Editing Force spectroscopy (section)

==Experimental techniques==
{{More citations needed section|date=May 2014}}
There are many ways to accurately manipulate single molecules. Prominent among these are optical or magnetic tweezers, atomic-force-microscope (AFM) cantilevers and acoustic force spectroscopy.  In all of these techniques, a biomolecule, such as protein or DNA, or some other biopolymer has one end bound to a surface or micrometre-sized bead 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.

===Atomic force microscope cantilevers===
Molecules [[adsorption|adsorbed]] on a [[Interface (matter)|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.<ref>{{cite journal | vauthors = Ott W, Jobst MA, Schoeler C, Gaub HE, Nash MA | title = Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox | journal = Journal of Structural Biology | volume = 197 | issue = 1 | pages = 3–12 | date = January 2017 | pmid = 26873782 | doi = 10.1016/j.jsb.2016.02.011 }}</ref> 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<sup>−11</sup> [[newton (unit)|N]]), the fundamental resolution limit is given by the cantilever's 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 (device)|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.

In biophysics, single-molecule force spectroscopy can be used to study the energy landscape underlying the interaction between two bio-molecules, like proteins. Here, one binding partner can be attached to a cantilever tip via a flexible linker molecule (PEG chain), while the other one is immobilized on a substrate surface. In a typical approach, the cantilever is repeatedly approached and retracted from the sample at a constant speed. In some cases, binding between the two partners will occur, which will become visible in the force curve, as the use of a flexible linker gives rise to a characteristic curve shape (see [[Worm-like chain]] model) distinct from adhesion. The collected rupture forces can then be analysed as a function of the bond loading rate. The resulting graph of the average rupture force as a function of the loading rate is called the ''force spectrum'' and forms the basic dataset for ''dynamic force spectroscopy''.<ref>{{cite book | vauthors = Bhushan B  |title=Springer handbook of nanotechnology |date=2017 |isbn=9783662543573 |oclc=1012104482 }}{{page needed|date=October 2019}}</ref><ref name=":1">{{cite book | vauthors = Hinterdorfer P, Van Oijen A |title=Handbook of single-molecule biophysics |date=2009 |publisher=Springer |isbn=9780387764979 |oclc=534951120 }}{{page needed|date=October 2019}}</ref>

In the ideal case of a single sharp energy barrier for the tip-sample interactions the dynamic force spectrum will show a linear increase of the rupture force as function of a logarithm of the loading rate, as described by a model proposed by Bell et al.<ref>{{cite journal | vauthors = Bell GI | title = Models for the specific adhesion of cells to cells | journal = Science | volume = 200 | issue = 4342 | pages = 618–627 | date = May 1978 | pmid = 347575 | doi = 10.1126/science.347575 | bibcode = 1978Sci...200..618B }}</ref>  Here, the slope of the rupture force spectrum is equal to the <math>\frac{k_BT}{x_\beta}</math>, where <math>x_\beta</math> is the distance from the energy minimum to the [[transition state]]. So far, a number of theoretical models exist describing the relationship between loading rate and rupture force, based upon different assumptions and predicting distinct curve shapes.<ref name=":1" /><ref>{{cite journal | vauthors = Petrosyan R | year = 2020 | title = Unfolding force definition and the unified model for the mean unfolding force dependence on the loading rate | journal = J. Stat. Mech. | volume = 2020 | number = 33201 | page = 033201 | doi = 10.1088/1742-5468/ab6a05|arxiv=1904.03925 | bibcode = 2020JSMTE..03.3201P | doi-access = free }}</ref>

For example, Ma X.,Gosai A. et al., utilized dynamic force spectroscopy along with molecular dynamics simulations to find out the binding force between thrombin, a blood coagulation protein, and its DNA aptamer.<ref>{{cite journal | vauthors = Ma X, Gosai A, Balasubramanian G, Shrotriya P |title=Force spectroscopy of the thrombin-aptamer interaction: Comparison between AFM experiments and molecular dynamics simulations |journal=Applied Surface Science |date=May 2019 |volume=475 |pages=462–472 |doi=10.1016/j.apsusc.2019.01.004 |bibcode=2019ApSS..475..462M |s2cid=104310868 }}</ref>

