Titania/Electro-Reduced Graphene Oxide Nanohybrid as an Efficient Electrochemical Sensor for the Determination of Allura Red

Titania/electro-reduced graphene oxide nanohybrids (TiO2/ErGO) were synthesized by the hydrolysis of titanium sulfate in graphene oxide suspension and in situ electrochemical reduction. It provides a facile and efficient method to obtain nanohybrids with TiO2 nanoparticles (TiO2 NPs) uniformly coated by graphene nanoflakes. TiO2/ErGO nanohybrids were characterized by transmission electron microscopy, X-ray diffraction, cyclic voltammogram, and electrochemical impedance spectroscopy in detail. Compared with pure ErGO and TiO2 NPs, TiO2/ErGO nanohybrids greatly enhanced the electrocatalytic activity and voltammetric response of Allura Red. In the concentration range of 0.5–5.0 μM, the anodic peak currents of Allura Red were linearly correlated to their concentrations. However, the linear relationship was changed to the semi-logarithmic relationship at a higher concentration region (5.0–800 μM). The detection limit (LOD) was 0.05 μM at a signal-to-noise ratio of 3. The superior sensing performances of the proposed sensor can be ascribed to the synergistic effect between TiO2 NPs and ErGO, which provides a favorable microenvironment for the electrochemical oxidation of Allura Red. The proposed TiO2/ErGO/GCE showed good reproducibility and stability both in determination and in storage, and it can accurately detect the concentration of Allura Red in milk drinks, providing an efficient platform for the sensitive determination of Allura Red with high reliability, simplicity, and rapidness.


Introduction
Nowadays, the safety of food additives has become an increasingly important concern worldwide, since most of these substances are poisonous and even carcinogenic. Among diverse food additives, synthetic colorants have been extensively added into various soft beverages and foodstuffs to make them more attractive and appealing. However, some synthetic colorants can lead to adverse health issues, especially if they are excessively consumed [1,2]. Therefore, the use of synthetic colorants in foodstuffs were rigorously controlled by legislation all over the world. Allura Red (E 129) is a typical water-soluble azo dye, which was usually added to candies, syrups, and beverages to make a fascinating red color. Despite its widespread applications, the health threats cannot be underestimated, since its aromatic rings and azo groups may harm the human body. The European Food Safety Authority (EFSA, Parma, Italy) recommended that the acceptable daily intake (ADI) of Allura Red is 7.0 mg/kg paracetamol [52], L-tryptophan [53], L-tyrosine [53], etc. However, to our best knowledge, TiO 2 /Gr nanohybrids have been rarely used for the detection of Allura Red.
Herein, TiO 2 /Gr nanohybrids were synthesized for the sensitive detection of Allura Red in soft drinks. TiO 2 /electro-reduced graphene oxide nanohybrids (TiO 2 /ErGO) were synthesized by the hydrolysis of titanium sulfate in graphene oxide (GO) suspension and in situ electrochemical reduction. In this way, the oxygen-containing functional groups present in graphene oxide can be reduced with the application of the appropriate potential. The electrical conductivity was greatly improved due to the restoration of carbon-conjugated networks after electrochemical reduction. Compared with chemical and thermal reduction methods, the electrochemical reduction is very simple and time-saving. Moreover, no any poisonous and potentially explosive chemicals such as hydrazine and sodium borohydride are not required. The synergistic effect between TiO 2 NPs and ErGO nanosheets greatly improved the electrochemical oxidation of Allura Red, with remarkably enhanced voltammetric response and decreased anodic peak potential. Moreover, the proposed TiO 2 /ErGO exhibited good selectivity, sensitivity, and stability for the detection of Allura Red.

Prepration of TiO 2 /GO Nanocomposites
TiO 2 NPs were synthesized by the hydrolysis of titanium sulfate in GO suspension. Typically, 40 mg of GO was dispersed in a mixed solvent of ethanol (10 mL) and water (20 mL) under 0.5 h of ultrasonication to obtain a homogenous suspension of exfoliated GO. Then, 6.2 mg of titanium sulfate was added to the GO suspension and ultrasonicated for 0.5 h. Afterwards, the resultant solution was transferred to a 50-mL autoclave and reacted at 180 • C for 4 h. After it was cooled to room temperature, the reaction solution was centrifuged at a rate of 11,000 r/min for 10 min. Subsequently, the solids were repeatedly washed with absolute ethanol and deionized water for 2-3 rounds; then, they were vacuum-dried at 60 • C overnight. The TiO 2 /GO nanohybrids were obtained as black powder. The as-obtained TiO 2 /GO nanohybrids (10 mg) were ultrasonically dispersed in 10 mL of deionized water for 2 h to get a homogenous dispersion (1 mg/mL).

