Luminescence by reacting an acridinium ester with superoxide

Abstract

A light emitting method of an acridinium ester, comprising reacting said acridinium ester and a superoxide anion, and a method of detecting a substance to be examined, comprising detecting a light emitted by reacting a superoxide anion with an acridinium ester used as a label am described. It is possible to carry out the reaction not under strongly alkaline conditions but around the neutral point and to generate strong luminescence which is stable over a long period of time.

1. A method for emitting chemiluminescence from an acridinium ester in an immunoassay, a hybridization assay or an immunoblot assay, wherein said ester is a compound of the formula (2): 
                 
 
       wherein R 11  is an alkyl, alkenyl, alkynyl or aryl group; R 12 , R 13 , R 15  and R 17  are independently a hydrogen atom, halide atom, amino, carboxyl, hydroxyl, alkoxyl or nitro group; R 14  and R 18  are independently an alkyl, alkenyl, alkynyl, aryl, alkoxyl, amino, amido, sulfonamido or sulfide group; R 16  is —R 19 —R 10 ; R 19  is not required but optionally can be an alkyl, aryl or aralkyl group; R 10  is selected from the group consisting of: 
                 
 
       X 1 is CH   3 SO 4   − , OSO 2 F − , a halide, OSO 2 CF 3   − , OSO 2 C 4 F 9   − , or 
                 
 
       and R is an alkyl, aryl or aralkyl group; or said ester is a compound of the formula (3a): 
                 
 
       wherein R 25  comprises one of the following: 
                 
 
       R 1 , and R 26  are independently a hydrogen atom or C 1 -C l0  optionally substituted alkyl, alkenyl, alkynyl or aryl group; R 22  and R 23  are independently a hydrogen atom, halide atom, amino, substituted amino carboxyl, hydroxyl, alkoxyl or nitro group; and X 2  is a halide atom, comprising reacting at a pH of 6 to 10 said acridinium ester and a superoxide anion produced in situ by electrochemical reduction of oxygen dissolved in an electrolyte using electrodes in the presence of a flavin compound, wherein said acridinium ester is attached as a label to a reagent in said immunoassay, hybridization may or immunoblot assay. 
     
     
       2. The method of  claim 1 , wherein the electrochemical reduction is performed in the presence of 1×10 −9 M to 1×10 ″4 M of the flavin compound at a potential of −0.3 to −0.7V (vs. Ag/AgCl). 
     
     
       3. The method of  claim 1  wherein the electrochemical reduction comprises use of an electrode comprising said flavin compound. 
     
     
       4. The method of  claim 1 , wherein the acridinium ester is  4 -[ 2 -succinimyzyloxycarbonyl)ethyl]phenyl- 10 -methylacridinium- 9 -carboxylate fluorosulfate. 
     
     
       5. A method of detecting a substance to be examined in a sample in an immunoassay, hybridization assay or immunoblot assay, comprising,
 binding a chemiluminescent labeled substance having affinity for said substance to be examined with said substance to be examined in a sample to produce a complex of said substance to be examined and said chemiluminescent labelled substance having affinity therefor, separating said complex;  
 reacting said chemiluminescent label with a superoxide anion at a pH of 6 to 10, said label being an acridinium ester of the formula (2): 
                 
 
 
       wherein R 11  is an alkyl, alkenyl, alkynyl or aryl group; R 12 , R 13 , R 15  and R 17  are independently a hydrogen atom, halide atom, amino, carboxyl, hydroxyl, alkoxyl or nitro group; R 14  and R 10  are independently an alkyl, alkenyl, alkynyl, aryl, alkoxyl, amino, amido, sulfonamido or sulfide group; R 14  is —R 19 —R 10 ; R 19  is not required but optionally can be an alkyl, aryl or aralkyl group; R 10  is selected from the group consisting of: 
                 
 
       X 1  is CH 3 SO 4 —, OSO 2 F—, a halide, OSO 2 CF 3 —, OSO 2 C 4 F 9 —, or 
                 
 
       and R is an alkyl, aryl or aralkyl group; or said label is an acridinium ester of the formula ( 3 a): 
                 
 
       wherein R 25  comprises one of the following: 
                 
 -halide or  (e)
   -azide;  (f)  
 
       R 21  and R 26  are independently a hydrogen atom or C 1 -C 10  optionally substituted alkyl, alkenyl, alkynyl or aryl group; R 22  and R 23  are independently a hydrogen atom, halide atom, amino, substituted amino, carboxyl, hydroxyl, alkoxyl or nitro group; and X 2  is a halide atom; wherein said superoxide anion is produced in situ by electrochemical reduction of oxygen dissolved in an electrolyte using electrodes in the presence of a flavin compound; and
 measuring luminescence of said chemiluminescent label to detect said substance to be examined.  
 
     
     
       6. The method of  claim 5  wherein said binding step in said hybridization assay is reaction between a nucleic acid and a nucleic acid complementary thereto. 
     
     
       7. The method of  claim 5  wherein the acridinium ester is  4 -[ 2 -succinimyzyloxycarbonyl)ethyl]phenyl- 10 -methylacridinium- 9 -carboxylate fluorosulfate. 
     
     
       8. A method for emitting chemiluminescence from an acridinium ester in an immunoassay, a hybridization assay or an immunoblot assay, where said ester is  4 -[ 2 -(succinimidyloxycarbonyl)ethyl]phenyl- 10 -methylacridinium- 9 -carboxylate fluorosulfate, comprising reacting at a pH of  6  to  10  said acridinium ester and a superoxide anion produced in situ by electrochemical reduction of oxygen dissolved in an electrolyte using electrodes in the presence of a flavin compound wherein said acridinium ester is attached as a label to a reagent in said immunoassay, hybridization assay or immunoblot assay. 
     
     
       9. A method of detecting a substance to be examined in a sample in an immunoassay, hybridization assay or immunoblot assay, comprising, binding a chemiluminescent labeled substance having affinity for said substance to be examined with said substance to be examined in a sample to produce a complex of said substance to be examined and said chemiluminescent labelled substance having affinity therefor; separating said complex; reacting said chemiluminescent label with a superoxide anion at a pH of  6  to  10 , said label being a  4 -[ 2 -(succinimidyloxycarbonyl)ethyl]phenyl- 10 -methylacridinium- 9 -carboxylate fluorosulfate; wherein said superoxide anion is produced in situ by electrochemical reduction of oxygen dissolved in an electrolyte using electrodes in the presence of a flavin compound; and measuring luminescence: of said chemiluminescent label to detect said substance to be examined.