Biosensors to Detect Phenolic Compounds

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Chapter 3 Preparation of PEDOT-Sensors

This chapter has been partially published in:
KARAOSMANOGLU, H., TRAVAS-SEJDIC, J. & KILMARTIN, P. A. 2014. Designing PEDOT-Based Sensors for Antioxidant Analysis. The International Journal of Nanotechnology, 11, 445-450.

Introduction

Cyclic voltammetry is a rapid, simple and efficient technique to evaluate antioxidants in real samples. This method has been used for research on antioxidant determination, making use of the redox properties of antioxidant compounds, and has been used with various samples from foods to bodily fluids. In order to compare and confirm the results, the most commonly used chromatographic and spectrophotometric methods for antioxidants have been performed alongside cyclic voltammetry (CV). Zielinska et al. have used CV of a bare electrode to test the antioxidant activity of plant extracts such as buckwheat (Zielinska et al., 2007b, Zielinska et al., 2007a), lupin sprouts (Zielinska et al., 2008a) and onions (Zielinska et al., 2008b).
Chevion et al. have worked on both edible plants (Chevion et al., 1999) and body fluids (Chevion et al., 1997a, Chevion et al., 1997b). The antioxidant activity of bodily fluids gives information about oxidative stress and the health condition of the body. Kohen et al. have performed CV with rat tissue homogenates and bodily fluids (Kohen et al., 1992). Evaluation of oxidative damage in biological samples (E. coli, rat jejunal mucosa and enzyme) has also been carried out by CV (Kohen, 1993). The results suggested that oxidative stress causes a decrease in the level of antioxidants, and this decrease can be determined by a change in the voltammetric response of samples. CV has been used in head injury cases for evaluation of antioxidant levels (Lomnitski et al., 1997, Beit-Yannai et al., 1997, Shohami, 1999).
Kilmartin et al. have undertaken electrochemical analyses of antioxidants in wines and teas. The main antioxidants present in wines and teas have been tested individually by cyclic voltammetry and compared with voltammograms of wine samples (Kilmartin et al.,2001, Kilmartin et al., 2002b, Zou et al., 2002, De Beer et al., 2004, Kilmartin and Hsu, 2003). The results were also compared with HPLC analyses and total phenolic content given by the Folin-Ciocalteu assay. The CV method was useful in the evaluation of the antioxidant content and for monitoring wine making processes.
However, these studies had been performed using a bare glassy carbon electrode, using an electrode that is at least 3 mm in diameter. Various modified electrodes have been investigated to improve the electrochemical analysis, including conducting polymers. Poly(3-methylthiophene), polypyrrole and polyaniline have been used to develop sensors for the quality control of olive oil (Guadarrama et al., 2000b, Guadarrama et al., 2001) and for wine analyses (Guadarrama et al., 2000a). In further research (Parra et al., 2006), an e-tongue has been developed using polypyrrole for wine analysis. The developed system showed good discrimination and recognition ability in terms of wine origin, grape variety etc. A multisensory system composed of six polypyrrole electrodes, using six doping agents and five sensors based on lanthanide bisphthalocyanines, has been developed for the evaluation of the phenolic content and bitterness of extra virgin olive oils (Rodríguez-Méndez et al., 2008). The amperometric measurements for wine analysis were found to be faster and simpler at PEDOT electrodes compared to bare metal electrodes (Pigani et al., 2008). In another study (Atta et al., 2011), a PEDOT electrode has been used for CV analysis in the selective determination of dopamine in the presence of ascorbic acid, and making use of a sodium dodecyl sulfate surfactant. Recently, electrochemical analysis for wine applications has been performed to determine concentrations of sulfur dioxide, polyphenol and ascorbic acid at a PEDOT-covered 1 mm dia. gold electrode (Türke et al., 2012).
The aim of this project is to design a PEDOT-based sensor to analyse antioxidants in beverages. Considering previous studies, a number of parameters were tested and compared to find the optimum conditions. The optimization experiments and results will be presented in this chapter.

