PEDOT Electrodes as Redox Mediators for Determination of Antioxidants in Beverages

Abstract

Antioxidants play important roles in food quality and the protection and the promotion of human health. The most commonly used antioxidant determination techniques are the Folin-Ciocalteu assay and HPLC analyses. However, these methods have various disadvantages such as long time requirements, low sensitivity, interfering agents etc. Cyclic voltammetry at inert electrodes is another method for antioxidant determination. Although this method is easy to apply and gives rapid results, the sensitivity of an electrode with a 1 mm dia. is not adequate to analyse antioxidants at levels present in beverages. In this study, the detection of beverage antioxidants was achieved by covering the electrode with a poly3,4-ethylenedioxythiophene (PEDOT) film, which is a conducting polymer. Polymerization was performed electrochemically with 0.1 M 3,4-ethylenedioxythiophene (EDOT) and 0.1 M lithium perchlorate in propylene carbonate (PC) using cyclic voltammetry at 1 mm dia. electrodes. Glassy carbon and gold electrodes substrates were tested and better results were obtained with the glassy carbon electrode. Polymerization was performed using different potential ranges and number of preparative cycles in order to find the optimum conditions. Green tea, catechin and epigallocatechin gallate solutions were used as test standards for system optimization. The optimum number of preparative cycles and potential range were determined to be cycling four cycles between -300 and 1200 mV vs. Ag/AgCl. Tea and coffee are among the most frequently consumed beverages, and are rich in polyphenol antioxidants. Green, black and herbal teas, different types of coffee and red wine samples, along with polyphenol standards, were tested by cyclic voltammetry using an electrochemically polymerized PEDOT-covered glassy carbon electrode. The voltammetric response of the coffee samples resembled that of chlorogenic acid, which was also determined to be the major polyphenol present by HPLC analysis. In the voltammograms of green tea, there were two oxidation peaks seen at around 200 and 300 mV (Ag/AgCl) at pH 5.5, assigned mainly to epigallocatechin gallate (EGCG). Moreover, simulation solutions of green, black teas and coffee were prepared depending on their phenolic compound profiles and were subjected to CV testing, and a similar response was obtained as with the real samples. The anodic peak area was used to estimate the total phenolic content (TPC) of the samples. When these TPC values were compared with alternative spectrophotometric assays, correlations of 0.87, 0.73 and 0.75 were obtained with the Folin-Ciocalteu, DPPH and ABTS assays, respectively. EDOT is poorly soluble in water; however, an aqueous environment was required for the next study involving an enzyme. In order to overcome this problem, two types of solutions were tested; the first one was an aqueous solution containing EDOT, LiClO4 and sodium dodecyl sulphate (SDS) and the second one had different ratios of acetonitrile/water containing EDOT and LiClO4. Polymerization was performed by cyclic voltammetry and the sensitivity of PEDOT-electrodes were compared by testing gallic acid and green tea solution, versus PEDOT prepared in propylene carbonate. The oxidation potential of EDOT decreased in the aqueous solutions compared to that in the organic solvent. Furthermore, the PEDOT electrode prepared in SDS solution system showed a higher signal for the tested antioxidants compared to electrodes prepared in acetonitrile/water solutions. In the final part of this study, an amperometric biosensor has been developed to quantify specifically the phenolic antioxidant content of beverages. Tyrosinase, which oxidizes polyphenols, was physically entrapped into PEDOT during electrochemical polymerization by a cyclic voltammetry preparation. Different CV parameters were used to find optimum conditions for enzyme entrapment and to obtain a better biosensor. The concentration of quinones formed by enzymatic reaction was determined by a constant potential application of –100 mV. Catechol was used as a model substrate. The biosensor prepared by 4 cycles in the polymerization solution containing 2 mg/mL tyrosinase gave the highest sensitivity (0.0020 μA/μM). The optimum pH and temperature of the biosensor were found to be pH 6.5 and 40 ⁰C. Enzyme kinetic studies showed that the entrapment of tyrosinase into the PEDOT film was favourable, since the Km value decreased compared to that of free enzyme. A few phenolic standards and beverages were also successfully tested with the biosensor.