Development of PEDOT-modified Electrochemical Sensors for Uric Acid Analysis in Milk

Abstract

Milk antioxidants such as ascorbic acid and uric acid are natural free radical scavengers, or reducing agents, which play a crucial role in milk quality. These two antioxidants can be quantified using conventional techniques such as HPLC and spectrophotometry. However, these methods are tedious, time-consuming, reagent-intensive and often of low sensitivity. An alternative technique is an electroanalytical method, cyclic voltammetry at inert electrodes, one that is fast and easy to use for the detection of low molecular weight antioxidants. However, with many electrodes, the electrochemical signals can be weak and poor separation of uric acid and ascorbic acid peaks is seen with milk samples. To improve the reponse, a conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), was used in this thesis to modify the electrode surface. PEDOT was electropolymerized as a thin layer on a 3 mm glassy carbon electrode using cyclic voltammetry in a propylene carbonate electrolyte containing 0.1 M EDOT and 0.1 M LiClO4, and then acclimatized to an aqueous buffer before sample analysis. Uric acid and ascorbic acid standard solutions were used to investigate the electrochemical processes that occur. Uric acid oxidized at around 350 mV (Ag/AgCl), in pH 6.6 buffers, typical of untreated milk samples. A separate peak due to ascorbic acid was observed close to 0 mV, allowing the simultaneous detection of the two antioxidants. Ascorbic acid showed a stronger response in pH 3 solutions, a pH value obtained after treating milk with acids to remove protein material prior to HPLC analysis. Uric acid oxidation can be considered as a form of stripping voltammetry, while ascorbic acid oxidized under a diffusion-controlled processes, with a diffusion coefficient of 6.9 x 10⁻⁶ cm² s⁻¹ obtained from cyclic voltammograms. Good linear relationships between concentrations and peak current intensities were obtained in the range of 6 to 100 μM for uric acid and 30 to 500 μM for ascorbic acid. The sensitivity and limit of detection for the PEDOTmodified electrode were 2.5 μA μM⁻¹ cm⁻² and 7 μM for uric acid, and 0.6 μA μM⁻¹ cm⁻² and 45 μM for ascorbic acid, respectively. The effect of accumulation time on the oxidation of uric acid and ascorbic acid was investigated by cyclic voltammetry. Pre-adsorption of uric acid at the PEDOT surface with time led to an enhanced current responses, which also allows a more sensitive response to be obtained with low concentrations of uric acid. Remarkably, no strict attention in terms of accumulation time was essential to analyse uric acid in untreated milk. The PEDOT modified sensor was also able to analyse ascorbic acid in milk when present. The developed PEDOT-modified glassy carbon electrode was then used to determine UA concentration directly in untreated milk by cyclic voltammetry using an anodic peak current at around 330 mV (Ag/AgCl). Another anodic peak at about 650 mV was ascribed to additional reducing agents present in the milk. The effect of different parameters such as dilution, temperature, uricase and ascorbate oxidase enzymes was investigated on the electrochemical responses of milk. A series of 36 milks comprised of commercial and raw milks were analysed by the modified sensor, and then the concentration of UA was compared to those obtained using reversed-phase high-performance liquid chromatography. Good agreement was achieved between the two approaches with some differences with particular samples. The electrochemical behavior of PEDOT modified glassy carbon electrode for the analysis of uric acid was also compared to PEDOT modified gold microelectrodes (ca. 10 μm diameter). PEDOT was electropolymerizaed on the bare gold microelectrode using two electropolymerisation methods, a water/acetonitrile system, and the propylene carbonate electrode already employed with glassy carbon macroelectrodes. More rapid electropolymerisation was seen for PEDOT polymerized in the propylene carbonate solution, which was used to quantify uric acid in standard solutions, milk and flavoured milks in comparison with a PEDOT-modified glassy carbon macroelectrode (3 mm diameter). The PEDOT-gold microelectrode was tested in the presence of a standard ferri-ferrocyanide redox couple at different scan rates. While a typical plateau current was at a bare gold microelectrode, a diffusion-controlled redox peak at around 0.1 V (Ag/AgCl) was seen for scan rates greater than 0.025 V s-1 at the PEDOT microelectrode. Pre-adsorption of uric acid on the modified microelectrode showed a linear relationship between anodic peak current and uric acid concentration range from 6.25 to 200 μM, and the limit of detection, limit of quantification, and sensitivity was determined to be 7 μM, 24 μM, and 397 μA μM-1 cm-2 , respectively. Cyclic voltammograms of milks, chocolate milks and espresso milks showed important contributions from different phenolic antioxidants. In conclusion, reliable analysis of UA in milk samples without any pre-treatment was achieved by cyclic voltammetry as a simple, fast, and inexpensive technique. The resulting voltammograms showed good peak separations and higher current densities due to increase in surface area after PEDOT deposition on the electrode surface.