chemosensors

Article A Simple but Efficient Voltammetric Sensor for Simultaneous Detection of Tartrazine and Based on TiO2/Electro-Reduced Graphene Oxide Nanocomposite

Zirong Qin †, Jinyan Zhang †, Ying Liu, Jingtao Wu, Guangli Li * , Jun Liu and Quanguo He College of Life Sciences and Chemistry, Hunan University of Technology, Zhuzhou 412007, China; [email protected] (Z.Q.); [email protected] (J.Z.); [email protected] (Y.L.); [email protected] (J.W.); [email protected] (J.L.); [email protected] (Q.H.) * Correspondence: [email protected]; Tel.: +86-189-7334-0268 The authors contributed equally to this work. †  Received: 17 July 2020; Accepted: 17 August 2020; Published: 19 August 2020 

Abstract: In this work, we report a simple but efficient voltammetric sensor for simultaneous detection of ponceau 4R and tartrazine based on TiO2/electro-reduced graphene oxide nanocomposites (TiO2/ErGO). TiO2/ErGO nanocomposites were prepared by ultrasonically dispersing TiO2 nanoparticles (TiO2 NPs) into graphene oxide (GO) solution followed by a green in-situ electrochemical reduction. TiO2 NPs were uniformly supported on ErGO nanoflakes, which provides a favorable interface for the adsorption and subsequent oxidation of target analytes. TiO2/ErGO showed remarkable electrocatalytic capacity for the oxidation of ponceau 4R and tartrazine, with minimized oxidation overpotentials and boosted adsorptive striping differential pulse voltammetric (AdSDPV) response peak currents. Under the optimal experimental conditions, the anodic peak currents of ponceau 4R and tartrazine increase linearly with the respective natural logarithm of concentrations from 0.01 to 5.0 µM. The detection limits (LOD = 3σ/s) for ponceau 4R and tartrazine are 4.0 and 6.0 nM, respectively. The extraordinary analytical properties of TiO2/ErGO/GCE are primarily attributed to the synergistic enhancement effect from ErGO nanoflakes and TiO2 NPs. Moreover, the proposed TiO2/ErGO/GCE achieves reliable determination of ponceau 4R and tartrazine in juice with excellent selectively, reproducibility and stability. Together with simplicity, rapidness, and low cost, the proposed sensor demonstrates great potential for on-site detection of azo colorants.

Keywords: ponceau 4R; tartrazine; TiO2; electro-reduced graphene oxide; voltammetric sensor

1. Introduction As two typical edible synthetic azo colorants, ponceau 4R and tartrazine always coexist together to give the orange color to food like orange juice. Due to their outstanding advantages such as relatively lower price, good water-solubility, low microbiological contamination and high stability, ponceau 4R and tartrazine have been widely added to make foodstuffs more appealing and appetizing [1]. However, ponceau 4R and tartrazine contain poisonous and carcinogenic aromatic ring structures and azo functional groups (–N=N–). Therefore, ponceau 4R and tartrazine likely lead to many adverse health effects such as allergies, neurobehavioral toxicities and cancers if they are excessively consumed [2–4]. To guarantee food safety, the dosages of ponceau 4R and tartrazine have been severely restricted by laws and regulations. For example, the joint FAO/WHO Expert Committee on Food Additives (JECFA) has set the acceptable daily intake (ADI) for ponceau 4R and tartrazine as 4.0 and 7.5 mg/kg bw/day, respectively [5,6]. In China, the maximum permitted dosages for ponceau 4R and

Chemosensors 2020, 8, 70; doi:10.3390/chemosensors8030070 www.mdpi.com/journal/chemosensors Chemosensors 2020, 8, 70 2 of 14 tartrazine in non-alcoholic beverages should not be more than 0.1 mg/mL (GB 2760–2007). Hence, the development of low cost but efficient analytical techniques for the sensitive determination of ponceau 4R and tartrazine is essential for food safety and human health. Currently, several analytical techniques have been developed for simultaneous detection of ponceau 4R and tartrazine, including capillary electrophoresis [7,8], spectrophotometry [9,10] and high-performance liquid chromatography [11–13]. These analytical methods are widely accepted and well proved, but they usually involve tedious and time-consuming analytical procedures and require costly equipment and high consumption of organic solvents. Owing to its low price, simple operation, excellent sensitivity and ease of miniaturization, the electroanalytical technique has recently been regarded as a competitive candidate for the in situ determination of azo colorants. Due to the presence of electrochemically active –N = N– groups, ponceau 4R and tartrazine can be readily detected by mercury electrodes based on their reduction signals [14–16]. However, the use of toxic mercury electrodes can inevitably cause adverse health effects and environmental pollution. Alternatively, a variety of mercury-free electrodes was fabricated for the individual determination of ponceau 4R [17–19] and tartrazine [20–23]. As far as we know, there are very few electrochemical sensors for simultaneous detection of ponceau 4R and tartrazine. Yang et al. [24] constructed an acetylene black film modified electrode for the simultaneous determination of ponceau 4R and tartrazine, but the proposed sensor suffered from lower sensitivity and inadequate detection limits (LODs). Ionic liquid modified expanded graphite paste electrode (IL-EGPE) [25] and ionic liquid-graphene oxide-multiwalled carbon nanotube nanocomposite (IL-GO-MWCNT) [26] showed extraordinary electrocatalytic oxidation capacity toward ponceau 4R and tartrazine. However, costly ionic liquids enormously hinder their practical applications. In our previous studies, TiO2/graphene nanocomposites exhibited high catalytic activity for tartrazine electrooxidation [27]. However, to the best of our knowledge, TiO2/graphene nanocomposite-based voltammetric sensors have not been used for simultaneous determination of ponceau 4R and tartrazine yet. Therefore, designing voltammetric sensors with simplicity, cost-effectiveness and excellent sensitivity is paramount for simultaneous determination of ponceau 4R and tartrazine. TiO2 nanoparticles (NPs) have been considered as the most promising candidate for advanced (photo)electrochemical sensors, owing to their attractive advantages such as more abundance, low cost, high electrical conductivity, strong adsorptive ability and remarkable catalytic activity [28–32]. However, TiO2 NPs cannot be firmly immobilized on the surface of carbon electrodes [33], thereby deteriorating electrode stability and sensing property. In addition, TiO2 NPs tend to aggregate without surfactant as a dispersant, which also degrades sensing performance. In order to address these issues, TiO2 NPs are often anchored on the graphene to improve the dispersibility and immobilization of TiO2 NPs onto carbon electrodes. Considering the advantageous features including huge specific surface area, excellent electroconductivity and extraordinary electron transfer capacity, graphene has become a versatile material to construct electrochemical sensing platforms [34–39]. Graphene with unique two-dimensional layered structures and intriguing electronic properties provide a favorable catalyst carrier for anchoring metal oxide nanoparticles [40], therefore achieving versatile selective catalytic or sensing performance. In addition, graphene can be readily anchored on the GCE surface through π–π interactions. For these reasons, TiO2/graphene nanocomposites have been exploited as efficient sensing films for voltammetric determination of biomolecules such as guanine [31], adenine [31], dopamine [41], glucose [42], L-tyrosine [43] and L-tryptophan [43]. However, to the best of our knowledge, TiO2/graphene nanocomposite-based voltammetric sensors have not been used for the simultaneous determination of ponceau 4R and tartrazine yet. Herein, a simple but efficient voltammetric sensor was designed for simultaneous determination of ponceau 4R and tartrazine in orange juice based on TiO2/ErGO. TiO2/ErGO nanocomposites were prepared by ultrasonically dispersing TiO2 NPs into graphene oxide (GO) solution followed by a green in-situ electrochemical reduction. TiO2 NPs were uniformly supported on ErGO nanoflakes, which provide a favorable interface for the adsorption and subsequent oxidation of target analytes. The resultant TiO2/ErGO generated synergistic effect toward the electrooxidation of ponceau 4R and Chemosensors 2020, 8, 70 3 of 14 tartrazine, which gives arise to amplified response signals. Furthermore, the proposed sensor was used for simultaneous determination of Ponceau 4R and Tartrazine in orange juice sample with satisfactory outcomes.

2. Materials and Methods

2.1. Chemicals and Materials Ponceau 4R, tartrazine, Allura Red, amaranth and sunset were bought from Aladdin Reagents Inc. (Shanghai, China). Other chemicals were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were directly used without further treatments. GO was supplied by Xianfeng Nanomaterial Technology Inc. (Nanjing, China). Orange juice samples were purchased from a local supermarket. Deionized water from a Millipore water purification system (Mini-Q, 18 MΩ cm) was used throughout the experiments.

2.2. Synthesis of TiO2 NPs

TiO2 NPs were prepared by a facile hydrothermal treatment. In a typical procedure, 2.4034 g of titanium sulfate were dissolved into 50 mL of deionized water, and then subjected to ultrasonication for 40 min. Afterwards, the reaction solution was transferred into a 100 mL Teflon-lined stainless-steel container and heated at 180 ◦C for 4 h. After being cooled into ambient temperature naturally, the resultant solution was centrifuged at 12,000 rpm for 15 min, and the supernatant was decanted gently. The as-collected white solid was alternately rinsed three times with absolute ethanol and deionized water, and then allowed to dry at 60 ◦C overnight.

