Determination of Uric Acid in Co-Presence of Dopamine and Ascorbic Acid Using Cuprous Oxide Nanoparticle-Functionalized Graphene Decorated Glassy Carbon Electrode

Cuprous oxide nanoparticles (Cu2O NPs) were dispersed into a graphene oxide (GO) solution to form a homogeneous Cu2O-GO dispersion. After this, the cuprous oxide nanoparticles were functionalized to electrochemically reduce the graphene oxide decorated glassy carbon electrode (Cu2O-ErGO/GCE). This was prepared by coating the Cu2O-GO dispersion onto the surface of the glassy carbon electrode (GCE), which was followed by a potentiostatic reduction process. An irreversible two-electron reaction of uric acid (UA) was observed at the voltammetric sensor. Moreover, the high concentrations of dopamine (DA) and ascorbic acid (AA) hardly affected the peak current of UA, which suggested that Cu2O-ErGO/GCE have excellent selectivity for UA. This is probably because the response peaks of the three compounds are well-separated from each other. The oxidation peak current was proportional to the concentration of UA in the ranges of 2.0 nM−0.6 μM and 0.6 μM−10 μM, respectively, with a low limit of detection (S/N = 3, 1.0 nM) after an accumulation time of 120 s. Cu2O-ErGO/GCE was utilized for the rapid detection of UA in human blood serum and urine samples with satisfactory results.


