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Facile Preparation of Cu2O Nanoparticles and Reduced Graphene Oxide Nanocomposite for Electrochemical Sensing of Rhodamine B

In this paper, the preparation, characterization, and electrochemical application of Cu2O nanoparticles and an electrochemical reduced graphene oxide nanohybrid modified glassy carbon electrode (denoted as Cu2O NPs‒ERGO/GCE) are described. This modified electrode was used as an electrochemical sensor for the catalytic oxidation of rhodamine B (RhB), and it exhibited an excellent electrochemical performance for RhB. The oxidation potential of RhB was decreased greatly, and the sensitivity to detect RhB was improved significantly. Under optimum conditions, a linear dynamic range of 0.01–20.0 μM and a low detection limit of 0.006 μM were obtained with the Cu2O NPs‒ERGO/GCE by using second‒order derivative linear sweep voltammetry. In addition, the selectivity of the prepared modified electrode was analyzed for the determination of RhB. The practical application of this sensor was investigated for the determination of RhB in food samples, and satisfactory results were obtained.


Introduction
Rhodamine B (RhB), as a synthetic organic dye, is widely used in paper, textile, porcelain, leather, and paint industries [1]. However, studies have shown that RhB is carcinogenic. According to the International Agency for Research on Cancer (IARC), RhB carries a carcinogenic risk: The inhalation, ingestion, and skin contact of RhB may lead to acute and chronic poisoning as well as injury [2]. The concentration of RhB in industrial wastewater is generally higher than 100 mg/L and difficult to biodegrade [3], which not only causes damage to plants and animals but also poses a serious health threat to human. Europe, the United States, and China have made it clear that it is not allowed to be used in foodstuffs [4][5][6]. However, due to the low price, bright color, and strong stability of RhB, the illegal use of RhB has still been found in food in recent years, endangering the health of consumers. Therefore, the determination of RhB in wastewater and foodstuffs is very important for ensuring the safety of human beings.

Reagents
Graphite powder, polyvinylpyrrolidone, cupric sulfate pentahydrate (CuSO 4 ·5H 2 O), and RhB were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). 0.0479 g of RhB was accurately weighed and dissolved in water, which was diluted to 100.0 mL to prepare the standard solution of 1.0 mM. The low concentration working solutions of RhB were prepared by diluting the standard solution. A 0.15 M HAc-NaAc buffer (pH 6.6) was used as a supporting electrolyte. All chemicals were of analytical grade and did not need further purification when used. All the water used was doubly distilled water.

Instruments
Cyclic voltammetry (CV) was carried out on a CHI 660E electrochemical workstation of Shanghai Chenhua Instruments Company, China. The second derivative linear scan voltammetry was determined on a JP-303E polarographic analyzer (Chengdu Instrument Factory, Chengdu, China). All electrochemical measurements were performed using a three-electrode system (including a working electrode (Cu 2 O NPs-ERGO/GCE), a reference electrode (saturated calomel electrode, SCE), and a counter electrode (platinum electrode). The pH value was measured on a pH-3c exact digital pH meter (Leichi Instrument Factory, Shanghai, China). A scanning electron microscope (EVO10, ZEISS, Jena, Germany) was used to obtain scanning electron microscopy (SEM) images at 2.0 KV of acceleration voltage. A powder X-ray diffractometer (PANalytical, Amsterdam, Holland) with Cu Kα radiation (0.1542 nm) was employed to analyze the crystal structure of Cu 2 O. High performance liquid chromatography (HPLC) analysis was carried out using an Agilent 1100 series HPLC system and fluorescence detector (Agilent Technology Co., Ltd., Beijing, China).

Preparation of Cu 2 O NPs-GO Dispersion
GO was synthesized according to our previous report [41]. Cu 2 O NPs were synthesized according to Xu et al. [33]. Typically, 100 mg of CuSO 4 ·5H 2 O and 50 mg of polyvinylpyrrolidone were dissolved in 20 mL of distilled water and stirred for 30 min, and then 4 mL of 0.2 M NaOH was added to the above solution slowly and stirred continuously for another 30 min to obtain a blue precipitate. Finally, 15 µL of hydrazine hydrate solution (80 wt%) was added to the mixture and stirred continuously for 20 min to obtain a brick red suspension. The suspension was centrifuged and washed with anhydrous ethanol and ultrapure water in turn, and then it was dried at room temperature. Finally, 1.0 mg of Cu 2 O-NPs were dispersed in 20 mL of a GO solution (1 mg/mL), and the uniform Cu 2 O-GO dispersion was obtained by ultrasonication for 2 h.

