An amperometric biosensor for polyphenolic compounds in red wine

Abstract

In the present work, a biosensor was developed with Laccase Coriolus Versicolor as the biological reconnaissance element immobilized on derivatized polyethersulphone membranes and applied to a Pt–Ag, AgCl US electrode base. Its application to several polyphenols usually found in red wine (caffeic acid, gallic acid, catechin, rutin, trans-resveratrol, quercetin and malvidin) was tested. It was observed that an amperometric response was obtained for catechin at +100 mV (versus Ag, AgCl) and caffeic acid at −50 mV in acetate buffer solutions (pH 4.5) having 12% ethanol. At pH 3.5 and +100 mV the biosensor was sensitive to both substrates and their response was additive. A limit of detection of 1.0 × 10⁻⁶ M, linearity ranging from 2.0 to 14.0 × 10⁻⁶ M, high sensitivity (0.0566 mAM⁻¹) and reproducibility (R.S.D. < 10%) were achieved for equimolar mixed solutions of catechin and caffeic acid.

Under the same experimental conditions the other polyphenols tested individually did not yield any biosensor response.

The application of the biosensor to red wine samples required a previous solid phase extraction for polyphenols enrichment. In fact, attempts to apply the biosensor in red wine using the “standard addition” methodology showed that large interferences occurred, as was to be expected. Reduction currents of −0.33 ± 0.03 nA were obtained when the biosensor was used with the wine extract at +100 mV. This current could be ascribed to catechin and caffeic acid, although some interference by other polyphenols at the matrix level seemed to persist. The present biosensor showed promising applications for the wine analysis in future.

Introduction

The development of new biosensors for analytical purposes is still progressing. It is recognized that a judicious choice of the biological receptor and applied potential greatly improves their selectivity. Biosensors will have a more central role in future due to their application in various fields, such as food and agriculture products processing, medicine and pollution monitoring. An amperometric biosensor is an attractive alternative to analytical methods, e.g. HPLC, used to monitor phenolic compounds in red wine. Some advantages of biosensors, relative to chromatographic techniques, are their rapid response, cost-effectiveness, simplicity of operation and manufacturing, minimal sample pretreatment involved and solvent requirements (Turner et al., 1987).

Laccase is a polyphenoloxidase that catalyses the oxidation of 1,2-dihydroxybenzene group on the flavanols, and is oxidized back to its oxidized form by oxygen, which reduces to water. The product of the enzyme oxidation: 1,2-benzoquinone is then reduced at the electrode (Cummings et al., 1998). The actual mechanism of reaction is still unclear. From spectroscopic and electron paramagnetic resonance (EPR) studies it has been shown that the enzyme is first completely reduced, then oxygen is reduced to water, concomitant with the formation of radical intermediates.

Immobilization of laccase in order to use it in electrochemical assays has been done on different solid bases: carbon fibres (Freire et al., 2002), redox hydrogel on glassy carbon (Leech and Daigle, 1998), graphite (Yaropolov et al., 1995), carbon paste (Leite et al., 2003), polyethersulphone membranes (Gomes and Rebelo, 2003) and others. While the present paper was being reviewed other papers appeared (Quan et al., 2004) where the immobilization was done on platinum.

Antioxidant components of wine including catechin, have been claimed to protect low-density lipoproteins (LDL) against oxidation more effectively than other antioxidants, such as alfa-tocopherol. Other polyphenols existing in wine: quercetin and rutin have anti-cancer effects (Goldberg et al., 1996). Polyphenols are also important for their organoleptic properties, giving astringency, odour and savour to wine, olive oil (Romani et al., 2000) tea and other fruits. Thus, the development of new biosensors for the polyphenolic fraction of red wines is a great ongoing challenge that will have a strong impact in the beverages industry.

The present contribution aimed at developing new amperometric biosensors for polyphenolic compounds and their potential application in red wines.