Scintillation proximity assay
Encyclopedia
Scintillation proximity assay, commonly known as SPA, is a type of technology that is used to assay development and biochemical screening which permit the rapid and sensitive measurement of a broad range of biological processes in a homogeneous system.
The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant
which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead. This interaction will trigger the bead to emit light, which can be detected using a photometer
.
, which releases light photon
s which can be detected via the use of some devices such as the photomultiplier tubes of scintillation counters or CCD imagers
. This is a very popular technique in practices that require detecting and quantifying radioactivity.
The process of converting radioactivity to light requires a liquid medium of scintillation combination consisting soluble organic
scintillators and organic solvents
. During the process of radioactive decay, a beta particle
will be released. While this particle travels in the medium, the energy it possesses is dissipated as it collides with the surrounding molecule
s in the solvent, exciting them while doing so. The excited molecules will transfer the energy they now possess to the scintillator molecules, where the energy will be emitted as light.
molecule is attached or is in proximity to bead, light emission is stimulated. However, if the bead does not become bound to the radio-labelled molecule, the bead will not be stimulated to emit light. This is because the energy released from the unbound molecule is not strong enough to excite the SPA bead which is not then stimulated to produce a signal.
The decay of radioactive atom releases electron
s as one of the subatomic particle
s. The energy of these particles influences the distance travelled by the particles itself through a medium such as water. This influence of the energy or the limitation of the water is what this SPA method is dependent upon.
For instance, the decay of a Tritium
atom releases a beta particle. Tritium is highly recommended as it suits SPA very well. It is due to the 1.5 µm path length
through water, which is very short. So, when the ß-particle is within that particular range of 1.5 µm with the scintillant bead, there is sufficient energy to stimulate the bead to emit light. If the distance between them is greater than 1.5 µm, then the ß-particle is incapable of travelling the required distance to stimulate the bead as it has insufficient energy.
The beads in SPA are formed from the incorporation of scintillant into small beads known as fluomicrospheres. These are specially designed to bind with specific molecules. When the bead is in close proximity to the radioactive molecule, light is stimulated.
The photonmultiplier tube (PMT) can be used to detect the emitted photons. This device is converts the emitted photon energy into electrical energy by a photocathode
via a series of other electrode
s. Another device is known as CCD Imager
, which is composed of a set of cooled digital camera
s with sensitive charge coupled device detectors and with some refined telecentric lens
es to convert the captured photon energy into high quality images.
There is also an assortment of bead coatings available that allows this method to be applied to a broad range of applications, such as enzyme
assays and radio-immuno ssay]s.
The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant
Scintillation (physics)
Scintillation is a flash of light produced in a transparent material by an ionization event. See scintillator and scintillation counter for practical applications.-Overview:...
which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead. This interaction will trigger the bead to emit light, which can be detected using a photometer
Photometer
In its widest sense, a photometer is an instrument for measuring light intensity or optical properties of solutions or surfaces. Photometers are used to measure:*Illuminance*Irradiance*Light absorption*Scattering of light*Reflection of light*Fluorescence...
.
Overview
The SPA technique is dependent on the energy conversion of radioactive decayRadioactive decay
Radioactive decay is the process by which an atomic nucleus of an unstable atom loses energy by emitting ionizing particles . The emission is spontaneous, in that the atom decays without any physical interaction with another particle from outside the atom...
, which releases light photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...
s which can be detected via the use of some devices such as the photomultiplier tubes of scintillation counters or CCD imagers
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...
. This is a very popular technique in practices that require detecting and quantifying radioactivity.
The process of converting radioactivity to light requires a liquid medium of scintillation combination consisting soluble organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
scintillators and organic solvents
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
. During the process of radioactive decay, a beta particle
Beta particle
Beta particles are high-energy, high-speed electrons or positrons emitted by certain types of radioactive nuclei such as potassium-40. The beta particles emitted are a form of ionizing radiation also known as beta rays. The production of beta particles is termed beta decay...
will be released. While this particle travels in the medium, the energy it possesses is dissipated as it collides with the surrounding 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 in the solvent, exciting them while doing so. The excited molecules will transfer the energy they now possess to the scintillator molecules, where the energy will be emitted as light.
