New glucose biosensor based on a poly( o-phenylendiamine)/glucose oxidase-glutaraldehyde/Prussian blue/Au electrode with QCM monitoring of various electrode-surface modifications

Abstract

The immobilization of glucose oxidase (GOD) via its dip-dry coating on a Prussian blue (PB) modified Au electrode followed by deposition of an outer poly( o-phenylenediamine) (P oPD) film and then glutaraldehyde (GA) cross-linking was investigated for amperometric glucose sensing, based on the reduction of enzymatically generated H 2O 2 catalyzed by the electroactive PB layer of the P oPD/GOD-GA/PB/Au electrode. The quartz crystal microbalance (QCM) was used to monitor various electrode-modification processes, and the effective enzymatic specific activity (ESA, defined as enzymatic activity per gram of enzyme) of immobilized GOD was estimated based on the mass of immobilized GOD (from QCM) and the quantity of hydrogen peroxide as the product of the enzymatic reaction (from amperometric detection) for the first time. The ESA of native GOD under our experimental conditions was also estimated via amperometric detection of enzymatically generated H 2O 2 at a PB-modified Au electrode. Effects of various experimental parameters on glucose sensing, including the applied potential, solution pH and electroactive interferents, were investigated. At an optimal potential of −0.05 V versus saturated KCl calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with glucose concentration from 0.05 to 10 mmol L −1, with detection limit of 8 μmol L −1, short response time (within ∼5 s) and good anti-interferent ability. The Michaelis constant ( K m app ) of the immobilized GOD was estimated as 19.6 mmol L −1. The biosensor exhibited good storage stability, i.e., 88% of its initial response was retained after 30-day storage in pH 7.0 phosphate buffer at 4 °C. Since the present sensor-fabrication platform is rather general and advantageous in saving biochemical reagents, it is recommended for the development of other biosensors based on other substrate electrode materials, e.g. glassy carbon that has been tested in this work with satisfactory results.