Materials and Methods

Reagents

3,4-ethyelenedioxythiophene (EDOT), lithium perchlorate (LiClO4), sodium perchlorate (NaClO4), propylene carbonate (PC), phosphate buffered saline (PBS) tablets, catechin and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich. To prepare PBS solution (pH 7.4), one PBS tablet was dissolved in 200 ml of Milli-Q water. The individual solutions at 0.2 M concentration were prepared in Milli-Q water and mixed to achieve the desired pH. Green tea was purchased from a local store in New Zealand and prepared by inserting a tea bag containing 2.0 g tea into 200 ml of Milli-Q water at 100 ⁰C for 10 min.

Electrodes

An Ag/AgCl electrode (BAS MF-2052) was used as reference electrode and kept in 3 M NaCl solution when not in use. A platinum wire (BAS MW-1032) was used as the counter electrode. A 1 mm dia. glassy carbon electrode (eDAQ ET-074) and a 1 mm dia. gold electrode (eDAQ ET-076) were used as the working electrode substrates. Before each polymerization, the surface of the electrode was polished using 0.05 µm alumina powder (BAS CF-1050) on a cloth pad (BAS MF-1040). After rinsing with Milli-Q water, the electrode was dipped into a small vial filled with Milli-Q water and kept in sonic bath for 2 min. In order to confirm the cleanliness of the gold electrode, cyclic voltammetry was also performed in 0.5 M H2SO4 solution between -400 and 1200 mV for 10 cycles (Fischer et al., 2009).

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Preparation of a PEDOT Electrode

A PEDOT-electrode was prepared by electrochemical polymerization of the EDOT monomer using cyclic voltammetry, as previously reported (Türke et al., 2012), but with slight modifications. After preparing the polymerization solution containing 0.1 M EDOT and 0.1 M LiClO4 in propylene carbonate, oxygen in the solution was removed by purging with nitrogen. Electropolymerization was performed by cycling between -300 and 1200 mV at a scan rate of 100 mVs-1 using a Bioanalytical Systems (BAS) 100A electrochemical analyser. A different number of preparative cycles were applied to check the effect of film thickness on sensor performance. The prepared electrode was then rinsed with Milli-Q grade water to remove unpolymerized monomer from the PEDOT film.

Cyclic Voltammetry of Samples

Green tea was used as a real test sample. Catechin and EGCG, two major antioxidants in green tea, were also tested as pure antioxidant compounds, and were prepared as solutions in PBS (pH 7.4). Electrochemical analysis of the samples was performed by cyclic voltammetry, and in comparison to a bare glassy carbon electrode. Before each measurement, the electrode was dipped into PBS solution containing 0.1 M sodium perchlorate. The potential was cycled between -200 and 800 mV at 100 mVs– 1 for 10 cycles in order to accustom the electrode to an aqueous environment before sample testing. After this step, CV was performed either from -200 to 600 mV or from -200 to 800 mV at 100 mVs-1 for 1 cycle. To create a background voltammogram, the first CV was performed in PBS containing 0.1 M sodium perchlorate.

Determination of Linear Concentration Range

In order to determine the linear concentration range of the antioxidant solutions, different dilutions were tested. Catechin solutions were prepared in PBS at concentrations of 0.1, 0.5, 0.75, 1.0, 1.5 and 2.0 mM. On the other hand, EGCG solutions were prepared in PBS at concentrations of 45, 90, 30, 450, 600 and 900 µM. The CVs were performed between -200 and 800 mV at a 100 mVs-1 scan rate. The plots of oxidation peak current versus concentration were drawn for evaluation.

Glassy Carbon vs. Gold Electrodes

Polymerization of EDOT was performed using 1 mm dia. gold and glassy carbon electrodes by cycling between -300 and 1200 mV for 4 cycles at a 100 mVs-1 scan rate. The prepared electrodes were used in testing catechin solutions (2 mM, pH 7.4) and the voltammograms were compared.