Preparation of TiO 2 /ErGO Modified Electrodes
Prior to electrode modification, bare GCEs were carefully polished to mirror-like surface at a cloth pad using 0.05 µm Al 2 O 3 slurry; then, they were alternately cleaned by anhydrous alcohol and deionized water. The freshly polished GCEs were dried under an infrared lamp. TiO 2 /ErGO/GCE were prepared by a simple drop-casting and in situ electrochemical reduction technique. Typically, 5 µL of TiO 2 /GO dispersion was dropped and casted on the surface of GCEs; then, it was allowed to dry under an infrared lamp. Finally, the TiO 2 /GO/GCEs were immersed into 0.1 M PBS solution (pH = 6.0) and electrochemically reduced at −1.2 V for 2 min. For comparison, individual TiO 2 NPs and ErGO-modified GCEs were also manufactured by the same procedure.

Electrochemical Detection of Allura Red
A classical three-electrode assembly was used in all the electrochemical measurements, consisting of a bare or modified GCE, a saturated calomel electrode (SCE), and a platinum wire as the working electrode, reference electrode, and auxiliary electrode, respectively. First, 0.1 M of phosphate buffer solution (PBS, pH 7.0) was used as the supporting electrolyte unless otherwise stated. To assess the electrochemical properties of the proposed sensor, cyclic voltammogram and electrochemical impedance spectroscopy was recorded in 0.1 mM [Fe(CN) 6 ] 3−/4− probe solution. Electrochemical impedance spectroscopy was scanned from 100,000 to 0.1 Hz at open circuit potential using a 5 mV (rms) AC sinusoid signal. After a suitable accumulation, the differential pulse voltammetry (DPV) was measured from 0.3 to 0.9 V. The pulse amplitude is 0.05 V, the pulse width is 0.05 s, the sample width is 0.0167 s, and the pulse period is 0.2 s.

Materials Chararazation
The microstructures of TiO 2 NPs and TiO 2 /ErGO nanohybrids were investigated by transmission electron microscopy (TEM). Cube-like structures were observed in Figure 1A, and the particle sizes of TiO 2 NPs are estimated to 30-50 nm. After introducing ErGO sheets onto the surface of TiO 2 NPs, TiO 2 NPs were wrapped and coated by ErGO nanoflakes (Figure 1B), confirming that TiO 2 NPs were successfully hybridized with ErGO nanoflakes. Electrochemical impedance spectroscopy was scanned from 100,000 to 0.1 Hz at open circuit potential using a 5 mV (rms) AC sinusoid signal. After a suitable accumulation, the differential pulse voltammetry (DPV) was measured from 0.3 to 0.9 V. The pulse amplitude is 0.05 V, the pulse width is 0.05 s, the sample width is 0.0167 s, and the pulse period is 0.2 s.

Materials Chararazation
The microstructures of TiO2 NPs and TiO2/ErGO nanohybrids were investigated by transmission electron microscopy (TEM). Cube-like structures were observed in Figure 1A, and the particle sizes of TiO2 NPs are estimated to 30-50 nm. After introducing ErGO sheets onto the surface of TiO2 NPs, TiO2 NPs were wrapped and coated by ErGO nanoflakes (Figure 1B), confirming that TiO2 NPs were successfully hybridized with ErGO nanoflakes. The crystalline structures of TiO2 NPs, GO nanoflakes, and TiO2/GO nanohybrids were further investigated by X-ray diffraction (XRD). As shown in Figure 2A, pronounced diffraction peaks appear at 25.26°, 36.92°, 47.94°, 53.98°, 54.90°, 62.60°, 68.86°, 70.08°, and 75.06°, respectively, which can be well assigned to (101), (004), (200), (105), (211), (213), (116), (220), and (215) crystal planes of anatase TiO2 (JCPDS card 21-1272), respectively. Moreover, no visible impurity peaks were observed. The results suggest that the as-obtained TiO2 NPs have anatase structure with good crystallinity. The XRD pattern of GO shows a sharp diffraction peak at 2Ɵ = 10.6° ( Figure 2B), which can be indexed to the (001) crystal plane of GO with a d-spacing of 0.80 nm [54]. In the XRD pattern of the TiO2/GO nanocomposite in Figure 2C, the major characteristic peaks of GO can also be identified in addition to TiO2 peaks. The results indicate that the TiO2/GO nanocomposite was successfully prepared. The crystalline structures of TiO 2 NPs, GO nanoflakes, and TiO 2 /GO nanohybrids were further investigated by X-ray diffraction (XRD). As shown in Figure (Figure 2B), which can be indexed to the (001) crystal plane of GO with a d-spacing of 0.80 nm [54]. In the XRD pattern of the TiO 2 /GO nanocomposite in Figure 2C, the major characteristic peaks of GO can also be identified in addition to TiO 2 peaks. The results indicate that the TiO 2 /GO nanocomposite was successfully prepared. The Fourier transform infrared spectroscopy (FTIR) of GO and ErGO are shown in Figure 3. In the FTIR of GO, apparent peaks at 1725, 1618, 1407, 1225, and 1057 cm −1 are assigned to the characteristic peaks of C=O (carbonyl or carboxy), C=C (aromatics), C-O (carboxy), C-O (epoxy), and C-O (alkoxy), respectively. As expected, these oxygen-containing functional groups almost disappear, indicating that graphene oxide was successfully electrochemically reduced into reduced graphene oxide.

