Antioxidant Determination with the Use of Carbon-Based Electrodes

: Antioxidants are compounds that prevent or delay the oxidation process, acting at a much smaller concentration, in comparison to that of the preserved substrate. Primary antioxidants act as scavenging or chain breaking antioxidants, delaying initiation or interrupting propagation step. Secondary antioxidants quench singlet oxygen, decompose peroxides in non-radical species, chelate prooxidative metal ions, inhibit oxidative enzymes. Based on antioxidants’ reactivity, four lines of defense have been described: Preventative antioxidants, radical scavengers, repair antioxidants, and antioxidants relying on adaptation mechanisms. Carbon-based electrodes are largely employed in electroanalysis given their special features, that encompass large surface area, high electroconduc-tivity, chemical stability, nanostructuring possibilities, facility of manufacturing at low cost, and easiness of surface modiﬁcation. Largely employed methods encompass voltammetry, amperometry, biamperometry and potentiometry. Determination of key endogenous and exogenous individual antioxidants, as well as of antioxidant activity and its main contributors relied on unmodiﬁed or modiﬁed carbon electrodes, whose analytical parameters are detailed. Recent advances based on modiﬁcations with carbon-nanotubes or the use of hybrid nanocomposite materials are described. Large effective surface area, increased mass transport, electrocatalytical effects, improved sensitivity, and low detection limits in the nanomolar range were reported, with applications validated in complex media such as foodstuffs and biological samples.


Defining, Classifying and Describing Modes of Action Antioxidants
Antioxidants are chemical species that prevent or delay oxidation processes. They originate from various sources and hamper lipid peroxidation following different mechanisms of intervention, acting at a much smaller concentration, in comparison to that of the preserved compound [1][2][3][4][5].
Primary antioxidants act as scavenging or chain breaking antioxidants, delaying initiation or disrupting propagation. Secondary antioxidants quench singlet oxygen, decompose peroxides in non-radical species, chelate prooxidative metal ions, inhibit oxidative enzymes or absorb UV radiation. It has been confirmed that they can exploit the above-described mechanisms to stabilize/regenerate primary antioxidants [1].
Considering antioxidants' reactivity, four lines of defense have been described. Antioxidants belonging to the first line of defense withhold radical species generation. The second Table 1. The main analytical methods applied to antioxidant assay .

Method's Principle
Detection of the End-Product Ref.
Absorbance of the blue molybdenumtungsten complex resulted is measured, versus gallic acid as reference antioxidant [33,34] ORAC Peroxyl radicals, induced by AAPH (2,2 -azobis-2-amidinopropane) decomposition are reduced by antioxidants Loss of fluorescence indicated by fluorescein [35,36] HORAC Co(II)-based Fenton systems result in OH radicals generation, followed by quenching by antioxidants Loss of fluorescence indicated by fluorescein [37,38] TRAP Luminol-derived radicals, formed by AAPH decomposition, are scavenged by antioxidants Quenching of chemiluminescence [39,40] Fluorimetry Emission of electromagnetic radiation (generally in the visible range) that follows an absorbtion process (generally in the UV domain) Recording of excitation/emission spectra of fluorescent reagent [41,42] Electrochemical Techniques

Potentiometry
Antioxidants interact with the oxidized form of a redox couple, changing the ratio between the oxidized form and the reduced form concentration The analytical signal recorded is the potential shift of the mediator system, resulting from interaction with antioxidants [43,44]

Method's Principle
Detection of the End-Product Ref.

Cyclic voltammetry (CV)
Linear variation of the potential of a working electrode following a triangular waveform, and recording of current intensity The intensity value corresponding the cathodic/anodic peak is measured [45,46] Differential pulse voltammetry (DPV) Voltage pulses are superimposed on the potential scan, that is varied linearly or stairstep-wise First intensity value is sampled before applying the pulse, and the second towards the end of the pulse [47,48] Square-wave voltammetry (SWV) A square wave is superimposed on a potential staircase sweep variation Current intensity recorded at the end of each potential change [49,50] Polarography Determination of the antioxidant potential of radical scavengers relied on the anodic oxidation of dropping mercury electrode Diminution of the anodic limiting current of the hydroxoperhydroxomercury(II) complex, [Hg(O 2 H) (OH)], generated in H 2 O 2 solution at alkaline pH, at the potential of Hg oxidation [51,52] Amperometry Measurement of the current intensity at a fixed potential value of the working electrode, with respect to a reference one The intensity of the current, occurring as result of oxidation/reduction of the analyte at constant potential, is measured [53,54] Biamperometry Reaction of the antioxidant with the oxidized form of a reversible indicator redox couple The current flowing between two identical working electrodes is measured, at a small potential difference; the measuring solution contains the antioxidant(s) in the presence of a reversible redox couple [55,56] Chromatography Gas chromatography (GC) The compounds to be separated and quantified are differentially distributed between a liquid stationary phase and a gaseous mobile phase Detection based on thermal conductivity or flame ionisation [57,58]

Method's Principle
Detection of the End-Product Ref.
High performance liquid chromatography (HPLC) The compounds to be separated suffer different repartition between a solid stationary phase and a liquid mobile phase with various polarities, at high values of pressure of the mobile phase and flow rate Diode array (UV-VIS), mass spectrometry, fluorescence, or electrochemical detection [59,60] Thin layer chromatography (TLC) Compound separation relies on the repartition between a solid stationary phase (silica gel, alumina) and a liquid mobile phase (methyl acetate/formic acid, ethanol/hexane, or methanol/chloroform/ hexane) UV-VIS vizualization, fluorescence or phosphorescence detection [61,62] The advantages and shortcomings of the methods applied in antioxidant assay have been described by Sadeer et al. [20]. Photometric techniques such as DPPH, ABTS are rapid, simple, and provide reproducible results, whereas TBARS assay is characterized by not so good sensibility and specificity [20]. Chromatographic techniques offer accurate and reproducible results, but are often laborious, time-consuming, and require specialized equipment and skilled personnel. Electroanalytical techniques benefit from the rapidity and sensitivity of the electrochemical detection, with specificity improvable by the use of mediators and enzymes in modified electrodes.

