Electrochemical Applications for the Antioxidant Sensing in Food Samples Such as Citrus and Its Derivatives, Soft Drinks, Supplementary Food and Nutrients

Although there are many definitions of antioxidants, the most general description; antioxidants are carried a phenolic function in their structure and prevent the formation of free radicals or intercept fromdamage to the cell by scavenging existing radicals. Moreover, they are one of themost effective substances that contain essential nutrients for healthy individuals. The importance of these antioxidants, which have an incredible effect on the body and increase the body’s resistance, is increasing day by day for healthy individuals. Numerous studies have been carried out for antioxidants with excellent properties and however new, reliable, selective, sensitive and green analytical methods are sought for their determination at trace levels in food samples. Along with the latest developments, electrochemical methods are of great interest in the world of science because they are fast, reliable, sensitive and environmentally friendly. Electrochemical methods have been frequently applied to analyze antioxidant capacity in many nutrients samples found in different forms such as solid, liquid without any pretreatment applications in the last decade. Furthermore, thesemethods are preferred because of the short analysis time, the ability to lower detection limits, reduction in a solvent, high sensitivity, portability, low sample consumption, wide working range, and more economical than existing other traditional analytical methods. The antioxidant sensing applications by modern electrochemical methods such as cyclic, square wave, differential pulse, and combined with stripping voltammetric techniques were used to deduce antioxidant capacity (AC) in critical nutrients. Moreover, this chapter includes a description of the classification of electrochemical methods according to the working electrode type, dynamic working range, limit of determination (LOD), limit of quantification (LOQ), sample type, and using standard analyte and so forth for each voltammetric methods. While many articles applied for the determination of antioxidant sensing by electrochemistry have gained momentum in the last two decades, we focused on the studies conducted over the last 4 years in this chapter.


Introduction
pigments, vitamins (A, B1, and C), and; organic acids such as ascorbic acid and citric acid, minerals and a number of active phytochemicals such as flavonoids and coumarins, as naringenin, naringin, hesperidin, neohesperidin, hesperetin, rutin, narirutin and tangeretin [9]. For example; polyphenol antioxidants such as flavanols (epicatechin, catechin), phenolic acids (caffeic acid and gallic acid), anthocyanins (e.g., malvidin-3-glucoside), oligomeric and polymeric proanthocyanidins, flavonols (myricetin, quercetin, and their glycosides), and many others polyphenols exist in wine, especially in red wine [10]. Flavonoids have an important role in scavenging reactive oxygen species, which can counteract lipid oxidation, decrease peroxide formation in vivo, and improve activity of the body's antioxidant enzyme. Citrus flavonoids such as naringin, naringenin, and hesperidin have antioxidant activity [11]. Naringenin is a flavonoid, particularly a flavanone, found in citrus fruits especially oranges and grape fruits and in vegetable's such as tomatoes and their preparations. The pharmacological and biological properties of phytoestrogen naringenin and its derivatives include, anticancer, anti-inflammatory, antiulcer, antifibrotic, diastolic, antioxidant and skin protective effects [8]. Also, citrus species are a rich source of flavanone glycosides such as hesperidin and narirutin, which have anticancer, antioxidant, antiobesity and anti-inflammatory activities [12].
Secondly, the antioxidant group is synthetic, that is a molecule that is obtained as a result of chemical reactions and is generally used as food preservatives [13]. Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroxyquinone (TBHQ) also extend the shelf life of foods [14]. However, natural antioxidants that can be taken from foods are less risky in terms of human health since synthetic antioxidants can have toxicity even if they are very little, they require high costs and have less capacity than natural antioxidants. Due to this reason, the investigations of foods types that can contain high levels of antioxidants in different types of endemic, organic and traditional food samples have been remarkably increased recently.