Electrode Preparation
The 3 mm in diameter glassy carbon electrode (GCE) was polished with 0.05 µm of alumina slurry and then washed with distilled water, absolute ethanol, distilled water in an ultrasonic bath-each for 1 min-and dried under an infrared lamp. A 5 µL dispersion of Cu 2 O NPs-GO was dried on the surface of the GCE at room temperature. The Cu 2 O NPs-ERGO/GCE was prepared by immersing the Cu 2 O NPs-GO/GCE in a phosphate buffer solution (pH 6.0) and reducing it for 120 s at a constant potential of −1.2 V. For comparison, other modified electrodes such as a GO/GCE, ERGO/GCE, and Cu 2 O NPs-GO/GCE were prepared in a similar way.

Electrochemical Measurement
One milliliter of a RhB standard solution with an appropriate concentration was transferred to a 10 mL electrochemical cell, and 1.5 mL of a 1.0 M HAc-NaAc buffer (pH 6.6) and 7.5 mL of H 2 O were added. Then, the Cu 2 O NPs-GO/GCE, an SCE, and a platinum electrode were inserted into Nanomaterials 2019, 9, 958 4 of 14 the electrochemical cell. the measuring parameters were adjusted, and cyclic voltammetry or second derivative linear scan voltammetry were recorded. The calibration curve was established by plotting the relationship between the measured current signal and the analyte concentration. The content of RhB in sample solutions was determined by the standard addition method.

SEM and XRD Analysis
The SEM images of GO, Cu 2 O NPs, ERGO, and Cu 2 O NPs-ERGO composites are illustrated in Figure 1. Figure 1A displays the image of the wrinkled multi-layer graphene oxide, which corrugated and scrolled like crumpled silk veils. Figure 1B is the SEM image of pure Cu 2 O NPs. The product is spherical in shape with an average diameter of about 50-100 nanometers. Figure 1C shows the layered structure of ERGO nanosheets. The layered structure of ERGO could effectively increase the specific surface area of the modified electrode. It can be clearly seen from Figure 1D that the ERGO were decorated by Cu 2 O NPs. These spherical crystals were distributed randomly on the surface and edges of the ERGO sheets. In addition, the SEM images in Figure 1D show that the morphology and size of Cu 2 O NPs are similar to those observed in Figure 1B, which indicates that Cu 2 O NPs-ERGO composites prepared by the electroreduction method would not change the structure of Cu 2 O. Nanomaterials 2019, 9, x FOR PEER REVIEW 4 of 15 the relationship between the measured current signal and the analyte concentration. The content of RhB in sample solutions was determined by the standard addition method.

SEM and XRD Analysis
The SEM images of GO, Cu2O NPs, ERGO, and Cu2O NPs-ERGO composites are illustrated in Figure 1. Figure 1A displays the image of the wrinkled multi-layer graphene oxide, which corrugated and scrolled like crumpled silk veils. Figure 1B is the SEM image of pure Cu2O NPs. The product is spherical in shape with an average diameter of about 50-100 nanometers. Figure 1C shows the layered structure of ERGO nanosheets. The layered structure of ERGO could effectively increase the specific surface area of the modified electrode. It can be clearly seen from Figure 1D that the ERGO were decorated by Cu2O NPs. These spherical crystals were distributed randomly on the surface and edges of the ERGO sheets. In addition, the SEM images in Figure 1D show that the morphology and size of Cu2O NPs are similar to those observed in Figure 1B, which indicates that Cu2O NPs-ERGO composites prepared by the electroreduction method would not change the structure of Cu2O.
The X-ray Diffraction (XRD) pattern of Cu2O NPs is as shown in Figure 2. Comparing with the standard document of Cu2O (JCPDS No. 05-0667) [42], the diffraction peaks of the prepared Cu2O are basically the same as that of the standard document. The main peaks are sharp, which proves that the crystallinity of Cu2O is relatively good. No diffraction peaks of other possible impurities (such as Cu and CuO) were detected, indicating that the product was pure Cu2O.

