2. Electrochemical Techniques

Electrochemistry offers a wide range of electroanalytical techniques. A typical electrochemical experiment includes a working electrode made of a solid conductive material, such as platinum, gold, or carbon, a reference electrode, and a counter electrode, all the electrodes are generally immersed in a solution with a supporting electrolyte to guarantee the conductivity in the solution ^[10].

Electrochemical sensors belong to the largest family of chemical sensors. A chemical sensor can be defined as ‘‘a small device that, as the result of a chemical interaction or process between the analyte and the sensor device, transforms chemical or biochemical information of a quantitative or qualitative type into an analytically useful signal” ^[11]. This definition can be extended to the electrochemical ones modifying it in this way: a small device that, as the result of an electrochemical interaction or process between the analyte and the sensing device, transforms electrochemical information of a quantitative or qualitative type into an analytically useful signal ^[11]. The use of a nanomaterial together with the analyte kind and nature have proven crucial for the sensor sensitivity, selectivity, and stability ^[12]. As for all the chemical sensors, the critical parameters of electrochemical sensors are sensitivity, detection limit, dynamic range, selectivity, linearity, response time, and stability ^[13].

Several electrochemical methods have been employed for the detection of food additives, biological contaminants, and heavy metals ^[9].

In general, an electrochemical reaction can generate different measurable data, depending on the electrochemical technique adopted. In fact, a measurable current can be generated, and in this case, the corresponding electrochemical techniques are the amperometric ones. Alternatively, a potential can be measured and/or controlled, and in this case, the corresponding electrochemical techniques are the potentiometric ones. Finally, the electrochemical techniques, involving measurements of impedance at the electrode/solution interface are included in the electrochemical impedance spectroscopy (EIS) method ^[14].

Starting from the presentation of EIS, we propose a brief presentation and overview of the best known and used electrochemical techniques.

EIS is an electroanalytical method used for the evaluation of electron-transfer properties of the modified surfaces and in understanding of surface chemical transformations. EIS analysis provides mechanistic and kinetic information on a wide range of materials, such as batteries, fuel cells, corrosion inhibitors, etc. ^[14].

The overall electrochemical behaviour of an electrode can be represented by an equivalent circuit comprising resistance, inductance, and capacitance. The equivalent circuit elements, useful for analyte detection are resistance to the charge transfer R_ct and the double layer capacitance C_dl. The measured capacitance usually arises from the series combination of several elements, such as analyte binding (C_anal) to a sensing layer (C_sens) on the electrode (C_el). The sensitivity is then determined by the relative capacitance of the analyte layer and the sensing layer. One difficulty with capacitive sensors is that their sensitivity depends on obtaining the proper thickness of the original sensing layer.

Voltammetry belongs to the class of the amperometric techniques because the current produced from an electrochemical reaction is measured whilst varying the potential window. Since there are many ways to vary the potential, we can consider many voltammetric techniques. Among others, the most common and employed are the following: cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) ^[15],^[16],^[17],^[18].

. In Figure 1, an overview of the electrochemical methods of analysis, namely voltammetry, amperometry, electrochemical impedance spectroscopy (EIS), and potentiometry, is reported.

All the above-mentioned techniques have been widely employed in the development of electrochemical sensors for different application fields.

3.1. Carbon-Based Nanomaterials

Carbon-based nanomaterials (single-walled carbon nanotubes (SWNTs), multi-walled carbon nanotubes (MWNTs), single-walled carbon nanohorns (SWCNHs), buckypaper, graphene, fullerenes (e.g., C₆₀), etc. present very interesting properties, such as high surface-to-volume ratio, high electrical conductivity, chemical stability/durability, and strong mechanical strength, and for these reasons they have found a large applicability in the sensing area ^[22],^[23],^[24],^[25],^[26],^[27],^[28].

Carbon nanotubes (CNTs) present several properties associated to their structure, functionality, morphology, and flexibility to be employed in synthesis of hybrid or composite materials due to their hollow cylindrical structure.

Carbon nanotubes can be classified as single-walled nanotubes (SWNTs), double-walled nanotubes (DWNTs), and multi-walled nanotubes (MWNTs) depending on the number of graphite layers. Functionalized CNTs have been used in several application fields. The chemical functionalities can easily be designed and tuned through the tubular structure modification.

Some interesting examples of carbon nanomaterials based electrochemical sensors, related to different application fields not only to food analysis, are illustrated below.