Introduction
Uric acid (UA) is a metabolite of nucleoprotein and nucleic acid.Having excessive amounts of UA in the human body can cause gout, renal failure and congenital hyperuricemia [1,2].Therefore, the quantitative analysis of UA in the physiological fluids is of great importance in drug control and clinical diagnosis.Various methods have been developed for UA detection, such as spectrofluorometry [3], chromatography [4,5], enzymatic methods [6,7], chemiluminescence [8] and electroanalytical methods.Among these techniques, electrochemical methods are very suitable for the practical detection of UA due to their unique superiorities, including low cost, high sensitivity and good selectivity.The electrochemical determination of UA is commonly based on monitoring the oxidation signal of UA on the electrode.The electrooxidation process of UA is illustrated in Scheme 1 [9].However, it is susceptible to interference from coexisting species, such as dopamine (DA) and ascorbic acid (AA), Scheme 1.The reaction mechanism of UA.
In the past few years, transition metals and metal oxides have received increasing attention in many fields, such as sensors and electrocatalysis, due to their unique sensing performance and superior electrocatalytic activity [9,11,23,24].Among these metal oxides, cuprous oxide (Cu2O) is a new type of p-type semiconductor with a narrow band gap, which can also be easily excited by visible light.Due to their outstanding advantages, such as non-toxic, low cost and stable photochemical properties, Cu2O nanoparticles have been extensively used in many fields, such as new energy, photocatalytic degradation, sterilization and sensing [25][26][27].The different morphologies of Cu2O nanomaterials, such as hollow spheres, cubes, wires, octahedrons and cages, can be synthesized by adjusting the synthesis conditions [28].At present, various electrochemical sensors based on Cu2O nanoparticles have been constructed for the detection of H2O2 [29,30], glucose [30][31][32], dopamine [33][34][35], herbicide paraquat [36], L-tyrosine [37], vanillin [38], sunset yellow [39] and NO2 [40].As far as we know, the use of Cu2O for the determination of UA has rarely been reported in the literature.However, Cu2O nanoparticles are easily agglomerated, which greatly limits their application in electrochemical sensors.How to reduce the agglomeration of nano-semiconductors is an urgent problem to be solved.
Graphene (GR) is a single-layer two-dimensional planar crystal with the specific surface area of 2630 m 2 /g, which enables it to be an ideal template for GR-based composites in the fields of battery, photocatalysis, sensing and so on [41].Xu et al. [29] successfully prepared Cu2O-reduced graphene oxide nanocomposites using three various methods (namely physical adsorption, in-situ reduction and one-pot synthesis).The composites were dispersed in 0.1% nafion solution and decorated on the glassy carbon electrode (GCE) by a drop-coating technique, which were constructed for nonenzymatic hydrogen peroxide sensing.Cu2O/GR nanocomposites were also successfully prepared by a solvothermal method [33], with Cu2O/GR/GCE having showed good electrocatalytic activity towards DA.As result, the proposed Cu2O/GR/GCE exhibited excellent sensing performances in terms of linear range (0.1−10 μM) and limit of detection (LOD, 10 nM).Ye et al. [36] developed a novel paraquat sensor based on Cu2O/polyvinyl pyrrolidone-functionalized GR nanosheet nanocomposite.However, the preparation of the above GR-based composites is complicated, which greatly affects the use of sensors in practical applications.Therefore, the development of a green routine for the creation of Cu2O-GR composite-based electrochemical sensors is very crucial.In the past few years, transition metals and metal oxides have received increasing attention in many fields, such as sensors and electrocatalysis, due to their unique sensing performance and superior electrocatalytic activity [9,11,23,24].Among these metal oxides, cuprous oxide (Cu 2 O) is a new type of p-type semiconductor with a narrow band gap, which can also be easily excited by visible light.Due to their outstanding advantages, such as non-toxic, low cost and stable photochemical properties, Cu 2 O nanoparticles have been extensively used in many fields, such as new energy, photocatalytic degradation, sterilization and sensing [25][26][27].The different morphologies of Cu 2 O nanomaterials, such as hollow spheres, cubes, wires, octahedrons and cages, can be synthesized by adjusting the synthesis conditions [28].At present, various electrochemical sensors based on Cu 2 O nanoparticles have been constructed for the detection of H 2 O 2 [29,30], glucose [30][31][32], dopamine [33][34][35], herbicide paraquat [36], L-tyrosine [37], vanillin [38], sunset yellow [39] and NO 2 [40].As far as we know, the use of Cu 2 O for the determination of UA has rarely been reported in the literature.However, Cu 2 O nanoparticles are easily agglomerated, which greatly limits their application in electrochemical sensors.How to reduce the agglomeration of nano-semiconductors is an urgent problem to be solved.
Graphene (GR) is a single-layer two-dimensional planar crystal with the specific surface area of 2630 m 2 /g, which enables it to be an ideal template for GR-based composites in the fields of battery, photocatalysis, sensing and so on [41].Xu et al. [29] successfully prepared Cu 2 O-reduced graphene oxide nanocomposites using three various methods (namely physical adsorption, in-situ reduction and one-pot synthesis).The composites were dispersed in 0.1% nafion solution and decorated on the glassy carbon electrode (GCE) by a drop-coating technique, which were constructed for nonenzymatic hydrogen peroxide sensing.Cu 2 O/GR nanocomposites were also successfully prepared by a solvothermal method [33], with Cu 2 O/GR/GCE having showed good electrocatalytic activity towards DA.As result, the proposed Cu 2 O/GR/GCE exhibited excellent sensing performances in terms of linear range (0.1−10 µM) and limit of detection (LOD, 10 nM).Ye et al. [36] developed a novel paraquat sensor based on Cu 2 O/polyvinyl pyrrolidone-functionalized GR nanosheet nanocomposite.However, the preparation of the above GR-based composites is complicated, which greatly affects the use of sensors in practical applications.Therefore, the development of a green routine for the creation of Cu 2 O-GR composite-based electrochemical sensors is very crucial.
Herein, a simple and green routine was proposed to construct an ultrasensitive sensor based on Cu 2 O/electrochemically reduced graphene oxide decorated glass carbon electrode (Cu 2 O-ErGO/GCE), which aimed to selectively detect UA even in the co-presence of DA and AA.The Cu 2 O-loaded graphene oxide (GO) nanocomposites were directly coated on the electrode surface, before being electrochemically reduced to obtain Cu 2 O-ErGO/GCE.The as-obtained Cu 2 O-ErGO has excellent electrocatalytic activity towards UA oxidation and thus, we have successfully realized the selective and sensitive detection of UA in human blood serum and urine samples.