Assessement of Electrochemical Properties
To assess the electrochemical properties, cyclic voltammograms for different modified electrodes were recorded in 0.1 M [Fe(CN)6] 3−/4− and KCl mixture solution. As shown in Figure 4A, the ratios of anodic and cathodic peak currents (ipa/ipc) approach to 1 for all electrodes, suggesting that all electrodes experience a quasi-reversible process. The redox peak currents increase in the following order: bare GCE, TiO2/GCE, ErGO/GCE, and TiO2/ErGO/GCE. Among these electrodes, TiO2/ErGO/GCE shows the largest electrochemical response, with ipa and ipc of 148.2 and 144.1 μA, respectively. Moreover, the peak separation between anodic and cathodic peaks (∆Ep = 67 mV) is the smallest. All these results demonstrate the synergistic effect between TiO2 NPs and ErGO. Referring to the Randles-Sevcik equation [18,46], the effective electroactive areas of bare GCE, TiO2/GCE, The Fourier transform infrared spectroscopy (FTIR) of GO and ErGO are shown in Figure 3. In the FTIR of GO, apparent peaks at 1725, 1618, 1407, 1225, and 1057 cm −1 are assigned to the characteristic peaks of C=O (carbonyl or carboxy), C=C (aromatics), C-O (carboxy), C-O (epoxy), and C-O (alkoxy), respectively. As expected, these oxygen-containing functional groups almost disappear, indicating that graphene oxide was successfully electrochemically reduced into reduced graphene oxide. The Fourier transform infrared spectroscopy (FTIR) of GO and ErGO are shown in Figure 3. In the FTIR of GO, apparent peaks at 1725, 1618, 1407, 1225, and 1057 cm −1 are assigned to the characteristic peaks of C=O (carbonyl or carboxy), C=C (aromatics), C-O (carboxy), C-O (epoxy), and C-O (alkoxy), respectively. As expected, these oxygen-containing functional groups almost disappear, indicating that graphene oxide was successfully electrochemically reduced into reduced graphene oxide.

Assessement of Electrochemical Properties
To assess the electrochemical properties, cyclic voltammograms for different modified electrodes were recorded in 0.1 M [Fe(CN)6] 3−/4− and KCl mixture solution. As shown in Figure 4A, the ratios of anodic and cathodic peak currents (ipa/ipc) approach to 1 for all electrodes, suggesting that all electrodes experience a quasi-reversible process. The redox peak currents increase in the following order: bare GCE, TiO2/GCE, ErGO/GCE, and TiO2/ErGO/GCE. Among these electrodes, TiO2/ErGO/GCE shows the largest electrochemical response, with ipa and ipc of 148.2 and 144.1 μA, respectively. Moreover, the peak separation between anodic and cathodic peaks (∆Ep = 67 mV) is the smallest. All these results demonstrate the synergistic effect between TiO2 NPs and ErGO. Referring to the Randles-Sevcik equation [18,46], the effective electroactive areas of bare GCE, TiO2/GCE,

Assessement of Electrochemical Properties
To assess the electrochemical properties, cyclic voltammograms for different modified electrodes were recorded in 0.1 M [Fe(CN) 6 ] 3−/4− and KCl mixture solution. As shown in Figure 4A, the ratios of anodic and cathodic peak currents (i pa /i pc ) approach to 1 for all electrodes, suggesting that all electrodes experience a quasi-reversible process. The redox peak currents increase in the following order: bare GCE, TiO 2 /GCE, ErGO/GCE, and TiO 2 /ErGO/GCE. Among these electrodes, TiO 2 /ErGO/GCE shows the largest electrochemical response, with i pa and i pc of 148.2 and 144.1 µA, respectively. Moreover, the peak separation between anodic and cathodic peaks (∆E p = 67 mV) is the smallest. All these results demonstrate the synergistic effect between TiO 2 NPs and ErGO. Referring to the Randles-Sevcik equation [18,46], the effective electroactive areas of bare GCE, TiO 2 /GCE, ErGO/GCE, and TiO 2 /ErGO/GCE were estimated to 0.0768, 0.0959, 0.1052, and 0.1308 cm 2 , respectively. The effective electroactive area of TiO 2 /ErGO/GCE is twice of that of bare GCE approximately. The results indicate that TiO 2 /ErGO nanohybrids greatly increase the electroactive surface area, which will eventually boost the electrochemical responses.
Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 15 ErGO/GCE, and TiO2/ErGO/GCE were estimated to 0.0768, 0.0959, 0.1052, and 0.1308 cm 2 , respectively. The effective electroactive area of TiO2/ErGO/GCE is twice of that of bare GCE approximately. The results indicate that TiO2/ErGO nanohybrids greatly increase the electroactive surface area, which will eventually boost the electrochemical responses.  Figure 4B represents the equivalent circuit model.
Electrochemical impedance spectroscopy is a valuable tool to investigate the charge transfer ability between electrode and electrolyte interfaces [18,46,55,56]. Nyquist plots includes the semicircular at high frequencies and linear portions at low frequencies, which are closely associated with the electron transfer-limited process and diffusion-controlled process, respectively. Generally, the charge transfer resistance (Rct) equals a semicircle diameter. As shown in Figure 4B, the Rct value decreases from 1562 Ω to 341 Ω and 211 Ω after loading ErGO nanosheets and TiO2 NPs onto the bare electrode, respectively, which means that the charge transfer between the electrode and solution is more efficient after the modification with electrode by ErGO and TiO2 NPs. In other words, both TiO2 NPs and ErGO nanosheets can significantly improve the electron transfer efficiency. As expected, a notable decrease in the semicircle diameter (Rct = 14.5 Ω) is found on the TiO2/ErGO/GCE. It is believed that the remarkably decreased Rct can promote the electrochemical sensing performance.

