Recent Advances in Electrochemical Chitosan-Based Chemosensors and Biosensors: Applications in Food Safety

Abstract

Chitosan is a biopolymer derived from chitin. It is a non-toxic, biocompatible, bioactive, and biodegradable polymer. Due to its properties, chitosan has found applications in several and different fields such as agriculture, food industry, medicine, paper fabrication, textile industry, and water treatment. In addition to these properties, chitosan has a good film-forming ability which allows it to be widely used for the development of sensors and biosensors. This review is focused on the use of chitosan for the formulation of electrochemical chemosensors. It also aims to provide an overview of the advantages of using chitosan as an immobilization platform for biomolecules by highlighting its applications in electrochemical biosensors. Finally, applications of chitosan-based electrochemical chemosensors and biosensors in food safety are illustrated.

1. Introduction

This review explores design, assembling and application of chitosan (CHI)-based electrochemical chemosensors and biosensors for the analysis of selected targets/molecules involved in food screening and safety.

Food safety is a central question to tackle for the food industry, because contaminants, bacteria, toxins, etc. can be developed and appear at each of the different stages in the food production chain for many reasons, e.g., unexpected reactions, degradation, and incorrect storage [1,2].

Moreover, food quality is essential for food industries and consumers, because such a process includes the screening of food contaminants/additives and the analysis of food composition/ingredients [2]. Consequently, the regulatory agencies around the world, such as the United States Food and Drug Administration (USFDA) and the European Food Safety Authority (EFSA), have legislated by imposing strict rules on the maximum levels of contaminants and additives in foodstuffs for guaranteeing consumer protection and mainly human health [3,4]. Generally, food analysis is carried out on the final food product using different well-known techniques, either individually or in combination, such as chromatography, mass spectrometry, and UV-Vis spectroscopy [2]. These traditional approaches have several limitations. In fact, these methods of analysis are demanding, expensive, time-consuming and require large amounts of samples and skilled personnel, and they are considered as routine analysis techniques. Moreover, these methods are not able to detect any contaminated or degraded products when they are formed during the production cycle, because they are only applied at the end of the production cycle itself.

In this context, electrochemical analysis methods and techniques are an interesting alternative route to food analysis as they are fast, equipment is relatively inexpensive, they do not require particularly experienced staff, and in many cases, miniaturization is possible. Moreover, good sensitivity, availability for real-time analysis and minimal matrix effect are achieved in many applications [2,5].

For these reasons, electrochemical chemosensors and biosensors have received increasing attention and thereby have been widely used in food analysis for food safety. In particular, they represent an alternative tool for the food screening, because they can be applied for contaminants detection before the end of the production cycle [6,7,8], providing real-time information about the production process through on-site food monitoring.

In this review, we would like to outline the principal characteristics of electrochemical chemosensors and biosensors, in particular evidencing among them those CHI-based and finally highlighting their strengths and weaknesses for application in food commodities analysis.

Before introducing and shaping electrochemical chemosensors and biosensors, we would like to highlight some considerations and comments about CHI evidencing its functional and structural properties which make it attractive for the preparation of electrochemical chemosensors and biosensors.

2. CHI: Structural and Functional Properties

CHI, a natural and linear polysaccharide, is considered one of the most important chitin derivatives and is the second most abundant natural polysaccharide after cellulose. Chitin has been obtained from the exoskeletons of arthropods, shrimp, and prawns and from beaks of cephalopods. On the other hand, CHI is also present in some microorganisms, such as fungi.

Unlike other natural polysaccharides that are neutral or negatively charged in an acidic environment, CHI looks like a polycationic polymer [9]. This allows it to form composites or multilayer structures with other polymers through an effective electrostatic interaction.

CHI is prepared from chitin through a N-deacetylation process in an alkaline medium, as shown schematically in Figure 1, and the degree of deacetylation modulates the properties and affects the molecular weight of CHI.

From a chemical point of view, CHI is composed of D-glucosamine and N-acetyl D-glucosamine copolymers joined by a β-1,4 glycosidic bond [10,11,12].

Figure 1. Extraction of chitin and chitosan (CHI) from different sources. Reprinted with the permission from reference [11]. Copyright 2013 American Chemical Society.

Figure 1. Extraction of chitin and chitosan (CHI) from different sources. Reprinted with the permission from reference [11]. Copyright 2013 American Chemical Society.

CHI presents different physical properties such as viscosity and solubility, depending on media under investigation. The solubility of CHI in aqueous solutions can be tuned by modifying the pH value (usually below 6.5), ionic strength, as well as the deacetylation degree. In addition, CHI presents reactive amino and hydroxyl groups in its linear structure, making it available for appropriate bio/chemical modifications [13]. As a result, the CHI structural and functional properties allow easily modifying the polymer, introducing specific functions and capabilities [14].

For these characteristics, CHI is a suitable material for (bio)sensing design, because of the presence of proper functional groups for biological/chemical binding and its high mechanical rigidity.

As well-known and reported in the literature [15], the natural micrometric porosity of CHI can increase the sensors surface area and the corresponding loading capacity, thus contributing to amplifying the output signal. Further, when detecting relatively small molecules, the CHI porous structures have a minimal effect on the analyte diffusion towards the electrodic surface.

Finally, CHI is non-toxic, and eco-friendly, being a raw biodegradable and renewable material. Being nontoxic is important as a sensing material component, because it produces low immunogenicity, avoiding and/or minimizing false responses due to the incompatibility between samples and sensor materials. Immunogenicity is defined as the ability of cells/tissues or a generic biological material sample to cause an immune response and is generally considered to be an undesirable physiological response.

3. Electrochemical Chemosensors and Biosensors: Definitions, Techniques, and Sensing Materials

First of all, we would like to introduce some definitions.

In general, a chemosensor is defined as “a sensory receptor that transduces a chemical signal to an action potential” [16]. In particular, when the receptor consists of chemical molecules coming from synthetic route processes, the corresponding sensor based on a synthetic receptor is broadly named chemosensor. If the receptor is based on biological units, i.e., natural macromolecules such as peptides, proteins, and nucleic acids, the corresponding sensor is properly named biosensor.

According to Wang, a biosensor is a chemosensor with a biological receptor [17]. Therefore, a biosensor is nothing more than a particular type of chemosensor, with a bioreceptor.

We would also like to point out that peptides, enzymes, and aptamers are nothing but “large” molecules, or in other words, chemical compounds of a natural origin with a high molecular weight. Perhaps then, the difference between chemosensors and biosensors is purely formal, and the origin and/or the process of obtaining the receptor is evidenced.

In the food analysis, chemosensors including biosensors play a crucial role, and in this review, particularly significant examples will be shown.

From an analytical point of view, a chemosensor and consequently a biosensor are also defined as “a device that transforms chemical information, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal”, according to the International Union of Pure and Applied Chemistry definition [18].

A typical chemosensor is composed of a receptor and a physical-chemical transducer, as schematized in Figure 2.

Analyzing the receptor role, the chemical information is transformed into a form of energy, measured by the transducer.

The transducer is a device capable of transforming the energy including the chemical information about the sample into a useful analytical signal.

The receptor of chemical sensors can be classified as:

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Physical receptor where no chemical reaction takes place;

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Chemical receptor where a chemical reaction involving the analyte produces the analytical signal;

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Biochemical receptor in which a biochemical process is the source of the analytical signal. The corresponding sensors may be regarded as a subgroup of the chemical ones. Such sensors are called biosensors and can be classified as catalytic or affinity ones, depending on the receptors typology [2,19,20,21].

The receptor turns the measured analyte concentration into a chemical or physical output signal, with a defined sensitivity and selectivity that allows the sensor application in a real matrix with potential interferents.

The transducer converts the signal generated by the receptor–analyte interaction to a readable value.

Depending on the type of transducer, both chemosensors and biosensors can be classified as electrochemical, optical, piezoelectric, and calorimetric sensors.

Particularly, in this review, we focused on electrochemical chemosensors and biosensors, providing some examples of application to food analysis.

Electrochemical sensors and biosensors may be classified according to the operating principle of the transducer. In general, electrochemical transducers are able to convert the result of the electrochemical analyte–electrode interaction into a measurable electrical signal (such as current, voltage, charge, and impedance).

Sensitivity, detection limit, selectivity, linearity, response time, and stability are the critical parameters to be optimized [22].

Different electrochemical techniques can be used for quantitative analysis, including food analysis [2]. The most common and mentioned in this review are the following: chronoamperometry (CA), cyclic voltammetry (CV), differential pulse voltammetry (DPV), squarewave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS).

For more details about theories, underlying the different electrochemical approaches used for electrochemical chemosensors and biosensors, several books and reviews in the literature are well-known [22,23,24,25,26,27,28].

The transducer is the most important component of a sensor, because it is closely related to its sensitivity and response time. The redox reaction occurring at the sensing interface is converted into an electrochemical signal. The electrode surface properties, the electron transfer rate, and the mass transfer affect directly the quality of the output signal. Thus, the quality and features of the electrode material highly affects the electrochemical sensors and biosensors performances. The most common types of electrodes and sensing materials used for CHI-based electrochemical chemosensors and biosensors concerning the food analysis are reported below.

First of all, we must stress the relevance of the nanomaterials introduction in sensing areas. In fact, their size- and shape-dependent chemical and physical properties can be exploited in particular for the fabrication of chemosensors and biosensors [5,29]. Nanomaterials also show a peculiar surface chemistry, enhanced conductivity properties, and high surface-to-volume ratios. These properties can help to improve the sensors performances. For these reasons, in recent years, we have assisted in the development of a wide range of nanomaterials with different structures, compositions, and properties to be used for different sensors assembling.

