Simultaneous electroanalytical sensing of dopamine and epinephrine using TiO2/reduced graphene oxide nanocomposite modified glassy carbon electrode

INTRODUCTION
The neurotransmitters dopamine (DA), norepinephrine (NE), and epinephrine (EP), belonging to the class of catecholamines, play essential roles in the central nervous system [1]. Given their crucial roles in mammals, they are of significant importance. Catecholamines, which are released into the blood during the body’s stress response, are generated in the adrenal glands, brainstem, and the brain. Functioning as hormones, they are broken down after only a few minutes [2], and then excreted in the urine [3]. Thus, the concentration of catecholamines must be monitored in biological fluids for neurochemical and medical purposes since they can signal the emergence of different diseases such as Parkinson’s disease, HIV, schizophrenia, tumors, diabetes, and cirrhosis [4-6].
Different methods including chromatography [7, 8], mass spectroscopy [9, 10], capillary electrophoresis [11], and electrochemical approaches [12, 13] have been used to determine catecholamines. Consequently, identifying catecholamines using modified electrodes via distinct electrochemical techniques has been increasing compared with other techniques, because the method does not need any sample pre-treatment and can be conducted in situ. Further, this method is highly sensitive, less time-consuming, and economical [14, 15].
The structures of DA and EP are similar, and these compounds often coexist in biological samples. Therefore, the simultaneous determination these species is difficult by the electro-oxidation process due to their mutual interference. Thus, developing an effective electrochemical sensor is of significant importance to monitor and determine catecholamines in authentic samples [3].
As a two-dimensional carbon sheet, atoms can refer to graphene, bonded by sp2 hybridization [16, 17]. Graphene has been widely used in manufacturing devices based on biosensors and sensors due to its high specific surface area, high mechanical strength, high thermal and electrical conductivity, and low cost [18, 19]. For example, Lee et al. made a nano-electrode from gold nanoparticles and graphene sheets coated on a glassy carbon electrode for determining dopamine. There are several methods for preparing graphene, including the reduction of graphene oxide (GO). Techniques such as chemical reduction [20], thermal reduction [21], and electrochemical reduction [22] can be used to reduce graphene. GO reduction is widely used for synthesizing graphene.
Peak current, sensitivity, and reproducibility are improved by preparing a working electrode using nanoparticles. Developing novel electrode surface modifiers is essential in current science and technology [23, 24]. Metal nanoparticles (MNPs) have been extensively used to develop electrochemical sensors by forming novel compounds with different properties. MNPs facilitate the electron transfer process on the surfaces of compounds used in electrocatalysis. Many metal nanoparticles, such as SiO2, Au, Cr2O3, CeO2, MnO2, and TiO2, have been applied to construct biosensors [25-29]. Among them, TiO2 nanoparticles have attracted considerable interest due to their superior properties, such as their large specific surface area, inexpensiveness, high uniformity, relatively high electrical conductivity, long-term stability, and good biocompatibility [30-32].
Graphene sheets can be used as good support for TiO2 nanoparticles due to their large surface area and unique electronic properties, leading to increased electrocatalytic activity and attaining a uniform distribution without association. Recent studies have shown that graphene oxide/TiO2 nanocomposite improves the electrochemical performance by providing a low-resistance charge transfer conduction pathway through the film. These results indicate that GR–TiO2 film is a suitable electrode material for electrochemical sensing applications [33].
A chemical method for the synthesis of reduced graphene oxide/TiO2 nanocomposite modified glassy carbon electrode is reported in this work, which will be used for the simultaneous determination of two catecholamines, dopamine and epinephrine, in real samples. The as-prepared RGO/TiO2 nanocomposite exhibits significant electrocatalytic activity towards dopamine and epinephrine reduction. These two species can be simultaneously detected on the RGO/TiO2 nanocomposite modified GCE with high selectivity in a wide linear range. This work presents an easy and efficient method to prepare RGO/TiO2 nanocomposite for electrochemical sensor applications.

