Force spectroscopy is a set of techniques for the study of the interactions and the binding forces between individual molecules.
These methods can be used to measure the mechanical properties of single
polymer
A polymer (; Greek '' poly-'', "many" + ''-mer'', "part")
is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...
molecule
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioche ...
s or
proteins, or individual
chemical bond
A chemical bond is a lasting attraction between atoms or ions that enables the formation of molecules and crystals. The bond may result from the electrostatic force between oppositely charged ions as in ionic bonds, or through the sharing of ...
s. 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
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
, 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
Ensemble may refer to:
Art
* Architectural ensemble
* Ensemble (album), ''Ensemble'' (album), Kendji Girac 2015 album
* Ensemble (band), a project of Olivier Alary
* Ensemble cast (drama, comedy)
* Ensemble (musical theatre), also known as the ...
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.
Experimental techniques
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 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
adsorbed
Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
on a
surface
A surface, as the term is most generally used, is the outermost or uppermost layer of a physical object or space. It is the portion or region of the object that can first be perceived by an observer using the senses of sight and touch, and is ...
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
Piezoelectricity (, ) is the electric charge that accumulates in certain solid materials—such as crystals, certain ceramics, and biological matter such as bone, DNA, and various proteins—in response to applied mechanical stress. The word '' ...
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
In physics, Hooke's law is an empirical law which states that the force () needed to extend or compress a spring by some distance () scales linearly with respect to that distance—that is, where is a constant factor characteristic of ...
, this deflection will be proportional to the force acting on the cantilever. Deflection is measured by the position of a
laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fi ...
beam reflected by the cantilever. This kind of set-up can measure forces as low as 10 pN (10
−11 N), the fundamental resolution limit is given by the cantilever's thermal
noise
Noise is unwanted sound considered unpleasant, loud or disruptive to hearing. From a physics standpoint, there is no distinction between noise and desired sound, as both are vibrations through a medium, such as air or water. The difference aris ...
.
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(s) may refer to:
Common uses
* Spring (season)
Spring, also known as springtime, is one of the four temperate seasons, succeeding winter and preceding summer. There are various technical definitions of spring, but local usage of ...
, 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
The worm-like chain (WLC) model in polymer physics is used to describe the behavior of polymers that are semi-flexible: fairly stiff with successive segments pointing in roughly the same direction, and with persistence length within a few orders o ...
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''.
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. Here, the slope of the rupture force spectrum is equal to the
, where
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.
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.
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.
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. Cells can be manipulated by the acoustic forces directly, or by using microspheres as handles. 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.
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
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fi ...
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.
Applications
Common applications of force spectroscopy are measurements of
polymer
A polymer (; Greek '' poly-'', "many" + ''-mer'', "part")
is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...
elasticity, especially biopolymers such as
RNA and
DNA. Another
biophysical
Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. Bi ...
application of polymer force spectroscopy is on
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
unfolding. Modular proteins can be adsorbed to a
gold
Gold is a chemical element with the symbol Au (from la, aurum) and atomic number 79. This makes it one of the higher atomic number elements that occur naturally. It is a bright, slightly orange-yellow, dense, soft, malleable, and ductile me ...
or (more rarely)
mica surface
A surface, as the term is most generally used, is the outermost or uppermost layer of a physical object or space. It is the portion or region of the object that can first be perceived by an observer using the senses of sight and touch, and is ...
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). Much information about protein elasticity and protein unfolding can be obtained by this technique. Many proteins in the living
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
must face mechanical stress.
Moreover, force spectroscopy can be used to investigate the enzymatic activity of proteins involved in
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
,
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
, organization and
repair
The technical meaning of maintenance involves functional checks, servicing, repairing or replacing of necessary devices, equipment, machinery, building infrastructure, and supporting utilities in industrial, business, and residential installa ...
. This is achieved by measuring the position of a bead attached to a DNA-protein complex stalled on a DNA tether that has one end attached to a surface, while keeping the force constant. This technique has been used, for example, to study transcription elongation inhibition by Klebsidin and Acinetodin.
The other main application of force spectroscopy is the study of
mechanical resistance
Mechanical impedance is a measure of how much a structure resists motion when subjected to a harmonic force. It relates forces with velocities acting on a mechanical system. The mechanical impedance of a point on a structure is the ratio of the for ...
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
In physics, energy (from Ancient Greek: ἐνέργεια, ''enérgeia'', “activity”) is the quantitative property that is transferred to a body or to a physical system, recognizable in the performance of work and in the form of hea ...
landscape of the chemical bond under mechanical force. This is leading to interesting results in the study of
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
-
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
, protein-protein, protein-living cell interaction and
catch bonds.
Recently this technique has been used in
cell biology to measure the aggregative
stochastic forces created by
motor proteins
Motor proteins are a class of molecular motors that can move along the cytoplasm of cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. Flagellar rotation, however, is powered by a proton pump.
Cellular function ...
that influence the motion of particles within the cytoplasm. In this way, force spectrum microscopy may be used better to understand the many cellular processes that require the motion of particles within cytoplasm.
References
Bibliography
*
*
*
*
*Smith, S.; Cui, Y.; Bustamante, C. Science (Washington, D.C.) 1996; 271:795.
*
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{{Branches of Spectroscopy
Spectroscopy
Scanning probe microscopy