Electrochemical Techniques
This sub-section provides a characterization of electroanalytical techniques applied to individual antioxidant content and antioxidant activity determination. Voltammetric and amperometric/biamperometric methods are the most broadly used.
These techniques are able to provide direct assay of the total antioxidant activity, even in the absence of reactive species. Voltammetric and amperometric techniques, including integration in flow injection analysis set-up and microfluidic chip configurations coupled with amperometric detection, relate oxidation potential to antioxidant activity [63,64]. Developing enzyme electrodes by biocatalyst incorporation enables viable assay of the total antioxidant profile or total phenolics, with quantitation in food and beverages, biological samples, pharmaceuticals [63].
Cyclic voltammetry (CV) as potentiodynamic technique involves linear variation applied to the working electrode's potential, in a triangular waveform, and the recording of the current intensity. The anodic oxidation and cathodic reduction potentials (Ea and Ec) furnish qualitative informations, whereas the intensities of the anodic and cathodic peaks (Ia, Ic) are related the analyte's amount. For reversible systems, the values of the intensities of the cathodic and anodic peaks are equal. For irreversible systems, the presence of one peak can be noticed on the voltammogram. Cyclic voltammetry has proved its analytical viability for the quantitation of low molecular weight antioxidant capacity of plant extracts, tissue homogenates and blood plasma. The oxidation potential and half-wave potential are linked to the nature of the antioxidant analyte(s); the intensity of the current measured for the anodic peak and the area of the anodic wave underlie quantitative assay [45].
Differential pulse voltammetry (DPV) implies one measurement before applying the potential pulse, and a second towards the end of the pulse period. Sampling the current just before the potential is changed, lowers the effect of the charging current and enhances faradaic current. In differential techniques, the advantage consists in measuring the ∆i/∆E value, where ∆i is the difference between the current intensity values, taken just before pulse application, and at the end of the pulse period. Another consequence of double intensity measurement is the presence of the analytical signals in the form of sharp peaks, with improved resolution and sensitivity [53].
Square-wave voltammetry (SWV): A square-wave is superimposed on the potential staircase variation, whereas the current is recorded at the end of each potential change, minimizing charging current, just as in differential pulse technique. Square-wave voltammetry facilitates data acquisition with high sensitivity and minimization of background signals. The fast potential scan enables repetitive measurements, with signals acquired at optimized signal-to-noise ratio. The technique benefits from high contribution of the faradaic current, increased resolution and sensitivity [53].
Staircase voltammetry is a derivative of the linear scan technique less applied in antioxidant assay, for which the potential sweep is a succession of stair steps. The current intensity is measured at the end of each potential change, just before the following step, so contribution of capacitive current is lowered.
Chronoamperometry relies on applying single or double potential steps, and the current resulting from faradaic processes that occur at the electrode is measured as a function of time. As in the case of other pulsed techniques, generated charging currents exponentially decline with time. The Faradaic current due to electron transfer diminishes as revealed by Cottrell equation, that illustrates the inverse dependence of the recorded intensity response, on the square root of time (seconds), under diffusion-controlled conditions. By integrating current intensity over longer periods of time, chronoamperometry improves signal to noise ratio versus other amperometric methods.
Chronocoulometry relies on analogous principles, but it records the variation of charge with time, instead of the current-time dependence. Nevertheless, in chronocoulometry, a signal increase in time is monitored instead of a decrease; signal integration diminishes noise, resulting in a smooth hyperbolic curve; the contributions of absorbed or double-layer charging species become readily noticeable.
Polarography: Polarography is a particular variant of linear sweep voltammetry, that makes use of mercury drop electrode [52]. As a technique with linear potential scan, it is controlled by mass transport, and the recorded polarograms (the current versus potential dependences) have a characteristic sigmoidal shape. The current oscillations noticed on the polarogram are assigned to the mercury drops that fall from the capillary. The limiting current is a diffusion one, as diffusion is the main contributor to the flux of electroactive compounds towards the electrode. The method may suffer from a significant capacitive current contribution, due to the continuous current measurement. Recently, a Clark's standard Pt electrode was employed for antioxidant activity assay, relying of the measurement of the rate of oxygen intake of a microsomal suspension [51].
The amperometric method: Amperometry involves the measurement of the intensity of the current generated by the oxidation/reduction of an electroactive analyte. During this type of electrochemical assay, the value of the potential is maintained at a fixed value with respect to a reference electrode [65]. The current measured at constant potential due to the oxidation/reduction of the electroactive analyte can be directly correlated to the concentration of the latter. The performances of this technique depend on the working potential. Lowering of the latter, with improvable sensitivity and selectivity, is possible by the use of mediators or enzyme incorporation [53].
The biamperometric method: Biamperometry relies on the measurement of the current flowing between two identical working electrodes, at a small potential difference. Biamperometric selectivity is dependent on the specificity of the reaction between the analyte and the oxidized/reduced form of the redox pair and the analyte. A redox pair largely used in biamperometric studies is DPPH•/DPPH. The recorded analytical signal is proportional to the residual concentration of DPPH•, after its reaction with the antioxidant. Other redox couples used in biamperometric antioxidant capacity assay are ABTS +· /ABTS, Fe 3+ /Fe 2+ , Fe(CN) 6 3− /Fe(CN) 6 4− , Ce 4+ /Ce 3+ , VO 3 − /VO 2+ , and I 2 /I − [56,66,67]. The assay of total antioxidant capacity has been applied to the analysis of alcoholic beverages [56] and juices [68].
Potentiometry: Potentiometric measurements logarithmically correlate the electrochemical cell's potential to the analyte concentration. Lowered potentials signify enhanced electron-donating abilities and, consequently, increased antioxidant potentials. Potentiometry does not need current or potential modulation as in voltammetry/amperometry [53]. It relies on the potential variation that results from a change in the ratio oxidized form/reduced form of an indicating redox species. With the increase of the antioxidant level, the concentration of the reduced form of the indicator increases, and the subsequent potential change is recorded [69]. Possible drawbacks in ion selective potentiometry can encompass deviations from Nernst's equation, caused by changes in ion activity or temperature.

Biosensor Methods
Biosensors have been applied to the assay of compounds endowed with reductive/antioxidant properties [70,71]. Oxido-reductases are often encountered in biosensor applications, due to the confirmed enhanced electron transfer ability. Biocatalysts act at low concentrations when compared to those of the target analytes (substrates) and do not always require the presence of cofactors. Laser-derived graphene sensors based on nanomaterials and conducting polymers were applied in environmental monitoring, food safety assay, and clinical diagnosis [72]. The use of multienzyme systems in electrochemical biosensors enables detection of a broad spectrum of compounds, as well as the improvement of electrochemical biosensor's analytical parameters, selectivity and sensitivity [73].
A series of review papers and books refer to antioxidant and antioxidant capacity assay by the use of biosensors [74][75][76][77][78][79]. Applications of biosensors for assessing antioxidant potential are based on monitoring superoxide anion radical (O 2 •− ), nitric oxide (NO), glutathione, uric acid, phenolics, or ascorbic acid [80]. A carbon paste biosensor developed by DNA incorporation relied on the partial damage of the DNA layer present on the electrode surface by OH• radicals, produced in a Fenton system. The electro-oxidation of the intact-remaining adenine nucleobases, generated an oxidation product able to catalyse NADH oxidation. Sample antioxidants scavenged OH•, so more adenine molecules were left unoxidized, resulting in an increase of the catalytic current given by NADH oxidation, quantified in differential pulse voltammetry. Ascorbic acid served as model antioxidant, enabling quantitation of levels as low as 50 nM ascorbic acid in aqueous media [81].
Amperometric biosensors applied in the assessment of phenolics, major contributors to the antioxidant capacity of plants, incorporated laccase, tyrosinase, or peroxidase [82][83][84][85]. Phenolic compounds could be quantitated by enzyme sensors developed by immobilizing polyphenol oxidase (PPO) into conducting copolymers obtained by coelectropolymerization of pyrrole with thiophene-capped polytetrahydrofuran [84]. Phenolic compounds are also determined using biosensors based on oxidases such as tyrosinase and laccase [86].
The principles of developing and particular applications of carbon-based sensors will be discussed in the next sections, given the increasing need for high performance analytical tools in the field of antioxidant assay, linked to food quality and health status monitoring.