Reusability of PEDOT Electrodes

Due to fouling on a bare electrode surface, the electrode needs to be cleaned before each measurement. To examine the reusability of PEDOT electrodes, a PEDOT electrode was first used for cyclic voltammetry in a 2 mM catechin solution. Then, after rinsing the electrode with Milli-Q water, it was dipped into a new buffer solution and CV was performed between -200 and 800 mV.

Results and Discussion
Effect of PEDOT Thickness

The PEDOT polymer film thickness is related to the number of cycles used in the electropolymerization. Polymerization of EDOT was performed on a glassy carbon electrode with 1, 2, 3 and 4 cycles between -300 and 1200 mV. CV was performed firstly in PBS solution containing 0.1 M NaClO4. The background CVs showed a capacitance-type response when the electrodes were cycled in an aqueous electrolyte with no redox-active species present in the solution, as has been observed previously for PEDOT electrodes (Liu et al., 2008). The comparison of background voltammograms obtained by a bare and PEDOT covered electrodes are shown in Figure 3.1. Psuedo-capacitance values were calculated by dividing the average of cathodic current with scan rate (100 mVs-1) (Khomenko et al., 2005). Therefore, it is found to be 70, 75, 88, 96 and 123 µF for the bare, PEDOT1, PEDOT2, PEDOT3 and PEDOT4, respectively. The larger values for the PEDOT electrode will include contributions from internal PEDOT redox processes, and an increase in the surface capacitance as the real surface area of the electrodes increased.
Figure 3.1. Cyclic voltammograms of the bare and PEDOT covered electrodes in PBS containing 0.1 M sodium perchlorate. They were prepared by one (PEDOT1), two (PEDOT2), three (PEDOT3) and four (PEDOT4) preparative electrochemical cycles.
The electrodes were then cycled in a 0.5 mM catechin solution from -200 to 800 mV and the intensity of the oxidation peaks was compared (Figure 3.2.). Voltammograms showed an oxidation peak for catechin at around 200 mV and the intensity of the peak current increased with a greater number of PEDOT preparative cycles. The PEDOT electrode prepared by 4 cycles gave better defined peaks than the other electrodes. As seen in Figure 3.2., the electrode with a PEDOT covering also provided a better separation and clarity of the two catechin oxidation peaks (at around 200 and 500 mV). Cycling to a higher number of cycles was also undertaken but no change in the response was obtained (data not shown).

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Adjustment of a PEDOT Electrode into an Aqueous Environment

Electrochemical analysis of beverages was needed to be performed in buffered solution to keep the pH at similar level to the respective samples. When the PEODT-electrode was cycled in PBS for 10 cycles, the current intensity changed gradually cycle-by-cycle until a near-steady response was obtained due to the counterion changing with phosphate ions instead of perchlorate ions (Figure 3.3A). A comparison of voltammogram at the first and the tenth cycles is shown in Figure 3.3B. Therefore, the baseline became same as the sample. Also, in a previous study (Pigani et al., 2011), the prepared PEDOT electrode was cycled in PBS containing LiClO4 between -0.5 and 0.5 V vs. Ag/AgCl at 0.05 Vs-1 for 10 cycles in order to reach to a steady state PEDOT conformation and to obtain repeatable results. Therefore, 10 cycles were performed in a PBS solution containing 0.1 M NaClO4 before recording the background response and undertaking sample analysis.

