A new strategy for simultaneous determination of 4-aminophenol, uric acid and nitrite based on a graphene/hydroxyapatite composite modified glassy carbon electrode

Abstract

A novel electrochemical sensor has been fabricated based on a graphene/hydroxyapatite nanocomposite modified glassy carbon electrode (GCE) for the selective and simultaneous detection of 4-aminophenol (4-AP), uric acid (UA) and nitrite ions (NO²⁻) in a phosphate buffer solution (PBS, pH 7.0) for the first time. The modified electrode exhibited improved electrocatalytic activity towards the oxidation of 4-AP, UA and NO²⁻ in the form of three strong peaks in both the cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques. Under optimum conditions, the ternary system comprising 4-AP, UA and NO²⁻ exhibited linear calibration plots over a wide range of 0.1–425 μM, 1–1000 μM and 3–950 μM with detection limits of 0.29 μM, 0.03 μM and 0.025 μM for 4-AP, UA and NO²⁻ respectively. The developed sensor displayed high sensitivity and low detection limits coupled with good stability and reproducibility which were attributed to the synergistic effect of graphene and HAP in the nanocomposite. In addition, the fabricated sensor was applied to the determination of UA, 4-AP and NO²⁻ in urine and tap water samples with satisfactory results.

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1. Introduction

4-Aminophenol (4-AP) is the primary hydrolytic degradation product of acetaminophen, and has been detected in the pharmaceutical preparation of acetaminophen as a synthetic intermediate. 4-AP is also broadly applied in the production of dyestuff, developers, chemical inhibitors and petroleum additives. As a result, large amounts of 4-AP may be inevitably released into the environment as a pollutant.¹ Due to the discovery of its significant nephrotoxicity and teratogenic effect, the maximum content of 4-AP in pharmaceuticals is limited to 50 ppm (0.005%, w/w) by the European² and United States³ pharmacopeia. The overdose of 4-AP is the foremost cause of acute liver failure and accounts for most morbidity and mortality in drug-poisoning cases. Hence, precise determination of 4-AP in matrices such as water, pharmaceutical formulations and human fluids is important.⁴

Nitrite (NO²⁻) is ubiquitous with environmental, food and life science and commonly used as food additive, fertilizing agents and corrosion inhibitor. Excessive nitrite in the body can lead to the irreversible oxidation of hemoglobin to methemoglobin; furthermore, nitrite can react with amine to form a carcinogenic nitrosamine, resulting in cancer and hypertension. The World Health Organization has reported that the fatal dose of nitrite ingestion is between 8.7 μM and 28.3 μM.^5,6 Recently, many studies have demonstrated that NO could act as a neurotransmitter or a neuromodulator in central nervous system, and also, NO could enhance or inhibit the release of dopamine.^7,8 Moreover, NO can be oxidized to NO²⁻ within a few seconds by reacting with the dissolved oxygen in solution and also in the biological environments.⁹ Hence, precise determination of NO²⁻ is important requirement for medical and environmental applications.

Uric acid (UA) is known to be an important natural antioxidant in blood and brain tissue scavenging superoxide, peroxynitrite and hydroxyl radical. The typical concentrations of UA is in millimolar range (120–450 μmol L⁻¹) in blood and about 2 mmol L⁻¹ in urine and its abnormality can lead to some diseases like gout, hyperpiesia, and Lesch-Nyhan disease. Higher serum UA levels have been associated with a significantly reduced risk of Parkinson disease, low levels linked to multiple sclerosis.¹⁰ Therefore, it is necessary to develop a simple, fast and sensitive analytical method for simultaneous detection of 4-AP, UA and NO²⁻ for both environmental and biomedical benefits. Recently, electrochemical techniques have been used extensively due to its advantageous features such as high sensitivity, fast response, inexpensive instrument, low energy consumption, simple operation, time saving, good reliability and possibility for in situ analysis. The 4-AP, UA and NO²⁻ contain electrochemically active groups which can be oxidized independently at different potentials. So, various electrochemical sensors were reported for their analysis. For instance, graphene/polyaniline,¹¹ graphene/chitosan,¹² single walled nanotube/nafion,¹³ hemim based molecular imprinted polymer¹⁴ modified electrodes were reported for the detection of 4-AP. As for the detection of UA and NO²⁻, their simultaneous determination was achieved using La(OH)₃ nanorods,¹⁵ poly(2-mercaptobenzothiazole),¹⁶ lanthanum multiwalled carbon nanotube nanocomposites,¹⁷ Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film,¹⁸ and thiazole-based copolymer¹⁹ modified electrodes. However, to the best of our knowledge, there has been no report for the simultaneous determination of all the three molecules viz. 4-AP, UA and NO²⁻. In this work, we have fabricated a graphene/hydroxyapatite composite modified glassy carbon electrode for the selective and simultaneous determination of 4-AP, UA and NO²⁻ for the first time.

