Edge-carboxylated graphene anchoring magnetite-hydroxyapatite nanocomposite for an efficient 4-nitrophenol sensor

Abstract

The surface chemistry and physical properties of edge-carboxylated graphene (ECG) have to date been over looked in terms of understanding the real world practical applications. The accurate identification of each possible oxygenated group on the surface of the basal plane as well as the edges of ECG is necessary to understand the properties for their potential multifunctional applications. Herein, we report the use of a simple high energy ball mill to prepare a large scale production of ECG from natural graphite flakes through interaction with aspartic acid under solid conditions. These 2 dimensional ECG sheets were anchored with magnetite-hydroxyapatite (m-HAp) using a simple hydrothermal process. The prepared materials were systematically investigated by various analytical techniques to realize the structural, morphological, compositional and functional properties. These m-HAp dispersed ECG sheets can be further used to modify the glassy carbon electrode (GCE) for the sensitive and selective detection of 4-nitrophenol (4-NP) by cyclic voltammogram (CV) and differential pulsed voltammetry (DPV). The high specific surface area of 130 m² g⁻¹ for the m-HAp on ECG displays an excellent catalytic activity with reversible redox behavior of 4-NP. The modified electrode possesses a good detection limit and high sensitivity of 0.27 μM and 0.587 μA μM⁻¹ cm⁻², respectively, towards 4-NP, rendering practical industrial applications.

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1. Introduction

Graphene is an increasingly popular unique two dimensional carbon material with outstanding indispensable electronic and mechanical properties such as high charge carrier mobility, optically transparent, high tensile strength, good thermal conductivity and an excellent theoretical surface area. These functional properties permit a wide range of applications such as in medicine, microelectronics, sensors, and batteries.^1,2 All these qualities stimulate researchers to prepare graphene by simple methods for the probable multifunctional applications. However, the large scale production of graphene is still a challenging task for researchers. Thereby, researchers focus on new synthetic strategies to make high quality graphene with large scale production in a cost effective manner. Several important methods have been already designated for the preparation of graphene such as chemical vapor deposition (CVD), epitaxial growth on SiC, scotch tape, mechanochemical cleavage, chemical exfoliation, unzipping of carbon nanotubes and other organic synthetic protocols.^3–8 The commonly affordable chemical vapor deposition (CVD) method can produce high quality graphene with minor defects that can be used for highly demanding electronics and sensing applications. The Hummers and modified Hummers method have been adopted to prepare bulk production of graphene oxide and graphene sheets. However, these methods exploit strong hazardous oxidizing agents and complicated multi-step processes.^9,10 The reduction of graphene oxide requires the reducing agents of hazardous hydrazine hydrate and sodium borohydride (NaBH₄). Moreover, the reduced graphene oxide in this method has structural defects induced at the basal planes and edges of the graphene sheets.

Recently, many researchers have produced graphene with less structural defects by novel and simple methods. The mechanochemical method is one example established for the fabrication of multi and few layers of graphene sheets with simple preparation, bulk production and cost effectiveness.¹¹ This mechanochemical process introduces various functional groups at the edges of the graphite. The high speed of the planetary rotation and the collision of stainless steel vials and balls generate sufficient kinetic energy for bond cleavages for the C–C aromatic graphite structure. The reactive carbon species, such as oxalic acid, dry ice, melamine, triazine, potassium hydroxide, carbon dioxide–sulfur trioxide mixture, N,N dimethylformamide (DMF) and sodium dodecyl sulphate (SDS) are the selective functionalized edges of cracked graphite.^11–16 The oxygen containing groups are possibly involved in the formation of nanoparticles anchored on the surface of negatively charged functionalized graphene. Nowadays, graphene and their nanocomposites show excellent electrochemical behavior. Graphene/metal oxides, graphene/conducting polymers and graphene/hydroxyapatite/Nafion nanocomposites are systematically explored for electrochemical biosensors and are used to determine H₂O₂, glucose, dopamine, ascorbic acid and uric acid.^15–21 Moreover, graphene based nanocomposite materials are widely used for the determination of toxic organic and inorganic compounds in contaminated water by various analytical techniques.^22–24

Graphene/graphene based nanocomposites and hydroxyapatites provide an enormous impact on the detection of toxic/polluted compounds for environmental remediation. In particular, graphene oxide, β-cyclodextrin functionalized graphene, N-doped graphene and molecularly imprinted polymer based graphene nanocomposite, hydroxyapatite, Fe₃O₄ incorporated HAp or HAp/Fe₃O₄ nanocomposites are important functional nanomaterials for the excellent detection of phenols-based aromatic nitro compounds in various contaminated environmental water samples by electrochemical detections.^22–29 It is important to detect the aromatic nitro compounds which are commonly produced from pharmaceuticals, pesticides, dyes and chemical industries, which are toxic to the human body, animals, plants and aquatic life. In particular, 4-nitrophenol (4-NP) is a toxic derivative cited in the list of priority pollutants by the U.S.A Environmental Production Agency (EPA). 4-NP has been found not only in industrial waste water but also in lake and sea water. The United States EPA will permit the allowed limit of 0.43 μM of 4-NP in drinking water. Therefore, the determination of highly toxic 4-NP is important and essential. A simple and reliable method is required for the determination of 4-NP in environmental water samples. Several electrochemical and spectroscopic techniques, such as cyclic voltammetry (CV), differential pulsed voltammetry (DPV), fluorescence, high-performance liquid chromatography and spectrophotometry, have been implemented for the determination of 4-NP in various environmental samples.^22–26 Among the various detection methods, the electrochemical analytical method shows excellent reliability, sensitivity, selectivity, simple operation, quick response and is cost effective. Moreover, most of the research focuses on developing higher electrocatalytic activity of electrode materials for the efficient detection of 4-NP by electrochemical analytical methods.