Characterization by CV
The cyclic voltammetric behaviors of 1.0 mM K3[Fe(CN)6] containing 0.5 M of KCl at different electrodes were studied at a potential scan rate of 100 mV s −1 . As shown in Figure 3, on the bare GCE, a pair of redox peaks of [Fe(CN)6] 3−/4− was observed with a peak-to-peak separation (ΔEp) of 158 mV (curve a). While on an ERGO/GCE, both cathodic and anodic peak currents increased obviously, while the ΔEp value decreased to 85 mV (curve b) due to the large surface area and excellent electrical conductivity of ERGO present on the electrode surface (curve b). On the Cu2O-ERGO/GCE (curve c), the electrochemical behavior of the [Fe(CN)6] 3−/4− was dramatically improved with a cathodic peak potential (Epc) of 0.204 V and anodic peak potential (Epa) of 0.292 V. The peak-to-peak separation (ΔEp) was 88 mV (curve c), indicating that the presence of the high conductivity of ERGO together with the good catalytic activity of Cu2O on the GCE surface can further promote electron transfer and improve the performance of the sensor. According to the Randles-Sevcik equation [43]: ipc = (2.69 × 10 5 ) n 3/2 D 1/2 v 1/2 AC, where ipc is the reduction peak current (A), n is the electrontransfer number, A is the electroactive surface area (cm 2 ), D is the diffusion coefficient of K3[Fe(CN)6] in the solution (7.6 × 10 -6 cm 2 s −1 [44]), C is the concentration of K3[Fe(CN)6] (mol cm −3 ), and v is the scan rate (V s −1 ). By exploring the redox peak current with scan rate, the average electroactive areas of the GCE, ERGO/GCE, and Cu2O-ERGO/GCE were calculated as 0.04711 cm 2 , 0.1142 cm 2 , and 0.1463 cm 2 , respectively. The results further indicate that the presence of ERGO and Cu2O NPs greatly improved the effective area of the electrode surface, which led to the great enhancement of the electrochemical response on the modified electrode.

Characterization by CV
The cyclic voltammetric behaviors of 1.0 mM K 3 [Fe(CN) 6 ] containing 0.5 M of KCl at different electrodes were studied at a potential scan rate of 100 mV s −1 . As shown in Figure 3, on the bare GCE, a pair of redox peaks of [Fe(CN) 6 ] 3−/4− was observed with a peak-to-peak separation (∆E p ) of 158 mV (curve a). While on an ERGO/GCE, both cathodic and anodic peak currents increased obviously, while the ∆E p value decreased to 85 mV (curve b) due to the large surface area and excellent electrical conductivity of ERGO present on the electrode surface (curve b). On the Cu 2 O-ERGO/GCE (curve c), the electrochemical behavior of the [Fe(CN) 6 ] 3−/4− was dramatically improved with a cathodic peak potential (E pc ) of 0.204 V and anodic peak potential (E pa ) of 0.292 V. The peak-to-peak separation (∆E p ) was 88 mV (curve c), indicating that the presence of the high conductivity of ERGO together with the good catalytic activity of Cu 2 O on the GCE surface can further promote electron transfer and improve the performance of the sensor. According to the Randles-Sevcik equation [43]: i pc = (2.69 × 10 5 ) n 3/2 D 1/2 v 1/2 AC, where i pc is the reduction peak current (A), n is the electrontransfer number, A is the electroactive surface area (cm 2 ), D is the diffusion coefficient of K 3 [Fe(CN) 6 ] in the solution (7.6 × 10 -6 cm 2 s −1 [44]), C is the concentration of K 3 [Fe(CN) 6 ] (mol cm −3 ), and v is the scan rate (V s −1 ). By exploring the redox peak current with scan rate, the average electroactive areas of the GCE, ERGO/GCE, and Cu 2 O-ERGO/GCE were calculated as 0.04711 cm 2 , 0.1142 cm 2 , and 0.1463 cm 2 , respectively. The results further indicate that the presence of ERGO and Cu 2 O NPs greatly improved the effective area of the electrode surface, which led to the great enhancement of the electrochemical response on the modified electrode.

