Determination of Trolox Equivalent Antioxidant Capacity in Berries Using Amperometric Tyrosine Biosensor Based on Multi-Walled Carbon Nanotubes  
[PERSON]  
1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Prishina, Str. Mother Teresa, 10000 Prishina, Kosovo; [EMAIL_ADDRESS] (A.F.); tahir.arbneshi@uni-predu (T.A.)  
[PERSON]  
2 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic; [EMAIL_ADDRESS] (A.M.A.); [EMAIL_ADDRESS] (V.A.); [EMAIL_ADDRESS] (L.R.)  
[PERSON]  
3 Central European Institute of Technology, Brno University of Technology, Purkynova 123, CZ-612 00 Brno, Czech Republic  
several spectrophotometric methods based on reductive properties of present antioxidants were developed. One of the most frequently performed method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, is based on 1,1-diphenyl-2-picrylhydrazyl radical [5]. Some methods, including an oxygen radical absorbance capacity (ORAC) [6], ferric reducing antioxidant power (FRAP) [7], and total radical trapping antioxidant potential (TRAP) [8], were also employed in the analysis of berries. L-ascorbic acid (AA), known as vitamin C, is a non-phenolic chemical compound, contained also in berries with high antioxidant activity [9]. It occurs commonly together with polyphenols in foodstuffs. AA has an interfering effect during electrochemical analysis of polyphenols due to its reduction properties (reduction of resulting quinones back to polyphenols). The interfering effect of AA on the determination of hydroquinone (HQ), which has a similar chemical structure to \(\alpha\)-tocohydroquinone [10], is known from previous studies [11]. The mechanism of HQ reduction by AA, which was proposed by [PERSON] and [PERSON], is shown in Scheme S1 [11]. However, it was found earlier that the presence of AA is not interfering up to the molar ratio of AA to HQ 1:1, in the case of the amperometric biosensor based on a carbon paste electrode modified with tyrosinase (TYR) [12].  
Mushroom TYR enzyme from _Agaricus bisporus_ (E.C. 1.14.18.1) is an oxidase enzyme which contains copper. It possesses a catalytic activity toward the oxidation of both catechols and cresols [13]. In the presence of TYR enzyme, the conversion of phenolic compounds to their quinone derivatives is catalyzed. Resultant products can then be detected by amperometric methods at constant reductive potential in phosphate buffer media of pH-7, where the enzyme exhibits optimum activity [14; 15]. TYR is capable of catalyzing an oxidation reaction of Trolox by oxygen, resulting in the formation of Trolox quinone [12], which can be electrochemically reduced by 2 e-/2 H+ to Trolox hydroquinone [16]. These reactions simply describe the principle of amperometric tyrosinase biosensor behavior (Scheme 1).  
As reported, the biosensor, prepared by covering the bare carbon paste electrode (CPE) with a thin layer of Nafion(r) and the TYR enzyme (CPE/Tyrosinase/Nafion(r)), provides a low sensitivity against Trolox in comparison to \(o\)-substituted polyphenols [12]. Hence, carbon nanotubes (CNTs) were used to improve this parameter, due to their high catalytic effect and large surface area. When considering the type of CNTs, multi-walled carbon nanotubes (MWCNTs) exhibited similar behavior to single-walled carbon nanotubes (SWCNTs), as shown in previously reported electrochemical study with Trolox [17]. Later, the CPE/MWCNTs/Tyrosinase/Nafion(r) biosensor was tested in analysis of polyphenols in various Moravian wines [18]. In this contribution, the previously introduced biosensor is employed in the determination of TEAC, in comparable matrices such as berries.  
Figure 1: Principle of amperometric tyrosinase biosensor for evaluation of TEAC.