• Overview
• Force Spectroscopy Methods
• References

Force Spectroscopy: Overview

The AFM can be operated in the force scan mode in which its extreme sensitivity can be used to measure interactions between two apposing surfaces down to the single-molecule level. In studies of ligand-receptor forces, the receptor is immobilized on the surface of a flexible AFM cantilever while the ligand is attached to a suitable substrate. The interaction force is acquired from the deflection of the cantilever during the approach and withdrawal of the cantilever from the substrate. Using this method (often referred to as force spectroscopy) we were able to directly measure the unbinding force of a single ligand-receptor interaction. This novel application led to the use of the AFM as an ultra-sensitive force transducer for probing other biomolecular interactions.

Before describing the methods employed in the AFM force spectroscopy experiment, we first discuss the steps of a simple force scan measurement. At the start of an experiment a flexible cantilever, coated with the receptor of interest, is brought in contact with proteins attached to an apposing substrate. The cantilever is then subsequently withdrawn until the final bond ruptures. The force between cantilever and the substrate, , is derived from the deflection of the flexible cantilever using Hooke’s law, i.e., , where is the spring of the cantilever and is the deflection of the cantilever. Throughout the measurement changes in cantilever’s deflection are monitored by a 2-segment photodiode that senses alterations in the reflective path of a laser spot off the back of the cantilever. The signals from the photodiodes are then amplified and sent to a computer for analysis. Thus, the essential components for an AFM include a piezoelectric translator that displaces the cantilever, a laser and photodiode to detect changes in cantilever deflection and a computer to control movement of the cantilever as well as to process and record data.