An amperometric biosensor for polyphenolic compounds in red wine
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.
Section snippets
Reagents, standards and samples
Potassium chloride (99.5%), H2SO4 pro-analysis (95–97%), HCl (5%) AnalaR were from Merck. (+)-Catechin hydrate (98%), Na2HPO4 (99.9%) and KH2PO4 (99.7%), NaCH3COO·3H2O (99.8%) were from Sigma. NaCl (99.8%) and glacial acetic acid (99.8%) and density 1.05 kg/L were from Riedel-de-Haën. trans-3,4-Dihydroxycinnamic acid (caffeic acid) (97%), quercetin (98%), gallic acid (97%), rutin (95%) and trans-resveratrol (99%) were from Aldrich. Malvidin was from Extrasynthese. Laccase from Coriolus Versicolor
Selecting the applied potential for biosensor operation
The laccase based biosensor prepared as above described allowed a reduction current to be measured at an applied potential of −200 mV (versus Ag, AgCl) when catechol was used as substrate, in acetate buffer at pH 4.5. Linearity of 1.0–11.0 × 10−5 M and sensitivity of 0.0221 mAM−1 were already reported (Gomes and Rebelo, 2003).
In a first approach caffeic acid, gallic acid, catechin, rutin, trans-resveratrol, quercetin and malvidin were selected for the present study, as a polyphenolic model for red
Conclusions
A laccase based biosensor, able of discriminating between catechin and caffeic acid (10−5 M) in acetate buffer solutions pH 4.5 having 12% ethanol was developed by application of potentials +100 and −50 mV (versus Ag, AgCl), respectively, to a Pt electrode of a US base sensor. Both polyphenols were detected by the biosensor at pH 3.5 and those potentials, the response of the mixture being additive for equimolar (10−5 M) standard solutions of the substrates. A limit of detection of 1.0 × 10−6 M as
Acknowledgements
This work is part of a project: POCTI/36177/AGR/2000 from Fundação para a Ciência e a Tecnologia. Financial support is gratefully acknowledged.
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2019, Microchemical JournalCitation Excerpt :The insert graph shows the voltammogram of the standard compound CA, which represents a typical quasi-reversible oxidation process with an anodic peak at +412 mV and a cathodic peak at +217 mV. CA is one of the widely used standards in the study of total phenolic compounds in different matrices [44,45], exhibiting a greater relative sensitivity for biosensors that use laccase [8,10]. It is also one of the main constituents of propolis [46].