Nanomaterials, nanoparticles, nanocomposites, and nanostructures play an important role in developing electrochemical (bio)sensors for food safety with improved performance in terms of stability and sensitivity.

Carbon has been recognized as one of the most common electrode materials used in electrochemical (bio)sensing areas. The most common forms of carbon used as electrode materials and/or as electrode modifiers are carbon paste, glassy carbon, carbon nanotubes, graphene, and carbon black. All these carbon materials are cheaper than noble metals [2,5].

Carbon paste is a hydrophobic mixture of graphite powder with an organic binder. CHI can be easily incorporated into the carbon paste for sensing applications. Similarly, glassy carbon electrodes have also been employed for electrochemical sensors using ad hoc modifications. They can be easily modified with CHI, but they require a cleaning pretreatment and this can represent a real problem for possible sensor commercialization.

Considering the carbon nanomaterials, carbon nanotubes (CNTs) are well-known, and they can be classified, depending on the number of graphite layers [30]. They present common nanomaterials properties, such as high conductivity, improving electron transfer, and a large surface area, and their chemical functionalization for application in (bio)sensing can easily be designed and performed, making appropriate modifications to their tubular structure.

Graphene is one of the most applied nanomaterials in sensing areas [31] and shows very similar properties to those of CNTS.

Graphene oxide (GO) is hydrophilic and can be dispersed in water solutions because of the presence of hydrophilic functional groups. It has found interesting and innovative applications in sensing areas [32], but it has a conductivity lower than graphene, so reduced GO (rGO) is more employed as an electrode modifier in electrochemical biosensing areas, because of its better conductivity [31,33].

Last but not least, we would like to introduce an old and low-cost carbon-based nanomaterial, i.e., carbon black (CB), evidencing a large surface area, an excellent electrical conductivity, dispersibility in solvents, possible easy functionalization [34,35,36,37].

The gold biocompatibility, stability, and conductivity have supported the use of this noble metal as a material electrode. The gold electrode sensitivity and functionality can be improved by introducing suitable molecules and polymers such as CHI on its surface.

In addition, gold nanomaterials such as nanoparticles, nanoclusters, nanowires, and nanotubes were employed in electrochemical (bio)sensing areas, not only for their high conductivity and compatibility, but also for their high surface-to-volume ratios [38,39,40,41,42,43].

Finally, nonconventional sensing platforms, such as screen-printed electrodes (SPEs) in many cases modified with nanomaterials and/or nanostructures, are employed as assembling platforms for electrochemical chemosensors and biosensors.

Screen-printing technology offers several advantages, including a wide range of geometries, mass production, disposability, and portability, which are very attractive for application in biosensing areas and in particular for real-time and on-site applications [44,45,46].

4. Applications in Food Safety

In this section, several examples of electrochemical chemosensors and biosensors are reported, evidencing different approaches to assembling smart CHI-based sensing platforms and applications to detect different analytical targets involved in food analysis. Before illustrating several applications, we would like to introduce different typologies of CHI-based sensing platforms.

As reported in Section 2, CHI is a functional material showing good adhesion, film-forming ability, and biocompatibility, so it is considered a good material to develop sensing platforms.

On the other hand, CHI is a non-conducting biopolymer; for this reason, it is generally combined with nanomaterials as well as with conducting polymers to improve its conductivity.

Moreover, this biopolymer presents its backbone several hydroxyl (OH) and amine (NH₂) groups, so it is a candidate for the immobilization of biomolecules. In fact, these reactive groups can be rapidly linked and/or coupled with different biomolecules, such as DNA, enzymes, and antibodies/antigens, using covalents, electrostatics, or entrapment immobilization approaches.

In particular, hydrogels of CHI are largely applied for entrapment immobilization. Hydrogels are a class of polymeric materials with a three-dimensional network structure, possessing a small amount of water. It can be also defined as a substance in which a liquid is dispersed at the microscopic level throughout a solid network [43]. Since hydrogels are highly compatible with most of biological molecules and materials, they have increased attraction in sensing areas [47].

CHI has been also employed as a molecularly imprinted polymer (MIP). As reported in the literature [48], the MIP is defined as an artificial recognition material of which the size and shape of the corresponding binding sites are complementary to the template molecules for the specific recognition. To generate an MIP with tailored cavities, a monomer is polymerized in the presence of the target molecule and a crosslinking agent promoting the formation of a highly crosslinked functional monomer/template complex. Cavities tailored to the target molecule are then obtained after extracting the template from the polymer. Furthermore, because of their high affinity and selectivity towards target molecules, MIPs can be considered comparable to natural receptors [48]. Finally, thanks to the CHI biocompatibility and film-forming capability, its MIPs are employed as receptors for assembling electrochemical chemosensors.

In the present review, recent examples of CHI-based electrochemical chemosensors and biosensors applied in food analysis to detect different analytical targets such as some phenolic antioxidants, caffeine (CAF), bacteria, contaminants, toxins, and pesticides were illustrated and discussed.

Recent reviews have reported and discussed the application of CHI in assembling electrochemical chemosensors and biosensors and different areas ranging from clinical to environmental ones, but generally very few examples are related to food analysis and safety, maybe because it seems a relatively new application field, requiring highly sensitive, specific, and rapid analysis and accuracy in analytical procedures.

Amine and his co-worker have investigated the advantages of CHI as an immobilization platform for biomolecules by highlighting its applications in electrochemical biosensors, but it should be stressed that examples of application in food analysis and real samples are very few and limited [49].

More recently, another review focuses on CHI as an innovative material to be applied in (bio)sensing areas for clinical and environmental analysis [50]. It is accurate and reports different examples, but those related to food analysis and safety are very few.

On the other hand, a review about the use of hybrid nanocomposites in sensors and biosensors for food analysis and safety reports several examples of different nanocomposites, but those including CHI are limited, because the introduction of CHI in nanocomposites synthesis and in general of polysaccharides is very recent and is linked to the requirement and need of green and sustainable materials in sensing areas [51].

4.1. Phenolic Antioxidants

Phenolic compounds are widespread in nature, and they are daily included in human diet. Actually, they are ubiquitously present in fruit, vegetables, and beverages worldwide consumed as coffee, tea, wine, and beer, and many beneficial effects on human health have been ascribed to their antioxidant properties [5,52,53,54]. Being naturally antioxidants, they are highly attractive in many areas, from food chemistry to health care and clinical applications, just to name a few. The class of hydroxycinnamic acids (HAs) is one of the most interesting, due to their well-known health benefits, their technological applications, and marketing [54].

Extensive electrochemical studies have been performed to investigate the redox properties of HAs and consequently to define an analytical strategy for their analysis and detection [55].

Chromatographic techniques, coupled with different kinds of detector, from spectrophotometric ones to mass spectrometry ones, are the most commonly used methods for the determination of phenolic compounds, including HAs [56,57,58], although sophisticated equipment and laborious analytical procedures are required. Nevertheless, the use of electrochemical methods for analytical purposes is receiving increasing interest [55,59], and interesting examples of CHI-based electrochemical sensors and biosensors are reported in Table 1.

As an example, the successful electrochemical determination of caffeic acid in complex matrices by using electrodes modified with nanocomposite films has been recently reported [60,61]. Antioxidant properties have been ascribed to caffeic acid (3,4-dihydroxycinnamic acid, CA; Figure 3), widely distributed in fruits and vegetables and found in consumer products as coffee, wine, and olive oil [54,55,59].

The nanocomposite films consist of gold nanoparticles (AuNPs) embedded into CHI, increasing the conductivity of the biopolymer.

Exploiting the nanocomposite peculiarities, an analytical method based on DPV for the determination of the polyphenol index in wines was proposed.

In this approach, AuNPs dispersed in a CHI film are prepared using a green method. The synthesis is carried out by reducing Au(III) to Au(0) in an aqueous solution of CHI and different organic acids [60]. Using this approach is possible to perform at the same time the synthesis and the surface modification of AuNPs.

The nanostructure and surface functional groups of AuNPs–CHI-modified electrodes also play a key role. In particular, the formation of a synergistic network with AuNPs and CHI films significantly affects the electron transfer, whereas the surface functional groups can foster the interaction with antioxidants, as shown in Figure 3.

AuNPs–CHI-modified electrodes have been tested for catechol, HAs, and flavonoids [62]. Good results were obtained for molecules containing a catechol unit in their structure and characterized by a low steric hindrance and molecular symmetry, i.e., caffeic acid, chlorogenic acid, and rosmarinic acid. The influence of the CHI functional groups as well the AuNPs size and distribution in the polymeric matrix was also evidenced, suggesting that controlling such parameters could significantly tune and improve the efficiency of the sensor.

DPV is successfully used to determine caffeic acid with sensors based on Au–CHI nanocomposites. They are sensitive, with a linear response over a wide range of concentration (5.00 × 10⁻⁸–2.00 × 10⁻³ M) and a limit of detection of 2.50 × 10⁻⁸ M, and selective, resulting from the test in the presence of phenolic and nonphenolic compounds as interferers. Caffeic acid is also quantified in real matrices, i.e., white and red wine, after a simple appropriate dilution with the supporting electrolyte, obtaining data in good agreement with the literature [60,61].

An electrochemical biosensor based on Laccase enzyme from Trametes versicolor for the total phenolic content evaluation in in vitro cultivated plants, i.e., Salvia officinalis (S. officinalis) and Mentha piperita (M. piperita), was reported by Diaconu and co-workers [63]. The enzyme immobilization is carried out by entrapment into a nanocomposite film during the electrodeposition process from a multiwall carbon nanotubes (MWCNTs)–CHI solution, in agreement with the literature on the use of CHI for immobilizing enzymes via crosslinking as well as dip-coating or electrodeposition processes [64,65,66,67]. The electrochemical deposition is an effective method, leading to stable enzyme immobilization and the controllable thickness of the CHI film.