Graphene has attracted considerable attention due to its extraordinary mechanical, thermal, electrical properties, fast electron transportation and good biocompatibility which makes it suitable for potential applications in the field of electrochemical sensors, solar cells and energy storage capacitors.^20–22 Hydroxyapatite (HAP, Ca₁₀(PO₄)₆(OH)₂) is a form of calcium phosphate that bears close chemical likeness with the mineral part of bone and teeth. It promotes tissue attachment and bone growth by spontaneously forming a biologically active bone-like apatite layer on its surface. Thus, HAP is classified as a biocompatible and bioactive material and crystalline HAP has been found in many biological applications such as dental or skeletal implants, bone repair scaffolds, and biosensors.²³ The HAP and graphene nanohybrids are important kinds of nanocomposite materials which successfully integrate the unique properties of two classes of materials and exhibit some new functions caused by the cooperative effects between the HAP and the graphene such as high electron mobility and excellent catalytic activity. Recently, graphene/HAP nanocomposites have been synthesized and their potential applications in bioactive and biomedical fields have been demonstrated.^24,25

Here, we report the synthesis of graphene/HAP composite by a simple chemical precipitation method and demonstrated its applicability as sensing material for the simultaneous detection of three important molecules 4-AP, UA and NO²⁻. The graphene/HAP modified GCE not only exhibits high electrocatalytic activities toward the oxidations of 4-AP, UA and NO²⁻ with enhanced oxidation peak currents, but also resolve the sluggish and over-lapped oxidation waves at GO/GCE into three well-defined anodic peaks. Thus, the simultaneous determinations of 4-AP, UA and NO²⁻ in human urine and tap water samples have been investigated as real sample applications.

2. Experimental

2.1 Materials

The reagents used were of analytical grade without further purification. Graphite, sulfuric acid, potassium permanganate, 4-aminophenol, uric acid, sodium nitrite and hydrogen peroxide (H₂O₂, 30% w/v solution) were purchased from Sigma Aldrich. Calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O), diammonium hydrogen phosphate ((NH₄)₂HPO₄), trisodium citrate and cetyltrimethyl ammonium bromide were obtained from Merck. 0.1 M phosphate buffer solution (PBS) was prepared by dissolving appropriate amounts of sodium hydrogen phosphate and sodium dihydrogen phosphate. The pH of the buffer was adjusted to appropriate value by adding hydrochloric acid. All electrochemical experiments were carried out in 0.1 M PBS at pH 7.0. Freshly prepared 4-AP, UA and NO²⁻ solution were used for each experiment.

2.2 Instrumentation

Powder X-ray diffraction (XRD) analyses were carried out on a Bruker D8-ADVANCE diffractometer with Cu Kα radiation of wavelength (λ) of 0.15418 nm. The morphology of the samples was checked by scanning electron microscope (SEM) (FEG QUANTA 250). The functional groups of graphene/HAP nanocomposite were identified by recording Fourier transform infrared (FTIR) spectra (Thermo Nicolect 380) and Raman spectra at room temperature (Princeton Instruments Acton SP2500 (0.5 m imaging triple grating monochramator/spectrograph)).