Venton and co-workers have used metal microelectrodes modified with CNTs for assembling an electrochemical sensor for detecting dopamine in vitro and in vivo. ^[29]. It has been found that CNTs-coated niobium (CNTs-Nb) microelectrode showed a low detection limit of 11 nM for dopamine. The CNTs-Nb sensor was also employed to detect stimulated dopamine release in anesthetized rats and showed high sensitivity for in vivo measurements.

The design and synthesis of functionalized CNTs for biological and biomedical applications are highly attractive because in vivo sensing requires high selectivity, accuracy, and long-term stability. Zhang et al. have prepared an electrochemical ascorbic acid sensor for measuring ascorbic acid in brain using aligned carbon nanotube fibers (CNF) as a microsensor ^[30] obtaining very interesting results. The sensor measured ascorbic acid concentration of 259.0 μM in the cortex, 264.0 μM in the striatum, and 261.0 μM in the hippocampus, respectively, under normal conditions.

Graphene is one of most applied nanomaterial in the sensing area. Different graphene-based materials have been produced (e.g., electrochemically and chemically modified graphene) using many procedures ^[31]. Graphene shows properties such as high conductivity, accelerating electron transfer, and a large surface area, very similar indeed to the corresponding properties of CNTS, so it is considered a good candidate for assembling sensors to determine several target molecules ^[9],^[31].

Graphene oxide (GO) is hydrophilic and can be dispersed in water solution because of hydrophilic functional groups (OH, COOH and epoxides) at the edge of the sheet and on the basal plane.

On the other hand, GO has a low conductivity in comparison to graphene, so reduced GO (rGO) is more employed as electrode modifier in electrochemical sensing/biosensing area ^[31]

Fluorine doped graphene oxide was used to prepare an electrochemical sensor for the detection of heavy metal ions such as Cd²⁺, Pb²⁺, Cu²⁺, and Hg²⁺. Square wave anodic stripping voltammetry was employed for the detection of the heavy metal ions. The authors have evidenced that the presence of fluorine builds a more appropriate platform for the stripping process, from the comparison between the sensor based on GO and the sensor based on F-GO ^[9].

Li and co-workers developed a based electrochemical sensor for the detection of metal ions, Pb²⁺ and Cd²⁺, employing a Nafion–graphene composite film. The synergistic effect of graphene nanosheets and Nafion gave rise to a better sensitivity for detecting metal ion and enhanced the electrochemical sensor selectivity ^[32].

Li and co-workers have reported a graphene potassium doped modified glassy carbon electrode, for the determination of sulphite in water solution. A linear response in the concentration range of 2.5 μM–10.3 mM with a detection limit of 1.0 μM for SO₃²⁻ has been obtained. The graphene electronic properties resulted modified by the K doping ^[33].

A glassy carbon electrode was modified by means of hexadecyl trimethyl ammonium bromide (CTAB) functionalized GO/multiwalled carbon nanotubes (MWCNTs) for the detection of ascorbic acid (AA) and nitrite. The combination of GO and MWCNTs provided a sensing platform with high electrocatalytic activity, increased surface area, as well as good stability and sensitivity, allowing for the determination of nitrite and AA at the same time ^[34].

3.2. Gold Based Nanomaterials

Since the first examples of gold nanoparticles (AuNPs) synthesis, AuNPs have been employed for the assembly of different sensors. From an electroanalytical point of view, Au nanoparticles and in general gold nanomaterials were employed in the electrochemical sensing area because of their high conductivity, their compatibility, and a high surface to volume ratio ^[35],^[36]. Gold nanomaterials have been used for the selective oxidation processes, or rarely, for the reduction ones.

Important improvements have been performed in the Au nanoparticles and nanomaterials synthesis for electrochemical sensing. However, researchers are dealt with several challenges/criticalities, such as size control, morphology, and suitable dispersion and/or stabilizing agents and/or media. The recent development of nanoporous Au materials seems to be fundamental in overcoming these challenges and hierarchical gold nanoporous materials have been recently reported for biomedical applications ^[37]. Some interesting examples of gold nanomaterials based electrochemical sensors, related to different application fields not only to food analysis, are illustrated below.

An electrochemical sensor using a gold microwire electrode for the detection of heavy metals ions, such as copper and mercury, in seawater was described by Salaün and coworkers. The sensor using gold microwires was able to detect the two metals ions at the same time by means of anodic stripping voltammetry ^[38].

Concerning the same analytical issue, i.e., the detection of different heavy metals ions at the same time, Soares and co-workers developed an electrochemical sensor using a vibrating gold microwire, and determined arsenic, copper, mercury, and lead ions in freshwater by means of stripping voltammetry ^[39].

