# Simultaneous Voltammetric Determination of Uric Acid, Xanthine, and Hypoxanthine Using CoFe2O4/Reduced Graphene Oxide-Modified Electrode

Authors: Nguyen Thi Vuong Hoan; Nguyen Ngoc Minh; Nguyen Thi Hong Trang; Le Thi Thanh Thuy; Cao Van Hoang; Tran Xuan Mau; Ho Xuan Anh Vu; Phan Thi Kim Thu; Nguyen Hai Phong; Dinh Quang Khieu
Journal: Journal of Nanomaterials
 (2020)

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## Abstract

In the present paper, the synthesis of cobalt ferrite/reduced graphene oxide (Co2Fe2O4/rGO) composite and its use for the simultaneous determination of uric acid (UA), xanthine (XA), and hypoxanthine (HX) is demonstrated. Cobalt ferrite hollow spheres were synthesized by using the carbonaceous polysaccharide microspheres prepared from a D-glucose solution as templates, followed by calcination. The CoFe2O4/rGO composite was prepared with the ultrasound-assisted method. The obtained material was characterized by using X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, EDX elemental mapping, and nitrogen adsorption/desorption isotherms. The electrochemical behavior of UA, XA, and HX on the CoFe2O4/rGO-modified electrode was studied with cyclic voltammetry and differential pulse voltammetry (DPV). The modified electrode exhibits excellent electrocatalytic activity towards the oxidation of the three compounds. The calibration curves for UA, XA, and HX were obtained over the range of 2.0–10.0 μM from DPV. The limits of detection for UA, XA, and HX are 0.767, 0.650, and 0.506 μM, respectively. The modified electrode was applied to the simultaneous detection of UA, XA, and HX in human urine, and the results are consistent with those obtained from the high-performance liquid chromatography technique.


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## Body

## 1. Introduction

Uric acid (UA: 7,9-dihydro-1H-purine-2,6,8(3H)-trione), xanthine (XA: 3,7-dihydropurine-2,6-dione), and hypoxanthine (HX: 1H-purin-6(9H)-one) are oxidation products of purine nucleotide and deoxynucleotide metabolisms in human beings. The concentration of these products in human serum and urine is of great importance for clinical diagnoses, such as gout, hyperuricemia, leukemia, and pneumonia [1]. The purine oxidation products are simultaneously determined with different techniques, such as capillary electrophoresis [2], enzymatic spectrophotometry [3], and high-performance liquid chromatography [4, 5]. However, these methods require complicated sample preparation, expensive material, and considerable time. As a result, they have limited applications. One of the alternatives to this challenge is electrochemical approaches that have attracted great interest owing to their inherent advantages, such as simplicity, high sensitivity, and low cost. The development of electrochemical analysis based on chemically modified electrodes is a major interest in current research [6, 7]. Several electrode modifiers, such as Ru (DMSO)4Cl2 nanoaggregated Nafion [7], poly(bromocresol purple) [8], and poly-(L-arginine)/graphene composite [9], have been used for the simultaneous determination of UA, XA, and HX.The synthesis and design of new electrode modifiers with numerous electrochemical sensing properties have been a great concern to many scientists. One of these modifiers is cobalt ferrite (CoFe2O4). Although cobalt ferrite has excellent magnetic properties, high coactivity and hardness, and moderate saturation magnetization and is used in magnetic devices, gas sensor application [10, 11], and surface-active Co(II), it has not attracted much attention in the electrochemical analysis [12]. Cobalt ferrite has a low surface area because aggregation usually occurs owing to the high surface energy of the nanoparticles, deteriorating the electrochemical activity of the material. To limit its aggregation, two approaches have usually been employed: (i) the synthesis of CoFe2O4 in hierarchical structures (e.g., rods, urchins, and flower-like structures) and (ii) dispersion of CoFe2O4 nanoparticles in carriers with a large surface area. Related to the latter, cobalt ferrite has usually been dispersed in organic or inorganic substrates. The cobalt ferrite-based materials have been employed to develop the novel electrodes for voltammetric determination of some compounds. Han et al. reported the synthesis of β-cyclodextrin-cobalt-ferrite nanocomposite to modify an electrode for catechol determination [13], Yardımcı et al. used cobalt ferrite/chitosan nanocomposite for H2O2 sensing [14], and Ensafi et al. determined H2O2 and nicotinamide adenine dinucleotide by using a cobalt ferrite/graphene oxide-modified electrode [15].Besides graphene oxide, a derivative of this material—reduced graphene oxide (rGO)—is also an excellent substrate to disperse cobalt ferrite. The rGO with smaller oxygen content is produced from graphene oxide via chemical, thermal, or other approaches. The rGO possesses good conductivity and thermal and chemical stability [16, 17] that makes it to be used as a novel material to develop the electrodes for electrochemical sensing biomolecules [18, 19], metal ions [20], and toxic chemicals [21]. Therefore, a combination of CoFe2O4 with rGO is expected to result in a composite with a high surface area and electrical conductivity and a possibility of application in electrochemistry. To the best of our knowledge, the use of the CoFe2O4/rGO composite as an electrode modifier for the determination of UA, XA, and HA by using the voltammetry method is very limited in the literature.In the present work, we prepared cobalt ferrite hollow spheres to fabricate a CoFe2O4/rGO-modified electrode. Then, we used this electrode to study the electrochemical behaviors on UA, XA, and HA oxidations by using cyclic and differential pulse voltammetry. We also addressed the analysis of real samples.


## 2. Experimental

### 2.1. Materials and Synthesis

#### 2.1.1. Materials

All reagents are of analytical grade. Graphite (C), cobalt nitrate hexahydrate (Co(NO3)·6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), uric acid (C5H4N4O3), xanthine (C19H16N4O2) and hypoxanthine (C5H4N4O), and hydrochloric acid (HCl) are from Sigma-Aldrich. Acetic acid (CH3COOH), phosphoric acid (H3PO4), boric acid (H3BO3), ammonia solution (NH3, 25%), ethanol (C2H5OH), hydroperoxide (H2O2, 30%), and potassium hydroxide (KOH) were purchased from Daejung (Korea). The Britton–Robinson buffer solution (B–RBS) in the range of pH 2.0–10.0 was prepared from 1.0 M H3BO3, 1.0 M H3PO4, and 1.0 M CH3COOH and was adjusted with 1 M KOH. The phosphate buffer solution (PBS) with pH 7 was prepared from 0.5 M Na2HPO4, 0.5 M KH2PO4, 0.5 M NaCl, and 0.5 M KCl. The UA, XA, and HX 2.0×10−5mol·L−1 standard aqueous solutions were prepared in a 0.2 mol·L−1 phosphate buffer solution (pH 5) or Britton–Robinson buffer solution. Double distilled water was used to prepare all the solutions.


#### 2.1.2. Synthesis of Cobalt Ferrite

The synthesis of CoFe2O4 hollow spheres was performed by adding 4 g of glucose, 1.477 g of Co(NO3)2·6H2O, and 2.808 g of FeSO4·7H2O to 40 mL of distilled water to give a homogeneous solution. This mixture was then transferred to a Teflon-lined autoclave (100 mL) for treatment at 185°C for 8 h. The black solid was separated via centrifugation and dried in an oven at 80°C for 5 h and calcined at 500°C for 5 h. The resulting product is cobalt ferrite (Co2Fe2O4).


#### 2.1.3. Synthesis of Reduced Graphene Oxide

Graphite oxide (GrO) was prepared by using Hummers’ process [22]. A mixture of 2 g of GrO and 500 mL of double distilled water was stirred under ultrasonication for 5 h to get a graphene oxide suspension (GO) (4 mg/mL GO). The GO suspension (12.5 mL GO in 250 mL distilled water) was adjusted to pH 9–10 with a 25% NH3 aqueous solution. Then, add 0.012 g of N2H4·H2O to the GO suspension and keep the mixture at 90°C for 60 min. To remove the residual N2H4·H2O, the mixture was washed with a 30% H2O2 aqueous solution several times. The resulting mixture was neutralized until pH 7, with a 5% HCl solution. The mixture was rinsed with distilled water five times (30 mL each time). The solid, which is a graphene oxide (rGO), was collected by centrifugation and dried at 60°C for 24 h.


#### 2.1.4. Synthesis of CoFe2O4/rGO

Add 10 mg of CoFe2O4 to 10 mL of pure ethanol and stir under the ultrasonic condition for 60 min to get a CoFe2O4 suspension (1 mg/mL). The rGO suspension was prepared in the same way as the CoFe2O4 suspension. The CoFe2O4/rGO suspension was obtained by mixing 10 mL of CoFe2O4 suspension (1 mg/mL) and 10 mL of rGO suspension (1 mg/mL) under ultrasonication for 5 h.


### 2.2. Apparatus

The crystal structure of the material was identified by using X-ray powder diffraction (XRD) on a Bruker D8 equipped with Cu Kα radiation (λ=1.5406Å). Infrared spectra were recorded on a Fourier mid-IR InfraLUM FT-08 between 4000 and 150 cm−1. Scanning electron microscopy (SEM) images were recorded on an SEM JMS-5300LV (Japan), equipped with energy-dispersive X-ray microanalysis Nova Nano SEM 450. TEM images were obtained on an FEI spirit instrument (120 kV) electron microscope. The thermal properties were measured on a Micromeritics Tristar 3000 (USA). Magnetic hysteresis loops were measured on a Vibrating Sample Magnetometer (Micro Sence Easy VSM 20130321-02) at room temperature. Energy-dispersive X-ray elemental mapping (EDX-elemental mapping) was conducted on a Horiba, Japan. Ultrasonic treatment was performed in a Cole-Parmer 8890. Electrochemistry was studied by using a CPA-HH5 in which the three-electrode system consisted of a glassy carbon electrode (GCE, a working electrode), an Ag/AgCl reference electrode (Model RE-5, BAS), and a platinum wire auxiliary electrode. The UA, XA, and HX determinations with high-performance liquid chromatography (HPLC) were performed on a Shimadzu 2030 HPLC system, with a UV-Vis detector set at 273 nm. An AC18 (6.0mm×150mm, 5 μm) chromatographic column was employed. The mobile phase is an acetonitrile/water mixture (25/75, v/v) at a flow rate of 1.5 mL·min−1, while the injection volume was 5 mL·min−1.


