Main Chemiluminescent Substrate：Luminol, Isoluminol and its Derivatives

Luminol, isoluminol and its derivatives which were first used in CLIA (chemiluminescent immunoassay), are common chemiluminescent chemicals.

Chemiluminescent mechanism
Take luminol as an example. Activated by hydroxide ions and the catalyst HRP, the luminol is oxidized by hydrogen peroxide (H2O2) into 3-Aminophthalic acid. When the 3-Aminophthalic acid changes from excited state to ground state, it releases energy as a photon. This emission produces the blue glow.

History
The luminescence quantum yield of luminol is 0.01%, and the wavelength can reach up to 425 nm. Luminol was directly used to mark the antigen or antibody in earlier time. But the luminescent intensity of luminol was reduced, which therefore influence the sensitivity. So recently HRP is used as the marker instead. After the immunoreaction, luminol reacts with the activator NaOH and H2O2, and exhibits the luminescence. The luminescent intensity of luminol depends upon the concentration of HRP in the immunoreaction.

Development
By adding a synthetic fluorescein in the system above, Whitehead and Thorpe extended the luminescent duration time to 7 minutes, strengthened the light signal intensity by 7 times and lowered the background signal intensity, increasing the SNR（Signalto Noise Ratio）to 80 times. Such was the Luminol/H2O2/HRP/Enhancer system.

In 1985, some substituted phenols such as p-Indophenol and p-Hydroxy-cinnamic acid were found out to be the best enhancer. They could extend the luminescent duration time to 30-60 minutes, and strengthen the luminescent intensity to more than 100 times. Since then, Luminol/H2O2/HRP/Enhancer system has developed rapidly.

Luminol, isoluminol and its derivatives are now applied in CLIA within Luminol/H2O2/HRP/Enhancer System: With HRP marking the antigen or antibody, Luminol, isoluminol or its
derivatives acts as the chemiluminescent substrate enhanced by p-Iodophenol or p-phenylphenol and activated by NaOH and H2O2. Two minutes after the chemiluminescence reaction, the luminescent intensity will reach the peak.

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