Recently, gold nanopores were synthesized by the alloying/dealloying method for increasing the electrochemical performance of an analyte under investigation and several examples are reported below.

Ding and co-workers prepared a nanoporous gold leaf by dealloying the Au/Ag alloy in nitric acid. The resulting 3D gold nanostructure allowed the small molecules movement inside. For example, it was employed to modify a GCE for detecting nitrite. The increased surface area and the high conductivity of the 3D nanostructured gold network justify the good performance of the sensor ^[40].

Other examples concerning the use of nanoporous gold have involved more properly the electrochemical investigation of different analytes with this new nanomaterial as proof of concept to apply it in the sensing area.

A green synthesis of nanoporous gold materials was proposed by Jia and co-workers by means of a cyclic alloying/de-alloying procedure. The resulting nanoporous gold film modified electrodes showed very interesting electrochemical performances in terms of a high surface area and good selectivity ^[41].

Lin et al. modified a GCE via the electrodeposition of Au nanoparticles on polypyrrole (PPy) nanowires. AuNPs enhanced the conductivity of the polymer nanowires and consequently the electron transfer rate resulted higher than that at bare GCE and/or at GCE just modified with the polymer nanowires ^[42].

Finally, a nanoporous Au 3D nanostructure was synthesized as a proof of concept to be applied as a sensing platform for detecting hydrazine, sulphite, and nitrite, present in the same sample. The nanostructured sensor showed good performances in terms of selectivity and sensitivity ^[43].

3.3. Hybrid Nanocomposites

To improve and amplify the performances of a sensor and/or a sensing platform, nanomaterials such as carbon and/or metal nanomaterials were incorporated in different polymers both natural (e.g., chitosan) or (electro)synthesized (e.g., PEDOT, polypyrrole). Some interesting examples of hybrid nanocomposite based electrochemical sensors, related to different application fields not only to food analysis, are illustrated below ^[44].

As a first example, we can introduce an electrochemical sensor using a polypyrrole–chitosan–titanium dioxide (PPy–CS–TiO₂) nanocomposite for glucose detection. Interactions between the TiO₂ nanoparticles and PPY enhanced the sensor properties in terms of sensitivity and selectivity ^[45].

Bimetallic Au–Pt nanoparticles have been incorporated in rGO and the electrochemical behaviour of resulting nanocomposite was investigated and compared with the electrochemical behaviour of the bimetallic nanoparticles and of the rGO. An enhanced electrochemical activity is observed, probably due to the increase of the surface area and to the increase of the nanocomposite conductivity respect to the bimetallic nanoparticles and to the rGO ^[46].

Feng et al. assembled a sensor for caffeic acid detection through a nanocomposite obtained by the combination of worm-like Au–Pd nanostructures and rGO ^[47]. From the spectroscopic characterization, the nanotubular worm-like Au–Pd nanostructures were uniformly distributed on rGO.

A carbon paste electrode was modified with a nanocomposite obtained by combining AuNPs and MWCNTs. The oxidation of nitrite was investigated at such a modified electrode and showed better performances in terms of electrocatalytic behaviour respect to those at the bare carbon paste electrode and/or to those at CPE modified with only AuNPs or with only MWCNTs ^[48].

A hybrid nanocomposite was prepared by assembling Pt nanoparticles on a graphene surface. The modified electrode was used for the detection of ascorbic acid (AA), uric acid (UA), and dopamine (DA), obtaining interesting results in terms of selectivity ^[49].

Chen and co-workers proposed a Pt nanocomposite combining Pt nanoparticles and single-walled carbon nanotubes (SWCNTs), instead of graphene or MWCNTs.

A GCE modified with this nanocomposite was used for the electrochemical detection of α-methylglyoxal. A good linearity in the concentration range of 0.1–100.0 μM, and a detection limit of 2.80 nM were obtained. The sensor was applied to detect α-methylglyoxal in real samples of wine and beer ^[50].

Yegnaraman and co-workers reported an Au based nanocomposite for the detection of AA, UA, and DA to test the selectivity for detecting analytes present in the same solution. The nanocomposite film was synthesized by introducing Au nanoparticles into the PEDOT polymer matrix. The modified GCE determined AA, UA, and DA simultaneously, with improved sensitivity and selectivity ^[51].