For antioxidant content and amount analyzes, oxygen radical absorbance capacity (ORAC) and radical-arrest antioxidant parameter (TRAP), ferric thiocyanate (FTC), Trolox equivalent antioxidant capacity (ABTS/TEAC), cupric ion (Cu 2+ ) reduction antioxidant capacity (CUPRAC), iron ion reducing antioxidant capacity (FRAP), DPPH radical scavenging activity determination and Folin-Ciocalteu methods are the most widely preferred as analytical methods [15][16][17]. Furthermore, to evaluate and characterize the antioxidant substances in food samples, various analytical methods such as high-pressure liquid chromatography (HPLC) combined with different detection, gas chromatography, micellar electrokinetic capillary chromatography, capillary electrophoresis includes different detection systems and UV-visible spectrophotometry have been used [18][19][20]. However, these classical methods have great shortcomings for fully validated analyzes such as long pretreatment, need for too much solvent, expensive equipment, long analysis time. They do not provide the necessary procedures for green chemistry, especially due to the use of too much solvent and too much waste in antioxidant analyses. For these reasons, scientists have turned to alternative methods for antioxidant quantification in food samples. Especially in recent years, they have focused on electrochemical techniques which are fast, inexpensive, reliable, non-pre-treatment, and environmentally friendly in the analysis of drugs, pesticides, metal ions and organic molecules such as antioxidants, vitamins and nucleic acid [21][22][23].
In this chapter, the applicability, sensitivity and reliable maintenance of electrochemical methods, which have attracted great attention in food and food samples, have been examined for the analysis of antioxidants. Moreover, which types of electrochemical methods are used and what advantages they provide have been investigated for the antioxidant sensing in food samples. It also describes the classification of each used in electrochemical methods by working electrode type, dynamic operating range, the limit of detection (LOD), measurement limit (LOQ), sample type, and standard analyte, etc. While many articles referenced for determining antioxidants by electrochemistry have gained momentum in the literature in the last two decades, we focused our study on the studies conducted in the last 4 years.

Electrochemistry
Electrochemistry is the branch of science which is investigating the physical and chemical changes coming from the interaction of the material with electrical factors such as current, potential, and electron charge. Electroanalytical chemistry is based on measuring the electrical properties of solutions containing analytes and switching to quantification using measured electrical signals a collection of electrochemical methods. Moreover, electroanalytical measurement methods are based on two basic points: potentiometric (static methods) and potentiostatic (dynamic methods). Electrode systems in both methods are immersed in the solution containing the analyte, called the electrochemical cell. Potentiostatic methods are widely used for routine analysis because they are less costly, high sensitive, and selective and have wider potential application areas than other electroanalytical methods. The basic principle of these methods is to measure the current that occurs during the oxidation or reduction of the analyte in the chemical reaction.
Electrochemical methods began with the Czech chemist Jaroslav Heyrovsky, discovering the basis of polarography in 1922 and took an important place among the analytical methods. Especially, since the 1980s, it has been possible to develop electrodes that have been modified mechanically or chemically with improved technology. In modification processes, polymers, organic ligands, inorganic clays, phthalocyanines and nanoparticles have been commonly used for the detection of electroactive substances in very small volume complex samples such as biological, environmental and human bodies. In the last twenty years, even very small quantities of substances that are electroactive have been additionally analyzed at high precision, selective by electrochemical methods by carbon-based or modified electrodes have wonderful properties. Electroanalytical methods have also an important place in quantification as well as in obtaining details such as determination, adsorption, reaction rate and equilibrium constants of the number of electrons transferred in the reduction or oxidation electrode reactions. In short, electroanalytical methods provide details on direct or indirect quantitative and qualitative analysis of electroactive species such as antioxidants, drugs, pesticides, etc.

Voltammetric application for the determination of antioxidant capacity
Voltammetry is a potentiostatic assay based on the recording of the peak current at controlled potential variation by the oxidation or reduction which enables qualitative and quantitative analysis by means in electrochemical reactions. Over the last two decades compared to other electroanalytical techniques, voltammetry has been intensely curious in all the electroanalytical methods due to their are used to analyze numerous compounds by anodic or cathodic scanning and to investigate their conceptual basis of electro-mechanism. There are four voltammetric techniques including cyclic (CV), linear (LSV), differential (DPV), and square (SWV) are commonly used to determination of antioxidant-type compounds.
Voltammetric techniques are an alternative analytical method, proved to have an excellent correlation compare with another conventional analytical process, for a while to study the AC in various food and beverage samples. They can be a benefit to characterize which species compounds have a greater contribution to the antioxidant capacity present for the real samples in terms of quantitative and qualitative by controlled the half-wave peak potential, peak current and the electron transfer number in reaction. The antioxidant capacity is related to the peak currents of oxidation species caused by hydroxyl groups (-OH) and antioxidant species contains many hydroxyl groups. They commonly give an electro-oxidation broad peak at a range of 400 mV-600 mV depend on pH. So that, almost all antioxidant substances have electro-activity compounds and their peak current and peak potential provide quantitative and qualitative details, respectively. Further, the voltammetric techniques allow investigating the electrochemical behavior of antioxidant agents and interaction with oxygenated species.