### 2.3. Analytical Procedures

The cyclic voltammetry (CV) technique was used for the preliminary studies on the electrochemical behavior of UA, XA, and HX. The differential pulse voltammetry (DPV) method was employed for the development of the electroanalytical method for the simultaneous determination of UA, XA, and HX in real samples.Before modification, the GCE was polished with 0.05μm alumina powder on a polishing pad, followed by sonication treatment for about two minutes in double distilled water and dried at room temperature and immediately used for modification. Two milligrams of CoFe2O4/rGO was added to 1 mL of methanol under ultrasonic agitation for 60 min, resulting in a homogeneous black suspension. Five microlitres of CoFe2O4/rGO suspension was dropped on the electrode surface. Then, the modified electrode was dried at ambient temperature to obtain a CoFe2O4/rGO glassy carbon electrode.


### 2.4. Real Sample Analysis

Three samples of human urine, provided by a clinical laboratory, were employed to test the method. In detail, 1.0 mL of the urine sample was spiked with UA, XA, and HX and mixed with 2 mL of the B–RBS buffer solution to make a 10.0 mL test solution. The proposed different pulse voltammetry method was used to detect UA, XA, and HX in the spiked solution.


## 2.1. Materials and Synthesis

### 2.1.1. Materials

All reagents are of analytical grade. Graphite (C), cobalt nitrate hexahydrate (Co(NO3)·6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), uric acid (C5H4N4O3), xanthine (C19H16N4O2) and hypoxanthine (C5H4N4O), and hydrochloric acid (HCl) are from Sigma-Aldrich. Acetic acid (CH3COOH), phosphoric acid (H3PO4), boric acid (H3BO3), ammonia solution (NH3, 25%), ethanol (C2H5OH), hydroperoxide (H2O2, 30%), and potassium hydroxide (KOH) were purchased from Daejung (Korea). The Britton–Robinson buffer solution (B–RBS) in the range of pH 2.0–10.0 was prepared from 1.0 M H3BO3, 1.0 M H3PO4, and 1.0 M CH3COOH and was adjusted with 1 M KOH. The phosphate buffer solution (PBS) with pH 7 was prepared from 0.5 M Na2HPO4, 0.5 M KH2PO4, 0.5 M NaCl, and 0.5 M KCl. The UA, XA, and HX 2.0×10−5mol·L−1 standard aqueous solutions were prepared in a 0.2 mol·L−1 phosphate buffer solution (pH 5) or Britton–Robinson buffer solution. Double distilled water was used to prepare all the solutions.


### 2.1.2. Synthesis of Cobalt Ferrite

The synthesis of CoFe2O4 hollow spheres was performed by adding 4 g of glucose, 1.477 g of Co(NO3)2·6H2O, and 2.808 g of FeSO4·7H2O to 40 mL of distilled water to give a homogeneous solution. This mixture was then transferred to a Teflon-lined autoclave (100 mL) for treatment at 185°C for 8 h. The black solid was separated via centrifugation and dried in an oven at 80°C for 5 h and calcined at 500°C for 5 h. The resulting product is cobalt ferrite (Co2Fe2O4).


### 2.1.3. Synthesis of Reduced Graphene Oxide

Graphite oxide (GrO) was prepared by using Hummers’ process [22]. A mixture of 2 g of GrO and 500 mL of double distilled water was stirred under ultrasonication for 5 h to get a graphene oxide suspension (GO) (4 mg/mL GO). The GO suspension (12.5 mL GO in 250 mL distilled water) was adjusted to pH 9–10 with a 25% NH3 aqueous solution. Then, add 0.012 g of N2H4·H2O to the GO suspension and keep the mixture at 90°C for 60 min. To remove the residual N2H4·H2O, the mixture was washed with a 30% H2O2 aqueous solution several times. The resulting mixture was neutralized until pH 7, with a 5% HCl solution. The mixture was rinsed with distilled water five times (30 mL each time). The solid, which is a graphene oxide (rGO), was collected by centrifugation and dried at 60°C for 24 h.


### 2.1.4. Synthesis of CoFe2O4/rGO

Add 10 mg of CoFe2O4 to 10 mL of pure ethanol and stir under the ultrasonic condition for 60 min to get a CoFe2O4 suspension (1 mg/mL). The rGO suspension was prepared in the same way as the CoFe2O4 suspension. The CoFe2O4/rGO suspension was obtained by mixing 10 mL of CoFe2O4 suspension (1 mg/mL) and 10 mL of rGO suspension (1 mg/mL) under ultrasonication for 5 h.


## 2.1.1. Materials

All reagents are of analytical grade. Graphite (C), cobalt nitrate hexahydrate (Co(NO3)·6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), uric acid (C5H4N4O3), xanthine (C19H16N4O2) and hypoxanthine (C5H4N4O), and hydrochloric acid (HCl) are from Sigma-Aldrich. Acetic acid (CH3COOH), phosphoric acid (H3PO4), boric acid (H3BO3), ammonia solution (NH3, 25%), ethanol (C2H5OH), hydroperoxide (H2O2, 30%), and potassium hydroxide (KOH) were purchased from Daejung (Korea). The Britton–Robinson buffer solution (B–RBS) in the range of pH 2.0–10.0 was prepared from 1.0 M H3BO3, 1.0 M H3PO4, and 1.0 M CH3COOH and was adjusted with 1 M KOH. The phosphate buffer solution (PBS) with pH 7 was prepared from 0.5 M Na2HPO4, 0.5 M KH2PO4, 0.5 M NaCl, and 0.5 M KCl. The UA, XA, and HX 2.0×10−5mol·L−1 standard aqueous solutions were prepared in a 0.2 mol·L−1 phosphate buffer solution (pH 5) or Britton–Robinson buffer solution. Double distilled water was used to prepare all the solutions.


## 2.1.2. Synthesis of Cobalt Ferrite

The synthesis of CoFe2O4 hollow spheres was performed by adding 4 g of glucose, 1.477 g of Co(NO3)2·6H2O, and 2.808 g of FeSO4·7H2O to 40 mL of distilled water to give a homogeneous solution. This mixture was then transferred to a Teflon-lined autoclave (100 mL) for treatment at 185°C for 8 h. The black solid was separated via centrifugation and dried in an oven at 80°C for 5 h and calcined at 500°C for 5 h. The resulting product is cobalt ferrite (Co2Fe2O4).


## 2.1.3. Synthesis of Reduced Graphene Oxide

Graphite oxide (GrO) was prepared by using Hummers’ process [22]. A mixture of 2 g of GrO and 500 mL of double distilled water was stirred under ultrasonication for 5 h to get a graphene oxide suspension (GO) (4 mg/mL GO). The GO suspension (12.5 mL GO in 250 mL distilled water) was adjusted to pH 9–10 with a 25% NH3 aqueous solution. Then, add 0.012 g of N2H4·H2O to the GO suspension and keep the mixture at 90°C for 60 min. To remove the residual N2H4·H2O, the mixture was washed with a 30% H2O2 aqueous solution several times. The resulting mixture was neutralized until pH 7, with a 5% HCl solution. The mixture was rinsed with distilled water five times (30 mL each time). The solid, which is a graphene oxide (rGO), was collected by centrifugation and dried at 60°C for 24 h.


## 2.1.4. Synthesis of CoFe2O4/rGO

Add 10 mg of CoFe2O4 to 10 mL of pure ethanol and stir under the ultrasonic condition for 60 min to get a CoFe2O4 suspension (1 mg/mL). The rGO suspension was prepared in the same way as the CoFe2O4 suspension. The CoFe2O4/rGO suspension was obtained by mixing 10 mL of CoFe2O4 suspension (1 mg/mL) and 10 mL of rGO suspension (1 mg/mL) under ultrasonication for 5 h.


## 2.2. Apparatus

The crystal structure of the material was identified by using X-ray powder diffraction (XRD) on a Bruker D8 equipped with Cu Kα radiation (λ=1.5406Å). Infrared spectra were recorded on a Fourier mid-IR InfraLUM FT-08 between 4000 and 150 cm−1. Scanning electron microscopy (SEM) images were recorded on an SEM JMS-5300LV (Japan), equipped with energy-dispersive X-ray microanalysis Nova Nano SEM 450. TEM images were obtained on an FEI spirit instrument (120 kV) electron microscope. The thermal properties were measured on a Micromeritics Tristar 3000 (USA). Magnetic hysteresis loops were measured on a Vibrating Sample Magnetometer (Micro Sence Easy VSM 20130321-02) at room temperature. Energy-dispersive X-ray elemental mapping (EDX-elemental mapping) was conducted on a Horiba, Japan. Ultrasonic treatment was performed in a Cole-Parmer 8890. Electrochemistry was studied by using a CPA-HH5 in which the three-electrode system consisted of a glassy carbon electrode (GCE, a working electrode), an Ag/AgCl reference electrode (Model RE-5, BAS), and a platinum wire auxiliary electrode. The UA, XA, and HX determinations with high-performance liquid chromatography (HPLC) were performed on a Shimadzu 2030 HPLC system, with a UV-Vis detector set at 273 nm. An AC18 (6.0mm×150mm, 5 μm) chromatographic column was employed. The mobile phase is an acetonitrile/water mixture (25/75, v/v) at a flow rate of 1.5 mL·min−1, while the injection volume was 5 mL·min−1.


## 2.3. Analytical Procedures

The cyclic voltammetry (CV) technique was used for the preliminary studies on the electrochemical behavior of UA, XA, and HX. The differential pulse voltammetry (DPV) method was employed for the development of the electroanalytical method for the simultaneous determination of UA, XA, and HX in real samples.Before modification, the GCE was polished with 0.05μm alumina powder on a polishing pad, followed by sonication treatment for about two minutes in double distilled water and dried at room temperature and immediately used for modification. Two milligrams of CoFe2O4/rGO was added to 1 mL of methanol under ultrasonic agitation for 60 min, resulting in a homogeneous black suspension. Five microlitres of CoFe2O4/rGO suspension was dropped on the electrode surface. Then, the modified electrode was dried at ambient temperature to obtain a CoFe2O4/rGO glassy carbon electrode.


## 2.4. Real Sample Analysis

Three samples of human urine, provided by a clinical laboratory, were employed to test the method. In detail, 1.0 mL of the urine sample was spiked with UA, XA, and HX and mixed with 2 mL of the B–RBS buffer solution to make a 10.0 mL test solution. The proposed different pulse voltammetry method was used to detect UA, XA, and HX in the spiked solution.


