Radioisotope Techniques

8/2/2019 Radioisotope Techniques

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Radioisotope Techniques

Mak Oi Tong

Department of Life SciencesNational Cheng Kung University


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Radioisotope

n Atoms with unstable nuclei transform

into other stable atoms.

n Results : Release of energetic particles

or radiant energy,


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Atoms, isotopes and radioisotopes

n Atomic number, mass (mass number or atomic weight)

n Carbon : 12C6n atomic number = 6 and atomic mass = 12

n Contains 6 protons, 6 neutrons and 6 electrons.

n Isotopes : atoms have identical chemical properties butdifferent atomic weight.

n e.g. 11C, 12C, 13C and 14C

n14C6 >

+ 14N7 +e- (antineutrino)

n11C6 >

+ + 11B5 +e (neutrino)

n Ratio of neutrons and protons

n Lighter atoms 1:1 and 1.5:1 at high atomic number


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Types of radioactive decay


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Negative-Beta (electron) emission

n Ejected electrons emit from the nucleus

which are identical with the orbital electron in

mass and charge (neutron to proton).n

3H1 >3He2 +

+e- (antineutrino)

n14C6 >

14N7 + +e- (antineutrino)

n

32

P15 >32

S16 +

+e- (antineutrino)n

35S16 >35Cl17 +

+e- (antineutrino)


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Positive-Beta (positron,+) emission

n + is same mass as electron but having opposite

charge, and it will emit as gamma (

) radiation.n + + e- > (1.02 MeV)

n11C6 >

11B5 ++ +e (neutrino)

n13N7 >

13C6 ++ +e (neutrino)


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Decay by electron capture

n Conversion of a nuclear proton into a neutron

by capture one of the two electrons in theinnermost K-shell, ie. K-capture (KC) and

emission of a positron+.


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Decay by-radiation

n -Ray emission occurs after or+

emission.

D b i l i i


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Decay by- particle emission

n Heavy atoms with more than 82 protons will

emit an-particle which is a helium nucleus

of atomic mass number of 4 (2 protons and 2

neutrons) with a charge of +2.

n226Ra88,

235U92 etc. are examples for-

particle emitters and are not for biochemical

studies but for radiological toxicity.


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radioactive isotopes

n Pierre and Marie Curie introduced the term

radioactivity to designate the process of spontaneousemission of radioisotope.

n Artificial production of radioisotopes is normallydone by neutron bombardment.

n Other methods: -particle, proton and deuterons


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n To-day, over 1500 isotopes are producedartificial produced.

n On earth, radioisotopes are produced byneutral bombardment, and 14C and 3H(tritium) are in equilibrium.

n Radioisotopes can be used for radiocarbon(14C) dating.


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Properties of radioactive emissions


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Energy of-particle emissions

n -

,+

and e-

travel near the speed of light, 3x 1010 cm/s or 3 x 108 m/s.

n Emax is the maximum energy value, and the

Eave Average) is equal to 1/3 of Emax.


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Interaction of particular with their


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Interaction of -particular with their

environment

n - will cause electron excitation and emit UV or

visible light.n In air, about 34 ev is required to produce one ion

pair.

n An ejected electron, with an energy of 1.5 MeV,will travel about 2 meters in air, and about 1 cm intissue.

n Eventually the ejected electron will be captured by

an empty electron orbit of a positive ion.n When a positron encounters an its electron, the two

particles annihilate (combine) each other and theirmasses are converted into electromagnetic-

radiation which is I the form of two 0.51-Mev

amma ra at on an ts nteract on


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amma ra at on an ts nteract onwith matter

n Gamma rays are high energy photons or light

particles that are electrically neutral with zero massand consequently can penetrate matter readily.

n -Ray is emitted from the nucleus with discreteenergy range between 10 KeV to 3 MeV.

n Many isotopes emit of- ray of several energies indiscrete steps (Fig. 6.2).

n Photoelectric effect is a- ray of relatively low

energy (


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n The Compton effect results when- rays of

medium energy (>0.1 MeV) undergo elastic

collisions with loosely bound orbital electron and aportion of the- ray energy is transferred to the

ejected electron.

n

The photon is deflected in a new direction withreduced energy and undergo several more similar

collisions before losing all its energy.

n Compton electrons have a wide range of energies

and these electrons will produce secondaryionization as they travel through the medium,

dissipating their energy.