Voltammetric methods have gained an important place among determinations of the antioxidant capacity in the last decade. Moreover, due to their great superiority, the use of complex samples such as food and beverages they have become widespread and widely found in the literature. Among these electroanalytical methods, square wave stripping, different pulse stripping, and cyclic voltammetric techniques are the most commonly preferred for the analysis of antioxidants by accuracy and precision. From past to these days, the compounds used as standard agents for the evolution of the AC by studies electrochemical methods are apigenin, ascorbic acid, caffeine, catechin, chlorogenic chrysin, p-coumarin acid, eugenol, fisetin, gallic acid, kaempferol, luteolin, morin, quercetin, rutin, t-resveratrol, Trolox and Malvidin-3-glucoside. As far as we have examined the literature, scientists have however preferred ascorbic acid, caffeic acid, gallic acid, catechin, rutin and quercetin which are often used as antioxidant standard substances due to excessive availability of these substances in food and drink. The chemical structures of some antioxidant molecules are given in Figure 1.

Cyclic voltammetric technique
Cyclic voltammetry (CV) is usually the first experiment in the electrochemical operation of a compound in biological materials as nature samples to get in details about the electro-behaviors. In particular, to study the thermodynamics, kinetic, electron transfer, substance transfer type, and as well as quantitative determinations of oxidation or reduction processes can be carried out by cyclic voltammetric technique. In addition to taking a single measurement with CV, sequential multiple measurements can be taken. The most common applications of cyclic voltammetry are additionally electro-polymerization, electrochemical characterization, and the design of modified electroanalytical systems. Two types of cyclic voltammograms can be obtained as irreversible or reversible, depending on the chemical components of the target molecules. In reversible voltammetry, there is a difference of about 59 mV between the reduction and oxidation peak potentials (Figure 2).
During the past years, cyclic voltammetry has been used as an alternative to existing methods to evaluate the antioxidant sensing in natural samples such as teas, biological fluids, beverage juices plants, foods and beverage juices on different working electrodes. The most using parameter is peak current because of its proportional to the concentration of the antioxidants. Peak current heights also provide quantitative information about the amount of antioxidant capacity in food samples. The carbonbased working electrodes such as glassy carbon electrode (GCE), carbon paste electrode (CPE), screen printed carbon electrode (SPCE), and modified electrodes (Nanoparticle/GCE, Nanoparticle/CPE, Fe 3 O 4 /GCE) have been widely preferred in electrochemical measurements for the analysis of total antioxidant capacity (TAC). Peak current and peak potential values of standard substances such as ascorbic acid, caffeic acid, catechin, coumarin, gallic acid, morin, quercetin and rutin were commonly taken care of for the evaluation of TAC. The amount of antioxidants in food samples is generally given as equivalent gallic acid, equivalent value quercetin, etc.
Even though the CV method raises doubts about sensitivity, it also has great advantages. Quick, simple, low detection limit, cheaper and easier application are summarized as great advantages. Interferences effect on antioxidant capacity by a nonantioxidant agent to reducing TAC and non-selective to a family of molecules between carotenoids and polyphenols unless the electrode is modified are drawbacks properties. Despite all of these disadvantages, CV attracts a great deal of attention among analytical methods, and a large number of studies deal with CV are also being undertaken. A large part of the work done up to day time to determine the antioxidant capacity by the CV method is summarized in Table 1. Table 1 includes the type of working electrode, working range, the limit of determination (LOD), the limit of quantification (LOQ), measurement parameter, standard compound and food sample.

Square wave voltammetric technique
Square wave voltammetry (SWV) can be used to perform a faster experiment than other voltammetric techniques. Commonly when the scanning speeds of other techniques are of 1-10 mV/second or more, in the square wave voltammetry a scanning speed is used at 1 V/second. Thus, the target molecule can be analyzed more quickly by SWS. The square wave voltammetry can combine with the stripping technique. Thus, a stripping voltammetric technique was developed to determine electroactive substances at high sensitive enables in ultra-trace concentration levels. Especially, ultra-trace target substances in complex samples can be analyzed by combining the technique with the enrichment stripping process. The working principle of the stripping technique is the same as square wave voltammetry and only two new parameters are more applied as the accumulation time and the accumulation potential (Figure 3).
