Reversible Optical Sensor Membrane for Hydrogen Peroxide Using an Immobilized Fluorescent Probe, and its Application to a Glucose Biosensor

Abstract.

An optical sensor for hydrogen peroxide (HP) based on the probe europium tetracycline (EuTc) incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix is described. Upon optical excitation with 400-nm light, the EuTc in the membrane displays fairly strong fluorescence peaking at 616 nm. The fluorescence intensity increases up to 3-fold if the sensor is exposed to solutions containing HP. The effect is reversible and can thus be used for continuous sensing. The largest signal changes with HP are observed at pH levels between 6.5 and 7.5, and the range of the response is between 10 and 300 ppm (w/w) of HP, equal to 0.3 to ∼10 mmol L⁻¹). At concentrations above 0.3% of HP, decomposition of the EuTc in the membrane is observed. The limit of detection is ∼15 ppm (0.45 mmol L⁻¹). The response is fully reversible and rather slow (∼10 min) in both directions, but the reverse response may be accelerated by addition of a reducing agent such as thiosulfate. Alkali ions and most anions remain inert, but phosphate and citrate interfere, as do Cu²⁺ ions, which quench fluorescence. In order to image the spatial distribution of HP concentrations, sensor membranes were placed on the bottom of the wells of a microtiter plate, and their fluorescence was imaged using an LED-based device based on the measurement of the luminescence decay time of EuTc. If glucose oxidase is immobilized on the sensor layer, a glucose sensor is obtained in which the HP sensor acts as the transducer and which can quantify glucose in concentrations between 0.1 and 5 mmol L⁻¹. The limit of detection for glucose is ∼0.2 mmol L⁻¹.