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. Capture the light while it is there!
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