Determination of Bisphenol A in Beverages by an Electrochemical Sensor Based on Rh 2 O 3 / Reduced Graphene Oxide Composites

A novel electrochemical sensor, based on a Rh2O3–reduced graphene oxide (rGO) composite modified carbon electrode, has been developed for detecting bisphenol A (BPA) in beverages. The prepared Rh2O3/rGO and its precursor materials were characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). Under optimum conditions, the sensor presented good electrochemical performance for analyzing BPA, with a linear range of 0.6–40 μM, detection limit of 0.12 μM, good reproducibility, and excellent stability. The good performance can be attributed to the combination of the good catalytic properties of Rh2O3 and good conductivity of rGO. The sensor is directly used for detecting BPA in the residual solutions of four beverages after simple filtration, with satisfactory recoveries of 93–99%.


Introduction
Bisphenol A (BPA), one of the most produced and used chemicals, has been widely used for preparing industrial polymers, such as polycarbonate, epoxy resin, and thermosensitive paper [1].Furthermore, it is often involved in various food contact materials (i.e., canned packaging, bottles, and lacquer coating).Its widespread usage, especially in these food contact materials, has inevitably lead to its exposure to human beings via food and drinking water [2,3].As an endocrine-disrupting chemical (EDC) [4,5], it can destroy natural or synthetic compounds with endocrine function by simulating or blocking endogenous hormones [6].To date, a temporary tolerable daily intake (t-TDI) of 4 µg kg −1 b.w.day −1 for BPA has been proposed by the European Food Safety Authority (EFSA) [7].Legislation against BPA usage in some infant food package and bottles has also been enacted, in many countries all over the world [8].
Compared with the methods mentioned above, electrochemical methods, with the advantages of easy preparation, high sensitivity, simplicity for operators, and in-situ monitoring [33], are often proposed as one of the preferable techniques for routine analysis.In order to analyze trace levels of chemicals, improving the response signals of substances is a vital part of these electrochemical methods.Modification of the electrodes with various functional materials is one of the most recommended strategies.For example, electrodes modified with graphene nanomaterials present excellent electric conductivity as a result of their good conductivity [33][34][35].Molybdenum-selenide (MoSe 2 ) electrodes showed much more sensitivity to BPA, as a result of increasing surface active sites [36].Noble metal oxides, such as RuO 2 , PdO, IrO 2 , and Rh 2 O 3 , are widely used in heterogeneous catalysis systems.Rh 2 O 3 has been proven to be an excellent catalyst for the oxidation of CO [37,38] and the deposition of H 2 O 2 [39] and N 2 O [40], but it is still rarely reported for sensors.
In this paper, a Rh 2 O 3 -GO composite nanomaterial was chosen, and a novel electrochemical sensor was fabricated with a modified glassy carbon electrode (GCE) for excellent catalytic performance and conductivity.The modified glassy carbon electrode (GCE), coated with a Rh 2 O 3 -rGO membrane with excellent stability and selectivity, was synthesized and was then used for analyzing BPA dissolved in drinks for the first time after simple filtration.

Instruments
All electrochemical experiments were carried out on a CHI660e electrochemical workstation (Chenhua Instruments, Shanghai, China) with a conventional three-electrode cell.A saturated Ag/AgCl electrode was used as reference electrode, platinum wire as a counter electrode, and a bare glassy carbon electrode (GCE) (or modified GCE) as the working electrode.
The morphology of the prepared nanocomposites was characterized by scanning electron microscopy (SEM, Hitachi S-4800, Tokyo, Japan) and X-ray diffraction (XRD, DX-2700 Bruker D8 Advance).