Characterization of Surface Morphologies
The morphologies of Cu 2 O, GO and Cu 2 O-ErGO nanocomposites were recorded by Scanning Electron Microscope (SEM) (Figure 1). Figure 1A shows the SEM image of the Cu 2 O nanoparticles.Most of these Cu 2 O nanoparticles had a spherical outline, with an average diameter of around 100 nm based on the SEM measurements of 500 nanoparticles.Figure 1B shows the low magnification SEM image of GO nanosheets, which clearly illustrates the flake-like shapes.Furthermore, the thickness of the wrinkled edges could be regarded as the thickness of the exfoliated GO nanosheets, which displayed a thickness about 2-20 nm.The GO at a high magnification (inset of Figure 1B) had a wrinkled surface, which corrugated and scrolled in a similar way to crumpled silk veils.This lamellar structure of GO can effectively increase the specific surface area of the modified electrode.

Voltammetric Responses of UA on Different Electrodes
The voltammetric behavior of 10 µM UA on the bare GCE, GO/GCE, ErGO/GCE and Cu 2 O-ErGO/GCE was measured in 0.1 M phosphate buffer solution (PBS, pH of 3.0) by cyclic voltammetry (CV).Only one oxidation peak was observed on all electrodes (Figure 2), which indicated that the electrochemical reaction of UA is completely irreversible.On the bare GCE, a broad and weak oxidation peak was observed at 0.683 V with a oxidation peak current (I pa ) of 5.413 µA, corresponding to a slow electron transfer kinetic process.On the GO/GCE, the oxidation potential (E pa ) moves positively to 0.705 V and the I pa decreases to 1.315 µA.This phenomenon is closely related to the retarded electron transfer due to the presence of poor electrical conductivity of GO.When decorated with ErGO, the I pa of UA increases obviously (I pa = 30.89µA) and the E pa shifts negatively to 0.580 V, showing that ErGO has good electrocatalytic activity towards UA.This is probably due to the large specific surface area and excellent electrical conductivity of ErGO, which eventually increases the response current of UA.When decorated with the ErGO-Cu 2 O nanocomposites, the sharp oxidation peak appears with the E pa further shifting negatively to 0.561 V and the I pa further increasing to 57.69 µA.The I pa for Cu 2 O-ErGO/GCE is about 10-fold higher than that on the bare GCE and almost twice as large as that at the ErGO/GCE, which is due to the synergistic effect of ErGO and Cu 2 O nanoparticles.

Voltammetric Responses of UA on Different Electrodes
The voltammetric behavior of 10 μM UA on the bare GCE, GO/GCE, ErGO/GCE and Cu2O-ErGO/GCE was measured in 0.1 M phosphate buffer solution (PBS, pH of 3.0) by cyclic voltammetry (CV).Only one oxidation peak was observed on all electrodes (Figure 2), which indicated that the electrochemical reaction of UA is completely irreversible.On the bare GCE, a broad and weak oxidation peak was observed at 0.683 V with a oxidation peak current (Ipa) of 5.413 μA, corresponding to a slow electron transfer kinetic process.On the GO/GCE, the oxidation potential (Epa) moves positively to 0.705 V and the Ipa decreases to 1.315 μA.This phenomenon is closely related to the retarded electron transfer due to the presence of poor electrical conductivity of GO.When decorated with ErGO, the Ipa of UA increases obviously (Ipa = 30.89μA) and the Epa shifts negatively to 0.580 V, showing that ErGO has good electrocatalytic activity towards UA.This is probably due to the large specific surface area and excellent electrical conductivity of ErGO, which eventually increases the response current of UA.When decorated with the ErGO-Cu2O nanocomposites, the sharp oxidation peak appears with the Epa further shifting negatively to 0.561 V and the Ipa further increasing to 57.69 μA.The Ipa for Cu2O-ErGO/GCE is about 10-fold higher than that on the bare GCE and almost twice as large as that at the ErGO/GCE, which is due to the synergistic effect of ErGO and Cu2O nanoparticles.