A glucose impedimetric biosensor ^[52] was assembled using a metal composite composed by a gold microtubes (AuμTs) architecture and polypyrrole overoxidized by Curulli and co-workers. A platinum (Pt) electrode was coated by gold microtubes, synthesized via electroless deposition within the pores of polycarbonate particle track-etched membranes (PTM). This platform was successfully used to deposit polypyrrole overoxidized film (OPPy) and to verify the possibility of developing a biosensor using OPPy, the characteristics of the H₂O₂ charge transfer reaction were studied before the enzyme immobilization. This composite material seems to be suitable in devices as biosensors based on oxidase enzymes, just because hydrogen peroxide is a side-product of the catalysis and could be directly related to the concentration of the analyte. Finally, a biosensor consisting of a Pt electrode modified with AuμTs, OPPy, and glucose oxidase was assembled to determine the glucose. The most important result of this biosensor was the wide linear range of concentration, ranging from 1.0 to 100 mM (18–1800 mg·dL⁻¹), covering the hypo- and hyperglycaemia range, useful in diabetes diagnosis, with limit of detection of 0.1 mM (1.8 mg·dL⁻¹) and limit of quantification of 1.0 mM (18 mg·dL⁻¹).

A wide range of glucose biosensor prototypes have been developed during these years, but the challenge is to obtain a biosensor capable of measuring the glucose concentration in the normal range (i.e., representative of healthy patients) and in the range of hyperglycaemia values (especially for diabetes patients, for clinical diagnosis of this widespread pathology). Very few examples propose biosensors with improved analytical performances, especially in terms of an extended linearity (still 50 mM ≈ 901 mg·dL⁻¹, useful for the hyperglycaemia pathology values), high selectivity toward the most common interferents and an improved stability ^[53],^[54],^[55].

4. Electrochemical Sensors for Food Analysis: Some Examples

4.1. Phenolic Antioxidants

Natural antioxidants are species of great interest in many areas, as food chemistry, health care and clinical applications. They have beneficial effects on human health and could play an important role in the prevention and treatment of many pathologies (such as cardiovascular disorders, cancer, etc.) and to protect from oxidative stress ^[56],^[57],^[58],^[59],^[60],^[61]. The classification of antioxidants is commonly carried out based on the chemical structure, determining their reactivity. However, their antioxidant action is also strictly related to the redox properties and consequently their knowledge is very crucial for a better understanding of antioxidant mechanisms.

Among the natural antioxidants found in fruits and plants, hydroxycinnamic acids (HAs) are very important and present in all parts of the fruit and/or plant ^[62],^[63],^[64]. Undoubtedly, these compounds in food provide added value for their well-known health benefits, for their technological role, and marketing. The electrochemical methods have been extensively used to investigate the redox properties of various species and as analytical tool for the determination of redox target molecules. At present, as for other classes of antioxidants, the analysis of HAs and phenolic antioxidants is usually carried out using chromatographic techniques, which require sophisticated equipment and laborious analytical procedures ^[57]. The use of electrochemical methods for analytical purposes is receiving increasing interest ^[57], since they are fast, accurate, sensitive and can be used for the analysis of different and complex matrices with a low cost.

Both electrochemical sensors and biosensors are widely used for the determination of HAs and phenolic antioxidants. However, the electrochemical responses have been studied only from an analytical point of view, whereas the relationship between the antioxidant chemical structure and electrochemical behaviour has been neglected ^[65]. The understanding of key factors that affect the electrochemical response of analytes could promote the design of highly efficient sensors. To this aim, the role of the chemical structural features of antioxidants and nature of the electrode surface must be evaluated.

Recently, electrodes modified with nanocomposite films were successfully used for the analysis of antioxidants, such as caffeic acid, in complex matrices ^[66],^[67]. These films consist of gold nanoparticles (AuNPs) embedded into chitosan, a biodegradable and biocompatible polymer containing many hydroxyl and amino groups that can interact with the analytes. Later, the electrochemical response of structurally related antioxidants on electrodes modified with different gold–chitosan nanocomposite films was investigated and discussed ^[65],^[66].

To evaluate how the chemical structural features of analytes affect the electrochemical behaviour, different types of antioxidants, such as catechols, hydroxycinnamic acids, and flavonoids, structurally correlated and bearing different functional groups and steric hindrances, have been studied. This investigation ^[66] has demonstrated that the electrochemical response for structurally related antioxidants at AuNPs–chitosan modified electrodes depends on several parameters. The chemical structural features of the analytes affect the interaction with the electrode surface. However, their electrochemical behaviour cannot be explained only on these bases.

