# ZIF-67/g-C3N4-Modified Electrode for Simultaneous Voltammetric Determination of Uric Acid and Acetaminophen with Cetyltrimethylammonium Bromide as Discriminating Agent

Authors: Huynh Truong Ngo; Le Thi Hoa; Nguyen Tan Khanh; Tran Thi Bich Hoa; Tran Thanh Tam Toan; Tran Xuan Mau; Nguyen Hai Phong; Ho Sy Thang; Dinh Quang Khieu
Journal: Journal of Nanomaterials
 (2020)

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## Abstract

In the present paper, the ZIF-67/g-C3N4 composite was synthesized and utilized as a modifier for a glassy carbon electrode for the simultaneous voltammetric determination of uric acid (URA) and acetaminophen (ACE) with cetyltrimethylammonium bromide (CTAB) as a discriminating agent. The composite was characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and nitrogen adsorption/desorption isotherms. The obtained ZIF-67/g-C3N4 composite exhibits good textural properties (specific surface area: 75 m2·g−1) and is stable in water with a pH range of 3 to 10. The ZIF-67/g-C3N4-modified electrode combined with CTAB as a discriminating agent possesses excellent catalytic electrochemistry towards URA and ACE with well-defined electrochemical responses. The electrochemical kinetics study is also addressed. The linear relation of the oxidation peak current of URA and ACE and the concentration ranging from 0.2 μM to 6.5 μM provide a detection limit of 0.052 μM for URA and 0.053 μM for ACE. The proposed method is well-suited to simultaneously analyze URA and ACE in human urine with comparable results with HPLC.


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## Body

## 1. Introduction

Uric acid (denoted as URA) is a heterocyclic compound with formula C5H4N4O3, which is the primary end product of purine metabolism. A high URA level in the blood can indicate the presence of numerous diseases and/or physiological disorders. A high concentration of URA in the urine and blood is observed in patients suffering from diseases such as gout and hyperuricaemia [1]. Acetaminophen (denoted as ACE) with formula C8H9NO2, also known as paracetamol, is an effective pain killer used to relieve pains related to many parts of the body [2]. An ACE overdose can cause toxic metabolite accumulation, which may cause serious hepatotoxicity and nephrotoxicity [3].Today, the speed, selectivity, sensibility, low detection limits, low cost, andin situ operation of electroanalytical techniques have been considered as the robust approaches to analyze organic or inorganic traces, especially in pharmaceutical compounds. Uric acid and ACE, as well as dopamine and ascorbic acid, exhibit the redox behaviour at similar potentials. The simultaneous detection of these compounds is sometimes difficult because of interfering overlapping effects. Employing separation steps such as chromatography can sometimes overcome this drawback, but it is usually an expensive option. Therefore, the search for simple, inexpensive, sensitive, and accurate analytical approaches for the simultaneous detection of URA and ACE would be necessary. There exist two approaches to overcome these issues in electrochemical analysis: (i) using an electrode modified with hybrid nanomaterials to improve the interaction of electrospecies and electrode and subsequently increase the peak-peak separation of analytes and electrochemical signals. Phong et al. [4] studied the simultaneous determination of ascorbic acid, paracetamol, and caffeine using an electrochemically rGO-modified electrode. Kutluay and Aslanoglu [5] reported the selective determination of ACE in the presence of ascorbic acid, dopamine, and uric acid using a glassy carbon electrode modified with multiwalled carbon nanotubes. (ii) The second approach is to use the surfactants as a discriminating agent to promote the peak-peak separation. Surfactants are amphiphilic molecules that contain a hydrophilic group at the one end and a hydrophobic group at the other. Below the critical micelle concentration on the solid-liquid interface, the surfactants form a bilayer or hemimicelle structures [6, 7]. These special structures initiate the interface properties of the electrodes and consequently act as discriminating agents to enhance the resolution of electrodes when the voltammetric peaks of two oxidation or reduction species occur at similar potentials. Alarcón-Angeles et al. [8] reported using sodium dodecyl sulfate as a discriminating agent for the electrochemical determination of dopamine in the presence of ascorbic acid. Liu et al. [9] studied the selective determination of dopamine in the presence of ascorbic acid using cetyltrimethylammonium bromide (denoted as CTAB) as a masking agent.Recently, graphitic carbon nitride (g-C3N4), which is a polymeric layered material, structurally analogous to graphene, has emerged as a prospective material for use in electrochemistry [10, 11]. Besides its thermal and chemical stability, graphitic carbon nitride possesses metal-free and multiple structural defects, tunable electronic structure, mechanical stability, and high electrical conductivity [12]. Zeolitic imidazolate frameworks (ZIFs) are a subclass of metal-organic frameworks (MOFs). ZIFs are topologically isomorphic with zeolites. They are formed from tetrahedral metal ions (e.g., Zn and Co) connected by imidazolate linkers [13]. ZIF-67 with isostructural SOD zeolitic topology is formed from cobalt ions and 2-methylimidazole. ZIF-67 has a porous structure, a large surface area, and a big amount of active sites, and therefore, it is applied to several fields such as as a catalyst, for separation, and for adsorption [14, 15]. However, ZIF-67 has poor stability and low electrical conductivity. This limits its application in electrochemistry. Combining the advantageous features of both ZIF-67 and g-C3N4, one can manufacture versatile materials for electrochemistry and other potential applications. Recently, Meng et al. [16] reported ZIF-67/g-C3N4 as an efficient photocatalyst for CO2 reduction. To the best of our knowledge, little is known about the use of ZIF-67/g-C3N4 as an electrode modifier in the electrochemical analysis.Responding to this gap of knowledge, this article presents the synthesis of the ZIF-67/g-C3N4 composite using the ultrasound/microwave-assisted approach. Then, the composite was employed as a modifier to develop a novel electrode for the simultaneous determination of URA and ACE with CTAB as a discriminating agent.


## 2. Experimental

### 2.1. Materials

Melamine (C3H6N6, 99%), cobaltous nitrate hexahydrate (Co(NO3)·6H2O, 99%), 2-methylimidazole (CH3C3H2N2H, 99%), uric acid (C5H4N4O3, >99%), acetaminophen (CH3CONHC6H4OH, >99%), and cetyltrimethylammonium bromide (denoted as CTAB, CH3(CH2)15N(Br)(CH3)3, >98%), sodium hydroxide (NaOH, ≥97%), hydrochloric acid (HCl, 37%), glucose (C6H12O6, ≥99%), sucrose (C12H22O11, ≥99%), sodium oxalate (Na2C2O4, ≥99%), sodium nitrate (NaNO3, ≥99%), calcium chloride (CaCl2, ≥98%), potassium sulfate (K2SO4, ≥98%), ammonium sulfate ((NH4)2SO4, ≥99%), and potassium bicarbonate (KHCO3, ≥99%) were obtained from Merck & Co., Germany. Phosphoric acid (H3PO4, 85%), acetic acid (CH3COOH, ≥99.8%), and boric acid (H3BO3, 99%) were purchased from Daejung Co., Korea. A Britton-Robinson buffer (denoted as BR-BS) is used for the pH range from 2 to 10. It was prepared by mixing equal volumes of 0.04 M H3BO3 (2.04 g/100 mL), 0.04 M H3PO4 (2.8 mL of 85% H3PO4/100 mL), and 0.04 M CH3COOH (2.3 mL of glacial CH3COOH/100 mL) that has been adjusted to the desired pH with 0.2 M NaOH or 0.2 M HCl. The stock solution of 1×10−2 M URA and 1×10−2 M ACE was prepared daily. Standard solutions were prepared by diluting the stock solution with the BR-BS.


### 2.2. Apparatus

X-ray diffraction (XRD) analysis was performed on a D8 Advance Bruker anode X-ray diffractometer with Cu Kα (λ=1.5406 Å) radiation. Nitrogen adsorption/desorption isotherms were performed using a Micromeritics 2020 volumetric adsorption analyzer system. Samples were degassed by heating under vacuum at 180°C for 3 hours. The specific surface area of the samples was calculated using the Brunauer-Emmett-Teller (BET) model. X-ray photoelectron spectroscopy (XPS) was recorded on a Shimadzu Kratos AXIS ULTRA DLD spectrometer equipped with a Theta Probe ARXPS System (Thermo Fisher Scientific, UK). The peak fitting was performed by CasaXPS software. The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images were collected using a JEOL JEM-2100F (USA) and an SEM JMS-5300LV (USA), respectively. Electrochemical measurements were performed using a CPA-HH5 Computerized Polarography Analyzer (Vietnam). Voltammetric measurements were performed using a glassy carbon electrode (GCE, 2.8 mm diameter) or a ZIF-67/g-C3N4-modified GCE (ZIF-67/C3N4-GCE) as a working electrode, an Ag/AgCl/3 M KCl as a reference electrode, and a platinum foil auxiliary electrode.High-performance liquid chromatography (HPLC) was also used to determine the concentration of URA and ACE. The measurements were performed on a Shimadzu 2030 HPLC system with the following parameters: UV-vis detector (λ=275 nm) and C18 (250×4 mm; 5 μm) chromatographic column; mobile phase: mixture of phosphate buffer pH 2.3/acetonitrile (35/65 v/v); flow rate of 1.5 mL·min−1; and injection volume: 5 mL.