pH-Dependent Electrochemical Response
The influence of the solution pH on the voltammetric responses of 10 μM UA at Cu2O-ErGO/GCE was investigated by CV.The variation of Epa and Ipa with the solution pH values are shown in Figure 3.The Epa of UA shifts negatively with an increase in the pH, suggesting that the Catalysts 2018, 8, 407 5 of 14

pH-Dependent Electrochemical Response
The influence of the solution pH on the voltammetric responses of 10 µM UA at Cu 2 O-ErGO/GCE was investigated by CV.The variation of E pa and I pa with the solution pH values are shown in Figure 3.The E pa of UA shifts negatively with an increase in the pH, suggesting that the protons (H + ) are involved in the oxidation of UA.Moreover, E pa is linearly related to the pH, while the corresponding regression equation is E pa (V) = −0.0548pH+ 0.7304 (r = 0.9977).The slope (54.8 mV/pH) approaches the theoretical value of 57.6 mV/pH, implying that the equal number of electrons and protons take part in the UA oxidation.The I pa of UA increases gradually with an increase in the pH, before reaching a maximum in the pH range of 2.7-3.1.After this, the I pa decreases with a further increase in the pH.It is well known that the electrochemical behavior of UA (pK a1 = 2.38 and pK a2 = 9.39) is dependent on the pH value of the aqueous solution.In an acidic solution, UA is positively charged due to the protonation of the amino group, which is available to form strong hydrogen bonds with oxygen-containing groups on ErGO surface, such as carboxyl groups and carbonyl groups.This ultimately improves the adsorption of UA onto the electrode surface.Considering the sensitivity of the detection, a pH of 3.0 is recommended for UA determination.

Sweep Rate and Electrochemical Kinetics
The electrochemical kinetics of UA oxidation at Cu2O-ErGO/GCE was investigated by CV, which was measured at various sweep rates.As presented in Figure 4, the Ipa has a positive linear relationship with the sweep rates (v), while the corresponding linear relationship can be expressed as Ipa (μA) = 497.32v(V s −1 ) + 5.2533 (r = 0.9984).This indicates that the oxidation of UA is controlled by the adsorption process.The diffusion or adsorption-controlled behavior was also cross-verified by plotting logIpa vs. logv, which yielded a linear regression equation of logIpa (μA) = 0.8841logv (V s −1 ) + 2.6319 (r = 0.9987).The obtained slope (0.8841) is close to 1.0, which further confirms that the reaction is adsorption-controlled.Furthermore, the Epa has a linear relationship to the Napierian logarithm of sweep rate (lnv).The linear regression equation is Ep (V) = 0.0219lnv (V s −1 ) + 0.6117 (r = 0.9974).For a completely irreversible and adsorption-controlled process, the electron transferred number (n) was calculated to be 2 according to Laviron's theory [43].Combining with the pHdependent response results, the UA oxidation is a 2-electron and 2-proton process.This is consistent with previous reports [12,21].

Sweep Rate and Electrochemical Kinetics
The electrochemical kinetics of UA oxidation at Cu 2 O-ErGO/GCE was investigated by CV, which was measured at various sweep rates.As presented in Figure 4, the I pa has a positive linear relationship with the sweep rates (v), while the corresponding linear relationship can be expressed as I pa (µA) = 497.32v(V s −1 ) + 5.2533 (r = 0.9984).This indicates that the oxidation of UA is controlled by the adsorption process.The diffusion or adsorption-controlled behavior was also cross-verified by plotting logI pa vs. logv, which yielded a linear regression equation of logI pa (µA) = 0.8841logv (V s −1 ) + 2.6319 (r = 0.9987).The obtained slope (0.8841) is close to 1.0, which further confirms that the reaction is adsorption-controlled.Furthermore, the E pa has a linear relationship to the Napierian logarithm of sweep rate (lnv).The linear regression equation is E p (V) = 0.0219lnv (V s −1 ) + 0.6117 (r = 0.9974).For a completely irreversible and adsorption-controlled process, the electron transferred number (n) was calculated to be 2 according to Laviron's theory [43].Combining with the pH-dependent response results, the UA oxidation is a 2-electron and 2-proton process.This is consistent with previous reports [12,21].
reaction is adsorption-controlled.Furthermore, the Epa has a linear relationship to the Napierian logarithm of sweep rate (lnv).The linear regression equation is Ep (V) = 0.0219lnv (V s −1 ) + 0.6117 (r = 0.9974).For a completely irreversible and adsorption-controlled process, the electron transferred number (n) was calculated to be 2 according to Laviron's theory [43].Combining with the pHdependent response results, the UA oxidation is a 2-electron and 2-proton process.This is consistent with previous reports [12,21].