Electrochemical measurements were carried out using a CHI 608d electrochemical workstation (CH Instruments, Austin, USA). A three-electrode system was employed with a 3 mm glassy carbon (GC) as the working electrode, a Pt wire as the auxiliary electrode and Ag/AgCl (saturated KCl) as the reference electrode in an electrochemical cell filled with 10 mL of PBS at room temperature. Electrochemical impedance spectroscopy (EIS) experiment was performed in 0.1 M KCl containing 1 mM [Fe(CN)₆]^3−/4−. The EIS experiments were carried out by applying an ac potential of amplitude 5 mV over the dc potential of 200 V in the frequency range of 100 kHz to 1 Hz. Cyclic voltammograms (CVs) were recorded between a potential window from −0.2 V to 1.2 V at the scan rate of 50 mV s⁻¹ in 0.1 M PBS (pH 7.0) containing 4-AP, UA and NO²⁻. Square wave voltammetry (SWV) measurements were performed in PBS (pH 7.0) in the potential region from −0.2 to 1.2 V with frequency of 10 Hz, amplitude of 50 mV and step potential of 5 mV.

2.3 Synthesis of graphene oxide (GO)

The graphene oxide (GO) was prepared according to modified Hummer's method.²⁶ Briefly, 1 g graphite was added to 50 mL of concentrated sulfuric acid cooled by an ice water bath. Then 4 g potassium permanganate was slowly added and the mixture was stirred at 30 °C for 1 h. Next, 150 mL water was added, and the mixture was stirred for 30 min while the temperature was raised to 90 °C. Finally, 200 mL water was added, followed by the slow addition of 3 mL of H₂O₂ (30%), which turned the color of the solution from dark brown to greenish yellow. The solid graphite oxide was separated by centrifugation, washed repeatedly with deionized water and finally dried under vacuum.

2.4 Synthesis of the graphene/HAP composite

Graphene/HAP nanocomposite was synthesized by a simple chemical precipitation method.²⁷ In brief, dried GO (2 mg) was dispersed in deionized water (1 mL). Then (40 wt%) 94 mg of Ca(NO₃).4H₂O was added into 90 mL of GO colloid solution with ultrasonic treatment for 60 min to form a colloidal suspension. Subsequently 40 mg of cetyltrimethylammonium bromide (CTAB) was added into solution and sonicated for 60 min to form solution A. Approximately 103.2 mg of trisodium citrate and 31.56 mg of (NH₄)₂HPO₄ were added into 15 mL of ultrapure water to form solution B which was poured into solution A. The combined solution was sonicated for 30 min and then kept in hot air oven at 180 °C for 12 h. The composite was washed several times with ethanol and water in order to remove CTAB and trisodium citrate ions and the product was dried at 60 °C for 24 h.

2.5 Fabrication of the modified electrodes

Glassy carbon electrode (GCE) was polished carefully with 1.0, 0.3 and 0.5 μM alumina on polishing cloth and was rinsed with deionized water which was followed by successive ultrasonic treatment to remove the physically adsorbed substance. Graphene/HAP composites (5.0 mg) were dispersed in 1 mL deionized water by using ultrasonicator. Then, 10 μL of the prepared suspension was dropped onto the GCE surface by drop casting method and dried in room temperature to obtain the graphene/HAP modified GCE (Scheme 1). In addition, GO and HAP modified GC electrodes were prepared by the above mentioned process for comparison.

Scheme 1 Schematic representation of the graphene/HAP modified GC electrode for simultaneous detection of 4-AP, UA and NO²⁻.

3. Results and discussion

3.1 Structural analysis

Powder X-ray diffraction pattern of GO shows a diffraction peak (Fig. 1A(a)) at around 10.87° (2θ), which corresponds to the (002) reflection of stacked GO sheets. HAP shows diffraction peaks corresponding to hexagonal structure with space group P63/m (ICDD card no. 09-0432) as shown in Fig. 1A(b).²³ No obvious diffraction peaks attributed to graphite in the XRD pattern of the graphene/HAP nanocomposite are observed. This result indicates that the HAP adhered on graphene sheets can prevent stacking into multilayers, favouring the maintenance of high surface area. Fig. 1B shows the SEM image of (a) GO, (b) HAP and (c) graphene/HAP nanocomposite. The characteristic plate like morphology was clearly seen for pure HAP. However, when GO is introduced, HAP plates anchored on a wrinkled GNS as shown in Fig. 1B(b).