Comparison CV of a Bare and PEDOT Covered Electrodes

A PEDOT electrode was prepared by cycling for four cycles with a potential range of -300 to 1200 mV at a 100 mVs-1 scan rate. After rinsing with Milli-Q water, the electrodes were dipped into a PBS solution containing 0.1 M NaClO4 and cycled for 10 cycles between -200 and 800 mV at 100 mVs-1. Green tea and catechin solutions were then tested, with catechin at a concentration of 2 mM in PBS. Green tea was tested without any dilution, which is not typically undertaken when using glassy carbon electrodes due to the effects of the very high flavonoids present (Kilmartin and Hsu, 2003). For both samples, the intensity of the anodic and cathodic peaks was greatly enhanced due to the presence of the PEDOT film (Figure 3.4A). Moreover, the peaks became more distinguishable and sharper. An oxidation peak at 500 mV and a smaller reduction peak at 300 mV were observed in the voltammogram of green tea (with a measured solution pH of 5.5), while the bare electrode did not show any distinguishable oxidation or reduction peak. In the voltammogram of catechin, three oxidation peaks (265, 510 and 725 mV) were observed at the PEDOT electrode, and one reduction peak (150 mV) (Figure 3.4B).
Figure 3.4. Cyclic voltammograms (background subtracted) of green tea (A), and a 2 mM catechin in pH 7.4 PBS solution (B), using bare and PEDOT covered glassy carbon electrodes.
While the main oxidation peak current of the green tea sample was measured as ̴2.5 µA at a bare glassy carbon electrode, it increased to ̴30 µA at the PEDOT electrode. Similarly, the first oxidation peak of catechin (at 240 mV) increased from ̴2.5 µA to ̴10.6 µA with an electrode covered with PEDOT. Moreover, the oxidation peaks shift to positive potential in solution at lower pH values (Janeiro and Oliveira Brett, 2004), and the pH of the green tea sample was found to be 5.5. Therefore, the potential range is important for cyclic voltammetry since the oxidation peak at high potentials might not be observed in the solutions at lower pH values when CV was performed to 600 mV.
Cyclic voltammetry has been typically been performed using a 3 mm dia. glassy carbon electrode to analyse beverage antioxidants in previous studies (Kilmartin and Hsu, 2003, Kilmartin et al., 2001). When a bare 1 mm dia. glassy was used, it did not give sufficiently well-defined signals. However, when the 1 mm electrode was covered with a PEDOT film, the conducting polymer enhanced the signal and the voltammograms showed measurable and well-defined peaks by the help of the redox mediator property of PEDOT.

TABLE OF CONTENTS
Abstract 
Dedication 
Acknowledgments 
Publications and Presentations 
Table of Contents 
List of Figures 
List of Tables 
List of Appendices 
List of Symbols and Abbreviations 
Chapter 1. Introduction
1. Introduction
1.1. The Outline of This Thesis
Chapter 2. Literature Review
2.1. Antioxidants in Beverages
2.2. Cyclic Voltammetry
2.3. Enzymes
2.4. Conducting Polymers
2.5. Conducting Polymer Based Sensors
2.6. Biosensors to Detect Phenolic Compounds
Chapter 3. Preparation and Optimization of PEDOT- 
Sensor
3.1. Introduction
3.2. Materials and Methods
3.3. Results and Discussion
3.4. Conclusion
Chapter 4. Analysis of Beverage Antioxidants Using a PEDOT-Based Sensor
4.1. Introduction
4.2. Materials and Methods
4.3. Results and Discussion
4.4. Conclusion
Chapter 5. Comparison of Cyclic Voltammetry with Spectrophotometric Methods
5.1. Introduction
5.2. Materials and Methods
5.3. Results and Discussion
5.4. Conclusion
Chapter 6. Polymerization of EDOT in Aqueous Solutions
6.1. Introduction
6.2. Materials and Methods
6.3. Results and Discussion
6.4. Conclusion
Chapter 7. Designing a Biosensor Using Tyrosinase and PEDOT
7.1. Introduction
7.2. Materials and Methods
7.3. Results and Discussion
7.4. Conclusion
Chapter 8. Conclusion and Future Work
8.1. Conclusion
8.2. Future perspectives
References
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PEDOT Electrodes as Redox Mediators for Determination of Antioxidants in Beverages

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