A kinetic approach to the formation of two-mediator systems for developing microbial biosensors as exemplified by a rapid biochemical oxygen demand assay

This work proposes a method of forming a microorganism–mediator(s) receptor system, in which the rates of separate stages of mediator bioelectrocatalysis are used as the basis for the development of biosensors for the biochemical oxygen demand (BOD) rapid assay. In the presence of a ferrocene mediator, the yeast Blastobotrys adeninivorans was shown to enable oxidation of a larger range of substrates as compared with other investigated microorganisms—bacteria Escherichia coli and yeast Ogataea polymorpha. The rate constants of the interaction of the yeast B. adeninivorans with nine compounds, electron transfer mediators, were determined; the best mediator for these microorganisms was found to be neutral red ( k int = 0.681 ± 0.009 dm 3 /g s). Neutral red possesses a high rate of interaction with the ferrocene mediator (14,200 ± 100 dm 3 /mol s) shown earlier to be the most promising acceptor of electrons at a carbon paste electrode (0.4 ± 0.1 cm/s). These features enabled the formation of a two-mediator ferrocene–neutral red system to be used in a biosensor. A two-mediator-based biosensor had a higher sensitivity (the lower limit of detected BOD concentrations, 0.16 mg/dm 3 ) than that of a one-mediator system based on neutral red and ferrocene. Analysis of ten samples from surface water reservoirs showed the combination of ferrocene, neutral red and the yeast B. adeninivorans to enable the data that highly correlated ( R = 0.9693) with those of the standard method.