Electrochemical Response of Allura Red on Different Modified Electrodes
DPV responses of 100 μM Allura Red were recorded at different electrodes ( Figure 5). Clearly, the anodic peak current of Allura Red at bare GCE is weakest (ipa = 0.607 μA). After introducing TiO2 NPs, the anodic peak current of Allura Red increased to 0.768 μA due to the good electrocatalytic activity and high adsorptive ability [36,[50][51][52]. When the GCE was decorated with TiO2/ErGO nanohybrids, the anodic peak current of Allura Red greatly increases (ipa = 0.890 μA), owing to the combined effect from good electrical conductivity and high specific surface area, which facilitates the absorption efficiency and electrochemical reactivity of Allura Red [52]. As expected, TiO2/ErGO/GCE shows the largest response signal (ipa = 1.187 μA). The anodic peak potentials (Epa) of bare GCE, TiO2/GCE, ErGO/GCE, and TiO2/ErGO/GCE are 0.648, 0.642, 0.624, and 0.620 V, respectively. Interestingly, the anodic peak potential at TiO2/ErGO/GCE is lower compared with previous reports [28,29,32,57], which can effectively avoid the co-oxidation of other interferences that may coexist in food with Allura Red. It probably because TiO2/ErGO nanohybrids can effectively accelerate the electron transfer between and analyte, thus minimizing the activated polarization potential. The enhancement on the peak current and lowering of oxidation overpotential demonstrate that TiO2/ErGO nanohybrids have superior electrocatalytic activity toward the oxidation of Allura Red. It  Figure 4B represents the equivalent circuit model.
Electrochemical impedance spectroscopy is a valuable tool to investigate the charge transfer ability between electrode and electrolyte interfaces [18,46,55,56]. Nyquist plots includes the semicircular at high frequencies and linear portions at low frequencies, which are closely associated with the electron transfer-limited process and diffusion-controlled process, respectively. Generally, the charge transfer resistance (R ct ) equals a semicircle diameter. As shown in Figure 4B, the R ct value decreases from 1562 Ω to 341 Ω and 211 Ω after loading ErGO nanosheets and TiO 2 NPs onto the bare electrode, respectively, which means that the charge transfer between the electrode and solution is more efficient after the modification with electrode by ErGO and TiO 2 NPs. In other words, both TiO 2 NPs and ErGO nanosheets can significantly improve the electron transfer efficiency. As expected, a notable decrease in the semicircle diameter (R ct = 14.5 Ω) is found on the TiO 2 /ErGO/GCE. It is believed that the remarkably decreased R ct can promote the electrochemical sensing performance.

Electrochemical Response of Allura Red on Different Modified Electrodes
DPV responses of 100 µM Allura Red were recorded at different electrodes ( Figure 5). Clearly, the anodic peak current of Allura Red at bare GCE is weakest (i pa = 0.607 µA). After introducing TiO 2 NPs, the anodic peak current of Allura Red increased to 0.768 µA due to the good electrocatalytic activity and high adsorptive ability [36,[50][51][52]. When the GCE was decorated with TiO 2 /ErGO nanohybrids, the anodic peak current of Allura Red greatly increases (i pa = 0.890 µA), owing to the combined effect from good electrical conductivity and high specific surface area, which facilitates the absorption efficiency and electrochemical reactivity of Allura Red [52]. As expected, TiO 2 /ErGO/GCE shows the largest response signal (i pa = 1.187 µA). The anodic peak potentials (E pa ) of bare GCE, TiO 2 /GCE, ErGO/GCE, and TiO 2 /ErGO/GCE are 0.648, 0.642, 0.624, and 0.620 V, respectively. Interestingly, the anodic peak potential at TiO 2 /ErGO/GCE is lower compared with previous reports [28,29,32,57], which can effectively avoid the co-oxidation of other interferences that may coexist in food with Allura Red. It probably because TiO 2 /ErGO nanohybrids can effectively accelerate the electron transfer between and analyte, thus minimizing the activated polarization potential. The enhancement on the peak current and lowering of oxidation overpotential demonstrate that TiO 2 /ErGO nanohybrids have superior electrocatalytic activity toward the oxidation of Allura Red. It can be reasonably ascribed to the synergistic effect between TiO 2 NPs and ErGO nanosheets, which combines the unique properties of two individual components. The electrocatalytic effect and cumulate accumulation capability of TiO 2 /ErGO nanohybrids accelerate electron transfer and eventually improve the electrochemical sensing performances [36,[50][51][52][53]. can be reasonably ascribed to the synergistic effect between TiO2 NPs and ErGO nanosheets, which combines the unique properties of two individual components. The electrocatalytic effect and cumulate accumulation capability of TiO2/ErGO nanohybrids accelerate electron transfer and eventually improve the electrochemical sensing performances [36,[50][51][52][53].