### 2.3. ZIF-67/g-C3N4 Preparation

g-C3N4 was synthesized according to the reference [17]. Briefly, melamine (10 g) was placed into a crucible with a cover under ambient pressure. Then, it was heated to 550°C for 4 h in nitrogen, and a yellow g-C3N4 powder was obtained. Co(NO3)·6H2O (2 mM) and 2-methylimidazole (2 mM) were completely dissolved in ethanol (15 mL) separately. 36 mg g-C3N4 was added into the cobaltous nitrate solution at ambient temperature and treated with ultrasound for 1 hour. Then, the 2-methylimidazole solution was added to the suspension of g-C3N4 and cobalt nitrate. Next, the mixture was placed into a microwave device and irradiated for 15 minutes, resulting in a light purple precipitate (ZIF-67/g-C3N4). Finally, the precipitate was washed with ethanol three times and dried at 80°C in air.


### 2.4. Preparation of Electrodes

A glassy carbon electrode (GCE) (2.8 mm diameter) was polished using 0.05 mm alumina slurry and rinsed thoroughly with distilled water. The electrode was then purified under ultrasonic agitation in ethanol for 5 min. 2 milligrams of ZIF-67/g-C3N4 was dispersed in 1 mL methanol under ultrasonic agitation for 60 min, resulting in a homogeneous purple suspension. 5 μL of ZIF-67/g-C3N4 suspension was dropped on the electrode surface. Then, the modified electrode was then dried at ambient temperature to obtain a ZIF-67/g-C3N4/GCE.


### 2.5. Electrochemical Measurements

The electrochemical measurements of URA and ACE were performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The DPVs were recorded in the potential range from −100 mV to 600 mV at ambient temperature with the pulse amplitude of 50 mV in all cases.


### 2.6. Real Sample Determination

Three samples of human urine were used to test the method. In detail, 1.0 mL of the urine sample was spiked with URA and ACE and mixed with 1.0 mL of BS buffer solution to produce a 2.0 mL test solution. 150μM CTAB was added. The proposed DPV method was applied to the determination of URA and ACE in the spiked solution.


## 2.1. Materials

Melamine (C3H6N6, 99%), cobaltous nitrate hexahydrate (Co(NO3)·6H2O, 99%), 2-methylimidazole (CH3C3H2N2H, 99%), uric acid (C5H4N4O3, >99%), acetaminophen (CH3CONHC6H4OH, >99%), and cetyltrimethylammonium bromide (denoted as CTAB, CH3(CH2)15N(Br)(CH3)3, >98%), sodium hydroxide (NaOH, ≥97%), hydrochloric acid (HCl, 37%), glucose (C6H12O6, ≥99%), sucrose (C12H22O11, ≥99%), sodium oxalate (Na2C2O4, ≥99%), sodium nitrate (NaNO3, ≥99%), calcium chloride (CaCl2, ≥98%), potassium sulfate (K2SO4, ≥98%), ammonium sulfate ((NH4)2SO4, ≥99%), and potassium bicarbonate (KHCO3, ≥99%) were obtained from Merck & Co., Germany. Phosphoric acid (H3PO4, 85%), acetic acid (CH3COOH, ≥99.8%), and boric acid (H3BO3, 99%) were purchased from Daejung Co., Korea. A Britton-Robinson buffer (denoted as BR-BS) is used for the pH range from 2 to 10. It was prepared by mixing equal volumes of 0.04 M H3BO3 (2.04 g/100 mL), 0.04 M H3PO4 (2.8 mL of 85% H3PO4/100 mL), and 0.04 M CH3COOH (2.3 mL of glacial CH3COOH/100 mL) that has been adjusted to the desired pH with 0.2 M NaOH or 0.2 M HCl. The stock solution of 1×10−2 M URA and 1×10−2 M ACE was prepared daily. Standard solutions were prepared by diluting the stock solution with the BR-BS.


## 2.2. Apparatus

X-ray diffraction (XRD) analysis was performed on a D8 Advance Bruker anode X-ray diffractometer with Cu Kα (λ=1.5406 Å) radiation. Nitrogen adsorption/desorption isotherms were performed using a Micromeritics 2020 volumetric adsorption analyzer system. Samples were degassed by heating under vacuum at 180°C for 3 hours. The specific surface area of the samples was calculated using the Brunauer-Emmett-Teller (BET) model. X-ray photoelectron spectroscopy (XPS) was recorded on a Shimadzu Kratos AXIS ULTRA DLD spectrometer equipped with a Theta Probe ARXPS System (Thermo Fisher Scientific, UK). The peak fitting was performed by CasaXPS software. The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images were collected using a JEOL JEM-2100F (USA) and an SEM JMS-5300LV (USA), respectively. Electrochemical measurements were performed using a CPA-HH5 Computerized Polarography Analyzer (Vietnam). Voltammetric measurements were performed using a glassy carbon electrode (GCE, 2.8 mm diameter) or a ZIF-67/g-C3N4-modified GCE (ZIF-67/C3N4-GCE) as a working electrode, an Ag/AgCl/3 M KCl as a reference electrode, and a platinum foil auxiliary electrode.High-performance liquid chromatography (HPLC) was also used to determine the concentration of URA and ACE. The measurements were performed on a Shimadzu 2030 HPLC system with the following parameters: UV-vis detector (λ=275 nm) and C18 (250×4 mm; 5 μm) chromatographic column; mobile phase: mixture of phosphate buffer pH 2.3/acetonitrile (35/65 v/v); flow rate of 1.5 mL·min−1; and injection volume: 5 mL.


## 2.3. ZIF-67/g-C3N4 Preparation

g-C3N4 was synthesized according to the reference [17]. Briefly, melamine (10 g) was placed into a crucible with a cover under ambient pressure. Then, it was heated to 550°C for 4 h in nitrogen, and a yellow g-C3N4 powder was obtained. Co(NO3)·6H2O (2 mM) and 2-methylimidazole (2 mM) were completely dissolved in ethanol (15 mL) separately. 36 mg g-C3N4 was added into the cobaltous nitrate solution at ambient temperature and treated with ultrasound for 1 hour. Then, the 2-methylimidazole solution was added to the suspension of g-C3N4 and cobalt nitrate. Next, the mixture was placed into a microwave device and irradiated for 15 minutes, resulting in a light purple precipitate (ZIF-67/g-C3N4). Finally, the precipitate was washed with ethanol three times and dried at 80°C in air.


## 2.4. Preparation of Electrodes

A glassy carbon electrode (GCE) (2.8 mm diameter) was polished using 0.05 mm alumina slurry and rinsed thoroughly with distilled water. The electrode was then purified under ultrasonic agitation in ethanol for 5 min. 2 milligrams of ZIF-67/g-C3N4 was dispersed in 1 mL methanol under ultrasonic agitation for 60 min, resulting in a homogeneous purple suspension. 5 μL of ZIF-67/g-C3N4 suspension was dropped on the electrode surface. Then, the modified electrode was then dried at ambient temperature to obtain a ZIF-67/g-C3N4/GCE.


## 2.5. Electrochemical Measurements

The electrochemical measurements of URA and ACE were performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The DPVs were recorded in the potential range from −100 mV to 600 mV at ambient temperature with the pulse amplitude of 50 mV in all cases.


## 2.6. Real Sample Determination

Three samples of human urine were used to test the method. In detail, 1.0 mL of the urine sample was spiked with URA and ACE and mixed with 1.0 mL of BS buffer solution to produce a 2.0 mL test solution. 150μM CTAB was added. The proposed DPV method was applied to the determination of URA and ACE in the spiked solution.