Optimization of Supporting Electrolytes
The influence of different supporting electrolytes on the I pa of 10 µM UA at Cu 2 O-ErGO/GCE was investigated.PBS (pH of 2.3-6.6),(CH 2 ) 6 N 4 -HCl buffer (pH of 4.0-6.0),HAc-NaAc buffer (pH of 3.0-6.0),HAc-NH 4 Ac buffer (pH of 3.0-6.0),NH 3 -NH 4 Cl buffer (pH of 8.0-10.0),H 2 SO 4 , HCl and H 3 PO 4 (all concentrations of 0.1 M) were used as the supporting electrolytes.The sharp oxidation peak with the largest I pa was found in 0.1 M PBS.

Optimization of Accumulation Conditions
As demonstrated in Section 2.4, the oxidation of UA at Cu 2 O-ErGO/GCE is an adsorption-controlled process.Therefore, the accumulation can increase the adsorption capacity of UA on the electrode surface and significantly enhance the sensitivity of the detection.The influence of the accumulation potential on the I pa of UA is plotted in Figure 5A.When the accumulation potential varied from −0.3 to 0.1 V, the I pa altered very slightly.However, the I pa of UA decreases rapidly when the accumulation potential shifts to the positive and exceeds 0.1 V.The influence of the accumulation time was also investigated, with the results presented in Figure 5B.During 0-120 s, the I pa of UA increases significantly over time, before leveling off.This is mainly related to the saturation adsorption of UA on the electrode surface.Therefore, the accumulation was performed at −0.2 V for 120 s before the electrochemical measurement or detection.

Calibration Curves, Dynamical Response Regions and Detection Limit
Under the optimal experimental conditions, the I pa of UA with different concentrations at Cu 2 O-ErGO/GCE was measured by Second-order derivative linear sweep voltammetry (SDLSV) (Figure 6).The I pa of UA increases linearly with an increase in the concentration in the ranges of 2.0 nM-0.6 µM and 0.6 µM-10 µM.The corresponding linear equations for these two ranges are I pa (µA) = 8.6267c (µM) + 0.1289 (r = 0.9971) and I pa (µA) = 13.467c(µM) − 4.3026 (r = 0.9996), respectively.The detection limit (S/N = 3) is found to be 1.0 nM.Compared with the existing nanocomposite-modified electrodes (Table 1), Cu 2 O-ErGO/GCE has a lower limit of detection and is suitable for the detection of UA at a lower concentration level.
As demonstrated in Section 2.4, the oxidation of UA at Cu2O-ErGO/GCE is an adsorptioncontrolled process.Therefore, the accumulation can increase the adsorption capacity of UA on the electrode surface and significantly enhance the sensitivity of the detection.The influence of the accumulation potential on the Ipa of UA is plotted in Figure 5A.When the accumulation potential varied from −0.3 to 0.1 V, the Ipa altered very slightly.However, the Ipa of UA decreases rapidly when the accumulation potential shifts to the positive and exceeds 0.1 V.The influence of the accumulation time was also investigated, with the results presented in Figure 5B.During 0-120 s, the Ipa of UA increases significantly over time, before leveling off.This is mainly related to the saturation adsorption of UA on the electrode surface.Therefore, the accumulation was performed at −0.2 V for 120 s before the electrochemical measurement or detection.

