Immobilized Cytochrome c Sensor in Organic/Aqueous Media for the Characterization of Hydrophilic and Hydrophobic Antioxidants

Abstract

A method for the characterization of antioxidants is introduced, which allows the measurement of pure hydrophilic and hydrophobic substances as well as complex cosmetic creams. The sensor is based on cytochrome c covalently immobilized on a gold wire electrode working in mixtures of phosphate buffer and organic solvents. It is combined with a superoxide generating enzyme system. The decrease of the superoxide concentration in the test solution by the added antioxidants is detected and used for the quantification of their antioxidative efficiency. Electrochemical properties of immobilized cytochrome c, such as formal potential and heterogeneous electron transfer rate constant, have been investigated in mixtures of aqueous buffer and DMSO, methanol, butanediol, and THF. The maximum organic solvent content for quasi-reversible electrode behavior was correlated to spectroscopic measurements. The activity of the radical producing enzyme in such media was determined and the radical generation characterized. The antioxidative properties of pure substance such as ascorbic acid and Biochanin A as well as of five anti-ageing cosmetic creams were studied. This showed also the influence of matrix composition on the efficiency of antioxidative supplements.

Electrochemical Biosensors in Nonaqueous Solutions and Their Applications

“…The sensitivity of the monolayer electrode was directly influenced by the surface density of protein on the electrode 60 and the electron transfer rate between the electrode surface and cyt c. In order to broaden the range of applications, sensors have been developed for use not only in aqueous buffer but also in mixtures of aqueous and organic solvents. 61 A new type of binary SAMs composed of T-COOH and T-NH2 to modify the gold electrode mentioned above 28 provided a superior gold surface for attaching cyt c. This was then used to develop a superoxide sensor in aqueous and organic/aqueous media. 29 The sensor works by electrochemically detecting cyt c reduced by the superoxide radical generated by a xanthine-XOD system.…”

A sensor for superoxide in aqueous and organic/aqueous media based on immobilized cytochrome c on binary self-assembled monolayers

“…The decrease in the electrochemical activity of cyt c adsorbed on the surface of the electrode results in lower oxidation of superoxide radical. Secondly, the activity of XOD itself decreases in the mixtures of aqueous buffer and organic solvents, which also leads to a decrease in the current response of superoxide radical (Beissenhirtz et al, 2003). Finally, lower oxidation of superoxide radical arises from the decrease of catalytic activity between superoxide and cyt c in the mixtures.…”

“…In order to broaden the range of applications, sensors have also been developed for use not only in aqueous buffer but also in mixtures of the aqueous and organic solvents of a system (Beissenhirtz et al, 2003).…”

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

“…Electrochemical biosensors use two main sources of ROS: OH Å and O ÅÀ 2 . The former can be generated photocatalytically [7] or by Fenton reaction in DNA-based antioxidant sensors [8,9], and the latter is mostly enzymatically [2,10,11] but also chemically [3,12,13] or electrochemically [14] formed for the determination of both superoxide radical and AOC. Sensors based on O ÅÀ 2 commonly rely on the immobilization of cytochrome c, which is reduced by superoxide radical, on gold [2][3][4], carbon [15] or screen printedAu-electrode [16] surfaces, where it is reoxidized.…”