Fig. 1 (A) XRD patterns of (a) GO, (b) HAP and (c) graphene/HAP and (B) SEM image of (a) GO, (b) HAP and (b) graphene/HAP; (C) Raman spectra of (a) GO and (b) graphene/HAP; (D) FTIR spectra of (a) GO, (b) graphene/HAP and (c) HAP.

Fig. 1C shows the Raman spectra of (a) GO and (b) graphene/HAP nanocomposite. The peak observed at about 1590 cm⁻¹ (G band) corresponding to E_2g mode of graphite, was related to the vibration of the sp²-bonded carbon atoms in a 2 dimensional hexagonal lattice, while the peak at about 1357 cm⁻¹ (D band) was related to the breathing of k point phonons of A_1g symmetry.²⁸ The intensity ratio of the D to G band (I_D/I_G) was calculated as 1.05 and 1.1 for GO and graphene/HAP nanocomposite respectively. Compared with GO, the intensity ratio of I_D/I_G for graphene/HAP is increased, which could be ascribed to the exfoliation of GO and the presence of HAP between the GO.

The FTIR spectra of (a) GO, (b) graphene/HAP and (c) HAP are shown in Fig. 1D. In case of composite sample, an absorption band located at ∼3420 cm⁻¹ is due to O–H vibrations of absorbed water and the bands located at 562 cm⁻¹, 603 cm⁻¹ are assigned to the PO₄³⁻. The oxygen-containing functional groups of GO were revealed by the presence of bands at 1089, 1227, 1633, and 1730 cm⁻¹. These bands can be assigned to the C–O stretching vibration, C–OH stretching vibration, C=C stretching vibration, and C=O stretching vibration of COOH group, respectively.¹⁷ The absorption bands at 1033, 565 and 602 cm⁻¹ are attributed to characteristic absorption of PO₄³⁻ in HAP.

3.2 Electrochemical activity of graphene/HAP nanocomposite modified GC electrode

The charge transport process of the graphene/HAP modified GC electrode was studied by monitoring charge transfer resistance (R_ct) at the electrode/electrolyte interface. The R_ct values were obtained by Randels equivalent circuit, R_s(Q_CPE(R_ctW)), where R_s is the solution resistance, R_ct is the charge transfer resistance, W is the Warburg impedance and Q_CPE is the constant phase element (CPE). Fig. S1A† shows the electrochemical impedance spectroscopy (EIS) responses of the bare and modified GC electrodes. The R_ct values for the bare GC (curve a), GO (curve b), HAP (curve c) and graphene/HAP (curve d) modified electrodes were estimated to be 705, 4125, 1506 and 310 Ω cm⁻² respectively. It can be noticed that the R_ct value of graphene/HAP/GCE is lower than that of bare GC, GO and HAP modified electrodes which demonstrate that the graphene/HAP modified electrode can form good electron pathways between the electrode and electrolyte and that it can be a good platform for sensing applications. These results are in good agreement with the peak current (i_pa) values obtained from CV measurements (Fig. S1B†).

Fig. S1B† shows the cyclic voltammograms (CVs) of different modified electrodes recorded in the presence of 1 mM Fe(CN)₆]^3−/4− in 0.1 M KCl at the scan rate of 50 mV s⁻¹. Well-resolved anodic and cathodic peaks are observed at the bare GCE (curve a), GO (curve b) HAP (curve c) and graphene/HAP (curve d) modified GC electrodes. Compared to those obtained at the bare GCE (17 μA), GO/GCE (2 μA) and HAP/GCE (16 μA), a significantly higher peak current was obtained at the graphene/HAP (35 μA) modified GC electrode. The peak separation potential (ΔE) of the redox peaks for graphene/HAP/GCE and HAP/GCE are obtained as 91 and 203 mV, respectively which indicate the fast electron transfer process capability of the graphene/HAP/GCE. These observations aptly suggest that the graphene/HAP based electrode is more facile than the bare, GO and HAP modified GC electrodes.