Effect of pH
The effect of pH on the response anodic peak current of Allura Red was explored by DPV. As displayed in Figure 6A, the anodic peaks of Allura Red shifts toward a negative potential direction, which demonstrate the proton participant in the redox process of Allura Red. The anodic peak currents of Allura Red gradually increase with pH varying from 5.6 to 7.0; then, they slowly decrease when the pH exceeds 7.0 ( Figure 6B). Therefore, pH 7.0 was selected as the optimal pH. In addition, the anodic peak potentials of Allura is inversely related to pH, with the linear expression of Epa =

Effect of pH
The effect of pH on the response anodic peak current of Allura Red was explored by DPV. As displayed in Figure 6A, the anodic peaks of Allura Red shifts toward a negative potential direction, which demonstrate the proton participant in the redox process of Allura Red. The anodic peak currents of Allura Red gradually increase with pH varying from 5.6 to 7.0; then, they slowly decrease when the pH exceeds 7.0 ( Figure 6B). Therefore, pH 7.0 was selected as the optimal pH. In addition, the anodic peak potentials of Allura is inversely related to pH, with the linear expression of E pa = −0.048 pH + 0.881 (R 2 = 0.987). The slope (48 mV/pH) is close to the theoretical value (59 mV/pH), indicating an electrochemical process involving an equal number of electrons (e − ) and protons (H + ). can be reasonably ascribed to the synergistic effect between TiO2 NPs and ErGO nanosheets, which combines the unique properties of two individual components. The electrocatalytic effect and cumulate accumulation capability of TiO2/ErGO nanohybrids accelerate electron transfer and eventually improve the electrochemical sensing performances [36,[50][51][52][53].

Effect of pH
The effect of pH on the response anodic peak current of Allura Red was explored by DPV. As displayed in Figure 6A, the anodic peaks of Allura Red shifts toward a negative potential direction, which demonstrate the proton participant in the redox process of Allura Red. The anodic peak currents of Allura Red gradually increase with pH varying from 5.6 to 7.0; then, they slowly decrease when the pH exceeds 7.0 ( Figure 6B). Therefore, pH 7.0 was selected as the optimal pH. In addition, the anodic peak potentials of Allura is inversely related to pH, with the linear expression of Epa = −0.048 pH + 0.881 (R 2 = 0.987). The slope (48 mV/pH) is close to the theoretical value (59 mV/pH), indicating an electrochemical process involving an equal number of electrons (e − ) and protons (H + ).

Effect of Scan Rates
To understand the electrochemical mechanism for the redox of Allura Red, cyclic voltammograms of 10 µM Allura Red were measured by TiO 2 /ErGO/GCE at various scan rates. Cyclic voltammograms at different scan rates are shown in Figure 7A. A pair of redox peaks are observed at all scan rates with the ratio of i pa /i pc approaching 1, demonstrating that a quasi-reversible process occurs at TiO 2 /ErGO/GCE. The anodic peak currents gradually increase with the increasing of scan rates. In addition, as the scan rates speed up, the anodic peaks of Allura Red shift positively, while the cathodic peaks move to the reverse direction. As shown in Figure 7B, there are good linear relationships between anodic/cathodic peak currents (i pa or i pc ) and the square root of scan rates (v 1/2 ). Their linear equations are i pa (µA) = 5.875v 1/2 (V/s) − 0.460 (R 2 = 0.999) and i pa (µA) = 5.875v 1/2 (V/s) − 0.460 (R 2 = 0.993), respectively. This result indicates that the redox of Allura Red is a typical diffusion-limited process. Moreover, the anodic or cathodic peak potentials are highly correlated to the Napierian logarithm of scan rates ( Figure 7C). Their linear regression equations can be expressed as E pa (V) = 0.036lnv (V/s) + 0.777 and E pc (V) = −0.023lnv (V/s) + 0.570, with a correlation coefficient (R 2 ) of 0.982 and 0.973, respectively. As for a diffusion-limited quasi-reversible process, the relationship between redox potential (E pa or E pc ) and lnv should follow Laviron's equations [58,59].
where α is the charge transfer coefficient, n is the electron transferred number, and other symbols have the usual meanings. Combining the linear relationships with Lavrion's equations, the charge transfer coefficient (α) is calculated as 0.6. Then, the electron transfer number (n) is found to 0.89 ≈ 1.0, indicating a one-electron transferred electrochemical process of Allura Red at TiO 2 /ErGO/GCE. Based on the earlier discussion that the electrochemical oxidation of Allura Red at TiO 2 /ErGO/GCE was a process related to an equal numbers of electrons and protons, it's deduced that the reaction mechanism of Allura Red involves one electron and one proton transfer, which is caused by the phenolic hydroxy (Scheme 1). The reaction mechanism is in good agreement with the previous report [4].