The nanostructure and surface functional groups of AuNPs-chitosan modified electrodes have also a key role. In particular, the formation of a collaborative network with interconnected metal nanoparticles in chitosan film significantly affects the electron transfer properties, whereas the surface functional groups can promote the interaction with the antioxidants. An overview of the behaviour of catechols, hydroxy cinnamic acids, and flavonoids derivatives at different AuNPs–chitosan modified electrodes has been illustrated by Curulli and co-workers ^[66].

A better response was observed for molecules with two hydroxyl groups in ortho position of the catechol ring, with a peculiar molecular symmetry (i.e., rosmarinic acid) and with a low steric hindrance, in particular for caffeic acid, chlorogenic acid, and rosmarinic acid. Moreover, the interaction with the antioxidants is also affected by the functional groups at modified electrode surface and the size and distribution of AuNPs into the polymeric matrix. The understanding of the parameters affecting the electrochemical behaviour of analytes at modified electrodes is a key issue and could significantly promote the design of highly efficient sensors.

4.2. Caffeine

Caffeine (CAF, chemical structure in Figure 2) a natural alkaloid, distributed in seeds, nuts, or leaves of a number of plants, mainly coffee, cocoa, tea, with the natural function as insecticide, is the most widely consumed psychoactive substance in human dietary. Many physiological effects of CAF are well known, from stimulation of the central nervous system, diuresis, and gastric acid secretion  to nausea, seizures, trembling, and nervousness. Mutation effects on DNA have been also reported. Moreover, it is considered a risk molecule for cardiovascular diseases. Recently, also an antioxidant activity has been suggested for CAF, showing protective effects against oxidative stress. The presence of CAF in many beverages and drug formulations of worldwide economic importance ^[68] makes it an analyte of great interest and although many different analytical methods are currently applied, novel analytical methods for fast, sensitive and reliable determination of CAF are always necessary, especially for particular purposes, as the determination in specific matrix in the presence of interfering agents, or in a specific concentration range, besides under beneficial conditions in terms of time consumption, material cost, and procedure ease ^[68].

Figure 2. Scheme of the approach and method used for the caffeine detection reprinted with the permission from ^[68]. Copyright 2017 Elsevier.

4.3. Ascorbic Acid

L-Ascorbic acid (AA) or Vitamin C is a water-soluble vitamin and antioxidant, present in many biological systems and food. The biochemically and physiologically active form is the L-enantiomer, with a γ-lactone structure ^[69]. AA is an ideal scavenger free-radical and singlet oxygen or act as a chelating agent. As a strong antioxidant, it acts as a two-electron donor involving hydrogen atom transfer, giving rise to the ascorbate radical ion first, and finally to dehydroascorbic acid. The AA action prevents the oxidation of several compounds present in food and/or beverages. The deficiency of AA can cause several diseases, such as rheumatoid arthritis, Parkinson’s and Alzheimer’s diseases, and even cancer ^[69]. The excess of AA can result to several other diseases such as gastric irritation. Moreover, in the presence of heavy metal cations, the excess of AA has other drawbacks, because it can act as a pro-oxidant, in other words it limits its own antioxidant action tills to produce the reactive oxygen species, causing oxidative stress. Therefore, the determination of AA in biological fluids is very important for the diagnosis of such diseases. The quantitative determination of AA is also necessary for different application fields, including among others, cosmetics, drugs, and food ^[70].

Conventional bare electrodes, like Pt, Au, and glassy carbon, were used for the ascorbic acid detection but because of the vitamin C overpotential, fouling of the electrode surface was observed, involving low sensitivity and selectivity. Nanomaterials were used for the modification of conventional electrode to reduce the overpotential and to enhance the electrode sensitivity, selectivity, and reproducibility.

4.4. Nitrite

Nitrite is an additive used to extend the shelf-life of beverages and foods such as ham, salami, and other cured meats ^[9]. On the other hand, it proves harmful for the human body if it is added to food and beverages at levels higher than those indicated by the safety standards. The World Health Organization (WHO) has established a concentration limit of 3.0 mg·L⁻¹ (65.22 μM) for nitrite in drinking water ^[71]. Therefore, the detection and quantification of nitrite is one of the critical issues of food analysis. Novel nanomaterials have been synthesized and used for the design of advanced sensors for the nitrite detection. It has been shown that nanomaterial-based electrocatalysts significantly improve analytical performances for the nitrite determination. We must evidence that the majority of them are concerned with the detection in water samples, but very significant examples are concerned with cured meats and milk.