There are three basic approaches to the measurement of glucose concentration: reducing methods, condensation methods, and enzymatic methods.
Today, we are going to use the enzymatic method. The glucose assay reagent contains glucose oxidase, peroxidase and o-dianisidine. The oxidase reacts with glucose to produce gluconic acid and hydrogen peroxide.
Peroxidase reacts with the hydrogen peroxide produced when glucose is oxidized, and the colorless reduced o-dianisidine, to make a brown colored product (oxidized o-dianisidine).
Since this product is unstable, sulfuric acid is added to stop the reaction by denaturing all enzymes, and a pink colored product (protonated oxidized o.dianisidine) is formed, that is stable and absorbs light at 540 nm. One molecule of this colored product is produced for each molecule of glucose in the solution.
Figure 1: Glucose assay reactions