Real-time quantification of methanol in plants using a hybrid alcohol oxidase-peroxidase biosensor

An amperometric biosensor immobilizing two enzymes and an electron mediator in an identical plane has been fabricated by the self-assembly technique for determination of methanol in crude plant samples. A self-assembled mixed monolayer of 4,4'-dithiodibutyric acid covalently attached two enzymes (Hansenula sp. alcohol oxidase and horseradish peroxidase) and 11-ferrocenyl-1-undecanethiol as an electron mediator on an Au electrode is exploited to produce a two-dimensional reaction matrix. The composition of the two enzymes and electron mediator molecules was optimized for detection of methanol in 0.1 M sodium phosphate buffer (pH 6.0). We successfully quantified methanol in low-purity tobacco (Nicotiana tabacum) plant extracts with the biosensor, which showed sensitivity comparable to that of gas chromatography/mass spectrometry. The redox-relay biosensor is quite simple and stable due to its covalent attachment to the Au surface, making it possible to downsize the construction. We fabricated a miniature methanol biosensor that fitted a well of a 96-well micro assay plate available for high-throughput assay. The biosensor is advantageous for the sensitive, continuous, and convenient determination of methanol.