2.3. Synthesis of TiO2/GO Nanocomposites Firstly, 0.2000 g of GO was dispersed into 10 mL deionized water under ultrasonication for 1 h to form a uniform GO dispersion (20 mg/mL). Then 0.0100 g TiO2 NPs were added into 10 mL GO dispersion and ultrasonically dispersed for 1 h to get a homogenous TiO2/GO dispersion.

2.4. Preparation of TiO2/ErGO/GCE Bare glass carbon electrode (GCE, diameter of 3 mm, GAOSSUNION, Tianjin, China) was polished to a shiny mirror-like surface with 0.05 µm Al2O3 powder. Then the GCE was alternately rinsed three times by absolute ethanol and deionized water under ultrasonication (each for 1 min), and was dried with an exposure of infrared light. TiO2/ErGO nanocomposite modified electrode (TiO2/ErGO/GCE) was fabricated by a convenient drop-casting technique followed by an in-situ potentiostatic reduction. In brief, 5 µL of TiO2/GO dispersion was firmly coated on the surface of the freshly polished GCE, and then allowed to form an intact sensing film with the radiation of an infrared light. Afterwards, the as-obtained TiO2/GO/GCE was immersed into 0.1 M phosphate buffer solution (PBS, pH 7.0), then electrochemically reduced into TiO /ErGO/GCE at 1.5 V for 2 min with stirring. 2 − 2.5. Voltammetric Determination of Ponceau 4R and Tartrazine All voltammetric measurements were implemented on a CHI 760E electrochemical workstation (Chenhua Inc., Shanghai, China) with a typical three-electrode setup. A bare or modified electrode was used as the working electrode. A saturated calomel electrode (SCE) and a platinum wire were served as the reference electrode and auxiliary electrode, respectively. Unless otherwise specified, 0.1 M PBS (pH 7.0) was used as the supporting electrolyte. The electrochemical properties of different electrodes were studied by cyclic voltammetry and AC impedance spectra in 0.1 M KCl solution containing 3 /4 5 mM [Fe(CN)6] − − probe. Cyclic voltammograms of different electrodes were recorded over the range of 0.2 – 0.6 V at a speed of 0.1 V s 1. AC impedance spectra were measured at open circuit − − potential from 100 kHz to 0.1 Hz with 5 mV (rms) AC sinusoid signal. To boost the voltammetric responses, a suitable deposition was applied before the voltammetric measurements. Considering its Chemosensors 2020, 8, x FOR PEER REVIEW 4 of 13

pulse amplitude, pulse width, sample width and pulse period for the DPV recording were set at 0.05 V, 0.05 s, 0.02 s and 0.2 s, respectively.

3. Results and Discussions

3.1. Physical Chararazation of TiO2/ErGO

The TEM images of TiO2 NPs and TiO2/GO are shown in Figure1A,B. TiO2 NPs shows cube-like Chemosensorsstructures2020 with, 8, 70the size of ca. 30~45 nm (Figure 1A). A large proportion of TiO2 NPs is uniformly and4 of 14 compactly embedded on the GO substrates without notable aggregation (Figure 1B). It can be seen from the TEM image of TiO2/GO that a great quantity of TiO2 NPs is attached onto the surface of GO advantageousnanoflakes. It featureswas reported of superior that GO resolution is heavily andoxyge sensitivity,nated, bearing diff erentialcarboxyl pulsegroups voltammetry at its basal edges (DPV) wasand used epoxide forthe or hydroxyl simultaneous groups determination on the basal planes of ponceau [44]. On one 4R andhand tartrazine., GO functions The as pulsean amphiphilic amplitude, pulsesurfactant width, to sample enhance width the dispersion and pulse periodof TiO2 forNPs, the due DPV to the recording presence were of massive set at 0.05 hydrophilic V, 0.05 s, oxygen- 0.02 s and 0.2containing s, respectively. functional groups (OxFGs) on the hydrophobic basal planes [45]. On the other hand, OxFGs promote the intercalation of Ti species into GO layers via interaction with the hard Ti4+ Lewis acid 3. Results[46,47]. Highly and Discussions dispersed TiO2 NPs on supports with large surface area GO would be conducive to enhance catalytic activity [30] and sensor sensitivity [48]. GO can be electrochemically reduced into 3.1. Physical Chararazation of TiO /ErGO ErGO with in-situ anchoring TiO2 2 NPs on the resultant graphene flakes. The crystalline structures of GO flakes, ErGO flakes, TiO2 NPs and TiO2/GO nanocomposites The TEM images of TiO2 NPs and TiO2/GO are shown in Figure1A,B. TiO 2 NPs shows cube-like were examined by X-ray diffraction (XRD). As depicted in Figure 1C, GO exhibited a sharp and structures with the size of ca. 30~45 nm (Figure1A). A large proportion of TiO 2 NPs is uniformly anddistinct compactly diffraction embedded peak at on the the 2 GOθ of substrates9.8°, attributing without to the notable (001) aggregation facet. After (Figurethe electrochemical1B). It can be reduction of GO, the (001) facet completely disappeared, and a new broad diffraction peak occurred seen from the TEM image of TiO2/GO that a great quantity of TiO2 NPs is attached onto the surface around 24° that corresponded to the (002) facet. This suggests an increase in the graphitic nature of of GO nanoflakes. It was reported that GO is heavily oxygenated, bearing carboxyl groups at its ErGO because of the decreased interlayer spacing to 3.55 Å after electrochemical reduction. The broad basal edges and epoxide or hydroxyl groups on the basal planes [44]. On one hand, GO functions diffraction peak indicates the loss of crystallinity in graphite. After incorporation of TiO2 NPs into as an amphiphilic surfactant to enhance the dispersion of TiO NPs, due to the presence of massive ErGO nanoflakes, the feature peak of ErGO (broad peak around2 24°) can be also recognized. In hydrophilic oxygen-containing functional groups (OxFGs) on the hydrophobic basal planes [45]. On the addition, apparent diffraction peaks were observed at 25.26°, 36.92°, 47.94°, 53.98°, 54.90° and 62.60°, other hand, OxFGs promote the intercalation of Ti species into GO layers via interaction with the hard respectively, which can be assigned to the anatase TiO2 NPs supported on ErGO substrates as (101), 4+ Ti (004),Lewis (200), acid (105), [46,47 (211)]. Highly and (213) dispersed crystal TiO planes2 NPs (tetragonal, on supports JCPDS with large21-1272). surface In addition, area GO wouldno any be conduciveevident impurity to enhance diffraction catalytic peaks activity are [found,30] and which sensor demonstrates sensitivity [that48]. the GO as-prepared can be electrochemically TiO2/ErGO reducednanocomposites into ErGO are with of high in-situ purity. anchoring TiO2 NPs on the resultant graphene flakes.

Figure 1. TEM images of TiO2 nanoparticles (NPs) (A) and TiO2/GO nanocomposites (B); (C) XRD Figure 1. TEM images of TiO2 nanoparticles (NPs) (A) and TiO2/GO nanocomposites (B); (C) XRD patterns of GO, ErGO, TiO2 and TiO2/ErGO nanocomposites. patterns of GO, ErGO, TiO2 and TiO2/ErGO nanocomposites.

3.2.The Electrochemical crystalline Properties structures of ofTiO GO2/ErGO flakes, ErGO flakes, TiO2 NPs and TiO2/GO nanocomposites were examinedFigure 2A by shows X-ray cyclic diffraction voltammograms (XRD). As depictedof variou ins electrodes Figure1C, in GO 0.1 exhibited M KCl solution a sharp containing and distinct diff5raction mM [Fe(CN) peak at6]3− the/4−. Obviously, 2θ of 9.8◦, attributinga pair of well-shaped to the (001) and facet. symmetrical After the redox electrochemical peaks occurred reduction at all of GO,electrodes the (001) with facet the completely anodic/cathodic disappeared, peak current and a new ratio broad (Ipa/Ipc di) ffofraction 1.0 approximately, peak occurred demonstrating around 24◦ that correspondedthat the redox to behavior the (002) of facet. FeIII/Fe ThisII corresponded suggests an to increase a quasi-reversible in the graphitic process. nature At bare of ErGO GCE, becausea pair of of therelative decreased weak interlayer redox peaks spacing (Ipa = to99.27 3.55μA; Å afterIpc = 99.90 electrochemical μA) appeared reduction. at 0.266 and The 0.121 broad V, di respectively.ffraction peak indicatesAfter loading the loss of ofTiO crystallinity2 NPs onto the in graphite.GCE surface, After the incorporation Ipa and Ipc increased of TiO to2 NPs125.1 into andErGO 119.5 μ nanoflakes,A while the feature peak of ErGO (broad peak around 24◦) can be also recognized. In addition, apparent diffraction peaks were observed at 25.26◦, 36.92◦, 47.94◦, 53.98◦, 54.90◦ and 62.60◦, respectively, which can be assigned to the anatase TiO2 NPs supported on ErGO substrates as (101), (004), (200), (105), (211) and (213) crystal planes (tetragonal, JCPDS 21-1272). In addition, no any evident impurity diffraction peaks are found, which demonstrates that the as-prepared TiO2/ErGO nanocomposites are of high purity.