## 3. Results and Discussion

### 3.1. Characterization of Materials

The ZIF-67/g-C3N4 composite was synthesized through mixing g-C3N4 with the Co (II)/imidazole solution in ethanol under the ultrasonic and microwave irradiation. The resulting solids were investigated using XRD analysis (Figure 1(a)). All characteristic peaks of ZIF-67 (Figure 1(a)) are indexed according to the simulated XRD pattern of ZIF-67 from the database (CCDC671073). As for C3N4 (Figure 1(b)), two characteristic diffraction peaks of the tetragonal phase for g-C3N4 appear at 2θ=13.4° and 27.5° corresponding to the crystal plane of (100) and (002), respectively, and are indexed according to JCPDS 87-1526 [18]. In the XRD pattern of ZIF-67/g-C3N4 (Figure 1(c)), all the characteristic peaks of g-C3N4 and ZIF-67 are reduced significantly but still clearly observed. During ultrasonic treatment, g-C3N4 could split into the small clusters of g-C3N4, and thus, its crystal structure practically collapses. Therefore, the XRD diffractions of g-C3N4 are not observed in Figure 1(c). The textural properties of g-C3N4, ZIF-67, and the composite were characterized using nitrogen adsorption/desorption isotherms. As represented in Figure 1(d), all the samples illustrate a type IV isotherm with an H3 hysteresis loop according to IUPAC classification. SBET of g-C3N4 is 5 m2·g−1, while ZIF-67 exhibits a large SBET of 1,330 m2·g−1 due to its highly ordered and uniform structure. It is worth noting that the specific surface area of ZIF-67/g-C3N4 increases significantly (SBET=75 m2·g−1) compared with that of pure g-C3N4. The large specific surface area results in the efficient adsorption of analytes during the electrochemical process.Figure 1
XRD patterns of (a) ZIF-67, (b)g-C3N4, and (c) ZIF-67/g-C3N4 and (d) nitrogen adsorption/desorption isotherms of ZIF-67, g-C3N4, and ZIF-67/g-C3N4.
(a)
(b)
(c)
(d)The morphologies of the resulting samples are investigated with SEM and TEM. The TEM image ofg-C3N4 (Figure 2(a)) exhibits nanorods with 50 nm diameters, while the morphology of ZIF-67 consists of uniform polyhedrons of 134.2±5.6 nm in size (counted for 140 particles) (Figure 2(b)). It is possible that the bonding of Co2+ to N in g-C3N4 is less strong than that in 2-methylimidazole. Therefore, Co2+ cations first coordinate with the nitrogen atoms from g-C3N4, and while 2-methylimidazole (MI) is added, it reacts with N to form ZIF-67 particles of around 10-20 nm which are highly dispersed on the g-C3N4 matrix as shown in Figure 3(c). The synthesis of ZIF-67 concurrently with g-C3N4 may suppress the growth of ZIF-67 crystals. Consequently, its size is rather small compared with that of ZIF-67 synthesized without g-C3N4.Figure 2
(a) TEM ofg-C3N4; (b) SEM of ZIF-67; (c) TEM of ZIF-67/g-C3N4.
(a)
(b)
(c)Figure 3
(a) CVs recording at ZIF-67/C3N4-GCE in 0.1 M BR-BS pH=7÷10 containing equal concentrations of 0.5 mM URA, ACE, and 150 μM CTAB; (b) the plot of Ep vs. pH; (c) the plot of Ip vs. pH.
(a)
(b)
(c)To determine the chemical composition and the elemental state of ZIF-67/g-C3N4, its XPS was performed (Figure 4). As can be seen in Figure 4(a), the composite mainly consists of C, N, and Co with binding energy at around 285, 400, and 795 eV, respectively. In the N1s core level spectrum (Figure 4(b)), two peaks at 399.03 and 400.9 eV are assigned to the sp2-bonded nitrogen and π excitation of g-C3N4, respectively [19, 20]. In the C1s core level spectrum (Figure 4(c)), the four deconvoluted peaks appear at 284.78, 285.36, 287.24, and 288.78 eV. These peaks can be assigned to C–C sp2 (284.78 eV) and C–C sp3 (287.24 eV) in imidazole [21, 22], sp2-bonded carbon (288.78 eV) in aromatic rings of g-C3N4 (N–C=N), and the C–C coordination of the surface adventitious carbon (285.36 eV) [12]. For the Co2p core level spectrum (Figure 4(d)), the duplex of Co2p appears at 779.85 eV for Co2p3/2 with a satellite peak at 783.94 eV and at 795.71 eV for Co2p1/2 with a satellite peak at 801.14 eV. The main peak-satellite peak separation is narrow at about ~4 and ~5.4 eV for Co2p3/2 and Co2p1/2, respectively. The difference between the main peaks and the satellites is an important characteristic of the oxidation state of the cobalt ion. A narrow separation of about 4~6 eV (found in our study) is typical for Co (II), whereas a larger difference of about 9~10 eV is often found in Co (III) [23]. Therefore, the Co ions in ZIF-67/g-C3N4 are divalent.Figure 4
XPS spectra of ZIF-67/g-C3N4: (a) survey spectrum; (b) N1s XPS spectrum; (c) C1s XPS spectrum; (d) Co2p XPS spectrum.
(a)
(b)
(c)
(d)The stability of the electrode modifier in different acidic media is critical for the application in the electrochemical analysis. In the present study, the ZIF-67/g-C3N4 composite was immersed in water with pH ranging from 1 to 11 for 10 hours (Figure 5). The stability of the composite was assessed via XRD measurement. At low pH (pH 1), the intensity of diffractions of this sample is reduced or even disappeared compared with the original ZIF-67/g-C3N4, and those of the samples at pH=3÷12 seem slightly changeable, indicating that the composite is stable in aqueous solutions in this pH range.Figure 5
The stability of ZIF-67/g-C3N4 in water with different pH.


### 3.2. Electrochemical Behaviour

Figure6(a) represents the CVs at bare GCE, g-C3N4/GCE, ZIF-67/GCE, and ZIF-67-g-C3N4/GCE electrodes. As seen in the figure, the oxidation of URA and ACE occurs at similar potentials, and as a result, these two peaks are overlapped. However, the peaks are resolved significantly at the modified electrodes. The peak-to-peak separation is 0.10 V, 0.07 V, and 0.11 V for g-C3N4/GCE, ZIF-67/GCE, and ZIF-67/g-C3N4/GCE, respectively. The intensity of the oxidation peak for URA and ACE at ZIF-67/g-C3N4-GCE is 3.06 and 3.11 times as high as that at g-C3N4/GCE and 2.21 and 2.35 times as high as that at ZIF-67/GCE. These figures reveal that ZIF-67-g/C3N4 significantly promotes the electron transfer and, thus, oxidation of URA and ACE.Figure 6
(a) CVs at different electrodes in 0.1 M BR-BS pH 9 containing equal concentrations of 0.5 mM URA and ACE; (b) effect of ZIF-67/g-C3N4 amount on anodic peak current.
(a)
(b)The peak current depends on the ZIF-67/g-C3N4 amount modified on the electrode surface (Figure 6(b)). ZIF-67/g-C3N4 enhances analyte adsorption. As a result, the peak current increases and reaches the maximum at the volume of the suspension of around 4 μL. Further increase of ZIF-67/g-C3N4 leads to a decrease in peak current because a thicker layer of adsorbed ZIF-67/g-C3N4 could reduce the electrical conductivity.


