Force spectroscopy
Force spectroscopy is a set of techniques for the study of the binding forces between individual molecules. These methods can be used to measure the mechanical properties of single polymer molecules or proteins, or individual chemical bonds. The name "force spectroscopy", although widely used in the scientific community, is somewhat misleading, because there is no true matter-radiation interaction.
Techniques that can be used to perform force spectroscopy include atomic force microscopy,optical tweezers,magnetic tweezers, acoustic force spectroscopy, microneedles, and biomembranes.
Force spectroscopy measures the behavior of a molecule under stretching or torsional mechanical force. In this way a great deal has been learned in recent years about the mechanochemical coupling in the enzymes responsible for muscle contraction, transport in the cell, energy generation (F1-ATPase), DNA replication and transcription (polymerases), DNA unknotting and unwinding (topoisomerases and helicases).
As a single-molecule technique, as opposed to typical ensemble spectroscopies, it allows a researcher to determine properties of the particular molecule under study. In particular, rare events such as conformational change, which are masked in an ensemble, may be observed.
There are many ways to accurately manipulate single molecules. Prominent among these are optical or magnetic tweezers and atomic-force-microscope (AFM) cantilevers. In all of these techniques, a biomolecule, such as protein or DNA, or some other biopolymer has one end bound to a surface and the other to a force sensor. The force sensor is usually a micrometre-sized bead or a cantilever, whose displacement can be measured to determine the force.
Molecules adsorbed on a surface are picked up by a microscopic tip (nanometres wide) that is located on the end of an elastic cantilever. In a more sophisticated version of this experiment (Chemical Force Microscopy) the tips are covalently functionalized with the molecules of interest. 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 (10−11N), and cannot achieve much better resolution only because of thermal noise. The so-called force curve is the graph of force (or more precisely, of cantilever deflection) versus the piezoelectric position on the Z axis. An ideal Hookean spring, for example, would display a straight diagonal force curve. Typically, the force curves observed in the force spectroscopy experiments consist of a contact (diagonal) region where the probe contacts the sample surface, and a non-contact region where the probe is off the sample surface. When the restoring force of the cantilever exceeds tip-sample adhesion force the probe jumps out of contact, and the magnitude of this jump is often used as a measure of adhesion force or rupture force. In general the rupture of a tip-surface bond is a stochastic process; therefore reliable quantification of the adhesion force requires taking multiple individual force curves. The histogram of the adhesion forces obtained in these multiple measurements provides the main data output for force spectroscopy measurement.
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