Ultra-sensitive chemiluminescent substrates for peroxidase

A method for increasing a light emission produced by a chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxidase enzyme or a conjugate thereof, an enhancer, a co-enhancer and a peroxide oxidizer, wherein the enhancer is an anionic N-alkylphenothiazine, wherein the co-enhancer is selected from the group consisting of imidazole, 1-methylimidazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinopyridine (PPY), the method comprising the following steps:

• i. preparing a chemiluminescent substrate by mixing together 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, the enhancer, the co-enhancer and the peroxide oxidizer, the chemiluminescent substrate having a pH in the range of 6.0 to 7.9, and
• ii. adding the peroxidase enzyme or a conjugate thereof to the chemiluminescent substrate.