Radioactivity in biological research
Encyclopedia
Radioactivity can be used in life sciences as a radiolabel to visualise components or target molecules in a biological system. Some radionuclei are synthesised in particle accelerator
s and have short half-lives
, giving them high maximum theoretical specific activities. This lowers the detection time compared to radionuclei with longer half-lives, such as carbon-14. In some applications they have been substituted by fluorescent dyes.
A good example of the difference in energy of the various radionuclei is the detection window ranges used to detect them, which are generally proportional to the energy of the emission, but vary from machine to machine: in a Perkin elmer TriLux Beta scintillation counter , the H-3 energy range window is between channel 5–360; C-14, S-35 and P-33 are in the window of 361–660; and P-32 is in the window of 661–1024.
, from the catalogues of the two major suppliers, Perkin Elmer NEG502H500UC http://las.perkinelmer.com/Catalog/ProductInfoPage.htm?ProductID=NEG502H500UC or GE AA0068-500UCI http://www6.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=25001746&moduleid=42364, in this case, the total activity is 500 μCi (other typical numbers are 250 μCi or 1 mCi). This is contained in a certain volume, depending on the radioactive concentration, such as 5 to 10 mCi/mL (185 to 370 TBq/m3); typical volumes include 50 or 25 μL.
Not all molecules in the solution have a P-32 on the last (i.e., gamma) phosphate: the "specific activity" gives the radioactivity concentration and depends on the radionuclei's half-life. If every molecule were labelled, the maximum theoretical specific activity is obtained that for P-32 is 9131 Ci/mmol. Due to pre-calibration and efficiency issues this number is never seen on a label; the values often found are 800, 3000 and 6000 Ci/mmol. With this number it is possible to calculate the total chemical concentration and the hot-to-cold ratio.
"Calibration date" is the date in which the vial’s activity is the same as on the label. "Pre-calibration" is the when the activity is calibrated in a future date to compensate for the decay occurred during shipping.
in the past three decades radioactivity was the most common label.
Advantages are:
Note: a channel
is similar to "colour" but distinct, it is the pair of excitation and emission filters specific for a dye, e.g. agilent microarrays are dual channel, working on cy3 and cy5, these are colloquially referred to as green and red.
Disadvantages are:
controls are maintained in a laboratory where radionuclides are used, it is unlikely that the overall radiation dose received by workers will be of much significance. Nevertheless the effects of low doses are mostly unknown so many regulations exist to avoid unnecessary risks, such as skin or internal exposure. Due to the low penetration power and many variables involved it is hard to convert a radioactive concentration to a dose.
1 μCi of P-32 on a square centimetre of skin (through a dead layer of a thickness of 70 μm) gives 7961 rads
(79.61 grays
) per hour . Similarly a mammogram gives an exposure of 300 mrem
(3 mSv) on a larger volume (in the US, the average annual dose is 620 mrem or 6.2 mSv ).
Particle accelerator
A particle accelerator is a device that uses electromagnetic fields to propel charged particles to high speeds and to contain them in well-defined beams. An ordinary CRT television set is a simple form of accelerator. There are two basic types: electrostatic and oscillating field accelerators.In...
s and have short half-lives
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...
, giving them high maximum theoretical specific activities. This lowers the detection time compared to radionuclei with longer half-lives, such as carbon-14. In some applications they have been substituted by fluorescent dyes.
Examples of radionuclei
- TritiumTritiumTritium 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...
(hydrogen-3) is a very low energy emitter that can be used to label proteinProteinProteins 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...
s, nucleic acidNucleic acidNucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s, drugs and toxins, but requires a tritium-specific film or a tritium-specific phosphor screen. In a liquid scintillation assay (LSA), the efficiency is 20–50%, depending on the scintillation cocktail used . The maximum theoretical specific activity of tritium is 28.8 CiCurieThe curie is a unit of radioactivity, defined asThis is roughly the activity of 1 gram of the radium isotope 226Ra, a substance studied by the pioneers of radiology, Marie and Pierre Curie, for whom the unit was named. In addition to the curie, activity can be measured using an SI derived unit,...
/mmolMole (unit)The mole is a unit of measurement used in chemistry to express amounts of a chemical substance, defined as an amount of a substance that contains as many elementary entities as there are atoms in 12 grams of pure carbon-12 , the isotope of carbon with atomic weight 12. This corresponds to a value...
(1.066 PBq/mol). However, there is often more than one tritium atom per molecule: for example, tritiated UTP is sold by most suppliers with carbons 5 and 6 each bonded to a tritium atom. C-14, S-35 and P-33 have similar emission energies. P-32 and I-125 are higher energy emitters -> inaccurate, see beta vs gamma radiation.
- Carbon-14Carbon-14Carbon-14, 14C, or radiocarbon, is a radioactive isotope of carbon with a nucleus containing 6 protons and 8 neutrons. Its presence in organic materials is the basis of the radiocarbon dating method pioneered by Willard Libby and colleagues , to date archaeological, geological, and hydrogeological...
has a long half-life of 5,730±40 years. Its maximum specific activity is 0.0624 Ci/mmol (2.31 TBq/mol). It is used in applications such as radiometric datingRadiometric datingRadiometric dating is a technique used to date materials such as rocks, usually based on a comparison between the observed abundance of a naturally occurring radioactive isotope and its decay products, using known decay rates...
or drug tests.