Nowadays SWV and square wave stripping voltammetry (SWSV) are frequently applied to deduce compounds such as drugs, heavy metals, pesticides and antioxidants, etc. in numerous specimen types because they have excellent analytical sensitivity and selectivity. Furthermore, SWV and its derivate combined technique can be applied for simultaneous determination of compounds which are close oxidation or reduction peak potentials like paracetamol, ascorbic acid, uric acid and dopamine. In the last decade, SWV and SWSV have been more effective in determining antioxidant substances in the complex matrix samples and are superior compared with analytical methods especially spectrophotometric to evaluate quantification and qualification. It is one of the most important electroanalytical methods for the determination of antioxidants since it is a wide working range, low detection limits, easy to apply, cheap and non-pretreatment. Furthermore, they have been successfully analyzed the phenols in food samples which is called a type of important antioxidant such as o-phenylenediamine, p-chlorophenol, paminophenol hydroquinone, pyrocatechol and phenol, etc. At the same time, various antioxidant substances such as gallate, gallic acid, quercetin and caffeine were easily studied in food or beverage samples at high precision, accuracy and selective on the carbon-based electrode. Besides, at nM concentration of antioxidant substances comparable to chromatographic techniques have been determined by modified electrodes which are increasing conductivity accurately and selectively in tea samples. Evaluation of antioxidant capacity by SWV or SWSV techniques in the last 4 years are summarized in Table 2 according to the type of working electrode, working range, the limit of detection (LOD), quantity limit (LOQ), measurement parameter, standard composition and food sample.

Differential pulse voltammetric technique
Differential pulse voltammetric technique (DPV) is one of the most widely used for the analysis of both organic and inorganic species. Pulse voltammetry techniques were proposed by Baker and Jenkin in 1952 as a more sensitive measurement electroanalytical method. Differential pulse voltammetry techniques can be used to determine up to 10 À8 M concentration of the target agents. The peak current (I p ) is a function of the concentration for the electroactive species and is linear as Ip = f (C). Also, it is possible to analyze substances not only quantitative analysis but also qualitative analysis with pulse technique. The peak currents are related to the concentration of the substance whereas the peak potential values are related to the selectivity. Thus, simultaneous determinations of the substances have been studied by DPV on bare or modified electrodes (Figure 4).
Nowadays, quite a lot of DPV studies can be found in the literature for the very sensitive detection of heavy metal, drug, pesticide, antioxidant agent and inorganic/ organic species on numerous bare and modified working electrodes. Besides, DPV is one of the most important candidates to determine the trace amount of target agents in analytical methods due to its high sensitivity and selective. Also, it can be applied to complex samples as biological and food samples such as blood and serum, beverages. Especially, DPV has an important place among antioxidant determination methods because of these advantages and the availability of low concentration.
In recent years, DPV has been used frequently in determining the total antioxidant capacity without any pretreatment of solid and liquid food samples. The complex matrix such as biological and food samples contain very dense different types of substances. For this reason, despite it is indeed very difficult to selectively and precisely determine the antioxidant capacity in some complex matrixes; DPV is the most applicable method for such species. There are also plenty of studies were published which deal with chlorogenic acid, caffeic acid, p-coumaric acid, quercetin, gallic acid and ferulic acid, etc. as illustrating the antioxidant properties were determined by DPV on bare or modified electrodes based on carbon nanomaterials. Several applications, based commonly on the used as a determination of antioxidant capacity are given in Table 3.
In amperometric techniques, the current produced during the reduction or oxidation of an electroactive species at a constant potential value that is applied between a working electrode and reference electrode is measured, in this way providing specific quantitative electroanalytical knowledge for the target analyte. Especially, amperometric, which is based on electrical current analysis, is commonly utilized in microchip electrophoresis applications owing to its high sensitivity, it also lets for the determination of electroanalytical active species without derivatization, accomplishing adjustable versatility and selectivity ( Table 4).
Ganesh et al., synthesized zinc oxide nanoparticles using mechanochemical synthesis technique. New ZnO nanoparticle as hexagonal prism was investigated by scanning electron microscopy, X-ray diffraction, particle size distribution, ultravioletvisible spectroscopy, and energy-dispersive X-ray spectroscopic methods. Electrochemical properties of the newly prepared electrode were characterized by using an amperometric method and cyclic voltammetry technique. The prepared electrode has  a wide working linear range between 0.1-130 μM with a detection limit of 0.02 μM.