#### 3.2.1. Effect of CTAB

The effects of CTAB concentration on the peak-to-peak separation of URA and ACE were performed by recording CVs of a series of solutions containingCURA=CACE=0.5 mM and various concentrations of CTAB (Figure 7(a)). As can be seen from Figure 7(b), the peak-to-peak separation (ΔE) increases with CTAB concentration and peaks at 150 μM CTAB (ΔE=0.18 V). Further increasing CTAB concentration leads to a slight reduction of ΔE. It is worth noting that the oxidation potential of ACE at 0.25 V seems to be constant, while the oxidation potential of URA shifts to less positive values with increasing CTAB concentration. The possible reason would be that CTAB molecules aggregate on the surface of the modified electrode to form micelles for discriminating ACE from URA. The concentration of 150 μM for CTAB is suitable for further experiments.Figure 7
(a) CVs at ZIF-67/g-C3N4/GCE in 0.1 M BR-BS containing CURA=CACE=0.5 mM and various concentrations of CTAB; (b) peak-to-peak separation as a function of CTAB concentration.
(a)
(b)


#### 3.2.2. Effect of pH

The CV curves at ZIF-67/g-C3N4-GCE were measured in the pH range from 7 to 10 (Figure 3(a)). The peak potential, Ep, reduces as pH increases, indicating that protons are involved in the redox processes (Figure 3(b)). The peak current, Ip, increases with pH and peaks at pH=9. Further increase in pH causes a negligible change in the peak current (Figure 3(c)).The anodic peak potential corresponds well to pH ranging from 7 to 10 with high correlation coefficients (r=0.985 and 0.999) (Equations (1) and (2)):
(1)Ep,URA=0.34±0.03+–0.030±0.004×pH,r=0.985,(2)Ep,ACE=0.74±0.01+–0.056±0,001×pH,r=0.999.The slope of the oxidation peak potential of ACE vs. pH is 0.056 V/pH, which is very close to the theoretical value of 0.059 V/pH, corresponding to the equal number of protons and electrons in the redox process. The electrochemical oxidation of URA proceeding by a 2e/2 proton mechanism to yield a diimine is nowadays well established. Meanwhile, the value of 0.030 V/pH deviates significantly from the theoretical value of 0.0599 V/pH and is about its half, indicating that the electrode process is more complex in the studied pH and the number of transferred electrons may be twice as much as that of protons.


#### 3.2.3. Effect of Scan Rate

Important information about the electrochemical mechanism can be derived from the relationship between the voltammetric signals (Ep and Ip) and the scan rate (denoted as ν). In the present study, the Ep and Ip dependence on the scan rate was investigated by using CVs (Figure 8(a)). If the electrooxidation reaction is reversible, Ep is independent on ν and vice versa. As can be seen from Figure 8(a), the peak potential increases with the scan rate. Therefore, the electron transfer in the URA and ACE electrooxidation is irreversible [24].Figure 8
Scan rate dependence of peak potential and peak current (a); the linear plots ofIp vs. ν1/2 (b); Ep vs. lnν (c); CCTAB=150×10−6 M and CURA=CACE=0.5×10−3 M in BR-BS buffer 0.1 M, pH 9.
(a)
(b)
(c)The linear plots ofIp vs. the square root of the scan rate (ν1/2) were established to assess whether the electrooxidation reaction is an adsorption-controlled or diffusion-controlled process (Figure 8(b)). If the linear plot of Ip vs. ν1/2 passes the origin, this process is controlled by diffusion; otherwise, it is an adsorption-controlled process [24]. The linear regression equations of Ip vs. ν1/2 are expressed as follows:
(3)Ip,URA=–0.160±0.033+0.100±0.006×v1/2,r=0.995,p<0.001,Ip,ACE=–0.023±0.019+0.091±0.011×v1/2,r=0.979,p=0.004.The linear relation ofIp,URA and Ip,ACE vs. ν1/2 is statistically significant (r=0.997÷0.999, p<0.05). The number in the parentheses represents the 95% confidence interval. The intercepts do not pass the origin because the 95% confidence interval for the intercept does not contain 0 (varying from −0.041 to −0.004 for ACE and from −0.072 to −0.006 for URA). This indicates that the electrode process of the URA and ACE electrooxidation is controlled by adsorption.The linear regression equations ofEp vs. lnν are as follows:
(4)Ep,URA=0.112+0.026×lnv,r=0.984,Ep,ACE=0.290+0.027×lnv,r=0.984.According to the Laviron theory [25], the relation of Ep vs. lnν can be expressed as Equation (5) in an irreversible system:
(5)Ep=E0−RT1−αnFlnRTKs1−αnF+RT1−αnFlnv,where α is the electron transfer coefficient, R is the universal constant (8.314 J/mol·K) at 298 K, and F is the Faraday constant (96,500 C·mol−1). The slope of the line of Ep vs. lnν provides the value of 1−α×n for ACE and URA being 0.95 and 0.99, respectively (Figure 8(c)). It is assumed that the value of α is 0.5. Then, the value of n is 1.9 for ACE and 1.98 for URA. Therefore, the equal number of electrons transferred is 2 for ACE. This means that two electrons and two protons are involved in the ACE oxidation to form N-acetyl-p-quinone-imine [26] at the modified electrodes. In the case of URA, the ratio of the number of protons and transferred electrons is not equal to one and involves less protons than electrons, e.g., two electrons and one proton. Although the mechanism of URA at the modified electrode is nuclear, this could be explained as the inference due to uncertainties introduced by the close proximity of voltammetric peak to the background discharge probably due to oxidation undergoing deprotonation or the adsorption of oxidation products blocking the electrode at the studied pH.The favorable signal-promoting effect indicates that CTAB enhances the discriminating peak current of ACE and URA. In this aspect, ZIF-67/g-C3N4 plays an important role in promoting the electron transfer rates of ACE and URA and brings out excellent electrocatalytic activity towards the redox reactions. Because ZIF-67 comprises imidazole rings of the sp2-conjugated bond (π–π interaction), the π–π stacking interaction between the phenyl structures of URA and ACE and the three-dimensional imidazolate structure of ZIF-67/g-C3N4 favors the adsorption on the modified electrode surface. The coordination of the nitrogen atoms in the analytes with Co (II) ions attracts ACE and URA to the modified electrode surface. In addition, g-C3N4 facilitates electron mobility in the redox reaction. In addition, the CTAB as a discriminating agent is also contributed to the well-defined separation of electrochemical signals. Therefore, the combination of these effects promotes the transfer of electrons and results in enhancing voltammetric signals. The oxidation mechanism for ACE and URA at the modified electrodes is proposed in Figure 9.Figure 9
Proposed mechanism of URA and ACE oxidation at the ZIF-67/g-C3N4-modified electrode.


### 3.3. Interference Study

Interferents commonly existent in biological samples include glucose, sucrose, oxalate, CaCl2, (NH4)2SO4, NaNO3, KHCO3, and K2SO4. Table 1 presents the tolerance limits of eight interferents. The tolerance limit, Ctol, is the concentration of the interferent that raises a relative error (RE) of 5% in the determination of 0.5 μM URA or 0.5 μM ACE. The findings show that the interference of inorganic salts is insignificant. However, some organic compounds, such as glucose, interfere but only at high concentrations. This indicates that the proposed method is likely to be free from common interferents in biological samples.Table 1
Tolerance limits of interferents,Ctot (5·10−7 M URA or 5·10−7 M URA in 0.1 M BR-BS pH 9 and 150 μM CTAB).
Interfering substances
Ctot (μM)
URA (0.5μM)
RE (%)
ACE (0.5μM)
RE (%)
Glucose
75
−4.55
75
4.9
Sucrose
75
−4.30
75
4.99
Oxalate
75
−4.79
150
4.85
NaNO3
150
−4.92
150
4.86
CaCl2
100
2.49
100
−3.40
K2SO4
150
4.2
150
−3.1
(NH4)2SO4
100
−3.26
100
4.38
KHCO3
150
−4.32
150
3.19


