The use of electrochemical methods like cyclic voltammetry and differential pulse voltammetry, in the quantification of various antioxidants has several benefits like simplicity, high sensitivity, rapidity of analytical measurements and analysis of colored samples without pretreatment, compared to traditional analytical methodologies 102. The electrochemical parameters of importance in the antioxidants determination via electrochemical methods are: i) the anodic (oxidation) peak potential, Epa; ii) the anodic (oxidation) peak current, ipa; and iii) the electric anodic charge, which is related to the area under the anodic oxidation wave, Qa. The Epa value is related to the electron donor capability of the measured antioxidant, while the ipa parameter refers to the concentration of the antioxidant. The charge of the anodic wave can be used in the assessment of the total antioxidant capacity. The electrochemical methods are usually applied in connection with metals (Pt, Au) or glassy carbon electrodes and semiconductors, as well as electrodes modified with various nanomaterials and organic polymers to improve the selectivity and the sensitivity of the analytical measurements. The judicious modification of conventional electrode substrates with a range of inorganic, organic and composite materials represents the most important achievement and development in the electrochemical science since 1975. The modification of the electrode surfaces revealed the possibility to achieve new properties like selectivity, sensitivity and polarity that greatly expand the final applications of obtained modified electrodes. Chemically modified electrodes (CMEs) are usually obtained by deposition of chemical modifiers, i.e. inorganic, organic and polymeric compounds, in forms of monomolecular, multimolecular and polymeric layers. The CMEs are actually functioning as electrochemical sensors and they have found several applications in the electroanalysis of antioxidants.
Another approach consists in the use of electrochemical biosensors based on oxidase enzymes such as tyrosinase, laccase and horseradish peroxidase for the quantification of various antioxidants. Electrochemical biosensors represent a sub-class of electrochemical sensors and they take the advantages of both the sensitivity of the electrochemical transducers (electrode substrates) and the selectivity of the biological recognition element: the enzyme. The immobilization of the enzymes onto electrode surfaces is of paramount importance for the proper functioning of the electrochemical biosensors. The main enzyme immobilization procedures are: adsorption, entrapment into a matrix, microencapsulation, cross-linking and covalent bonding. The entrapment of enzymes within conducting polymers has attracted great interest because of the simplicity and reproducibility of this procedure. Polypyrrole (PPy), polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT), and their derivatives have been successfully and extensively used in the fabrication of enzymatic electrochemical biosensors 103–105. The use of tyrosinase 106–108 and laccase 109,110 for the detection of antioxidants like polyphenols is very convenient since the enzymatically generated quinone derivative may be reduced at low potentials; thus the electrochemical interferences are considerably minimized. The reduction current of the generated quinone derivative is measured and it is related to the concentration of the investigated polyphenols. The functioning principle of the electrochemical biosensors based on oxidase enzymes for polyphenols detection is schematically depicted in Figure 2.
FIGURE 2 WILL BE NEAR HERE.
Tyrosinase (polyphenol oxidase, E.C. 22.214.171.124), in the presence of oxygen, catalyses the hydroxylation of mono-phenols to o-diphenols (Reaction 1), and the oxidation of o-diphenols to o-quinones (Reaction 2) 111. The resulted quinone derivatives can be easily detected by electrochemical reduction at low potential values (Reaction 3). This is the basic principle of tyrosinase-based amperometric biosensors and can be described by the following reactions scheme :
mono-phenol + Tyrosinase (O2) ? o-diphenols (1)
o-diphenols + Tyrosinase (O2) ? o-quinones + H2O (2)
o-quinones + 2e? + 2H+ ? o-diphenols (3)
Laccase (EC 126.96.36.199) catalyses the oxidation of phenol, diphenols and various polyphenols to quinone derivatives and does not require the hydrogen peroxide as a co-substrate 112,113. Similarly to tyrosinase-based amperometric biosensors, the reduction of the enzymatically generated quinone derivatives provides the electrochemical signal in laccase-based biosensors. Actually, the antioxidant capacity is measured using a standard compound like caffeic acid, catechin, chlorogenic acid or catechol, and this compound displays good electrochemical behavior at the electrode surface.
The analytical performance of tyrosinase- and laccase-based amperometric biosensors depends mainly on the enzyme immobilization method, the enzyme loading and activity, and pH of the sample solution. The immobilization method is the most important parameter in the development of enzyme-based electrochemical biosensors. The main achievements in this topic will be discussed taking into account the enzyme immobilization procedure. For instance, the adsorption of enzyme is a simply and versatile approach in biosensors construction 106. In this study, it was demonstrated the beneficial role of the immobilization matrix alongside to the enzyme adsorption procedure by covering the adsorbed tyrosinase enzyme with polyethylene glycol and/or the ion-exchanger Nafion®. The use of Nafion® coating ensured the highest analytical performance in synthetic samples (low detection limit of 0.064 µM, sensitivity of 8×103 nA ?mol?1 L?1 cm?2, Michaelis-Menten constant of 67.1 ?mol L?1 for catechol), as well as in real samples. In another study 114, a tyrosinase-based biosensor was constructed by the immobilization of the enzyme onto screen-printed electrode using various methods such as cross-linking with glutaraldehyde, entrapment with polyvinyl alcohol, and cross-linking with glutaraldehyde and human serum albumin. The best analytical performance in terms of the lowest detection and quantification limits (1.5 µM and 5.1 µM catechol, respectively) were obtained for the biosensor prepared via cross-linking of tyrosinase with glutaraldehyde. This biosensor was successfully applied in the determination of trolox equivalent antioxidant capacity of infusions prepared with various medicinal plants and the results were compared with a well-established method, namely the DPPH spectrophotometric method.