Carbon Electrodes-General Overview
Carbon electrodes are largely employed in electroanalysis due to their special features, that encompass large surface area, tunable porosity, high electroconductivity, chemical stability, temperature resistance, nanostructuring possibilities, facility of manufacturing at low cost, and easiness of surface modification. The carbonaceous electrodes were classified as carbon paste, glassy carbon, fullerenes, graphite, diamond, and screen-printed electrodes [87]. Carbon Paste Electrodes: Carbon paste electrodes are synthesized from graphite powder and various water-immiscible nonelectrolytic organic pasting liquids, such as mineral (paraffin) oil, [88,89]. Most often high purity mineral oil (Nujol) is employed, nevertheless quasi-solid binders such as silicone grease or polypropylene could replace commonly used pasting liquids, but the developed structure has much higher density and becomes less easy to handle [90]. The advantages of carbon paste electrodes are facility of including modifiers (for developing novel, redox-mediated sensors), very low ohmic resistance, minimized toxicity of this environmentally compatible material, reduced background current, individual polarizability [87]. Obtaining carbon paste electrodes can also be rely on alternative carbon-based materials, replacing graphite powder with glassy carbon powder, carbon nanotubes, porous carbon foam, acetylene black [90].
Glassy Carbon Electrodes: Glassy carbon, also called vitreous carbon is a type of nongraphitizing, solid, three-dimensional carbon material, broadly used in electro-assay. It is obtained at temperatures above 2000 • C, to decompose pyrolysis intermediates that generally exhibit a scarce thermal conductivity [91]. The surface of glassy carbon electrodes can be modified with functional nanomaterials (metals, alloys, or metal oxides) [92,93]. Glassy carbon and glassy carbon-based electrodes provide excellent electroconductivity, mechanical resistance, broad potential range, and gas impermeability [87]. They have large potential window and chemical stability, prove better resistance to solvents than metal electrodes, been confirmed for their viability at the assay of organic compounds. Bare glassy carbon electrodes are characterized by facility of use, being mechanically cleaned by mere polishing on alumina slurry, procedure that can be followed by sonication in aqueous medium. Nevertheless, residual alumina particles on the electrode surface can affect the recorded electrochemical profile of electroactive analytes endowed with reductive (antioxidant) potential, such as phenolics [94].
Glassy carbon modification with alumina particles aimed at improving sensitivity in the case of dopamine [95]. The application of alumina-modified glassy carbon electrode promotes sensitivity, detectability and selectivity in the case of nitroaromatic compounds. Enhancement of dissolved oxygen electrochemical reduction in the presence of alumina was also reported [96]. Employing alumina suspensions for modifying glassy carbon electrodes by abrasive polishing, resulted in successful electro-assay of various phenolics. Modification of glassy carbon electrodes with α-alumina resulted in improved electrochemical response in comparison with θ and γ-alumina, and it was confirmed that alumina structure, and not the particle size or surface area, can exert notable effects [97].
Glassy carbon modification with alumina enhanced the voltammetric current response of gallic acid, caffeic acid, chlorogenic acid, catechin, quecetin, and rutin. By applying this simple procedure of electrode modification, lowered DPV relative standard deviation (RSD < 3%, n = 5) for repetitive assays and high inter-electrode precision (RSD < 4%, n = 3) were reported. Nevertheless, when use of unmodified glassy carbon electrodes is chosen, the application of the sonication step to remove all residual alumina is compulsory, as alumina remaining on the electrode surface can significantly affect the voltammetric profile of sample antioxidants [98].
The use of other metal oxides for modification, may also provide performance improvement in the electrochemical determination of antioxidant species. Modification with metal (gold, silver and platinum) or metal oxide nanoparticles imparts distinctive size-dependent electrochemical features. The sol-gel chemistry served for developing silica-modified electrodes, exploiting silica adsorption properties [98,99].
Carbon nanomaterials were divided into: Zero-dimensional fullerenes [100], one dimensional carbon nanotubes [101], two-dimensional graphene [102], and the threedimensional porous carbons [103]. Porous carbon materials are fabricated using precursors named template composite materials which are synthesized, with subsequent carbonization and template removal [104]. Nevertheless, such a technique is laborious and necessitates a series of synthetic steps, the first stage of template injection, the etching process and the long solidification time [105], which may restrict mass applications of porous carbon materials [106].
Fullerene Electrodes: Fullerenes represent a class of carbon compounds in which carbon atoms form closed cage or cylinder-shaped structures. In the first case the compound is called Buckminsterfullerene (C60, named after the American architect R. Buckminster Fuller, whose geodesic dome was constructed relying on the same structural principles), and in the second case, the obtained structure is called carbon-nanotube [107][108][109]. Single-walled and multi-walled carbon nanotubes are widely employed in electrode modification, being biocompatible, having large surface area to volume ratio, enhanced electro-conductivity, chemical and mechanical resistance.
In the structure of graphene, atoms constitute a single layer and are placed in a twodimensional honeycomb lattice. Each atom uses sp 2 hybridized orbitals to connect by sigma bonds to its three nearest neighbors, and contributes with one electron (belonging to the p unhybridized orbital) to the conduction band that is common for the whole sheet. This type of bond is also encountered in carbon nanotubes, fullerenes and glassy carbon. Graphene oxide nanoparticles, alongside metal oxide nanoparticles are largely used in electrode modification, aiming at antioxidant determination. The oxygenated groups can lead to improvement in the electrochemical responses and mechanical properties. The polarity of these groups present at the surface of graphene oxide results in high dispersibility in polar solvents, enabling applications in biosensing systems.
Graphite Electrodes: Graphite is an allotropic form of carbon, where sp 2 -hybridized atoms form planes of hexagonal bonds. Graphite is a stable crystalline form of carbon, which can be employed as such, or in the form of composites [110][111][112][113]. Electrodes are inexpensive, commercially available and easy to modify, so they benefit from selectivity enhancement through various modifications, renewable surfaces and hazard-free polishing [114]. The high delocalization degree of pi electrons and the weak van der Waals interactions between the layers, result in good electro-conductivity.
Diamond Electrodes: Diamond is an allotrope form of carbon with insulating properties, very good mechanical resistance, the hardness being due to the bonds established between sp 3 carbon atoms. Diamond is a non-easily accessible material, whose preparation require high temperatures and pressures [115]. By doping diamond with boron in different proportions, conductive, superconducting or semiconductor materials can be synthesized. These electrodes are chemically inert and are endowed with excellent electrical features. High boron-doped (10 3 -10 4 ppm) diamond has metal-like conductivity and can be applied as electrode material [116]. Boron-doped polycrystalline diamond exhibits a rougher morphology, a higher sp 3 content, a broader water potential window, and a lower background current [117].
Screen-Printed Electrodes: Such electrodes are developed by printing inks on ceramic or plastic surfaces. The inks, depending on the composition (carbon, gold, platinum) will determine the characteristics of screen-printed electrodes, that can be part of a three electrode set-up in a measuring cell. Depending on the target analyte, the ink can be modified by incorporation of metal powders, redox complexes or biocatalysts, to promote electron transfer [118][119][120].
Glassy carbon, vitreous carbon, carbon nanotubes, fullerenes, screen-printed, graphite, and diamond electrodes are considered homogenous carbon electrodes, whereas carbon paste and modified carbon pastes are heterogeneous carbon electrodes.
In a recent study, a detailed description of advantages and shortcomings of several broadly employed types of carbon-based electrodes is provided [121].
Carbon paste electrodes offer an analytical response on wide potential ranges, with small background current, reduced ohmic resistance, and facility of tuning pretreatment methods and surface modification. These materials do not harm the environment and operate at low cost. Nevertheless, they were characterized as not stable for functioning in flow systems, are not compatible with organic solvents, and often require recalibration. When using organic compounds as binders, the use of such sensors results in irreversibility of recorded voltammograms, and the surface roughness can influence reproducibility of the response.
Glassy carbon electrodes exhibit high mechanical resistance and excellent electroconductivity. They are characterized by chemical inertness and function on large potential domains, over wide pH ranges, from strongly acidic to alkaline environment. Their large size and difficulty to manufacture at large scale may constitute inconvenients. The electron transfer occurs slower than at electrodes based on noble metals.
Carbon fiber microdisk electrodes are easy to use due to their small diameter and benefit from high speed of electron transfer. They give a rapid analytical response and exhibit high sensitivity for small concentration changes. Being compatible with biological media and non-toxic to cells, can be applied successfully to in vivo determinations. Nevertheless, they may not exhibit resistance to the mechanical force or to the high temperatures applied during the step of capillary pulling. Low selectivity and the possibility to break glass insulation during in vivo measurements are other disadvantages.
Basal plane pyrolytic graphite electrodes are characterized by high speed electrode reaction kinetics, with lowered background signal. Shortcomings may be the irreversible behavior and the large dimensions.
Screen-printed carbon electrodes are easy to employ, due to portability and facility to apply modifications. A broad series of geometries can be obtained, and the determinations can be performed with the possibility to eliminate surface fouling, and low cost. Nevertheless, the incorporated binders may alter the shape of voltammograms. Fouling of the electrode surface may be caused by products of redox reactions. Other shortcomings may be the rough surface and the slow reaction kinetics. The organic solvents present in the buffer may dissolve the ink, diminishing sensitivity.
Given the confirmed advantages of boron-doped diamond (excellent electroconductivity, mechanical resistance), the behavior of this type of electrode has been investigated in cyclic voltammetry of gallic acid. At low potentials, when the electrolytes are stable, deactivation of boron-doped diamond has been reported. It was found that gallic acid electro-oxidation generated the occurrence of a polymeric film on the anodic surface, causing boron-doped diamond deactivation [122].