Characterization by CV
The cyclic voltammetric behaviors of 1.0 mM K3[Fe(CN)6] containing 0.5 M of KCl at different electrodes were studied at a potential scan rate of 100 mV s −1 . As shown in Figure 3, on the bare GCE, a pair of redox peaks of [Fe(CN)6] 3−/4− was observed with a peak-to-peak separation (ΔEp) of 158 mV (curve a). While on an ERGO/GCE, both cathodic and anodic peak currents increased obviously, while the ΔEp value decreased to 85 mV (curve b) due to the large surface area and excellent electrical conductivity of ERGO present on the electrode surface (curve b). On the Cu2O-ERGO/GCE (curve c), the electrochemical behavior of the [Fe(CN)6] 3−/4− was dramatically improved with a cathodic peak potential (Epc) of 0.204 V and anodic peak potential (Epa) of 0.292 V. The peak-to-peak separation (ΔEp) was 88 mV (curve c), indicating that the presence of the high conductivity of ERGO together with the good catalytic activity of Cu2O on the GCE surface can further promote electron transfer and improve the performance of the sensor. According to the Randles-Sevcik equation [43]: ipc = (2.69 × 10 5 ) n 3/2 D 1/2 v 1/2 AC, where ipc is the reduction peak current (A), n is the electrontransfer number, A is the electroactive surface area (cm 2 ), D is the diffusion coefficient of K3[Fe(CN)6] in the solution (7.6 × 10 -6 cm 2 s −1 [44]), C is the concentration of K3[Fe(CN)6] (mol cm −3 ), and v is the scan rate (V s −1 ). By exploring the redox peak current with scan rate, the average electroactive areas of the GCE, ERGO/GCE, and Cu2O-ERGO/GCE were calculated as 0.04711 cm 2 , 0.1142 cm 2 , and 0.1463 cm 2 , respectively. The results further indicate that the presence of ERGO and Cu2O NPs greatly improved the effective area of the electrode surface, which led to the great enhancement of the electrochemical response on the modified electrode. .

Electrochemical Behaviors of RhB on Cu 2 O NPs-ERGO/GCE
The electrochemical behaviors of RhB at five different working electrodes in a 0.15 M HAc-NaAc buffer of pH 6.6 were compared by cyclic voltammetry, and the results are shown in Figure 4. As can be seen (in the inset in Figure 4), no redox peaks were observed on the Cu 2 O-ERGO/GCE in a blank solution, indicating that the Cu 2 O-ERGO/GCE is nonelectroactive in the selected potential region. On the other hand, when 10 µM of RhB was added into the blank solution, an oxidation peak was observed at each electrode in the scanning window of 0-1.2 V, indicating that RhB undergoes an irreversible redox process. On a bare GCE (curve a), RhB shows a poorly defined peak at 1.024 V with a very low current response (i p = 0.6031 µA), which indicates a slow electron transfer. It can be seen that on the GO/GCE (curve b), the current response of RhB (i p = 1.105 µA) was larger than that of the GCE, which may be due to the fact that the oxygen-containing functional groups on GO nanosheets often act as catalytic active sites for some substances in the electrochemical reaction process [45,46]. On the Cu 2 O NPs-GO/GCE (curve c), the oxidation peak current was bigger (i p = 2.935 µA) than that of the GO/GCE, and the peak potential was negatively shifted to 0.995 V, indicating that Cu 2 O NPs could catalyze the electrochemical oxidation of RhB. An oxidation current peak of RhB at 0.986 V was obtained with the ERGO/GCE (curve d). The peak current (i p = 5.338 µA) was enhanced by about nine times in comparison to that of the bare GCE. These findings indicate the excellent catalytic ability and good conductive performance of ERGO on the oxidation of RhB. With the Cu 2 O NPs-ERGO/GCE (curve e), the charging current was obviously higher than that at the above electrodes, and the peak current of RhB was significantly higher (i p = 13.884 µA). The peak current of RhB was enhanced by about 23 folds in comparison to that of the bare GCE. The peak current enhancement and the negative shift of the peak potential of RhB (0.970 V) were undoubtedly attributed to the characteristics of Cu 2 O NPs and ERGO. Specifically, the large specific surface area of ERGO increased the adsorption of RhB on the electrode surface. In addition, ERGO had good conductivity, which made up for the shortcomings of the Cu 2 O semiconductor. On the other hand, Cu 2 O loaded on the ERGO surface accelerated the electron exchange between RhB and the electrode surface, and it promoted the electrocatalytic reaction. Thus, these nanocomposites can be utilized to the maximum extent in the limited surface area of the electrode, providing an electron transfer microenvironment and realizing the sensitive determination of RhB.