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n Pair production results when a relativelyenergetic (>1.02 MeV)- ray interacts with

a nuclear force field.n The photon has all its energy converted into a

positron and an electron.

n This is essentially the conversion of energyinto mass.

n Both the ejected positron and electron may

cause ionization of atoms in their paths, andcollies together.


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Kinetics of radioactive decay

n The process of decay of a radioactive nucleus into a

stable, nonradioactive nucleus is irreversible.

n Rate of decay (particle emission) is equal to the rate

of disintegraton of the nuclei, which is proportional

to the number of radioactive nuclei present.

n Rate of decay decreases exponentially with time.

n It is independent of pressure, temperature and other

chemical and physical changes.n The rate is first order reaction.


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n Rate of decay:

n N = amount of radioactive nuclei present

n = decay constant


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n Activity is the quantitative measurement of

radioactive and if the activity of sample is A

(by detector), it becomes the counting rate ofthe sample.

Efficient of detector


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Half-life (t )


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Units used in radioactive measurements

n The electron volt

n One electron volt (1 eV) is the kinetic energydeveloped by an electron when it is acceleratedthrough a potential difference of 1 volt in a

vacuum.n 1 eV is equal to 3.85 x 10-20 calories or 1.602 x

10-19joule.

n It is normally expressed in KeV or MeV.

n The electron emitted from 14C has an averageenergy of 4500 eV per nucleus (atom).

Th i h i f di i di i i


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n The curie: the unit of radioactive disintegration

n One curie is equal to the number of disintegration per

second in 1 gram of pure radium 226.

n It is also equal to 2.22 x 1012 disintegration per minute

(dpm).

n One becquerel (one disintegration per second) is equal to

3.70 x 1010 becquerels.


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Safety in handling radioisotopes


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Safety in handling radioisotopes

n First to know whether it is- or+ -ray isotopes.

n Prevent injection, inhalation or absorption ofradioisotopes in human body.

n Laboratory coat, plastic gloves etc. Should be worn for

body protection.n Restricted areas for handling radioisotopes must be

labeled, clean and neat.

n Covered with plastic absorbent paper with a tray line.

n Have special containers for any radioisotope disposal

such as glasses, plastic, paper, water and organic

solvent.


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n Monitor the working place routinely for any

contamination of radioactivity by using Geiger-

Muler survey monitor for strong and ray.

n Wear a personal dosimeter for high energy

radioisotopes.

n Wash immediately if any contamination is present.n If skin has contacted with radioisotope, wash with

immediately for at least 2 minutes.

n Special care is required for anyone handling 125I or131I because of the effects on thyroid gland in

human body.

Statistics of counting radioisotopes


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Statistics of counting radioisotopes

n Compare Fig. 6.1 and Fig. 6.5.

n Standard deviation s:

n A more common situation is:

n Confidence of the true value.


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P l i d d d i i (% RSD)


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n Percent relative standard deviation (% RSD)

n For sample comparison, % RSD is generally

required.


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Measurement of radioactivity

n By gas ionization.

n By scintillation counting

By gas ionization


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By gas ionization

n When a energetically charged particle, such

as- particle, passes through a gas, itselectrostatic field displaces orbital electron

fro the gas molecules close to its path.

n This displacement produce a positive ionfrom the other part of the molecule, resulting

an electron pair.