## 3. Results and Discussion

### 3.1. Characterization of CoFe2O4/rGO

Figure1(a) shows the TG/dTG curves of CoFe2O4/carbon hollow spheres recorded in airflow from 40 to 800°C. Clearly, a weight decrease of about 2 wt.% is observed from 30 to 120°C, which is ascribed to the desorption of physically adsorbed water in the precursor. Another weight loss of about 50 wt.% at around 400°C is assigned to the combustion of rGO in the CoFe2O4/rGO nanohybrid. This weight loss is close to the initial CoFe2O4/rGO ratio of 1 : 1. The carbon residues are completely removed at temperatures higher than 400°C.Figure 1
(a) TG/dTG diagram of CoFe2O4/carbon spheres. (b) XRD patterns of rGO, CoFe2O4/carbon spheres, CoFe2O4, and CoFe2O4/rGO.
(a)(b)The XRD pattern of rGO in Figure1(b) presents a broad (002) diffraction peak between 20 and 35°, which corresponds to a short-range order in the stacked graphene sheets. The largely reduced interlayer spacing of about 0.342 nm (at 2 theta of 26°), compared with 0.780 nm (at 2 theta of 11.3°) for graphene oxide [23], indicates the formation of reduced graphene oxide, in which the oxygen functional groups are removed significantly during the reduction process. It is worth noting that the characteristic X-ray diffraction indexed as a spinel type according to JCPDS No. 00-002-1045 in CoFe2O4/carbon spheres is observed. This means that the cobalt ferrite phase is formed during the hydrothermal treatment. Cobalt ferrite with high crystallinity is formed after the removal of the carbon template through calcination at 500°C (Figure 1(b)). The characteristic peaks of the cobalt ferrite phase in CoFe2O4/rGO are observed in Figure 1(b). However, the large background indicates a large amorphous phase resulting from reduced graphene oxide. These results confirm the successful synthesis of the CoFe2O4/rGO composite.The EDX analysis shows that the molar composition of Co, Fe, O, and C in CoFe2O4 is 13.56, 26.81, 47.18, and 12.03%, respectively, and in CoFe2O4/rGO is 4.81, 9.21, 34.52, and 51.46%, respectively. Correspondingly, the Co/Fe molar ratio is 1 : 2, which is very close to the stoichiometric ratio in the two samples. In contrast, the excessive carbon content and low Co and Fe content in CoFe2O4/rGO indicate the presence of rGO. These results further confirm the presence of stoichiometric cobalt ferrite (CoFe2O4), and this is probably assigned to the high dispersion of CoFe2O4 on the rGO surface.The formation of the CoFe2O4/rGO composite was also studied by using FT-IR spectroscopy (Figure 2). On the FT-IR spectrum of rGO (Figure 2(a)), we can see vibration at 3444 cm−1 characteristic to OH groups and at 1641–1127 cm−1, attributed to carbonyl (C=O) and epoxy (C–O–C) groups in rGO [24]. However, these peaks have very low intensities, implying that they are removed significantly during the reduction. The typical inverse spinel ferrite structure includes two adsorption bands: one at around 339 cm−1, representing the stretching vibration of the tetragonal group Fe3+–O2−, and the other at around 526 cm−1, attributed to the stretching vibration of the octahedral group complex Co2+–O2− [24] (Figure 2(b)). The characteristic vibration bands of both CoFe2O4 and rGO are observed in Figure 2(c), indicating successful synthesis of the CoFe2O4/rGO composite.Figure 2
IR spectra of (a) rGO, (b) CoFe2O4, and (c) CoFe2O4/rGO.The material is formed in a hollow spherical shape (Figure3). Figure 3(a) shows the hollow spheres (2–10 μm) with flocculent substances (CoFe2O4·nH2O) on the carbon surface. These particles remain unaltered after removing the carbon template (Figure 3(b)). The TEM image of rGO shows a stacked and crumpled morphology due to the exfoliation and restacking process [25] (Figure 3(c)). The CoFe2O4/rGO composite consists of CoFe2O4 hollow spheres embroiled homogeneously with rGO sheets to form a hierarchical structure that favors the diffusion and adsorption of the analytes (Figure 3(d)).Figure 3
SEM images of (a) CoFe2O4/carbon spheres and (b) CoFe2O4. (c) TEM image of rGO. (d) SEM image of CoFe2O4/rGO.
(a)(b)(c)(d)The EDX elemental mapping in Figure4(a) shows the SEM bright field image of the CoFe2O4/rGO composite. The images in Figure 4 reveal that CoFe2O4 clusters of around 500 nm in size are embedded in the rGO matrix.Figure 4
Elemental mapping of the CoFe2O4/rGO (a) bright field image, (b) mapping of oxygen, (c) mapping of carbon, (d) mapping of cobalt, and (e) mapping of iron.
(a)(b)(c)(d)(e)The XPS survey curve presents the existence of Co, Fe, O, and C in CoFe2O4/rGO at 793.28, 721.48, 536.78, and 288.78 eV, respectively (Figure 5(a)). The XPS spectrum Co2p possesses two main peaks at 780.67 and 796.28 eV, which are assigned to Co2p3/2 and Co2p1/2, respectively (Figure 5(b)). The energy gap between the Co2p main peak and the satellite peak can be employed to decide whether cobalt exists as Co(II) or Co(III). If the gap is ca. 6.0 eV, cobalt exists as Co(II), and while the gap is 9–10 eV, the cation is Co(III) [26, 27]. In our case, the energy gap of the cobalt cation is 6.62 eV for Co2p1/2 and 5.96 eV for Co2p3/2, and as a result, Co(II) is the main form in CoFe2O4. The Fe2p spectrum could be deconvoluted into two main peaks at 724.35 eV for Fe2p1/2 and 711.13 eV for Fe2p3/2. This spectrum has two satellite peaks with a binding energy of 733.02 and 718.83 eV (Figure 5(c)), indicating the presence of Fe(III) in the sample, which is consistent with the valence of Fe in CoFe2O4 [28–30]. In Figure 5(d), the O1s peak is deconvoluted into two shoulder peaks at 532.56 eV and 530.46 eV, corresponding to the hydroxyl groups adsorbed on the surface and the Fe–O bond in the crystal lattice, respectively [31]. The C1s spectrum could be fitted to four carbon species at 284.58, 285.55, 286.9, and 289.46 eV, corresponding to C/C=C groups in the nonoxygenated rings, C–OH, epoxy C–O–C, and carboxyl group COOH, respectively (Figure 5(e)) [32].Figure 5
XPS spectrum of CoFe2O4/rGO: (a) survey spectrum; (b) XPS spectrum Co2p; (c) XPS spectrum Fe2p; (d) XPS spectrum O1s; (e) XPS spectrum C1s.
(a)(b)(c)(d)(e)The magnetic hysteresis curves of the CoFe2O4 and CoFe2O4/rGO composite, measured at 298 K with the field sweeping from −10000 to 10000 Oe, indicate that the two materials are ferromagnetic (Figure 6). The saturation magnetization is 60.2 emu·g−1 for CoFe2O4 and 48.6 emu·g−1 for CoFe2O4/rGO. These values are slightly higher than those of other cobalt-ferrite-based materials, reported previously [32–34].Figure 6
Magnetic saturation curves of (a) CoFe2O4 and (b) CoFe2O4/rGO.The specific surface area and pore volume of the materials were determined by using the nitrogen sorption technique, with a typical isotherm shown in Figure7. The isotherms of rGO, CoFe2O4, and CoFe2O4/rGO exhibit type IV with hysteresis loops at high relative pressures. This indicates the presence of interparticle and nonordered mesoporosity in the materials. The BET surface area is 319 m2·g−1 for rGO, 16 m2·g−1 for CoFe2O4, and 77 m2·g−1 for CoFe2O4/rGO. The surface area of the CoFe2O4/rGO composite is nearly fivefold compared with that of pure CoFe2O4. These findings indicate that cobalt ferrite particles are highly dispersed on the rGO matrix, and the material possesses a large surface area.Figure 7
Nitrogen adsorption/desorption of (a) rGO, (b) CoFe2O4, and (c) CoFe2O4/rGO.


### 3.2. Electrochemical Performance of CoFe2O4/rGO-Modified Electrode

#### 3.2.1. Voltammetric Behavior of Different Electrodes

As seen in Figure8, all electrodes provide an anodic peak current (Ia) for the analytes, and the highest peak current with low standard deviation is observed on CoFe2O4-rGO/GCE for all the analytes.Figure 8
Anodic peak current (Ip) of UA, XA, and HX at bare GCE (A), CoFe2O4/GCE (B), rGO/GCE (C), and CoFe2O4-rGO/GCE (D). Conditions: Eacc=−0.3V, tacc=20s, ΔE=120mV, and v=20mV·s−1 (CUA=CXA=CHX=2×10−5M; 0.2 M PBS pH 7, n=4).The CoFe2O4-rGO/GCE is favorable for electron transfer and oxidation. Therefore, this modified electrode was selected for further experiments.


#### 3.2.2. Effect of Amount of Electrode Modifier

The amount of CoFe2O4/rGO on the electrode surface is related to the thickness of the cast film and responsible for the total specific surface area. Therefore, this amount affects the adsorption of UA, XA, and HX on the electrode surface and, thus, the anodic peak current of UA, XA, and HX. To study this effect, we changed the volume of the CoFe2O4/rGO suspension (1.0 mg·mL−1) during the measurements. At the beginning of the volume range, the Ia increases with the volume of the CoFe2O4/rGO suspension, owing to the increasing amount of UA, XA, and HX adsorbed on the modified electrode surface. Later, the Ia changes slightly when the volume of the CoFe2O4/rGO suspension exceeds 5 μL, indicating an insignificant influence of the film thickness on the adsorption of the analytes (Figure 9). This also suggests a rapid electron transfer within the CoFe2O4/rGO film. However, when the volume of the CoFe2O4/rGO suspension is greater than 7.5 μL, the Ia tends to decrease due to larger film thickness and the increasing mass transfer resistance against the electron transfer. In this work, the amount of CoFe2O4/rGO suspension on the GCE surface was selected at 5 μL.Figure 9
Effects of the amount of CoFe2O4/rGO on the GCE on the anodic peak currents (Ip) of CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS pH 7. Other conditions are as in Figure 8.