Electrochemical Behaviors of RhB on Cu2O NPs-ERGO/GCE
The electrochemical behaviors of RhB at five different working electrodes in a 0.15 M HAc-NaAc buffer of pH 6.6 were compared by cyclic voltammetry, and the results are shown in Figure 4. As can be seen (in the inset in Figure 4), no redox peaks were observed on the Cu2O-ERGO/GCE in a blank solution, indicating that the Cu2O-ERGO/GCE is nonelectroactive in the selected potential region. On the other hand, when 10 μM of RhB was added into the blank solution, an oxidation peak was observed at each electrode in the scanning window of 0-1.2 V, indicating that RhB undergoes an irreversible redox process. On a bare GCE (curve a), RhB shows a poorly defined peak at 1.024 V with a very low current response (ip = 0.6031 μA), which indicates a slow electron transfer. It can be seen that on the GO/GCE (curve b), the current response of RhB (ip = 1.105 μA) was larger than that of the GCE, which may be due to the fact that the oxygen-containing functional groups on GO nanosheets often act as catalytic active sites for some substances in the electrochemical reaction process [45,46]. On the Cu2O NPs-GO/GCE (curve c), the oxidation peak current was bigger (ip = 2.935 μA) than that of the GO/GCE, and the peak potential was negatively shifted to 0.995 V, indicating that Cu2O NPs could catalyze the electrochemical oxidation of RhB. An oxidation current peak of RhB at 0.986 V was obtained with the ERGO/GCE (curve d). The peak current (ip = 5.338 μA) was enhanced by about nine times in comparison to that of the bare GCE. These findings indicate the excellent catalytic ability and good conductive performance of ERGO on the oxidation of RhB. With the Cu2O NPs-ERGO/GCE (curve e), the charging current was obviously higher than that at the above electrodes, and the peak current of RhB was significantly higher (ip = 13.884 μA). The peak current of RhB was enhanced by about 23 folds in comparison to that of the bare GCE. The peak current enhancement and the negative shift of the peak potential of RhB (0.970 V) were undoubtedly attributed to the characteristics of Cu2O NPs and ERGO. Specifically, the large specific surface area of ERGO increased the adsorption of RhB on the electrode surface. In addition, ERGO had good conductivity, which made up for the shortcomings of the Cu2O semiconductor. On the other hand, Cu2O loaded on the ERGO surface accelerated the electron exchange between RhB and the electrode surface, and it promoted the electrocatalytic reaction. Thus, these nanocomposites can be utilized to the maximum extent in the limited surface area of the electrode, providing an electron transfer microenvironment and realizing the sensitive determination of RhB.

The Effect of Potential Scan Rate
The electrode reaction kinetics of RhB on the Cu 2 O NPs-ERGO/GCE can be investigated by exploring the relationship between the scan rate and the electrochemical response. The cyclic voltammograms of 10 µM of RhB on the Cu 2 O NPs-ERGO/GCE at different scan rates are shown in Figure 5. It can be seen form Figure 5 that when the scan rate increased from 0.03 V·s −1 to 0.3 V·s −1 , the peak current increased gradually and had a linear relationship with the square root of the scan rate. The linear regression equation can be expressed as i p = 14.851 v 1/2 + 0.9307 (i p : µA, v: V s −1 ), and the correlation coefficient R 2 was 0.9928, which indicates that the electrochemical process of RhB on the Cu 2 O NPs-ERGO/GCE was controlled by diffusion. The diffusion control behavior was also confirmed by plotting logi vs. logv, and the corresponding linear equation is logi = 0.4338 logv + 1.185 (R 2 = 0.996). The slope of 0.4338 was close to 0.5, which confirms the diffusion control characteristics of the electrode process. The electron transfer number of the electrode reaction can be calculated from the relationship between the peak potential and the scan rate [41]. As shown in the inset of Figure 5, the peak potential was linearly related to lnv in the range of 0.03-0.3 V·s −1 . The linear regression equation was E p = 0.0273 lnv + 1.0308 (Ep: V, v: V s −1 ), and the correlation coefficient R 2 was 0.9977. For a totally irreversible diffusion-controlled process, the slope of 0.0273 was equal to RT/2αnF [43], so αn = 0.47 can be calculated. α is generally considered to be 0.5 in a completely irreversible electrode process [46], so the number of electron transferred (n) that were involved in the oxidation process of RhB was about one.

