Analytica Chimica Acta 463 (2002) 39–51 Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation Mihaela Niculescu a,∗ , Thomas Erichsen b , Valentin Sukharev c , Zoltan Kerenyi d , Elisabeth Csöregi a , Wolfgang Schuhmann b a Department of Biotechnology, Lund University, P.O. Box 124, S-22100 Lund, Sweden b Analytische Chemie-Elektroanalytik & Sensorik, Ruhr-Universität Bochum, D-44780 Bochum, Germany c Department of Chemistry, Moscow State University, Ru-119899 Moscow, Russia d Research Institute for Viticulture and Enology of the Ministry of the Agriculture, H-6001 Kecskemet, Hungary Received 11 December 2001; received in revised form 15 April 2002; accepted 22 April 2002 Abstract This paper describes the development and optimization of an amperometric biosensor for monitoring ethanol in beverages. The biosensor is constructed by cross-linking a quinoprotein alcohol dehydrogenase (QH-ADH) to an Os-complex-modified poly(vinylimidazole) redox polymer using poly(ethylene glycol) diglycidyl ether. The optimum biosensor configuration was evaluated by changing the ratio between enzyme, redox polymer, and cross-linker using conventional graphite rods as basis electrodes. The optimized sensor showed a sensitivity of 0.336 ±0.025 A M -1 cm 2 for ethanol and a detection limit (calculated as three times the signal-to-noise ratio) of 1 M. This biosensor configuration was further evaluated in a conventional flow-injection system and the applicability for the determination of ethanol in diverse wine samples could be successfully demonstrated. Adaptation of this sensor configuration to screen-printed (SP) electrodes allowed their integration into an automated sequential-injection analyzer and the successful on-line monitoring of ethanol during wine fermentation processes. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Alcohol dehydrogenase; Ethanol; Biosensor; Fermentation; On-line process control; Redox polymer; Sequential-injection analysis 1. Introduction Analysis of ethanol is of great importance in medicine, biotechnology and food industry, due to its toxicological and psychological effects. In certain industrial processes, such as fermentation and dis- tillation, the ethanol concentration can reach toxic levels, causing inflammation of the nasal mucous membrane and conjunctiva, irritation of the skin and, ∗ Corresponding author. Fax: +46-46-2224613. E-mail address: mihaela.niculescu@biotek.lu.se (M. Niculescu). at high levels, even alcohol poisoning [1]. Moreover, a continuous, simultaneous and selective monitor- ing of glucose and ethanol (desirably together with other key compounds) is highly needed in the wine industry, since these parameters together do not only determine the quality and special flavor of a particular wine, but also allow, if necessary, a rapid intervention in the fermentation process, thus avoiding serious economical losses. Therefore, sensitive and selective analytical meth- ods are required for ethanol determination. Com- monly used methods are based on chromatographic 0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0003-2670(02)00344-6