Calculating the output signal parameters of a lactose bienzymatic biosensing system from the transient phase response

Abstract

We constructed for the determination of lactose a bienzymatic biosensing system based on a fibre-optical oxygen sensor and two enzymes – β-galactosidase (β-gal, from Aspergillus oryzae, Sigma Aldrich, EC 3.2.1.23) and glucose oxidase (GOD, from A. niger, Sigma Aldrich, EC 1.1.3.4) and analysed how the calculation of biosensor output signal parameters, used for the calibration of lactose biosensors, is influenced by the data collection period during the transient phase of the signal rising in case no preliminary incubation period with β-gal was applied. The calculation of reaction steady state and kinetic parameters from the biosensor signal revealed that longer data collection periods resulted in more accurate biosensor calibration curves with bigger slopes, while in case of slower reactions the calculated reaction parameters had their maximal values already if data were collected for 600 seconds. For reactions where enzyme concentrations were higher (0.027–0.071 IU/mL β-gal and 2.03– 5.33 IU/mL GOD), the steady state signal was not achieved even within 1 hour from the initiation of the reaction and the calculated reaction parameters continued to change. Although the sensor signal was decreasing continuously, the reaction parameters calculated from the transient phase data were suitable for biosensor calibration if the data of at least 500 seconds were taken into consideration.