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 (H2O2), which is closely related to people's daily lives. Enzyme activity (micromoles of H2O2 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 H2O2/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 (H2O2) to react with KI through redox reaction to generate I2, Excess I− reacts with I2 to form negatively charged and water-soluble iodo-ions (I3−), 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

Keywords

• Glucose oxidase
• Glucose
• Fluorescence quenching