3.2. Electrochemical Properties of TiO2/ErGO Figure2A shows cyclic voltammograms of various electrodes in 0.1 M KCl solution containing 3 /4 5 mM [Fe(CN)6] − −. Obviously, a pair of well-shaped and symmetrical redox peaks occurred at all Chemosensors 2020, 8, 70 5 of 14

electrodes with the anodic/cathodic peak current ratio (Ipa/Ipc) of 1.0 approximately, demonstrating that the redox behavior of FeIII/FeII corresponded to a quasi-reversible process. At bare GCE, a pair of relative weak redox peaks (Ipa = 99.27µA; Ipc = 99.90 µA) appeared at 0.266 and 0.121 V, respectively. Chemosensors 2020, 8, x FOR PEER REVIEW 5 of 13 After loading of TiO2 NPs onto the GCE surface, the Ipa and Ipc increased to 125.1 and 119.5 µA while thethe peak peak separation separation (∆ (∆EEp)p) decreased decreased fromfrom 0.145 to to 0.109 0.109 V. V. This This phenomenon phenomenon indicates indicates TiO TiO2 NPs2 NPs own own electrocatalyticelectrocatalytic capacity. capacity. Likewise, Likewise, the the Ipa Ipaand and I pcIpc alsoalso increased (I pa == 137.3137.3 μµA;A; Ipc Ipc = 137.5= 137.5 μA)µA) on onthe the ErGOErGO modified modified electrode electrode meanwhile meanwhile the ∆theEp declined∆Ep declined to 0.101 to V,0.101 which V, suggestswhich suggests that ErGO that nanoflakes ErGO acceleratednanoflakes an accelerated electron transfer an electron process transfer significantly process significantly due to its due good to electricalits good electrical conductivity conductivity and high specificand high surface specific area. surface As expected, area. As expected, the best voltammetricthe best voltammetric responses responses (Ipa = (I148.2pa = 148.2µA; μ IA;pc =Ipc144.1 = 144.1µ A) wereμA) achieved were achieved at TiO 2at/ErGO TiO2/ErGO/GCE/GCE with minimizedwith minimized∆Ep (0.097∆Ep (0.097 V), mainly V), mainly due du toe the to synergisticthe synergistic effect fromeffect ErGO from nanoflakes ErGO nanoflakes and TiO2 andNPs. TiO Based2 NPs. on theBased Randles–Sevcik on the Randles–Sevcik equation [36equation,49], the [36,49], electroactive the electroactive surface areas of bare, TiO2 NPs, ErGO nanoflakes and TiO2/ErGO modified GCEs were surface areas of bare, TiO2 NPs, ErGO nanoflakes and TiO2/ErGO modified GCEs were calculated calculated as 0.072, 0.084, 0.10 2and 0.13 cm2, respectively. TiO2/ErGO nanocomposites greatly as 0.072, 0.084, 0.10 and 0.13 cm , respectively. TiO2/ErGO nanocomposites greatly enlarged the electroactiveenlarged the surface electroactive area, which surface could area, provide which could more provide available more reactive available active reactive sites for active the adsorptionsites for andthe oxidation adsorption of and azo colorants,oxidation of thus azoultimately colorants, thus boosting ultimately the voltammetric boosting the responses.voltammetric responses.

Figure 2. Cyclic voltammograms (A) and Nyquist plots (B) recorded at various electrodes in 0.1 M KCl Figure 2. Cyclic voltammograms (A) and Nyquist3 /4 plots (B) recorded at various electrodes in 0.1 M solution (pH 7.0) containing 5 mM [Fe(CN)6] − −. The inset in Figure2B represents the equivalent KCl solution (pH 7.0) containing 5 mM [Fe(CN)6]3−/4−. The inset in Figure 2B represents the equivalent circuit model for impedance fitting. circuit model for impedance fitting. AC impedance spectra have become the most popular technique to acquire the electrode interfacial AC impedance spectra have become the most popular technique to acquire the electrode characteristic [36,49–51]. Nyquist plots of different electrodes are illustrated in Figure2B. Typically, interfacial characteristic [36,49–51]. Nyquist plots of different electrodes are illustrated in Figure 2B. the diameter of semicircle is equivalent to the charge transfer resistance (Rct). The largest semicircle was Typically, the diameter of semicircle is equivalent to the charge transfer resistance (Rct). The largest observed at bare GCE (Rct = 1562 Ω), indicating sluggish electrode kinetics occurred at the unmodified semicircle was observed at bare GCE (Rct = 1562 Ω), indicating sluggish electrode kinetics occurred at electrode. After decoration with TiO NPs and ErGO nanoflakes, the R value decreased to 366 Ω the unmodified electrode. After decoration2 with TiO2 NPs and ErGO ctnanoflakes, the Rct value anddecreased 233 Ω, respectively, to 366 Ω and indicating 233 Ω, respectively, that TiO2 indicatingNPs or ErGO thatnanoflakes TiO2 NPs or promoted ErGO nanoflakes the electron promoted transfer effitheciently. electron The transfer Nyquist efficien plottly. of TiO The2 /NyquistErGO/GCE plot wasof TiO almost2/ErGO/GCE a straight was line, almost which a straight demonstrates line, which that thedemonstrates TiO2/ErGO nanocompositethat the TiO2/ErGO minimizes nanocomposite the Rct value minimizes (Rct = 44.9the ΩRct). value This is(R primarilyct = 44.9 Ω attributed). This is to theprimarily fact that theattributed synergistic to the eff ectfact fromthat ErGOthe syne nanoflakesrgistic effect and from TiO2 NPsErGO expedites nanoflakes the and electron TiO2transfer. NPs It isexpedites believed the that electron the significant transfer. It reduction is believed in that Rct theis a significant benefit to reduction improve thein R sensorct is a benefit sensitivity. to improve the sensor sensitivity. 3.3. Enrichment in Voltammetric Responses of Ponceau 4R and Tartrazine 3.3. Enrichment in Voltammetric Responses of Ponceau 4R and Tartrazine The voltammetric responses of 1.0 µM ponceau 4R and tartrazine were investigated in 0.1 M PBS (pHThe 7.0). voltammetric Figure3 shows responses the DPV of 1.0 curves μM ponceau of 1.0 4RµM and ponceau tartrazine 4R were and tartrazineinvestigated (1:1) in 0.1 at M di ffPBSerent modified(pH 7.0). electrodes. Figure 3 shows At the the bare DPV electrode, curves twoof 1.0 weak μM andponceau ill-shaped 4R and anodic tartrazine peaks (1:1) appeared at different at 0.628 andmodified 0.872 V, electrodes. corresponding At the tobare the electrode, oxidation two of weak ponceau and 4Rill-shaped and tartrazine. anodic peaks In this appeared case, the at 0.628 anodic peakand currents 0.872 V, of corresponding ponceau 4R and to the tartrazine oxidation were of 0.327ponceau and 4R 0.0540 and µtartrazine.A, respectively. In this Aftercase, loadingthe anodic TiO 2 NPspeak alone, currents the anodicof ponceau peak 4R currents and tartrazine of ponceau were 4R 0.327 and and tartrazine 0.0540 μ increasedA, respectively. to 0.383 After and loading 0.176 µ A, respectively.TiO2 NPs alone, The the poor anodic voltammetric peak currents responses of ponceau of ponceau 4R and 4Rtartrazine and tartrazine increased are to 0.383 attributed and 0.176 to the μA, respectively. The poor voltammetric responses of ponceau 4R and tartrazine are attributed to the offset effect between their poor dispersibility and excellent electrocatalytic capacity [31,41,42,52]. Two well-shaped and pronounced anodic peaks occurred at ErGO nanoflakes modified electrodes. In addition, the voltammetric responses of ponceau 4R and tartrazine boost significantly, with their