During electrodeposition, compounds, such as redox mediators [68] and/or nanomaterials (e.g., carbon nanotubes) [64,65,66,67], can be incorporated into the CHI film, producing bio-nanocomposites with enhanced electrical conductivity. It was evidenced from the FTIR spectroscopy data that the Laccase enzyme retains its conformational structure, so the entrapment in nanocomposite preserves the enzyme catalytic properties. The optimization of the biosensor was carried out with respect to rosmarinic acid (RA) as a Laccase-specific substrate because it is one of the main components of Salvia [69] and Mint extracts, so the total polyphenolic content from real samples are expressed in equivalent RA. Under optimized conditions and by means of amperometry, a limit of detection 2.33 × 10⁻⁷ M and a linear range from 9.10 × 10⁻⁷ to 1.21 × 10⁻⁵ M are obtained. It should be underlined that the value of the apparent Michaelis–Menten constant (K_mapp) of 1.8 × 10⁻⁴ M is comparable with that one reported in the literature [63], indicating that the CHI–MWCNT nanocomposite layer provides a friendly environment for enzyme immobilization, preserving its catalytic properties. Finally, the biosensor is applied to determine the total polyphenolic content (TPC) of real samples (S. officinalis and M. piperita extracts), and the obtained results are expressed in equivalent RA but they are not validated with an independent method.

An electrochemical sensor based on a CHI/carbon nanotube composite-modified carbon paste electrode covered with DNA is prepared for the detection of RA (Figure 4) [70], a major secondary metabolite of plants, with antiseptic, antiviral, antibacterial, anti-inflammatory, and antioxidant properties, of interest in food and cosmetics industry besides medicine [71].

The bulk of a carbon paste electrode is modified with carbon nanotubes and CHI, and then, the surface of the obtained electrode is covered with DNA. CHI can form a strong complex with the polyanionic phosphodiester backbone of DNA, making the immobilization very stable [72]. The sensing approach is based on the interaction between RA from solutions and DNA immobilized on the electrode surface; consequently, this strong interaction gives rise to an accumulation RA on the electrode surface. This resulting efficient preconcentration produces a high sensitivity of the sensor for RA determination.

All the several experimental parameters affecting the sensor response are optimized. Under the optimized conditions and using squarewave stripping voltammetry as an analytical technique, a linear concentration range of 0.040–1.5 μM with a detection limit of 0.014 μM is obtained. The proposed method is successfully applied to the analysis of a rosemary extract. The obtained data are in good agreement with those ones obtained from HPLC analysis with a recovery ranging from 103% to 98%.

Table 1. Recent examples of CHI-based electrochemical sensors for hydroxycinnamic acids (HAs) detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
AuE	Electrochemical sensor based on AuNPs/CHI nanocomposites	DPV	Caffeic acid/red and white wines	5.00 × 10−8/2.00 × 10−3 M	2.50 × 10−8 M	[60,61]
AuE	Electrochemical biosensor based on the Laccase enzyme immobilized by entrapment in MWCNTs/CHI nanocomposites	CA	Rosmarinic acid/Mint and Salvia officinalis extracts	9.1 × 10−7/1.21 × 10−5 M	2.33 × 10−7 M	[63]
CPE	Electrochemical sensor based on a CPE modified with MWCNTs and chitosan and covered with DNA	SWSV	Rosmarinic acid/Rosmarinus officinalis extracts	0.040–1.50 μM	0.014 μM	[70]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CPE, carbon paste electrode; DPV, differential pulse voltammetry; MWCNTs, multiwalled carbon nanotubes; SWSV, squarewave stripping voltammetry.

Table 1. Recent examples of CHI-based electrochemical sensors for hydroxycinnamic acids (HAs) detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
AuE	Electrochemical sensor based on AuNPs/CHI nanocomposites	DPV	Caffeic acid/red and white wines	5.00 × 10−8/2.00 × 10−3 M	2.50 × 10−8 M	[60,61]
AuE	Electrochemical biosensor based on the Laccase enzyme immobilized by entrapment in MWCNTs/CHI nanocomposites	CA	Rosmarinic acid/Mint and Salvia officinalis extracts	9.1 × 10−7/1.21 × 10−5 M	2.33 × 10−7 M	[63]
CPE	Electrochemical sensor based on a CPE modified with MWCNTs and chitosan and covered with DNA	SWSV	Rosmarinic acid/Rosmarinus officinalis extracts	0.040–1.50 μM	0.014 μM	[70]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CPE, carbon paste electrode; DPV, differential pulse voltammetry; MWCNTs, multiwalled carbon nanotubes; SWSV, squarewave stripping voltammetry.

4.2. CAF

CAF (1,3,7-trimethylxantine; Figure 5) is a widespread natural alkaloid, present in highly consumed beverages as coffee and tea, as well as in many popular soft drinks, which is the reason why it is the psychoactive compound that most consumed worldwide. Besides many known physiological effects, both positive and negative, and therapeutic properties, antioxidant potentialities have been also suggested for CAF [73,74,75,76]. On the basis of the above statements, it is clear that CAF can be considered an analyte of great interest.

The electrochemical determination of CAF on different electrode materials, ranging from carbon-based materials to nanomaterials, has been reported in the literature [5]. In this review, we focused our attention on CHI-based electrochemical sensors, and interesting examples are reported in Table 2.

As the first example, we would like to introduce a sensor for CAF and theophylline (TP) detection based on a nanohybrid film containing anisotropic AuNPs, CHI, and an ionic liquid (IL) such as 1-butyl-3-methylimidazoliumtetrafluoroborate [BMIM][BF₄] [77]. The nanocomposite is drop-casted on a glassy carbon electrode (GCE) coated with reduced graphene oxide (rGO). We remind that TP can be used for treating respiratory diseases, but it also can cause nausea, diarrhea, arrhythmias, etc. [77]. Consequently, its use must be monitored to avoid toxicity.

Anisotropic AuNPs are prepared by reducing HAuCl₄ in an aqueous CHI–IL solution. The resulting solution is directly casted on the rGO-modified GCE to form a nanocomposite film. This is the first time that IL and CHI play a controlling and shaping role in the synthesis of anisotropic AuNPs. In fact, it has to be underlined that if the reaction was performed only in a CHI aqueous solution, anisotropic AuNPs are obtained and the diameters range from 30 to 50 nm, indicating that the CHI is a crucial factor in synthesizing AuNPs with an anisotropic structure. When the reaction is performed in an aqueous CHI–IL solution, the diameter of major nanoparticles range from 5 to 30 nm, which means that IL plays an important role in controlling the dimensions/diameters of AuNPs. The hybrid film has a three-dimensional structure, and small anisotropic AuNPs are well distributed in it. All the factors influencing the sensor performances are investigated, including the ingredients of the hybrid film, the concentrations of rGO, HAuCl₄, and IL, and the pH of the buffer solution.

Under the optimized conditions, the linear response ranges are 2.50 × 10⁻⁸–2.10 × 10⁻⁶ M and 2.50 × 10⁻⁸–2.49 × 10⁻⁶ M for TP and CAF, respectively. The detection limits are 1.32 × 10⁻⁹ M and 4.42 × 10⁻⁹ M for TP and CAF, respectively. The electrochemical sensor shows good reproducibility, stability, and selectivity, and it is applied to the determination of TP and CAF in real samples, including tea, energy drink, and pharmaceutics. The obtained results are in agreement with those obtained with HPLC. The recoveries for the TP and CAF standards added are 97–107% and 98–104%, respectively.

A simple, sensitive, and selective method was proposed for the determination of CAF in teas, coffee, and soft and energy drinks using a gold electrode modified with AuNPs synthetized in a CHI matrix in the presence of oxalic acid [76].

The electrochemical behaviors of CAF at both bare gold and gold-modified electrodes are investigated and compared by means of CV, DPV, and EIS.

The peculiar performance of the AuNPs–CHI-modified electrode can be explained with the simultaneous and synergistic action of AuNPs and CHI: in fact, AuNPs guarantee a more efficient electron transfer, whereas the CHI functional groups facilitate a more effective interaction between CAF and the electrodic surface.

The performance of the sensor is then evaluated in terms of a linearity range (2.0 × 10⁻⁶–5.0 × 10⁻² M), operational and storage stability, reproducibility, limit of detection (1.0 × 10⁻⁶ M), and selectivity towards interfering compounds such as ascorbic acid, citric acid, gallic acid, caffeic acid, ferulic acid, chlorogenic acid, glucose, catechin, and epicatechin. The sensor is then successfully applied to determine CAF in commercial drinks and beverages, and the results are in agreement with those obtained with HPLC–PDA as an independent method and those with those declared from manufacturers.

Table 2. Recent examples of CHI-based electrochemical sensors for caffeine detection.

Electrode	Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical sensor based on AuNPs/CHI/IL/rGO nanocomposites	DPV	Caffeine/tea and energy drink	2.50 × 10−8/2.49 × 10−6 M	4.42 × 10−9 M	[77]
AuE	Electrochemical sensor based on AuNPs/CHI nanocomposites	DPV	Caffeine/Coca Cola, Pepsi Cola, energy drink, green tea, and tea	2.00 × 10−6/5.00 × 10−2 M	1.00 × 10−6 M	[76]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CHI, chitosan; DPV, differential pulse voltammetry; GCE, glassy carbon electrode; IL, ionic liquid; rGO, reduced graphene oxide.

