Chemiluminescence ...an Introduction

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Dr M Muzaffar Mir

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Chemiluminescence :

Chemiluminescence بسم الله الرحمن الرحيم Ref. Book : TEITZ text book of Clinical Chemistry and Molecular Diagnostics 4 th Edition

Luminescence :

Luminescence is the generation of light when an excited molecule returns to a ground state . The excitation is not caused by photo illumination [ الإضاءة من الضوء ] as in case of fluorescence. Luminescence

Luminescence :

It may be caused by : A chemical Reaction : Chemi luminescence A biochemical Reaction : Bio Luminescence An electrochemical reaction: Electrochemi luminescence While the light can, in principle, be emitted in the ultraviolet, visible or infrared region , those emitting visible light are the most common and useful. Luminescence

Chemiluminescence :

Chemiluminescence Chemiluminescence is the emission of light when an electron returns from an excited or high energy level to a ground or low energy level. The excitation event is caused by a chemical reaction. It involves the oxidation of an organic compound such as luminal, isoluminol, acridium esters or luciferin by an oxidant [ e.g. hydrogen peroxide, hypochlorite or oxygen ]. The light is emitted from the excited product formed in the oxidation process.

Chemiluminescence :

These reactions occur in presence of catalysts such as : Enzymes : alkaline phosphatase, horseradish peroxidase and microperoxidase Metal ions: Cu 2+ , Fe 3+ Metal Complexes: Cu 2+ phthalo-cynide complex , Fe 3+ phthalo-cynide complex , Hemin etc. Chemiluminescence

Luminol --- As an example:

Luminol is a chemical that exhibits blue chemiluminescence, when mixed with an appropriate oxidizing agent. It is a white to slightly yellow crystalline solid that is soluble in water and most polar organic solvents. To exhibit its luminescence, the luminol must first be activated with an oxidant. Usually, a solution of hydrogen peroxide and a hydroxide salt in water is used as the activator. Luminol --- As an example

Bioluminescence :

Bioluminescence is a special form of Chemiluminescence found in biological systems. Light-emitting reactions arising from living organisms such as the firefly or jellyfish , are commonly termed bioluminescent reactions. In bioluminescence an enzyme or a photo -protein increases the efficiency of the luminescence reaction. Luciferase and aequorin are two examples of these biological catalysts. Bioluminescence

Different types of Jelly Fish:

Different types of Jelly Fish

Quantum Yield and Sensitivity :

Quantum yield is defined as total photons emitted per total reacting moles. The Quantum yield is approximately 0.1 to 10 % for Chemiluminescence and 10 to 30% for bioluminescence. Chemiluminescence assays are ultrasensitive and are widely used in automated immunoassays and DNA probe assays. Their sensitivity is in the atto to zepto mole range [ 10 - 18 to 10 -2 1 mole]. Quantum Yield and Sensitivity

Electrochemiluminescence [EC]:

EC differs from Chemiluminescence. In Electroluminescence, the reactive species that produce the Chemiluminescence reaction are electrically generated from stable precursors at the surface of an electrode . A commonly used label is Ruthenium[Ru2+]-tris(bipyridyl)chelate and EC is genetated at an electrode via a REDOX reaction with tripropylamine . Electrochemiluminescence [EC]

Electrochemiluminescence [EC]:

This chelate is very stable and relatively small and can be used to label haptens or large molecules [ e.g proteins or oligonucleotides ]. The EC has been used in both immunoassays and nucleic acid assays. The advantages of EC include improved reagent stability , simple reagent preparation and enhanced sensitivity. Electrochemiluminescence [EC]

Instrumentation::

The instrument which is used to measure Chemiluminescence is called as a Luminometer. The basic componets of Luminometer are : The sample cell housed in a light tight chamber. The injection system to add reagents to the sample cell. The detector [ The detector is usually a Photomultiplier tube . However a CCD, X ray film or photographic film has also been used to image Chemiluminescence reactions. For Electrochemiluminescence, the reaction vessel also incorporates an electrode at which the Chemiluminescence is generated. The other accessory components include a digital read out device , a temperature control device , a monochromator[only under special conditions], a microprocessor or computer and a printer . Instrumentation:

Applications:

The Chemiluminescence is extremely sensitive technique and is very widely used. It is used in immunoassays for : Hormones Metabolites Vitamins Biomarkers for cancer and other diseases. It is used in DNA probe assays for genetic testing and forensic medicine. It is used for sensitive drug assays. Applications

Limitations:

The extreme sensitivity of Chemiluminescence requires stringent controls on the purity of reagents and the solvents [e.g. water]. The other factors which may degrade analytical performance: Light leaks Light piping High background luminescence from reagents and reaction vessels Limitations

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