3.3 Electrochemical behavior of 4-AP, UA and NO²⁻

The individual electrochemical behavior of 4-AP, UA and NO²⁻ in PBS (pH 7.0) at bare GCE (curve a), GO/GCE (curve b), HAP/GCE (curve c) and graphene/HAP/GCE (curve d) were first investigated by cyclic voltammetry (CV) studies. In the case of 0.1 mM 4-AP (Fig. 2A), a small defined redox wave appeared at the bare electrode (curve a). On the other hand, modification GC electrode with graphene/HAP significantly enhances the oxidation peak current of 4-AP (7.5 μA) which is 2-fold higher relative to that of bare GCE (3.7 μA) and HAP (2.6 μA) modified GCE. The modified graphene/HAP/GCE not only improves the redox peak currents but also makes the redox reaction of 4-AP more reversible which shows that the modified GCE plays an important role in accelerating the electron transfer toward the detection of 4-AP. For UA (0.5 mM) oxidation (Fig. 2B), a sluggish and much smaller anodic peak response was observed at the bare GCE. On the other hand, the graphene/HAP/GCE exhibits a sharp and strong oxidation peak at 0.30 V with greatly increased peak current response (16.2 μA), which is 18-fold higher than that of the bare GCE (0.9 μA). In the case of 0.5 mM NO²⁻ (Fig. 2C), the bare GCE shows a small and broad oxidation peak at 0.9 V; where as, a strong anodic peak appears at a more negative potential of 0.8 V at the graphene/HAP modified GC electrode. Moreover, about 1.2-fold increase in anodic peak current (14.7 μA) is observed in comparison with that at bare GCE (12 μA). These results indicate that, among the three investigated electrodes, the graphene/HAP/GCE possesses the best electrocatalytic properties towards the oxidation of 4-AP, UA and NO²⁻, which demonstrate the synergistic effect of the reduced GO and HAP in the hybrid composite.

Fig. 2 (A) Cyclic voltammograms of (A) 0.1 mM 4-AP (B) 0.5 mM UA and (C) 0.5 mM NO²⁻ in 0.1 M PBS (pH 7.0) at (a) bare, (b) GO, (c) HAP and (d) graphene/HAP composite modified GCE at a scan rate of 50 mV s⁻¹; (D) CVs obtained for the mixture of 25 μM 4-AP, 50 μM UA and 50 μM NO²⁻ in 0.1 M PBS (pH 7.0) at (a) GO (b) HAP and (c) graphene/HAP composite modified GCE at a scan rate of 50 mV s⁻¹.

The effect of scan rate on the electrochemical responses of 4-AP, UA and NO²⁻ at the graphene/HAP modified GCE was studied by CV. As demonstrated in Fig. S2,† the anodic peak currents of the analytes increase linearly with the scan rate (ν) over the range of 50–500 mV s⁻¹. The linear equations for 4-AP, UA and NO²⁻ have been deduced as (i_pa = 0.8221ν^1/2 − 1.088, R² = 0.9935), (i_pa = 0.17696ν^1/2 + 3.8244, R² = 0.9955), (i_pa = 1.2426ν^1/2 + 2.586, R² = 0.9974) respectively. The results indicate that the electrode reactions of all the three investigated analytes at the graphene/HAP/GCE are of diffusion-controlled processes.