The Effect of Potential Scan Rate
The electrode reaction kinetics of RhB on the Cu2O NPs-ERGO/GCE can be investigated by exploring the relationship between the scan rate and the electrochemical response. The cyclic voltammograms of 10 μM of RhB on the Cu2O NPs-ERGO/GCE at different scan rates are shown in Figure 5. It can be seen form Figure 5 that when the scan rate increased from 0.03 V·s −1 to 0.3 V·s −1 , the peak current increased gradually and had a linear relationship with the square root of the scan rate. The linear regression equation can be expressed as ip = 14.851 v 1/2 + 0.9307 (ip: μA, v: V s −1 ), and the correlation coefficient R 2 was 0.9928, which indicates that the electrochemical process of RhB on the Cu2O NPs-ERGO/GCE was controlled by diffusion. The diffusion control behavior was also confirmed by plotting logi vs. logv, and the corresponding linear equation is logi = 0.4338 logv + 1.185 (R 2 = 0.996). The slope of 0.4338 was close to 0.5, which confirms the diffusion control characteristics of the electrode process. The electron transfer number of the electrode reaction can be calculated from the relationship between the peak potential and the scan rate [41]. As shown in the inset of Figure 5, the peak potential was linearly related to lnv in the range of 0.03-0.3 V·s −1 . The linear regression equation was Ep = 0.0273 lnv + 1.0308 (Ep: V, v: V s −1 ), and the correlation coefficient R 2 was 0.9977. For a totally irreversible diffusion-controlled process, the slope of 0.0273 was equal to RT/2αnF [43], so αn = 0.47 can be calculated. α is generally considered to be 0.5 in a completely irreversible electrode process [46], so the number of electron transferred (n) that were involved in the oxidation process of RhB was about one.

Optimization of Determination Parameters
In order to obtain the best determination conditions, the effects of various parameters on the electrochemical oxidation of RhB were studied. Firstly, the effects of the type and concentration of the supporting electrolyte were studied. The electrochemical responses of 10 μM of RhB were measured in different supporting electrolytes, which include an HAc-NaAc buffer (pH 3.0-8.0), an HAc-NH4Ac buffer (pH 3.0-8.0), a Britton-Robinson buffer (pH 3.0-8.0), a phosphate buffer (pH

Optimization of Determination Parameters
In order to obtain the best determination conditions, the effects of various parameters on the electrochemical oxidation of RhB were studied. Firstly, the effects of the type and concentration of the supporting electrolyte were studied. The electrochemical responses of 10 µM of RhB were measured in different supporting electrolytes, which include an HAc-NaAc buffer (pH 3.0-8.0), an HAc-NH 4 1 M). It was found that the largest oxidation peak current and the best peak shape of RhB were obtained in an HAc-NaAc Nanomaterials 2019, 9, 958 8 of 14 buffer. In addition, the effect of the concentration of the HAc-NaAc buffer on RhB oxidation was evaluated in the range of 0.02-0.6 M. It was found that with the increase of the concentration from 0.02-0.15 M, the peak oxidation current of RhB increased gradually, and then the current decreased when increasing the concentration from 0.15-0.6 M ( Figure 6A). Therefore, 0.15 M was used as the best concentration of the HAc-NaAc buffer in this study. The effect of solution pH on the peak current of 10 µM of RhB was also investigated. The experimental results are shown in Figure 6B. It can be seen that when the pH value of the HAc-NaAc buffer increased from 3.21 to 6.60, the peak current of RhB continued to increase. However, when the pH value was higher than 6.60, the peak current began to decrease. Therefore, the optimal pH value of the HAc-NaAc buffer was 6.60. The effects of accumulation potential and time on 10 µM of RhB were also studied. Firstly, the accumulated potential was changed in the range from −0.3 to 0.3 V with a fixed accumulation time of 120 s. It was found that when the accumulated potential increased from −0.3 V to −0.1 V, the peak current increased, and then the peak current decreased dramatically with the further positive shift of the accumulation potential from −0.1 V to 0.3 V ( Figure 6C). Therefore, the best accumulation potential was chosen as −0.1 V. The effect of accumulation time on the oxidation peak current of RhB was investigated at −0.1 V. It was found that the peak current of RhB increased with the increase of accumulation time. However, increasing accumulation time after 120 s did not lead to significant changes in peak current ( Figure 6D). Therefore, considering the sensitivity and analysis speed, 120 s was chosen for quantitative analysis.  1 M). It was found that the largest oxidation peak current and the best peak shape of RhB were obtained in an HAc-NaAc buffer. In addition, the effect of the concentration of the HAc-NaAc buffer on RhB oxidation was evaluated in the range of 0.02-0.6 M. It was found that with the increase of the concentration from 0.02-0.15 M, the peak oxidation current of RhB increased gradually, and then the current decreased when increasing the concentration from 0.15-0.6 M ( Figure 6A). Therefore, 0.15 M was used as the best concentration of the HAc-NaAc buffer in this study. The effect of solution pH on the peak current of 10 μM of RhB was also investigated. The experimental results are shown in Figure 6B. It can be seen that when the pH value of the HAc-NaAc buffer increased from 3.21 to 6.60, the peak current of RhB continued to increase. However, when the pH value was higher than 6.60, the peak current began to decrease. Therefore, the optimal pH value of the HAc-NaAc buffer was 6.60. The effects of accumulation potential and time on 10 μM of RhB were also studied. Firstly, the accumulated potential was changed in the range from −0.3 to 0.3 V with a fixed accumulation time of 120 s. It was found that when the accumulated potential increased from −0.3 V to −0.1 V, the peak current increased, and then the peak current decreased dramatically with the further positive shift of the accumulation potential from −0.1 V to 0.3 V ( Figure 6C). Therefore, the best accumulation potential was chosen as −0.1 V. The effect of accumulation time on the oxidation peak current of RhB was investigated at −0.1 V. It was found that the peak current of RhB increased with the increase of accumulation time. However, increasing accumulation time after 120 s did not lead to significant changes in peak current ( Figure 6D). Therefore, considering the sensitivity and analysis speed, 120 s was chosen for quantitative analysis.