Determination of Individual Antioxidants with Carbon-Based Electrodes
The determination of individual key antioxidants relied on a series of unmodified or modified carbon-based electrodes. An overview of the analytical parameters and applications on real samples, at the assay of some individual antioxidants, using carbonaceous working electrodes is presented in Table 2  . Table 2. Electroanalytical techniques applied to the assay of key individual antioxidants .

Carbon-Based Working Electrode
Analytical Characteristics Ref.
β-carotene Chronoamperometry;  -the anodic peak area in the range −100 to 1200 mV accounted for about 70% of total phenolics that absorbed at 280 nm; -catechol and galloyl containing polyphenols present in wine were quantitated relying on the size of the first anodic peak at around 450 mV after treatment with acetaldehyde; -flavonols were quantitated on the basis of the anodic peak current at 1120 mV; -good correlation of total flavanols with HPLC; [137] 16.
μmol L −1 (DPV); -LOQ for butylated hydroxyanisole 0.26 μmol L −1 (SWV) and 0.23 μmol L −1 (DPV); -determination in mayonnaise, margarine, biodiesel; A series of irreversible cyclic voltammograms recorded for increasing ascorbic acid concentrations, at a carbon paste electrode are given in Figure 1. A 50 mV s −1 cyclic voltammetric scan rate could enable analyte diffusion to the electrode and promoted electron transfer. In the case of irreversible or quasi-reversible voltammograms [123,168], at elevated scan rates, electron transfer is slow relative to the applied potential sweep rate, so the rate of establishing the equilibrium at the electrode is diminished. In the case of very elevated scan rates, peaks have high current intensities, but distortions may occur on the voltammogram [168]. In differential pulse voltammetry at carbon paste electrode (Figure 2), the optimum value of 75 mV was used for the pulse amplitude, and 125 ms was chosen for the pulse period, allowing a good compromise between promoting analytical signal, minimizing noise and good resolution [123]. A 50 mV s −1 cyclic voltammetric scan rate could enable analyte diffusion to the electrode and promoted electron transfer. In the case of irreversible or quasi-reversible voltammograms [123,168], at elevated scan rates, electron transfer is slow relative to the applied potential sweep rate, so the rate of establishing the equilibrium at the electrode is diminished. In the case of very elevated scan rates, peaks have high current intensities, but distortions may occur on the voltammogram [168]. In differential pulse voltammetry at carbon paste electrode (Figure 2), the optimum value of 75 mV was used for the pulse amplitude, and 125 ms was chosen for the pulse period, allowing a good compromise between promoting analytical signal, minimizing noise and good resolution [123].
-LOQ for butylated hydroxyanisole 0.26 μmol L −1 (SWV) and 0.23 μmol L −1 (DPV); -determination in mayonnaise, margarine, biodiesel; A series of irreversible cyclic voltammograms recorded for increasing ascorbic acid concentrations, at a carbon paste electrode are given in Figure 1. A 50 mV s −1 cyclic voltammetric scan rate could enable analyte diffusion to the electrode and promoted electron transfer. In the case of irreversible or quasi-reversible voltammograms [123,168], at elevated scan rates, electron transfer is slow relative to the applied potential sweep rate, so the rate of establishing the equilibrium at the electrode is diminished. In the case of very elevated scan rates, peaks have high current intensities, but distortions may occur on the voltammogram [168]. In differential pulse voltammetry at carbon paste electrode (Figure 2), the optimum value of 75 mV was used for the pulse amplitude, and 125 ms was chosen for the pulse period, allowing a good compromise between promoting analytical signal, minimizing noise and good resolution [123].