- Sodium-22 and chlorine-36Chlorine-36Chlorine-36 is an isotope of chlorine. Chlorine has two stable isotopes and one radioactive environmental isotope: the cosmogenic isotope 36Cl. The ratio of 36Cl to stable 37Cl in the environment is ~700 × 10−15. Its half-life is 301,000 ± 4,000 years. The long half-life of 36Cl makes it...
are commonly used to study ion transporters. However, sodium-22 is hard to screen off and chlorine-36, with a half-life of 300,000 years, has low activity.
- Sulfur-35 is used to label proteins and nucleic acids. CysteineCysteineCysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
is an amino acidAmino acidAmino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
containing a thiol group which can be labeled by S-35. For nucleotideNucleotideNucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s that do not contain a sulfur group, the oxygen on one of the phosphate groups can be substituted with a sulfur. This thiophosphateThiophosphateA thiophosphate is a family of compounds and anions with the general chemical formula PS4-xOx3- . The state of protonation is usually not specified. They could be bound to as many as three protons for the neutral H3PS4-xOx species. Two protons correspond to the related monoanions, and one...
acts the same as a normal phosphate group, although there is a slight bias against it by most polymerasePolymeraseA polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
s. The maximum theoretical specific activity is 1,494 Ci/mmol (55.28 PBq/mol).
- Phosphorus-33 is used to label nucleotides. It is less energetic than P-32 and does not require protection with plexi glass. A disadvantage is its higher cost compared to P-32, as most of the bombarded P-31 will have acquired only one neutronNeutronThe neutron is a subatomic hadron particle which has the symbol or , no net electric charge and a mass slightly larger than that of a proton. With the exception of hydrogen, nuclei of atoms consist of protons and neutrons, which are therefore collectively referred to as nucleons. The number of...
, while only some will have acquired two or more. Its maximum specific activity is 5,118 Ci/mmol (189.4 PBq/mol).
- Phosphorus-32Phosphorus-32Phosphorus-32 is a radioactive isotope of phosphorus. The nucleus of phosphorus-32 contains 15 protons and 17 neutrons, one more neutron than the most common isotope of phosphorus, phosphorus-31...
is widely used for labeling nucleic acids and phosphoproteins. It has the highest emission energy (1.7 MeV) of all common research radioisotopes. This is a major advantage in experiments for which sensitivity is a primary consideration, such as titrations of very strong interactions (i.e., very low dissociation constant), footprinting experiments, and detection of low-abundance phosphorylated species. 32P is also relatively inexpensive. Because of its high energy, however, a number of safety and administrative controls are required (e.g., acrylic glassAcrylic glassPoly is a transparent thermoplastic, often used as a light or shatter-resistant alternative to glass. It is sometimes called acrylic glass. Chemically, it is the synthetic polymer of methyl methacrylate...
). The half-life of 32P is 14.2 days, and its maximum specific activity is 9131 Ci/mmol.
- Iodine-125Iodine-125Iodine-125 is a radioisotope of iodine which has uses in biological assays, nuclear medicine imaging and in radiation therapy as brachytherapy to treat prostate cancer and brain tumors. It is the second longest-lived radioisotope of iodine, after iodine-129.Its half-life is around 59 days and it...
is commonly used for labeling proteins, usually at tyrosine residues. Unbound iodine is volatile and must be handled in a fume hood. Its maximum specific activity is 2,176 Ci/mmol (80.51 PBq/mol).
A good example of the difference in energy of the various radionuclei is the detection window ranges used to detect them, which are generally proportional to the energy of the emission, but vary from machine to machine: in a Perkin elmer TriLux Beta scintillation counter , the H-3 energy range window is between channel 5–360; C-14, S-35 and P-33 are in the window of 361–660; and P-32 is in the window of 661–1024.
Quantitative
- In a liquid scintillation assay (LSA), or liquid scintillation countingLiquid scintillation countingLiquid scintillation counting is a standard laboratory method in the life-sciences for measuring radiation from beta-emitting nuclides. Scintillating materials are also used in differently constructed "counters" in many other fields....
(LSC), a small aliquot, filter or swab is added to scintillation fluid and the plate or vial counter in a scintillation counterScintillation counterA scintillation counter measures ionizing radiation. The sensor, called a scintillator, consists of a transparent crystal, usually phosphor, plastic , or organic liquid that fluoresces when struck by ionizing radiation. A sensitive photomultiplier tube measures the light from the crystal...
. - A Geiger counterGeiger counterA Geiger counter, also called a Geiger–Müller counter, is a type of particle detector that measures ionizing radiation. They detect the emission of nuclear radiation: alpha particles, beta particles or gamma rays. A Geiger counter detects radiation by ionization produced in a low-pressure gas in a...
is a quick and rough approximation of activity. Lower energy emitters such as tritium can not be detected.
