Review—Electrochemical Sensors and Biosensors Modified with Binary Nanocomposite for Food Safety

Binary nanocomposites containing metal (oxide) nanoparticles were used to modify on electrochemical sensor for the detection of food safety such as toxic chemicals, antibiotic and food preservative. Gold nanoparticle (AuNP) was the most used nanoparticle in nanocomposite. A molecularly imprinted polymer (MIP) electrochemical sensor with Au and polyaniline composites (Au/PANI) was used for the rapid detection of melamine in milk and feed.¹⁹ Polyaniline, as one of the most common conductive polymers, has the advantages of chemical stability and reversibility of doping and dedoping as well as polyaminothiophenol (PATP). AuNP was also functionalized with a PATP coating which was obtained by p-aminothiophenol (p-ATP) electropolymerizing on the electrode surface to form binary composite for the modification of sensors. In the MIP sensor toward tetracycline (Fig. 1),²⁰ the signal of composite increased with the increase of target concentration due to template molecule enhance the electron transfer. Acted as a π-donor, PATP with Au could concentrate tetracycline at the electrode surface by π-donor acceptor interactions. The sensor with a linear range from 2.24 × 10⁻⁴ to 22.4 nM and a detection limit of 2.2 × 10⁻⁷ nM, was proposed to determine tetracycline in honey. As for the oxidation of nitrite,²¹ the nanostructured film of AuNP with average diameters of 75 nm and PATP enhanced the active surface area of Au electrode and promoted electron transfer because of Au-S covalent binding contributed to metal nanoparticle immobilization. Aminobenzenesulfonic acid which is a derivative of aniline can be polymerized for the utilization in composite with AuNP in virtue of sulfonic functional groups in its structure. The oxidation peak current of propyl gallate at modified sensor with a porous composite of AuNP/poly(p-aminobenzenesulfonic acid) [poly(p-ABSA)] increased almost five fold, and the potential reduced nearly by 100mV compared to the unmodified sensor, resulting that AuNP/poly(p-ABSA) showed excellent electron transfer ability for the oxidation of propyl gallate.²² The linear response range of the designed sensor was 1.0 × 10⁻⁴ – 9.0 × 10⁻⁶ M, the detection limit was 1.9 × 10⁻⁷ M, and diffusion coefficient was 2.3 × 10⁻⁶ cm² s⁻¹.

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Other metals such as Cd, Rh and Co have been merged into ionic liquids (ILs), inorganic substances and polymers. Cadmium oxide nanoparticles (CdO NPs) have been reported to unite with some kinds of ILs such as imidazolium bromide and phosphate. IL/CdO NPs has been used as a high sensitive sensor for the quantitative determination of Sudan I in chilli, tomato and strawberry sauce,²³ in which the voltammetric oxidation peak current of Sudan I showed linear dynamic ranges with a detection limit of 0.05 μM. Vanadium family and copper group combined with hexafluorophosphate showed linear ranges of 0.1–700.0 μM for vanillin²⁴ and 0.08–500 μM for kojic acid.²⁵ In ILs, the electrochemical generation of charge can produce the transfer for components across liquid-liquid interface, and then increase the active surface area for modified sensor. A nanostructured electrocatalytic composite of rhodium nanoparticles and polyethyleneimine²⁶ which is a partial branched polymer containing primary amine, secondary amine and tertiary amine, has been fabricated on graphite screen-printed electrode through coulombic attraction to determine hydrogen peroxide in tea extracts. CoS nanorods with nafion (CoS NR@Nafion),²⁷ of which the peak at 452 cm⁻¹ corresponded to the Co-S bond while the peak at 471 cm⁻¹ was attributed to that of polysulfide, exhibited efficient electro-oxidation of vanillin and became a promising material for the sensors for monitoring vanillin.

In addition, the nanocomposite of metal oxide-metal oxide has been developed to construct electrochemical sensors. In Zhao's work,²⁸ cobalt-nickel oxide nanorods decorated molybdenum disulfide nanosheets (NiCo₂O₄/MoS₂) nanocomposite material was synthesized by a simple ionothermal synthesis method in deep eutectic solvents, after which, a glucose sensor based on the NiCo₂O₄/MoS₂ nanocomposites was formed. At the potential of +0.5823 V, the electrochemical response of the sensor was linear with the glucose concentration ranging from 0.70 μM to 13.78 mM, and it exhibited excellent electrocatalytic performance toward the oxidation of glucose with a low detection limit of 0.23 μM.

