Radioactivity in the life sciences
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 accelerators 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.
Examples of radionuclei
- Tritium (hydrogen-3) is a very low energy emitter that can be used to label proteins, nucleic acids, 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 Ci/mmol (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-14 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 dating or drug tests.
- Sodium-22 and chlorine-36 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.[1]
- Sulfur-35 is used to label proteins and nucleic acids. Cysteine is an amino acid containing a thiol group which can be labeled by S-35. For nucleotides that do not contain a sulfur group, the oxygen on one of the phosphate groups can be substituted with a sulfur. This thiophosphate acts the same as a normal phosphate group, although there is a slight bias against it by most polymerases. 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 neutron, while only some will have acquired two or more. Its maximum specific activity is 5,118 Ci/mmol (189.4 PBq/mol).
- Phosphorus-32 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 glass). The half-life of 32P is 14.2 days, and its maximum specific activity is 9131 Ci/mmol.
- Iodine-125 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.
Detection
Quantitative
- In a liquid scintillation assay (LSA), or liquid scintillation counting (LSC), a small aliquot, filter or swab is added to scintillation fluid and the plate or vial counter in a scintillation counter.
- A Geiger counter 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 blot 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 PMT. When two different radiolabels are used, a computer can be used to discriminate the two.
Scientific methods
- Schild regression 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 ATP, from the catalogues of the two major suppliers, Perkin Elmer NEG502H500UC or GE AA0068-500UCI , 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 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 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 (tritium 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. ).
- Several properties are extremely useful (cf. next section)
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:
- the dye may be a hindrance or toxic
Safety
If good health physics 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[2] ).
See also
References
- Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter (2007). Molecular Biology of the Cell, Fifth Edition, Garland Science, 1268 pages. ISBN 0-8153-4105-9.