Antioxidant capacity is an ability of organisms or food to catch free radicals and prevent their harmful effects. Substances with antioxidative properties are called antioxidants and have received much attention in recent years. They have ability to fight against the oxidative processes [1
], to promote health and to prevent a wide variety of diseases: atherosclerosis, type 2 diabetes, neurodegeneration (Alzheimer’s and Parkinson’s diseases) [2
] and cancer [4
]. Epidemiological studies recommend to introduce in the diet substances as fruits, vegetables and less processed staple foods ensure the best protection against possible diseases caused by oxidative stress, such as coronary heart disease, obesity, hypertension, and cataracts [5
]. The explanation consists in the beneficial health effect, due to antioxidants present in fruit and vegetables [6
]. In particular, polyphenols are naturally-occurring antioxidants found widely in the fruits, vegetables, cereals, dry legumes, chocolate and beverages, such as tea, coffee, or wine. Polyphenols and other food phenolics are the subject of increasing scientific interest because offer a great hope for the prevention of human diseases [7
Total antioxidant capacity (TAC) is the measure of the quantity of free radicals scavenged by a test solution [8
], for evaluating the antioxidant capacity in samples of different nature. Several methods have been proposed for the determination of the TAC of body fluids [9
], of biological samples [14
], and food extracts [16
]. Usually, the antioxidant capacity is given as the Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalent antioxidant capacity (TEAC) [18
], as the ferric reducing antioxidant power (FRAP) [20
], as the cupric reducing antioxidant capacity (CUPRAC) [22
], as the oxygen radical absorption capacity method (ORAC) [25
], as the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) [25
], and as the DPPH (2,2-diphenyl-1-picrylhydrazyl) method [27
], respectively based on the different spectrophotometric methods which have been used to estimated it.
Recently, also electrochemical methods have greatly contributed to measure of TAC based on biosensors [28
] and sensors [32
]. They offer sensitivity, inexpensive instrumentation, fast response, small volumes [34
In the last years, the application of nanomaterials resulted in many advantages for the sensing systems, including the observation of enhanced electrocatalytic phenomena with benefits in chemical and biosensing and improving analytical performance of classical sensing platforms [37
]. Some authors reported nanomaterial based sensor for determination of polyphenols in foodstuffs [47
Different kinds of nanomaterials have been used as stable and low-cost alternatives to biomolecules in (bio)analytical methods. The materials comprise metal/metal oxides, metal complexes, nanocomposites, porphyrins, phthalocyanines, smart polymers, and carbon nanomaterials [56
]. Cerium oxide nanoparticles (CeNPs) or nanoceria particles have attractive catalytic and electrochemical features [58
]. Moreover, ceria is an excellent co-immobilization material for a variety of oxidase and peroxidase enzymes such as horseradish peroxidase [59
], glutamate oxidase [61
]. Its catalytic activity can be exploited to develop highly sensitive, enzymeless H2
] and for the fabrication of third generation biosensors [63
]. Ceria has high oxygen mobility at its surface [64
] and a large oxygen diffusion coefficient, which facilitates the conversion between valance states Ce4+
] that allow oxygen to be released or stored in its crystalline structure [67
Recently, CeNPs have also been reported to have multienzyme, including superoxide oxidase, catalase and oxidase, and mimetic properties [69
], and have emerged as a fascinating material in biological fields, such as in bioanalysis [72
], biomedicine [75
], and drug delivery [78
]. Catalytically active nanoceria offer several advantages over natural enzymes, such as controlled synthesis at low cost, tunable catalytic activities and high stability against severe physiological conditions [80
In the present work, we developed an electrochemical disposable sensor modified with nanoceria particles for determination of total antioxidant capacity (TAC) in real samples. Firstly, the sensor was characterized electrochemically and then has been tested for some common antioxidants present in wines, such as gallic acid, caffeic acid, ascorbic acid, quercetin, trans-resveratrol and, finally, for the detection of TAC in six white and red commercial wine samples. The results relative to wine samples obtained with the electrochemical method were compared to those carried out with the spectrophotometric method (ABTS-based method). The modification procedure was demonstrated to be very simple and the developed sensor resulted to be easy-to-use, robust and cheap without involving labeled reagents.