Carbon nanotube (CNT) has been utilized with polymer to modify many sensors. In CNT/polymer composites, the polymer can bind the CNT reinforced body together and give it a certain rigidity and specific geometry. The matrix phase in the composite is not completely separated from the reinforced phase, but a transition region which is a quasi three dimensional exists between the two phases. The CNT/polypyrrole composite had a great ability to analyze the isomers of Amaranth and Ponceau 4R²⁹ on account of synthetic dye more accumulated to the modified sensor due to the increasing dispersibility and effective surface area. As for heavy metal, the sites in MWCNT/ion-imprinted polymer are highly selective to lead ions and presented good analytical electrochemical response when this composite was modified to a platinum electrode,³⁰ resulting in a low detection limit of 2 × 10⁻² μM.

The metal or metal oxide nanoparticles are modified to the surface of carbon nanotubes to obtain superior functional nanocomposites. The use of carbon nanotubes as the carrier of metal catalyst can make the precursor of active metal fully dispersed, not only can improve the utilization of metal and prevent the sintering of metal particles, but also greatly promote the selectivity and stability of the metal catalyst because of the strong interaction between carbon nanotube and active metal. The sensor with MWCNT/CeO₂ nanocomposite had the concentration range of 10⁻⁸ to 10⁻⁵ M with a detection limit of 7.4 × 10⁻⁹ M toward acetaldehyde,³¹ in which CeO₂ donated its labile oxygen atoms so that acetaldehyde was transformed into acetic acid. Specifically, tetravalent Ce is reduced to trivalent by oxidizing the molecules on its surface, and oxygen atoms can easily move around the crystal in the fluorite structure of CeO₂. The introduction of hexafluorophosphate IL to CNT can obviously enhance the current response of target substance leading to an increase in the sensitivity of sensor and decrease in the detection limit. The MWCNTs/[BMIM][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) composite in which the electrostatic interaction exists between the electron–rich π system of MWCNTs and the positively charged species [BMIM]⁺,³² was used to modify the electrochemical sensor for the detection of pyrimethanil in fruits. An ultrasensitive electrochemical sensor based on a GCE modified by MWCNT@rGONR (reduced graphene oxide nanoribbon) composite was used for simultaneous determination of hydroquinone (HQ), catechol (CC) and resorcinol (RS). It displayed with the concentration range of 15 to 921 μM, 15 to 1101 μM, and 15 to 1301 μM and detection limit of 3.89 μM, 1.73 μM and 5.77 μM for HQ, CC and RS respectively.³³

Graphene (Gr) and reduced graphene oxide (rGO) have been employed as the carriers of metal and polymer to form the composites which can significantly improve the stacking phenomenon of Gr. The composite can also realize the uniform dispersion of alternative non-nanomaterial because of the interconnected porous structure of graphene.³⁴ A poly (diallyldimethylammonium chloride) (PDDA) functionalized rGO nanocomposite (rGO/PDDA) showed a well dispersed morphology with some wrinkles preventing agglomeration but persisting a large surface area, nevertheless the rGO displayed a stack layered morphology in the previous study.³⁵ The proposed sensor also exhibited excellent anti-interference property, repeatability and stability toward detection of quinoline yellow in soft drink.