Synthesis of Rh
Rh 2 O 3 nanoparticles were prepared, according to the method in the literature [41] with slight modification.Typically, RhCl 3 (63 mg) and poly (allylamine hydrochloride) (200 mg) were dissolved in 100 mL of water.The pH of the solution was adjusted to 7.0 by KOH.After that, 10 mL of a HCHO solution was added to the solution.Then, the reaction solution was transferred to a teflon-lined high-pressure vessel and heated at 120 • C for 6 h.Then, 15 mL of NaClO solution was added to the mixture and mechanically stirred for 72 h at room temperature.Finally, the suspension was centrifuged and then washed with water several times, to eliminate the residues and obtain Rh 2 O 3 -NPs.
Graphene oxide (GO) was prepared, according to previous reports [42].Briefly, graphite (0.5 g) and NaNO 3 (0.5 g) were added into concentrated H 2 SO 4 (23 mL) in an ice bath, followed by slow addition of 3 g KMnO 4 .The mixture was heated at 35 • C for two hours, and then 40 mL of H 2 O was added.After that, the reaction temperature was raised to 95 • C, kept for 30 min, and then followed by the addition of 100 mL H 2 O and 20 mL of H 2 O 2 .The final suspension was filtered, washed with 1.2 M of HCl and ultrapure water three times, and dried at 60 • C to obtain graphene oxide (GO).The schematic diagram of the preparation and action mechanism of the Rh 2 O 3 -GO/GCE is given as Scheme 1.The Rh 2 O 3 -NP solution was prepared by dispersing the above NPs into 10 mL water, then ultrasound treatment for 2 h.The GO suspension was dispersed into water to prepare 1 mg mL −1 GO solution.Then, 100 µL of Rh 2 O 3 nanoparticle solution was dispersed into 10 mL GO solution (1 mg mL −1 ).After ultrasound treatment for 30 min, the Rh 2 O 3 -GO nanoparticle composites were synthesized successfully.Before modifying the electrode, the GCE was polished to a mirror-like finish using 0.3 mm alumina slurry, and sonicated in distilled water and ethanol.The Rh 2 O 3 -GO solution (5 µL) was coated on the GCE surface and left to dry in air.Reduction of GO/GCE to rGO/GCE and Rh 2 O 3 -GO/GCE to Rh 2 O 3 -rGO/GCE was performed by cyclic voltammetry (CV) at a reduced potential of −1.2 V for 120 s in phosphate buffer solution.by the addition of 100 mL H2O and 20 mL of H2O2.The final suspension was filtered, washed with 1.2 M of HCl and ultrapure water three times, and dried at 60 °C to obtain graphene oxide (GO).

Preparation of Reduced Graphene Oxide-Rhodium Nanoparticle Electrode (Rh2O3-rGO/GCE)
The schematic diagram of the preparation and action mechanism of the Rh2O3-GO/GCE is given as Scheme 1.The Rh2O3-NP solution was prepared by dispersing the above NPs into 10 mL water, then ultrasound treatment for 2 h.The GO suspension was dispersed into water to prepare 1 mg mL −1 GO solution.Then, 100 μL of Rh2O3 nanoparticle solution was dispersed into 10 mL GO solution (1 mg mL −1 ).After ultrasound treatment for 30 min, the Rh2O3-GO nanoparticle composites were synthesized successfully.Before modifying the electrode, the GCE was polished to a mirror-like finish using 0.3 mm alumina slurry, and sonicated in distilled water and ethanol.The Rh2O3-GO solution (5 μL) was coated on the GCE surface and left to dry in air.Reduction of GO/GCE to rGO/GCE and Rh2O3-GO/GCE to Rh2O3-rGO/GCE was performed by cyclic voltammetry (CV) at a reduced potential of −1.2 V for 120 s in phosphate buffer solution.

Real Sample Preparation
Four beverage samples and one thermal paper sample were purchased from a local supermarket, to evaluate the performance of Rh2O3-rGO/GCE.After simple filtration for removing the fruit flesh, 800 μL beverage was mixed with PBS solution (9.2 mL) and directly analyzed by the proposed sensors.Thermal paper (1 g) was cut to pieces and extracted by ultrasound with 100 mL ethanol for 2 h.Then, 100 μL of extraction agent was mixed with PBS (9.9 mL) and analyzed by the proposed sensors.Afterward, the samples were spiked with BPA at three levels (5, 10 and 15 μM), and treated, following the above procedures, for recovery evaluation.

Material Characterization
Pure GO exhibited a typical silk fold surface (Figure 1a), corresponding to a much higher surface area than GO.As for Rh2O3-NP, it displayed uniform three-dimensional characteristics composed of network structures (Figure 1b).The Rh2O3 nanoparticles were wrapped by the gauze-like GO, while a relatively smoother surface was observed in the Rh2O3-GO composite, compared with that of the Rh2O3-NPs (Figure 1c).In XRD spectra of rGO (Figure 1d), the peak at 2θ = 28.4°belonged to the characteristic reflection of graphite (002).The wide diffraction peaks at 2θ = 40° and 70° can be attributed to the diffractions of ( 100) and (214) planes of Rh2O3 (Figure 1d).