EXPERIMENTAL
Chemical and Reagents
Dopamine (DA) and epinephrine (EP) were purchased from Sigma-Aldrich. Sulfuric acid (H2SO4), phosphoric acid (H3PO4), acetic acid (HOAC), sodium hydroxide (NaOH), sodium borohydride (NaBH4), titanium dioxide (TiO2), boric acid (H2BO3), and dimethylformamide (DMF) were from Merck. The graphene oxide powder with the purity of 99% was supplied from Nanoscale. All of these materials were analytical grade and used without re-purification. Additionally, all the solutions were prepared with distilled water. Britton–Robinson buffer solution (BRS, 0.04 M, pH 7.0) was employed as a supporting electrolyte, and all the experiments were conducted at room temperature.

Apparatus
A Metrohm Autolab B. V.s Autolab PGSTAT 101 potentiostat/galvanostat (Utrecht, UT, the Netherlands) was used for electrochemical measurements, including CV and DPV. A three-electrode system including a glassy carbon electrode (GCE) with a diameter of 2.5 mm as the working electrode, platinum wire as the counter electrode, and Ag/AgCl as the reference electrode was applied (from the Azar electrode, Urmia, Iran). Field emission scanning electron microscopy (MIRA 3, TESCAN, Czech Republic) was used at 20 kV accelerating voltage to observe the morphology of the studied surfaces. Raman tests by a Thermo Nicolet Dispersive Raman Spectrometer were performed (equipped to a 532 nm Laser beam at 30 mW; a charge-coupled device detector with a 4 cm-1 resolution). X-ray diffraction (XRD) (Model: X’Pert MPD, Company: Philips, Netherlands) was utilized for studying the crystal structure. Xray photoelectron spectroscopy (XPS) was performed using a Gammadata-Scienta ESCA 200 hemispherical analyzer equipped with an Al Ka X-ray source (1486.6 eV) with a monochromator. The binding energies were calibrated based on the C 1s peak at 285 eV.

Preparation of RGO and RGO/TiO2 nanocomposite
The following procedure was implemented for preparing the reduced graphene oxide. First, 100 mg of graphene oxide was mixed with 75 ml of water and placed in an ultrasonic bath for 1 hour. Then, 200 mg of NaBH4 was added to this solution and stirred vigorously for 30 minutes; the solution was then heated at 135 ° C for 6 hours. After that, it was centrifuged, washed with distilled water, dried by heating at 60 ° C for 6 hours, and named RGO [34]. 
As shown in scheme 1, RGO/TiO2 was synthesized through a two-step method. At first, 0.05 g of RGO and 0.02 g of TiO2 were separately added to ethanol, each followed by ultrasonication for 30 min. Then, RGO and TiO2 solutions were mixed and sonicated for 30 min, followed by half an hour of stirring. After that, the solution was refluxed for 5 hours at 70˚C and centrifuged after cooling. The final product was washed with ethanol, dried by heating at 60 °C for 3 hours, and named RGO/TiO2 nanocomposite.

Fabrication of the modified electrodes
First, the glassy carbon electrode (GCE) was thoroughly polished onto a soft cloth with 0.05 μm alumina powder. Then, the GCE was placed in a solution of distilled water and sulfuric acid at a ratio of 1:10 for 5 minutes in an ultrasonic bath to thoroughly clean the alumina particles on the electrode surface. After that, it was completely washed and utilized in a 0.1 M sulfuric acid solution in a cyclic range of -1.0 to 1.0 V vs. Ag/AgCl to obtain reproducible voltammograms. 
0.5 mg of RGO and 0.5 mg of RGO/TiO2 were dissolved separately in 0.5 ml DMF and placed in an ultrasonic bath for 1 hour to open the graphene plates [35, 36]. Then, 8 µL of these solutions were dropped on the surface of the clean bare GCE to modify the surface of the electrode. These modified electrodes were named GCE/RGO and GCE/RGO/TiO2.