Determination of Total Antioxidant Activity with Carbon-Based Electrodes
Bare and modified carbon-based electrodes have been used for the antioxidant activity and its main contributors' assessment. The electrochemical measurements are performed as per reference to a standard antioxidant, Trolox, gallic acid, ascorbic acid or quercetin, etc. In most of the cases, the peak intensities or peak areas are considered, providing quantitative informations.
Often, authors report indexes illustrating antioxidant activity: IC 30 and IC 50 [169]. IC 50 corresponds to the antioxidant amount that induces a change of a model signal by 50%. The lower the concentration required for 50% increase or inhibition of the considered model signal, the more active the antioxidant. Korotkova et al. [170] relied on the modifications in the oxygen electro-reduction current in the presence of antioxidants, as criteria to determine IC 50 by voltammetry. Catalase and superoxide dismutase yielded an increase of the oxygen electro-reduction current, when compared to the signal obtained in supported electrolyte. Superoxide dismutase presented an IC 50 value of 1.08 µM [170]. Wei et al. evaluated IC 50 , as the antioxidant amount that diminishes the oxidation peak of superoxide by 50%. They reported an IC 50 value of ascorbic acid of 5 × 10 -4 mol/L [171].
Blasco et al. [172] define the "Electrochemical Index" as the total polyphenolic content measured by an electrochemical technique. In this approach, the corresponding total phenolics concentration obtained from the "total electrochemical signal" was named "Electrochemical Index". The total electrochemical signal was assimilated to the total amperometric current measured at controlled potential (800 mV), to achieve oxidation of all polyphenolics at neutral pH of 7.5. So, the "Electrochemical Index" became an approach to "total polyphenols" in samples without ascorbic acid (or alpha-tocopherol), or an approach to "total natural antioxidant" content in those samples where ascorbic acid (or alpha-tocopherol) could be present [172].
An overview of the analytical parameters and real sample applications regarding the assay of total antioxidant activity by the use of carbon-based working electrodes is given in Table 3  . Table 3. Some relevant examples of electrochemical assay of antioxidant activity and its key contributors .

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

9.
Cyclic voltammetry; Differential pulse voltammetry; -carbon black nanoparticles press imprinted films; -oxidation of ascorbic acid at 0.90 V in cyclic voltammetry and around 0.75 V vs. Ag/AgCl in differential pulse voltammetry; -in the CVs of the bark extract, a very well contoured peak was observed at 1.3 V, corresponding to meta-diphenols and isolated phenols; -in the CVs of the root and leaf extracts, an additional peak at 0.9 V indicates the presence of phenolics with ortho-or para-diphenol groups, in low amounts; -determination of the antioxidant capacity of Bunchosia glandulifera (Jacq.) Kunth (Malpighiaceae) extracts, using ascorbic acid as standard; [182] 11.
Chrono-amperometry; Differential pulse voltammetry; -glassy carbon electrode modified with multi-walled carbon nanotubes; -glassy carbon polyquercetinmodified electrode; -supporting electrolyte: Phosphate buffer pH 7.0; -antioxidant capacity using gallic acid as reference; -DPVs recorded from 0 to 0.8 V (pulse amplitude 50 mV, pulse time 50 ms and potential scan rate 10 mV/s); -DPVs of tea on the polyquercetin-modified electrode exhibited oxidation peaks at 0.080 and 0.19 V depending on the type of tea and a less configured oxidation step between 0.55 and 0.62 V vs Ag/AgCl; -chronoamperograms recorded at a constant potential of 0.2 V, potential corresponding to oxidation of tea antioxidants; -RSD = 0.5-20%, as function of the tea type (chronoamperometry); -determination of the antioxidant capacity of tea, highest content for Green Sencha; [183]

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

12.
Cyclic voltammetry; -glassy carbon electrode; -supporting electrolyte: Phosphate buffer pH = 7.0; -cyclic voltammetric scans performed between 0.0 and 0.5 V vs Ag/AgCl at a scanning rate of 5 mV/s; -anodic peak at 244 mV for pomace and its parts (skins and stems), and 252 mV for seeds; -analyse of winemaking by-products (pomace, skins, seeds and stems separated from pomace); [184] 13. Cyclic voltammetry; -glassy carbon electrode; -supporting electrolyte: 0.1 M sodium acetate-acetic acid buffer at pH 3.6; -all the grapes revealed peak I at 0.26-0.31 V, peak II between 0.42 and 0.55 V, and peak III at approximately 0.66 V vs Ag/AgCl; -correlations of anodic peak area with phenolic content and antioxidant activity were assessed; -determination of phenolic contents and antioxidant capacity in 12 grape cultivars; [185] 14.
Cyclic voltammetry; Square-wave voltammetry; Differential pulse voltammetry -glassy carbon electrode; -laccase-modified carbon paste electrode; -supporting electrolyte: 0.1 M phosphate buffer solution, pH 6.0, using Ag/AgCl reference; -CV: Scan rate of 100 mV s −1 within the range 0-1.4 V; -SWV: Pulse amplitude 50 mV, frequency 50 Hz and a potential increment of 2 mV, scan rate of 100 mV s −1 ; -first peak present between 100 and 400 mV, second between 0.55 and 0.7, and third at around 1 V, in CV/SWV; -solutions of the extracts yielded highest DPV peaks at 0.2 V, alongside peaks present at 0.6 and 0.9 V; -electrochemical indexes were calculated based on the sum of ratios of peak currents to peak potentials in DPV; -antioxidant activity evaluation of dried herbal extracts; -highest electrochemical indexes obtained for Gingko biloba and Hypericum perforatum, consistent with the results obtained by spectrophotometry; [186] 15.

Cyclic voltammetry;
Differential pulse voltammetry; -glassy carbon electrode; -supporting electrolyte 0.1 M KCl; -CV scan from 0 to +1000 mV at a scan rate of 100 mV s −1 ; -DPV scan from 0 to +1000 mV at a scan rate of 100 mV s −1 ; -the first peak of mature-phase milk occurred at around 400 mV; colostrum, had oxidation peaks at very high potential, around 800 mV (DPV) vs Ag/AgCl; -mature-phase milk yielded a peak at around 400 mV; pasteurized milk had a peak at around 500 mV (CV); -areas below oxidation peaks proportional to the amount of antioxidant compounds; -free radical scavenging activity was highest for fresh breast milk and lowest for pasteurized breast milk, confirming the results obtained in DPPH assay; -correlation between DPV and CV (r = 0.602, p < 0.001); correlation between DPV and DPPH method (r = 0.339, p = 0.003); correlation between CV and DPPH method (r = 0.468 p < 0.000); [187] Table 3. Cont.

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

16.
Cyclic voltammetry; Differential pulse voltammetry; -screen-printed carbon electrodes; -supporting electrolyte 0.1 M HCl; -in CV, the potential recorded between 0.0 V and +1.2 V, at 100 mV s −1 scan rate, using silver pseudo-reference electrode; -optimum DPV parameters: 100 mV modulation amplitude, 10 mV step potential, 0.05 s modulation time and 0.5 s interval time; -ortho-diphenols (oleuropein, hydroxytyrosol and caffeic acid) show one anodic peak between 0.5 and 0.6 V, and one cathodic peak between 0.4 and 0.6 V (CV); same compounds present an anodic peak at +0.5 V, in standard and real sample (DPV); -ferulic acid gave an oxidation peak at higher potential (0.7 V), in the standard solution, whereas this signal was almost negligible in the real sample (DPV); -tyrosol is oxidized at +0.93 V, alongside other mono-phenols (such as vanillic acid) that suffer oxidation around this potential value, in standard and real sample (DPV); -LOD of 0.022 mg L −1 for caffeic acid and tyrosol, in DPV; -determination of hydrophilic phenols in olive oil; [188] 17.