Qualitative
- Autoradiography: A membrane such as a Northern blotNorthern blotThe northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
or a hybridised slot blot is put against a film that is then developed. - Phosphor storage screen: The membrane is placed against a phosphor storage screen which is then scanned in a phosphorimager. This is ten times faster and more precise than film and the result is already in digital form.
Microscopy
- Electron microscopy: The sample is not exposed to a beam of electrons but detectors picks up the expelled electrons from the radionuclei.
- Micro-autoradiography imager: A slide is put against scintillation paper and in a PMTPMTPMT may refer to:* Parent Management Training* Photomultiplier tube, an extremely sensitive light detector* Program Map Table, program specific information in an MPEG transport stream* Place–manner–time*Post-glacial marine transgression*Pearl milk tea...
. When two different radiolabels are used, a computer can be used to discriminate the two.
Scientific methods
- Schild regressionSchild regressionSchild regression analysis, named for Heinz Otto Schild, is a useful tool for studying the effects of agonists and antagonists on the cellular response caused by the receptor or on ligand-receptor binding....
is a radioligand binding assay. It is used for DNA labelling (5' and 3'), leaving the nucleic acids intact.
Radioactivity concentration
A vial of radiolabel has a "total activity". Taking as an example γ32P ATPAdenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
, from the catalogues of the two major suppliers, Perkin Elmer NEG502H500UC http://las.perkinelmer.com/Catalog/ProductInfoPage.htm?ProductID=NEG502H500UC or GE AA0068-500UCI http://www6.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=25001746&moduleid=42364, in this case, the total activity is 500 μCi (other typical numbers are 250 μCi or 1 mCi). This is contained in a certain volume, depending on the radioactive concentration, such as 5 to 10 mCi/mL (185 to 370 TBq/m3); typical volumes include 50 or 25 μL.
Not all molecules in the solution have a P-32 on the last (i.e., gamma) phosphate: the "specific activity" gives the radioactivity concentration and depends on the radionuclei's half-life. If every molecule were labelled, the maximum theoretical specific activity is obtained that for P-32 is 9131 Ci/mmol. Due to pre-calibration and efficiency issues this number is never seen on a label; the values often found are 800, 3000 and 6000 Ci/mmol. With this number it is possible to calculate the total chemical concentration and the hot-to-cold ratio.
"Calibration date" is the date in which the vial’s activity is the same as on the label. "Pre-calibration" is the when the activity is calibrated in a future date to compensate for the decay occurred during shipping.
Comparison with fluorescence
Prior to the widespread use of fluorescenceFluorescence in the life sciences
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence....
in the past three decades radioactivity was the most common label.
Advantages are:
- fluorescence is much safer and more convenient to use
- Several fluorescent molecules can be used simultaneously (given that they do not overlap, cf. FRET), whereas with radioactivity two isotopes can be used (tritiumTritiumTritium 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...
and a low energy isotope, e.g. 33P due to different intensities) but require special machinery (a tritium screen and a regular phosphor-imaging screen or a specific dual channel detector, e.g. http://www.biospacelab.com/html/microimager.html). - Several properties are extremely useful (cf. next section)
Note: a channel
Channel (digital image)
Color digital images are made of pixels, and pixels are made of combinations of primary colors. A channel in this context is the grayscale image of the same size as a color image, made of just one of these primary colors. For instance, an image from a standard digital camera will have a red, green...
is similar to "colour" but distinct, it is the pair of excitation and emission filters specific for a dye, e.g. agilent microarrays are dual channel, working on cy3 and cy5, these are colloquially referred to as green and red.
Disadvantages are:
- the dye may be a hindrance or toxic
Safety
If good health physicsHealth physics
Health physics is a field of science concerned with radiation physics and radiation biology with the goal of providing technical information and proper techniques regarding the safe use of ionizing radiation...
controls are maintained in a laboratory where radionuclides are used, it is unlikely that the overall radiation dose received by workers will be of much significance. Nevertheless the effects of low doses are mostly unknown so many regulations exist to avoid unnecessary risks, such as skin or internal exposure. Due to the low penetration power and many variables involved it is hard to convert a radioactive concentration to a dose.
1 μCi of P-32 on a square centimetre of skin (through a dead layer of a thickness of 70 μm) gives 7961 rads
Rad (unit)
The rad is a unit of absorbed radiation dose. The rad was first proposed in 1918 as "that quantity of X rays which when absorbed will cause the destruction of the malignant mammalian cells in question..." It was defined in CGS units in 1953 as the dose causing 100 ergs of energy to be absorbed by...
(79.61 grays
Gray (unit)
The gray is the SI unit of absorbed radiation dose of ionizing radiation , and is defined as the absorption of one joule of ionizing radiation by one kilogram of matter ....
) per hour . Similarly a mammogram gives an exposure of 300 mrem
Röntgen equivalent man
Named after Wilhelm Röntgen , the roentgen equivalent in man or rem is a unit of radiation dose equivalent...
(3 mSv) on a larger volume (in the US, the average annual dose is 620 mrem or 6.2 mSv ).