By gas ionization


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y ga a

n If an electric field is applied in the chamber,

the displaced electrons are accelerated towardthe anode and the positive ions move towardcathode slowly (because of their larger mass).

n

If higher electric fields are applied, theelectron displaced by the first- particlecollision is accelerated and collides with othergas molecules to produce additional ion pairs.

n This result is an amplification of the numberof electrons from the first electron, giving ashower of electrons.

monitor


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n A type of radioactive detector.

n It is a gas-filled tube with cylindrical cathode

and a fine wire anode.

n A common gas mixture is the so called Q-gas

which is 1.3 % isobutane in helium.

n One end of the tube has a window through

which the- particles from the radioactive

sample.

monitor


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n A voltage potential is applied between the electrodes

of the tube and produces shower

n The electrons are collected at the anode within amicrosecond, and a strong electrical pulse is formed.

n The number of electrons collected is proportional to

the number of initial ionization, and is proportionalto the number of- particles entering the chamber.

n The region is called proportional region.

n

Efficient, for

14

C about 1 to 5 % and for

3

H is nearlyzero.


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By scintillation counting

n Use scintillator or fluors for- and- ray counting.

n Scintillator can absorb radiant energy to form excited atoms

or molecules and return rapidly to ground state, releasing

excitation energy, to form photon and heat.

n Amount of photon released directly related to the amount of

radiant energy.

n

Liquid scintillation counting is that the scintillators aredissolved in a solvent called cocktail.

By scintillation counting


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y g

n Steps for photon formation:

n - Radiant energy is absorbed by solvent and become excited.

n The solvent transfers the the excited energy to the scintillator to

cause excitation.

n The scintillators release photon which is detected by the phototubes.

n The phototube converts photons into amplified numbers of electron,and then becomes voltage pulse by a pulse height analyzer, and this

is called- and- scintillation counter.

n - energy spectrum refers to Fig. 6.12.

n

Dual-labeled mixture activity determination refers to Fig.6.13.

Cocktail composition and samplepreparation


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preparationn Primary fluor is 2,5-diphenyloxazole (PPO).

n Secondary fluor is 1,4-bis-2-(5-phenyloxazolyl) benzene(POPOP).

n Common solvents: toluene and dioxanen Toluene is used for organic compounds.

n

Dioxane is used for aqueous samples.n Heterogeneous counting is that the radioactive samples are

in solid support.

n Homogeneous counting is that the radioactive samples arein dissolved form.

n Background counting is caused by electronic noise, cosmicradiation and other natural radioisotopes present.

n Cerenkov counting is used for direct- particle countingwithout fluor, only for high energy- emittor, e.g. 32P.

Quenching


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n It is the process that causes a reduction in the amount of

fluorescence produced by

- emission in a liquidscintillation cocktail, reduction amount of light reaching the

photomultipler tube.

n Chemical quenching is that the transfer of energy is

interfered from solvent to fluor molecules (fluorescencereduction).

n By acid

n By dipole-dipole interaction of quenching agent

n Electron capture by quenching agent.

n Color quenching is the presence of colored compounds

which can absorb the photons emitted by the fluor, e.g.

Pigment, blood.


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Quench correctionn

Internal standard method:n Add another non quenching radioactive standard and

recounted.

n External standard method:

n Use of a highly radioactive- radiation source, e.g.226Ra or 137Cs, which is brought near to the test

sample vial and the sample is recounted in a short

time.

n Efficient by ES refers to 6-19.

S lid i till ti ti f di ti


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Solid scintillation counting of radiation

n

A crystalline inorganic fluor, sodium iodide (NaI), withsmall amount of thallium (Tl) as activator.

n - Rays interact with crystalline fluor to form ion pair, the

Compton effect and induce photoelectric and excite

crystalline fluor, finally produce photon to reachphotomultiplier tube.

n The crystalline fluor is sealed in a metal cylinder from

external light.

n The cylinder must be hermetically sealed because ofhygroscopic of NaI.

n It is designed for the counting of125I, 51Cr and 60Co.