### 3.4. Long-Term Stability, Repeatability, and Linear Range

The long-term stability of the electrochemical response is of special interest for automatic monitoring of biological analytes. Hence, the response of ZIF-67/g-C3N4 was performed for a ten-day period by immersing the electrode in a solution of spiked water with 0.1 M BR-BS pH 9 containing 150 μM CTAB, 0.2 μM URA, and 0.2 μM ACE (10 measurements were performed during the working-day period). The electrode was stored in the buffer solution between each analysis. The changes of average Ip versus time are presented in Figure 10. The RSDs (relative standard deviations) of Ip for URA and ACE were 7.72 and 7.02%, respectively, using the same electrode for all the measurements. These values were lower than 1/2RSDH [27] indicating that the proposed DP-ASV method exhibits high stability.Figure 10
Long-term stability test for a ten-day period.The repeatability of the DP-ASV responses was evaluated by using a RSD for nine consecutive determinations of5×10−7 M URA and 5×10−7 M ACE. The RSD of URA and ACE is 1.03 and 1.52, which is lower than 1/2RSDHozit, indicating that the modified electrode shows good stability. The stability of the ZIF-67/g-C3N4-modified electrode was also tested by leaving the electrode in a desiccator under atmospheric conditions for 10 days. The DP-ASV peak currents for 5×10−7 M URA and 5×10−7 M ACE decrease by less than 4.61 and 4.90%, respectively. The high stability of the ZIF-67/g-C3N4-modified electrode contributed to its high mechanical strength and high stability in water, making it a potential for practical applications.The detection of each compound in the presence of the other was conducted. Figure11(a) shows the DPV curves recorded when adding URA or ACE and keeping the other constant. The anodic peak current increases linearly (Ip,URA=0.09+5.69CURA, r=0.995) with the concentration of URA from 0.02 to 0.65 μM in the presence of ACE with the limit detection (LOD) of 0.055 μM (Figure 11(b)). A similar behaviour is observed with the detection of ACE (Ip,ACE=0.15+5.63CACE, r=0.997) in the same concentration range with the detection limit of 0.056 μM (Figures 11(c) and 11(d)). Figure 12(a) represents the DP-DVS curves recorded for the simultaneous addition of URA and ACE in the concentration range between 0.02 and 0.65 μM. The plots of Ip,URA and Ip,ACE vs. the URA and ACE concentrations are shown in Figure 12(b). The linear regression equations are Ip,URA=0.06+5.81CURA, r=0.998, and Ip,ACE=0.14+5.84CACE, r=0.999. The LODs of URA and ACE are 0.052 μM and 0.053 μM, respectively. The similarity in LOD of URA and ACE in the mixture and as an individual infers that no remarkable interference due to the oxidation of the compounds occurs.Figure 11
Differential pulse voltammetric responses at ZIF-67/g-C3N4-GCE in 0.1 M BR-BS pH 9 containing (a) 150 μM CTAB and 0.2 μM ACE; the URA concentration varying from 0.02 to 0.65 μM; (b) plot of anodic peak current as a function of URA concentration; (c) 150 μM CTAB, 0.2 μM URA; the ACE concentration varying from 0.02 to 0.65 μM; (d) plot of anodic peak current as a function of ACE concentration.
(a)
(b)
(c)
(d)Figure 12
(a) DPVs recorded at ZIF-67/g-C3N4/GCE in 0.2 BR-BS pH 9 containing equal concentrations varying from 0.2 to 6.5 μM; (b) plot of anodic peak current as a function of analyte concentration.
(a)
(b)The linear regression equations of the peak current vs. the analyte concentration are expressed as follows:(6)Ip,URA=0.06±0.05+5.82±0.03×CURA,r=0.998,Ip,ACE=0.14±0.04+5.84±0.11×CACE,r=0.999.In the range from 0.02 to 0.65μM for URA and ACE, the LODs of URA and ACE are 0.052 μM and 0.053 μM, respectively. Table 2 shows the ability of the ZIF-67/g-C3N4 electrode for the URA and ACE determination compared with other reported electrodes. The present electrode has a much lower limit of detection for the determination of URA and ACE in comparison with most modified materials.Table 2
Comparison of some LOD and linear range of different modified electrodes for the determination of URA and ACE.
Electrode
Linear range (μM) (URA/ACE)
LOD (μM)
Reference
URA
ACE
Ionic liquid/CNT/chitosan-CGE
(2–450)/(1–400)
0.34
0.24
[28]
Thiol functional ferrocene derivative stabilized Au NP/carbon dot nanocomposite coupling with graphene-GCE
(0.6–9.2)/(0.5–46)
0.2
0.1
[29]
SWCNT/chitosan/ionic liquid nanocomposite-GCE
(3–320)/(2–200)
0.27
0.11
[30]
MCNT/chitosan composite-GCE
(10–400)/(2–250)
0.4
0.16
[31]
ZIF-67/g-C3N4-GCE
(0.2–6.5)/(0.2–6.5)
0.052
0.053
The present study
Notes: MCNT: multiwalled carbon nanotube; SWCNT: single-walled carbon nanotube; GCE: glassy carbon electrode.The ZIF-67/g-C3N4-modified electrode was utilized in the real sample analysis. Urine samples were collected from three healthy volunteers. The DPV results of the urine samples were obtained for URA and ACE in the BS solution, to which 10 μL of a URA and ACE stock solution was spiked to an electrochemical cell without any preliminary pretreatment. The amount of URA and ACE in the samples was determined with the calibration method using DPV and is presented in Table 3. The recovery of the proposed method varies in the acceptable range of 90–110%. The URA and ACE level in the samples was also tested using HPLC for comparison. The paired-sample t-test with α=0.05 shows that there is no significant difference between the DPV proposed method and HPLC (t5=0.215; p=0.839). This suggests that the proposed method enables to determine the URA and ACE level in the human urine with satisfactory results.Table 3
URA and ACE detection in urine samples obtained using the proposed method and HPLC.
Sample
Analytes
Added (μM)
Founda (μM)
Rev. (%)
HPLC (μM)
Urine #1
URA
0
22.69
99.4
—a
10
32.63
32.77
ACE
0
0
96.7
—
10
9.67
9.81
Urine #2
URA
0
26.11
97.8
—
10
35.89
35.92
ACE
0
0
98.0
—
10
9.80
9.85
Urine #3
URA
0
25.03
96.9
—
10
34.72
34.81
ACE
0
0
104.0
—
10
10.4
9.80
aNo analysis.


## 3.1. Characterization of Materials

The ZIF-67/g-C3N4 composite was synthesized through mixing g-C3N4 with the Co (II)/imidazole solution in ethanol under the ultrasonic and microwave irradiation. The resulting solids were investigated using XRD analysis (Figure 1(a)). All characteristic peaks of ZIF-67 (Figure 1(a)) are indexed according to the simulated XRD pattern of ZIF-67 from the database (CCDC671073). As for C3N4 (Figure 1(b)), two characteristic diffraction peaks of the tetragonal phase for g-C3N4 appear at 2θ=13.4° and 27.5° corresponding to the crystal plane of (100) and (002), respectively, and are indexed according to JCPDS 87-1526 [18]. In the XRD pattern of ZIF-67/g-C3N4 (Figure 1(c)), all the characteristic peaks of g-C3N4 and ZIF-67 are reduced significantly but still clearly observed. During ultrasonic treatment, g-C3N4 could split into the small clusters of g-C3N4, and thus, its crystal structure practically collapses. Therefore, the XRD diffractions of g-C3N4 are not observed in Figure 1(c). The textural properties of g-C3N4, ZIF-67, and the composite were characterized using nitrogen adsorption/desorption isotherms. As represented in Figure 1(d), all the samples illustrate a type IV isotherm with an H3 hysteresis loop according to IUPAC classification. SBET of g-C3N4 is 5 m2·g−1, while ZIF-67 exhibits a large SBET of 1,330 m2·g−1 due to its highly ordered and uniform structure. It is worth noting that the specific surface area of ZIF-67/g-C3N4 increases significantly (SBET=75 m2·g−1) compared with that of pure g-C3N4. The large specific surface area results in the efficient adsorption of analytes during the electrochemical process.Figure 1
XRD patterns of (a) ZIF-67, (b)g-C3N4, and (c) ZIF-67/g-C3N4 and (d) nitrogen adsorption/desorption isotherms of ZIF-67, g-C3N4, and ZIF-67/g-C3N4.
(a)
(b)
(c)
(d)The morphologies of the resulting samples are investigated with SEM and TEM. The TEM image ofg-C3N4 (Figure 2(a)) exhibits nanorods with 50 nm diameters, while the morphology of ZIF-67 consists of uniform polyhedrons of 134.2±5.6 nm in size (counted for 140 particles) (Figure 2(b)). It is possible that the bonding of Co2+ to N in g-C3N4 is less strong than that in 2-methylimidazole. Therefore, Co2+ cations first coordinate with the nitrogen atoms from g-C3N4, and while 2-methylimidazole (MI) is added, it reacts with N to form ZIF-67 particles of around 10-20 nm which are highly dispersed on the g-C3N4 matrix as shown in Figure 3(c). The synthesis of ZIF-67 concurrently with g-C3N4 may suppress the growth of ZIF-67 crystals. Consequently, its size is rather small compared with that of ZIF-67 synthesized without g-C3N4.Figure 2
(a) TEM ofg-C3N4; (b) SEM of ZIF-67; (c) TEM of ZIF-67/g-C3N4.
(a)
(b)
(c)Figure 3
(a) CVs recording at ZIF-67/C3N4-GCE in 0.1 M BR-BS pH=7÷10 containing equal concentrations of 0.5 mM URA, ACE, and 150 μM CTAB; (b) the plot of Ep vs. pH; (c) the plot of Ip vs. pH.
(a)
(b)
(c)To determine the chemical composition and the elemental state of ZIF-67/g-C3N4, its XPS was performed (Figure 4). As can be seen in Figure 4(a), the composite mainly consists of C, N, and Co with binding energy at around 285, 400, and 795 eV, respectively. In the N1s core level spectrum (Figure 4(b)), two peaks at 399.03 and 400.9 eV are assigned to the sp2-bonded nitrogen and π excitation of g-C3N4, respectively [19, 20]. In the C1s core level spectrum (Figure 4(c)), the four deconvoluted peaks appear at 284.78, 285.36, 287.24, and 288.78 eV. These peaks can be assigned to C–C sp2 (284.78 eV) and C–C sp3 (287.24 eV) in imidazole [21, 22], sp2-bonded carbon (288.78 eV) in aromatic rings of g-C3N4 (N–C=N), and the C–C coordination of the surface adventitious carbon (285.36 eV) [12]. For the Co2p core level spectrum (Figure 4(d)), the duplex of Co2p appears at 779.85 eV for Co2p3/2 with a satellite peak at 783.94 eV and at 795.71 eV for Co2p1/2 with a satellite peak at 801.14 eV. The main peak-satellite peak separation is narrow at about ~4 and ~5.4 eV for Co2p3/2 and Co2p1/2, respectively. The difference between the main peaks and the satellites is an important characteristic of the oxidation state of the cobalt ion. A narrow separation of about 4~6 eV (found in our study) is typical for Co (II), whereas a larger difference of about 9~10 eV is often found in Co (III) [23]. Therefore, the Co ions in ZIF-67/g-C3N4 are divalent.Figure 4
XPS spectra of ZIF-67/g-C3N4: (a) survey spectrum; (b) N1s XPS spectrum; (c) C1s XPS spectrum; (d) Co2p XPS spectrum.
(a)
(b)
(c)
(d)The stability of the electrode modifier in different acidic media is critical for the application in the electrochemical analysis. In the present study, the ZIF-67/g-C3N4 composite was immersed in water with pH ranging from 1 to 11 for 10 hours (Figure 5). The stability of the composite was assessed via XRD measurement. At low pH (pH 1), the intensity of diffractions of this sample is reduced or even disappeared compared with the original ZIF-67/g-C3N4, and those of the samples at pH=3÷12 seem slightly changeable, indicating that the composite is stable in aqueous solutions in this pH range.Figure 5
The stability of ZIF-67/g-C3N4 in water with different pH.


