MICROBIAL O-2-ELECTRODE AND H2O2-ELECTRODE SENSORS FOR ALCOHOL ASSAYSBASED ON THE USE OF PERMEABILIZED MUTANT YEAST-CELLS AS THE SENSITIVEBIOELEMENTS

Two types of alcohol-specific microbial/electrochemical biosensors hav e been developed using specially constructed mutant cells of the methy lotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O-2 electrode based bio sensor contained mutant cells with strongly elevated alcohol oxidase a ctivity. The peroxide electrode based biosensor consisted of catalase- defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized for m in order to release some components of the cellular respiration syst em, thus increasing the selectivity of the cellular respiration respon se to alcohol (cell/O-2-biosensor). Permeabilization also increased se nsitivity of the signal and shortened the response time (cell/H2O2-bio sensor). Cell/O-2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM fo r ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operatio nal and storage stability. The use of sucrose, dulcitol or inositol du ring the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Bot h biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates. (C) 1998 Elsevier Sci ence S.A. All rights reserved.