#### 3.2.3. . Effects of pH

The pH of the solution has a remarkable influence on the UA, XA, and HX electrooxidation on the CoFe2O4/rGO-GCE. Figure 10(a) illustrates the DPV curves of UA, XA, and HX on the modified electrode in the pH range of 2–10. As shown in Figure 10(b), when the pH of the solution is lower than 4, the Ia of all analytes decreases with pH. At a pH higher than 6, only the Ia of XA increases with pH and reaches a maximum at pH 6. However, the Ia of UA and HX tends to reduce until pH reaches 7. Afterward, they change irregularly as pH increases. The dependence of the peak current of UA, XA, and HX on pH is complicated, but a decreasing tendency is observed with all the analytes (Figure 10(b)). This may result from the adsorption behavior of UA, XA, and HX on the CoFe2O4/rGO-GCE. UA, XA, and HX are known as protic aromatic molecules and can become deprotonated to form negatively charged species (anions) at higher pH. Simultaneously, the surface of CoFe2O4/rGO-GCE also becomes negatively charged at high pH. As a result, less intensive adsorption of the analytes on the electrode might take place, thus reducing the Ia.Figure 10
(a) DPV curves of UA, XA, and HX oxidations on the modified electrode at different pH. (b) Effects of pH on the anodic peak currents (Ip). (c) The linear plot of anodic peak current (Ip) vs. pH (CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS). Other conditions are as in Figure 8.
(a)(b)(c)The pH effects on peak potentials (Ep) for UA, XA, and HX oxidations on the CoFe2O4/rGO-GCE were also studied. The Ep values shift to more negative potential with pH, and the plots of Ep vs. pH exhibit high linearity with high determination coefficients (>0.99) (Figure 10(c)). The regression equations are expressed as follows:
(1)UA:Ep,UA=0.607±0.010+−0.061±0.001×pH,r=0.9978.XA:Ep,XA=0.976±0.010+−0.056±0.001×pH,r=0.9976.HX:Ep,HX=1.346±0.012+−0.060±0.002×pH,r=0.9970.The slopes of the lines are −0.061, −0.056, and −0.060 for UA, XA, and HX, respectively, and they are close to the theoretical slope of −0.0599 V/pH. This indicates that the oxidation of UA, XA, and HX involves an equal number of electrons and protons.


#### 3.2.4. Effects of Scan Rate

The effect of the scan rate on electrochemical signals of UA, XA, and HX was also assessed by changing the scan rate from 50 to 500 mV·s−1 (Figure 11). Figure 11(a) presents the scan rate effect of electrochemical responses studied with the CV method. The linear plot of the peak current vs. the square root of the scan rate was conducted to estimate whether the electrooxidation reaction is controlled by diffusion or adsorption. If the plot of Ip vs. v1/2 passes the origin, this process is controlled by diffusion; otherwise, it is controlled by adsorption [35, 36]. The plots of Ip,UA, Ip,XA, and Ip,HX vs. v1/2 are highly linear (r=0.9588÷0.9866, p<0.001) (Figure 11(b)). The number in the parentheses presents the 95% confidence interval. No lines pass the origin because all the intercepts are greater than zero (varying from 0.690 to 6.770 for UA, 10.913 to 16.767 for XA, and 1.799 to 7.843 for HX at 95% confidence—Equations (2), (3), and (4)). This indicates that the electrode process is controlled by adsorption.
(2)Ip,UA=3.730±3.040+1.276±0.189×v1/2,r=0.9588,(3)Ip,XA=13.840±2.927+2.182±0.182×v1/2,r=0.9863,(4)Ip,HX=4.771±3.072+1.435±0.191×v1/2,r=0.9663.Figure 11
(a) DPV curves of UA, XA, and HX oxidations at the CoFe2O4/rGO-GCE. (b) The plot of anodic peak current (Ia) vs. v1/2. (c) The plot of anodic peak potential (Ea) vs. lnv. Conditions: Eacc=−0.3V,tacc=20s,Erange=−0.0÷+1.4V (CUA=CXA=5×10−4M and CHX=10−3M in 0.2 M B–RBS pH 5).
(a)(b)(c)The relationship of the anodic potential vs. the natural logarithm of the scan rate is expressed by the Laviron equation [37].
(5)Ep=E°−R×T1−α×n×FlnR×T×Ks1−α×n×F+R×T1−α×n×F×lnv,where Ks is the electron rate constant of the surface-confined redox couple, n is the number of electrons transferred, α is the charge transfer coefficient, v is the scan rate (V·s−1), E° is the formal redox potential, T=298K, R=8.314J·mol·K−1, and F=96480C·mol−1.The plots of the anodic peak potential vs. the natural logarithm of the scan rate are presented in Figure11(c). The regression equation is expressed in Equations (6)–(8).
(6)Ep,UA=0.270±0.012+0.021±0.002×lnv,r=0.977(7)Ep,XA=0.654±0.006+0.027±0.001×lnv,r=0.996,(8)Ep,HX=0.994±0.019+0.030±0.004×lnv,r=0.978.The values ofn×α for UA, XA, and HX are 1.222, 0.950, and 0.856, respectively. For the irreversible system, α is assumed to be 0.5 [38]. Therefore, the average values of n are 2.444, 1.900, and 1.912. Because the number of electrons transferred is an integer, this number should be 2 in this case for all analytes. Therefore, the oxidation of UA, XA, and HX involves two electrons and two protons. This is consistent with previous work [8]. The mechanism for the oxidation of the analytes is proposed in Scheme 1.Scheme 1
Proposed mechanism for the oxidation of UA, XA, and HX.The CoFe2O4/rGO film possesses a large number of negatively charged surface functional groups (–OH) and electron-rich oxygen atoms (epoxide, OH, C=O), and they could interact with the purine derivatives (in this case, they are UA, XA, and HX). Furthermore, the CoFe2O4/rGO-modified electrode exhibits a larger real surface, π-π conjugated bonds, an abundant number of active sites, and better electronic conductivity, which leads to a strong interaction between the purine derivatives and the electrode interface. Therefore, the electrochemical oxidation of UA, XA, and HX may be substantially accelerated in the presence of the CoFe2O4/rGO film. This might result from the enhanced rate of electron transfer and electrocatalytic activity towards the oxidation of UA, XA, and HX. The argument is illustrated in Scheme 2.Scheme 2
Proposed mechanism for the electrode process of UA, XA, and HX oxidations.


#### 3.2.5. Interferent Study

For evaluating the selectivity of the modified electrode, several possible interferents were evaluated for their behavior in the determination of UA, XA, and HX (equal concentration of2.0×10−5mol·L−1). The tolerance limit is the maximum concentration of a foreign substance that causes an approximately ±5% relative error in the determination. It is found that 10-fold of paracetamol and 50-fold of ascorbic acid do not interfere with the peak current of XA and HX but significantly affect that of UA. However, 1000-fold of D-glucose, 20-fold of KCl and Na2CO3, and 100-fold of MgSO4 do not interfere with the determination of UA, XA, and HX (Table 1). These findings indicate that the present method can be used for real samples.Table 1
Tolerance limit of interfering species atCUA=CXN=CHX=10−5M in 0.2 M PBS pH 5.
InterferentInterferent/analyte molar ratioUAXAHXIp (μA)RE (%)Ip (μA)RE (%)Ip (μA)RE (%)Paracetamol10 : 14.2517.493.68−5.001.96−1.23Ascorbic acid50 : 14.8128.053.75−7.572.03−4.86D-Glucose1000 : 13.40−1.923.650.061.96−1.11KCl20 : 11.4292.154.5741.642.600.902Na2CO320 : 10.562−5.233.662.522.2624.31MgSO4100 : 10.620−1.273.530−3.292.085−5.51


#### 3.2.6. Repeatability, Linear Range, and Limit of Detection

The repeatability ofIp at the CoFe2O4/rGO-GCE was estimated by comparing the relative standard deviation (RSD) with the relative standard deviation calculated from Horwitz’s equation: RSDH=21−0.5×lgC, where C is the concentration in mole fraction. If the RSD is less than 1/2×RSDH, the repeatability is acceptable [39]. Each signal of Ip was obtained by conducting a series of four successive measurements at three concentrations. Table 2 displays the values of RSD and the 1/2×RSDH predicted, and we can see that all the measurements are repeatable. Therefore, the CoFe2O4/rGO-GCE can be used repeatedly for the detection of UA, XA, and HX at low or high concentrations.Table 2
Mean values ofIp, RSD, and RSDH.
C (M)Ip,average (μA)SDRSD (%)1/2×RSDH (%)EPX 1CUA=10−51.9680.042.175.66CXA=10−50.9180.022.045.66CHX=10−50.3810.012.335.66EPX 2CUA=5×10−53.2990.051.494.44CXA=5×10−52.7880.031.184.44CHX=5×10−51.3380.011.054.44EPX 3CUA=10−42.6040.062.294.00CXA=10−43.4230.041.114.00CHX=10−41.7180.031.504.00SD: standard deviation; RSD: relative standard deviation. B–RBSpH=5; n=4.Under optimum conditions, the peak current in stripping voltammetry exhibits two linear ranges: 2μM to 10 μM (P1) and 10 μM to 35 μM (P2) (Figure 12). The linear regression equations are as follows:
(9)UA:Ip1,UA=1.301±0.039+0.145±0.006×CUA,r=0.9976,Ip2,UA=2.387±0.055+0.038±0.002×CUA,r=0.9928.XA::Ip1,XA=0.277±0.029+0.126±0.004×CXA,r=0.9982,Ip2,XA=1.017±0.074+0.059±0.003×CXA,r=0.9945.HX::Ip1,HX=0.119±0.008+0.044±0.001×CHX,r=0.9989,Ip2,HX=0.270±0.025+0.031±0.0001×CHX,r=0.9976.Figure 12
(a) DPV curves for UA, XA, and HX with different concentrations in B–RBS 0.2 M pH 5. (b) The plot ofIa vs. concentration. Other conditions are as in Figure 8.
(a)(b)Limit of detection (LOD) is calculated using the formula [40]
(10)LOD=3Sb,whereS is the standard deviation of the lowest concentration of UA, XN, and HP and b is the slope of calibration curve obtained from the DPV. The detection limits of UA, XA, and HX obtained in the first range are 0.767, 0.650, and 0.506 μM, respectively. A comparison of the proposed method with other electrochemical approaches for UA, XA, and HX is presented in Table 3. It is notable that the LOD of UA, XA, and HX from the proposed methods is compatible with those based on the modified electrodes reported previously. From the analysis above, we can conclude that CoFe2O4/rGO is a potential electrode modifier for determining UA, XA, and HX.Table 3
Comparison of the proposed method with other electrochemical approaches for the simultaneous determination of UA, XA, and HX.
Electrode modifierAnalytesLinear range (μM)LOD (μM)ReferencesRu (DMSO)4Cl2 nanoaggregated NafionUA100–7000.372[7]XA50–5002.35HX50–3002.37Preanodized nontroniteUA2.0–400.42[6]XA2.0–400.07HX4.0–300.34Poly(bromocresol purple)UA0.5–1200.20[8]XA0.1–1000.06HX20–800.12Poly-(L-arginine)/graphene compositeUA0.1–100.05[41]XA0.1–100.05HX0.2–200.1Pyrolytic graphiteUA10–50010[42]XA10–50010HX10–50010CoFe2O4/rGOUA2–100.767The present workXA2–100.650The present workHX2–100.506The present work