RESULTS AND DISCUSSION
Characterization of the RGO/TiO2 nanocomposite
Field emission scanning electron microscopy was used to evaluate the structure, amount, and manner of particle aggregation and average particle size, as well as the presence of amorphous materials in the synthesized nanocomposite. Fig. 1A shows the structure and wrinkle-like morphology of the chemically reduced graphene. Graphene has a layered structure that forms intertwined graphene plates which have folds and waves and increase surface conductivity. Fig. 1B indicates the structure of the RGO/TiO2 nanocomposite. Titanium dioxide nanoparticles with a particle size of about 140-180 nm are evenly and compactly dispersed on the graphene plates [37]. As indicated in Fig. 1C, the distribution of particles is homogeneous, within which the graphene nanosheets are hardly visible. Furthermore, the surface of these nanosheets is saturated with TiO2 nanoparticles, which, due to their conductivity, can change the stability and sensitivity of the modified electrode to oxidize and reduce biological molecules.
Energy-dispersive X-ray spectroscopy (EDX) and elemental mapping spectroscopy were recorded to confirm and investigate the uniform distribution of elements in the RGO/TiO2 nanocomposite structure (Fig. 2(A-E)). The results indicate that the elements carbon (C), oxygen (O), and titanium (Ti) are distributed in the nanocomposite structure.
The FT-IR spectra of GO, RGO, and RGO/TiO2 nanocomposite are shown in Fig. 3. The presence of different types of oxygenated functional groups in the FT-IR spectrum of graphene oxide (curve (A)) confirms the successful oxidation of graphite. The stretching and bending vibrations of the hydroxyl (O-H) groups of water-adsorbed molecules on graphene oxide can be proved by the presence of a wide peak at 3420 cm-1. These polar groups form a hydrogen bond between graphene oxide and water molecules, confirming that the nature of graphene oxide is hydrophilic. The stretching vibrations of the C=C groups in the benzene ring (non-oxidized graphite skeleton vibrations) and the C=O groups of Carboxylic and carbonyl ions present at the edges of graphene oxide appeared in the middle frequency regions, at 1649 and 1699 cm-1, respectively. The two absorption peaks at 1421 and 1458 cm-1 are attributed to the C-OH stretching vibrations of the carboxyl groups. Additionally, the sharp peak in 1064 cm-1 is consistent with the C-O stretching vibrations of CO2 and alkoxy groups. Finally, the absorption peak at 1170 cm-1 is ascribed to the stretching vibrations of the epoxy groups. The absorption peaks at 2854 and 2925 cm-1 demonstrate the symmetric and asymmetric stretching vibrations of CH2. These results confirm that the oxygenated functional groups are present on the surface of graphene oxide [38].
In the FT-TR spectrum of the RGO (curve (B)) the peaks at 1421 and 1458 cm-1 belonging to the C-OH stretching vibrations of the carboxyl groups have almost disappeared. Furthermore, the peaks at 1649 and 1170 cm-1 are disappeared, which belong to the stretching vibrations of the C=C groups of the benzene ring (vibrations of the non-oxidized graphite skeleton) and epoxy groups, respectively. These results confirm the successful reduction of graphene oxide to graphene.
In the FT-IR spectrum of the RGO/TiO2 nanocomposite (curve (C)) shifts are observed in several peaks with the change in peak intensity compared to RGO. Moreover, the presence of a peak at 670 cm-1 proved TiO2 presence. All these observations confirm the accuracy of RGO/TiO2 nanocomposite synthesis.
Raman spectroscopy is a helpful technique for surveying the chemical structures of carbon-based materials. Fig. 4 shows the Ramon spectra of RGO and RGO/TiO2 in the range of 0-3000 cm-1. Curve (A) indicates the spectrum of reduced graphene oxide in which two sharp and intense peaks at 1345 and 1593 cm -1 belong to the D and G bands, respectively. The G-band corresponds to the intraplate vibrations of the sp² hybrid in carbon chains, and the D-band indicates the sp3 carbon [38]. The D-to-G bond strength ratio indicates the size of the sp² chains and the structural irregularities in the graphene sheets. In the RGO Raman spectrum, the ratio of the D to G band is 1.7. 
Curve (B) the Ramon spectrum of RGO/TiO2, demonstrates that the ratio of the D to G band is 1.3, which decreases compared to RGO, indicating an increase in sp² chain size and a decrease in structural irregularities in reduced graphene oxide. This is a reason for the interaction between the reduced graphene oxide and the titanium dioxide nanoparticles. Further, in RGO/TiO2 nanocomposite, in comparison to RGO, the D and G band have shifted from 1345 to 1337 cm-1 and 1593 to 1575 cm-1, respectively. These shifts towards lower wavenumbers indicate an interaction between reduced RGO and TiO2 nanoparticles [39]. Furthermore, in the RGO/TiO2 Raman spectrum, there are peaks at 205, 424, and 599 cm-1, corresponding to Eg, B1g, and A1g, respectively. These peaks represent the anatase phase structure of TiO2 and the presence of a peak at 2648 cm-1, indicating the presence of RGO as monolayer graphene in the nanocomposite structure [40].
The crystal structure of the nanocomposite was investigated by X-ray diffraction (XRD). Fig. 5 shows the XRD spectrum of the RGO/TiO2 nanocomposite in which the peaks located at 2Ɵ values equal to 42.2°, 45°, 48.3°, 51.6°, 64.0°, 66.9°, 74.4°, 76.0° correspond to the (004), (100), (200), (105), (204), (116), (220), and (215) planes of RGO/TiO2 nanocomposite, respectively [37, 40]. The peak observed at 45.9° is related to the reduced graphene oxide, and the other peaks are attributed to the structure of the TiO2 anatase form. The peaks at 31° and 32° are related to the NaBH4 which was used to reduce the GO to RGO.
XPS is a powerful tool for identifying the states of elements in bulk materials. Fig. 6 shows the XPS spectra of the RGO/TiO2 nanocomposite. Fig. 6A indicates the survey spectrum of RGO/TiO2 nanocomposite and the surface of this sample containing the elements C, O, and Ti. The spectrum C 1s, shown in Fig. 6B, can deconvolve into five types of carbon bonds in the RGO/TiO2 nanocomposite including 284.23 eV (C-C (graphite carbon)), 285.28 eV (C-OH), 286.41 eV (C-O), 287.58 eV (C=O), and 288.42 eV (O-C=O) [41]. These results confirm the presence of carbon in the RGO/TiO2 nanocomposite. The O 1s spectrum (Fig. 6C) can deconvolve into three peaks at 530.76 eV, 532.14 eV, and 533.44 eV, which correspond to Ti-O, HO-C=O, and C-OH, respectively. These results confirm the presence of oxygen in the RGO/TiO2 nanocomposite. Additionally, the peak observed at 530.76 eV can be related to oxygen networks in TiO2 because oxygen in metal oxides usually appears in the range of 530-532 eV. Fig. 6D shows the deconvolved spectrum for Ti 2p with two peaks in the range of 458.02 eV and 463.84 eV, corresponding to 2p3/2 and 2p1/2, respectively [42].