Real Sample Preparation
Four beverage samples and one thermal paper sample were purchased from a local supermarket, to evaluate the performance of Rh 2 O 3 -rGO/GCE.After simple filtration for removing the fruit flesh, 800 µL beverage was mixed with PBS solution (9.2 mL) and directly analyzed by the proposed sensors.Thermal paper (1 g) was cut to pieces and extracted by ultrasound with 100 mL ethanol for 2 h.Then, 100 µL of extraction agent was mixed with PBS (9.9 mL) and analyzed by the proposed sensors.Afterward, the samples were spiked with BPA at three levels (5, 10 and 15 µM), and treated, following the above procedures, for recovery evaluation.

Material Characterization
Pure GO exhibited a typical silk fold surface (Figure 1a), corresponding to a much higher surface area than GO.As for Rh 2 O 3 -NP, it displayed uniform three-dimensional characteristics composed of network structures (Figure 1b).The Rh 2 O 3 nanoparticles were wrapped by the gauze-like GO, while a relatively smoother surface was observed in the Rh 2 O 3 -GO composite, compared with that of the Rh 2 O 3 -NPs (Figure 1c).In XRD spectra of rGO (Figure 1d), the peak at 2θ = 28.4• belonged to the characteristic reflection of graphite (002).The wide diffraction peaks at 2θ = 40 • and 70 • can be attributed to the diffractions of (100) and (214) planes of Rh 2 O 3 (Figure 1d).Totally, the XRD pattern of Rh 2 O 3 -rGO indicated the successful preparation of the Rh 2 O 3 -rGO composite.

Electrochemical Properties of the Modified Electrodes
Electrochemical characterizations of the electrodes were investigated using [Fe(CN)6] 3−/4− as probes.The cyclic voltammograms (CVs) of different electrodes in 1.0 mM [Fe(CN)6] 3−/4− solution containing 0.1 M KCl at a scan rate of 100 mV s −1 are shown in Figure 2A.The smaller peak-to-peak separations (Ep) of Rh2O3-rGO/GCE (90 mV) than that of bare GCE (149 mV) and rGO/GCE (148 mV) can be referred to the faster electron transfer on the surface of Rh2O3-rGO/GCE.Additionally, the redox current of rGO/GCE was much larger than GCE.When Rh2O3-NPs were modified on rGO, the peak current increased rapidly.This indicated that Rh2O3-NPs should be involved for the enhanced electron transfer rate.

Electrochemical Properties of the Modified Electrodes
Electrochemical characterizations of the electrodes were investigated using [Fe(CN) 6 ] 3−/4− as probes.The cyclic voltammograms (CVs) of different electrodes in 1.0 mM [Fe(CN) 6 ] 3−/4− solution containing 0.1 M KCl at a scan rate of 100 mV s −1 are shown in Figure 2A.The smaller peak-to-peak separations (Ep) of Rh 2 O 3 -rGO/GCE (90 mV) than that of bare GCE (149 mV) and rGO/GCE (148 mV) can be referred to the faster electron transfer on the surface of Rh 2 O 3 -rGO/GCE.Additionally, the redox current of rGO/GCE was much larger than GCE.When Rh 2 O 3 -NPs were modified on rGO, the peak current increased rapidly.This indicated that Rh 2 O 3 -NPs should be involved for the enhanced electron transfer rate.