## 3.2. Electrochemical Behaviour

Figure6(a) represents the CVs at bare GCE, g-C3N4/GCE, ZIF-67/GCE, and ZIF-67-g-C3N4/GCE electrodes. As seen in the figure, the oxidation of URA and ACE occurs at similar potentials, and as a result, these two peaks are overlapped. However, the peaks are resolved significantly at the modified electrodes. The peak-to-peak separation is 0.10 V, 0.07 V, and 0.11 V for g-C3N4/GCE, ZIF-67/GCE, and ZIF-67/g-C3N4/GCE, respectively. The intensity of the oxidation peak for URA and ACE at ZIF-67/g-C3N4-GCE is 3.06 and 3.11 times as high as that at g-C3N4/GCE and 2.21 and 2.35 times as high as that at ZIF-67/GCE. These figures reveal that ZIF-67-g/C3N4 significantly promotes the electron transfer and, thus, oxidation of URA and ACE.Figure 6
(a) CVs at different electrodes in 0.1 M BR-BS pH 9 containing equal concentrations of 0.5 mM URA and ACE; (b) effect of ZIF-67/g-C3N4 amount on anodic peak current.
(a)
(b)The peak current depends on the ZIF-67/g-C3N4 amount modified on the electrode surface (Figure 6(b)). ZIF-67/g-C3N4 enhances analyte adsorption. As a result, the peak current increases and reaches the maximum at the volume of the suspension of around 4 μL. Further increase of ZIF-67/g-C3N4 leads to a decrease in peak current because a thicker layer of adsorbed ZIF-67/g-C3N4 could reduce the electrical conductivity.


### 3.2.1. Effect of CTAB

The effects of CTAB concentration on the peak-to-peak separation of URA and ACE were performed by recording CVs of a series of solutions containingCURA=CACE=0.5 mM and various concentrations of CTAB (Figure 7(a)). As can be seen from Figure 7(b), the peak-to-peak separation (ΔE) increases with CTAB concentration and peaks at 150 μM CTAB (ΔE=0.18 V). Further increasing CTAB concentration leads to a slight reduction of ΔE. It is worth noting that the oxidation potential of ACE at 0.25 V seems to be constant, while the oxidation potential of URA shifts to less positive values with increasing CTAB concentration. The possible reason would be that CTAB molecules aggregate on the surface of the modified electrode to form micelles for discriminating ACE from URA. The concentration of 150 μM for CTAB is suitable for further experiments.Figure 7
(a) CVs at ZIF-67/g-C3N4/GCE in 0.1 M BR-BS containing CURA=CACE=0.5 mM and various concentrations of CTAB; (b) peak-to-peak separation as a function of CTAB concentration.
(a)
(b)


### 3.2.2. Effect of pH

The CV curves at ZIF-67/g-C3N4-GCE were measured in the pH range from 7 to 10 (Figure 3(a)). The peak potential, Ep, reduces as pH increases, indicating that protons are involved in the redox processes (Figure 3(b)). The peak current, Ip, increases with pH and peaks at pH=9. Further increase in pH causes a negligible change in the peak current (Figure 3(c)).The anodic peak potential corresponds well to pH ranging from 7 to 10 with high correlation coefficients (r=0.985 and 0.999) (Equations (1) and (2)):
(1)Ep,URA=0.34±0.03+–0.030±0.004×pH,r=0.985,(2)Ep,ACE=0.74±0.01+–0.056±0,001×pH,r=0.999.The slope of the oxidation peak potential of ACE vs. pH is 0.056 V/pH, which is very close to the theoretical value of 0.059 V/pH, corresponding to the equal number of protons and electrons in the redox process. The electrochemical oxidation of URA proceeding by a 2e/2 proton mechanism to yield a diimine is nowadays well established. Meanwhile, the value of 0.030 V/pH deviates significantly from the theoretical value of 0.0599 V/pH and is about its half, indicating that the electrode process is more complex in the studied pH and the number of transferred electrons may be twice as much as that of protons.


### 3.2.3. Effect of Scan Rate

Important information about the electrochemical mechanism can be derived from the relationship between the voltammetric signals (Ep and Ip) and the scan rate (denoted as ν). In the present study, the Ep and Ip dependence on the scan rate was investigated by using CVs (Figure 8(a)). If the electrooxidation reaction is reversible, Ep is independent on ν and vice versa. As can be seen from Figure 8(a), the peak potential increases with the scan rate. Therefore, the electron transfer in the URA and ACE electrooxidation is irreversible [24].Figure 8
Scan rate dependence of peak potential and peak current (a); the linear plots ofIp vs. ν1/2 (b); Ep vs. lnν (c); CCTAB=150×10−6 M and CURA=CACE=0.5×10−3 M in BR-BS buffer 0.1 M, pH 9.
(a)
(b)
(c)The linear plots ofIp vs. the square root of the scan rate (ν1/2) were established to assess whether the electrooxidation reaction is an adsorption-controlled or diffusion-controlled process (Figure 8(b)). If the linear plot of Ip vs. ν1/2 passes the origin, this process is controlled by diffusion; otherwise, it is an adsorption-controlled process [24]. The linear regression equations of Ip vs. ν1/2 are expressed as follows:
(3)Ip,URA=–0.160±0.033+0.100±0.006×v1/2,r=0.995,p<0.001,Ip,ACE=–0.023±0.019+0.091±0.011×v1/2,r=0.979,p=0.004.The linear relation ofIp,URA and Ip,ACE vs. ν1/2 is statistically significant (r=0.997÷0.999, p<0.05). The number in the parentheses represents the 95% confidence interval. The intercepts do not pass the origin because the 95% confidence interval for the intercept does not contain 0 (varying from −0.041 to −0.004 for ACE and from −0.072 to −0.006 for URA). This indicates that the electrode process of the URA and ACE electrooxidation is controlled by adsorption.The linear regression equations ofEp vs. lnν are as follows:
(4)Ep,URA=0.112+0.026×lnv,r=0.984,Ep,ACE=0.290+0.027×lnv,r=0.984.According to the Laviron theory [25], the relation of Ep vs. lnν can be expressed as Equation (5) in an irreversible system:
(5)Ep=E0−RT1−αnFlnRTKs1−αnF+RT1−αnFlnv,where α is the electron transfer coefficient, R is the universal constant (8.314 J/mol·K) at 298 K, and F is the Faraday constant (96,500 C·mol−1). The slope of the line of Ep vs. lnν provides the value of 1−α×n for ACE and URA being 0.95 and 0.99, respectively (Figure 8(c)). It is assumed that the value of α is 0.5. Then, the value of n is 1.9 for ACE and 1.98 for URA. Therefore, the equal number of electrons transferred is 2 for ACE. This means that two electrons and two protons are involved in the ACE oxidation to form N-acetyl-p-quinone-imine [26] at the modified electrodes. In the case of URA, the ratio of the number of protons and transferred electrons is not equal to one and involves less protons than electrons, e.g., two electrons and one proton. Although the mechanism of URA at the modified electrode is nuclear, this could be explained as the inference due to uncertainties introduced by the close proximity of voltammetric peak to the background discharge probably due to oxidation undergoing deprotonation or the adsorption of oxidation products blocking the electrode at the studied pH.The favorable signal-promoting effect indicates that CTAB enhances the discriminating peak current of ACE and URA. In this aspect, ZIF-67/g-C3N4 plays an important role in promoting the electron transfer rates of ACE and URA and brings out excellent electrocatalytic activity towards the redox reactions. Because ZIF-67 comprises imidazole rings of the sp2-conjugated bond (π–π interaction), the π–π stacking interaction between the phenyl structures of URA and ACE and the three-dimensional imidazolate structure of ZIF-67/g-C3N4 favors the adsorption on the modified electrode surface. The coordination of the nitrogen atoms in the analytes with Co (II) ions attracts ACE and URA to the modified electrode surface. In addition, g-C3N4 facilitates electron mobility in the redox reaction. In addition, the CTAB as a discriminating agent is also contributed to the well-defined separation of electrochemical signals. Therefore, the combination of these effects promotes the transfer of electrons and results in enhancing voltammetric signals. The oxidation mechanism for ACE and URA at the modified electrodes is proposed in Figure 9.Figure 9
Proposed mechanism of URA and ACE oxidation at the ZIF-67/g-C3N4-modified electrode.