3.4 Voltammetric separation of 4-AP, UA and NO²⁻

In order to understand the electrochemical behavior of 4-AP, UA and NO²⁻ in the mixture solution, CVs were recorded at GO/GCE (curve a), HAP/GCE (curve b) and graphene/HAP/GCE (curve c) respectively and the results are shown in Fig. 2D. At the GO/GCE, the oxidation peaks of 4-AP and UA completely overlap with each other leading to a broad oxidation peak at 0.6 V which revealed that it is impossible to deduce any information from this peak. On the other hand, when the graphene/HAP modified GC electrode was used, the overlapped voltammetric peak was resolved into three well-defined CV peaks at 0.01 V, 0.38 V and 0.75 V, corresponding to 4-AP, UA and NO²⁻ respectively. The peak potential separations for 4-AP–UA and UA–NO²⁻ were 0.37 mV and 0.37 mV at the graphene/HAP/GCE, which were large enough for the selective and simultaneous determinations of 4-AP, UA and NO²⁻ in their mixture. These results reaffirm that the graphene/HAP/GCE not only enhances the oxidation of 4-AP, UA and NO²⁻, but also dramatically enlarge the peak separation among 4-AP, UA and NO²⁻. The enlarged separation of the anodic peak potential, coupled with the increased sensitivity, indicates that the simultaneous determination of 4-AP, UA and NO²⁻ in the co-existence system is feasible. Hence we have used of graphene/HAP modified GC electrode for further investigations.

3.5 Selective determinations of 4-AP, UA and NO²⁻ by SWV

SWV technique was employed for the selective determinations of 4-AP, UA and NO²⁻ in their mixture due to its higher sensitivity and better resolution than the CV method. For individual determination of 4-AP, UA and NO²⁻ at the graphene/HAP/GCE, SWV was carried out in the potential range of −0.2 to 1.0 V in PBS (pH 7.0). As shown in Fig. 3, a series of SWV curves were obtained by changing the concentration of one molecule while keeping the concentrations of the other two as constant. The results shown in Fig. 3A–C indicate that the peak currents for the oxidations of 4-AP, UA and NO²⁻ increase proportionally with their concentrations. For 4-AP, the linear relationship between the peak current and its concentration is obtained in the range of 0.1–50 μM and 50–425 μM (Fig. 3D), and the linear regression equation is calibrated as (0.1–50 μM) i_pa = 0.112c_4-AP + 4.27 (R² = 0.997); (50–420 μM) i_pa = 0.067c_4-AP + 6.88 (R² = 0.996). In Fig. 3E, the oxidation peak current of UA increases with the increase of its concentration in the range of 1–50 μM and 50–1000 μM. The corresponding linear function is (1–50 μM) i_pa = 0.0467c_UA + 0.928 (R² = 0.996); (5–0.1 mM) i_pa = 0.0208c_UA + 2.90 (R² = 0.995). Similarly (Fig. 3F), the peak current of NO²⁻ increases linearly with the increase of its concentration with the linear function (3–50 μM) i_pa = 0.0527c_NO²⁻ + 1.43 (R² = 0.997); (50–950 μM) i_pa = 0.0122c_NO²⁻ + 3.72 (R² = 0.995). The lowest detection limits (LODs) are estimated as 0.29 μM, 0.03 μM and 0.025 μM for 4-AP, UA and NO²⁻ respectively based on the lower concentration range. The linear range and detection limit for 4-AP, UA and NO²⁻ at graphene/HAP/GCE were compared with the recently reported values for similar chemically modified electrodes (Table 1). These results demonstrate that individual determination of 4-AP, UA and NO²⁻ can be achieved with high sensitivity and selectivity at the graphene/HAP/GC electrode.

Fig. 3 (A) SWVs obtained for various concentrations 4-AP (0.1 to 425 × 10⁻⁶ M) at graphene/HAP modified GCE in the presence of 200 μM each UA and NO²⁻ in 0.1 M PBS (pH 7.0), (B) SWVs obtained for various concentrations of UA (1 to 1000 × 10⁻⁶ M) at graphene/HAP modified GCE in the presence of 200 μM each 4-AP and NO²⁻ in 0.1 M PBS (pH 7.0), (C) SWVs obtained for various concentrations NO²⁻ (3 to 950 × 10⁻⁶ M) at graphene/HAP modified GCE in the presence of 200 μM each 4-AP and UA in 0.1 M PBS (pH 7.0) and (D–F) represents the corresponding calibration curves.

