Luminescence by reacting an acridinium ester with superoxide
View Patent ↗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.