## 3.2.1. Effect of CTAB

The effects of CTAB concentration on the peak-to-peak separation of URA and ACE were performed by recording CVs of a series of solutions containingCURA=CACE=0.5 mM and various concentrations of CTAB (Figure 7(a)). As can be seen from Figure 7(b), the peak-to-peak separation (ΔE) increases with CTAB concentration and peaks at 150 μM CTAB (ΔE=0.18 V). Further increasing CTAB concentration leads to a slight reduction of ΔE. It is worth noting that the oxidation potential of ACE at 0.25 V seems to be constant, while the oxidation potential of URA shifts to less positive values with increasing CTAB concentration. The possible reason would be that CTAB molecules aggregate on the surface of the modified electrode to form micelles for discriminating ACE from URA. The concentration of 150 μM for CTAB is suitable for further experiments.Figure 7
(a) CVs at ZIF-67/g-C3N4/GCE in 0.1 M BR-BS containing CURA=CACE=0.5 mM and various concentrations of CTAB; (b) peak-to-peak separation as a function of CTAB concentration.
(a)
(b)


## 3.2.2. Effect of pH

The CV curves at ZIF-67/g-C3N4-GCE were measured in the pH range from 7 to 10 (Figure 3(a)). The peak potential, Ep, reduces as pH increases, indicating that protons are involved in the redox processes (Figure 3(b)). The peak current, Ip, increases with pH and peaks at pH=9. Further increase in pH causes a negligible change in the peak current (Figure 3(c)).The anodic peak potential corresponds well to pH ranging from 7 to 10 with high correlation coefficients (r=0.985 and 0.999) (Equations (1) and (2)):
(1)Ep,URA=0.34±0.03+–0.030±0.004×pH,r=0.985,(2)Ep,ACE=0.74±0.01+–0.056±0,001×pH,r=0.999.The slope of the oxidation peak potential of ACE vs. pH is 0.056 V/pH, which is very close to the theoretical value of 0.059 V/pH, corresponding to the equal number of protons and electrons in the redox process. The electrochemical oxidation of URA proceeding by a 2e/2 proton mechanism to yield a diimine is nowadays well established. Meanwhile, the value of 0.030 V/pH deviates significantly from the theoretical value of 0.0599 V/pH and is about its half, indicating that the electrode process is more complex in the studied pH and the number of transferred electrons may be twice as much as that of protons.


## 3.2.3. Effect of Scan Rate

Important information about the electrochemical mechanism can be derived from the relationship between the voltammetric signals (Ep and Ip) and the scan rate (denoted as ν). In the present study, the Ep and Ip dependence on the scan rate was investigated by using CVs (Figure 8(a)). If the electrooxidation reaction is reversible, Ep is independent on ν and vice versa. As can be seen from Figure 8(a), the peak potential increases with the scan rate. Therefore, the electron transfer in the URA and ACE electrooxidation is irreversible [24].Figure 8
Scan rate dependence of peak potential and peak current (a); the linear plots ofIp vs. ν1/2 (b); Ep vs. lnν (c); CCTAB=150×10−6 M and CURA=CACE=0.5×10−3 M in BR-BS buffer 0.1 M, pH 9.
(a)
(b)
(c)The linear plots ofIp vs. the square root of the scan rate (ν1/2) were established to assess whether the electrooxidation reaction is an adsorption-controlled or diffusion-controlled process (Figure 8(b)). If the linear plot of Ip vs. ν1/2 passes the origin, this process is controlled by diffusion; otherwise, it is an adsorption-controlled process [24]. The linear regression equations of Ip vs. ν1/2 are expressed as follows:
(3)Ip,URA=–0.160±0.033+0.100±0.006×v1/2,r=0.995,p<0.001,Ip,ACE=–0.023±0.019+0.091±0.011×v1/2,r=0.979,p=0.004.The linear relation ofIp,URA and Ip,ACE vs. ν1/2 is statistically significant (r=0.997÷0.999, p<0.05). The number in the parentheses represents the 95% confidence interval. The intercepts do not pass the origin because the 95% confidence interval for the intercept does not contain 0 (varying from −0.041 to −0.004 for ACE and from −0.072 to −0.006 for URA). This indicates that the electrode process of the URA and ACE electrooxidation is controlled by adsorption.The linear regression equations ofEp vs. lnν are as follows:
(4)Ep,URA=0.112+0.026×lnv,r=0.984,Ep,ACE=0.290+0.027×lnv,r=0.984.According to the Laviron theory [25], the relation of Ep vs. lnν can be expressed as Equation (5) in an irreversible system:
(5)Ep=E0−RT1−αnFlnRTKs1−αnF+RT1−αnFlnv,where α is the electron transfer coefficient, R is the universal constant (8.314 J/mol·K) at 298 K, and F is the Faraday constant (96,500 C·mol−1). The slope of the line of Ep vs. lnν provides the value of 1−α×n for ACE and URA being 0.95 and 0.99, respectively (Figure 8(c)). It is assumed that the value of α is 0.5. Then, the value of n is 1.9 for ACE and 1.98 for URA. Therefore, the equal number of electrons transferred is 2 for ACE. This means that two electrons and two protons are involved in the ACE oxidation to form N-acetyl-p-quinone-imine [26] at the modified electrodes. In the case of URA, the ratio of the number of protons and transferred electrons is not equal to one and involves less protons than electrons, e.g., two electrons and one proton. Although the mechanism of URA at the modified electrode is nuclear, this could be explained as the inference due to uncertainties introduced by the close proximity of voltammetric peak to the background discharge probably due to oxidation undergoing deprotonation or the adsorption of oxidation products blocking the electrode at the studied pH.The favorable signal-promoting effect indicates that CTAB enhances the discriminating peak current of ACE and URA. In this aspect, ZIF-67/g-C3N4 plays an important role in promoting the electron transfer rates of ACE and URA and brings out excellent electrocatalytic activity towards the redox reactions. Because ZIF-67 comprises imidazole rings of the sp2-conjugated bond (π–π interaction), the π–π stacking interaction between the phenyl structures of URA and ACE and the three-dimensional imidazolate structure of ZIF-67/g-C3N4 favors the adsorption on the modified electrode surface. The coordination of the nitrogen atoms in the analytes with Co (II) ions attracts ACE and URA to the modified electrode surface. In addition, g-C3N4 facilitates electron mobility in the redox reaction. In addition, the CTAB as a discriminating agent is also contributed to the well-defined separation of electrochemical signals. Therefore, the combination of these effects promotes the transfer of electrons and results in enhancing voltammetric signals. The oxidation mechanism for ACE and URA at the modified electrodes is proposed in Figure 9.Figure 9
Proposed mechanism of URA and ACE oxidation at the ZIF-67/g-C3N4-modified electrode.