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

23.
Cyclic voltammetry; -glassy carbon electrode; -supporting electrolyte: Sodium acetate-acetic acid buffer (0.1 mol L −1 , pH = 4.5) in acidified 80% methanol; -analytical cyclic voltammetric signals for all target phenolic compounds present between 0 mV and 800 mV, at a scan rate of 100 mV s −1 ; -cyclic voltamogramms of berry fruits presented anodic peaks between 310 mV (quercetin) and 0.756 mV (coumaric acid) vs. Ag/AgCl -antioxidant capacity quantification relied on the area underneath the anodic peak, corresponding to the charge up to a potential value of 500 mV (Q 500 ); -evaluation of antioxidant activity of 15 berry samples (strawberries, blackberries, blueberries and red raspberries); [194] 24. Amperometry; -glassy carbon electrode integrated in a flow injection system with sequential diode array and amperometric detection; -supporting electrolyte: Ethanol 12% v/v and tartaric acid 2 g/L, pH 3.6; -amperometric determinations at 800 mV vs. Ag/AgCl; -calibration curve over the range 0-0.19 mM gallic acid equivalents; -determination of total polyphenol content and antioxidant activity of white, red wines and oenological tannins; -total wine phenolic content between 1.08 and 15.4 mM gallic acid; -concentration range 0.07-0.34 mM gallic acid obtained for tannin solutions; [195] 25.

No. Analytical Method Carbon-Based Working Electrode
Analytical Characteristics Ref.

27.
Cyclic voltammetry; Differential pulse voltammetry; -glassy carbon electrodes modified with carbon nanotubes and chitosan; -supporting electrolyte: Britton-Robinson electrolyte buffer, at pH 3.0; -chicoric acid anodic peak at 0.610 ± 0.060 V and cathodic peak at 435 ± 0.055 V vs Ag/AgCl, at 300 mV s −1 scan rate; -the intensities of oxidation and reduction currents linearly vary with the square root of the scanning speed, in cyclic voltammetry; -DPVs showed oxidation peaks for caftaric acid at 0.505 ± 0.002 V, and for chicoric acid at 0.515 ± 0.001 V vs Ag/AgCl, which are consistent with the results obtained at the assay of pharmaceutical forms; -determination of total polyphenol content and antioxidant activity of Echinacea purpurea extracts in 3 different pharmaceutical forms (capsules, tablets and tincture); [198] 28. Staircase voltammetry; glassy carbon electrode; -supporting electrolyte-100 mM KNO 3 ; -staircase voltammograms recorded successively for 4 cycles between +1.0 and −1.2 V vs Ag/AgCl; -scan rate 50 mV/s, starting and ending in +1.0 V; -the half-wave potential (E 1/2 ), or the potential corresponding to half the anodic peak current (Ipa) was considered; lower E 1/2 values are correlated to higher antioxidant potential; -the peak intensity or, more accurately the surface area under the oxidation peak, provided quantitative informations: Antioxidant concentration/antioxidant capacity; -analytical peaks present between −0.6 and 0 V, and around 0.5 V; -evaluation of antioxidant activity for teas, wines and (superfood) juices; -antioxidant index calculated relying on the maximum charge of oxidation (Qmax), the standard potential of the oxygen evolution reaction (vs. Ag/AgCl) and the standard potential of hydrogen evolution reaction (vs. Ag/AgCl); [199] 29.  -carbon electrode modified with guanine-, polythionine-, and nitrogen-doped graphene; -determinations performed in PBS pH = 1.5; -1.0 mg/mL −1 guanine solution as optimum for modification of the electrode; -a pair of redox peaks found between 0 and 0.3 V in CV; peak currents increased with increasing scan times; a thin blue membrane formed on the surface of electrode, showed that thionine was successfully polymerized; -oxidation peak for ascorbic acid at about 1.1 V (SWV); -linear range for ascorbic acid (standard antioxidant) analytical response ranged from 0.5 to 3.0 mg L −1 in SWV; -LOD 0.21 mg L −1 (SWV); -RSD 3.1% (SWV); -determination of antioxidant capacity of fruit juices (grape juice, guava juice, and orange juice) and jute leaves extract, ramie leaves extract, and hemp leaves extract; [202] 32 Differential pulse stripping voltammetry (DPSV); Cyclic voltammetry; -glassy carbon electrode modified with polyglycine; -supporting electrolyte: Britton-Robinson electrolyte buffer, pH 3.0; -well-configured oxidation peak of quercetin (model antioxidant) occurs at around +460 mV, a corresponding cathodic peak being visible at 420 mV vs Ag/AgCl; -peaks shifted towards less positive potentials when the scan rates increased from 20 to 400 mV/s in CV; -oxidation peak current assigned to phenolic compounds of yam, at 430 mV, consistent to the peak potential of quercetin, on differential pulse stripping