Calibration Curves, Dynamical Response Regions and Detection Limit
Under the optimal experimental conditions, the Ipa of UA with different concentrations at Cu2O-ErGO/GCE was measured by Second-order derivative linear sweep voltammetry (SDLSV) (Figure 6).The Ipa of UA increases linearly with an increase in the concentration in the ranges of 2.0 nM-0.6 μM and 0.6 μM-10 μM.The corresponding linear equations for these two ranges are Ipa (μA) = 8.6267c (μM) + 0.1289 (r = 0.9971) and Ipa (μA) = 13.467c(μM) − 4.3026 (r = 0.9996), respectively.The detection limit (S/N = 3) is found to be 1.0 nM.Compared with the existing nanocomposite-modified electrodes (Table 1), Cu2O-ErGO/GCE has a lower limit of detection and is suitable for the detection of UA at a lower concentration level.As we all know, AA, DA and UA always coexist in the biological fluids, such as blood and urine.Hence, the interference effect of AA and DA on UA detection was first studied.The CV response of 10 µM UA in the presence of 1 mM AA and 50 µM DA is illustrated in Figure 7A.A broad and small peak appears at 430 mV at the bare GCE, indicating the very close peak potentials of AA, DA and UA at the bare electrode so that the simultaneous detection of the three compounds is difficult to achieve.On the contrary, the oxidation peaks of the three species were well separated at both ErGO/GCE and ErGO-Cu 2 O/GCE.For ErGO-Cu 2 O/GCE, the peak potential separation (∆E p ) between DA and UA is greater, while the I pa of UA is much higher than DA and AA.The SDLSV of DA, UA and AA at ErGO-Cu 2 O/GCE is shown in Figure 7B.The ∆E p for AA-DA, DA-UA are 0.262 V and 0.152 V, respectively.The large ∆E p is favorable for the accurate detection of UA without interference.Other possible interferents, such as glucose, purine, oxalate, citrate and urea (all concentrations of 1.0 mM), were also examined.The variation of I pa is not beyond ±5%, indicating no significant interference even in the presence of these potential interfering substances.Therefore, Cu 2 O-ErGO/GCE exhibits good selectivity for the detection of UA.As we all know, AA, DA and UA always coexist in the biological fluids, such as blood and urine.Hence, the interference effect of AA and DA on UA detection was first studied.The CV response of 10 μM UA in the presence of 1 mM AA and 50 μM DA is illustrated in Figure 7A.A broad and small peak appears at 430 mV at the bare GCE, indicating the very close peak potentials of AA, DA and UA at the bare electrode so that the simultaneous detection of the three compounds is difficult to achieve.On the contrary, the oxidation peaks of the three species were well separated at both ErGO/GCE and ErGO-Cu2O/GCE.For ErGO-Cu2O/GCE, the peak potential separation (∆Ep) between DA and UA is greater, while the Ipa of UA is much higher than DA and AA.The SDLSV of DA, UA and AA at ErGO-Cu2O/GCE is shown in Figure 7B.The ∆Ep for AA-DA, DA-UA are 0.262 V and 0.152 V, respectively.The large ∆Ep is favorable for the accurate detection of UA without interference.Other possible interferents, such as glucose, purine, oxalate, citrate and urea (all concentrations of 1.0 mM), were also examined.The variation of Ipa is not beyond ±5%, indicating no significant interference even in the presence of these potential interfering substances.Therefore, Cu2O-ErGO/GCE exhibits good selectivity for the detection of UA.

Repeatability, Reproducibility and Stability
The repeatability of Cu2O-ErGO/GCE was validated by ten parallel measurements of the Ipa of 10 μM UA.The relative standard deviation (RSD) is 2.2%, suggesting the good repeatability of Cu2O-ErGO/GCE.The precision of multiple Cu2O-ErGO/GCEs was also investigated.Seven Cu2O-ErGO/GCEs were fabricated by the same procedure independently and the RSD is calculated as 4.8%, which suggested that the electrode fabrication method has good reproducibility.Finally, the stability of Cu2O-ErGO/GCE was also evaluated.The ErGO-Cu2O/GCE was placed at 4 °C in a refrigerator for refrigerator for 2 weeks.After two weeks, the I pa of 10 µM UA retains 92.4% of the initial response current, indicating that the Cu 2 O-ErGO/GCE has good stability.