Detail
In more detail, when the radio-labelledIsotopic labeling
Isotopic labeling is a technique for tracking the passage of a sample of substance through a system. The substance is 'labeled' by including unusual isotopes in its chemical composition...
molecule is attached or is in proximity to bead, light emission is stimulated. However, if the bead does not become bound to the radio-labelled molecule, the bead will not be stimulated to emit light. This is because the energy released from the unbound molecule is not strong enough to excite the SPA bead which is not then stimulated to produce a signal.
The decay of radioactive atom releases electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...
s as one of the subatomic particle
Subatomic particle
In physics or chemistry, subatomic particles are the smaller particles composing nucleons and atoms. There are two types of subatomic particles: elementary particles, which are not made of other particles, and composite particles...
s. The energy of these particles influences the distance travelled by the particles itself through a medium such as water. This influence of the energy or the limitation of the water is what this SPA method is dependent upon.
For instance, the decay of a Tritium
Tritium
Tritium is a radioactive isotope of hydrogen. The nucleus of tritium contains one proton and two neutrons, whereas the nucleus of protium contains one proton and no neutrons...
atom releases a beta particle. Tritium is highly recommended as it suits SPA very well. It is due to the 1.5 µm path length
Path length
In chemistry, the path length is defined as the distance that light travels through a sample in an analytical cell. Typically, a sample cell is made of quartz, glass, or a plastic rhombic cuvette with a volume typically ranging from 0.1 mL to 10 mL or larger used in a spectrophotometer. For the...
through water, which is very short. So, when the ß-particle is within that particular range of 1.5 µm with the scintillant bead, there is sufficient energy to stimulate the bead to emit light. If the distance between them is greater than 1.5 µm, then the ß-particle is incapable of travelling the required distance to stimulate the bead as it has insufficient energy.
The beads in SPA are formed from the incorporation of scintillant into small beads known as fluomicrospheres. These are specially designed to bind with specific molecules. When the bead is in close proximity to the radioactive molecule, light is stimulated.
The photonmultiplier tube (PMT) can be used to detect the emitted photons. This device is converts the emitted photon energy into electrical energy by a photocathode
Photocathode
A photocathode is a negatively charged electrode in a light detection device such as a photomultiplier or phototube that is coated with a photosensitive compound...
via a series of other electrode
Electrode
An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit...
s. Another device is known as CCD Imager
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...
, which is composed of a set of cooled digital camera
Digital camera
A digital camera is a camera that takes video or still photographs, or both, digitally by recording images via an electronic image sensor. It is the main device used in the field of digital photography...
s with sensitive charge coupled device detectors and with some refined telecentric lens
Telecentric lens
A telecentric lens is a compound lens which has its entrance or exit pupil at infinity; in the prior case, this produces an orthographic view of the subject. This means that the chief rays are parallel to the optical axis in front of or behind the system, respectively...
es to convert the captured photon energy into high quality images.
There is also an assortment of bead coatings available that allows this method to be applied to a broad range of applications, such as enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
assays and radio-immuno ssay]s.
Advantages over previous methods
In comparison to the previous over-coated plate-based methods, SPA has a number of advantages that makes it more popular:- Assay flexibility - the concentration of the components in the assay can be adjusted to suit the user due to the higher surface area design of the SPA beads, hence providing the user flexibility in designing assay.
- Radioactive labelRadioactive tracerA radioactive tracer, also called a radioactive label, is a substance containing a radioisotope that is used to measure the speed of chemical processes and to track the movement of a substance through a natural system such as a cell or tissue...
reduction - the SPA beads allows a potential reduction in the quantity of radioactive labeling required due to its higher binding capacity, which gives a higher signal. This method also allows the user to optimize the sensitivity of the assay by altering the quantity of SPA beads.
- Convenient - the key component of the SPA assay, the beads, do not depend on a particular plate type or supplier, hence its wide availability.
- Bead assortment - there is a diversity of bead types to choose from to suit the need of the user and application.
- No separation step - allows binding measurement without separation step associated with earlier methods, which reduces errors and allows higher throughput.