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Chemiluminescence - biostep

Chemiluminescence

What is chemiluminescence?

Chemiluminescence (CL) means that molecules emit light in the visible or UV range (sometimes also in the IR range) below their glowing temperature. The molecules are not excited physically (laser, light, heat, electricity), but reach the excited state due to a chemical reaction. The emittance of energy occures by light quanta not by heat. Therefore, chemiluminescence is called “cold luminescence“.

Several hundreds of organic and anorganic compounds are capable of chemiluminescence.

You can prove the following suitable components of a reaction quantitatively with the help of the chemiluminescence with great sensitivity:

  • Substrates
  • Catalysts
  • Quenchers (CL-reducing)
  • Intermediate and end products.
luziferin

Luciferin

If chemiluminescence processes take place in living organisms, it is called bioluminescence, e.g. the luciferin/luciferase system of the firefly which is applied variously in biochemistry and molecular biology [1,2].

Chemiluminescence appears at oxidation processes, the reaction product is transferred into an energetically excited level and stabilizes itself by energy emittance in the form of light. The reaction product is chemically different from the initial components.

luminol

Luminol (3-Aminophthalic hydrazide)

The most famous example is the luminescence of white phosphor when exposed to air and reacting to phosphorus pentoxide by emitting white light. A further well known example is the blood proof with luminol (3-aminophthalic hydrazide). The iron containing hemin complex of the blood serves as a catalyst to transform the luminol with the oxidant H2O2 to 3-aminophthalic acid while blue light with a characteristic maximum at 425 nm is emitted [3]. This forensic blood proof was consistantly optimized in its long history and is still the most sensitive proof method for blood to today’s science state [4, 5].

 

luminolreaktion

Reaction course of luminol with hydrogen peroxide (*indicates the excited transfer state of the 3-aminophthalic acid resulting by oxidation, which changes to normal state emitting light quanta).

The chemiluminescence of luminol and of other luminophores is the basis of many analytic proof methods and systems in biochemistry and is used for biosensors, immunoassays and microarrays (e.g. ELISA, Western Blot). In contrast to the forensic blood proof in most cases the enzyme horseradish peroxidase (HRP) is used as catalyst for chemiluminescence reaction [6]. This enzyme e.g. is linked at a secondary antibody in immuno reactions in a solid or fluid phase (see Fig. 6) or serves as proof for hydrogen peroxide which was generated before in a specific biochemical or biological reaction [7 – 10].

For detection of the chemiluminescence camera, scanner or film-based methods can be used. The Celvin® S is our own development and the smallest camera-based CL system for Western-Blots.

proteinnachweis

Specific proof of proteins after SDS-PAGE and blotting by chemiluminescence immundetection. The second antibody is covalently linked with horseradish peroxidase = HRP. For development a complex composed CL reagent mixture is applied on the blot. This results in the oxidation of luminol and the emission of light. The detection of the labeled antigen can be made by a CCD-camera.