Interference Studies
In order to evaluate the selectivity of the Cu2O NPs-ERGO/GCE, the effect of some interfering substances on the electrochemical oxidation of RhB was studied. The response of the modified electrode to 10 μM of RhB in the presence of a 100-fold concentration of Na + , Zn 2+ , K + , Mg 2+ , Ca 2+ , Cu 2+ , Cl − , SO4 2− , glucose, sucrose, citric acid, and glycine were detected. The results showed that the

Interference Studies
In order to evaluate the selectivity of the Cu 2 O NPs-ERGO/GCE, the effect of some interfering substances on the electrochemical oxidation of RhB was studied. The response of the modified electrode to 10 µM of RhB in the presence of a 100-fold concentration of Na + , Zn 2+ , K + , Mg 2+ , Ca 2+ , Cu 2+ , Cl − , SO 4 2− , glucose, sucrose, citric acid, and glycine were detected. The results showed that the above species did not cause significant interference (signal change ≤ ±5%). In addition, the interference of other dyes was tested. The experimental results indicated that a 100-fold concentration of quinoline yellow; 10-fold concentration of amaranth, ponceau 4R, allura red, sunset yellow, and lemon yellow  Figure 7 (curve a) and the oxidation peak appeared at 135 mV (vs. an SCE). Compared with the peak of RhB in Figure 7 (curve b), it can be seen that the oxidation peak was located at the different potential position (970 mV), and it did not interfere with the other peaks. Curve c was the cyclic voltammogram of AA and an RhB mixture solution on the Cu 2 O NPs-ERGO/GCE, and two separated oxidation peaks appeared on the cyclic voltammogram with the potentials at 74 and 987 mV (vs. an SCE), which was attributed to that of AA and RhB, respectively (curve c). The oxidation peak potential separation was 913 mV for AA and RhB. These separations were large enough for simultaneous determinations of AA and RhB in the mixed solution. The above experimental data indicate that the proposed method has a good anti-jamming ability.
Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 15 above species did not cause significant interference (signal change ≤ ±5%). In addition, the interference of other dyes was tested. The experimental results indicated that a 100-fold concentration of quinoline yellow; 10-fold concentration of amaranth, ponceau 4R, allura red, sunset yellow, and lemon yellow did not interfere with the determination of 10 μM of RhB (signal change ≤ ±5%). Ascorbic acid(AA) is the main coexisting substance in food samples, so the electrochemical responses of RhB in the presence of AA on the Cu2O NPs-ERGO/GCE were studied. The typical cyclic voltammogram of AA on a Cu2O NPs-ERGO/GCE is shown in Figure 7 (curve a) and the oxidation peak appeared at 135 mV (vs. an SCE). Compared with the peak of RhB in Figure 7 (curve b), it can be seen that the oxidation peak was located at the different potential position (970 mV), and it did not interfere with the other peaks. Curve c was the cyclic voltammogram of AA and an RhB mixture solution on the Cu2O NPs-ERGO/GCE, and two separated oxidation peaks appeared on the cyclic voltammogram with the potentials at 74 and 987 mV (vs. an SCE), which was attributed to that of AA and RhB, respectively (curve c). The oxidation peak potential separation was 913 mV for AA and RhB. These separations were large enough for simultaneous determinations of AA and RhB in the mixed solution. The above experimental data indicate that the proposed method has a good anti-jamming ability.

