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Sensors and Materials, Volume 34, Number 6(2) (2022)
Copyright(C) MYU K.K.
pp. 2105-2124
S&M2959 Research Paper
Published: June 15, 2022

A Thiol-reactive Phenazine Ethosulfate – A Novel Redox Mediator for Quasi-direct Electron-transfer-type Sensors [PDF]

Maya Fitriana, Kentaro Hiraka, Kazunori Ikebukuro, Koji Sode, and Wakako Tsugawa

(Received March 22, 2022; Accepted May 17, 2022)

Keywords: thiol-reactive phenazine ethosulfate, quasi-direct electron transfer, redox probe, enzyme modification, lactate oxidase, electrochemical enzyme sensor, biomedical engineering

A novel redox mediator, thiol-reactive phenazine ethosulfate (trPES), was used to modify an enzyme for the first time for biosensor development. Aerococcus viridans-lactate oxidase (LOx), widely used to study lactate biosensors, was modified with a single trPES molecule. A cysteine mutation was introduced into the vicinity of the LOx cofactor to enable the modification by trPES. LOx cysteine mutants were then successfully modified using trPES, thus acquiring quasi-direct electron transfer ability. An electrode immobilized with the trPES-LOx A96L/S210C mutant showed the highest amperometric response currents among the modified LOx cysteine mutants, indicating efficient electron transfer. The position around residues S210 to N212 on the LOx surface (distance <14 Å from FMN N5) is important for the mediator to access the reduced flavin. Then, the performances of the lactate sensor were improved by utilizing LOx A96L/S210C modified with trPES and arPES, another redox mediator used to modify a lysine residue. The lactate sensor has a detection range of up to 1 mM, a sensitivity of 6.62 µA/mM.cm2, and a limit of detection of 9.9 µM. Furthermore, Aspergillus flavus-derived FAD glucose dehydrogenase was successfully modified with trPES and the response currents were obtained, showing the versatility of trPES for modifying other oxidoreductases.

Corresponding author: Wakako Tsugawa

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Cite this article
Maya Fitriana, Kentaro Hiraka, Kazunori Ikebukuro, Koji Sode, and Wakako Tsugawa, A Thiol-reactive Phenazine Ethosulfate – A Novel Redox Mediator for Quasi-direct Electron-transfer-type Sensors, Sens. Mater., Vol. 34, No. 6, 2022, p. 2105-2124.

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