2. Materials and Methods
2.1. Chemicals and Reagents
All chemicals used were analytical grade and were used as received without any further purification. In particular: sodium monobasic phosphate (Na2HPO4), sodium dibasic phosphate NaH2PO4, potassium chloride (KCl), potassium ferricyanide (III) (K3[Fe(CN)6]), cerium (IV) oxide NPs (20 wt% colloidal dispersion in acetic acid 2.5 wt%, d = 30–60 nm), gallic acid (GA), caffeic acid (CA), quercetin (Q), trans-resveratrol (t-R), ascorbic acid (AA), and dimethyl sulfoxide were purchased from Sigma-Aldrich (Buchs, Switzerland). For the TEAC assay: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and potassium persulfate (K2S2O8) were also supplied by Sigma-Aldrich (Buchs, Switzerland).
All the solutions were prepared in phosphate buffer 0.1 M, KCl 0.1 M, pH 7.4 (PBS buffer) high-purity deionized water (resistance: 18.2 MΩ × cm at 25 °C; TOC < 10 µg L−1) obtained from Millipore (Molsheim, France) has been used to prepare all the solutions.
A solution of 1.1 mM K3[Fe(CN)6] in PBS buffer was used in cyclic voltammetric experiments for determination of electroactive area using the Randles–Ševćik equation. Stock solutions of 10 mM of several phenolic (GA, CA, Q and t-R) and not phenolic antioxidants (AA) were prepared daily before use: all substances in PBS buffer only the quercitin had need of few drops of DMSO.
The value of pH 7.4 was chosen because from early studies in literature was reported that at this value the particles have the highest oxidase-like activity against phenolic compounds [81
]. Moreover, all the experiments were carried out at room temperature, approximately 25 °C.
2.2. Electrochemical Measurements
Electrochemical measurements were performed using a portable PalmSens potentiostat (PalmSens, Houten, The Netherlands) controlled by means of the PSTrace 4 program (Vers. 4.4 PalmSens BV). All the experiments were conducted using a three screen-printed electrodes system from Orion High Technologies S.L. (Parla, Madrid, Spain). In particular: Nanostructured carbon (OHT-000), carboxylic acid functionalized multi-walled carbon nanotubes (OHT-069) and Carboxylic acid functionalized multi-walled carbon nanotubes-Fe3O4 superparamagnetic nanoparticles (OHT-102) screen printed electrodes, respectively. The working electrodes were different for each sensor (with a surface diameter of 4 mm) but the counter electrode (graphite) and the reference one (Ag/AgCl) were the same for all the SPEs.
All absorbance measurements were made at the specified wavelength (731 nm) of the selected spectrophotometric method (ABTS-based method) using a T60U Spectrometer PG Instruments Ltd. (Wibtoft Leicestershire, Lutterworth, UK).
2.3. Electrochemical Method
The procedure of square wave voltammetry (SWV) was carried out as analytical technique to develop the calibration curve of GA used as standard and to test the selected wine samples and for the other antioxidant compounds. The frequency (f) and pulse amplitude at 25 Hz and 50 mV, respectively. The OHT-069 modified SPE was used as sensor.
2.4. Sensor Modification by Using CeNPs
The surface modification procedure was realized by two steps: first, a colloidal NPs suspension of 2% (w/v) nanoceria was prepared by dispersing particles in distilled water, then 5 µL of the solution were dropped onto the working electrode surface of the SPE and let it dry for two days at room temperature until use. Electrodes were stored at room temperature.
2.5. ABTS-Based Method
The ABTS antioxidant assay was slightly modified and carried out as described in literature [82
]. Briefly, 7 mM ABTS solution prepared in 2.5 mM K2
was incubated in the dark for about 15 h at room temperature to generate ABTS radicals. Then, the solution was diluted 400 times with distilled water. The white wines and the red ones were diluted 10 and 100 times respectively with distilled water. Next 100 µL of each sample were mixed with 2.5 mL of ABTS radical solution and 0.4 mL H2
O and the absorbance has read after 3 min at 731 nm. The phenolic antioxidant gallic acid (GA) was used as standard, and triplicates were analysed for each sample.