Electrochemical behavior of DA and EP at the surface of various electrodes
The electrochemical behavior of DA (Fig. 7A), EP (Fig. 7B), and simultaneous detection of DA and EP (Fig. 7C) were investigated on the bare GCE, GCE/RGO, and GCE/RGO/TiO2 in BRS 0.04 M with pH = 7.0 by cyclic voltammetry. DA manifested a pair of redox peaks (ΔEp=310 mV) with the cathodic peak current of -1.45 µA at the bare GCE (Fig. 7A). The CV DA at GCE/RGO and GCE/RGO/TiO2 has two of redox peaks with the potential differences (ΔEp) of 110 mV and 80 mV, respectively. Moreover, the peak intensity significantly increased, which can be attributed to the large specific area of RGO and the electrocatalytic performance of TiO2 nanoparticles. For the GCE/RGO/TiO2, a sharp cathodic peak current of -22.42 µA was observed, approximately 15.5 times larger than the bare GCE. The enhanced electrocatalytic feature may be due to the synergistic effect of RGO and TiO2 nanoparticles. 
Similar results for the electrochemical reaction of EP are also observed in Fig. 7B. Only a small cathodic peak of EP was found at the bare GCE while a pair of EP peaks for GCE/RGO and GCE/RGO/TiO2 were observed, the cathodic peak potential of which were -0.228 and -0.218 V, respectively; the peak currents were about -13.80 and -19.63 µA. 
The simultaneous detection of DA and EP was also performed (Fig. 7C). At bare GCE, two cathodic peaks for DA (0.144 V) and EP (-0.263 V) with small current intensity were detected. The cathodic peak currents of DA and EP significantly increased for the GCE/RGO and GCE/RGO/TiO2, and the cathodic peak potentials positively shifted. Moreover, two separated reduction peaks were obtained for DA (0.155 V) and EP (-0.233 V); the peak currents were more than 30-fold higher for DA (Ipc = -18.4 µA) and seven-fold higher for EP (Ipc = -28.3 µA) when compared to that of bare GCE. The significant potential separation between DA and EP (0.388 V) allows for the simultaneous determination of DA and EP in mixture samples without interference from the two catecholamines [43]. The composite of RGO and TiO2 with high conductivity may offer more active sites and higher surface area, which may be beneficial for electrocatalysis. Moreover, increasing current could be explained by the interaction between the TiO2 nanoparticles and the –OH groups of DA and EP, leading to an increase in the analyte concentration on the surface of the electrode [44].