Table 2. Recent examples of CHI-based electrochemical sensors for caffeine detection.

Electrode	Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical sensor based on AuNPs/CHI/IL/rGO nanocomposites	DPV	Caffeine/tea and energy drink	2.50 × 10−8/2.49 × 10−6 M	4.42 × 10−9 M	[77]
AuE	Electrochemical sensor based on AuNPs/CHI nanocomposites	DPV	Caffeine/Coca Cola, Pepsi Cola, energy drink, green tea, and tea	2.00 × 10−6/5.00 × 10−2 M	1.00 × 10−6 M	[76]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CHI, chitosan; DPV, differential pulse voltammetry; GCE, glassy carbon electrode; IL, ionic liquid; rGO, reduced graphene oxide.

4.3. Pathogenic Bacteria

Bacteria are the most diffuse cause of food-origin illness worldwide [78]. Because of bacteria’s potential hazard for human health and considering the very low infective dose for the majority of them, some pathogens must be totally absent from food. For example, see the Salmonella case [79].

Consequently, developing accurate, simple, rapid, low-cost analysis methods of sensors is crucial for achieving an effective food safety.

There are many kinds of pathogens causing foodborne diseases [80]; among them Escherichia coli (E. coli), Salmonella, and Listeria monocytogenes (L. monocytogenes) are common, and recent examples of electrochemical CHI-based (bio)sensors have been reported and are discussed in Table 3.

As the first example, we introduced an electrochemical immunosensor for the detection of Salmonella, developed using AuNPs which are well distributed in a CHI hydrogel and a modified GCE [81]. As the CHI solution is protonated and positively charged in an acid medium, AuNPs can be easily adsorbed onto the surfaces of CHI and form a biocompatible film. However, the electron transfer between the electrode and biomolecules is prevented because of the low conductivity of the chitosan film [82]. Consequently, for guaranteeing an effective electron transfer, CHI and AuNPs are mixed together for improving the performance of the composite film. AuNPs are well dispersed in the biopolymer film, and then the nanocomposite is casted on the electrode surface. It has been demonstrated that a chitosan film can be oxidized in a NaCl solution, forming reactive carbonyl groups capable of reacting with proteins [83]. Considering the biocompatibility of CHI and AuNPs, the film can provide a good interface and microenvironment for the conjugation of proteins, so this process is carried out by incubating the activated films with a Salmonella antibody solution.

A sandwich immunosensor is obtained, after the modified electrode is incubated in the Salmonella suspension and the horseradish peroxidase (HRP)-conjugated secondary antibody (Ab2) solution.

The immunosensor shows a linear range from 10 to 10⁵ CFU mL⁻¹ with a low detection limit of 5 CFU mL⁻¹. Furthermore, the sensor is applied to spiked tap water and milk samples. The results are compared and validated with those obtained by the plate count method, indicating that the immunosensor is suitable for food safety analysis.

An electrochemical aptasensor [84] is assembled where a thiol functionalized aptamer is immobilized onto an electrochemically reduced graphene–oxide–chitosan composite (ERGO-CHI) as a sensing platform for the Salmonella detection.

The rGO–CHI composite is prepared by mixing together the biopolymer and rGO. We remind that the characteristic low chemical reactivity, high conductivity, and hydrophobicity at the nanometer level of rGO make it a conductive platform for the development of a (bio)sensor [85]. rGO exhibits a wide electrochemical potential window and reduced charge transfer resistance compared to glassy carbon. The combination of rGO with CHI further enhances the properties of rGO by producing a suitable microenvironment for sensors [85]. In the literature, it is well-known [86] that the coupling of these two materials increases the mechanical and tensile properties and produces a strengthened matrix for coating/modifying an electrode surface and/or immobilizing biomolecules. In this case, the rGO–CHI composite forms a conductive nanocomposite stable for immobilizing the modified aptamer without degrading.

In fact, a thiol-functionalized aptamer specific for a surface membrane protein of Salmonella is selected as a biorecognition element and is immobilized on the rGO–CHI composite using glutaraldehyde as a crosslinker. The aptasensor against Salmonella is electrochemically characterized and tested by means of CV and DPV, and a limit of detection of 10 CFU mL⁻¹ is found. The developed aptasensor is specific to Salmonella and selective, distinguishing Salmonella enterica cells and non-Salmonella bacteria. The aptasensor is applied to raw spiked chicken samples, and the results are in agreement with those ones obtained using pure cultures.

An electrochemical immunosensor for the common food pathogen Escherichia coli (E. coli) is developed, based on a hybrid bionanocomposite including CHI, polypyrrole, AuNPs, and MWCNTs (PPy/AuNPs/MWCNTs/CHI) [87]. The nanohybrid composite is used to modify a pencil graphite electrode (PGE), and an anti-E. coli monoclonal antibody is immobilized on it.

Considering the ingredients of the nanocomposite, AuNPs and MWCNTs have high surface areas as well as peculiar electrical and mechanical properties and promote the electron transfer between electrochemically active compounds and electrodes. In addition, CHI is adopted due to its film-forming ability, surface good adhesion, non-toxicity and biocompatibility, crosslinking capability, and the ability of being a proper matrix for biomaterial immobilization. As mentioned above, the combination of CHI with nanomaterials such as AuNPs and carbon nanomaterials produces a suitable microenvironment for immobilizing biomolecules and for enhancing the performances of the resulted sensor. Moreover, PPy is used in synthesizing nanocomposites because of its high electrical conductivity.

Under the optimum conditions, concentrations of E. coli from 30 to 3 × 10⁷ CFU/mL⁻¹ are detected with a detection limit of 30 CFU mL⁻¹ in a PBS buffer. Good results in terms of specificity and stability are also achieved.

On the other hand, it should be mentioned that this immunosensor is not tested in real samples, and in our opinion, this would be a very important element for the effective evaluation of the sensor performance.

A rapid, label-free aptasensor based on a hybrid nanomaterial using the actuation of CHI-based nanobrushes on graphene/nanoplatinum electrodes is developed to detect L. monocytogenes cells through a combination of electrostatic interactions and receptor–cell binding [88], as illustrated in Figure 6.

The following is a brief explanation about a biomimetic material including CHI nanobrushes with various receptors immobilized, which is used for capturing bacteria cells.

The bio-inspired material and sensing strategy simulate natural systems, where low levels of bacteria are selectively captured from complex matrices. To prepare this biomimetic system, reduced graphene oxide/nanoplatinum (rGO–nPt) electrodes are involved, and their electrochemical behaviors in the presence and absence of CHI nanobrushes are investigated during a pH-stimulated osmotic swelling.

To characterize the electrostatic interactions during nanobrush actuation, a CV investigation of the modified electrode is performed using differently charged redox probes at various pH values.

In addition, the electrochemical behavior of the nanobrush is investigated, when receptors (antibodies or DNA aptamers) are immobilized and conjugated onto the electrodic surface.

CHI nanobrushes are preferred to other materials because of their low cost and biocompatibility.

The maximum cell capture is obtained, when aptamers conjugated to the nanobrush link the bacteria cells in the extended nanobrush conformation (at pH < 6), followed by EIS measurement in the collapsed nanobrush conformation (at pH > 6). The aptamer–nanobrush hybrid material is more efficient than the antibody–nanobrush material, probably because of the relatively high adsorption capacity of the aptamers. The analytical characterization of the aptasensor for L. monocytogenes evidences a linear concentrations range from 9 to 10⁷ CFU mL⁻¹, with a limit of detection of 3.0 CFU mL⁻¹ in buffer, and in the presence of other bacteria such as Listeria innocua and Staphylococcus aureus. The sensor is applied to detect L. monocytogenes in a vegetal broth, but the calibration curves are non-linear, evidencing a saturation effect probably due to a non-specific binding. This non-specific binding is expected, since a vegetable broth can contain different components and ingredients, such as tomato paste and yeast extract, thus underlying the importance of calibrating pathogen sensors in media specific to the application of interest.

As the last and more recent example, we introduced an electrochemical immunosensor [89] for the determination of Lactobacillus brevis, which is the most common bacteria that causes beer spoilage.

The electrochemical sensor is an HRP-labelled sandwich immunosensor, based on a nanocomposite including AuNPs, CHI, and an IL (1-Butyl-3-methylimidazolium hexafluorophosphate).

In this case, AuNPs are firstly electrodeposited on a GCE for improving the conductivity and the specific surface area. IL is used for boosting the immobilization performance of the immunosensor.

Unlike the electrochemical sensor, mentioned in Section 4.2 [77], CHI is not used to control the synthesis of nanoparticles, but it has been used for further enhancing the stability of antibody because of the binding and blanketing effects [90]. After optimization, a linear concentration range is observed from 10⁴ to 10⁹ CFU mL⁻¹. The limit of detection is estimated to be 10³ CFU mL⁻¹. As a proof-of-concept, the proposed immunosensor is successfully applied to spiked beer samples, obtaining good results in terms of relative standard deviation (RSD%).

