Construction as well as EQCM and SECM characterizations of a novel Nafion/glucose oxidase-glutaraldehyde/poly(thionine)/Au enzyme electrode for glucose sensing

A novel glucose biosensor based on the electrochemical detection of enzymatically generated H2O2 was constructed by the effective immobilization of glucose oxidase (GOD) via glutaraldehyde cross-linking with a poly(thionine) (PTH)-modified Au electrode, followed by coating with a Nafion outer layer to obtain high selectivity. An electrochemical quartz crystal microbalance (EQCM) was used to track various modification procedures. The mass of the deposited PTH via cyclic voltammetric electropolymerization of thionine in aqueous H2SO4 was estimated from the “dry” frequency shift after fully removing the PTH layer via its acidic degradation, allowing us to evaluate the electroactivity per unit mass of PTH. A Nafion/glucose oxidase-glutaraldehyde/poly(thionine)/Au electrode was thus constructed for glucose sensing. We found that the PTH film is a good matrix to immobilize more GOD, and the performance of the constructed sensor is significantly improved compared with its absence, though its mediator effect on the enzyme electrode is negligible. Influence of various experimental parameters on glucose sensing, including the applied potential, solution pH and electroactive interferents, was investigated. At an optimal potential of 0.7 V versus the KCl-saturated calomel electrode (SCE), the glucose biosensors were linear with glucose concentration from 0.005 to ∼5 mmol L−1, with high sensitivity (13.5 A mol−1 cm−1), short response time (within ca. 10 s) and good anti-interferent ability. The Michaelis constant ( K m app ) of the immobilized GOD was estimated to be 5.47 mmol L−1. The biosensor exhibited good storage stability, i.e., 83% of its initial response was retained after 1-month storage in a pH 7.0 phosphate buffer at 4 °C. The effective enzymatic specific activity (ESA, defined as enzymatic activity per gram of enzyme) of immobilized GOD was estimated to be 6.85 kU g−1 on the basis of the mass of immobilized GOD (from EQCM) and the quantity of hydrogen peroxide as the product of the enzymatic reaction (from amperometric detection), and the leakage of H2O2 from the Nafion/GOD-GA/PTH/Au enzyme electrode at the detection potential, as characterized by a scanning electrochemical microscope (SECM), was found to be an important factor influencing the ESA estimation.