Optimization of the experimental conditions
One of the most important and effective parameters in studying the electrochemical behavior of EP and DA is pH. CVs were recorded in a solution of EP and DA at a concentration of 0.5 mM to investigate the pH. As shown in Fig. 8A and B, the maximum currents were observed at the pH of 6.0 and 5.0 for EP and DA, respectively. Further, the potential of both species shifted to a negative value with an increase in pH from 4.0 to 9.0. This negative shift in the potential indicates that protons are involved in the EP and DA reactions. The reduction peak potential obeys the following equations Epc = - 0.059pH + 0.5856 (R2 = 0.9981) and Epc = -0.0522 pH + 0.1793 (R2 = 0.9963) for DA and EP, receptively. Due to the proximity of the slopes (-0.059 and -0.0522 for DA and EP, respectively) to the theoretical value of -0.059 mV per unit pH, we can conclude that an equal number of electrons and protons were involved in the electrochemical oxidation of EP and DA. Since our goal is to determine of EP and DA at the physiological pH of the body, all the electrochemical tests are performed at the pH of 7.0.
The effect of the scan rate on the electrochemical behavior of 0.5 mM DA and EP in BRS (0.04 M, pH = 7.0) was investigated. Cyclic voltammograms recorded at different scan rates in the range of 20-200 mVs-1 at GCE/RGO/TiO2 surface are shown in Fig. 9A and B, for DA and EP, respectively. As shown, both currents of anodic and cathodic peaks of DA and EP were enhanced with increasing the scan rate. The linear relationship between the anodic and cathodic peak currents and the squqre root of the scan rate indicates that the electrochemical reactions of both species are under diffusion control, and the GCE/RGO/TiO2 surface is not destroyed by oxidation and reduction of these compounds.

Individual and simultaneous determination of DA and EP
Differential pulse voltammetry (DPV) was conducted to measure DA and EP reduction peaks separately by changing the concentration of each species at the GCE/RGO/TiO2 surface (Fig. 10). DPV curves for the individual detection of DA and EP at GCE/RGO/TiO2 are shown in Fig. 10A and, respectively. B. As their concentrations rise, their currents increase, and the reduction potential remained constant. As shown in Fig. 10A, there are two linear relationships for DA ranging 3-180 and 180-1000 µM with linear equations calibrated as: I (µA) = -0.0561CDA (µM) – 1.2506, (R2 = 0.9904) and I (µA) = -0.0026CDA (µM) – 10.408, (R2 = 0.995). Based on Fig. 10B, the reduction peak currents of EP increases with the increase of EP concentration in the two linear relationships ranging from 4-160 and 160-1000 µM with linear equations calibrated as: I (µA) = -0.0346CEP (µA) – 1.5286, (R2 = 0.9904) and I (µA) = -0.0044CEP (µA) – 5.8838, (R2 = 0.9977).
Furthermore, DPV was investigated at GCE/RGO/TiO2 for the simultaneous determination of DA and EP. In a mixture of DA and EP, when the concentration of either DA or EP was changed, the other three (GCE/RGO/TiO2) remained constant. As shown in Fig. 11A, the reduction peak currents of DA containing 15 µM EP were enhanced with the increase of the DA concentration and two linear ranges were 3-180 and 180-1000 µM with linear equations calibrated as: I (µA) = -0.048CDA (µM) – 1.6935, (R2 = 0.9903) and I (µA) = -0.0026CDA (µM) – 9.5644, (R2 = 0.993). Fig. 11B shows DPV responses for different concentrations of EP in the presence of a fixed concentration of 15 µM DA. The reduction peak currents of EP increases linearly with the elevation of the EP concentration in two ranges of 5-170 and 170-1000 µM. The linear equations for EP were obtained as: I (µA) = -0.0169CEP (µM) -2.3889, (R2 = 0.9969) and I (µA) = -0.0051CEP (µM) – 4.5428, (R2= 0.9915). The detection limits (S/N = 3) for DA and EP were 1.6 and 1.7 µM, respectively. 
Fig. 11C shows the DPVs of DA and EP, the concentrations of which increased simultaneously in 0.04 M BRS at pH 7.0. The electrochemical responses of the two species increased linearly with their concentrations. The results indicated that the proposed electrode enabled the simultaneous determination of the two species, and the response of peak currents of DA and EP at GCE/RGO/TiO2 were linear with the concentration of each in the ranges of 5-180 and 180-1000 µM for DA and 5-20 and 20-1000 µM for EP. As a result, the linear regression equations for DA and EP were Ip (µ) = -0.0503CDA (µM) – 1.7779 (R2 = 0.9902), Ip (µ) = -0.0087CDA (µM) – 9.0808 (R2 = 0.9946), Ip (µA) = -0.022CEP (µM) – 2.1506(R2 = 0.9955), and Ip (µA) = -0.0069CEP (µM) -2.5666 (R2 = 0.9979). The limits of detection for DA and EP were 1.3 µM and 1.4 µM (S/N = 3). 
The linear dynamic range and detection limit of this sensor with other sensors were compared, and the results are listed in Table 1.

