Determination of glucose oxidase activity based on the H2O2/KI/RhB fluorescence quenching system

Abstract Glucose oxidase (GOx) is a specific enzyme that can catalyze the oxidation of glucose to produce gluconic acid and hydrogen peroxide (H 2 O 2 ), which is closely related to people's daily lives. Enzyme activity (micromoles of H 2 O 2 produced per minute by the catalytic oxidation of glucose in a unit volume of enzyme solution) is the sole indicator for evaluating the efficacy of glucose oxidase. This study proposes a novel strategy for determining glucose oxidase activity based on the H 2 O 2 /KI/RhB fluorescence quenching system. Firstly, the enzyme-containing sample is mixed with a certain concentration of glucose and incubated for a certain period of time. Then, dilute hydrochloric acid is added to terminate the enzyme reaction, allowing the enzyme-catalyzed oxidation product hydrogen peroxide (H 2 O 2 ) to react with KI through redox reaction to generate I 2 , Excess I − reacts with I 2 to form negatively charged and water-soluble iodo-ions (I 3 − ), which then form an association complex with the cationic fluorophore Rhodamine B through electrostatic interaction, resulting in fluorescence quenching of Rhodamine B. Since the quenching degree is related to the activity of glucose oxidase, a new method for detecting the activity of glucose oxidase has been established. Using this method, the GOx activity can be detected in the range of 1–20 U/L, with a detection limit of 2.6 × 10 −3 U/L. due to its advantages of simplicity, safety, sensitivity, speed, and reliability, this strategy has practical application value