Critical Perspectives and Comparative Conclusions
Analytical parameters of carbon-based sensors, analyte oxidation-reduction steps and potentials are influenced by analyte chemical structure (number and position of -OH groups or other substituents), electrode type, its development and interaction with the antioxidant molecule, as well as working conditions (matrix characteristics such pH or presence of interferences).
Studies performed on ascorbic acid electro-oxidation reveal irreversible voltammograms, consistent with the reported mechanism describing electrochemically reversible electron transfer, and a subsequent irreversible chemical process. During the first step, the oxidation of ascorbic acid involves liberation of two electrons and two protons, yielding dehydroascorbic acid. This electron transfer is followed by an irreversible solvation reaction at pH < 4.0. At pH values smaller than the first pKa value of L-ascorbic acid (around 4.5), two protons are exchanged during the process, whereas at higher pH values (4.5-8.0), a single proton is released, giving ascorbate anion as electroactive compound [206,207]. These described oxidation/solvation steps are consistent with the variation of the peak potential with pH, noticed up to pH 8.0, at a gold electrode: A pH increase from strong acidic to mild acidic and neutral values, results in anodic peak displacement to more positive values. Reported mechanistics aspects underlying ascorbic acid oxidation at pH > 8.0 imply ascorbate anion oxidation to a diketolactone, which by dehydration gives dehydroascorbic acid, that eventually suffers isomerisation to an ene-diol oxidizable at higher potentials [207].
Studies performed at a glassy carbon electrode modified by single-walled carbon nanotube/zinc oxide, proved that ascorbic acid anodic peak current increased with the increase of pH from 2 to 5, and reached a maximal value for pH comprised between pH 4 and 5. Then, the peak current progressively diminished as the pH increased from 6 to 10 and then increased again, for pH values found between 11 and 12. This confirms the involvement of deprotonation step in the oxidation of ascorbic acid. The oxidation peak shifted towards less positive potential values as the pH increased from 2 to 13. On the irreversible voltammograms obtained at the modified electrode, the oxidation potential shifted by approximately 240 mV towards a lower potential and the peak current doubled, when compared to the bare glassy carbon electrode, confirming electrocatalytical effect in a diffusion-controlled process (linear dependence of the current intensity on the square root of scan rate was observed) [208].
At a p-phenylenediamine film-holes modified glassy carbon electrodes, the cyclic voltammetric oxidation peak current of ascorbic acid increased, as the pH increased from pH 2 to 5. A further pH increase of the buffer resulted in a decrease of the analytical signal. This observation is consistent with the described analyte-electrode interaction: Within the pH range for which the modifier film is positively charged and the analyte is found in anionic form, the interaction of the modified glassy carbon electrode with ascorbic acid promotes the redox signal. With increasing pH value, the film may adopt a negative charge (caused by adsorption of free OH − ), causing rejection of the anionic form of ascorbic acid. Therefore, the pH value of 5.0 was chosen as optimum, privileging interaction between the cationic film of modifier and the anionic form of analyte [209].
The analysis of anthocyanins by cyclic voltammetry at pH 7.0 revealed for kuromanine (cyanidin-3-O-glucoside) chloride and cyanin (cyanidin-3,5-O-diglucoside) chloride a first reversible peak at around 300 mV, due to the oxidation of catechol moiety present on the B-ring. The oxidized molecule can be subject to further oxidation at higher potential values yielding a second oxidation peak corresponding to the oxidation of the 5,7-dihydroxyl structure of the A-ring (resorcinol moiety). Given the different sensitivities of the techniques, the second peak obtained in cyclic voltammetry is smaller than that obtained in differential pulse voltammetry [129].
Studies on flavonoids reconfirmed first oxidation of the most redox active -OH groups present on ring B. Oxidations of the -OH groups present at C3 on ring C, and on the ring A (in resorcinol group) occur at more positive potentials. Studies on delphinidin anthocyanidin firstly reveal two peaks corresponding to oxidative processes involving -OH groups at 3 ,4 ,5 positions (ring B), followed by a third peak corresponding to oxidation of -OH present on position 3 on ring C, and a fourth corresponding to oxidation of -OH groups present at positions 5 and 7 on ring A (resorcinol moiety) [128].
Oxidation of most ortho-diphenols occurs at close potentials, while mono-phenols are oxidized at higher potentials. Phenolic compounds that possess two −OH groups in ortho position on the aromatic ring, are reversibly oxidized to ortho-quinones: They give first peaks appearing on voltammograms, consistent with enhanced electron donating ability typically assigned to catechol-like polyphenols, endowed with highest antioxidant power. Most monophenols are subject to irreversible oxidation, due to only one -OH group. Ortho-diphenols present in olive oil, such as caffeic acid, hydroxytyrosol, and oleuropein are subject to reversible oxidation due to the presence of two hydroxyl groups in ortho position, exhibiting one anodic peak and one corresponding cathodic peak at the reverse scan. Tyrosol was irreversibly oxidized generating one anodic peak, due to the oxidation of only one −OH group present on the benzene ring, with the absence of the corresponding cathodic peak. Ferulic acid, a cinnamic acid derivative presenting only one −OH group, has one oxidation peak, and a much less configured reverse cathodic peak [188].
A cyclic voltammetric study performed on wine phenolics confirmed first oxidation of catechol moieties. Catechol-containing hydroxycinnamic acids, as main phenolics present in white wines exhibit a first peak at around 480 mV, followed by polyphenols with more positive potentials (900-1000 mV) such as coumaric acid and their derivatives. High molecular mass compounds resulting from oxidation of original white wine phenolics during storage can contribute to IInd peak. Catechin-type flavonoids, oligomeric and polymeric tannins present in red wines give a first peak around 440 mV; the second peak at around 680 mV was assigned to malvidin, with slight contribution from other phenolics such as trans-resveratrol; second oxidation of the catechin-type flavonoids resulted in a third anodic peak for red wines at 890 mV [137].
Yakovleva et al. performed an electroactivity-based general classification of the phenolic compounds under study (benzenediols, phenolic acids, flavonoids) into groups: Quercetin, dihydroquercetin, and phenolic acids with two −OH groups at ortho positions (2,3-dihydroxybenzoic, protocatechuic, and caffeic acid) are subject to reversible electrooxidation at pH 6.0, below 400 mV; polyphenols with -OCH 3 substituents present oxidation peaks in the range 400-600 mV; monophenols such as monohydroxyphenolic acids are oxidized at lower rate, having anodic peak potentials greater than 600 mV; the electrondonating groups −OH, −OCH 3 or −CH 3 present on the aromatic ring, were confirmed to render studied phenols more oxidizable. Monohydroxylated phenolics with methoxy substituents could be faster oxidized by electron donation than monohydroxyphenols lacking methoxy groups. It was stipulated that the mechanism underlying this trend is the stabilizing effect of the methoxy groups in the phenoxyl radical formed after electron loss by phenolic compounds [136].
In a study focused on the electrochemical behavior of anthocyanins and anthocyanidins, comparing myrtillin chloride (that has a pyrogallol group on the B-ring) with oenin chloride (that has a hydroxyl group on C4 , placed in ortho position with respect to two methoxy groups), the methoxy groups could not impart the previously discussed oxidation facility, the reverse effect being noticed: Hydroxyl group from pyrogallol moiety on the B-ring (the case of myrtillin chloride) was oxidized with greater facility than the hydroxyl group placed in the ortho position with respect to two methoxyl groups (the case of oenin chloride). Moreover, the hydroxyl group found in the ortho-position with respect two methoxyl groups (in oenin chloride), was more oxidizable than the catechol group placed in the ortho position with respect to a single methoxy group (in petunidin chloride) [129].
Electrochemical studies on phenolic antioxidants are performed in acid media or, most of them, close to physiological pH values, to hamper irreversible conversion of polyphenols at alkaline values. Cyclic voltammograms recorded for some phenolic acids and flavones at pH higher than 8.0 were characterized by irreversibility, and the anodic oxidation potential could not be precisely assessed [136]. It was asserted that phenolic acids can suffer dimerization and polymerization, synchronous with their oxidation by one-electron loss [210]. An increase of the pH value was corroborated with a linear diminution of the oxidation potentials in the case of anthocyanins. Moreover, an enhanced adsorption of the oxidation products (with fouling the electrode surface) was also noticed, correlated to a dramatic anthocyanins' oxidation peaks decrease during second scan, at all pH values [129].