Interference, Reproducibility, and Stability of the electrode
Possible interferences in the detection of DA and EP at GCE/RGO/TiO2 by adding different species (500 µM of ascorbic acid, uric acid, hypoxanthine, xanthine, tryptophan, lysine, glutamic acid, glucose) to BRS ((0.04 M, pH = 7.0)) in the presence of 10 µM of DA and EP (n=3) were investigated in order to evaluate the selectivity of the proposed method. The tolerance limit, defined as the maximum concentration of the interfering substance, resulted in an error of less than ±5% for determining DA and EP. The results showed that the interferences are only significant at relatively high concentrations, and we can conclude that this sensor is free from common interfering species.
The reproducibility of GCE/RGO/TiO2 was evaluated for the detection of DA, and EP. The standard deviations (RSD) for the five different electrodes produced under the same conditions were 1.20% and 2.30% for DA and EP, respectively, suggesting that GCE/RGO/TiO2 is highly reproducible.
The stability of the fabricated electrode was investigated by storing it in the air at room temperature for two weeks. The peak current maintained at 98.2% for DA and 97.0% for EP from its initial value. These results suggest that the GCE/RGO/TiO2 has good selectivity, reproducibility, and stability.
Validation of proposed sensor using real samples
GCE/RGO/TiO2 was utilized for determining DA and EP in human blood serum and Epinephrine ampoule (1 mg ml-1) to evaluate the reliability of the method for the analysis of DA and EP in pharmaceutical products. For the purpose, appropriate amounts of diluted samples were transferred into the electrochemical cell using DPV. The analytical results are summarized in Table 2. The recovery ranged from 90.0% to 104.2% for DA and 90.0% to 102.7% for EP; the RSD (n=3) was less than 3.0%. The results are acceptable, demonstrating that the proposed method can be used effectively to determine DA and EP in real samples.

CONCLUSIONS
In this work, we synthesized RGO/TiO2 nanocomposite by a chemical method and used it to modify a GCE. Thus, we built a sensor and used it for the simultaneous determination of EP and DA. The proposed sensor had excellent characteristics such as high levels of sensitivity, selectivity, and reproducibility, and with an acceptable detection limit and linear dynamic range. The structure of the RGO/TiO2 nanocomposite was investigated by FE-SEM, EDX, FTIR, Raman spectroscopy, XRD, and XPS. In addition, the sensor was successfully applied for determining EP, and DA in pharmaceutical formulation samples.

ACKNOWLEDGMENT 
The authors gratefully acknowledge partial financial support from the Research Council of Alzahra University.