Analytical Application
Under the optimum conditions, the second derivative linear sweep voltammetry was used to check the linear range and detection limit. Compared with square wave voltammetry (SWV) and differential pulse voltammetry (DPV), the main advantages of second derivative linear sweep voltammetry are a small background current and a sharp peak shape, both of which can significantly improve the sensitivity of determination and the overlapping resolution [47][48][49]. The second derivative linear sweep voltammograms of RhB at different concentrations are shown in Figure 8A. The linear range was between 0.01 and 20 μM. The linear regression equation can be expressed as ip = 1.3511 c + 0.959 (ip: μA, c: μM), and the correlation coefficient R 2 was 0.9979 ( Figure 8B). Because of the large active surface area and strong accumulation ability, the detection limit of RhB was as low as 0.006 μM.

Analytical Application
Under the optimum conditions, the second derivative linear sweep voltammetry was used to check the linear range and detection limit. Compared with square wave voltammetry (SWV) and differential pulse voltammetry (DPV), the main advantages of second derivative linear sweep voltammetry are a small background current and a sharp peak shape, both of which can significantly improve the sensitivity of determination and the overlapping resolution [47][48][49]. The second derivative linear sweep voltammograms of RhB at different concentrations are shown in Figure 8A. The linear range was between 0.01 and 20 µM. The linear regression equation can be expressed as i p = 1.3511 c + 0.959 (i p : µA, c: µM), and the correlation coefficient R 2 was 0.9979 ( Figure 8B). Because of the large active surface area and strong accumulation ability, the detection limit of RhB was as low as 0.006 µM.
In order to compare with other electrochemical methods for RhB determination, Table 1 summarizes the performance of different modified electrodes. The results show that this method can provide a comparable linear range and detection limit with other modified electrodes. In addition, the Cu 2 O NPs-ERGO/GCE described in our paper has the characteristics of fast response, simple electrode preparation, and good analytical performance.
The successive measurements of 10 µM of RhB were examined on a same Cu 2 O NPs-ERGO/GCE. Unfortunately, the oxidation peak current of RhB continued to decrease, mainly due to the strong surface adsorption and fouling. However, the used Cu 2 O NPs-ERGO/GCE could be recovered by cyclic scanning in a 0.1 M HNO 3 solution in the range of 0-1.2 V for three to five cycles at a scanning rate of 0.1 V s −1 . The relative standard deviation (RSD) was calculated to be 2.4% (n = 8). In addition, an RSD of 2.8% was obtained on 10 different Cu 2 O NPs-ERGO/GCEs. These results showed that the method has good rrepeatability and reproducibility. In addition, the long-term stability of the Cu 2 O NPs-ERGO/GCE was tested. The experimental results showed that the current response of the modified electrode was 94.28% of the initial value after two weeks, which indicated that the modified electrode has high stability and can be used for RhB determination. In order to compare with other electrochemical methods for RhB determination, Table 1 summarizes the performance of different modified electrodes. The results show that this method can provide a comparable linear range and detection limit with other modified electrodes. In addition, the Cu2O NPs-ERGO/GCE described in our paper has the characteristics of fast response, simple electrode preparation, and good analytical performance.

Detection of RhB in Real Samples
Using tomato juice, chili sauce, chili powder, and soy sauce as real samples, the practical application of the Cu 2 O NPs-ERGO/GCE was evaluated. The samples were obtained from a local supermarket. The preparation of sample solutions was done according to reference [14]. A standard additional method was used to obtain the the ttypical results. In order to verify the accuracy of this method, the content of RhB was also analyzed by high performance liquid chromatography (HPLC). The results determined by HPLC and the Cu 2 O NPs-ERGO/GCE are in good agreement (Table 2). In addition, a known amount of RhB was added to the sample solution, and a recovery test was carried out. The recoveries ranged from 96.3% to 103.0%, indicating that the Cu 2 O NPs-ERGO/GCE has good analytical performance and can meet the requirements of RhB determination in food samples.

Conclusions
A sensitive and simple electrochemical method for the determination of RhB was developed using a Cu 2 O NPs-ERGO/GCE. Based on the advantages of Cu 2 O NPs and ERGO, a Cu 2 O NPs-ERGO/GCE has high electrocatalytic activity for the oxidation of RhB. Under optimum conditions, RhB was determined by second-order derivative linear scan voltammograms with a wide linear range and a low detection limit. The method has good accuracy, acceptable precision, and reproducibility. This method provides a useful tool for on-site monitoring of RhB in food samples.