Chemosensors 2020, 8, 70 6 of 14 offset effect between their poor dispersibility and excellent electrocatalytic capacity [31,41,42,52]. TwoChemosensors well-shaped 2020, 8,and x FOR pronounced PEER REVIEW anodic peaks occurred at ErGO nanoflakes modified electrodes.6 of 13 In addition, the voltammetric responses of ponceau 4R and tartrazine boost significantly, with their respective anodic peak currents of 0.418 and 1.21 μA, respectively. This may be strongly benefited respective anodic peak currents of 0.418 and 1.21 µA, respectively. This may be strongly benefited from from the intrinsic properties of ErGO nanoflakes such as a large surface area and high conductivity, the intrinsic properties of ErGO nanoflakes such as a large surface area and high conductivity, which which promote the absorption and subsequent electrooxidation of azo colorants effectually [52]. promote the absorption and subsequent electrooxidation of azo colorants effectually [52]. When loading When loading TiO2/ErGO onto the GCE surface, two sharp and well-defined anodic peaks occurred TiOat2 /0.616ErGO and onto 0.868 the GCEV, respectively. surface, two Moreover, sharp and th well-definede voltammetric anodic responses peaks of occurred ponceau at 4R 0.616 and and 0.868tartrazine V, respectively. enhanced Moreover,prominently, the with voltammetric their respective responses anodic of peak ponceau currents 4R and of 2.03 tartrazine and 0.541 enhanced μA, prominently,respectively. with The their anodic respective peak currents anodic of peakponceau currents 4R and of tartrazine 2.03 and 0.541 were µ5-foldA, respectively. and 9-fold higher The anodic as peakcompared currents to ofbare ponceau GCE, respectively. 4R and tartrazine The lowered were oxidation 5-fold and overpotentials 9-fold higher and as comparedremarkably to enhanced bare GCE, respectively.peak currents The suggest lowered that oxidation TiO2/ErGO overpotentials nanocomposites and remarkably generated enhancedthe conspicuous peak currents synergistic suggest effect that TiOtoward2/ErGO the nanocomposites electrooxidation generated of ponceau the conspicuous4R and tartrazine. synergistic To be especific,ffect toward the excellent the electrooxidation adsorption of ponceaucapability 4R andand tartrazine.electrocatalytic To be effect specific, of TiO the2/ErGO excellent nanocomposites adsorption capability greatly improve and electrocatalytic electron transfer effect ofefficiency, TiO2/ErGO thus nanocomposites ultimately amplifying greatly improvethe voltamme electrontric responses transfer e ffi[31,41–43,52].ciency, thus In ultimately addition, amplifyingthe high theelectrochemical voltammetric active responses area [and31,41 low–43 charge,52]. In transfer addition, resistance the high also electrochemical contributed to activethe extraordinary area and low chargeelectrocatalytic transfer resistance performances. also contributed to the extraordinary electrocatalytic performances.

FigureFigure 3. 3.Di Differentialfferential pulse pulse voltammetry voltammetry (DPV) curves of of 1.0 1.0 μµMM ponceau ponceau 4R 4R and and tartrazine tartrazine (1:1) (1:1) at at diffdifferenterent modified modified electrodes. electrodes. SupportingSupporting electrolyte: 0.1 0.1 M M PBS PBS (p (pHH == 7.0);7.0); deposition deposition parameters: parameters: 0.1 V, 120 s. − −0.1 V, 120 s. 3.4. Exploration of Voltammetric Parameters 3.4. Exploration of Voltammetric Parameters 3.4.1. Deposition Parameters 3.4.1. Deposition Parameters DepositionDeposition parameters parameters directly directly determine determine the voltammetricthe voltammetric responses responses of ponceau of ponceau 4R and 4R tartrazine. and Therefore,tartrazine. the Therefore, dependences the dependences of deposition of potential deposition as well potential as time as onwell the as voltammetric time on the voltammetric responses were alsoresponses investigated. were also Both investigated. the anodic Both peak the current anodic of peak ponceau current 4R of and ponceau tartrazine 4R and (I patartrazine(PR) and (Ipa Ipa(PR)(TZ)) increased gradually as the deposition potentials moved from 0.4 to 0.1 V, then reached maximum and Ipa(TZ)) increased gradually as the deposition potentials moved− from− −0.4 to −0.1 V, then reached valuesmaximum at 0.1 values V, afterwards at −0.1 V, itafterwards sharply decreased it sharply decreased with more with positive more potentialspositive potentials (Figure4 (FigureA). Likewise, 4A). − bothLikewise, Ipa(PR) both and IIpapa(PR)(TZ) sharplyand Ipa(TZ) increased sharply within increased the first within 120 the s, then first they 120 decreaseds, then they dramatically decreased as thedramatically deposition as time the exceededdeposition 120 time s exceeded (Figure4B). 120 So, s (Figure the optimal 4B). So, deposition the optimal parameters deposition parameters were 0.1 V, − 120were s. −0.1 V, 120 s.

3.4.2. Medium pH Figure5A displays the DPV curves of ponceau 4R and tartrazine at various medium pH. The anodic peaks of ponceau 4R and tartrazine shifted negatively as medium pH increased,

Chemosensors 2020, 8, x FOR PEER REVIEW 7 of 13

Chemosensors 2020, 8, 70 7 of 14 demonstrating that the redox reactions of ponceau 4R and tartrazine involve protons (H+). Furthermore, the anodic peak potentials of ponceau 4R and tartrazine were inversely proportional to medium pH 2 (Figure5B). Their corresponding linear equations were E pa (V) = 0.0676pH + 1.38 (R = 0.986) and 2 − Epa (V) = 0.0474pH + 0.892 (R = 0.989) for ponceau 4R and tartrazine, respectively. Their respective − slopes (67.6 and 47.4 mV/pH) closely approached the Nernstian values (59 mV/pH), implying that + the numbers of electrons (e−) and protons (H ) was 1:1. Besides, both Ipa (PR) and Ipa (TZ) increased gradually as the medium pH increased from 5.8 to 7.0, then they suddenly reduced when medium pH was beyond 7.0 (Figure5C). So, the optimal pH was set at 7.0. ChemosensorsFigure 2020 4. Effect, 8, x FOR of deposition PEER REVIEW potential (A) and deposition time (B) on the DPV anodic peak currents7 of 13 of ponceau 4R and tartrazine. (A) Deposition time was set at 150 s and (B) deposition potential was fixed at −0.1 V.