#### 3.2.7. Determination of Purine Derivatives in Human Samples

The proposed DPV method was applied to determining the human purine derivatives. The standard addition technique was employed. The recovery experiments were conducted with human urine samples using the CoFe2O4/rGO-CGE, and the findings are listed in Table 4. The recoveries found in the range of 95%–105.3% indicate the accuracy and efficiency of the proposed method for the urine samples. The results obtained with HPLC for the real samples are given for comparison. The results from HPLC are comparable with those obtained from the DPV method proposed in this paper (paired sample t-test: α=0.05, t5=0.5, p=0.637>0.05).Table 4
Determination of UA, XA, and HX in human urine sample.
SamplesAnalyteDetected (mM)Ave±SDn=4Added (mM)Found (mM)Ave±SDn=4Rev (%)Detected by HPLC (mM)Urine 01UA4.31±0.471.505.820±0.270101.04.250XA0.106±0.0040.0450.149±0.01695.40.111HX<LODn/an/an/an/aUrine 02UA1.990±0.1701.5003.440±0.3096.42.180XA0.059±0.0090.0450.105±0.015101.90.077HX<LOD0.7500.786±0.042104.8n/aUrine 03UA5.57±0.081.5007.15±0.75105.35.390XA0.007±0.0010.0450.053±0.003101.30.012HX<LOD0.7500.712±0.03795.0n/an/a: not applicable; Ave: average value; SD: standard deviation;n: the number of repetitions of experiments.


## 3.1. Characterization of CoFe2O4/rGO

Figure1(a) shows the TG/dTG curves of CoFe2O4/carbon hollow spheres recorded in airflow from 40 to 800°C. Clearly, a weight decrease of about 2 wt.% is observed from 30 to 120°C, which is ascribed to the desorption of physically adsorbed water in the precursor. Another weight loss of about 50 wt.% at around 400°C is assigned to the combustion of rGO in the CoFe2O4/rGO nanohybrid. This weight loss is close to the initial CoFe2O4/rGO ratio of 1 : 1. The carbon residues are completely removed at temperatures higher than 400°C.Figure 1
(a) TG/dTG diagram of CoFe2O4/carbon spheres. (b) XRD patterns of rGO, CoFe2O4/carbon spheres, CoFe2O4, and CoFe2O4/rGO.
(a)(b)The XRD pattern of rGO in Figure1(b) presents a broad (002) diffraction peak between 20 and 35°, which corresponds to a short-range order in the stacked graphene sheets. The largely reduced interlayer spacing of about 0.342 nm (at 2 theta of 26°), compared with 0.780 nm (at 2 theta of 11.3°) for graphene oxide [23], indicates the formation of reduced graphene oxide, in which the oxygen functional groups are removed significantly during the reduction process. It is worth noting that the characteristic X-ray diffraction indexed as a spinel type according to JCPDS No. 00-002-1045 in CoFe2O4/carbon spheres is observed. This means that the cobalt ferrite phase is formed during the hydrothermal treatment. Cobalt ferrite with high crystallinity is formed after the removal of the carbon template through calcination at 500°C (Figure 1(b)). The characteristic peaks of the cobalt ferrite phase in CoFe2O4/rGO are observed in Figure 1(b). However, the large background indicates a large amorphous phase resulting from reduced graphene oxide. These results confirm the successful synthesis of the CoFe2O4/rGO composite.The EDX analysis shows that the molar composition of Co, Fe, O, and C in CoFe2O4 is 13.56, 26.81, 47.18, and 12.03%, respectively, and in CoFe2O4/rGO is 4.81, 9.21, 34.52, and 51.46%, respectively. Correspondingly, the Co/Fe molar ratio is 1 : 2, which is very close to the stoichiometric ratio in the two samples. In contrast, the excessive carbon content and low Co and Fe content in CoFe2O4/rGO indicate the presence of rGO. These results further confirm the presence of stoichiometric cobalt ferrite (CoFe2O4), and this is probably assigned to the high dispersion of CoFe2O4 on the rGO surface.The formation of the CoFe2O4/rGO composite was also studied by using FT-IR spectroscopy (Figure 2). On the FT-IR spectrum of rGO (Figure 2(a)), we can see vibration at 3444 cm−1 characteristic to OH groups and at 1641–1127 cm−1, attributed to carbonyl (C=O) and epoxy (C–O–C) groups in rGO [24]. However, these peaks have very low intensities, implying that they are removed significantly during the reduction. The typical inverse spinel ferrite structure includes two adsorption bands: one at around 339 cm−1, representing the stretching vibration of the tetragonal group Fe3+–O2−, and the other at around 526 cm−1, attributed to the stretching vibration of the octahedral group complex Co2+–O2− [24] (Figure 2(b)). The characteristic vibration bands of both CoFe2O4 and rGO are observed in Figure 2(c), indicating successful synthesis of the CoFe2O4/rGO composite.Figure 2
IR spectra of (a) rGO, (b) CoFe2O4, and (c) CoFe2O4/rGO.The material is formed in a hollow spherical shape (Figure3). Figure 3(a) shows the hollow spheres (2–10 μm) with flocculent substances (CoFe2O4·nH2O) on the carbon surface. These particles remain unaltered after removing the carbon template (Figure 3(b)). The TEM image of rGO shows a stacked and crumpled morphology due to the exfoliation and restacking process [25] (Figure 3(c)). The CoFe2O4/rGO composite consists of CoFe2O4 hollow spheres embroiled homogeneously with rGO sheets to form a hierarchical structure that favors the diffusion and adsorption of the analytes (Figure 3(d)).Figure 3
SEM images of (a) CoFe2O4/carbon spheres and (b) CoFe2O4. (c) TEM image of rGO. (d) SEM image of CoFe2O4/rGO.
(a)(b)(c)(d)The EDX elemental mapping in Figure4(a) shows the SEM bright field image of the CoFe2O4/rGO composite. The images in Figure 4 reveal that CoFe2O4 clusters of around 500 nm in size are embedded in the rGO matrix.Figure 4
Elemental mapping of the CoFe2O4/rGO (a) bright field image, (b) mapping of oxygen, (c) mapping of carbon, (d) mapping of cobalt, and (e) mapping of iron.
(a)(b)(c)(d)(e)The XPS survey curve presents the existence of Co, Fe, O, and C in CoFe2O4/rGO at 793.28, 721.48, 536.78, and 288.78 eV, respectively (Figure 5(a)). The XPS spectrum Co2p possesses two main peaks at 780.67 and 796.28 eV, which are assigned to Co2p3/2 and Co2p1/2, respectively (Figure 5(b)). The energy gap between the Co2p main peak and the satellite peak can be employed to decide whether cobalt exists as Co(II) or Co(III). If the gap is ca. 6.0 eV, cobalt exists as Co(II), and while the gap is 9–10 eV, the cation is Co(III) [26, 27]. In our case, the energy gap of the cobalt cation is 6.62 eV for Co2p1/2 and 5.96 eV for Co2p3/2, and as a result, Co(II) is the main form in CoFe2O4. The Fe2p spectrum could be deconvoluted into two main peaks at 724.35 eV for Fe2p1/2 and 711.13 eV for Fe2p3/2. This spectrum has two satellite peaks with a binding energy of 733.02 and 718.83 eV (Figure 5(c)), indicating the presence of Fe(III) in the sample, which is consistent with the valence of Fe in CoFe2O4 [28–30]. In Figure 5(d), the O1s peak is deconvoluted into two shoulder peaks at 532.56 eV and 530.46 eV, corresponding to the hydroxyl groups adsorbed on the surface and the Fe–O bond in the crystal lattice, respectively [31]. The C1s spectrum could be fitted to four carbon species at 284.58, 285.55, 286.9, and 289.46 eV, corresponding to C/C=C groups in the nonoxygenated rings, C–OH, epoxy C–O–C, and carboxyl group COOH, respectively (Figure 5(e)) [32].Figure 5
XPS spectrum of CoFe2O4/rGO: (a) survey spectrum; (b) XPS spectrum Co2p; (c) XPS spectrum Fe2p; (d) XPS spectrum O1s; (e) XPS spectrum C1s.
(a)(b)(c)(d)(e)The magnetic hysteresis curves of the CoFe2O4 and CoFe2O4/rGO composite, measured at 298 K with the field sweeping from −10000 to 10000 Oe, indicate that the two materials are ferromagnetic (Figure 6). The saturation magnetization is 60.2 emu·g−1 for CoFe2O4 and 48.6 emu·g−1 for CoFe2O4/rGO. These values are slightly higher than those of other cobalt-ferrite-based materials, reported previously [32–34].Figure 6
Magnetic saturation curves of (a) CoFe2O4 and (b) CoFe2O4/rGO.The specific surface area and pore volume of the materials were determined by using the nitrogen sorption technique, with a typical isotherm shown in Figure7. The isotherms of rGO, CoFe2O4, and CoFe2O4/rGO exhibit type IV with hysteresis loops at high relative pressures. This indicates the presence of interparticle and nonordered mesoporosity in the materials. The BET surface area is 319 m2·g−1 for rGO, 16 m2·g−1 for CoFe2O4, and 77 m2·g−1 for CoFe2O4/rGO. The surface area of the CoFe2O4/rGO composite is nearly fivefold compared with that of pure CoFe2O4. These findings indicate that cobalt ferrite particles are highly dispersed on the rGO matrix, and the material possesses a large surface area.Figure 7
Nitrogen adsorption/desorption of (a) rGO, (b) CoFe2O4, and (c) CoFe2O4/rGO.


