the Accuracy of Atomic Force Microscopy Based Single-Molecule Force Spectroscopy

Area of Science:

• Biophysics
• Materials Science
• Nanotechnology

Background:

• Single-molecule force spectroscopy (SMFS) using atomic force microscopy (AFM) is crucial for studying biomolecular mechanics.
• Current AFM-based SMFS accuracy is limited by uncorrected lateral stretching angles, especially for stiff molecules like DNA.
• This inaccuracy hinders precise characterization of molecular properties and interactions.

Purpose of the Study:

• To develop and validate an automated algorithm for precise tip alignment in AFM-based SMFS.
• To correct for lateral offsets in force-extension measurements of DNA molecules.
• To improve the accuracy, precision, and throughput of AFM-based SMFS.

Main Methods:

• Developed an automated algorithm for aligning the AFM tip to DNA attachment points on a coverslip.
• Performed alignment at low force (10-20 pN) and short duration (15-25 s) to maintain molecular connection.
• Acquired and analyzed force-extension curves for DNA, polyprotein, and PEG-based assays.

Main Results:

• Identified significant uncorrected lateral offsets in DNA measurements (24 ± 18 nm for 100 nm DNA, 180 ± 110 nm for 650 nm DNA).
• Correcting these offsets improved accuracy and precision by 3-fold for DNA overstretching transitions.
• Demonstrated high throughput (88 curves in 40 min) and versatility across different molecular systems.

Conclusions:

• The automated alignment algorithm significantly enhances AFM-based SMFS accuracy and precision.
• This method is broadly applicable to commercial AFMs, improving biomolecular characterization.
• Sophisticated data-acquisition protocols are key to advancing AFM-based SMFS capabilities.