Real Sample Analysis
Under the optimal analytical conditions, Cu 2 O-ErGO/GCE was used for the detection of UA in human serum and urine by SDLSV.The serum samples was provided by a local hospital and the urine samples were collected from students in our group.A total of 1.0 mL was taken from each sample, before being diluted with 0.1 M PBS (pH 3.0) in a 100-mL and 500-mL flask.The sample solutions were measured by the SDLSV method.The results are listed in Table 2.The samples were spiked with UA standard solution at levels similar to those found in the samples.The recoveries ranged from 97.5% to 102.8%, which indicates that this method can meet the requirements for the detection of UA in the actual samples.a The results are expressed as mean value ± S.D. based on four replicates (n = 4).b The value of content is obtained by multiplying the detected value by the dilution factor of 100 for serum samples and 500 for urine samples.

Reagents and Solutions
CuSO 4 •5H 2 O, NaOH, hydrazine hydrate solution (80 wt%), graphite powder, polyvinyl pyrrolidone (PVP) and H 2 O 2 solution (30 wt%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).AA, DA and UA were supplied by Sigma-Aldrich Co. (St.Louis, MO, USA).All the reagents were of analytical grade.The stock solution of 0.01 M UA was prepared by dissolving the UA solid into a small volume of 0.1 M NaOH solution, before this was diluted to reach the desired concentration.Doubly distilled water was used in all experiments.

Synthesis of Cu 2 O Nanoparticles
According to a previous report [38], Cu 2 O nanoparticles (Cu 2 O NPs) were synthesized by the wet-chemical method.Following a typical process, 100 mg of CuSO 4 •5H 2 O and 50 mg of PVP were added into 20 mL of H 2 O with stirring.After this, 4 mL of NaOH (0.2 M) was added dropwise to form a blue precipitate.In the end, 15 µL of hydrazine hydrate solution (80 wt%) was added and the mixture was agitated at room temperature for 20 min.The solid product was separated from the solution by centrifugation.After this, the product was cleaned with anhydrous alcohol and water, before being vacuum-dried at 60 • C overnight.

Preparation of GO and Cu 2 O-GO Composite
Graphene oxide (GO) was synthesized according to our previous report [34].Typically, concentrated H 2 SO 4 (23 mL) was cooled to 0 • C, before graphite powder (0.5 g) and NaNO 3 (0.5 g) were added with mechanical stirring.KMnO 4 (3.0 g) was added slowly at a control temperature below 5 • C, before the temperature was raised to 35 • C and the mixture was stirred for 2 h to form a mash.Subsequently, 40 mL of water was slowly added into the solution at a temperature below 50 • C, before the temperature was increased to 95 • C and the mixture was stirred for 0.5 h.After 100 mL of water was added, the above solution was added into 20 mL of 30% H 2 O 2 in batches.The suspension was treated by suction filtration in time and the precipitate was washed with 150 mL of hydrochloric acid (1:10) and 150 mL of H 2 O, respectively.The graphite oxide was obtained by drying under a vacuum at 50 • C overnight.Finally, 100 mg pf graphite oxide were dispersed in 100 mL of water and exfoliated to GO by ultrasonication for 2 h.After this, the golden-yellow supernatant with a concentration of about 1 mg/mL was obtained after centrifugation.A total of 1.0 mg of Cu 2 O NPs was added into 20 mL pf GO (1 mg/mL) and mixed under ultrasonication for 2 h.Finally, a homogeneous dispersion of Cu 2 O-GO was obtained.

Fabrication of Cu 2 O-ErGO/GCE
First, bare GCE was polished by 0.3 µm of alumina slurry, before being rinsed by absolute ethanol and water in an ultrasonic bath for 3 min and dried in N 2 blowing.Five µL of Cu 2 O-GO dispersion was dropped on the freshly prepared GCE.After drying under ambient conditions, the Cu 2 O-GO/GCE was obtained.The Cu 2 O-GO/GCE was immersed in 0.1 M PBS (pH of 6.0), before being electrochemically reduced at −1.2 V for 120 s to obtain Cu 2 O-ErGO/GCE.For comparison, GO/GCE and ErGO/GCE were fabricated by the similar procedures.

