Monitoring the activity of glucose oxidase during the cultivation of Aspergillus niger using novel amperometric sensor with 1, 1′-dimethylferricinium as a mediator

1, 1′-dimethylferricinium (DMF⁺), a deep blue, and stable mediator, was prepared from a water-soluble 1, 1′-dimethylferrocene(DMF):2-hydroxypropyl-β-cyclodextrin complex via enzymatic oxidation using immobilised bilirubin oxidase. This mediator was superior to other soluble ferrocenes, notably carboxyferrocene, in terms of both solubility (110 mM vs 0·5 mM) and oxidation potential (150 mV vs 300 mV against Ag/AgCl). Although the cyclic voltammogram of DMF⁺ was electrochemically equivalent to DMF, the use of the former resulted in a significantly lower background current (<10 nA vs 30 nA). Because of its higher solubility, concentrated stock solutions of DMF⁺ can be prepared and supplied to the electrode. This is of particular importance when the signal is severely limited by the rate at which the working electrode can oxidise DMF to DMF⁺. A linear response of current versus units of glucose oxidase (GOD) was obtained up to 0·5 unit/ml. The detection limit was estimated to be 0·03 unit/ml and the response time was 2·5 min or less. The amperometric system was used successfully to follow the GOD activity during the growth of Aspergillus niger a well-known GOD producer. The results obtained correlated well with a standard absorbance-based assay using dichlorophenol-indophenol (DCPIP). The K_M of GOD for the glucose in the lysate was measured as 38 mM. A reduced response and higher K_M (48 mM) of the cell homogenate, compared to the lysate, illustrated the requirement for the DMF⁺ and glucose to diffuse across the cell membrane to interact with GOD in whole cells.