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.
Hasunuma
Tomohisa
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
Kuwabata
Susumu
S
Fukusaki
Ei-ichiro
E
Kobayashi
Akio
A
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002138:Calibration; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010936:Plant Extracts / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014026:Nicotiana / Q000737:chemistry*
D004800:Enzymes, Immobilized; D010936:Plant Extracts; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D006735:Horseradish Peroxidase; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D000432:Methanol