## 3.2. Electrochemical Performance of CoFe2O4/rGO-Modified Electrode

### 3.2.1. Voltammetric Behavior of Different Electrodes

As seen in Figure8, all electrodes provide an anodic peak current (Ia) for the analytes, and the highest peak current with low standard deviation is observed on CoFe2O4-rGO/GCE for all the analytes.Figure 8
Anodic peak current (Ip) of UA, XA, and HX at bare GCE (A), CoFe2O4/GCE (B), rGO/GCE (C), and CoFe2O4-rGO/GCE (D). Conditions: Eacc=−0.3V, tacc=20s, ΔE=120mV, and v=20mV·s−1 (CUA=CXA=CHX=2×10−5M; 0.2 M PBS pH 7, n=4).The CoFe2O4-rGO/GCE is favorable for electron transfer and oxidation. Therefore, this modified electrode was selected for further experiments.


### 3.2.2. Effect of Amount of Electrode Modifier

The amount of CoFe2O4/rGO on the electrode surface is related to the thickness of the cast film and responsible for the total specific surface area. Therefore, this amount affects the adsorption of UA, XA, and HX on the electrode surface and, thus, the anodic peak current of UA, XA, and HX. To study this effect, we changed the volume of the CoFe2O4/rGO suspension (1.0 mg·mL−1) during the measurements. At the beginning of the volume range, the Ia increases with the volume of the CoFe2O4/rGO suspension, owing to the increasing amount of UA, XA, and HX adsorbed on the modified electrode surface. Later, the Ia changes slightly when the volume of the CoFe2O4/rGO suspension exceeds 5 μL, indicating an insignificant influence of the film thickness on the adsorption of the analytes (Figure 9). This also suggests a rapid electron transfer within the CoFe2O4/rGO film. However, when the volume of the CoFe2O4/rGO suspension is greater than 7.5 μL, the Ia tends to decrease due to larger film thickness and the increasing mass transfer resistance against the electron transfer. In this work, the amount of CoFe2O4/rGO suspension on the GCE surface was selected at 5 μL.Figure 9
Effects of the amount of CoFe2O4/rGO on the GCE on the anodic peak currents (Ip) of CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS pH 7. Other conditions are as in Figure 8.


### 3.2.3. . Effects of pH

The pH of the solution has a remarkable influence on the UA, XA, and HX electrooxidation on the CoFe2O4/rGO-GCE. Figure 10(a) illustrates the DPV curves of UA, XA, and HX on the modified electrode in the pH range of 2–10. As shown in Figure 10(b), when the pH of the solution is lower than 4, the Ia of all analytes decreases with pH. At a pH higher than 6, only the Ia of XA increases with pH and reaches a maximum at pH 6. However, the Ia of UA and HX tends to reduce until pH reaches 7. Afterward, they change irregularly as pH increases. The dependence of the peak current of UA, XA, and HX on pH is complicated, but a decreasing tendency is observed with all the analytes (Figure 10(b)). This may result from the adsorption behavior of UA, XA, and HX on the CoFe2O4/rGO-GCE. UA, XA, and HX are known as protic aromatic molecules and can become deprotonated to form negatively charged species (anions) at higher pH. Simultaneously, the surface of CoFe2O4/rGO-GCE also becomes negatively charged at high pH. As a result, less intensive adsorption of the analytes on the electrode might take place, thus reducing the Ia.Figure 10
(a) DPV curves of UA, XA, and HX oxidations on the modified electrode at different pH. (b) Effects of pH on the anodic peak currents (Ip). (c) The linear plot of anodic peak current (Ip) vs. pH (CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS). Other conditions are as in Figure 8.
(a)(b)(c)The pH effects on peak potentials (Ep) for UA, XA, and HX oxidations on the CoFe2O4/rGO-GCE were also studied. The Ep values shift to more negative potential with pH, and the plots of Ep vs. pH exhibit high linearity with high determination coefficients (>0.99) (Figure 10(c)). The regression equations are expressed as follows:
(1)UA:Ep,UA=0.607±0.010+−0.061±0.001×pH,r=0.9978.XA:Ep,XA=0.976±0.010+−0.056±0.001×pH,r=0.9976.HX:Ep,HX=1.346±0.012+−0.060±0.002×pH,r=0.9970.The slopes of the lines are −0.061, −0.056, and −0.060 for UA, XA, and HX, respectively, and they are close to the theoretical slope of −0.0599 V/pH. This indicates that the oxidation of UA, XA, and HX involves an equal number of electrons and protons.


### 3.2.4. Effects of Scan Rate

The effect of the scan rate on electrochemical signals of UA, XA, and HX was also assessed by changing the scan rate from 50 to 500 mV·s−1 (Figure 11). Figure 11(a) presents the scan rate effect of electrochemical responses studied with the CV method. The linear plot of the peak current vs. the square root of the scan rate was conducted to estimate whether the electrooxidation reaction is controlled by diffusion or adsorption. If the plot of Ip vs. v1/2 passes the origin, this process is controlled by diffusion; otherwise, it is controlled by adsorption [35, 36]. The plots of Ip,UA, Ip,XA, and Ip,HX vs. v1/2 are highly linear (r=0.9588÷0.9866, p<0.001) (Figure 11(b)). The number in the parentheses presents the 95% confidence interval. No lines pass the origin because all the intercepts are greater than zero (varying from 0.690 to 6.770 for UA, 10.913 to 16.767 for XA, and 1.799 to 7.843 for HX at 95% confidence—Equations (2), (3), and (4)). This indicates that the electrode process is controlled by adsorption.
(2)Ip,UA=3.730±3.040+1.276±0.189×v1/2,r=0.9588,(3)Ip,XA=13.840±2.927+2.182±0.182×v1/2,r=0.9863,(4)Ip,HX=4.771±3.072+1.435±0.191×v1/2,r=0.9663.Figure 11
(a) DPV curves of UA, XA, and HX oxidations at the CoFe2O4/rGO-GCE. (b) The plot of anodic peak current (Ia) vs. v1/2. (c) The plot of anodic peak potential (Ea) vs. lnv. Conditions: Eacc=−0.3V,tacc=20s,Erange=−0.0÷+1.4V (CUA=CXA=5×10−4M and CHX=10−3M in 0.2 M B–RBS pH 5).
(a)(b)(c)The relationship of the anodic potential vs. the natural logarithm of the scan rate is expressed by the Laviron equation [37].
(5)Ep=E°−R×T1−α×n×FlnR×T×Ks1−α×n×F+R×T1−α×n×F×lnv,where Ks is the electron rate constant of the surface-confined redox couple, n is the number of electrons transferred, α is the charge transfer coefficient, v is the scan rate (V·s−1), E° is the formal redox potential, T=298K, R=8.314J·mol·K−1, and F=96480C·mol−1.The plots of the anodic peak potential vs. the natural logarithm of the scan rate are presented in Figure11(c). The regression equation is expressed in Equations (6)–(8).
(6)Ep,UA=0.270±0.012+0.021±0.002×lnv,r=0.977(7)Ep,XA=0.654±0.006+0.027±0.001×lnv,r=0.996,(8)Ep,HX=0.994±0.019+0.030±0.004×lnv,r=0.978.The values ofn×α for UA, XA, and HX are 1.222, 0.950, and 0.856, respectively. For the irreversible system, α is assumed to be 0.5 [38]. Therefore, the average values of n are 2.444, 1.900, and 1.912. Because the number of electrons transferred is an integer, this number should be 2 in this case for all analytes. Therefore, the oxidation of UA, XA, and HX involves two electrons and two protons. This is consistent with previous work [8]. The mechanism for the oxidation of the analytes is proposed in Scheme 1.Scheme 1
Proposed mechanism for the oxidation of UA, XA, and HX.The CoFe2O4/rGO film possesses a large number of negatively charged surface functional groups (–OH) and electron-rich oxygen atoms (epoxide, OH, C=O), and they could interact with the purine derivatives (in this case, they are UA, XA, and HX). Furthermore, the CoFe2O4/rGO-modified electrode exhibits a larger real surface, π-π conjugated bonds, an abundant number of active sites, and better electronic conductivity, which leads to a strong interaction between the purine derivatives and the electrode interface. Therefore, the electrochemical oxidation of UA, XA, and HX may be substantially accelerated in the presence of the CoFe2O4/rGO film. This might result from the enhanced rate of electron transfer and electrocatalytic activity towards the oxidation of UA, XA, and HX. The argument is illustrated in Scheme 2.Scheme 2
Proposed mechanism for the electrode process of UA, XA, and HX oxidations.