Table 3. Recent examples of CHI-based electrochemical (bio)sensors for pathogenic bacteria detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical immunosensor based on highdensity gold nanoparticles (AuNPs), dispersed in a CHI hydrogel, and a modified glassy carbon electrode (GCE)	DPV	Salmonella/milk, water	10–105 CFU mL−1	5 CFU mL−1	[81]
GCE	Electrochemical aptasensor developed using an electrochemically reduced graphene oxide–chitosan (ERGO–CHI) composite deposited onto GCE	DPV	Salmonella/chicken	10–106 CFU mL−1	10 CFU mL−1	[84]
PGE	Electrochemical immunosensor based on a hybrid PPy/AuNP/MWCNT/CHI nanocomposite-modified pencil graphite electrode (PGE)	Amperometry	Escherichia coli (E. coli)/no real samples	30–306 CFU mL−1	30 CFU mL−1	[87]
Pt/Ir	Impedimetric label-free aptasensor based on a Pt/Ir electrode modified with graphene nanoplatinum “sandwich” transducer layer and rGO suspension	EIS	Listeria monocytogenes /vegetal broth	9–107 CFU mL−1	3 CFU mL−1	[88]
GCE	Horseradish peroxidase-labelled sandwich immunosensor, based on a nanocomposite including gold nanoparticles, CHI, and IL	CV	Lactobacillus brevis/ beer	104–109 CFU mL−1	103 CFU mL−1	[89]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CHI, chitosan; CPE, carbon paste electrode; CV, cyclic voltammetry; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy; IL, ionic liquid; MWCNTs, multiwalled carbon nanotubes; PGE, pencil graphite electrode; rGO, reduced graphene oxide.

Table 3. Recent examples of CHI-based electrochemical (bio)sensors for pathogenic bacteria detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical immunosensor based on highdensity gold nanoparticles (AuNPs), dispersed in a CHI hydrogel, and a modified glassy carbon electrode (GCE)	DPV	Salmonella/milk, water	10–105 CFU mL−1	5 CFU mL−1	[81]
GCE	Electrochemical aptasensor developed using an electrochemically reduced graphene oxide–chitosan (ERGO–CHI) composite deposited onto GCE	DPV	Salmonella/chicken	10–106 CFU mL−1	10 CFU mL−1	[84]
PGE	Electrochemical immunosensor based on a hybrid PPy/AuNP/MWCNT/CHI nanocomposite-modified pencil graphite electrode (PGE)	Amperometry	Escherichia coli (E. coli)/no real samples	30–306 CFU mL−1	30 CFU mL−1	[87]
Pt/Ir	Impedimetric label-free aptasensor based on a Pt/Ir electrode modified with graphene nanoplatinum “sandwich” transducer layer and rGO suspension	EIS	Listeria monocytogenes /vegetal broth	9–107 CFU mL−1	3 CFU mL−1	[88]
GCE	Horseradish peroxidase-labelled sandwich immunosensor, based on a nanocomposite including gold nanoparticles, CHI, and IL	CV	Lactobacillus brevis/ beer	104–109 CFU mL−1	103 CFU mL−1	[89]

Abbreviations: AuE, gold electrode; AuNPs, gold nanoparticles; CA, chronoamperometry; CHI, chitosan; CPE, carbon paste electrode; CV, cyclic voltammetry; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy; IL, ionic liquid; MWCNTs, multiwalled carbon nanotubes; PGE, pencil graphite electrode; rGO, reduced graphene oxide.

4.4. Toxins

Toxins are naturally synthesized by microbes and algae. Toxins contamination can occur during the entire food production chain. It is unpredictable and inevitable and trigger serious economic and public health problems. Based on the survey from the World Health Organization (WHO), humans are exposed to toxins through the ingestion of contaminated foods, producing cases of severe poisoning [91,92].

Herein, we would like to show examples of electrochemical biosensors for toxins detection by employing CHI, which are summarized in Table 4.

As the first example, we presented a sensitive electrochemical immunosensor for aflatoxin B1 (AFB1) detection based on single-walled carbon nanotubes/chitosan (SWCNTs/CHI) nanocomposites [93].

Aflatoxins are detected in several foods and beverages such as corn, peanuts, cottonseeds, nuts, almonds, figs, pistachios, spices, milk, and cheese. Being stable even at high temperatures, they may resist the cooking processes [94]. Four types of aflatoxins were identified as following: AFB1, AFB2, AFG1, and AFG2, plus two additional metabolites (i.e., AFM1 and AFM2), with AFB1 classified as the most abundant and toxic.

Among these, AFB1 is highly toxic, carcinogenic, mutagenic, genotoxic and immunosuppressive. It is ranked as a group 1 carcinogen by International Agency for Research on Cancer (IARC), and a dose of more than 20 mg kg⁻¹ body weight (bw) per day was associated with acute aflatoxicosis in adults [95].

SWCNTs exhibit a considerable specific superficial area, increasing the immobilizing enzymes loading, amplifying the electroactive area, improving electrical conductivity, and the (bio)sensors performance. On the other hand, due to the presence of strong attractive forces between nanotubes, SWCNTs cannot be dispersed in aqueous solutions without an appropriate dispersing agent. To overcome this problem, CHI is therein adopted. When combined with SWCNTs, CHI could enhance the dispersion of SWCNTs.

The proposed immunosensor is based on an indirect competitive binding for anti-AFB1 between free AFB1 and AFB1–bovine serum albumin, immobilized on a nanotubes/CHI layer casted on a GCE [93]. Then, the anti-mouse immunoglobulin G secondary antibody labeled with alkaline phosphatase is linked to the electrode surface through the reaction with a primary antibody. Finally, alkaline phosphatase catalyzes the hydrolysis of the substrate α-naphthyl phosphate, generating an electrochemical signal. A scheme of the strategy for AFB1 sensing using a GCE modified with an SWCNTs/CHI film is illustrated in Figure 7.

Under the optimized conditions, a linear concentrations range from 0.01 to 100 ng mL⁻¹ is found, with a detection limit of 3.5 pg mL⁻¹. Moreover, the immunosensor is successfully applied to assay AFB1 in corn powder, and a good correlation with the results obtained from HPLC has been evidenced.

A novel label-free impedimetric electrochemical aptasensor for the detection of ochratoxin A (OTA) is finally reported [96].

OTA belongs to the ochratoxins family. The ochratoxins are secondary metabolites produced by fungi species (e.g., Aspergillus and Penicillium) during their growth. They are present in different foods and beverages, including coffee, wine, grape juice, and dried fruits [97]. Among them, OTA has been classified as a possible carcinogen by the IARC due to its severe toxicity [95], and the EFSA Panel on Contaminants in the Food Chain stated a tolerable daily intake (TDI) of OTA is 0.4 mg kg⁻¹ bw [98]. In addition, OTA is chemically stable, so that it is metabolized very slowly, being stable for more than 30 days in the body.

Coming back to the impedimetric sensor, a thin film of a nanofibrous CHI/dipeptide hydrogel is used as a sensing interface and a holder for hybridization chain reaction (HCR) of an OTA aptamer and a DNA2 strand to form a DNA concatemer.

A concatemer is a long continuous DNA molecule containing multiple copies of the same DNA sequence linked in series.

The (tert-Butoxycarbonyl)(Boc)-phenylalanine-tryptophan-OH dipeptide hydrogel has been used as a nanofibrous dipeptide hydrogel and acts as a supporting material for its properties of biocompatibility and biodegradation.

Considering the CHI role in the bionanocomposite, CHI is introduced to stabilize the immobilization of the dipeptide hydrogel film and provide a biointerface with many and proper –NH₂ groups for immobilizing the carboxyl-modified DNA1. As a result, the working electrode is a GCE modified with the CHI/nanofibrous dipeptide hydrogel bio-nanocomposite.

The electrode surface modification is implemented through a layer-by-layer thin film deposition of the nanofibrous dipeptide hydrogel and CHI composite.

In the presence of the target OTA, the concatemer is dissociated to single-stranded DNA (ssDNA), while the signal amplification is also achieved by introducing RecJf exonuclease, for digesting the single-stranded DNA. A scheme of the described impedimetric electrochemical sensor for the OTA detection is summarized in Figure 8.

EIS has been employed as an electrochemical technique to determine OTA. A linear detection range of 0.1–100 ng mL⁻¹ is obtained for OTA, with a low detection limit of 0.03 ng mL⁻¹. The sensor is applied to real spiked samples of white wine, obtaining recoveries ranging from 96% to 102.58% with the RSD values in the range of 4.58–7.8%. These results indicate that the proposed sensor could effectively recognize the target molecule in a complex matrix.

Table 4. Recent examples of CHI-based electrochemical sensors for toxins detection.

Electrode	Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical immunosensor based on single-walled carbon nanotubes/chitosan (SWCNTs/CHI) nanocomposites	SWV	AFB1/corn powder	0.01–100 ng mL−1	3.5 pg mL−1	[93]
GCE	Label-free impedimetric aptasensor based on a chitosan/dipeptide nanofibrous hydrogel and immobilized DNA probes with OTA aptamer	EIS	OTA/white wine	0.1–100 ng mL−1	0.03 ng mL−1	[96]

Abbreviations: AFB1, aflatoxin B1; CHI, chitosan; EIS, electrochemical impedance spectroscopy; GCE, glassy carbon electrode; OTA, ochratoxin A; SWCNTs, single-walled carbon nanotubes; SWV, squarewave voltammetry.

Table 4. Recent examples of CHI-based electrochemical sensors for toxins detection.

Electrode	Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical immunosensor based on single-walled carbon nanotubes/chitosan (SWCNTs/CHI) nanocomposites	SWV	AFB1/corn powder	0.01–100 ng mL−1	3.5 pg mL−1	[93]
GCE	Label-free impedimetric aptasensor based on a chitosan/dipeptide nanofibrous hydrogel and immobilized DNA probes with OTA aptamer	EIS	OTA/white wine	0.1–100 ng mL−1	0.03 ng mL−1	[96]

Abbreviations: AFB1, aflatoxin B1; CHI, chitosan; EIS, electrochemical impedance spectroscopy; GCE, glassy carbon electrode; OTA, ochratoxin A; SWCNTs, single-walled carbon nanotubes; SWV, squarewave voltammetry.

