Ultra-sensitive chemiluminescent substrates for peroxidase

Abstract

A method for increasing the light emission produced by the 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 and the co-enhancer is selected from a 4-dialkylaminopyridine or an N-azole, and wherein the method comprises the following steps: i. realizing a chemiluminescent substrate by means of 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, and ii. adding the peroxidase enzyme or a conjugate thereof to the chemiluminescent substrate.

1 . 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.   
     
     
         2 . The method according to  claim 1 , wherein the anionic N-alkylphenothiazine is selected from the group consisting of 3-(10H-phenothiazin-10-yl)propane-1-sulfonic acid, 4-(10H-phenothiazin-10-yl)butane-1-sulfonic acid, 3-(10H-phenothiazin-10-yl)propanoic acid and 4-(10H-phenothiazine-10-yl)butanoic acid, and their salts. 
     
     
         3 . The method according to  claim 1 , wherein the peroxidase enzyme is selected from the group consisting of horseradish peroxidase, soybean peroxidase and sweet potato peroxidase. 
     
     
         4 . The method according to  claim 1 , wherein the peroxide oxidizer is selected from the group consisting of hydrogen peroxide, urea and hydrogen peroxide complex in a molar ratio 1:1, a perborate salt, a percarbonate salt. 
     
     
         5 . The method according to  claim 1 , wherein the concentration of the anionic N-alkylphenothiazine enhancer in the chemiluminescent substrate is comprised between 0.1 and 10 mM. 
     
     
         6 . The method according to  claim 1 , wherein the concentration of the co-enhancer in the chemiluminescent substrate is comprised between 0.1 and 100 mM. 
     
     
         7 . The method according to  claim 1 , wherein the concentration of the peroxide in the chemiluminescent substrate is comprised between 0.1 and 10 mM. 
     
     
         8 . The method according to  claim 1 , wherein the concentration of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione in the chemiluminescent substrate is comprised between 0.01 mM and 1 mM. 
     
     
         9 . The method according to  claim 1 , wherein the chemiluminescent substrate contains:
 a. 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione in a concentration between 0.01 mM and 1 mM;   b. the anionic N-alkylphenothiazine enhancer in a concentration between 0.1 and 10 mM;   c. the co-enhancer in a concentration between 0.1 and 100 mM;   d. the peroxide oxidizer in a concentration between 0.1 and 10 mM, and   e. a buffer solution to maintain the pH of the chemiluminescent substrate in the range of 6.0 to 7.9.   
     
     
         10 . The method according to  claim 1 , wherein pH is maintained in the range of 6.0 to 7.9 by a buffer solution. 
     
     
         11 . The method according to  claim 10 , wherein the buffer solution is a TRIS/phosphate buffer solution. 
     
     
         12 . The method according to  claim 1 , wherein the method further comprises a step of detecting an analyte in a sample through measuring the light emission produced by the 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. 
     
     
         13 . A kit for performing a method according to  claim 1 , wherein the kit comprises 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxide oxidizer, an enhancer and a co-enhancer, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from the group consisting of imidazole, 1-methylimidazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinopyridine (PPY). 
     
     
         14 . The kit according to  claim 13 , wherein the kit further comprises a peroxidase enzyme or a conjugate thereof. 
     
     
         15 . The kit according to  claim 13 , wherein the anionic N-alkylphenothiazine is selected from the group consisting of 3-(10H-phenothiazin-10-yl)propane-1-sulfonic acid, 4-(10H-phenothiazin-10-yl)butane-1-sulfonic acid, 3-(10H-phenothiazin-10-yl)propanoic acid and 4-(10H-phenothiazine-10-yl)butanoic acid, and their salts. 
     
     
         16 . The kit according to  claim 13 , wherein the peroxidase enzyme is selected from the group consisting of horseradish peroxidase, soybean peroxidase and sweet potato peroxidase. 
     
     
         17 . The kit according to  claim 13 , wherein the peroxide oxidizer is selected from the group consisting of hydrogen peroxide, urea/hydrogen peroxide complex, a perborate salt, a percarbonate salt. 
     
     
         18 . The kit according to  claim 13 , wherein 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione is present in a first vial and the peroxide oxidizer is present in a second vial, and wherein the enhancer and the co-enhancer are present in the first vial or in the second vial or in both vials.