3.4.2. Medium pH Figure 5A displays the DPV curves of ponceau 4R and tartrazine at various medium pH. The anodic peaks of ponceau 4R and tartrazine shifted negatively as medium pH increased, demonstrating that the redox reactions of ponceau 4R and tartrazine involve protons (H+). Furthermore, the anodic peak potentials of ponceau 4R and tartrazine were inversely proportional to medium pH (Figure 5B). Their corresponding linear equations were Epa (V) = −0.0676pH + 1.38 (R2 = 0.986) and Epa (V) = −0.0474pH + 0.892 (R2 = 0.989) for ponceau 4R and tartrazine, respectively. Their respective slopes (67.6 and 47.4 mV/pH) closely approached the Nernstian values (59 mV/pH), implying that the numbers of electrons (e−) and protons (H+) was 1:1. Besides, both Ipa (PR) and Ipa (TZ) increasedFigure gradually 4. EffectEffect of as deposition the medium potentialpotential pH (A(increasedA)) and and deposition deposition from 5.8 time time to ( B 7.0,(B) on) onthen the the DPV they DPV anodic suddenlyanodic peak peak currentsreduced currents of when ponceauof ponceau 4R 4R and and tartrazine. tartrazine. (A) (A) Deposition Deposition time time was was set atset 150 at s150 and s (B)and deposition (B) deposition potential potential was fixedwas medium pH was beyond 7.0 (Figure 5C). So, the optimal pH was set at 7.0. fixedat 0.1 at V.−0.1 V. − 3.4.2. Medium pH Figure 5A displays the DPV curves of ponceau 4R and tartrazine at various medium pH. The anodic peaks of ponceau 4R and tartrazine shifted negatively as medium pH increased, demonstrating that the redox reactions of ponceau 4R and tartrazine involve protons (H+). Furthermore, the anodic peak potentials of ponceau 4R and tartrazine were inversely proportional to medium pH (Figure 5B). Their corresponding linear equations were Epa (V) = −0.0676pH + 1.38 (R2 = 0.986) and Epa (V) = −0.0474pH + 0.892 (R2 = 0.989) for ponceau 4R and tartrazine, respectively. Their respective slopes (67.6 and 47.4 mV/pH) closely approached the Nernstian values (59 mV/pH), implyingFigure that 5. ( the(AA)) DPV numbers DPV curves curves of of ofelectrons ponceau ponceau 4R(e 4R− )and and and tartrazine protons tartrazine on (H onTiO+) was TiO2/ErGO/GCE2 1:1./ErGO Besides,/GCE measured measured both Iatpa different(PR) at di andfferent pH; Ipa (TZ) increasedpH;(B) the ( Bgradually )dependence the dependence as of the anodic medium of anodic peak potential pH peak increased potential of ponceau from of ponceau4R 5.8 and to tartrazine 4R7.0, and then tartrazineon they pH and suddenly on(C) pHthe dependence and reduced (C) the when dependenceof anodic peak of anodiccurrent peak of ponceau current 4R of and ponceau tartrazine 4R and on tartrazine pH. Deposition on pH. parameters: Deposition parameters:−0.1 V, 120 s. 0.1 V, medium pH was beyond 7.0 (Figure 5C). So, the optimal pH was set at 7.0. − 120 s. 3.4.3. Scan Rate 3.4.3. Scan Rate Cyclic voltammograms of ponceau 4R and tartrazine at various scan rates are illustrated in Figure Cyclic voltammograms of ponceau 4R and tartrazine at various scan rates are illustrated in Figure S1. S1. The symmetric redox peaks of ponceau 4R suggest that ponceau 4R underwent a quasi-reversible The symmetric redox peaks of ponceau 4R suggest that ponceau 4R underwent a quasi-reversible process. In contrast, no cathodic peak of tartrazine was found at the reverse scan, indicating tartrazine process. In contrast, no cathodic peak of tartrazine was found at the reverse scan, indicating tartrazine underwent a totally irreversible process. Notably, both Ipa (PR) and Ipa (TZ) increased steadily as the scan underwent a totally irreversible process.−1 Notably, both Ipa (PR) and Ipa (TZ) increased steadily as the rates increased from 0.03 to 0.3 Vs . In addition,1 the redox peak currents of ponceau 4R and tartrazine scan rates increased from 0.03 to 0.3 Vs− . In addition, the redox peak currents of ponceau 4R and (Ipa or Ipc) increased linearly with the square root of scan rates (Figure 6A,B), suggesting the reactions of tartrazine (I or I ) increased linearly with the square root of scan rates (Figure6A,B), suggesting ponceau 4R paand tartrazinepc are typical diffusion-controlled processes. As the potential scan rate grew the reactions of ponceau 4R and tartrazine are typical diffusion-controlled processes. As the potential up, the anodic peaks shifted positively while the cathodic peaks moved towards the reverse direction. scanFigure rate grew 5. (A) up,DPV the curves anodic of ponceau peaks 4R shifted and tartrazine positively on TiO while2/ErGO/GCE the cathodic measured peaks at different moved towardspH; The redox peak potentials (Epa or Epc) were highly correlated with natural logarithm of scan rates (Figure the reverse(B) the dependence direction. of The anodic redox peak peak potential potentials of ponceau (Epa 4Ror and E tartrazinepc) were on highly pH and correlated (C) the dependence with natural 6C,D). According to Laviron theory [53], the slope values of Epa-lnv and Epc-lnv plots are equivalent to logarithmof anodic of scanpeak current rates (Figure of ponceau6C,D). 4R and According tartrazine to on Laviron pH. Deposition theory parameters: [ 53], the slope −0.1 V, values 120 s. of E -lnv RT/2αnF. Generally, α is assumed as 0.5 for a quasi-reversible or irreversible electrode reaction.pa and Epc-lnv plots are equivalent to RT/2αnF. Generally, α is assumed as 0.5 for a quasi-reversible or 3.4.3.irreversible Scan Rate electrode reaction. Therefore, the electron transfer number (n) was estimated to 0.95 ( 1) ≈ Cyclic voltammograms of ponceau 4R and tartrazine at various scan rates are illustrated in Figure S1. The symmetric redox peaks of ponceau 4R suggest that ponceau 4R underwent a quasi-reversible process. In contrast, no cathodic peak of tartrazine was found at the reverse scan, indicating tartrazine underwent a totally irreversible process. Notably, both Ipa (PR) and Ipa (TZ) increased steadily as the scan rates increased from 0.03 to 0.3 Vs−1. In addition, the redox peak currents of ponceau 4R and tartrazine (Ipa or Ipc) increased linearly with the square root of scan rates (Figure 6A,B), suggesting the reactions of ponceau 4R and tartrazine are typical diffusion-controlled processes. As the potential scan rate grew up, the anodic peaks shifted positively while the cathodic peaks moved towards the reverse direction. The redox peak potentials (Epa or Epc) were highly correlated with natural logarithm of scan rates (Figure 6C,D). According to Laviron theory [53], the slope values of Epa-lnv and Epc-lnv plots are equivalent to RT/2αnF. Generally, α is assumed as 0.5 for a quasi-reversible or irreversible electrode reaction.

Chemosensors 2020, 8, x FOR PEER REVIEW 8 of 13

Therefore, the electron transfer number (n) was estimated to 0.95 (≈ 1) and 1.19 (≈ 1) for ponceau 4R and ChemosensorsChemosensors2020 2020, 8, 8 70, x FOR PEER REVIEW 8 of8 13 of 14 tartrazine, respectively, implying one electron participation in the electrooxidation of ponceau 4R and tartrazine.Therefore, Therefore, the electron it transfercan be numberinferred (n that) was th estimatede electrooxidation to 0.95 (≈ 1) andmechanisms 1.19 (≈ 1) forof ponceauponceau 4R 4R and and tartrazineand 1.19( involved1) for ponceau one electron 4R and tartrazine,(1 e−) transfer respectively, coupled implying with one one proton electron (1 participationH+). The possible in the tartrazine,≈ respectively, implying one electron participation in the electrooxidation of ponceau 4R and electrooxidationelectrooxidationtartrazine. Therefore, mechanisms of ponceau it can 4R arebe and illustinferred tartrazine.rated that in Figureth Therefore,e electrooxidation 7. On itthe can TiO be 2mechanisms/ErGO/GCE, inferred that of the the ponceau hydroxyl electrooxidation 4R groups and − + inmechanisms ponceautartrazine 4R involved of and ponceau tartrazine one 4R andelectron were tartrazine oxidized (1 e−) involvedtransfer to carbonyl onecoupled electrongroups with with (1 eone− )releasingtransfer proton coupled1(1 e Hand+). with1The H , onepossiblewhich proton are + in(1 goodelectrooxidation H ). Theagreement possible mechanisms with electrooxidation the previous are illust reports mechanismsrated in[19,20,22,54]. Figure are 7. illustrated On the TiO in2/ErGO/GCE, Figure7. On the the hydroxyl TiO 2/ErGO groups/GCE, thein hydroxyl ponceau 4R groups and tartrazine in ponceau were 4R oxidized and tartrazine to carbonyl were groups oxidized with toreleasing carbonyl 1 e− groups and 1 H with+, which releasing are + 1 ein− andgood 1 agreement H , which with are the in good previous agreement reports with[19,20,22,54]. the previous reports [19,20,22,54].

Figure 6. Linear plots of cyclic voltammograms peak currents of ponceau 4R (A) and tartrazine (B) Figure 6. Linear plots of cyclic voltammograms peak currents of ponceau 4R (A) and tartrazine (B) againstFigure the 6. squareLinear plotsroot ofof thecyclic scan voltammograms rate (v1/2) and peaklinear currents plots of of redox ponceau peak 4R potentials (A) and tartrazine of ponceau (B )4R against the square root of the scan rate (v1/21/2) and linear plots of redox peak potentials of ponceau 4R (C) (C)against and tartrazine the square (D root) against of the the scan natural rate (v logarithm) and linear of the plots scan of redoxrate (ln peak v). potentials of ponceau 4R and(C tartrazine) and tartrazine (D) against (D) against the natural the natural logarithm logarithm of the of the scan scan rate rate (ln (lnv). v).

2 FigureFigureFigure 7. 7. 7.ElectrooxidationElectrooxidation Electrooxidation mechanism mechanismmechanism ofof ponceauponceau 4R 4R andand and tartrazinetartrazine tartrazine at at at TiO TiO TiO/ErGO/GCE.22/ErGO/GCE./ErGO/GCE.

