Which of the following is a substrate for horseradish peroxidase (HRP) in luminescent assays?

• A. Luminol
• B. Dioxetane phosphate
• C. Luciferin
• D. Ruthenium tris chelate

Answer: (A)

In luminescent assays with horseradish peroxidase, the substrate must be something that HRP can oxidize in the presence of hydrogen peroxide to emit light. Luminol fits this perfectly. When HRP catalyzes luminol’s oxidation, 3-aminophthalate is produced in an excited state and releases a photon as it returns to ground state, giving a measurable flash of light. This chemiluminescent reaction is widely used because it is highly sensitive and compatible with many immunoassays.

The other options belong to different luminescent systems. Dioxetane phosphate substrates are designed for alkaline phosphatase; they produce light after dephosphorylation, not via HRP. Luciferin is the substrate for luciferase and requires ATP and oxygen in that enzyme's specific reaction. Ruthenium tris chelate is used in other luminescence contexts, such as electrochemiluminescence, not as a substrate for HRP.