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Force spectroscopy

Force spectroscopy is a dynamic analytical technique that allows the study of the mechanical properties of polymer molecules and/or chemical bonds. It is exceptional in being a single molecule technique[?], i.e. it gains structural information by manipulating molecules one by one. It is a direct and recent application of atomic force microscopy[?].

The name "force spectroscopy", although widely used in the scientific community, is somewhat misleading, because there is no true matter-radiation interaction[?]. Force spectroscopy measures the behaviour of a molecule under stretching mechanical force.

A common experimental set-up is as following. Molecules adsorbed on a surface are picked up by a microscopic tip (few microns wide) that is located on the end of an elastic cantilever. A piezoelectric controller then pulls up the cantilever. If some force is acting on the elastic cantilever (for example because some molecule is being stretched between the surface and the tip) , this will deflect upward (repulsive force) or downward (attractive force). According to Hooke's law, this deflection will be proportional to the force acting on the cantilever. Deflection is measured by the position of a laser beam reflected by the cantilever. This kind of set-up can measure forces as low as 10 pN (10-11 N), and cannot achieve much better resolution only because of thermal noise. The so-called force spectrum is the graph of force (or more precisely, of cantilever deflection) versus the piezoelectric position on the Z axis. An ideal Hookian spring, for example, would display a straight diagonal line spectrum.

Common applications of force spectroscopy are measurements of polymer elasticity. Polymer chains tend to be randomly coiled at rest. They resist to stretching forces because the more the stretching , the less the configurations accessible to the chain. This corresponds to a loss of entropy, and therefore generates a restoring force. Polymers generally don’t behave like Hookian springs, but are well described by more sophisticated models like the worm-like chain model[?] of entropic elasticity. If during stretching a conformational change occurs, the elasticity will also gain an enthalpic component (i.e. a component due to the breaking/forming/reshaping of chemical bonds). This is readily seen in the force spectra as deviations from the expected entropic elasticity curve. For example, DNA double helices show a marked conformational change when stretched at forces around 60 pN.

An exciting biophysical application of polymer force spectroscopy is on protein unfolding. Modular proteins can be adsorbed to a gold or (more rarely) mica surface and then stretched. The sequential unfolding[?] of modules is observed as a very characteristic sawtooth pattern of the force vs elongation graph; every tooth corresponds to the unfolding of a single protein module (apart from the last that is generally the detachment of the protein molecule from the tip) A lot of information about protein elasticity and protein unfolding can be obtained by this technique. This is even more interesting if we consider the fact that a lot of proteins in the living cell must face mechanical stress.

The other main application of force spectroscopy is the study of mechanical resistance of chemical bonds. In this case generally the tip is functionalized with a ligand that binds to another molecule bound to the surface. The tip is pushed on the surface, allowing for contact between the two molecules, and then retracted until the newly formed bond breaks up. The force at which the bond breaks up is measured. Since mechanical breaking is a kinetic, stochastic[?] process, the breaking force is not an absolute parameter, but it is a function of both temperature and pulling speed. Low temperatures and high pulling speeds correspond to higher breaking forces. By careful analysis of the breaking force at various pulling speeds, it is possible to map the energy landscape of the chemical bond under mechanical force. This is leading to interesting results in the study of antibody-antigen, protein-protein and even protein-living cell interaction.



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