4.5. Pesticides

Pesticides are among the most used products to fight and eliminate insects, fungi, and weeds in agriculture. According to the target pest, they can be classified as insecticides, fungicides, herbicides, etc. The main classes of pesticides are carbamates, organophosphates, pyrethroids, or triazines, among others [99,100], and all these compounds are highly toxic. According to the WHO, they can be classified as carcinogenic, neurotoxic, or teratogenic [99].

The maximum residual limits (MRLs) legally permitted in the European Union are 0.1 μg L⁻¹ for a single pesticide and 0.5 μg L⁻¹ for total pesticides [101].

Herein, some recent examples of application of CHI in (bio)sensors for pesticides detection are reported and summarized in Table 5.

A stable electrochemical acetylcholinesterase (AChE) biosensor [102] for the detection of organophosphorus pesticides (OPs) was developed by adsorbing AChE on a multilayered immobilization matrix including CHI, TiO₂ sol-gel, and rGO (CH@TiO₂–CHI/rGO). The matrix has a mesoporous nanostructure. In fact, the rGO nanosheets have a typical wrinkle surface morphology of graphene. The TiO₂ sol-gel film shows a homogeneous mesoporous morphology. With the incorporation of CHI, the surface becomes rougher, and the nanoparticles get bigger. The content of TiO₂ has no particular effect on the surface morphology. The electrodeposited CHI layer induces the formation of nanoparticle aggregates, but the surface is still rough and porous. At the end, the incorporation of CHI and the electrodeposition of a CHI layer into/on the TiO₂ sol-gel improve the gel mechanical properties.

The AChE loading fills the pores, and the resulting surface is more regular. The rough and porous surface morphology would facilitate a stable enzyme immobilization.

The linear concentrations range for the detection of dichlorvos, as a model OP compound, is from 0.036 μM (7.9 ppb) to 22.6 μM, with a limit of detection of 29 nM (6.4 ppb). Finally, the biosensor is applied to the detection of dichlorvos in spiked cabbage juice samples. The recovery ratios are all within the range of 100% ± 3%, and the RSDs are less than 10.0%, indicating a possible accurate OPs detection with the biosensor.

Liu and co-workers developed an electrochemical sensor [103] for the detection of another organophosphorus pesticide, such as methyl parathion, based on an indium tin oxide (ITO) electrode modified with a nanocomposite including zirconia oxide nanoparticles (ZrO₂ NPs), poly(3,4-ethylenedioxythiophene) (PEDOT), and CHI.

Firstly, PEDOT is electrodeposited onto the electrode surface, and then, a colloid containing ZrO₂ NPs and CHI is dropped on it.

CHI improves the stability of zirconia nanoparticles. As can be seen from the morphology of the ZrO₂ NPs–CHI–PEDOT–ITO nanocomposite, it induces an aggregation of the zirconia nanoparticles. Moreover, the CHI membrane is flat, but it has some cracks, which produce channels between the solution and the PEDOT/ITO nanocomposite. The aggregation of zirconia nanoparticles on PEDOT supports the adsorption of MP, and the cracks of the CHI membrane allow accessing the polymer and then the electrode surface.

Even if CHI decreases the background current, the coupling of ZrO₂ NPs and the conductivity and electrocatalytic activity of PEDOT improve the total electrochemical process. Under the optimized conditions, a limit of detection of 2.8 ng mL⁻¹ and a linear range of 5–2000 ng mL⁻¹ were obtained. Furthermore, the sensor was applied to spiked tap water real samples, with interesting results in terms of recovery, ranging from 93.5% to 104.7%.

Another electrochemical sensing tool using a bioelectrode based on haemoglobin (Hb), a redox protein, is reported for the determination of methyl parathion [104]. A scheme of the methyl parathion electrochemical sensor assembling is illustrated in Figure 9.

The bioelectrode is assembled by immobilizing Hb on a fluorine-doped tin oxide (FTO) electrode modified with ERGO–CHI/Hb layers. CHI is mixed with rGO, as previously mentioned in Section 4.3 for a Salmonella aptasensor [84], to increase its conductivity. In addition, in this case, the CHI properties, such as hydrophilicity, biodegradability, good film-forming ability, and large mechanical strength, are integrated by those of graphene oxide, such as high surface areas, high electron transfer rates, together with lower background currents and wide potential windows. Finally, ERGO–CHI layers are used as an immobilizing platform of Hb. The biocompatible ERGO–CHI matrix promotes the direct electron transfer from the protein to the electrode surface without the breakdown of the native protein structure.

The sensor showed a low limit of detection of 79.77 nM, with a good reproducibility. The modified biosensor is applied to detect MP in spiked vegetable samples, with good results in terms of recovery ranging from 94% to 101%.

A simple method for the simultaneous electrochemical determination of two phenylurea herbicides (diuron and isoproturon) with differential pulse adsorptive stripping voltammetry (DPAdSV) by using a modified platinum/CHI electrode [105] has been reported. CHI can be a good choice to stabilize metallic nanoparticles by stereoelectronic effects, providing nanocomposite with different functional groups. This can occur, because CHI has a great affinity for transition metals and good film-forming ability. In addition, its high permeability enables the diffusion process of the analyte at the modified electrode surface.

PtNPs are synthesized by a one-pot reaction using CHI as a stabilizer agent. The resulting bionanocomposite is applied in the assembly of a modified GCE (PtNPs/CHI/GCE) for the rapid, selective, and low-cost simultaneous analysis of diuron and isoproturon in river water samples.

Under the optimized conditions, the limits of detection are 7 μg L⁻¹ for isoproturon and 20 μg L⁻¹ for diuron. The proposed method is successfully applied for the determination of both analytes in spiked river water samples, with a recovery range of 90–110%.

A very particular and ecofriendly method [106] was developed to determine trifluralin. Trifluralin is an herbicide affecting endocrine function, and it is listed as an endocrine disruptor in the European Union list [107].

The trifluralin sensor is based on the herbicide electrochemical oxidation at a three-electrode system designed directly on the surface of an agricultural product, generally a fruit, using a Ag-citrate/graphene quantum dots (GQDs) nanoink. The sensor is prepared by writing directly on the fruit skin, obtaining a Ag-citrate/GQDs nano-ink/leaf electrode.

CHI is present in the nanoink with the role of dispersing and supporting the matrix of GQDs and Ag nanoparticles. It has to be pointed out that the interaction mechanism of trifluralin with the leaf electrode surface is probably due to the electrostatic interactions between the negatively charged dipole nitro group of trifluralin and the protonated amine group of CHI, involving Ag⁺ cation of Ag-citrate present in the conductive nanoink [108].

Under the optimized experimental conditions, this sensor exhibits good sensitivity and specificity for the trifluralin detection. The obtained linear range is between 0.008 to 1 mM, and the limit of quantification is 0.008 mM, using CV. In addition, the obtained linear range using DPV and SWV is 0.005–0.04 mM with the limit of quantification of 0.005 mM. For the further validation of the applicability of the proposed method, it is also used to detect trifluralin on the surface of apple and/apricot skin.

A sensitive and selective electrochemical sensor was reported based on the modification of activated glassy carbon electrode (GCE_ox) with GQDs, CHI, and nickel molybdate (NiMoO₄) nanocomposites for diazinon determination. The sensor assembling strategy is based on the covalent immobilization of polymers and nanoparticles on the GCE_ox surface [109].

Diazinon is classified as an organophosphorus pesticide and acts as an inhibitor of cholinesterase (ChE) enzyme. As already mentioned and discussed, CHI is selected as a supporting matrix for its film-forming capability, its water permeability, adhesion to hydrophilic surfaces, easy chemical modification, and the presence of suitable functional groups. NiMoO₄ is selected because of its interesting properties for application in an electrochemical sensor design such as high electrical conductivity, chemical stability, low-cost, and catalytic properties.

The GCE was activated by means of CV oxidative scans in an acidic medium: functional groups such as hydroxyl oxygen (O⁻) and carbonyl oxygen (COO⁻) groups are inserted onto the GCE surface, introducing negative charges on the electrode surface [110]. CHI is immobilized on the surface of GCE_ox, through the electrostatic attraction between ammonium groups of CHI and functional groups of the electrode surface (carboxyl and hydroxyl groups) to prepare CHI/GCE_ox. Next, the CHI/GCE_ox composite is immersed in the GQDs solution to form the GQDs/CHI/GCE_ox composite. The plentiful presence of functional groups, especially carboxyl groups in the GQDs, provides a strong covalent bonding between GQDs and amino groups of CHI. Finally, NiMoO₄ nanoparticles suspension is casted on the modified electrode surface. Under the optimized conditions, a linear concentrations range from 0.1 to 330 μM and a limit of detection of 30 nM using DPV are obtained. The sensor is applied for the determination of diazinon in real spiked samples of tomato and cucumber, obtaining acceptable results in terms of recovery ranging from 106% to 101%.