### 3.2.5. Interferent Study

For evaluating the selectivity of the modified electrode, several possible interferents were evaluated for their behavior in the determination of UA, XA, and HX (equal concentration of2.0×10−5mol·L−1). The tolerance limit is the maximum concentration of a foreign substance that causes an approximately ±5% relative error in the determination. It is found that 10-fold of paracetamol and 50-fold of ascorbic acid do not interfere with the peak current of XA and HX but significantly affect that of UA. However, 1000-fold of D-glucose, 20-fold of KCl and Na2CO3, and 100-fold of MgSO4 do not interfere with the determination of UA, XA, and HX (Table 1). These findings indicate that the present method can be used for real samples.Table 1
Tolerance limit of interfering species atCUA=CXN=CHX=10−5M in 0.2 M PBS pH 5.
InterferentInterferent/analyte molar ratioUAXAHXIp (μA)RE (%)Ip (μA)RE (%)Ip (μA)RE (%)Paracetamol10 : 14.2517.493.68−5.001.96−1.23Ascorbic acid50 : 14.8128.053.75−7.572.03−4.86D-Glucose1000 : 13.40−1.923.650.061.96−1.11KCl20 : 11.4292.154.5741.642.600.902Na2CO320 : 10.562−5.233.662.522.2624.31MgSO4100 : 10.620−1.273.530−3.292.085−5.51


### 3.2.6. Repeatability, Linear Range, and Limit of Detection

The repeatability ofIp at the CoFe2O4/rGO-GCE was estimated by comparing the relative standard deviation (RSD) with the relative standard deviation calculated from Horwitz’s equation: RSDH=21−0.5×lgC, where C is the concentration in mole fraction. If the RSD is less than 1/2×RSDH, the repeatability is acceptable [39]. Each signal of Ip was obtained by conducting a series of four successive measurements at three concentrations. Table 2 displays the values of RSD and the 1/2×RSDH predicted, and we can see that all the measurements are repeatable. Therefore, the CoFe2O4/rGO-GCE can be used repeatedly for the detection of UA, XA, and HX at low or high concentrations.Table 2
Mean values ofIp, RSD, and RSDH.
C (M)Ip,average (μA)SDRSD (%)1/2×RSDH (%)EPX 1CUA=10−51.9680.042.175.66CXA=10−50.9180.022.045.66CHX=10−50.3810.012.335.66EPX 2CUA=5×10−53.2990.051.494.44CXA=5×10−52.7880.031.184.44CHX=5×10−51.3380.011.054.44EPX 3CUA=10−42.6040.062.294.00CXA=10−43.4230.041.114.00CHX=10−41.7180.031.504.00SD: standard deviation; RSD: relative standard deviation. B–RBSpH=5; n=4.Under optimum conditions, the peak current in stripping voltammetry exhibits two linear ranges: 2μM to 10 μM (P1) and 10 μM to 35 μM (P2) (Figure 12). The linear regression equations are as follows:
(9)UA:Ip1,UA=1.301±0.039+0.145±0.006×CUA,r=0.9976,Ip2,UA=2.387±0.055+0.038±0.002×CUA,r=0.9928.XA::Ip1,XA=0.277±0.029+0.126±0.004×CXA,r=0.9982,Ip2,XA=1.017±0.074+0.059±0.003×CXA,r=0.9945.HX::Ip1,HX=0.119±0.008+0.044±0.001×CHX,r=0.9989,Ip2,HX=0.270±0.025+0.031±0.0001×CHX,r=0.9976.Figure 12
(a) DPV curves for UA, XA, and HX with different concentrations in B–RBS 0.2 M pH 5. (b) The plot ofIa vs. concentration. Other conditions are as in Figure 8.
(a)(b)Limit of detection (LOD) is calculated using the formula [40]
(10)LOD=3Sb,whereS is the standard deviation of the lowest concentration of UA, XN, and HP and b is the slope of calibration curve obtained from the DPV. The detection limits of UA, XA, and HX obtained in the first range are 0.767, 0.650, and 0.506 μM, respectively. A comparison of the proposed method with other electrochemical approaches for UA, XA, and HX is presented in Table 3. It is notable that the LOD of UA, XA, and HX from the proposed methods is compatible with those based on the modified electrodes reported previously. From the analysis above, we can conclude that CoFe2O4/rGO is a potential electrode modifier for determining UA, XA, and HX.Table 3
Comparison of the proposed method with other electrochemical approaches for the simultaneous determination of UA, XA, and HX.
Electrode modifierAnalytesLinear range (μM)LOD (μM)ReferencesRu (DMSO)4Cl2 nanoaggregated NafionUA100–7000.372[7]XA50–5002.35HX50–3002.37Preanodized nontroniteUA2.0–400.42[6]XA2.0–400.07HX4.0–300.34Poly(bromocresol purple)UA0.5–1200.20[8]XA0.1–1000.06HX20–800.12Poly-(L-arginine)/graphene compositeUA0.1–100.05[41]XA0.1–100.05HX0.2–200.1Pyrolytic graphiteUA10–50010[42]XA10–50010HX10–50010CoFe2O4/rGOUA2–100.767The present workXA2–100.650The present workHX2–100.506The present work


### 3.2.7. Determination of Purine Derivatives in Human Samples

The proposed DPV method was applied to determining the human purine derivatives. The standard addition technique was employed. The recovery experiments were conducted with human urine samples using the CoFe2O4/rGO-CGE, and the findings are listed in Table 4. The recoveries found in the range of 95%–105.3% indicate the accuracy and efficiency of the proposed method for the urine samples. The results obtained with HPLC for the real samples are given for comparison. The results from HPLC are comparable with those obtained from the DPV method proposed in this paper (paired sample t-test: α=0.05, t5=0.5, p=0.637>0.05).Table 4
Determination of UA, XA, and HX in human urine sample.
SamplesAnalyteDetected (mM)Ave±SDn=4Added (mM)Found (mM)Ave±SDn=4Rev (%)Detected by HPLC (mM)Urine 01UA4.31±0.471.505.820±0.270101.04.250XA0.106±0.0040.0450.149±0.01695.40.111HX<LODn/an/an/an/aUrine 02UA1.990±0.1701.5003.440±0.3096.42.180XA0.059±0.0090.0450.105±0.015101.90.077HX<LOD0.7500.786±0.042104.8n/aUrine 03UA5.57±0.081.5007.15±0.75105.35.390XA0.007±0.0010.0450.053±0.003101.30.012HX<LOD0.7500.712±0.03795.0n/an/a: not applicable; Ave: average value; SD: standard deviation;n: the number of repetitions of experiments.


## 3.2.1. Voltammetric Behavior of Different Electrodes

As seen in Figure8, all electrodes provide an anodic peak current (Ia) for the analytes, and the highest peak current with low standard deviation is observed on CoFe2O4-rGO/GCE for all the analytes.Figure 8
Anodic peak current (Ip) of UA, XA, and HX at bare GCE (A), CoFe2O4/GCE (B), rGO/GCE (C), and CoFe2O4-rGO/GCE (D). Conditions: Eacc=−0.3V, tacc=20s, ΔE=120mV, and v=20mV·s−1 (CUA=CXA=CHX=2×10−5M; 0.2 M PBS pH 7, n=4).The CoFe2O4-rGO/GCE is favorable for electron transfer and oxidation. Therefore, this modified electrode was selected for further experiments.


## 3.2.2. Effect of Amount of Electrode Modifier

The amount of CoFe2O4/rGO on the electrode surface is related to the thickness of the cast film and responsible for the total specific surface area. Therefore, this amount affects the adsorption of UA, XA, and HX on the electrode surface and, thus, the anodic peak current of UA, XA, and HX. To study this effect, we changed the volume of the CoFe2O4/rGO suspension (1.0 mg·mL−1) during the measurements. At the beginning of the volume range, the Ia increases with the volume of the CoFe2O4/rGO suspension, owing to the increasing amount of UA, XA, and HX adsorbed on the modified electrode surface. Later, the Ia changes slightly when the volume of the CoFe2O4/rGO suspension exceeds 5 μL, indicating an insignificant influence of the film thickness on the adsorption of the analytes (Figure 9). This also suggests a rapid electron transfer within the CoFe2O4/rGO film. However, when the volume of the CoFe2O4/rGO suspension is greater than 7.5 μL, the Ia tends to decrease due to larger film thickness and the increasing mass transfer resistance against the electron transfer. In this work, the amount of CoFe2O4/rGO suspension on the GCE surface was selected at 5 μL.Figure 9
Effects of the amount of CoFe2O4/rGO on the GCE on the anodic peak currents (Ip) of CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS pH 7. Other conditions are as in Figure 8.


## 3.2.3. . Effects of pH

The pH of the solution has a remarkable influence on the UA, XA, and HX electrooxidation on the CoFe2O4/rGO-GCE. Figure 10(a) illustrates the DPV curves of UA, XA, and HX on the modified electrode in the pH range of 2–10. As shown in Figure 10(b), when the pH of the solution is lower than 4, the Ia of all analytes decreases with pH. At a pH higher than 6, only the Ia of XA increases with pH and reaches a maximum at pH 6. However, the Ia of UA and HX tends to reduce until pH reaches 7. Afterward, they change irregularly as pH increases. The dependence of the peak current of UA, XA, and HX on pH is complicated, but a decreasing tendency is observed with all the analytes (Figure 10(b)). This may result from the adsorption behavior of UA, XA, and HX on the CoFe2O4/rGO-GCE. UA, XA, and HX are known as protic aromatic molecules and can become deprotonated to form negatively charged species (anions) at higher pH. Simultaneously, the surface of CoFe2O4/rGO-GCE also becomes negatively charged at high pH. As a result, less intensive adsorption of the analytes on the electrode might take place, thus reducing the Ia.Figure 10
(a) DPV curves of UA, XA, and HX oxidations on the modified electrode at different pH. (b) Effects of pH on the anodic peak currents (Ip). (c) The linear plot of anodic peak current (Ip) vs. pH (CUA=CXA=CHX=2×10−5M in 0.2 M B–RBS). Other conditions are as in Figure 8.
(a)(b)(c)The pH effects on peak potentials (Ep) for UA, XA, and HX oxidations on the CoFe2O4/rGO-GCE were also studied. The Ep values shift to more negative potential with pH, and the plots of Ep vs. pH exhibit high linearity with high determination coefficients (>0.99) (Figure 10(c)). The regression equations are expressed as follows:
(1)UA:Ep,UA=0.607±0.010+−0.061±0.001×pH,r=0.9978.XA:Ep,XA=0.976±0.010+−0.056±0.001×pH,r=0.9976.HX:Ep,HX=1.346±0.012+−0.060±0.002×pH,r=0.9970.The slopes of the lines are −0.061, −0.056, and −0.060 for UA, XA, and HX, respectively, and they are close to the theoretical slope of −0.0599 V/pH. This indicates that the oxidation of UA, XA, and HX involves an equal number of electrons and protons.