During a study performed on major phenolics present in coffee, an inverse linear dependence of oxidation peak potential vs pH was observed until reaching the pKa value, with a slope of approximately 59.2 mV, close to the Nerstian theoretical value, describing an electron:proton transfer processes. On the differential pulse voltammograms obtained for 0.5% w/v coffee sample, highest peak currents were noticed at middle-acid pH values (ranging between 5.0 and 6.0), while the slope was broken and peak currents diminished at alkaline pH of 8.0. The same observation was valid for voltammograms of hydroxycinnamic acid derivatives. It was concluded that the redox behavior of electroactive species from coffee samples is dominated by catechol-like main redox contributors, endowed with most enhanced antioxidant features [177].
A series of electroanalytical methods have proved their viability in antioxidant and antioxidant activity assay, largely employed being cyclic, differential pulse and amperometric techniques. In differential pulse and square wave techniques, the influence of capacitive currents is hampered, promoting sensitivity and detection of irreversible oxidation-reduction processes linked to the presence of minor electroactive contributors. Moreover, linear scan (cyclic voltammetric) techniques proved their viability in the comparative study of antioxidants' electroactivity (relying on peak position), as well as in antioxidant capacity assay (based on the area under the anodic peak).
Carbonaceous electrode properties affecting electrochemical response are represented by surface properties, electronic structure, adsorption, electrocatalytical behavior and surface preparation [211]. The performances of carbon electrodes as working electrodes depend on the structure of the electrode, on the chemical bonds established between the carbon atoms, as these factors influence mechanical, conductive and chemical features. Considering allotropic carbon forms, diamond, whose structure is ensured by sp 3 bonds, has poor conductivity, thus requiring boron dopping. Carbon allotropic forms whose structure is ensured by sp 2 bonds are endowed with convenient electrical features, mainly conductivity.
Electrochemical determinations rely on surface phenomena and thus, electrode surface represents an important characteristic influencing analytical performances, reaction kinetics and interactions with the compounds to be analyzed. Electrode surfaces are commonly polished using alumina, mainly in the case of glassy carbon. A significant voltammetric current increase was reported in the case of gallic acid, chlorogenic acid, caffeic acid, catechin, rutin and quercetin, as consequence of surface modification with alumina, by mere abrasive polishing [98]. It was found that modification of the glassy carbon electrode surface with small amounts of alumina can result in enhanced apparent catalysis of electron transfer involving catechol at low pH value. It was reported that catechol suffers adsorbtion on alumina, not directly on the electrode, so the process involves the triple boundary present between alumina particles, analyzed solution and the electrode. Enhancement of catechol redox process takes place at low pH, as such values facilitate the proton-coupled electron transfer at the previously-mentioned three-phase boundary [94]. The antioxidant capacity and activity, as well as the electrochemical index are significantly influenced by the presence of alumina in the case of tea, wine and phytotherapics samples. The improvement in detectability and sensitivity recommends the use of alumina-modified glassy carbon electrode for this assay. The shift of the anodic peak potentials towards less-positive values was reported, thus indicating that the electron transfer is promoted by surface modified with alumina. Nevertheless, the reported diminution in the difference between peak potentials (affecting peak-to-peak discrimination), may constitute a shortcoming in the case of surface modification with alumina [98].
Glassy carbon electrodes are prone to other modification methods: Electrode coating with ionic polymers was applied due to their ability to enhance conductive properties. A glassy carbon electrode coated with a thin film of poly(trihexylvinylbenzylammonium chloride) proved permselectivity to uric acid, that presented a linear voltammetric analytical response in the concentration range of 1-10 µM [212]. A glassy carbon electrode modified with multi-walled carbon nanotubes dispersed in polyhistidine allowed for simultaneous differential pulse voltammetric determination of ascorbic acid and paracetamol with improved analytical responses at minimized overvoltage and under diffusion control, benefiting from large electroactive area and enhanced electrocatalytical properties [213].
Electrolytes influence the surface of carbonaceous electrodes. Studies performed in sulfuric acid 4.5 M confirmed a change in composition of surface compounds, removal of finely crystalline graphite, as well as of unstable functional groups from the surface of activated carbon [214].
Conventional carbon-based sensors include glassy carbon, carbon fiber or pyrolytic graphite electrodes. Developing sensors at sizes below 100 nm is often a key characteristic that typically results in high surface area and improved surface/volume ratio. Other distinctive, attractive properties are swift electron transfer, enhanced electrocatalytic activity, improved interfacial adsorption and biocompatibility, when compared to other conventional materials [215,216]. Carbon-based nanomaterials composed of graphitic-type atoms linked by sp 2 bonds, include fullerenes, carbon nanotubes and graphene. Their behavior depends on the interactions established with other materials and atomic or molecular structures [215,[217][218][219][220]. These nanomaterials possess hollow or layered structures, and can establish non-covalent linkages with organic species through π-π stacking, hydrophobic interactions, hydrogen bonding, van der Waals or electrostatic forces [215,221].
Carbon nanomaterials are prone to combination with a series of other nanomaterials (based on noble metal nanocrystals, ceramics or Teflon) giving rise to nanocomposites with improved characteristics in a distinctive, novel material [215]. Previously reported novel application reveals that oxidation peaks of key analytes such as ascorbic acid, dopamine and uric acid at a ZnO nanosheet arrays/graphene foam sensor, are higher than those obtained at metal oxide-based (indium tin oxide) electrode, proving enhanced electrochemical features in the assay of both analytes, linked to the high conductivity of 3D porous graphene foam and large specific surface imparted by ZnO nanosheet arrays [222]. Diminished ascorbic acid overpotential and good peak-to-peak DPV separation for ascorbic acid vs dopamine (218.0 mV), dopamine vs paracetamol (218.0 mV), and ascorbic acid vs paracetamol (436.0 mV) were reported for platinum nanoparticles-decorated graphene nanocomposite electrode, with performances improved versus graphene-modified glassy carbon electrode and bare glassy carbon electrode [125]. A ZnO nanorods/graphene nanosheets/indium tin oxide hybrid electrode allowed good discrimination between uric acid and ascorbic acid, peaks at ZnO nanorods/indium tin oxide being not configured [150].
Carbon paste electrodes are synthesized by combining carbon-based materials (carbon nanotubes, microspheres or nanofibers, acetylene black, graphite and diamond,) with a binder (silicon oil or mineral oil) and largely used in antioxidant assay. A highly rigorous control over pasting liquid amount is required, as large amounts, although able to lower background currents, may diminish electron transfer rates [223,224]. These materials are easy to synthesize, inexpensive, possess reduced ohmic resistance, have broad potential range. They are prone to facile modification by using redox complexes, metal oxides or ionic surfactants, to improve performances in voltammetric assays [88]. The use of a carbon paste electrode modified with copper oxide-decorated reduced graphene enabled simultaneous voltammeric assay of ascorbic acid, uric acid and cholesterol, with sensitivity and good separation in SWV [127].
Employing printing technologies for rapid assay of electroactive species benefits from adaptability, compactibility, reduced costs, facility of large scale production, onsite application and easy modifier incorporation. Characteristics such as the employed ink material, the type of substrate, the choice of a particular electrochemical technique, the extent of waste generation, the obtained analytical performances, as well as shortcomings of each application of printed electrode should be considered. Researches should be focused on developing printed electrodes characterized by best analytical performances, lacking hazardous effects to the environment, to successfully replace conventional electrochemical cells [225].
The applications of chemical and biochemical carbon-based sensors encompass swift evaluation of key antioxidants and antioxidant activity in foodstuffs and dietary supplements, as well as in various biological media, these aspects being tightly related to screening/maintaining health status, hence the permanent interest in improving analytical performances and adaptability.
Electrode modification with single-, multiwalled carbon nanotubes or noble metal nanoparticles promotes electrocatalytical features, facilitating electron transfer: Peaks appear at lower potential and have higher corresponding current intensity. Hybrid nanosensors made of carbonaceous materials and metal/metal oxides enable for antioxidant signal separation and enhanced sensitivity in complex media.