New bioorganometallic ferrocene derivatives as efficient mediators for glucose and ethanol biosensors based on PQQ-dependent dehydrogenases

One known and two new ferrocene-containing mediators incorporating the organometallic moiety and the fragments of natural substrates of oxidative enzymes, viz. 4-ferrocenylphenol (FP), 2-ferrocenyl-4-nitrophenol (FNP), and N-(4-hydroxybenzylidene)-4-ferrocenylaniline (HBFA), were studied as electron transfer mediators between the coenzyme pyrroloquinoline quinone (PQQ) of glucose (GDH) and alcohol (ADH) dehydrogenases and the carbon electrode surface. A screen-printed carbon electrode (SPCE) suitable for ADH and GDH immobilization served as a transducer. The electrodes were integrated into a flow-through amperometric cell. All data were obtained at a flow rate of 1 ml min−1. The maximal currents (jmax) obtained from the calibration curves for the oxidation of ethanol and d-glucose by ADH and GDH of 2.3 and 3.0 μA, respectively, were obtained when SPCE was modified with HBFA, i.e. with a mediator with a longer arm and a high degree of conjugation. The biosensors were used for ethanol and d-glucose measurements in beverages. There was a good correspondence (r=0.978 for d-glucose and r=0.920 for ethanol) between the data obtained by using the biosensors, on one hand, and by the refractometric or hydrometric methods, on the other. The operational stability of biosensors is determined by the inactivation of the immobilized enzymes rather than by leakage of a mediator from an electrode.