Table 5. Recent examples of CHI-based electrochemical (bio)sensors for pesticides detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical biosensor based on CHI, a TiO2 solgel, and a reduced graphene oxide (rGO) multilayered immobilization matrix (CHI@TiO2-CHI/rGO) for AChE	DPV	Dichlorvos/cabbage juice	0.036–22.6 μM	29 nM	[102]
ITO	Electrochemical sensor using an ITO electrode modified with a poly-3,4-ethylenedioxythiophene (PEDOT) membrane and zirconia nanoparticles (ZrO2 NPs)	CV	Parathion/water	5–2000 ng mL−1	2.8 ng mL−1	[103]
Fluorine-tinoxide glass electrode (FTO)	Electrochemical sensor developed by immobilizing haemoglobin (Hb) on a electrochemically reduced graphene oxide–chitosan (ERGO–CHI/Hb/FTO) nanocomposite	SWV	Parathion/onion, lettuce	0.076–0.988 μM	79.77 nM	[104]
GCE	Electrochemical sensor based on a platinum nanoparticles and chitosan (PtNPs/CHI) nanocomposite	DPAdSV	Diuron/river water	-	20 μg L−1	[105]
GCE	Electrochemical sensor based on a platinum nanoparticles and chitosan (PtNPs/CHI) nanocomposite	DPAdSV	Isoproturon/river water	-	7 μg L−1	[105]
Ag-citrate/GQDs nano-ink/leaf or skin	Electrochemical sensor prepared by direct writing on the surface of the samples, using a Ag-citrate/graphene quantum dots (GQDs) nanoink	DPV, SWV	Trifluralin/apple skin	0.005–0.04 mM	0.005 mM	[106]
GCE	Electrochemical sensor based on a quantum dots, chitosan, and nickel molybdate (GQDs/CHI/NiMoO4) nanocomposite	DPV	Diazinon/tomato and cocumber	0.1–330 μM	30 nM	[109]

Abbreviations: AChE, acetylcholinesterase; CHI, chitosan; CV, cyclic voltammetry; DPAdSV, differential pulse adsorptive stripping voltammetry; DPV, differential pulse voltammetry; ERGO, electrochemically reduced graphene oxide; FTO, fluorine-tin-oxide; GCE, glassy carbon electrode; GQDs, graphene quantum dots; ITO, indium tin oxide; Hb, haemoglobin; MWCNTs, multiwalled carbon nanotubes; rGO, reduced graphene oxide; SWV, squarewave voltammetry.

Table 5. Recent examples of CHI-based electrochemical (bio)sensors for pesticides detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical biosensor based on CHI, a TiO2 solgel, and a reduced graphene oxide (rGO) multilayered immobilization matrix (CHI@TiO2-CHI/rGO) for AChE	DPV	Dichlorvos/cabbage juice	0.036–22.6 μM	29 nM	[102]
ITO	Electrochemical sensor using an ITO electrode modified with a poly-3,4-ethylenedioxythiophene (PEDOT) membrane and zirconia nanoparticles (ZrO2 NPs)	CV	Parathion/water	5–2000 ng mL−1	2.8 ng mL−1	[103]
Fluorine-tinoxide glass electrode (FTO)	Electrochemical sensor developed by immobilizing haemoglobin (Hb) on a electrochemically reduced graphene oxide–chitosan (ERGO–CHI/Hb/FTO) nanocomposite	SWV	Parathion/onion, lettuce	0.076–0.988 μM	79.77 nM	[104]
GCE	Electrochemical sensor based on a platinum nanoparticles and chitosan (PtNPs/CHI) nanocomposite	DPAdSV	Diuron/river water	-	20 μg L−1	[105]
GCE	Electrochemical sensor based on a platinum nanoparticles and chitosan (PtNPs/CHI) nanocomposite	DPAdSV	Isoproturon/river water	-	7 μg L−1	[105]
Ag-citrate/GQDs nano-ink/leaf or skin	Electrochemical sensor prepared by direct writing on the surface of the samples, using a Ag-citrate/graphene quantum dots (GQDs) nanoink	DPV, SWV	Trifluralin/apple skin	0.005–0.04 mM	0.005 mM	[106]
GCE	Electrochemical sensor based on a quantum dots, chitosan, and nickel molybdate (GQDs/CHI/NiMoO4) nanocomposite	DPV	Diazinon/tomato and cocumber	0.1–330 μM	30 nM	[109]

Abbreviations: AChE, acetylcholinesterase; CHI, chitosan; CV, cyclic voltammetry; DPAdSV, differential pulse adsorptive stripping voltammetry; DPV, differential pulse voltammetry; ERGO, electrochemically reduced graphene oxide; FTO, fluorine-tin-oxide; GCE, glassy carbon electrode; GQDs, graphene quantum dots; ITO, indium tin oxide; Hb, haemoglobin; MWCNTs, multiwalled carbon nanotubes; rGO, reduced graphene oxide; SWV, squarewave voltammetry.

4.6. Contaminants and Additives

Endocrine-disrupting chemicals (EDCs) are environmental contaminants/pollutants and are also known as hormone-disrupting compounds [111].

The WHO is particularly sensitive to the problem of the presence and determination of endocrine disruptors [112].

Furthermore, EDCs represent a broad class of molecules, such as pesticides (for example trifluralin [107]) and industrial chemicals, plastics, plasticizers, and fuels.

Herein, we focused our attention on bisphenol A (BPA), one of the most important endocrine disruptors.

BPA is a synthetic chemical compound, classified as non-biodegradable with high chemical resistance and widely used as a monomer in the synthesis of epoxy resins and polycarbonate. BPA has a similar structure to those of estradiol and diethylstilbestrol and thus can stimulate a cellular response, binding with estrogen receptors. For this reason, it is classified as an endocrine disrupting chemical.

Polycarbonates have different applications, particularly in the fabrication of water bottles and infant-feeding bottles, and they can be usually transferred into drinks and food from plastic products. On the other hand, epoxy resins are widely used as protective coatings for food and beverage containers, so BPA can produce the contamination of food commodities and water.

An MIP based on MWCNTs is synthesized and used for modifying GCEs in order to detect BPA [113]. The MIP is synthesized on silica-coated CNTs. In addition, glycidoxy propyl trimethoxy silane (GLYMO), as a bifunctional silylating agent, is used because of its mechanical and adhesion properties [114]. The MIP is prepared by co-polymerization of vinylpyridine (4-VPy) and methacrylic acid (MAA) as functional monomers, with ethylene glycol dimethylacrylate (EDGMA) as a crosslinker.

Finally, the selectivity and stability of the prepared MIP is improved by the H-bond interactions between the monomers and the π–π stacking interactions between pyridinyl and phenyl rings. CHI acts as a supporting matrix for the MIP, integrating its mechanical and tensile properties and producing a strengthened matrix for electrode coating. A well-defined electrochemical response is obtained for BPA by CV and DPV. Under the electrochemically optimized conditions, a linear concentration range from 400.00 mM to 0.10 nM with a detection limit of 0.02 nM is obtained. The MIP/GCE sensor is evaluated towards the determination of BPA in commercially available baby-feeding bottle samples. BPA is extracted in ethanol at 55 °C for 24 h, and then the exctracts are concentrated and analyzed. The results are in good agreement with those obtained by HPLC.

Graphene nanoplatelets (GNPs), MWCNTs, and CHI are self-assembled by means of one-step hydrothermal reaction to obtain a GNPs–MWCNTs–CHI nanocomposite [115]. CHI acts as a supporting and dispersing matrix for GNPs and MWCNTs, and the nanocomposite stability is improved by the electrostatics interactions among CHI, GNPs and MWCNTs. In addition, the GNPs–MWCNTs nanocomposite also shows better stability than GNPs or MWCNTs alone due to the π–π stacking interactions between GNPs and MWCNTs.

The GNPs–MWCNTs–CHI–GCE nanocomposite was electrochemically characterized using CV and EIS. The GNPs–MWCNTs–CHI–GCE nanocomposite is used for the determination of BPA by means of DPV. Under the optimum conditions, the linear BPA concentration range is 0.1–100 μM, with a detection limit of 0.05 nM.

The proposed GNPs–MWCNTs–CHI–GCE-based sensor shows good selectivity, repeatability, and reproducibility. The sensor is applied to determine BPA in real milk samples including liquid milk samples and milk powders. The results are promising and acceptable in terms of recoveries ranging from 85.0% to 113.8%.

A three-dimensional hierarchical nickel nanoparticles/nitrogen-doped carbon nanosheet/CHI nanocomposite (NiNPs/NCN/CHI) is used for modifying a GCE to assemble an electrochemical sensor for the BPA detection [116], as shown in Figure 10.

As reported in the literature [117,118], NiNPs exhibit similar properties to noble metal nanoparticles. Hence, nickel nanoparticles can be applied as substitutes for noble metals for assembling electrochemical sensors. However, nickel nanoparticles on the electrode surface are easily oxidized, reducing active surface areas and decreasing conductivity and stability [117,118]. Consequently, the sensor performances such as sensitivity and reproducibility get worse. The immobilization of NiNPs onto carbon-supporting nanomaterials prevents the oxidation of NiNPs and thus improved the electrochemical sensors performances [112,119]. In addition, CHI is involved in the nanocomposite formulation, not only because of its film-forming capability but also because of its good antifouling property [120]. Therefore, nickel nanoparticles/carbon nanocomposites with CHI are selected as electrode modifiers to assemble an electrochemical sensor for the BPA detection. Two linear concentration ranges, from 0.1 to 2.5 μM and from 2.5 to 15.0 μM, are obtained. The detection limit for BPA is 45 nM. Additionally, the BPA sensor is selective and stable. It is applied to detect BPA in spiked milk samples, obtaining recoveries ranging from 96% to 105% and RSD values less than 5%, indicating that this proposed sensor could be applied for BPA detection in milk samples.

We now turn to some examples of CHI-based sensors for the determination of additives. Food additives/preservatives play an essential role in the food industry, as well as the protective mechanism as a retarder of degradation and/or oxidation reactions in food products, even at low concentrations.