Electrocatalytic Behaviors of BPA
The CVs were performed in 0.1 M PBS (pH = 7.0) solutions containing 20 µM BPA, to investigate the electrochemical behaviors of the prepared electrodes at a scan rate of 100 mV s −1 .There was only a relatively small oxidation peak (4.5 µA) at the potential of 0.57 V (Figure 2B, curve a).After coating with rGO (curve b), the peak current dramatically increased, due to the good adsorption efficiency of BPA onto rGO.Rh 2 O 3 -rGO/GCE (curve c) presented the greatest peak current of 30.5 µA, at the peak potential of about 0.52 V. Though peak potential shifted from 0.57 to 0.52 V, the results still indicated that Rh 2 O 3 -rGO could accelerate the electrocatalytic oxidation of BPA.This enhancement can be attributed to both the effective specific surface area of rGO, and the good electrocatalytic properties of Rh 2 O 3 -NPs.containing 0.1 M KCl at a scan rate of 100 mV s −1 are shown in Figure 2A.The smaller peak-to-peak separations (Ep) of Rh2O3-rGO/GCE (90 mV) than that of bare GCE (149 mV) and rGO/GCE (148 mV) can be referred to the faster electron transfer on the surface of Rh2O3-rGO/GCE.Additionally, the redox current of rGO/GCE was much larger than GCE.When Rh2O3-NPs were modified on rGO, the peak current increased rapidly.This indicated that Rh2O3-NPs should be involved for the enhanced electron transfer rate.

Effects of Solution pH
The electrochemical responses of BPA on prepared Rh 2 O 3 -rGO/GCE under different pH were also studied by CV (Figure 3).The oxidation peak current increased gradually with pH from 5.5 to 7.0, and then it began to decrease with the pH continually increasing to 8.5.So, in the following studies, a pH of 7.0 was selected for the supporting electrolyte.

Electrocatalytic Behaviors of BPA
The CVs were performed in 0.1 M PBS (pH = 7.0) solutions containing 20 μM BPA, to investigate the electrochemical behaviors of the prepared electrodes at a scan rate of 100 mV s −1 .There was only a relatively small oxidation peak (4.5 μA) at the potential of 0.57 V (Figure 2B, curve a).After coating with rGO (curve b), the peak current dramatically increased, due to the good adsorption efficiency of BPA onto rGO.Rh2O3-rGO/GCE (curve c) presented the greatest peak current of 30.5 μA, at the peak potential of about 0.52 V. Though peak potential shifted from 0.57 to 0.52 V, the results still indicated that Rh2O3-rGO could accelerate the electrocatalytic oxidation of BPA.This enhancement can be attributed to both the effective specific surface area of rGO, and the good electrocatalytic properties of Rh2O3-NPs.

Effects of Solution pH
The electrochemical responses of BPA on prepared Rh2O3-rGO/GCE under different pH were also studied by CV (Figure 3).The oxidation peak current increased gradually with pH from 5.5 to 7.0, and then it began to decrease with the pH continually increasing to 8.5.So, in the following studies, a pH of 7.0 was selected for the supporting electrolyte.

Effects of Scan Rate
To assess the electrocatalytic behaviors of BPA at Rh2O3-rGO/GCE, scan rates from 10 to 270 mV s −1 were studied (Figure 4).Oxidation peak current (I, μA) of BPA was linearly increased with the scan rate (Ʋ, mV s −1 ) with R 2 = 0.9938, and the relationship can be expressed by the linear regression Equation (1).

Effects of Scan Rate
To assess the electrocatalytic behaviors of BPA at Rh 2 O 3 -rGO/GCE, scan rates from 10 to 270 mV s −1 were studied (Figure 4).Oxidation peak current (I, µA) of BPA was linearly increased with the scan rate (υ, mV s −1 ) with R 2 = 0.9938, and the relationship can be expressed by the linear regression Equation (1).
The oxidation of BPA on the Rh 2 O 3 -rGO/GCE surface was an adsorption-controlled process.Meanwhile, the oxidation peak potential (E, V) increased linearly with natural logarithm of the scan rate (lnυ, mV s −1 ) with R 2 = 0.9931, and the Equation ( 2) is as follows: Appl.Sci.2018, 8, 2535 6 of 11 As for an adsorption-controlled and totally irreversible electrode process, E was defined by Equation ( 3) [43].
where α, k θ , n, υ, E θ , R, T and F represent transfer coefficient, standard rate constant of the reaction, electron transfer number (involved in rate determining step), scan rate, formal redox potential, the gas constant, the absolute temperature, and the Faraday constant, respectively.According to Equation ( 2), the value of RT/αnF was 0.0246.Generally, α is assumed to be 0.5 in a totally irreversible electrode process [44].Therefore, n was calculated to be 2.09, indicating that the electron transfer number for BPA oxidation is 2. The oxidation of BPA on the Rh2O3-rGO/GCE surface was an adsorption-controlled process.Meanwhile, the oxidation peak potential (E, V) increased linearly with natural logarithm of the scan rate (lnƲ, mV s −1 ) with R 2 = 0.9931, and the Equation ( 2) is as follows: As for an adsorption-controlled and totally irreversible electrode process, E was defined by Equation (3) [43].
where α, k θ , n, υ, E θ , R, T and F represent transfer coefficient, standard rate constant of the reaction, electron transfer number (involved in rate determining step), scan rate, formal redox potential, the gas constant, the absolute temperature, and the Faraday constant, respectively.According to Equation ( 2), the value of RT/αnF was 0.0246.Generally, α is assumed to be 0.5 in a totally irreversible electrode process [44].Therefore, n was calculated to be 2.09, indicating that the electron transfer number for BPA oxidation is 2.