Obtained results showed that the prepared electrode has numerous active surface sites, good electronic activity, and surface area. They applied the proposed electrode to the determination of gallic acid in samples as wine successfully [40]. Kumar and coworkers successfully synthesized NiO nanoparticles from natural fruit using an efficient, simple, and low-cost technique. The obtained NiO nanoparticles were investigated with various methods such as FTIR, XRD, TEM, SEM, UV, and PL. XRD studies showed that NiO nanoparticles have cubic geometry. The band of Ni-O bond was shown at 430 cm À1 . Photocatalytic properties of the obtained NiO nanoparticles were applied to photodegrade the methylene blue dye. They used the prepared electrode to the determination of dopamine with the LOD of 11 μM [93].
Koçak et al. prepared a new composite electrode using carbon nanotube and polyl-methionine onto the glassy carbon electrode. Electrochemical properties and surface structure of the prepared electrode were studied using electrochemical impedance spectroscopy and scanning electron microscopy. Electrochemical properties of gallic acid with the proposed electrode were investigated in various techniques such as differential pulse voltammetry, cyclic voltammetry and amperometry. The obtained results of electrochemical studies exhibited that the prepared electrode shows a suitable method of determination for gallic acid in pH 2.2 BR buffer solution. The prepared sensor has a wide working linear range with two linear segments between 4nM-1.1μM and 1.7-20.0 μM with LOD of 3.1 nM. They used the prepared new sensor for the detection of gallic acid in various samples as black tea, green tea and wine samples. The experimental results showed that the proposed sensor exhibit high selectivity, reproducibility, stability and catalytic effect [88].
Potentiometry is an electrochemical technique based on measuring the potential difference between two electrodes called working and reference electrodes. The working basis of the potentiometry technique is the potential difference based on the concentration of an analyte in the sample solution relative to a reference electrode ( Table 5).
Brainina and coworkers developed a new, simple, reliable and fast potentiometric method for the determination of plant total antioxidant activity. Plant micro suspension and extracts were analyzed by the proposed method. The experimental conditions for acquiring plant extracts were selected for the highest antioxidant activity as extraction time 20 min at +80°C. The characterization of plant micro suspensions reduces the duration of plant total antioxidant activity evaluation. Comparison of the obtained results of antioxidant activity of green tea and black tea micro suspensions samples with the results of the investigations of extracts prepared by a certified method showed no difference [95] (Tables 6 and 7).

Conclusion
Electrochemistry is a powerful and versatile analytical technique for the determination of numerous substances such as drugs, pesticides, inorganic, antioxidanttype compounds and electroactive compounds by rapidly possible applications in a lot of fields. Electroanalytical methods besides providing details on quantitative and qualitative of analyte that offer validation parameters such as sensitivity, accuracy and precision, selective and linear working range. Moreover, it is superior to determine the target analyte by electroanalytical methods lack of interferences effect especially in a complex matrix such as biological and food samples contain countless substances. The improvement of simultaneous determination of analytes considerably has been carried out to be applied in biological and environmental systems by the sensitive and selective electrochemistry methods. Because of this, the use of many areas of electrochemistry is widespread.
Nowadays, electrochemical methods, especially voltammetry from medicine to the determination of antioxidants, have made an important place especially in the world of science. Not only analytical chemists but also biology, food engineering and all people who are engaged in food have been used electrochemical methods to determine the antioxidant capacity in plants, tea, beverages, carbonated beverages and solid food samples, etc. Compounds such as ascorbic acid, caffeic, catechin, ascorbic acid, quercetin, gallic acid and coumarin have been widely used as reference standard agents to an evaluation of antioxidant capacity by electrochemical methods have been carried out until today. Due to advances in electronics and computer science have provided significant benefits in terms of electrochemical instrumentation such as accuracy, sensitivity and easy application, the electro-analysis of antioxidant compounds is successfully applied by stripping voltammetric techniques at nM concentration level. The purpose of this review is to show that electroanalytical methods for commonly used antioxidant types may be the best analytical method for the quantitative and qualitative analyte and that they can successfully compete with more conventional methods especially spectrometric methods. Consequently, voltammetric techniques supply that even at low concentrations, the antioxidant capacities of food samples can be determined to be very fast, simple, non-pretreatment and highly sensitive compared to conventional analytical methods. The review presented that the antioxidant capacity of various food samples can be carried out by voltammetric techniques in the estimation in real samples.