An amperometric sensor based on palladium nanoparticles (PdNPs) supported by graphene oxide nanosheets modified glassy carbon electrode (GCE) was fabricated for bromate detection. It has been illustrated that oxygen-containing groups offer optimal nucleation sites for the growth of PdNPs, and also stabilize the PdNPs by increasing the interaction with graphene oxide.³⁶ AuNPs were used to fabricate the composites with Gr for diethylstilboestrol in fresh meat³⁷ and rGO for methylmercury (CH₃Hg⁺) in fish.³⁸ For the detection of CH₃Hg⁺, the AuNPs/rGO modified electrode showed an obvious stripping peak of CH₃Hg⁺ in contrast to the bare GCE and rGO modified electrode, indicating the affinity of Au for CH₃Hg⁺ attributed to the amalgam formation and preconcentration. The sensors with AuNPs/rGO had a good linear relation with the peak current of CH₃Hg⁺ in the range from 3 to 24 μg L⁻¹. A composite of graphene nanosheets (GS) in the presence of Bi³⁺ remarkably enlarged the response signals of Cd²⁺ and Pb²⁺.³⁹ The higher accumulation efficiency for Cd²⁺ and Pb²⁺ onto the GS/ Bi³⁺ composite was the result of the greatly-enhanced surface roughness of composite certainly brought more active sites. Metal oxides such as MnO₂,⁴⁰ CeO₂⁴¹ and MnCo₂O₄⁴² have been combined with graphene family to fabricate nanocomposites for the sensitive detection of synthetic pigments, antioxidants and harmful chemical substances in food samples.

Core-shell nanocomposites are universally combined with magnetic metal particle as core, noble metal or inorganic medium as shell. Magnetic metal particle in nanoscale has good saturation magnetization but is easily oxidized in air due to large specific surface area. However, the main use of magnetic metal oxide has been delivered, of which the saturation magnetization is generally lower but antioxidant stability is better. The Fe₃O₄@SiO₂ ⁴³ consisted with shielding of the magnetite by the polymeric coating in virtue of a low saturation magnetization, and obtained rapid magnetic separation due to enough magnetic response providing massive binding sites. The Fe₃O₄@SiO₂-based magnetic MIP sensor developed for the quantitative measurement of Gram-negative bacterial quorum in a linear detection range of 2.5 × 10⁻⁹–1.0 × 10⁻⁷ M has potential applications in food analysis.

Core-shell nanocomposites have better optical properties compared to their separate components, which can improve the optical stability and fluorescence quantum yield. The semiconductor nanoparticles with wider bandgap on the surface than the core⁴⁴ can greatly increase the fluorescence quantum yield of the core, enhance the stability, and realize the adjustable bandgap energy in a certain light band. In the presence of bimetallic Ni-Sn-oxide nanospheres,⁴⁵ the surface area and porosity of the electrode were greatly increased due to its tight arrangement and possession of many nanopores, thereby optimize the efficiency of modified sensor for detection of erythrosine.

Antibody is considered as one of the most common biological elements for immunosensors. AuNPs have been applied in immunosensors due to their high specific surface and the ability for immobilizing antibody.⁴⁶ A composite of AuNPs and ILs was successfully fabricated for the immunosensor, as a result of the roles of each component, that is, AuNPs captured antibodies against S. Pullorum and S. gallinarum to enhance the electrochemical signals as ILs reduced the influence of external adverse factors to maintain the biological activity of antibodies.⁴⁷ Two immunosensors were applied for the determination of Aflatoxin B1 (AFB1) using AFB1 antibody, as shown in Fig. 2. The AuNPs/chitosan nanocomposite prepared by one-step electrodeposition⁴⁸ provided plentiful amine groups, high surface energy and excellent biocompatibility for antibody immobilization owing to its porous three-dimensional morphology increasing the effective surface of microelectrode. The disposable and label-free immunosensor obtained the linear range from 1.6-32 ng mL⁻¹ in wheat. In another immunoassay,⁴⁹ AFB1 antibody fixed on the Au NPs was utilized to detect AFB1 based on stripping voltammetric detection of copper ions released from Cu-apatite through acidolysis which further amplified the electrochemical signal.

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The CeO₂ nanowires-based immunosensor was studied via different antibody immobilization methods for the detection of Vibrio cholerae O1.⁵⁰ Three methods including absorption, protein A-mediated and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/ Nhydroxysuccinimide (NHS)- activated were employed in this immunosensor. The detection limits and sensitivities for Vibrio cholerae O1 detection using three methods were in the order as followed: protein A > EDC/NHS-activated > absorption, conforming that the protein A-mediated method had the highest cell binding efficiency. The anti-tetracycline monoclonal antibody was immobilized on the electrode modified by carboxyl-Fe₃O₄ nanoparticles (cFe₃O₄ NPs) with chitosan for the detection of tetracycline.⁵¹ In this case, cFe₃O₄ NPs promoted the electron transfer by means of the decrease in resistance so that the modified biosensor showed a good linear current response to the target concentration.