=== Acoustic force spectroscopy ===
A recently developed technique, acoustic force spectroscopy (AFS), allows the force manipulation of hundreds of single-molecules and single-cells in parallel, providing high experimental throughput.<ref name=":0" /> In this technique, a piezo element resonantly excites planar acoustic waves over a microfluidic chip. The generated acoustic waves are capable of exerting forces on microspheres with different density than the surrounding medium. Biomolecules, such as DNA, RNA or proteins, can be individually tethered between the microspheres and a surface and then probed by the acoustic forces exerted by the piezo sensor. With AFS devices it is possible to apply forces ranging from 0 to several hundreds of picoNewtons on hundreds of microspheres and obtain force-extension curves or histograms of rupture forces of many individual events in parallel.

This technique is mostly utilized to study DNA-bindings protein. For example, AFS was used to examine bacterial transcription with presence of antibacterial agents.<ref>{{cite journal | vauthors = Metelev M, Arseniev A, Bushin LB, Kuznedelov K, Artamonova TO, Kondratenko R, Khodorkovskii M, Seyedsayamdost MR, Severinov K | display-authors = 6 | title = Acinetodin and Klebsidin, RNA Polymerase Targeting Lasso Peptides Produced by Human Isolates of Acinetobacter gyllenbergii and Klebsiella pneumoniae | journal = ACS Chemical Biology | volume = 12 | issue = 3 | pages = 814–824 | date = March 2017 | pmid = 28106375 | doi = 10.1021/acschembio.6b01154 }}</ref> Viral proteins also can be studied by AFS, for instance this technique was used to explore DNA compaction along with other single-molecule approaches.<ref>{{cite journal | vauthors = Marchetti M, Kamsma D, Cazares Vargas E, Hernandez García A, van der Schoot P, de Vries R, Wuite GJ, Roos WH | display-authors = 6 | title = Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level | journal = Nano Letters | volume = 19 | issue = 8 | pages = 5746–5753 | date = August 2019 | pmid = 31368710 | pmc = 6696885 | doi = 10.1021/acs.nanolett.9b02376 }}</ref>

Cells also can be manipulated by the acoustic forces directly, or by using microspheres as handles.<ref>{{cite journal | vauthors = Sorkin R, Bergamaschi G, Kamsma D, Brand G, Dekel E, Ofir-Birin Y, Rudik A, Gironella M, Ritort F, Regev-Rudzki N, Roos WH, Wuite GJ | display-authors = 6 | title = Probing cellular mechanics with acoustic force spectroscopy | journal = Molecular Biology of the Cell | volume = 29 | issue = 16 | pages = 2005–2011 | date = August 2018 | pmid = 29927358 | pmc = 6232971 | doi = 10.1091/mbc.E18-03-0154 }}</ref> 

===Optical tweezers===
Another technique that has been gaining ground for single molecule experiments is the use of [[optical tweezers]] for applying mechanical forces on molecules. A strongly focused [[laser]] beam has the ability to catch and hold particles (of dielectric material) in a size range from nanometers to micrometers. The trapping action of optical tweezers results from the dipole or optical gradient force on the dielectric sphere. The technique of using a focused laser beam as an atom trap was first applied in 1984 at Bell laboratories. Until then experiments had been carried out using oppositely directed lasers as a means to trap particles. Later experiments, at the same project at Bell laboratories and others since, showed damage-free manipulation on cells using an infrared laser. Thus, the ground was made for biological experiments with optical trapping.

Each technique has its own advantages and disadvantages. For example, AFM cantilevers, can measure angstrom-scale, millisecond events and forces larger than 10 pN. While glass microfibers cannot achieve such fine spatial and temporal resolution, they can measure piconewton forces. Optical tweezers allow the measurement of piconewton forces and nanometer displacements which is an ideal range for many biological experiments. Magnetic tweezers can measure femtonewton forces, and additionally they can also be used to apply torsion. AFS devices allow the statistical analysis of the mechanical properties of biological systems by applying picoNewton forces to hundreds of individual particles in parallel, with sub-millisecond response time.