Force spectroscopy
Encyclopedia
Force spectroscopy is a dynamic analytical technique that allows the study of the mechanical properties of single polymer
molecule
s or proteins, or individual chemical bond
s. It is performed by pulling on the system under scrutiny with controlled forces. 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.
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 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), and so on.
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 (10−11 N), 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.
Quite often researchers repeat the measurements 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 the dynamic force spectroscopy. 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. The slope of the line is equal to the
, where 
is the distance from the energy minimum to the transition state.
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.
elasticity
, especially biopolymers such as RNA
and DNA
. Another 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 many 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.
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
s or proteins, or individual chemical bond
Chemical bond
A chemical bond is an attraction between atoms that allows the formation of chemical substances that contain two or more atoms. The bond is caused by the electromagnetic force attraction between opposite charges, either between electrons and nuclei, or as the result of a dipole attraction...
s. It is performed by pulling on the system under scrutiny with controlled forces. As a single-molecule
Single-molecule
A single-molecule experiment investigates the properties of an individual molecule that can be distinguished for the purpose of an experiment or analysis. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior can...
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.
The name "force spectroscopy
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...
", although widely used in the scientific community, is somewhat misleading, because there is no true matter-radiation interaction. Force spectroscopy measures the behavior of a molecule under stretching or torsional mechanical force
Force
In physics, a force is any influence that causes an object to undergo a change in speed, a change in direction, or a change in shape. In other words, a force is that which can cause an object with mass to change its velocity , i.e., to accelerate, or which can cause a flexible object to deform...
. In this way a great deal has been learned in recent years about the mechanochemical coupling in the enzymes responsible for muscle
Muscle
Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...
contraction, transport in the cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
, energy generation (F1-ATPase), DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
replication and transcription (polymerases), DNA unknotting and unwinding (topoisomerases and helicases), and so on.
Experimental techniques
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.Atomic force microscope cantilevers
Molecules adsorbedAdsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
on a surface
Surface
In mathematics, specifically in topology, a surface is a two-dimensional topological manifold. The most familiar examples are those that arise as the boundaries of solid objects in ordinary three-dimensional Euclidean space R3 — for example, the surface of a ball...
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
Hooke's law
In mechanics, and physics, Hooke's law of elasticity is an approximation that states that the extension of a spring is in direct proportion with the load applied to it. Many materials obey this law as long as the load does not exceed the material's elastic limit. Materials for which Hooke's law...
, this deflection will be proportional to the force acting on the cantilever. Deflection is measured by the position of a laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
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
Noise
In common use, the word noise means any unwanted sound. In both analog and digital electronics, noise is random unwanted perturbation to a wanted signal; it is called noise as a generalisation of the acoustic noise heard when listening to a weak radio transmission with significant electrical 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
Spring (device)
A spring is an elastic object used to store mechanical energy. Springs are usually made out of spring steel. Small springs can be wound from pre-hardened stock, while larger ones are made from annealed steel and hardened after fabrication...
, 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.
Quite often researchers repeat the measurements 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 the dynamic force spectroscopy. 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. The slope of the line is equal to the
, where is the distance from the energy minimum to the transition state.Optical tweezers
Another technique that has been gaining ground for single molecule experiments is the use of optical tweezersOptical tweezers
Optical tweezers are scientific instruments that use a highly focused laser beam to provide an attractive or repulsive force , depending on the refractive index mismatch to physically hold and move microscopic dielectric objects...
for applying mechanical forces on molecules. A strongly focused laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
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.
Applications
Common applications of force spectroscopy are measurements of polymerPolymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
elasticity
Elasticity (physics)
In physics, elasticity is the physical property of a material that returns to its original shape after the stress that made it deform or distort is removed. The relative amount of deformation is called the strain....
, especially biopolymers such as RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. Another exciting biophysical
Biophysics
Biophysics is an interdisciplinary science that uses the methods of physical science to study biological systems. Studies included under the branches of biophysics span all levels of biological organization, from the molecular scale to whole organisms and ecosystems...
application of polymer force spectroscopy is on protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
unfolding. Modular proteins can be adsorbed to a gold
Gold
Gold is a chemical element with the symbol Au and an atomic number of 79. Gold is a dense, soft, shiny, malleable and ductile metal. Pure gold has a bright yellow color and luster traditionally considered attractive, which it maintains without oxidizing in air or water. Chemically, gold is a...
or (more rarely) mica
Mica
The mica group of sheet silicate minerals includes several closely related materials having highly perfect basal cleavage. All are monoclinic, with a tendency towards pseudohexagonal crystals, and are similar in chemical composition...
surface
Surface
In mathematics, specifically in topology, a surface is a two-dimensional topological manifold. The most familiar examples are those that arise as the boundaries of solid objects in ordinary three-dimensional Euclidean space R3 — for example, the surface of a ball...
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 many proteins in the living cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
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
Stochastic process
In probability theory, a stochastic process , or sometimes random process, is the counterpart to a deterministic 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
Energy
In physics, energy is an indirectly observed quantity. It is often understood as the ability a physical system has to do work on other physical systems...
landscape of the chemical bond under mechanical force. This is leading to interesting results in the study of antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
-antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
, protein-protein and even protein-living cell interaction.