Chemosensors 2020, 8, 70 9 of 14 Chemosensors 2020, 8, x FOR PEER REVIEW 9 of 13

3.5.3.5. Determination Determination ofof Ponceau Ponceau 4R 4R and and Tartrazine Tartrazine FigureFigure 88AA showsshows thethe DPVDPV curvescurves ofof ponceauponceau 4R4R andand tartrazinetartrazine withwith theirtheir concentrationsconcentrations changingchanging from from 0.01 0.01 to to 5.0 5.0 μM.µM. Apparently, Apparently, the the response response anodic anodic peak peak currents currents of ponceau of ponceau 4R and 4R tartrazineand tartrazine increased increased gradually gradually as their ascorresponding their corresponding concentrations. concentrations. In the concentration In the concentration range from 0.01 to 5.0 μM, both Ipa(PR) and Ipa(TZ) increased linearly with the respective natural logarithm of range from 0.01 to 5.0 µM, both Ipa(PR) and Ipa(TZ) increased linearly with the respective natural concentrations (Figure 8B,C). The linear functions of Ipa−lnC can be expressed as Ipa(μA) = 0.150 lnC logarithm of concentrations (Figure8B,C). The linear functions of I pa lnC can be expressed as (μM) + 1.22 (R2 = 0.991) and Ipa(μA) =2 0.0774 lnC (μM) + 0.386 (R2 = 0.991) for −ponceau 4R and2 tartrazine, Ipa(µA) = 0.150 lnC (µM) + 1.22 (R = 0.991) and Ipa(µA) = 0.0774 lnC (µM) + 0.386 (R = 0.991) for respectively. It is noted that both Ipa(PR) and Ipa (TZ) also increased linearly with their respective ponceau 4R and tartrazine, respectively. It is noted that both Ipa(PR) and Ipa (TZ) also increased linearly concentrationswith their respective at the concentrationslow concentration at the domain low concentration (0.01~0.3 μM). domain The (0.01~0.3correspondingµM). The linear corresponding regression equations of ponceau 4R and tartrazine were Ipa(μA) = 0.290C (μM) + 0.608 (R2 = 0.989) and I2pa(μA) = linear regression equations of ponceau 4R and tartrazine were Ipa(µA) = 0.290C (µM) + 0.608 (R = 0.989) 0.165C (μM) + 0.0505 (R2 = 0.986), respectively.2 At higher concentration, the linear relationship and Ipa(µA) = 0.165C (µM) + 0.0505 (R = 0.986), respectively. At higher concentration, the linear changed to a semi-log relationship, probably because more ponceau 4R and tartrazine molecules relationship changed to a semi-log relationship, probably because more ponceau 4R and tartrazine were adsorbed on the electrode surface at high concentrations, which reduced the reversibility of the molecules were adsorbed on the electrode surface at high concentrations, which reduced the reversibility redox of ponceau 4R and tartrazine, thereby resulting in a sluggish electrode kinetics. The LODs of the redox of ponceau 4R and tartrazine, thereby resulting in a sluggish electrode kinetics. The LODs (LOD = 3σ/s) for the determination of ponceau 4R and tartrazine were estimated to 4.0 and 6.0 nM, (LOD = 3σ/s) for the determination of ponceau 4R and tartrazine were estimated to 4.0 and 6.0 nM, respectively. As summarized in Table 1, the sensing properties (including dynamics response range respectively. As summarized in Table1, the sensing properties (including dynamics response range and and LOD) were almost comparable or even exceeded previously reported electrodes. Unlike costly LOD) were almost comparable or even exceeded previously reported electrodes. Unlike costly ionic ionic liquids [25,26], the electrode preparation only involves readily available and low cost raw liquids [25,26], the electrode preparation only involves readily available and low cost raw materials. materials. Compared with aluminum microfiber/CPE and porous carbon-2/GCE [54,55], the Compared with aluminum microfiber/CPE and porous carbon-2/GCE [54,55], the fabrication of the fabrication of the proposed TiO2/ErGO/GCE was simpler and more convenient. In short, the proposed TiO2/ErGO/GCE was simpler and more convenient. In short, the comparatively excellent comparatively excellent sensing performances, together with low cost and ease of fabrication, make sensing performances, together with low cost and ease of fabrication, make TiO2/ErGO/GCE an attractive TiO2/ErGO/GCE an attractive candidate for on-site detection of ponceau 4R and tartrazine. candidate for on-site detection of ponceau 4R and tartrazine.

FigureFigure 8. 8. (A(A) )DPV DPV curves curves of of ponceau ponceau 4R 4R and and tartrazine tartrazine wi withth their their concentrations concentrations varying varying from from 0.01 0.01 to to 5.0 µM, a k: 0.01, 0.003, 0.05, 0.08, 0.10, 0.30, 0.50, 0.80, 1.0, 3.0 and 5.0 µM; (B) plots of the anodic peak 5.0 μM, a→k: 0.01, 0.003, 0.05, 0.08, 0.10, 0.30, 0.50, 0.80, 1.0, 3.0 and 5.0 μM; (B) plots of the anodic peakcurrents currents of ponceau of ponceau 4R as 4R the as functionthe function of the of the natural natural logarithm logarithm of itsof its concentrations concentrations in in the the range range of µ of0.01–5.0 0.01–5.0 M(μMn (n= =3) 3) and and ( C(C)) plots plots of of the the anodic anodic peakpeak currentscurrents ofof tartrazinetartrazine as the function of of natural natural µ logarithmlogarithm of of its its concentrations concentrations in in the the range range of of 0.01 0.01–5.0–5.0 μMM( (n = 3).3). Table 1. Comparison on analytical performance of the simultaneous detection of ponceau 4R and Tabletartrazine 1. Comparison with previously on analytical reported electrodes.performance of the simultaneous detection of ponceau 4R and tartrazine with previously reported electrodes. Detection Range (µM) LOD (µM) Electrode Method Detection Range (μM) LOD (μM) Ref Electrode Method Ref PonceauPonceau 4R 4R Tartrazine Tartrazine PonceauPonceau 4R 4R Tartrazine Tartrazine AcetyleneAcetylene black/GCE black/GCE AASV AASV 0.083–6.60.083–6.6 0.28–340.28–34 0.0500.050 0.1870.187 [24[24]] IL-EGPEIL-EGPE SWSV SWSV 0.01–5.00.01–5.0 0.01–2.00.01–2.0 0.00140.0014 0.003 0.003 [25[25]] IL-GO/MWCNT/GCEIL-GO/MWCNT/GCE SWV SWV 0.008–0.0150.008–0.015 0.02–0.0130.02–0.013 0.0060.006 0.01 0.01 [26[26]] Al Almicrofiber/CPE microfiber/CPE DPV DPV 0.001–0.100.001–0.10 0.005–0.140.005–0.14 0.00080.0008 0.002 0.002 [54[54]] PorousPorous carbon-2/GCE carbon-2/GCE DPV DPV 0.004–1.650.004–1.65 0.009–0.560.009–0.56 0.00350.0035 0.0065 0.0065 [55[55]] TiO /ErGO/GCE AdSDPV 0.01–5.0 0.01– 5.0 0.004 0.006 This work TiO2/ErGO/GCE2 AdSDPV 0.01–5.0 0.01– 5.0 0.004 0.006 This work

3.6. Interference, Reproducibility and Stability Test The interferences from common metal ions and other azo colorants were investigated at the TiO2/ErGO/GCE for the simultaneous determination of ponceau 4R and tartrazine. The anodic peak

Chemosensors 2020, 8, 70 10 of 14

3.6. Interference, Reproducibility and Stability Test The interferences from common metal ions and other azo colorants were investigated at the TiO2/ErGO/GCE for the simultaneous determination of ponceau 4R and tartrazine. The anodic peak currents of 10 µM ponceau 4R and tartrazine coexisting with various interfering substances are tabulated in Table S1. The DPV responses of ponceau 4R and tartrazine were not influenced by the additions of 100-fold concentrations of Na+,K+, Mg2+ and Ca2+ or 10-fold concentrations of sunset yellow, Allura Red and amaranth. This indicates that it is feasible to analyze ponceau 4R and tartrazine at TiO2/ErGO/GCE with high selectivity. To examine the electrode reproducibility, the DPV responses of 10 µM ponceau 4R and tartrazine were parallelly recorded on five TiO2/ErGO/GCEs (Figure S2). The relative standard deviations (RSD) for ponceau 4R and tartrazine were 5.04% and 4.01%, respectively, indicating that the electrode preparation was highly reproducible. To evaluate electrode stability, the DPV responses of ponceau 4R and tartrazine were regularly monitored during one week. When it was not in use, the TiO2/ErGO/GCE was stored in the air. After each measurement, the adsorbed target analytes on the TiO2/ErGO/GCE were removed by ten successive scans of cyclic voltammograms in 0.1 M PBS (pH=7.0). The anodic peak currents of 1.0 µM ponceau 4R and tartrazine keep 89.5% and 88.6% of their respective initial values after one-week storage (Figure S3), which demonstrated extraordinary storage stability.

3.7. Applications for Real Samples The concentrations of ponceau 4R and tartrazine in orange juice samples were determined by the DPV technique using the TiO2/ErGO/GCE. Orange juices were first diluted to 100-fold with 0.1 M PBS (pH = 7.0), then the DPV responses of ponceau 4R and tartrazine were recorded under the optimal experimental conditions. The concentrations of ponceau 4R and tartrazine were figured out from the related calibration curves. As tabulated in Table2, the concentrations of ponceau 4R and tartrazine in orange juice were 0.112 (67.7 µg/L) and 0.423 µM (226 µg/L), respectively, which are much lower than their maximum allowable dosages (100 mg/L) specified in GB 2760–2014. To further confirm the detection reliability, known concentration solutions of ponceau 4R and tartrazine were separately spiked to the sample solutions. The satisfactory recoveries were obtained in the recovery assays with the accepted RSDs. These results suggest that the TiO2/ErGO/GCE could obtain reliable detection results without visible interference from the complicated matrix. The promising analytical properties, along with low cost, simple fabrication, and facile operation, were expected to broaden the application prospects of TiO2/ErGO/GCE.

Table 2. Detection of ponceau 4R and tartrazine in samples by the TiO2/ErGO/GCE.