An electrochemical biosensor based on the Nafion/TB composite of Nafion nano polymer and toluidine blue constructed with catalase enzyme (Fig. 3) was proposed for the determination of hydrogen peroxide in different beverage.⁵² The immobilization of catalase on the Nafion/TB surface decreased the overpotential of H₂O₂ and increased cathodic peak current compared to the case of the surface merely with Nafion/TB. The electron transfer coefficient (α) and the electron transfer rate constant (k_s) were found to be 0.48 and 12.1 ± 0.3 s⁻¹ in pH 7.0, respectively. The proposed biosensor presented two linear dynamic ranges with a detection limit of 0.25 μM for H₂O₂.

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Acetylcholinesterase (AChE) enzymatic activity and inhibition has been reported for the fabrication of biosensors. The enzymatic activity of AChE was blocked while organophosphorus and carbosulfan pesticides or their active metabolites can exert their detrimental effects, resulting in the accumulation of the neurotransmitter acetylcholine in synapses.⁵³ Bimetallic Pt-AuNPs performed a strong conductivity as an electron-transfer channel on account of the high conducting Au and Pt NPs, and offered substantial anchoring sites leading to the significant enhancement of electrochemiluminescence signal.⁵⁴ The electrochemiluminescence (ECL) intensity of the sensor with Pt-AuNPs/MWCNT decreased accordingly with the increase in concentration of organophosphorus pesticides including malathion, methyl parathion and chlorpyrifos, and the inhibition rates were proportional to the concentrations of 0.1–50 nmol L⁻¹ for each. The AChE bio-electrode with ZnO/Chitosan was employed to detect carbosulfan ranging from 5 to 30 nM in rice.⁵⁵

An aptamer is a highly affinity nucleic acid sequence (DNA or RNA) that can specifically recognize a target substance. It usually contains 15–40 bases and its molecular weight is approximately 5–25 kD.⁵⁶ The aptamer on the composite of Gr and AuNP that enhanced loading capacity of biomolecules⁵⁷ offered ultrasensitive electrochemical probe with high affinity and excellent specificity for oxytetracycline, whereas for the detection of tetracycline in milk and honey,⁵⁸ the Fe₃O₄/oleic acid composite was made due to the presence of a magnetic bar useful for the incorporation of nanoparticles and aptamer. The AuNPs-based composites^59,60 have been entrapped by DNA for food biosensors as a result of cooperative interactions among the AuNPs of high surface area, polymers of introducing additional functional groups and DNA. The DNA-based nanocomposite⁶¹ explored the interaction of the dsDNA with zearalenone and detected DNA damages caused by zearalenone, resulting in the DNA sensor for zearalenone determination as low as 0.005 ng mL⁻¹.

Besides hydrophobic forces, the electrostatic interactions between the positively charged amino groups of the aptamer and the negatively charged citrate groups surrounding iridium oxide nanoparticles (IrO2 NPs) which acted as a barrier between polythionine and the redox probe contributed to the immobilization of aptamer on the composite selective to ochratoxin A (OTA).⁶² The aptasensor displayed the lowest detection limit (5.65 ng kg⁻¹) reported so far for label-free impedimetric detection of OTA. Similarly, for the sensitive detection of OTA,⁶³ the integration of MoSe₂ nanoflowers and AuNPs was employed with aptamer and the guanine-rich complementary DNA sequence to develop a biosensor with a low detection limit. MoSe₂ nanoflowers owned large specific area and highly porous channels due to the hierarchical structure formed by many nanosheets stretching out toward the outside of the flowers. Another OTA aptamer was based on an as-synthesized nanocomposite of AgNPs and polydopamine nanospheres (PDANSs) through in-situ deposition.⁶⁴ In the presence of OTA, this aptamer was preferentially bonded with OTA because of their high affinity, and resulted in the decrease of signal tags on the aptasensor surface, which reduced the electrochemical response signal of AgNPs.