Table 1 Comparison of different chemically modified electrodes for the determination of 4-AP, UA, NO²⁻ with graphene/HAP/GCE

Electrode	4-AP	UA	NO^2−	4-AP	UA	NO^2−	Ref.
	Linear range (μM)			Detection limit (μM)
a Graphene–polyaniline modified GCE.b Graphene–chitosan modified GCE.c Singled walled carbon nanotube modified GCE.d Hemim based molecular imprinted polymer modified GCE.e La(OH)3 nanorods modified GCE.f Poly(2-mercaptobenzothiazole) modified GCE.g Lanthanum/multiwalled carbon nanotube modified GCE.h Au nanoclusters/3-amino-5-mercapto-1,2,4-triazole modified GCE.i Thiazole-based copolymer modified GCE.j Graphene/hydroxyapatite modified GCE.
GR/polyaniline^a	0.2–100	—	—	0.065	—	—	11
GR chitosan^b	0.2–550	—	—	0.050	—	—	12
SWNT-nafion^c	0.005–2	—	—	0.008	—	—	13
MIP^d	10–90	—	—	3	—	—	14
La(OH)3^e	—	0.05–790	0.55–720		0.001	0.18	15
PMBT^f	—	1–165	60–1000	—	0.10	0.30	16
La/MWCNT^g	—	0.04–810	0.4–710	—	0.015	0.13	17
Au-p-A/GCE^h	—	1.6–110	15.9–277	—	0.08	0.89	18
PATA^i	—	1.9–1200	2–1240	—	0.25	0.5	19
GR/HAP^j	1–140	1–840	1–840	0.90	0.35	0.52	This work

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3.6 Simultaneous determinations of 4-AP, UA and NO²⁻

To investigate the possibility of using the graphene/HAP modified GC electrode for simultaneous determinations of 4-AP, UA and NO²⁻, the SWV peak current responses of these species for various concentrations in the mixture have been measured. It can be seen from Fig. 4A that the peak currents for oxidations of 4-AP, UA and NO²⁻ increase proportionally with their concentrations. The linear ranges for the determinations of 4-AP, UA and NO²⁻ are found to be 1–140 μM, 1–840 μM and 1–840 μM with detection limits of 0.90 μM, 0.35 μM and 0.52 μM respectively. The linear regression equations for 4-AP, UA and NO²⁻ were obtained as i_pa = 0.146c_4-AP + 3.261 (R² = 0.995); i_pa = 0.0319c_UA + 3.17 (R² = 0.995) and i_pa = 0.112c_NO²⁻ + 3.50 (R² = 0.995) respectively. These results demonstrated that the graphene/HAP composite modified GC electrode provide a good electrocatalytic activity towards simultaneous detection of the three important chemical species 4-AP, UA and NO²⁻. Moreover, graphene/HAP composite preparation and electrode modification procedure adopted in the present study are very simple and highly reproducible.

Fig. 4 (A) SWVs obtained for various concentrations of 4-AP (1–140 μM), UA (1–840 μM) and NO²⁻ (1–840 μM) at graphene/HAP/GCE in 0.1 M PBS (pH 7.0) and (B), (C) & (D) are plots of the oxidation peak currents as a function of various concentrations of 4-AP, UA and NO²⁻.

3.7 Interference, reproducibility and stability studies

The anti-interferent ability of the graphene/HAP/GCE was investigated by detecting its response to the mixture of 4-AP, UA and NO²⁻ in the presence of several common co-exiting substances. The results indicate that the presence of 10 times ascorbic acid and 100 times of K⁺, Na⁺, Ca²⁺, Mg²⁺ and glucose have no influence on peak currents of 4-AP, UA and NO²⁻. The reproducibility of the proposed biosensor was determined with four different electrodes. The relative standard deviations (RSD) of the current responses were 1.6%, 2.5% and 1.8% for 4-AP, UA and NO²⁻ respectively. The storage stability of the modified electrode was investigated over a period of 30 days by storing it in 0.1 mol L⁻¹ PBS (pH 7.0) at room temperature. There were no obvious changes in the current response during the first week, and the response decreased to 97.0% of the initial value after 10 days, and it further got reduced to 92.5% after 20 days and this response remained nearly stable thereafter.