Detected RSD Added Found RSD Recovery Samples Analytes (µM) (%) (µM) (µM) (%) (%) Ponceau 4R 0.112 4.04 0.090 0.197 3.02 94.4 A Tartrazine 0.423 2.21 0.338 0.654 3.04 97.9 Ponceau 4R 0.112 3.21 0.112 0.223 4.06 99.1 B Tartrazine 0.423 5.01 0.583 1.056 4.86 108.6

4. Conclusions A simple but efficient voltammetric sensor was constructed for the simultaneous detection of ponceau 4R and tartrazine based on TiO2/ErGO composites. TiO2 NPs were uniformly anchored on the ErGO nanoflakes, which provide a favorable electrode interface for the electrochemical oxidation of target species. Electrochemical experiments show TiO2/ErGO composites decreased charge transfer resistance and enlarged the effective electrochemical active area significantly. The TiO2/ErGO/GCE generated a synergistic enhancement effect for the electrooxidation of ponceau 4R and tartrazine, which gave a rise in greatly amplified voltammetric responses. Compared with bare GCE, the anodic Chemosensors 2020, 8, 70 11 of 14 peak currents increased by 5-fold and 9-fold for ponceau 4R and tartrazine, respectively. Under the optimal analytical conditions, the TiO2/ErGO/GCE achieved sensitive determination of ponceau 4R and tartrazine at the nanomolar level, with the low LODs of 4.0 and 6.0 nM for ponceau 4R and tartrazine over a wide dynamical response range (0.01 0.50 µM). Finally, the proposed TiO /ErGO/GCE enabled − 2 simultaneous determination of ponceau 4R and tartrazine in orange juices with a satisfactory outcome. Combined with portable electrochemical devices, the proposed TiO2/ErGO/GCE is expected to achieve in-situ detection of azo colorants in various drinks and foodstuffs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9040/8/3/70/s1, Figure S1: Cyclic voltammograms of ponceau 4R (A) and tartrazine (B) at various scan rates, Figure S2: Anodic AdSDPV peak currents of ponceau 4R and tartrazine parallelly recorded on five TiO2/ErGO/GCEs, Figure S3: AdSDPVs responses of ponceau 4R and tartrazine in dependence on storage time, Table S1: Anodic peak currents of ponceau 4R and tartrazine in the presence of various interfering substances. Author Contributions: Conceptualization, Z.Q. and J.Z.; methodology, Z.Q. and J.Z.; formal analysis, Z.Q., J.Z. and J.L.; investigation, Z.Q., J.Z., Y.L. and J.W.; writing—original draft preparation, Z.Q. and J.Z.; writing—review and editing, G.L.; visualization, Z.Q., J.Z., Y.L. and J.W.; supervision, G.L. and Q.H.; funding acquisition, G.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was financially supported by Scientific Research Foundation of Hunan Provincial Education Department (18C0522), Undergraduates’ Innovation Experiment Program of Hunan Province (2018649), Natural Science Foundation of Hunan Province (2018JJ3134), and National Natural Science Foundation of China (61703152). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Yoshioka, N.; Ichihashi, K. Determination of 40 synthetic food colors in drinks and by high performance liquid chromatography using a short column with photodiode array detection. Talanta 2008, 74, 1408–1413. [CrossRef] 2. Tanaka, T. Reproductive and neurobehavioural toxicity study of Ponceau 4R administered to mice in the diet. Food Chem. Toxicol. 2006, 44, 1651–1658. [CrossRef] 3. Tanaka, T. Reproductive and neurobehavioural toxicity study of tartrazine administered to mice in the diet. Food Chem. Toxicol. 2006, 44, 179–187. [CrossRef] 4. Sun, H.; Sun, N.; Li, H.; Zhang, J.; Yang, Y. Development of multiresidue analysis for 21 synthetic colorants in meat by microwave–assisted extraction–solid–phase extraction–reversed–phase ultrahigh performance liquid chromatography. Food Anal. Methods 2013, 6, 1291–1299. [CrossRef] 5. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS). Scientific Opinion on the re–evaluation Tartrazine (E 102). EFSA J. 2009, 7, 1331. [CrossRef] 6. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS). Scientific Opinion on the re–evaluation of Ponceau 4R (E 124) as a food additive. EFSA J. 2009, 7, 1328. [CrossRef] 7. Huang, H.-Y.; Shih, Y.-C.; Chen, Y.-C. Determining eight colorants in milk beverages by capillary electrophoresis. J. Chromatogr. 2002, 959, 317–325. [CrossRef] 8. Chou, S.S.; Lin, Y.H.; Cheng, C.C.; Hwang, D.F. Determination of synthetic colors in soft drinks and by micellar electrokinetic capillary chromatography. J. Food Sci. 2002, 67, 1314–1318. [CrossRef] 9. Altιnöz, S.; Toptan, S. Determination of Tartrazine and Ponceau–4R in various food samples by Vierordt‘s method and ratio spectra first–order derivative UV spectrophotometry. J. Food Compost. Anal. 2002, 15, 667–683. [CrossRef] 10. Capitán-Vallvey, L.; Fernández, M.; De Orbe, I.; Avidad, R. Simultaneous determination of the colorants tartrazine, ponceau 4R and sunset yellow FCF in foodstuffs by solid phase spectrophotometry using partial least squares multivariate calibration. Talanta 1998, 47, 861–868. [CrossRef] 11. García-Falcón, M.S.; Simal-Gándara, J. Determination of food dyes in soft drinks containing natural pigments by liquid chromatography with minimal clean–up. Food Control 2005, 16, 293–297. [CrossRef] 12. Bonan, S.; Fedrizzi, G.; Menotta, S.; Elisabetta, C. Simultaneous determination of synthetic dyes in foodstuffs and beverages by high–performance liquid chromatography coupled with diode–array detector. Dyes Pigments 2013, 99, 36–40. [CrossRef] Chemosensors 2020, 8, 70 12 of 14

13. Liao, Q.G.; Li, W.H.; Luo, L.G. Applicability of accelerated solvent extraction for synthetic colorants analysis in meat products with ultrahigh performance liquid chromatography–photodiode array detection. Anal. Chim. Acta 2012, 716, 128–132. [CrossRef][PubMed] 14. López-de-Alba, P.L.; López-Martínez, L.; De-León-Rodríguez, L.M. Simultaneous determination of synthetic dyes tartrazine, allura red and sunset yellow by differential pulse polarography and partial least squares. A multivariate calibration method. Electroanalysis 2002, 14, 197–205. [CrossRef] 15. Chanlon, S.; Joly-Pottuz, L.; Chatelut, M.; Vittori, O.; Cretier, J.L. Determination of carmoisine, allura red and ponceau 4R in sweets and soft drinks by differential pulse polarography. J. Food Compost. Anal. 2005, 18, 503–515. [CrossRef] 16. Combeau, S.; Chatelut, M.; Vittori, O. Identification and simultaneous determination of Azorubin, Allura red and Ponceau 4R by differential pulse polarography: Application to soft drinks. Talanta 2002, 56, 115–122. [CrossRef] 17. Huang, J.; Zeng, Q.; Wang, L. Ultrasensitive electrochemical determination of Ponceau 4R with a novel

ε–MnO2 microspheres/chitosan modified glassy carbon electrode. Electrochim. Acta 2016, 206, 176–183. [CrossRef] 18. Wang, Z.; Zhang, H.; Wang, Z.; Zhang, J.; Duan, X.; Xu, J.; Wen, Y. Trace analysis of Ponceau 4R in soft drinks using differential pulse stripping voltammetry at SWCNTs composite electrodes based on PEDOT:PSS derivatives. Food Chem. 2015, 180, 186–193. [CrossRef] 19. Zhang, Y.; Zhang, X.; Lu, X.; Yang, J.; Wu, K. Multi–wall carbon nanotube film–based electrochemical sensor for rapid detection of Ponceau 4R and Allura Red. Food Chem. 2010, 122, 909–913. [CrossRef] 20. Gan, T.; Sun, J.; Meng, W.; Song, L.; Zhang, Y. Electrochemical sensor based on graphene and mesoporous

TiO2 for the simultaneous determination of trace colourants in food. Food Chem. 2013, 141, 3731–3737. [CrossRef] 21. Ghoreishi, S.M.; Behpour, M.; Golestaneh, M. Simultaneous determination of Sunset yellow and Tartrazine in soft drinks using gold nanoparticles carbon paste electrode. Food Chem. 2012, 132, 637–641. [CrossRef] [PubMed] 22. Ye, X.; Du, Y.; Lu, D.; Wang, C. Fabrication of β–cyclodextrin–coated poly (diallyldimethylammonium chloride)–functionalized graphene composite film modified glassy carbon–rotating disk electrode and its application for simultaneous electrochemical determination colorants of sunset yellow and tartrazine. Anal. Chim. Acta 2013, 779, 22–34. [PubMed] 23. Zhang, W.; Liu, T.; Zheng, X.; Huang, W.; Wan, C. Surface–enhanced oxidation and detection of Sunset Yellow and Tartrazine using multi–walled carbon nanotubes film–modified electrode. Colloids Surf. B 2009, 74, 28–31. [CrossRef] 24. Yang, X.; Qin, H.; Gao, M.; Zhang, H. Simultaneous detection of Ponceat 4R and tartrazine in food using adsorptive stripping voltammetry on an acetylene black nanoparticle–modified electrode. J. Sci. Food Agric. 2011, 91, 2821–2825. [CrossRef][PubMed] 25. Zhang, J.; Wang, X.; Zhang, S.; Wang, W.; Hojo, M.; Chen, Z. An electrochemical sensor for simultaneous determination of ponceau 4R and tartrazine based on an ionic liquid modified expanded graphite paste electrode. J. Electrochem. Soc. 2014, 161, H453–H457. [CrossRef] 26. Wang, M.; Zhao, J. A facile method used for simultaneous determination of ponceau 4R, allura red and tartrazine in alcoholic beverages. J. Electrochem. Soc. 2015, 162, H321–H327. [CrossRef] 27. He, Q.; Liu, J.; Liu, X.; Li, G.; Deng, P.; Liang, J.; Chen, D. Sensitive and selective detection of tartrazine based

on TiO2–electrochemically reduced graphene oxide composite–modified electrodes. Sensors 2018, 18, 1911. [CrossRef] 28. Zhou, W.-Y.; Liu, J.-Y.; Song, J.-Y.; Li, J.-J.; Liu, J.-H.; Huang, X.-J. Surface–electronic–state–modulated,

single–crystalline (001) TiO2 nanosheets for sensitive electrochemical sensing of heavy–metal ions. Anal. Chem. 2017, 89, 3386–3394. [CrossRef]