Characterization and Measurements
The SEM photos of Cu 2 O NPs, GO nanosheets and Cu 2 O-ErGO nanocomposites were obtained by scanning electron microscopy (FESEM, EVO10, Carl Zeiss, Jena, Germany).The electrochemical behaviors of UA on the Cu 2 O-ErGO/GCE was measured by cyclic voltammetry (CV) and second-order derivative linear sweep voltammograms (SDLSV).Both the CV and SDLSV swept at a rate of 100 mV/s.The potential sweep ranges were 0.0-1.2V for CV and 0.3-0.8V for SDLSV.The supporting electrolyte used was 0.1 M PBS (pH of 3.0) except where stated otherwise.All the electrochemical tests were performed on a CHI 660D electrochemical workstation (Chenhua Instrument Co. Ltd., Shanghai, China).The SDLSV were obtained from the JP-303E polarographic analyzer (Chengdu Instrument Factory, Chengdu, China).All the electrochemical experiments were carried out with a three-electrode system where the Cu 2 O-ErGO/GCE (3-mm inner diameter) served as the working electrode, while Pt wire and saturated calomel electrode (SCE) acted as the counter electrode and reference electrode.The pH was measured by a pH meter (Shanghai Leichi Instrument Factory, Shanghai, China).The measurements were carried out at room temperature.

Conclusions
A novel electrochemical sensor based on cuprous oxide nanoparticles-electrochemically reduced graphene oxide nanocomposites decorated electrode (Cu 2 O-ErGO/GCE) was developed for the sensitive and selective detection of UA.The Cu 2 O-ErGO/GCE was prepared by a green and convenient routine that combined the drop-casting method with the electrochemical reduction method.The graphene can effectively load Cu 2 O NPs and promote the electron transfer between electrode and analytes due to its specific surface area and good conductivity.The proposed sensor shows high electrocatalytic activity towards the oxidation of UA.Moreover, the Cu 2 O-ErGO/GCE can selectively detect UA even in the presence of high concentrations of DA and AA.Considering its distinct advantages, including simplicity, rapidness, high selective and sensitive as well as good repeatability, reproducibility and stability, the proposed Cu2O-ErGO/GCE shows great potential for the detection of UA in the physiological samples.

Scheme 1 .
Scheme 1.The reaction mechanism of UA.

15 Figure 3 .
Figure 3.The influence of pH on the voltammetric response of 10 μM UA on Cu2O-ErGO/GCE.The plots of the Epa against pH (a) and Ipa against pH (b).Supporting electrolyte: 0.1 M PBS; Accumulation potential: −0.2 V, accumulation time: 30 s and scan rate: 0.1 V s −1 .

Figure 3 .
Figure 3.The influence of pH on the voltammetric response of 10 µM UA on Cu 2 O-ErGO/GCE.The plots of the E pa against pH (a) and I pa against pH (b).Supporting electrolyte: 0.1 M PBS; Accumulation potential: −0.2 V, accumulation time: 30 s and scan rate: 0.1 V s −1 .

Figure 5 . 15 Figure 5 .
Figure 5.The influence of accumulation potential (A, accumulation time = 120 s) and accumulation time (B, accumulation potential = 120 s) on the peak current of 10 µM UA.Sweep rate: 0.1 V s −1 .

Figure 7 .
Figure 7. (A) CV measured on the bare GCE (a), ErGO/GCE (b) and Cu 2 O-ErGO/GCE (c) in 0.1 M PBS (pH = 3.0) containing the mixture of 1.0 mM AA, 50 µM DA and 10 µM UA; (B) SDLSV of the mixture of 1.0 mM AA, 50 µM DA and 10 µM UA obtained at Cu 2 O-ErGO/GCE.2.6.3.Repeatability, Reproducibility and Stability The repeatability of Cu 2 O-ErGO/GCE was validated by ten parallel measurements of the I pa of 10 µM UA.The relative standard deviation (RSD) is 2.2%, suggesting the good repeatability of Cu 2 O-ErGO/GCE.The precision of multiple Cu 2 O-ErGO/GCEs was also investigated.Seven Cu 2 O-ErGO/GCEs were fabricated by the same procedure independently and the RSD is calculated as 4.8%, which suggested that the electrode fabrication method has good reproducibility.Finally, the stability of Cu 2 O-ErGO/GCE was also evaluated.The ErGO-Cu 2 O/GCE was placed at 4 • C in a

Table 2 .
Determination of uric acid in human serum and urine samples.