The immobilizations of myoglobin⁶⁵ and hemoglobin⁶⁶ were respectively conducted to the SWCNT/Nafion and Fe₃O₄ NPs/poly(indole-co-thiophene) composite providing suitable matrices for the electron exchange between the globin proteins and the modified electrodes. The myoglobin-SWCNT/Nafion biosensor exhibited a great potential for the determination of nitrite via a one-step redox reaction of the surface-confined myoglobin. The Fe₃O₄ NPs/poly(indole-co-thiophene) composite provided a suitable matrix for immobilization of hemoglobin so that the biosensors showed bioelectrocatalytic performances of biomolecule and sensitive quantitation of H₂O₂ in food samples.

Melamine (1,3,5-triazine-2,4,6-triamine) is an industrial chemical of triazine analogue together with three amino groups. It becomes an illegal food additive to fake the high content of protein for milk products, leading to the damage of human urinary system and increase the risk of some diseases. Compared to traditional methods, the modified sensors had the better properties for its detection. A MIP sensor with Au/PANI composites¹⁹ was used for the rapid detection of melamine (MEL) in the range of 1 × 10⁻⁵–10 μM with a low detection limit of 1.39 × 10⁻⁶ μM. The Au/PANI with a mean size of diameter 100 nm assembled with template molecule by hydrogen bonds were deposited on the surface of GCE. The modified MIP sensor increased 340.36 μA in the redox peak current compared to the bare electrode, indicating that it can amplify the electrode signal and improve the sensitivity for detection of MEL, in which AuNPs caught the wider plasmon peak and lost the optical effect. The MEL imprinted electrode⁶⁷ was prepared through electropolymerization process of phenol as monomer in the presence of phosphate buffer solution, showing the linearity range of 1.0 × 10⁻¹⁰–5.0 × 10⁻⁹ M and the detection limit of 1.0 × 10⁻¹¹ M. In the composite based on carbon nitride nanotubes (C₃N₄NTs), the tubular nanostructure is shaped by curling utg-C₃N₄ nanosheets. The AuNPs/rGO nanocomposite by co-reduction of Au(III) and graphene oxide⁶⁸ was developed to detect MEL attributed to competitive adsorption of MEL at the composite through the interaction between the amino groups of MEL and AuNPs. The modified electrode was applied to the concentration range of 5–50 nM and the detection limit of 1.0 nM. For the determination of MEL using diverse sensors with different modifiers shown in Table II, binary nanocomposites showed the lowest detection limit compared to other modifiers such as single nanomaterial, polymer, organic substance, etc. Thus, these electrochemical sensors modified with binary nanocomposites can meet the growing demands for monitoring the trace of MEL.

Bisphenol A (BPA) is widely used in the synthesis of polycarbonate plastics and epoxy resins for food and drink packaging applications, which negatively effects human health. A ruthenium-mediated photoelectrochemical sensor using MIP as the recognition element, SnO₂ nanoparticle-modified ITO as the electrode and a blue LED as the excitation light source,⁶⁹ was proposed for the detection of BPA in the linear range of 2–500 nM with the detection limit of 1.2 nM. It produced a photocurrent response of 134 nA while photocurrent of NIP electrode was measured to be 123 nA, causing by the formation of cavities in the polypyrrole film for the acceleration of ruthenium. The composite of CdO NPs and ionic liquid had the ability to lower over-potential and high sensitivity for BPA, resulting in the range of 0.3–650 μM with a detection limit of 0.1 μM.⁷⁰ The electrooxidation of BPA in the modified electrode generated at a potential about 50 mV less positive than the unmodified electrode in neutrality. Ordered mesoporous carbon CMK-3 modified nano-carbon ionic liquid paste electrode was fabricated as a BPA sensor⁷¹ in the range from 0.2–150 μM with the detection limit of 0.05 μM. Its conductive performance was improved by filling the interstice between graphite layers and bridging the carbon flakes owing to the high viscosity of IL. On the Gr/IL modified GCE, the peak current of BPA was the largest and the peak potential was the most negative compared to the bare GCE and the Gr modified GCE.⁷² Its linear range for BPA was from 20 to 2 × 10⁶ nM, and the detection limit was 8 nM. To be concluded, for pH effect in the electrooxidation of BPA on the modified electrodes, the shifts found in binary nanocomposites were closer to the theoretical value 57.6 mV pH⁻¹ than other modifiers, indicating that the electron transfer in modified electrode is accompanied by an equal number of protons. Further increasing the accumulation time, the current response increased slightly, due to the saturated adsorption of BPA at the modified electrode surface.