Sulfites are among the most used additives and preservatives for food and pharmaceutical industries, because of their antioxidizing and antiseptic effects against food degradation. In addition, sulfites are added to fruits and vegetables to prevent “browning” and decomposition [121]. Further, they are also employed to maintain the effectiveness and stability of some drugs [122].

Despite the aforementioned utilization of sulfites, their excessive concentration in the body has a lot of negative effects such as anaphylaxis, urticaria, angioedema, and gastro-intestinal problem [122]. For this reason, the USFDA advised placement of caution on any food and drugs containing 10 mg kg⁻¹ sulfite [123].

A non-enzymatic sensor for sulfite has been developed by Marwani [124], based on a PPy–CHI biocomposite-modified GCE.

PPy and CHI are specifically selected because of their functionalities. For instance, PPy is a conducting polymer because of delocalized π electrons, enhancing the mobility of ions, polarons, solitons, and bipolarons [124]. The electrical conductivity could be further improved including dopants (p-doing) into the polymeric matrix [124]. CHI is a natural biopolymer with good biocompatibility, and its amine group and hydroxyl moieties could produce hydrogen bonding to the PPy backbone [124]. Further, the combination of PPY with CHI increases its adhesion onto the electrodic surface. The synthesized PPY–CHI film is used for the sulfite detection using differential pulse voltammetric, with a detection limit of 0.21 μM and a linearity ranging from 50 to 1100 μM.

Real samples of malt drinks and fruit juices are used to verify the applicability of the PPY–CHI–GCE nanocomposite for determining sulfites in drinks. Promising and interesting results in terms of recoveries, ranging from 92% to 104%, are obtained.

BHA is an additive which deserves attention as a chemical used as a phenolic preservative and an antioxidant in food, cosmetics, animal feeds, and pharmaceutical [125].

BHA is added to foods containing fat, because it prevents the fat rancidification, producing unpleasant odors. BHA is known as an E320 food additive. Nevertheless, BHA is irritating to the skin and eyes. It has been considered as possible carcinogenic, producing also other toxic effects, if its maximum permitted level is exceeded [126]. Indeed, the amount of BHA is limited to 100 μg g⁻¹ when used alone and 200 μg g⁻¹ of the total amount when used in a mixture with other additives [127].

An electrochemical sensor for the analysis of BHA in foodstuffs was developed using CHI capped with AuNPs [128], as illustrated in Figure 11.

The nanocomposite is self-assembled on a screen-printed carbon electrode (SPCE) and is used as an MIP by employing BHA as a template. An electrochemical polymerization process is chosen to design the MIP, using CV as an electrochemical technique, as illustrated in Figure 12.

As already mentioned, CHI presents a large number of amino (–NH₂) and hydroxyls (–OH) groups in the polymer backbone, several reaction sites, and highly crosslinking degree as well as good stability. On the other hand, AuNPs nanocomposites possess a large surface area and a high conductivity. In this example, the appropriate choice of the CHI polymer is based on its –NH₂ groups, possibly interacting with the –OH groups of the BHA through a hydrogen bond. This allows forming peculiar self-assembly network provided with several recognition sites for imprinted polymers [129].

The BHA-imprinted MIP sensor exhibits a good sensitivity and selectivity compared to those interfering species such as ascorbic acid and citric acid. Under the optimal experimental conditions, it shows a linear concentrations range from 0.01 to 20 μg mL⁻¹, with a low detection limit of 0.001 μg mL⁻¹. Besides, the reproducibility, stability, and repeatability of the MIP sensor are tested. The MIP sensor is applied for detecting BHA in real spiked food samples such as chewing-gum, mayonnaise, and potato chips, with a RSD of ≤8%. Spectrophotometry is utilized as a validation method with acceptable results.

Table 6 summarizes the examples of CHI-based sensors for the determination of contaminants and additives.

Table 6. Recent examples of CHI-based electrochemical (bio)sensors for contaminants and additives detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical sensor based on an MWCNT/MIP/CHI nanocomposite	DPV	BPA/baby-feeding bottle	400.00 μM–0.10 nM	0.02 nM	[113]
GCE	Electrochemical sensor using as sensing platform with MWCNTs–CHI nanocomposites self-assembled on graphene nanoplatelets GNPs (GNPs– MWCNTs–CHI)	DPV	BPA/milk	0.1–100 μM	0.05 nM	[115]
GCE	Electrochemical sensor based on a three-dimensional hierarchical cylinder-like nickel nanoparticle/nitrogen-doped carbon nanosheets/chitosan (NiNP/NCN/CHI) nanocomposite	DPV	BPA/milk	0.1–2.5 μM and 2.5–15.0 μM	45 nM	[116]
GCE	Electrochemical sensor based on a PPY/CHI composite	DPV	sulfite/malt drink and orange juice	50–1100 μM	0.21 μM	[124]
GCE	Electrochemical sensor based on MIP-included CHI capped with gold nanoparticles	DPV	BHA/chewing-gum, mayonnaise, potato chips	0.01–20 μg mL−1	0.001 μg mL−1	[128]

Abbreviations: CHI, chitosan; CV, cyclic voltammetry; BHA, butylated hydroxyanisole; BPA, bisphenol A; DPV, differential pulse voltammetry; GCE, glassy carbon electrode; GNPs, graphene nanoplatelets; MWCNTs, multiwalled carbon nanotubes; NCN, nitrogen-doped carbon nanosheet; PPY, polypyrroe.

Table 6. Recent examples of CHI-based electrochemical (bio)sensors for contaminants and additives detection.

Electrode	(Bio)Sensor Format	Electrochemical Technique	Analyte/Sample	L.R.	LOD	References
GCE	Electrochemical sensor based on an MWCNT/MIP/CHI nanocomposite	DPV	BPA/baby-feeding bottle	400.00 μM–0.10 nM	0.02 nM	[113]
GCE	Electrochemical sensor using as sensing platform with MWCNTs–CHI nanocomposites self-assembled on graphene nanoplatelets GNPs (GNPs– MWCNTs–CHI)	DPV	BPA/milk	0.1–100 μM	0.05 nM	[115]
GCE	Electrochemical sensor based on a three-dimensional hierarchical cylinder-like nickel nanoparticle/nitrogen-doped carbon nanosheets/chitosan (NiNP/NCN/CHI) nanocomposite	DPV	BPA/milk	0.1–2.5 μM and 2.5–15.0 μM	45 nM	[116]
GCE	Electrochemical sensor based on a PPY/CHI composite	DPV	sulfite/malt drink and orange juice	50–1100 μM	0.21 μM	[124]
GCE	Electrochemical sensor based on MIP-included CHI capped with gold nanoparticles	DPV	BHA/chewing-gum, mayonnaise, potato chips	0.01–20 μg mL−1	0.001 μg mL−1	[128]

Abbreviations: CHI, chitosan; CV, cyclic voltammetry; BHA, butylated hydroxyanisole; BPA, bisphenol A; DPV, differential pulse voltammetry; GCE, glassy carbon electrode; GNPs, graphene nanoplatelets; MWCNTs, multiwalled carbon nanotubes; NCN, nitrogen-doped carbon nanosheet; PPY, polypyrroe.

5. Conclusions

The development of highly sensitive, reliable, robust, portable, and cost-effective sensing approaches has become fundamental to guarantee food safety, addressing the critical issues of infection/contamination of food commodities due to several causes, such as bacteria, contaminants, and toxins.

Considering the drawbacks of the conventional analytical approaches, such as complex analytical protocols, long duration of the analytical procedure, costly operation, and skilled personnel, it is quite clear that the electrochemical (bio)sensing approach is very attractive for many reasons, such as easiness to handle, relatively low cost, good sensitivity, and easy miniaturization.

In this context, CHI is considered a good ingredient for the development of electrochemical sensors and biosensors. Indeed, the combination of CHI with nanoparticles and conductive polymers has provided a sensitive determination of an analyte, by increasing the surface area and the electron transfer. In addition, CHI has demonstrated its efficiency to be employed as an immobilization platform for biomolecules using different approaches. It is to underline that the developed CHI-incorporated sensors show a relatively wide linear dynamic range and interesting results in terms of the limit of detection. Hence, we concluded that all these sensors have the prospective to be applied in other fields of applications and in real-time investigation of target molecules, because CHI shows high film-forming capability, non-toxicity, biocompatibility, and biodegradability.

Even if many of the described sensors evidence the possibility to be applied on real complex matrices, they have been validated only in the laboratory involving artificially spiked samples. For this reason, precise and accurate validation protocols would be highly desirable to carefully evaluate real effects due to the matrix effect and/or interferences. Statistically relevant numbers of samples, comparability, and inter-laboratory studies are consequently mandatory and required for a correct validation protocol and for eventually achieving regulatory approvals.

As a general comment, the development of performant sensors is linked to the development of portable devices. Smart portability can be obtained by integrating electrochemical sensors with ICT devices such as smartphones and tablets. Such a integration of sensors and ICT research fields could allow introducing the next generation of smart sensors into the food industries to increase the quality and safety of food and beverages.

As a final and general comment, taking into account the broad involvement of nanomaterials in the design and development of CHI-based sensors, studies of the toxicity and degradation of the nanomaterials are to be further addressed before introducing them in the sensors market. As a consequence of the large introduction of nanomaterials in the sensor design and development, the following issues should be investigated: the development of actually sustainable nanostructures, the related synthesis routes, and the toxicity, which represents a serious shortcoming for a wide range of applications of chemosensors and biosensors in food monitoring. The synthesis of nanocomposites involving sustainable, degradable, and green materials such as CHI can limit the effects of the toxicity of nanomaterials, but not permanently eliminate them. A rigorous study of the toxicity of nanomaterials is therefore urgent and mandatory and cannot be further neglected.