Effect of Scan Rates
To understand the electrochemical mechanism for the redox of Allura Red, cyclic voltammograms of 10 μM Allura Red were measured by TiO2/ErGO/GCE at various scan rates. Cyclic voltammograms at different scan rates are shown in Figure 7A. A pair of redox peaks are observed at all scan rates with the ratio of ipa/ipc approaching 1, demonstrating that a quasi-reversible process occurs at TiO2/ErGO/GCE. The anodic peak currents gradually increase with the increasing of scan rates. In addition, as the scan rates speed up, the anodic peaks of Allura Red shift positively, while the cathodic peaks move to the reverse direction. As shown in Figure 7B, there are good linear relationships between anodic/cathodic peak currents (ipa or ipc) and the square root of scan rates (v 1/2 ). Their linear equations are ipa (μA) = 5.875v 1/2 (V/s) − 0.460 (R 2 = 0.999) and ipa (μA) = 5.875v 1/2 (V/s) − 0.460 (R 2 = 0.993), respectively. This result indicates that the redox of Allura Red is a typical diffusionlimited process. Moreover, the anodic or cathodic peak potentials are highly correlated to the Napierian logarithm of scan rates ( Figure 7C). Their linear regression equations can be expressed as Epa (V) = 0.036lnv (V/s) + 0.777 and Epc (V) = −0.023lnv (V/s) + 0.570, with a correlation coefficient (R 2 ) of 0.982 and 0.973, respectively. As for a diffusion-limited quasi-reversible process, the relationship between redox potential (Epa or Epc) and lnv should follow Laviron's equations [58,59].
where α is the charge transfer coefficient, n is the electron transferred number, and other symbols have the usual meanings. Combining the linear relationships with Lavrion's equations, the charge transfer coefficient (α) is calculated as 0.6. Then, the electron transfer number (n) is found to 0.89 ≈ 1.0, indicating a one-electron transferred electrochemical process of Allura Red at TiO2/ErGO/GCE. Based on the earlier discussion that the electrochemical oxidation of Allura Red at TiO2/ErGO/GCE was a process related to an equal numbers of electrons and protons, it's deduced that the reaction mechanism of Allura Red involves one electron and one proton transfer, which is caused by the phenolic hydroxy (Scheme 1). The reaction mechanism is in good agreement with the previous report [4].

Effect of Accumulation Parameters
The effect of accumulation time as well as potential were also investigated ( Figure 7). As shown in Figure 8A, the anodic peak currents of Allura Red gradually increase for 150 s; then, they suddenly decrease with the further extension of accumulation time. So, 150 s was recommended as the optimal accumulation time. When the accumulation potentials were changed from −0.5 V to −0.3 V, their anodic peak currents gradually increase. However, they slowly decrease when the accumulation potential went beyond −0.3 V ( Figure 8B). Hence, −0.3 V was chosen as the optimal accumulation potential. The optimal accumulation parameters (−0.3 V, 150 s) were adopted in the following measurements.

Selectivity, Reproducibility, and Stability
To assess the selectivity of the proposed sensor, the DPV responses of Allura Red in the presence of common inorganic metal ions (e.g., K + , Na + , Ca 2+ , Mg 2+ ) and amaranth were also measured. The relative errors lower than 5% in the coexistence of 100-fold of these potential interfering substances ( Figure 9A), indicating excellent anti-jamming performance. It is worth pointing out that the anodic peaks of Allura Red and amaranth are well separated with rather wide peak-to-peak separation (∆Ep) of 0.252 V. Moreover, the andic peak current of Allura Red is hardly changed even in the existence of 100-fold amaranth. The phenomenon also shows that it is feasible to detect Allura Red and amaranth simultaneously. To check repeatability, seven successive measurements were also carried out. The relative standard deviation (RSD) is 5.46%, suggesting that TiO2/ErGO/GCE have good repeatability Scheme 1. Schematic diagram of the electrochemical mechanism for the redox of Allura Red at TiO 2 /ErGO/GCE.

Effect of Accumulation Parameters
The effect of accumulation time as well as potential were also investigated ( Figure 7). As shown in Figure 8A, the anodic peak currents of Allura Red gradually increase for 150 s; then, they suddenly decrease with the further extension of accumulation time. So, 150 s was recommended as the optimal accumulation time. When the accumulation potentials were changed from −0.5 V to −0.3 V, their anodic peak currents gradually increase. However, they slowly decrease when the accumulation potential went beyond −0.3 V ( Figure 8B). Hence, −0.3 V was chosen as the optimal accumulation potential. The optimal accumulation parameters (−0.3 V, 150 s) were adopted in the following measurements. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 15 Scheme 1. Schematic diagram of the electrochemical mechanism for the redox of Allura Red at TiO2/ErGO/GCE.