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A toradiograph


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Autoradiography

n A technique for the determination the location of

radioisotopes in tissue, tissue sections, chromatograms and

gels.

n It is a non-destructive method for radioactive compounds,

and can be isolated and recounted.

n Results are shown on a X-ray film and developed.

n The- ray hits the silver bromide of the film and reducing

to metallic silver from silver ions, and black spots will be

appeared.n For best darkening of the film, at least 1 x 106 (- particle)

passing through the film per cm2 per minute.

A di h


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Autoradiography

n If sample contains 1 x 103 cpm/cm2, it will take about 100

minutes or 17 hours to induce the black spot.

n For weak ray, enhancer ( fluorography, PPO) is need to

enhance the sensitivity, particularly 3H.

n The photoelectric effect are more stable under -50 to -80C

about 4x more stable.

n The resolution can be increased by decreasing the thicknes

of the sample and the distance between sample and the fil


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M h d f l b li bi h i l


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Methods of labeling biochemicalcompounds

n Two general methods:

n biosynthesis and

n chemical synthesis.


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Biosynthesis

n Used by either whole organisms (cells) or specific enzymesto synthesize radioactive compounds.

n The common used radioisotopes are 14C, 32P and 35S.

n Factors for the successful use of biosynthesis:

n By using whole organism, labeled compound(s) must beaccumulated in large number from starting compound.

n For using a specific enzyme, the enzyme must be easily isolatedand the substrate is readily available, and the labeled compoundmust be isolated and purified from the reaction mixtures.

n

For using microorganism, labeled compound is isolated from themicroorganism or the cell-free culture supernatant fluid.

n Whole organism biosynthesis gives a relatively largenumber of labeled products but enzyme synthesis gives onlyone or specific labeled products.

Ch i l h i


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Chemical synthesisn

Products can be labeled in one or few positions.n Starting materials always be scarce and expensive.

n Results give low yield.

n Give a racemic mixture of optical isomers, e.g. D- and L-

isomers of amino acids.n Types of labeling of compounds:

n Labeling of proteins.

n Labeling of nucleic acids.

n Formation of14C carbon-carbon bonds.

n Synthesis of tritium labeling [3H] compounds from NaB3H4.

n Synthesis of35S-labeled L-cysteine

T f di h i l l b li


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Types of radiochemical labelingn

Isotopically: one or more radioactive atoms have replacedstable atoms of the same element, e.g.

n3H1

1H1,14C6

12C6,35S15

31P15

n They are the same molecules.

n

Nonisotopically: a radioactive isotopes has beensubstituted for a stable atom of another element, e.g.

n125I for 1H1

n Three forms of labeling positions:

n

Specific: [1-14

C]-D-glucosen random: randomly distributed (G-).

n *CH3-CH2COOH, or CH3-*CH2COOH or CH3-CH2*COOH

n Uniform: all related atoms are labeled, [U-], e.g. [U-14C]-

sucrose.


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Radioimmunoassayn Ab (antibody) + Ag (antigen) Ab-Ag complex

n Present an abstract about the basic principle and

application of radioimmunoassay.


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Designing a radioisotope experiment


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n

First and foremost in the design of anyexperiment is the formulation of the question tobe studied, the purpose and nature of theexperiment.

n Next is what experimental techniques toanswering the question.

n Is radioisotope necessary ? What type ofradioisotope ?

n

Which position and and which type of labeledcompound.

n Availability of the labeled compound.

n How much specific activity and amount of

radioactivity required



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n What is the cost of the labeled compound ?n Experimental methods.

n What type of detection system and its efficiency ?

n The dilution effects and the yield of theradioisotope.

n Control and correction factors needed.

n

Expression and interpretation of the results, andfinally

n The possible isotope effects and possible effects

of radiation damage.

Documents

Radioisotope Techniques