29. Shehata, M.; Azab, S.; Fekry, A.; Ameer, M.A. Nano–TiO2 modified carbon paste sensor for electrochemical nicotine detection using anionic surfactant. Biosens. Bioelectron. 2016, 79, 589–592. [CrossRef]

30. Bao, S.J.; Li, C.M.; Zang, J.F.; Cui, X.Q.; Qiao, Y.; Guo, J. New nanostructured TiO2 for direct electrochemistry and glucose sensor applications. Adv. Funct. Mater. 2008, 18, 591–599. [CrossRef]

31. Fan, Y.; Huang, K.-J.; Niu, D.-J.; Yang, C.-P.; Jing, Q.-S. TiO2–graphene nanocomposite for electrochemical sensing of adenine and guanine. Electrochim. Acta 2011, 56, 4685–4690. [CrossRef] Chemosensors 2020, 8, 70 13 of 14

32. Benvenuto, P.; Kafi, A.; Chen, A. High performance glucose biosensor based on the immobilization of glucose oxidase onto modified titania nanotube arrays. J. Electroanal. Chem. 2009, 627, 76–81. [CrossRef]

33. Daniel, D.; Gutz, I.G. Microfluidic cell with a TiO2–modified gold electrode irradiated by an UV–LED for in situ photocatalytic decomposition of organic matter and its potentiality for voltammetric analysis of metal ions. Electrochem. Commun. 2007, 9, 522–528. [CrossRef]

34. Wan, X.; Yang, S.; Cai, Z.; He, Q.; Ye, Y.; Xia, Y.; Li, G.; Liu, J. Facile synthesis of MnO2 nanoflowers/N–doped reduced graphene oxide composite and its application for simultaneous determination of dopamine and uric acid. Nanomaterials 2019, 9, 847. [CrossRef] 35. Cai, Z.; Ye, Y.; Wan, X.; Liu, J.; Yang, S.; Xia, Y.; Li, G.; He, Q. Morphology–dependent electrochemical sensing properties of iron oxide–graphene oxide nanohybrids for dopamine and uric acid. Nanomaterials 2019, 9, 835. [CrossRef] 36. He, Q.; Liu, J.; Liu, X.; Li, G.; Chen, D.; Deng, P.; Liang, J. A promising sensing platform toward dopamine

using MnO2 nanowires/electro–reduced graphene oxide composites. Electrochim. Acta 2019, 296, 683–692. [CrossRef] 37. Li, G.; Xia, Y.; Tian, Y.; Wu, Y.; Liu, J.; He, Q.; Chen, D. Recent developments on graphene–based electrochemical sensors toward nitrite. J. Electrochem. Soc. 2019, 166, B881–B895. [CrossRef]

38. Li, Q.; Xia, Y.; Wan, X.; Yang, S.; Cai, Z.; Ye, Y.; Li, G. Morphology–dependent MnO2/nitrogen–doped graphene nanocomposites for simultaneous detection of trace dopamine and uric acid. Mater. Sci. Eng. C 2020, 109, 110615. [CrossRef] 39. Li, G.; Zhong, P.; Ye, Y.; Wan, X.; Cai, Z.; Yang, S.; Xia, Y.; Li, Q.; Liu, J.; He, Q. A highly sensitive and stable

dopamine sensor using shuttle–like α–Fe2O3 nanoparticles/electro–reduced graphene oxide composites. J. Electrochem. Soc. 2019, 166, B1552–B1561. [CrossRef] 40. Kamat, P.V. Graphene–based nanoarchitectures. Anchoring semiconductor and metal nanoparticles on a two–dimensional carbon support. J.Phys.Chem. Lett. 2009, 1, 520–527. [CrossRef] 41. Fan, Y.; Lu, H.-T.; Liu, J.-H.; Yang, C.-P.; Jing, Q.-S.; Zhang, Y.-X.; Yang, X.-K.; Huang, K.-J. Hydrothermal

preparation and electrochemical sensing properties of TiO2–graphene nanocomposite. Colloids Surf. B 2011, 83, 78–82. [CrossRef][PubMed] 42. Jang, H.D.; Kim, S.K.; Chang, H.; Roh, K.-M.; Choi, J.-W.; Huang, J. A glucose biosensor based on

TiO2–graphene composite. Biosens. Bioelectron. 2012, 38, 184–188. [CrossRef][PubMed] 43. Fan, Y.; Liu, J.-H.; Lu, H.-T.; Zhang, Q. Electrochemistry and voltammetric determination of l–tryptophan

and l–tyrosine using a glassy carbon electrode modified with a Nafion/TiO2–graphene composite film. Microchim. Acta 2011, 173, 241–247. [CrossRef] 44. Park, S.; Ruoff, R.S. Chemical methods for the production of graphenes. Nat. Nanotechnol. 2009, 4, 217. 45. Gan, T.; Shi, Z.; Deng, Y.; Sun, J.; Wang, H. Morphology–dependent electrochemical sensing properties of manganese dioxide–graphene oxide hybrid for guaiacol and vanillin. Electrochim. Acta 2014, 147, 157–166. [CrossRef] 46. Tang, Y.-B.; Lee, C.-S.; Xu, J.; Liu, Z.-T.; Chen, Z.-H.; He, Z.; Cao, Y.-L.; Yuan, G.; Song, H.; Chen, L.

Incorporation of graphenes in nanostructured TiO2 films via molecular grafting for dye–sensitized solar cell application. ACS Nano 2010, 4, 3482–3488. [CrossRef] 47. Lambert, T.N.; Chavez, C.A.; Hernandez-Sanchez, B.; Lu, P.; Bell, N.S.; Ambrosini, A.; Friedman, T.; Boyle, T.J.; Wheeler, D.R.; Huber, D.L. Synthesis and characterization of titania graphene nanocomposites. J. Phys. − Chem. C 2009, 113, 19812–19823. [CrossRef] 48. Luo, X.; Morrin, A.; Killard, A.J.; Smyth, M.R. Application of nanoparticles in electrochemical sensors and biosensors. Electroanal 2006, 18, 319–326. [CrossRef] 49. He, Q.; Liu, J.; Liu, X.; Li, G.; Deng, P.; Liang, J. Manganese dioxide nanorods/electrochemically reduced graphene oxide nanocomposites modified electrodes for cost–effective and ultrasensitive detection of Amaranth. Colloids Surf. B 2018, 172, 565–572. [CrossRef] 50. Li, G.; Wang, S.; Duan, Y.Y. Towards conductive–gel–free electrodes: Understanding the wet electrode, semi–dry electrode and dry electrode–skin interface impedance using electrochemical impedance spectroscopy fitting. Sens. Actuators B Chem. 2018, 277, 250–260. [CrossRef] 51. Li, G.; Wu, J.; Xia, Y.; Wu, Y.; Tian, Y.; Liu, J.; Chen, D.; He, Q. Towards emerging EEG applications: A novel printable flexible Ag/AgCl dry electrode array for robust recording of EEG signals at forehead sites. J. Neural Eng. 2020, 17, 026001. [CrossRef][PubMed] Chemosensors 2020, 8, 70 14 of 14

52. Fan, Y.; Liu, J.-H.; Lu, H.-T.; Zhang, Q. Electrochemical behavior and voltammetric determination of

paracetamol on Nafion/TiO2–graphene modified glassy carbon electrode. Colloids Surf. B 2011, 85, 289–292. [CrossRef][PubMed] 53. Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. 1979, 101, 19–28. [CrossRef] 54. Zhang, Y.; Hu, L.; Liu, X.; Liu, B.; Wu, K. Highly–sensitive and rapid detection of ponceau 4R and tartrazine in drinks using alumina microfibers–based electrochemical sensor. Food Chem. 2015, 166, 352–357. [CrossRef] [PubMed] 55. Cheng, Q.; Xia, S.; Tong, J.; Wu, K. Highly–sensitive electrochemical sensing platforms for food colourants based on the property–tuning of porous carbon. Anal. Chim. Acta 2015, 887, 75–81. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).