Effect of Accumulation Parameters
The effect of accumulation time as well as potential were also investigated ( Figure 7). As shown in Figure 8A, the anodic peak currents of Allura Red gradually increase for 150 s; then, they suddenly decrease with the further extension of accumulation time. So, 150 s was recommended as the optimal accumulation time. When the accumulation potentials were changed from −0.5 V to −0.3 V, their anodic peak currents gradually increase. However, they slowly decrease when the accumulation potential went beyond −0.3 V ( Figure 8B). Hence, −0.3 V was chosen as the optimal accumulation potential. The optimal accumulation parameters (−0.3 V, 150 s) were adopted in the following measurements.

Selectivity, Reproducibility, and Stability
To assess the selectivity of the proposed sensor, the DPV responses of Allura Red in the presence of common inorganic metal ions (e.g., K + , Na + , Ca 2+ , Mg 2+ ) and amaranth were also measured. The relative errors lower than 5% in the coexistence of 100-fold of these potential interfering substances ( Figure 9A), indicating excellent anti-jamming performance. It is worth pointing out that the anodic peaks of Allura Red and amaranth are well separated with rather wide peak-to-peak separation (∆Ep) of 0.252 V. Moreover, the andic peak current of Allura Red is hardly changed even in the existence of 100-fold amaranth. The phenomenon also shows that it is feasible to detect Allura Red and amaranth simultaneously. To check repeatability, seven successive measurements were also carried out. The relative standard deviation (RSD) is 5.46%, suggesting that TiO2/ErGO/GCE have good repeatability

Selectivity, Reproducibility, and Stability
To assess the selectivity of the proposed sensor, the DPV responses of Allura Red in the presence of common inorganic metal ions (e.g., K + , Na + , Ca 2+ , Mg 2+ ) and amaranth were also measured. The relative errors lower than 5% in the coexistence of 100-fold of these potential interfering substances ( Figure 9A), indicating excellent anti-jamming performance. It is worth pointing out that the anodic peaks of Allura Red and amaranth are well separated with rather wide peak-to-peak separation (∆E p ) of 0.252 V. Moreover, the andic peak current of Allura Red is hardly changed even in the existence of 100-fold amaranth. The phenomenon also shows that it is feasible to detect Allura Red and amaranth simultaneously. To check repeatability, seven successive measurements were also carried out.
The relative standard deviation (RSD) is 5.46%, suggesting that TiO 2 /ErGO/GCE have good repeatability ( Figure 9B). Then, anodic peak currents of Allura Red were recorded at seven TiO 2 /ErGO/GCEs that were fabricated by the same procedure, aiming to evaluate electrode reproducibility ( Figure 9C). The RSD of 4.83% is acceptable, demonstrating electrode fabrication that has good reproducibility. Finally, the anodic peak currents of Allura Red were monitored during two weeks. After two weeks, the anodic peak current of Allura Red retains 90.9% of its original value, demonstrating good stability ( Figure 9D). Nanomaterials 2020, 10, x FOR PEER REVIEW 10 of 15 ( Figure 9B). Then, anodic peak currents of Allura Red were recorded at seven TiO2/ErGO/GCEs that were fabricated by the same procedure, aiming to evaluate electrode reproducibility ( Figure 9C). The RSD of 4.83% is acceptable, demonstrating electrode fabrication that has good reproducibility. Finally, the anodic peak currents of Allura Red were monitored during two weeks. After two weeks, the anodic peak current of Allura Red retains 90.9% of its original value, demonstrating good stability ( Figure 9D).  Figure 9A is DPV of Allura Red in the existence of 100-fold amaranth and all interfering specifies. GCE: glass carbon electrodes.

Standard Curves, Detection Range, and Detection Limit
DPV curves of different concentrations of Allura Red are plotted in Figure 10A. Clearly, the response anodic peak currents of Allura Red increase with their concentrations. At a low concentration domain (0.3-5 μM), the anodic peak currents of Allura Red are linearly correlated to their concentrations ( Figure 10B). The corresponding linear equation can be expressed as ipa (μA) = 0.0756CAR (μM) + 0.0498 (R 2 = 0.991). However, the anodic peak currents of Allura Red are positively proportional to the Napierian logarithm of their concentrations (lnCAR) ranging from 5 to 800 μM ( Figure 10C). The standard deviation (σ) for 10 parallel measurements in bank solution is 0.001276. Therefore, the detection limit (LOD) is estimated to 0.05 μM (LOD = 3σ/s). The analytical performances including detection range and detection limit are almost comparable to previous reported modified electrodes (Table 1). In addition, TiO2/ErGO nanohybrids possess the characteristics of low cost, available raw materials, and easy preparation, which is very advantageous for commercial applications. presence of 100-fold K + , Na + , Ca 2+ , Mg 2+ , and amaranth; (B) Seven successive measurements of Allura Red at the same TiO 2 /ErGO/GCE; (C) Anodic peak currents of Allura Red recorded at seven TiO 2 /ErGO/GCEs; (D) Anodic peak currents of Allura Red were regularly recorded during two weeks. The inset in Figure 9A is DPV of Allura Red in the existence of 100-fold amaranth and all interfering specifies. GCE: glass carbon electrodes.

