Radioactivity in biological research

Radioactivity can be used in life sciences as a radiolabel to easily visualise components or target molecules in a biological system. 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.

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Examples of radionuclei

• Tritium (Hydrogen-3) is a very low energy emitter 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/mmole. 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. Tritium waste disposal regulations depend on the institution: in some places it is acceptable to dispose of it down the drain. C-14, S-35 and P-33 have similar emission energies. P-32 and I-125 are higher energy emitters.

• Carbon-14 is not used very often as a radiolabel due to its long half-life. Its maximum specific activity is 0.0624 Ci/mmole. It is instead used in many other applications, such as radiometric dating or drug tests.

• Sulfur-35 is used to label proteins and nucleic acids. Cysteine is an amino acid with a thiol group. For nucleotides that do not contain a sulfur group, the oxygen on one of the phosphate groups can be substituted with a sulphur. 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 1494 Ci/mmole.

• Phosphorus-33 is used to label nucleotides. It is less energetic than P-32, giving a better resolution. 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 5118 Ci/mmole.

• Phosphorus-32 is a radionucleus of choice for nucleotides. Its high energy and low half-life result in lower autoradiography exposure times. Unfortunately its higher energy requires acrylic glass protection and a film badge. Its maximum specific activity is 9131 Ci/mmole.

• Iodine-125 is used for labelling tyroxine. Due to the fact that it can become airborne, where the iodine is absorbed, heavy precautions must be taken. Its maximum specific activity is 2176 Ci/mmole.

In a 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, 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. 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 [1] or GE AA0068-500UCI [2], 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 or 10 mCi/mL; 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/mmole. 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/mmole. 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.

Safety

Radionuclei used in a biology lab are extremely faint compared to well-known radioactive samples such as uranium. 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 rad per hour. Similarly a mammogram gives an exposure of 300 mrem on a larger volume (in the US, the average annual dose is 360 mrem).

References