## 3.2.4. Effects of Scan Rate

The effect of the scan rate on electrochemical signals of UA, XA, and HX was also assessed by changing the scan rate from 50 to 500 mV·s−1 (Figure 11). Figure 11(a) presents the scan rate effect of electrochemical responses studied with the CV method. The linear plot of the peak current vs. the square root of the scan rate was conducted to estimate whether the electrooxidation reaction is controlled by diffusion or adsorption. If the plot of Ip vs. v1/2 passes the origin, this process is controlled by diffusion; otherwise, it is controlled by adsorption [35, 36]. The plots of Ip,UA, Ip,XA, and Ip,HX vs. v1/2 are highly linear (r=0.9588÷0.9866, p<0.001) (Figure 11(b)). The number in the parentheses presents the 95% confidence interval. No lines pass the origin because all the intercepts are greater than zero (varying from 0.690 to 6.770 for UA, 10.913 to 16.767 for XA, and 1.799 to 7.843 for HX at 95% confidence—Equations (2), (3), and (4)). This indicates that the electrode process is controlled by adsorption.
(2)Ip,UA=3.730±3.040+1.276±0.189×v1/2,r=0.9588,(3)Ip,XA=13.840±2.927+2.182±0.182×v1/2,r=0.9863,(4)Ip,HX=4.771±3.072+1.435±0.191×v1/2,r=0.9663.Figure 11
(a) DPV curves of UA, XA, and HX oxidations at the CoFe2O4/rGO-GCE. (b) The plot of anodic peak current (Ia) vs. v1/2. (c) The plot of anodic peak potential (Ea) vs. lnv. Conditions: Eacc=−0.3V,tacc=20s,Erange=−0.0÷+1.4V (CUA=CXA=5×10−4M and CHX=10−3M in 0.2 M B–RBS pH 5).
(a)(b)(c)The relationship of the anodic potential vs. the natural logarithm of the scan rate is expressed by the Laviron equation [37].
(5)Ep=E°−R×T1−α×n×FlnR×T×Ks1−α×n×F+R×T1−α×n×F×lnv,where Ks is the electron rate constant of the surface-confined redox couple, n is the number of electrons transferred, α is the charge transfer coefficient, v is the scan rate (V·s−1), E° is the formal redox potential, T=298K, R=8.314J·mol·K−1, and F=96480C·mol−1.The plots of the anodic peak potential vs. the natural logarithm of the scan rate are presented in Figure11(c). The regression equation is expressed in Equations (6)–(8).
(6)Ep,UA=0.270±0.012+0.021±0.002×lnv,r=0.977(7)Ep,XA=0.654±0.006+0.027±0.001×lnv,r=0.996,(8)Ep,HX=0.994±0.019+0.030±0.004×lnv,r=0.978.The values ofn×α for UA, XA, and HX are 1.222, 0.950, and 0.856, respectively. For the irreversible system, α is assumed to be 0.5 [38]. Therefore, the average values of n are 2.444, 1.900, and 1.912. Because the number of electrons transferred is an integer, this number should be 2 in this case for all analytes. Therefore, the oxidation of UA, XA, and HX involves two electrons and two protons. This is consistent with previous work [8]. The mechanism for the oxidation of the analytes is proposed in Scheme 1.Scheme 1
Proposed mechanism for the oxidation of UA, XA, and HX.The CoFe2O4/rGO film possesses a large number of negatively charged surface functional groups (–OH) and electron-rich oxygen atoms (epoxide, OH, C=O), and they could interact with the purine derivatives (in this case, they are UA, XA, and HX). Furthermore, the CoFe2O4/rGO-modified electrode exhibits a larger real surface, π-π conjugated bonds, an abundant number of active sites, and better electronic conductivity, which leads to a strong interaction between the purine derivatives and the electrode interface. Therefore, the electrochemical oxidation of UA, XA, and HX may be substantially accelerated in the presence of the CoFe2O4/rGO film. This might result from the enhanced rate of electron transfer and electrocatalytic activity towards the oxidation of UA, XA, and HX. The argument is illustrated in Scheme 2.Scheme 2
Proposed mechanism for the electrode process of UA, XA, and HX oxidations.


## 3.2.5. Interferent Study

For evaluating the selectivity of the modified electrode, several possible interferents were evaluated for their behavior in the determination of UA, XA, and HX (equal concentration of2.0×10−5mol·L−1). The tolerance limit is the maximum concentration of a foreign substance that causes an approximately ±5% relative error in the determination. It is found that 10-fold of paracetamol and 50-fold of ascorbic acid do not interfere with the peak current of XA and HX but significantly affect that of UA. However, 1000-fold of D-glucose, 20-fold of KCl and Na2CO3, and 100-fold of MgSO4 do not interfere with the determination of UA, XA, and HX (Table 1). These findings indicate that the present method can be used for real samples.Table 1
Tolerance limit of interfering species atCUA=CXN=CHX=10−5M in 0.2 M PBS pH 5.
InterferentInterferent/analyte molar ratioUAXAHXIp (μA)RE (%)Ip (μA)RE (%)Ip (μA)RE (%)Paracetamol10 : 14.2517.493.68−5.001.96−1.23Ascorbic acid50 : 14.8128.053.75−7.572.03−4.86D-Glucose1000 : 13.40−1.923.650.061.96−1.11KCl20 : 11.4292.154.5741.642.600.902Na2CO320 : 10.562−5.233.662.522.2624.31MgSO4100 : 10.620−1.273.530−3.292.085−5.51


## 3.2.6. Repeatability, Linear Range, and Limit of Detection

The repeatability ofIp at the CoFe2O4/rGO-GCE was estimated by comparing the relative standard deviation (RSD) with the relative standard deviation calculated from Horwitz’s equation: RSDH=21−0.5×lgC, where C is the concentration in mole fraction. If the RSD is less than 1/2×RSDH, the repeatability is acceptable [39]. Each signal of Ip was obtained by conducting a series of four successive measurements at three concentrations. Table 2 displays the values of RSD and the 1/2×RSDH predicted, and we can see that all the measurements are repeatable. Therefore, the CoFe2O4/rGO-GCE can be used repeatedly for the detection of UA, XA, and HX at low or high concentrations.Table 2
Mean values ofIp, RSD, and RSDH.
C (M)Ip,average (μA)SDRSD (%)1/2×RSDH (%)EPX 1CUA=10−51.9680.042.175.66CXA=10−50.9180.022.045.66CHX=10−50.3810.012.335.66EPX 2CUA=5×10−53.2990.051.494.44CXA=5×10−52.7880.031.184.44CHX=5×10−51.3380.011.054.44EPX 3CUA=10−42.6040.062.294.00CXA=10−43.4230.041.114.00CHX=10−41.7180.031.504.00SD: standard deviation; RSD: relative standard deviation. B–RBSpH=5; n=4.Under optimum conditions, the peak current in stripping voltammetry exhibits two linear ranges: 2μM to 10 μM (P1) and 10 μM to 35 μM (P2) (Figure 12). The linear regression equations are as follows:
(9)UA:Ip1,UA=1.301±0.039+0.145±0.006×CUA,r=0.9976,Ip2,UA=2.387±0.055+0.038±0.002×CUA,r=0.9928.XA::Ip1,XA=0.277±0.029+0.126±0.004×CXA,r=0.9982,Ip2,XA=1.017±0.074+0.059±0.003×CXA,r=0.9945.HX::Ip1,HX=0.119±0.008+0.044±0.001×CHX,r=0.9989,Ip2,HX=0.270±0.025+0.031±0.0001×CHX,r=0.9976.Figure 12
(a) DPV curves for UA, XA, and HX with different concentrations in B–RBS 0.2 M pH 5. (b) The plot ofIa vs. concentration. Other conditions are as in Figure 8.
(a)(b)Limit of detection (LOD) is calculated using the formula [40]
(10)LOD=3Sb,whereS is the standard deviation of the lowest concentration of UA, XN, and HP and b is the slope of calibration curve obtained from the DPV. The detection limits of UA, XA, and HX obtained in the first range are 0.767, 0.650, and 0.506 μM, respectively. A comparison of the proposed method with other electrochemical approaches for UA, XA, and HX is presented in Table 3. It is notable that the LOD of UA, XA, and HX from the proposed methods is compatible with those based on the modified electrodes reported previously. From the analysis above, we can conclude that CoFe2O4/rGO is a potential electrode modifier for determining UA, XA, and HX.Table 3
Comparison of the proposed method with other electrochemical approaches for the simultaneous determination of UA, XA, and HX.
Electrode modifierAnalytesLinear range (μM)LOD (μM)ReferencesRu (DMSO)4Cl2 nanoaggregated NafionUA100–7000.372[7]XA50–5002.35HX50–3002.37Preanodized nontroniteUA2.0–400.42[6]XA2.0–400.07HX4.0–300.34Poly(bromocresol purple)UA0.5–1200.20[8]XA0.1–1000.06HX20–800.12Poly-(L-arginine)/graphene compositeUA0.1–100.05[41]XA0.1–100.05HX0.2–200.1Pyrolytic graphiteUA10–50010[42]XA10–50010HX10–50010CoFe2O4/rGOUA2–100.767The present workXA2–100.650The present workHX2–100.506The present work


## 3.2.7. Determination of Purine Derivatives in Human Samples

The proposed DPV method was applied to determining the human purine derivatives. The standard addition technique was employed. The recovery experiments were conducted with human urine samples using the CoFe2O4/rGO-CGE, and the findings are listed in Table 4. The recoveries found in the range of 95%–105.3% indicate the accuracy and efficiency of the proposed method for the urine samples. The results obtained with HPLC for the real samples are given for comparison. The results from HPLC are comparable with those obtained from the DPV method proposed in this paper (paired sample t-test: α=0.05, t5=0.5, p=0.637>0.05).Table 4
Determination of UA, XA, and HX in human urine sample.
SamplesAnalyteDetected (mM)Ave±SDn=4Added (mM)Found (mM)Ave±SDn=4Rev (%)Detected by HPLC (mM)Urine 01UA4.31±0.471.505.820±0.270101.04.250XA0.106±0.0040.0450.149±0.01695.40.111HX<LODn/an/an/an/aUrine 02UA1.990±0.1701.5003.440±0.3096.42.180XA0.059±0.0090.0450.105±0.015101.90.077HX<LOD0.7500.786±0.042104.8n/aUrine 03UA5.57±0.081.5007.15±0.75105.35.390XA0.007±0.0010.0450.053±0.003101.30.012HX<LOD0.7500.712±0.03795.0n/an/a: not applicable; Ave: average value; SD: standard deviation;n: the number of repetitions of experiments.


## 4. Conclusions

In this paper, the CoFe2O4/rGO-GCE is fabricated by using a simple method and employed as an electrochemical sensor for the simultaneous detection of UA, XA, and HX with the differential pulse voltammetry technique. The modified electrode has good stability and sensitivity. The results obtained with the proposed method are consistent with those from standard HPLC analysis. Therefore, CoFe2O4/rGO is expected to become a potential tool for the assay of UA, XA, and HX both in research and in clinical diagnosis owing to its good precision, high speed, and low cost of analysis.



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