AAT Bioquest

What is enhanced chemiluminescence?

Posted June 1, 2020


Enhanced chemiluminescence (ECL) is a detection technique based on the chemiluminescence of substrates such as luminol and acridan. Due to its high sensitivity, wide dynamic range, and high signal-to-noise ratio, ECL is one of the most popular detection methods for a variety of western blotting applications, and is also widely used for quantifying biological analytes such as DNA, RNA as well as cells.

In a typical ECL assay, antibodies that specifically recognize the molecule of interest are first labeled with horseradish peroxidase (HRP). A chemiluminescent substrate and an oxidizing agent (hydrogen peroxide) are then catalyzed by HRP to produce excited intermediates, which release a strong blue emission at 450 nm wavelength upon decaying to the ground state. The light emissions can be captured with an x-ray film and/or detected by a luminescent signal instrument.

The term “enhanced” is derived from the enhancer being used together with the chemiluminescent substrates. Without an enhancer, the light emitted is usually of low intensity and decays too fast to make an accurate detection and analysis. With an enhancer (e.g. modified phenol, naphthol, aromatic amine or benzothiazole), the reactions can proceed for prolonged duration (up to several minutes) without significant reduction in light output, allowing for accurate and sensitive detections.

Additional resources

D-Luciferin, sodium salt *CAS#: 103404-75-7*

Lumiwox™ acridinium NHS ester

Cormier, M. (Ed.). (2013). Chemiluminescence and bioluminescence. Springer Science & Business Media.

Díaz, A. N., Sanchez, F. G., & González Garcia, J. A. (1998). Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence. Journal of bioluminescence and chemiluminescence, 13(2), 75-84.