The development of a generic hydrogen peroxide sensor with application to the detection of glucose

A generic sensor for hydrogen peroxide producing metabolites was developed. The sensor consists of a small bore nylon tube fitting closely over two UV-transmitting fibers in an optrode configuration. In the case of glucose sensing, horseradish peroxidase and glucose oxidase were coimmobilized on the inside surface of the nylon tube. Glucose oxidase catalyzes the production of hydrogen peroxide from glucose and horseradish peroxidase catalyzes the reaction of hydrogen peroxide (H₂O₂) and p-hydroxyphenylacetic acid (HPA) into 6-6’-dihydroxy(1,1′-biphenyl) 3,3′-diacetic acid (DBDA) which is measured by laser-assisted fluorometry. The amount of fluorescing DBDA is directly proportional to the amount of glucose present. Michaelis-Menten kinetics for soluble and immobilized enzymes were established for the H₂O₂/HPA/HRP system to determine the range of hydrogen peroxide concentrations for which sensor linear response is observed. Sensors were found to be stable at 4°C in pH 7 phosphate buffer for a period of thirty days with no significant loss in enzyme activity. Glucose levels in buffer at physiological pH were measured and sensor linearity was established. Applicability of the sensor to the measurement of glucose levels in serum was established. Further studies will involve in vitro sensor calibration.