Integrated Electrochemical Gluconic Acid Biosensor Based on Self-Assembled Monolayer-Modified Gold Electrodes. Application to the Analysis of Gluconic Acid in Musts and Wines S. CAMPUZANO, M. GAMELLA, B. SERRA, A. J. REVIEJO, AND J. M. PINGARRO Ä N* Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain An integrated amperometric gluconic acid biosensor constructed using a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which gluconate dehydrogenase (GADH, 0.84 U) and the mediator tetrathiafulvalene (TTF, 1.5 μmol) were coimmo- bilized by covering the electrode surface with a dialysis membrane is reported. The working conditions selected were E app )+0.15 V and 25 ( 1 °C. The useful lifetime of one single TTF-GADH-MPA- AuE was surprisingly long. After 53 days of continuous use, the biosensor exhibited 86% of the original sensitivity. A linear calibration plot was obtained for gluconic acid over the 6.0 × 10 -7 to 2.0 × 10 -5 M concentration range, with a limit of detection of 1.9 × 10 -7 M. The effect of potential interferents (glucose, fructose, galactose, arabinose, and tartaric, citric, malic, ascorbic, gallic, and caffeic acids) on the biosensor response was evaluated. The behavior of the biosensor in a flow-injection system in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining gluconic acid in wine and must samples, and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit. KEYWORDS: Self-assembled monolayers; enzyme biosensors; gluconic acid; wines INTRODUCTION Gluconic acid is one of the predominant organic acids found in honey, vinegars, and noble rot wine, which are produced from grapes infected with the fungi Botrytis cinerea (1). This infection, called gray rot disease, produces skin contraction and dehydration of grape berry, with the subsequent increase in polysaccharides concentration (sugar amounts up to 30-40° Brix are attained). B. cinerea produces fissures in the berry allowing proliferation of acetic acid bacteria (Acetobacter and Gluconobacter), which are fed by the sweet juice that escapes from the berry, thus increasing the gluconic acid concentration above 3 g/L and the volatile acidity of musts (2). Moreover, the contents of tartaric acid, malic acid, and available nitrogen decrease, and the concentration of glycerol increases in grapes affected by B. cinerea (2). The gray rot disease develops during growth of grape berry and is dependent on meteorological factors, such as moisture and rainfall, and on physiological factors, such as grape variety, bunch shape, and viticultural practices (3). This disease causes substantial losses to wine farmers, as it decreases the quality and size of crops, and to winemakers, as a result of microbio- logically induced changes in grape composition. The rot extent affecting grapes is usually related to the gluconic acid concentration in the resulting must. Thus, concentrations above ∼1 g/L reflect a substantial proportion of rotten grapes. Recently, Couto et al. (4) have evidenced that levels of gluconic acid up to 1 g/L indicate an initial stage of grape infection by fungi, whereas higher levels (up to 2-3 g/L) might be taken as indication of the activity of acetic acid bacteria. D-Gluconate has been usually determined by high perfor- mance liquid chromatography (5-7), gas chromatography (8), capillary electrophoresis (9), near-infrared reflectance spectro- scopy (10), and enzyme assays with spectrophotometric (11) or fluorimetric determination (1), but these methods are time- consuming. Although some enzyme electrodes using D-gluconate dehydrogenase have been developed, they were not applied to determine gluconic acid in foodstuffs (12-14). Recently, we have reported on the construction and perfor- mance of robust integrated amperometric enzyme biosensors in which the biomolecules were coimmobilized, together with the mediator tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop 3-mercaptopropionic acid (MPA) self- assembled monolayers (SAMs) on gold disk electrodes (AuEs) (15, 16). Therefore, following a somewhat similar approach, we report, in this Article, the construction and performance of an integrated * Author to whom correspondence should be addressed [telephone ++0034 91 394 4315. Fax: ++0034 91 394 4329. E-mail: pingarro@quim.ucm.es]. J. Agric. Food Chem. 2007, 55, 2109-2114 2109 10.1021/jf063073w CCC: $37.00 © 2007 American Chemical Society Published on Web 02/27/2007 Downloaded by KING MONGKUT UNIV TECH THONBUR on September 15, 2009 | http://pubs.acs.org Publication Date (Web): February 27, 2007 | doi: 10.1021/jf063073w