Analytical Performance of the Rh2O3-rGO/GCE
As shown in Figure 5, a linear relationship between oxidation current and BPA concentration within the range of 0.6-40 μM could be expressed as:

Analytical Performance of the Rh 2 O 3 -rGO/GCE
As shown in Figure 5, a linear relationship between oxidation current and BPA concentration within the range of 0.6-40 µM could be expressed as: where I is oxidation peak current of BPA (µA), and C is the concentration of BPA in solution (µM).The limit of detection (LOD) was estimated to be 0.12 µM using the Equation ( 5) where k is a constant, SD is the standard deviation of the intercept, and b is the slope of the calibration plot.In this paper, the values of k, SD, and b are 3, 0.1315 and 3.1569, respectively.Compared with some other electrodes, based on gold and copper oxide-graphene nanoparticles [42,45], the Rh 2 O 3 -rGO/GCE presented comparable performance in similar linear detection ranges (Table 1).
where k is a constant, SD is the standard deviation of the intercept, and b is the slope of the calibration plot.In this paper, the values of k, SD, and b are 3, 0.1315 and 3.1569, respectively.Compared with some other electrodes, based on gold and copper oxide-graphene nanoparticles [42,45], the Rh2O3-rGO/GCE presented comparable performance in similar linear detection ranges (Table 1).
(a) (b)  The stability, reproducibility, and selectivity of the Rh2O3-rGO/GCE were also evaluated.To test the stability of prepared electrode, Rh2O3-rGO/GCE was stored at 4 °C and examined three times per week by CV.After two weeks, the peak current retained 77.2% of the initial response.The Rh2O3-GO solution displayed excellent stability, being deposited at 4 °C within 30 days.Using this Rh2O3-GO solution to prepare Rh2O3-rGO/GCE, the response current for detecting BPA still remained within 98% of the initial currents.The good stability and reproducibility indicated the sensor should be appropriate for routine measurements of real samples.The relative standard deviation (RSD) for detecting BPA (n = 7) by one as-prepared sensor was less than 2.73%.The RSD for seven Rh2O3-rGO/GCEs fabricated in parallel was less than 3.22%.
The i-t curves of bisphenol A, by the addition of different inorganic ions and BPA analogs, are shown in Figure 6.The concentrations of inorganic ions, BPA, and BPA analogs are 200 μM, 20 μM, and 20 μM, respectively.Common inorganic ions (Ba 2+ , Cu 2+ , Ca 2+ , K + , Na + , OH − , SO4 2− and Cl − ) have no effect on analyzing BPA, even when their levels are 10 times higher than BPA.Meanwhile, no obvious interferences from BPA analogs have been observed, such as phenol, hydroquinone, bisphenol B, bisphenol F, and bisphenol S. The RSDs for different interferences were all less than 3.53%.The stability, reproducibility, and selectivity of the Rh 2 O 3 -rGO/GCE were also evaluated.To test the stability of prepared electrode, Rh 2 O 3 -rGO/GCE was stored at 4 • C and examined three times per week by CV.After two weeks, the peak current retained 77.2% of the initial response.The Rh 2 O 3 -GO solution displayed excellent stability, being deposited at 4 • C within 30 days.Using this Rh 2 O 3 -GO solution to prepare Rh 2 O 3 -rGO/GCE, the response current for detecting BPA still remained within 98% of the initial currents.The good stability and reproducibility indicated the sensor should be appropriate for routine measurements of real samples.The relative standard deviation (RSD) for detecting BPA (n = 7) by one as-prepared sensor was less than 2.73%.The RSD for seven Rh 2 O 3 -rGO/GCEs fabricated in parallel was less than 3.22%.
The i-t curves of bisphenol A, by the addition of different inorganic ions and BPA analogs, are shown in Figure 6.The concentrations of inorganic ions, BPA, and BPA analogs are 200 µM, 20 µM, and 20 µM, respectively.Common inorganic ions (Ba 2+ , Cu 2+ , Ca 2+ , K + , Na + , OH − , SO 4 2− and Cl − ) have no effect on analyzing BPA, even when their levels are 10 times higher than BPA.Meanwhile, no obvious interferences from BPA analogs have been observed, such as phenol, hydroquinone, bisphenol B, bisphenol F, and bisphenol S. The RSDs for different interferences were all less than 3.53%.