Nitrite is a widely used food preservative due to its good antimicrobial property, which has the capability to retard the development of rancidity during storage of foodstuff. It has been evidenced that nitrite can cause cancer in humans as a result of the reaction with amines to produce carcinogenic N-nitrosamines inside the body. A new cobalt phthalocyanine supported palladium nanoparticle composite (Pd/CoPc)⁷³ displayed the relocation of the electrons from Pd nanoparticles with the diameters of 4–6 nm to the phthalocyanine group of CoPc, and thereby reducing the gap between lowest unoccupied molecular orbital and highest occupied molecular orbital. The fabricated sensor for nitrite detection had two different linear concentration ranges of 0.2–50 μM and 500–5000 μM with a low detection limit of 0.10 μM and a sensitivity of 0.01 μA μM⁻¹. For the AuNP/PATP-modified electrode,²¹ the anodic and cathodic peaks shifted to slightly smaller potentials, whereas the corresponding peaks of PATP-modified electrode occurred with the displacement of 15.3 mV and 11.5 mV. Its linear range was 0.5–50 mg L⁻¹ for nitrite, and the detection limit was 0.12 mg L⁻¹. Another composite composed of Mn₃O₄ microcubes and thin sheets of graphene oxide has been developed to modified a screen-printed electrode⁷⁴ for amperometric determination of nitrite in the range of 0.1–1300 μM with a detection limit of 20 nM. In this composite, Mn₃O₄ microcube (Fig. 4) manifested the desired elements Mn and O via field emission scanning electron microscopy (FESEM), and the distribution of O and Mn in the cubes were weighted percentages of 58.47 and 41.53 respectively via energy-dispersive X-ray (EDX) profile. The low detection limit at nanomolar level illustrated the outstanding performance of the modified electrode and its parameters compared with other electrodes.

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Synthetic colorants, as additives in foodstuff, have the advantages of lower production cost, higher color uniformity and better stability compared with natural dyes. Sunset yellow (SY) and tartrazine (TZ) are two of widely used synthetic colorants containing azo functional groups and aromatic ring structures (Fig. 5),⁷⁷ which can lead to adverse health effects. The nanocomposites of MWCNT and MIP for different sensors^75,76 were used to detect SY in different linear range (0.0022 –4.64 μM for MWCNT/MIP-PDA and 0.05–100 μM for MIP/f-MWCNT) with different detection limit (1.4 nM and 5.0 nM, respectively). The electron transfer between the hydroxyl group and MWCNTs occurred smoothly when the target molecule is in a suitable configuration near the interface of the MIP and MWCNTs. There were massive shape-matched cavities for SY in the MIP layer, and the hydroxyl groups that captured SY molecules were located close to the surface of the MWCNTs to allow fast electron transfer.

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For the detection of TZ, some nanocomposites containing zinc oxide nanoparticles (ZnO NPs) were utilized for the modification of sensors while the other component causing the difference of detection range and limit. The prepared sensor with ZnO/Cysteic acid was employed in the sensitive simultaneous determination of SY and TZ in the concentration ranges of 0.1–3.0 μM and 0.07–1.86 μM, and detection limits of 0.03 μM and 0.01 μM, respectively.⁷⁷ ZnO NPs and p-ABSA were integrated a composite to modify voltammetric electrode for the determination of TZ in the range of 0.0349–5.44 μM with a detection limit of 80 nM.⁷⁸ The composite promoted the kinetics of the electrochemical process due to the high conductivity, good antifouling property and fast electron transfer rate of single material. In binary nanocomposite, the redox peak currents of SY and TZ increased with increasing accumulation time and reached a platform in a few minutes, indicating the saturated rebinding of analyte onto the surface of modified electrode with binary nanocomposite is quickly obtained. And, compared to other modifiers, binary nanocomposite exhibited a smaller pH range (3.0–5.0) in which the best discrimination and highest sensitivity were achieved.