3.8 Determination of 4-AP, UA and NO²⁻ in real samples

Human urine and tap water samples were selected for real sample analysis using the standard addition method. The developed sensor was applied to test 4-AP and NO²⁻ in tap water and UA in urine samples respectively. Solutions were prepared by adding 4-AP and NO²⁻ in tap water and analyzes were done without any prior treatment. The results are shown in Table 2. The human urine samples were diluted 50 times with 0.1 M PBS without any other treatment. The dilution process helps to reduce the matrix effect of real samples. SWV response was measured after different concentrations of UA standard solution was added into 10 mL 0.1 M PB solution (pH 7.0) and the results are presented in Table 3. Fig. 5 shows typical SWV responses obtained at graphene/HAP/GCE from tap water and urine samples containing 4-AP, NO²⁻ and UA respectively. The peak current responses were found to regularly increase upon standard addition of 4-AP, NO²⁻ and UA. It can be clearly seen that a good recovery has occurred in all the three investigated samples. The results suggest that the proposed method could be effectively used for the determination of 4-AP, UA and NO²⁻ in real samples.

Table 2 Determination of 4-AP and NO²⁻ in tap water using graphene/HAP/GCE

Sample	Added (μM)	Found (μM)	RSD (%) (n = 3)	Recovery (%)
4-Aminophenol (4-AP)
1	2	2.1	3.61	98.2
2	4	3.8	2.70	97.6
3	10	9.7	0.59	101.2
Nitrite (NO^2−)
1	1	1.8	2.46	102.3
2	5	5.7	2.04	97.5
3	10	9.8	1.57	98.5

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Table 3 Determination of UA in human urine sample using graphene/HAP/GCE

Sample	Added (μM)	Found (μM)	RSD (%) (n = 3)	Recovery (%)
1	5	6.2	1.61	104.5
2	10	12.4	0.96	99.8
3	15	17.5	0.88	102.3

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Fig. 5 SWVs obtained at graphene/HAP/GCE from (A) tap water and (B) human urine samples containing 4-AP, NO²⁻ and UA respectively.

4. Conclusions

An electrochemical sensor for the simultaneous determination of 4-AP, UA and NO²⁻ have been realized for the first time using graphene/HAP hybrid composite modified GCE. The fabricated electrode exhibited high electrocatalytic activities towards the oxidation of 4-AP, UA and NO²⁻ with well separated strong peaks at 0.02, 0.27 and 0.77 V respectively. Compared with the electrodes made of either GO and HAP independently, their hybrid graphene/HAP composite modified GCE facilitate the simultaneous determinations of 4-AP, UA and NO²⁻ with high sensitivity and selectivity. The oxidation peak currents of the three compounds depend linearly on the concentrations over the range of 0.1–420 μM for 4-AP, 1–1000 μM for UA and 3–900 μM for NO²⁻ with the lowest detection limits of 0.29 μM, 0.03 μM and 0.025 μM respectively. With such excellent features as wide linear dynamic range, high sensitivity and selectivity, the developed sensor provides a new strategy for determination of 4-AP, UA and NO²⁻ in biomedical and environmental samples with satisfactory results.

Acknowledgements

Authors NL & CS acknowledge the Council of Scientific and Industrial Research (CSIR no. 03(1203)/12/EMR-II) and University Grants Commission (UGC. F. no. 40-2/2011) for the financial assistance. P.K. acknowledges the University Grants Commission (UGC) for providing the Post Doctoral Fellowship (no. F.15-1/2011-12/PDFWM-2011-12-OB-TAM-2867 (SA-II)). N. Sudhan acknowledges the DST-PURSE for financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra06763d

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