Sample Analysis
The real samples spiked with BPA at 0-15 μM were successfully analyzed by the prepared Rh2O3-rGO/GCE.Before analysis, these drinks were filtered with an 0.45 μM hydrophilic membrane to remove the suspended particles.The detailed procedure has been described in Section 2.5.The recoveries of BPA from four kinds of beverages and thermal paper ranged from 93% to 98% (Table

Sample Analysis
The real samples spiked with BPA at 0-15 µM were successfully analyzed by the prepared Rh 2 O 3 -rGO/GCE.Before analysis, these drinks were filtered with an 0.45 µM hydrophilic membrane to remove the suspended particles.The detailed procedure has been described in Section 2.5.The recoveries of BPA from four kinds of beverages and thermal paper ranged from 93% to 98% (Table 2), indicating that the prepared Rh 2 O 3 -rGO modified electrode should be a useful sensor for analyzing BPA in aqueous solutions containing relatively complex interferences.

Scheme 1 .
Scheme 1. Schematic diagram for electrochemical synthesis of the Rh2O3-rGO electrode, and the electrochemical oxidation process of BPA by Rh2O3-rGO/GCE.

Scheme 1 .
Scheme 1. Schematic diagram for electrochemical synthesis of the Rh 2 O 3 -rGO electrode, and the electrochemical oxidation process of BPA by Rh 2 O 3 -rGO/GCE.

Figure 3 .
Figure 3. (a) CVs of Rh2O3-rGO/GCE detected BPA with different pH; (b) effects of pH on the peak current of BPA at Rh2O3-rGO/GCE.

Figure 3 .
Figure 3. (a) CVs of Rh 2 O 3 -rGO/GCE detected BPA with different pH; (b) effects of pH on the peak current of BPA at Rh 2 O 3 -rGO/GCE.

Figure 4 .
Figure 4. (a) CVs of Rh2O3-rGO/GCE detected 20 μM BPA with scan rate from 10 to 270 mV s −1 ; (b) Dependence of the oxidation peak current on the scan rate; (c) The relationship between E and ln Ʋ.

Figure 4 .
Figure 4. (a) CVs of Rh 2 O 3 -rGO/GCE detected 20 µM BPA with scan rate from 10 to 270 mV s −1 ; (b) Dependence of the oxidation peak current on the scan rate; (c) The relationship between E and lnυ.

Figure 5 .
Figure 5. (a) CVs of Rh2O3-rGO/GCE with BPA concentration from 0.6 to 40 μM; (b) Relationship between the oxidation peak current and the concentration of BPA (from 0.6 to 40 μM).

Figure 5 .
Figure 5. (a) CVs of Rh 2 O 3 -rGO/GCE with BPA concentration from 0.6 to 40 µM; (b) Relationship between the oxidation peak current and the concentration of BPA (from 0.6 to 40 µM).

Appl. Sci. 2018, 8 ,Figure 6 .
Figure 6.The i-t curves of bisphenol A by the addition of (a) different inorganic ions and (b) BPA analogs.

Figure 6 .
Figure 6.The i-t curves of bisphenol A by the addition of (a) different inorganic ions and (b) BPA analogs.

Table 1 .
Comparison of the Rh2O3-rGO and other sensors for BPA determination.

Table 1 .
Comparison of the Rh 2 O 3 -rGO and other sensors for BPA determination.

Table 2 .
The results of determined BPA in practical samples.