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The Age of Chemiluminescent Detection

In a crime scene, large pools of spilled blood are easy to see. But what about blood that
has been deliberately washed away? Enter luminol! You've probably seen luminol on TV.
Luminol is often used in the visualization of blood. It is very sensitive, capable of detecting
blood at 1 part per million. This is because luminol is a chemical that glows greenish-blue
when it comes into contact with blood — even traces that are years old. This light-emitting
property of luminol began to be explored by life science research laboratories some 20
years ago, and now constitutes the basis of “enhanced chemiluminescent (ECL) detection”.

To explain this phenomenon, we’ll first tear apart its name. The first, chemi, means that it
has to do with chemicals, and the second, luminescence, means that it emits light. Put
together then, chemiluminescence means giving off light through a chemical reaction. For
instance, the addition of bleach to a luminol solution causes the oxidation of luminol, which
results in the production of the excited state of luminol. The decay of this excited state
molecule to the ground state causes the release of a photon. This release is the observed
"chemiluminescence". In crime scene investigations, luminol reacts to hemoglobin and is
oxidized by heme, resulting in the release of greenish-blue light that can be picked up by
various detection instruments.

The two chemical reactions described above represent the basic reaction, in which
chemiluminescent emission is caused by oxidative degradation of luminol. In this basic
reaction, the light output is not sustained and therefore is not easily harnessed for
laboratory use. To overcome this limitation, various “enhanced” chemiluminescence
systems were later developed that demonstrated a large increase (>1000-fold) in the
amount and duration of the light output. The maximum quantity of light is emitted at a
wavelength of 428 nm (blue light) and can be captured on x-ray film or on a suitable light-
capturing instrument such as a charge-coupled device (CCD) camera.

The most popular ECL detection is based on direct detection of horse radish peroxidase
(HRP)-labeled probes, typically HRP-conjugated secondary antibodies in Western blotting
applications. The basic reaction scheme for the unenhanced reaction involves a cyclical
process, in which the iron center of the HRP enzyme, using hydrogen peroxide as a
substrate, becomes oxidized. The altered iron center then returns to the original state with
the production of a luminol radical, which further proceeds through a number of oxidation
reactions leading ultimately to the formation of an excited form of 3-aminophthalate. You
can picture what would happen next when this excited molecule returns to the ground state.
In the ECL system, the “enhancer” is thought to directly react with HRP, forming an
enhancer radical, which more efficiently reacts with luminol to form luminol radicals and
allows the reaction to cycle rapidly. Eventually, however, a buildup of free radicals damages
the HRP enzyme and light emission ceases. As the old saying goes- Strikes the iron while it
is hot. Try our enhanced chemiluminescent detection system, and capture the light while it
is there!

Biomedical Research Service