## 3.3. Interference Study

Interferents commonly existent in biological samples include glucose, sucrose, oxalate, CaCl2, (NH4)2SO4, NaNO3, KHCO3, and K2SO4. Table 1 presents the tolerance limits of eight interferents. The tolerance limit, Ctol, is the concentration of the interferent that raises a relative error (RE) of 5% in the determination of 0.5 μM URA or 0.5 μM ACE. The findings show that the interference of inorganic salts is insignificant. However, some organic compounds, such as glucose, interfere but only at high concentrations. This indicates that the proposed method is likely to be free from common interferents in biological samples.Table 1
Tolerance limits of interferents,Ctot (5·10−7 M URA or 5·10−7 M URA in 0.1 M BR-BS pH 9 and 150 μM CTAB).
Interfering substances
Ctot (μM)
URA (0.5μM)
RE (%)
ACE (0.5μM)
RE (%)
Glucose
75
−4.55
75
4.9
Sucrose
75
−4.30
75
4.99
Oxalate
75
−4.79
150
4.85
NaNO3
150
−4.92
150
4.86
CaCl2
100
2.49
100
−3.40
K2SO4
150
4.2
150
−3.1
(NH4)2SO4
100
−3.26
100
4.38
KHCO3
150
−4.32
150
3.19


## 3.4. Long-Term Stability, Repeatability, and Linear Range

The long-term stability of the electrochemical response is of special interest for automatic monitoring of biological analytes. Hence, the response of ZIF-67/g-C3N4 was performed for a ten-day period by immersing the electrode in a solution of spiked water with 0.1 M BR-BS pH 9 containing 150 μM CTAB, 0.2 μM URA, and 0.2 μM ACE (10 measurements were performed during the working-day period). The electrode was stored in the buffer solution between each analysis. The changes of average Ip versus time are presented in Figure 10. The RSDs (relative standard deviations) of Ip for URA and ACE were 7.72 and 7.02%, respectively, using the same electrode for all the measurements. These values were lower than 1/2RSDH [27] indicating that the proposed DP-ASV method exhibits high stability.Figure 10
Long-term stability test for a ten-day period.The repeatability of the DP-ASV responses was evaluated by using a RSD for nine consecutive determinations of5×10−7 M URA and 5×10−7 M ACE. The RSD of URA and ACE is 1.03 and 1.52, which is lower than 1/2RSDHozit, indicating that the modified electrode shows good stability. The stability of the ZIF-67/g-C3N4-modified electrode was also tested by leaving the electrode in a desiccator under atmospheric conditions for 10 days. The DP-ASV peak currents for 5×10−7 M URA and 5×10−7 M ACE decrease by less than 4.61 and 4.90%, respectively. The high stability of the ZIF-67/g-C3N4-modified electrode contributed to its high mechanical strength and high stability in water, making it a potential for practical applications.The detection of each compound in the presence of the other was conducted. Figure11(a) shows the DPV curves recorded when adding URA or ACE and keeping the other constant. The anodic peak current increases linearly (Ip,URA=0.09+5.69CURA, r=0.995) with the concentration of URA from 0.02 to 0.65 μM in the presence of ACE with the limit detection (LOD) of 0.055 μM (Figure 11(b)). A similar behaviour is observed with the detection of ACE (Ip,ACE=0.15+5.63CACE, r=0.997) in the same concentration range with the detection limit of 0.056 μM (Figures 11(c) and 11(d)). Figure 12(a) represents the DP-DVS curves recorded for the simultaneous addition of URA and ACE in the concentration range between 0.02 and 0.65 μM. The plots of Ip,URA and Ip,ACE vs. the URA and ACE concentrations are shown in Figure 12(b). The linear regression equations are Ip,URA=0.06+5.81CURA, r=0.998, and Ip,ACE=0.14+5.84CACE, r=0.999. The LODs of URA and ACE are 0.052 μM and 0.053 μM, respectively. The similarity in LOD of URA and ACE in the mixture and as an individual infers that no remarkable interference due to the oxidation of the compounds occurs.Figure 11
Differential pulse voltammetric responses at ZIF-67/g-C3N4-GCE in 0.1 M BR-BS pH 9 containing (a) 150 μM CTAB and 0.2 μM ACE; the URA concentration varying from 0.02 to 0.65 μM; (b) plot of anodic peak current as a function of URA concentration; (c) 150 μM CTAB, 0.2 μM URA; the ACE concentration varying from 0.02 to 0.65 μM; (d) plot of anodic peak current as a function of ACE concentration.
(a)
(b)
(c)
(d)Figure 12
(a) DPVs recorded at ZIF-67/g-C3N4/GCE in 0.2 BR-BS pH 9 containing equal concentrations varying from 0.2 to 6.5 μM; (b) plot of anodic peak current as a function of analyte concentration.
(a)
(b)The linear regression equations of the peak current vs. the analyte concentration are expressed as follows:(6)Ip,URA=0.06±0.05+5.82±0.03×CURA,r=0.998,Ip,ACE=0.14±0.04+5.84±0.11×CACE,r=0.999.In the range from 0.02 to 0.65μM for URA and ACE, the LODs of URA and ACE are 0.052 μM and 0.053 μM, respectively. Table 2 shows the ability of the ZIF-67/g-C3N4 electrode for the URA and ACE determination compared with other reported electrodes. The present electrode has a much lower limit of detection for the determination of URA and ACE in comparison with most modified materials.Table 2
Comparison of some LOD and linear range of different modified electrodes for the determination of URA and ACE.
Electrode
Linear range (μM) (URA/ACE)
LOD (μM)
Reference
URA
ACE
Ionic liquid/CNT/chitosan-CGE
(2–450)/(1–400)
0.34
0.24
[28]
Thiol functional ferrocene derivative stabilized Au NP/carbon dot nanocomposite coupling with graphene-GCE
(0.6–9.2)/(0.5–46)
0.2
0.1
[29]
SWCNT/chitosan/ionic liquid nanocomposite-GCE
(3–320)/(2–200)
0.27
0.11
[30]
MCNT/chitosan composite-GCE
(10–400)/(2–250)
0.4
0.16
[31]
ZIF-67/g-C3N4-GCE
(0.2–6.5)/(0.2–6.5)
0.052
0.053
The present study
Notes: MCNT: multiwalled carbon nanotube; SWCNT: single-walled carbon nanotube; GCE: glassy carbon electrode.The ZIF-67/g-C3N4-modified electrode was utilized in the real sample analysis. Urine samples were collected from three healthy volunteers. The DPV results of the urine samples were obtained for URA and ACE in the BS solution, to which 10 μL of a URA and ACE stock solution was spiked to an electrochemical cell without any preliminary pretreatment. The amount of URA and ACE in the samples was determined with the calibration method using DPV and is presented in Table 3. The recovery of the proposed method varies in the acceptable range of 90–110%. The URA and ACE level in the samples was also tested using HPLC for comparison. The paired-sample t-test with α=0.05 shows that there is no significant difference between the DPV proposed method and HPLC (t5=0.215; p=0.839). This suggests that the proposed method enables to determine the URA and ACE level in the human urine with satisfactory results.Table 3
URA and ACE detection in urine samples obtained using the proposed method and HPLC.
Sample
Analytes
Added (μM)
Founda (μM)
Rev. (%)
HPLC (μM)
Urine #1
URA
0
22.69
99.4
—a
10
32.63
32.77
ACE
0
0
96.7
—
10
9.67
9.81
Urine #2
URA
0
26.11
97.8
—
10
35.89
35.92
ACE
0
0
98.0
—
10
9.80
9.85
Urine #3
URA
0
25.03
96.9
—
10
34.72
34.81
ACE
0
0
104.0
—
10
10.4
9.80
aNo analysis.


## 4. Conclusions

ZIF-67/g-C3N4 was synthesized using the ultrasonic-assisted approach. The obtained material exhibits a large specific surface area and high stability in pH ranging of 3 to 12. The ZIF-67/g-C3N4 electrode shows high stability and reproducibility in repetitive measurements. The proposed method provides satisfactory results for the detection of uric acid and acetaminophen in human urine. The method is time-competitive, easy to perform, highly stable, and sensitive with high detectability. All these features suggest that the proposed method is a potential candidate for practical applications.



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*Source: 7915878-2020-01-27.xml*