Process for the enzymatic determination of glucose with a glucose-oxydazed/peroxidazed enzyme system

Abstract

Process for enzymatic determination of glucose with a glucose-oxidase-peroxidase enzyme system involving the use of a reduced acceptor which is oxidized. As reduced acceptor in the enzymatic reaction catalyzed by peroxidase, a ferrocyanide salt is oxidized to ferricyanide; then a further reducing system is introduced, which system is oxidized by the ferricyanide, with the formation of a colored quinone compound, the reaction between ferricyanide and the reducing system taking place spontaneously.

I claim: 
     
       1. A process for the enzymatic determination of the amount of glucose present in body fluids which comprises the steps: i. adding to a sample of a body fluid an enzymatic system containing glucose-oxidase and peroxidase, to which has been added sodium or potassium ferrocyanide,   ii. incubating the sample and enzyme system at room temperature whereby the ferrocyanide is converted to ferricyanide equivalent to the amount of glucose present in the sample, and   iii. reacting the ferricyanide with a reducing system which forms a colored product on oxidation, the color being a measure of the ferricyanide present, said reducing system consisting of a phenolic compound and an aminoantipyrine.   
     
     
       2. A process according to claim 1 wherein the phenolic compound is selected from the group consisting of phenol, salicylamide, barbituric acid, and esters of hydroxybenzoic acid. 
     
     
       3. A process according to claim 2 wherein the phenolic compound is phenol. 
     
     
       4. A process according to claim 3 wherein the aminoantipyrine is 4-aminoantipyrine. 
     
     
       5. A composition for use in the determination of glucose in body fluids by the method of claim 1, which comprises i. an enzymatic system consisting of glucose-oxidase and peroxidase,   ii. sodium or potassium ferrocyanide,   iii. a phenolic compound, and   iv. an aminoantipyrine.   
     
     
       6. A composition according to claim 5 wherein said composition is buffered at a pH between about 5.5 and 8. 
     
     
       7. A composition according to claim 5 which is in two parts, the first part comprising i. an enzymatic system consisting of glucose-oxidase and peroxidase, and   ii. sodium or potassium ferrocyanide, and the second part comprising   iii. a phenolic compound and   iv. an aminoantipyrine.   
     
     
       8. A composition according to claim 5 which is in two parts, the first part comprising i. an enzymatic system consisting of glucose-oxidase and peroxidase,   ii. sodium or potassium ferrocyanide, and   iii. a phenolic compound, and the second part comprising an aminoantipyrine.   
     
     
       9. A composition according to claim 8 wherein the part containing the enzyme system is buffered at a pH between about 5.5 to 8. 
     
     
       10. A composition according to claim 6 wherein the phenolic compound is phenol. 
     
     
       11. A composition according to claim 10 wherein the aminoantipyrine is 4-aminoantipyrine. 
     
     
       12. A composition according to claim 9 wherein the phenolic compound is phenol. 
     
     
       13. A composition according to claim 12 wherein the aminoantipyrine compound is 4-aminoantipyrine. 
     
     
       14. A composition according to claim 7 wherein the part containing the enzyme system is buffered at a pH between about 5.5 to 8. .Iadd. 15. A process for the enzymatic determination of the amount of glucose in an undeproteinized body fluid which consists in adding to said undeproteinized body fluid an aqueous solution of an enzymatic system consisting of glucose-oxidase and peroxidase, an aminoantipyrine and a phenolic compound which is a member of the group consisting of salicylamide, barbituric acid, para- and meta-hydroxy-benzoate and esters thereof, and thereafter measuring color change photometrically. .Iaddend. .Iadd. 16. A composition for use in the enzymatic determination of the amount of glucose in a body fluid, consisting essentially of a glucose-oxidase and peroxidase enzyme system, a phenolic compound selected from the group consisting of salicylamide, barbituric acid, para- and meta-hydroxybenzoate and esters thereof, and an aminoantipyrine. .Iaddend..Iadd. 17. A dry composition as claimed in claim 16. .Iaddend.