Standard Curves, Detection Range, and Detection Limit
DPV curves of different concentrations of Allura Red are plotted in Figure 10A. Clearly, the response anodic peak currents of Allura Red increase with their concentrations. At a low concentration domain (0.3-5 µM), the anodic peak currents of Allura Red are linearly correlated to their concentrations ( Figure 10B). The corresponding linear equation can be expressed as i pa (µA) = 0.0756C AR (µM) + 0.0498 (R 2 = 0.991). However, the anodic peak currents of Allura Red are positively proportional to the Napierian logarithm of their concentrations (lnC AR ) ranging from 5 to 800 µM ( Figure 10C). The standard deviation (σ) for 10 parallel measurements in bank solution is 0.001276. Therefore, the detection limit (LOD) is estimated to 0.05 µM (LOD = 3σ/s). The analytical performances including detection range and detection limit are almost comparable to previous reported modified electrodes (Table 1). In addition, TiO 2 /ErGO nanohybrids possess the characteristics of low cost, available raw materials, and easy preparation, which is very advantageous for commercial applications.

Determination of Allura Red in Actual Samples
In order to verify the workability, the level of Allura Red in milk drink samples was detected by our proposed TiO2/ErGO/GCE using DPV. Liziyuan drinking was diluted to 100-fold with 0.1 M PBS solution (pH = 7.0). The determination results are summarized in Table 2. The concentration of Allura Red in the Liziyuan milk drink sample is 14.28 μM (7088 μg/L). Obviously, the level of Allura Red in soft drink is far lower than the maximum permitted dosage (0.1 g/kg) specified in GB 2760-2014. To further verify the accuracy and precision of the proposed TiO2/ErGO/GCE, a series of known concentration solutions of Allura Red were spiked to the milk drink samples. The RSD is lower than 5.0% and recoveries range from 98.42% to 106.0%, confirming the feasibility for the Allura Red determination in real samples. In short, the proposed TiO2/ErGO/GCE shows tremendous application prospects on the determination of Allura Red in real samples such as beverages and food.
Based on the detected values, it is easy to estimate the amount of Liziyuan milk drink that an individual must drink to reach Allura Red's ADI. For instance, if an individual weighs 60 kg, the amount of Liziyuan milk drink that must consume is 59 L per day. This means that the volume

Determination of Allura Red in Actual Samples
In order to verify the workability, the level of Allura Red in milk drink samples was detected by our proposed TiO 2 /ErGO/GCE using DPV. Liziyuan drinking was diluted to 100-fold with 0.1 M PBS solution (pH = 7.0). The determination results are summarized in Table 2. The concentration of Allura Red in the Liziyuan milk drink sample is 14.28 µM (7088 µg/L). Obviously, the level of Allura Red in soft drink is far lower than the maximum permitted dosage (0.1 g/kg) specified in GB 2760-2014. To further verify the accuracy and precision of the proposed TiO 2 /ErGO/GCE, a series of known concentration solutions of Allura Red were spiked to the milk drink samples. The RSD is lower than 5.0% and recoveries range from 98.42% to 106.0%, confirming the feasibility for the Allura Red determination in real samples. In short, the proposed TiO 2 /ErGO/GCE shows tremendous application prospects on the determination of Allura Red in real samples such as beverages and food. Based on the detected values, it is easy to estimate the amount of Liziyuan milk drink that an individual must drink to reach Allura Red's ADI. For instance, if an individual weighs 60 kg, the amount of Liziyuan milk drink that must consume is 59 L per day. This means that the volume required to reach the ADI value is so large that the potential side effects on consumers health are negligible, even if it is consumed daily.

Conclusions
In this paper, a novel electrochemical sensor based on TiO 2 /ErGO nanocomposites were developed for the sensitive detection of Allura Red. TiO 2 /ErGO nanocomposites were synthesized by the hydrolysis of titanium sulfate in graphene oxide suspension and in situ electrochemical reduction. The electrochemical characterization of the proposed sensor was performed by cyclic voltammetry and electrochemical impedance spectroscopy with [Fe(CN) 6 ] 3−/4− as the redox pair. TiO 2 /ErGO nanohybrids showed the largest electroactive surface area and lowest charge transferred resistance (R ct ). As a result, TiO 2 /ErGO/GCE showed remarkable electrocatalytic activity toward Allura Red, which was mainly due to the synergistic effect between TiO 2 NPs and ErGO nanosheets. The anodic peak currents of Allura Red were linearly correlated with their concentrations in the range of 0.3 to 5.0 µM. However, the linear relationship was changed to a semi-logarithmic relationship at a higher concentration region (5.0-800 µM). The detection limit (LOD) was estimated to 0.05 µM at SNR = 3. Finally, the Allura Red in soft milk drink was detected by the proposed TiO 2 /ErGO/GCE with satisfactory recovery. Featured with the fascinating merits including high sensitivity as well as good anti-jamming performance, repeatability, reproducibility, and stability, the proposed TiO 2 /ErGO/GCE nanocomposites have become an attractive candidate for the detection of food colorants.