Enzyme and Microbial Technology 40 (2007) 661–668 A green bean homogenate immobilized on chemically crosslinked chitin for determination of caffeic acid in white wine Suellen Cadorin Fernandes, Inˆ es Rosane W. Zwirtes de Oliveira, Iolanda Cruz Vieira ∗ Departamento de Qu´ ımica, Universidade Federal de Santa Catarina, CEP 88040-900, Florian´ opolis-SC, Brazil Received 19 December 2005; received in revised form 25 May 2006; accepted 25 May 2006 Abstract A new green bean (Phaseolus vulgaris) tissue homogenate-based biosensor was developed for the square-wave voltammetric determination of caffeic acid in white wine. The biosensor was constructed by immobilization of green bean tissue homogenate, as a source of peroxidase, in a chemically crosslinked chitin matrix with epichlorohydrin and glutaraldehyde that was incorporated in a carbon paste electrode. In the presence of hydrogen peroxide the peroxidase catalyses the oxidation of caffeic acid to quinone and the electrochemical reduction of the product was obtained at a fixed potential of +0.10 V versus Ag/AgCl (3.0 mol L -1 KCl). The response characteristics and optimization of the bioelectrode design were evaluated. The recovery of caffeic acid from three samples ranged from 91.0 to 103.1% and a rectilinear calibration curve for caffeic acid concentrations from 2.0 × 10 -5 to 2.0 × 10 -4 mol L -1 (r = 0.9990) was obtained. The detection limit was 2.0 × 10 -6 mol L -1 and the relative standard deviation was 2.2% for a solution containing 1.2 × 10 -4 mol L -1 caffeic acid and 2.0 × 10 -3 mol L -1 hydrogen peroxide in 0.1 mol L -1 phosphate buffer solution at pH 7.0 (n = 10). The long-term stability of the biosensor was 300 days. © 2006 Elsevier Inc. All rights reserved. Keywords: Green bean (Phaseolus vulgaris); Chitin; Caffeic acid; Epichlorohydrin 1. Introduction After cellulose, chitin, -(1–4)-n-acetyl-d-glucosamine is the most important natural polysaccharide found in shells of crus- taceans or in fungal cell walls. It is a white, hard, inelastic, nitrogenous polysaccharide and the major source of surface pollution in coastal areas. This biopolymer is recommended as suitable for functional materials because of specific characteris- tics that include biocompatibility, biodegradability, non-toxicity, and adsorption. It is highly hydrophobic and is insoluble in water and most organic solvents, with poor solubility being the major limiting factor in its application. Despite this limitation, various uses of chitin and modified chitin have been reported [1–4]. Peroxidase (EC 1.11.1.7) is a member of a large group of enzymes called the oxido-reductases and it is considered to have an empirical relationship with off-flavors and off-colors in raw and unblanched vegetables. In post-harvest plant foods, peroxidases are believed to be responsible for deterioration in flavor, color, texture and nutritional qualities of raw and pro- cessed foods [5–7]. ∗ Corresponding author. Tel.: +55 48 3331 6844; fax: +55 48 3331 9711. E-mail address: iolanda@qmc.ufsc.br (I.C. Vieira). The development of biosensors and analytical procedures involving the use of vegetable tissues as biocatalysts has received considerable interest for the replacement of isolated enzymes. For example, Rechnitz’s group introduced tissue as a biocatalyst into the analysis via an electrochemical device [8]. However, the first biosensor based on a vegetable homogenate was constructed by Signori and Fatibello-Filho [9]. In this work, an amperomet- ric biosensor for the determination of phenols was developed by using a homogenate of yam (Alocasia macrohiza) as a source of polyphenol oxidase. The biosensor was constructed by immo- bilization of this homogenate with glutaraldehyde and bovine serum albumin on an oxygen membrane and this was used for the determination of phenols in industrial wastewaters. Enzymes are immobilized onto or into a solid support to increase their thermostability, operational stability and recov- ery. Several different methods have been employed for enzyme immobilization on materials, including entrapment in polymeric gels, encapsulation in membranes, crosslinking with bifunc- tional or multifunctional reagents and linking to an insoluble carrier [10–12]. Phenolic acids are widely distributed in the plant kingdom and present in, for example, tea, wine, fruits, beverages and various medicinal plants. Caffeic acid is a derivative of cin- 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.05.023