However, there are two major challenges obstructing the wide scale application of environmental remediation and targeted drug delivery. For example, graphene and hydroxyapatite have excellent adsorption capability, but it is not easy to separate and recycle them after treatment. Magnetite nanoparticles can guide in targeted drug delivery and they have a high recovery ratio, stable performance and are easy to recycle. Therefore, we designed the magnetic electro catalytic systems to be an effective way to resolve the aforementioned problems. The magnetic separation technique possesses the advantages of high efficiency, rapidity, and cost-effectiveness. In addition, it effectively eliminates the requirement for either solvent swelling before or catalyst filtration after the reaction.³⁰

In the present work, a high energy ball mill can exfoliate the edge-carboxylated graphene (ECG) sheets from natural graphite flakes. Furthermore, the mHAp nanocomposites were dispersed on the prepared two dimensional ECG sheets using a one pot facile hydrothermal process. The possible synthesis strategies of edge-carboxylated graphene through a high energy ball mill and the formation mechanism of m-HAp nanocomposites on the two dimensional ECG sheets are proposed in detail. The glassy carbon electrode modified with this novel m-HAp/ECG nanocomposite exhibits admirable electrocatalytic activity for developing sensitive sensors for the electrochemical detection of 4-NP. The fabricated electrochemical sensor exhibits high sensitivity, good selectivity and desired low detection limit.

2. Experimental section

2.1. Materials

Graphite flakes (∼105 μm), aspartic acid, di-ammonium hydrogen phosphate ((NH₄)₂(HPO₄)), ferrous chloride tetra hydrate (FeCl₂·4H₂O), ferric chloride hexahydrate (FeCl₃·6H₂O), 4-nitrophenol and calcium chloride dihydrate (CaCl₂·2H₂O) were supplied by Sigma Aldrich. Acetone, hydrochloric acid 5% (HCl) and ethanol were purchased from Himedia Laboratory Pvt. Ltd, India. All these chemicals were of analytical grade and used without further purification.

2.2. Synthesis of edge-carboxylated graphene sheets by high energy ball mill

In a typical process, natural graphite was milled in a planetary ball mill (Fritsch Pulverisette-P7) in the presence of aspartic acid at 300 rpm for 40 h in a stainless steel vial and ball with the ball to powder weight ratio of 10 : 1 in air. The obtained product was washed with double distilled water and 5% of HCl solution to remove metal impurities. Furthermore, it was washed with deionized water several times until the pH became neutral and was dried in vacuum oven at 7 °C for 24 h.

2.3. Synthesis of m-HAp on ECG sheets by hydrothermal process

In this process, 50 mg of ECG sheets were suspended in 50 ml of deionized water by ultrasonic dispersion for 30 min to form a stable black colour graphene solution, and the supernatant was collected for further process. Furthermore, 30 mM of calcium chloride was dissolved in 30 ml of supernatant graphene solution. Subsequently, 18 mM of di-ammonium hydrogen phosphate was dissolved in 20 ml of deionized water and gradually added to the abovementioned mixture. The pH was adjusted to 10–10.5 by adding ammonium hydroxide solution (30%) and it is represented as solution A. Moreover, 5 mM of ferrous chloride and 10 mM of ferric chloride were dissolved in 20 ml of aqueous solution to prepare the 1 : 1.5 molar ratio of Fe²⁺ and Fe³⁺. In addition, the pH was adjusted to 10–11 by adding ammonium hydroxide solution (30%) to start the nucleation growth of Fe₃O₄ and it is represented as solution B. Subsequently, the solution B was added to solution A with constant stirring for 30 min. Finally, the mixed solution was transferred to a Teflon-lined stainless steel autoclave for hydrothermal treatment. The autoclave was sealed and placed in an oven at 18 °C for 12 h and then cooled to room temperature naturally. The obtained precipitate was washed with deionized water and ethanol several times and was dried in vacuum at 7 °C overnight.

2.4. Characterization

X-ray diffraction (XRD) was performed at room temperature using a PANalytical (X'Pert-Pro) diffractometer with Cu Kα₁ radiation (λ = 1.5406 Å). The average crystallite sizes were estimated using the Scherrer formula. The morphology and elemental analysis of the composite were determined by high resolution transmission electron microscopy (HRTEM) and field emission scanning electron microscopy (FEI Quanta – 250) with EDX and elemental mapping. The infrared spectrum of the samples was obtained using a Fourier transform infrared (FTIR) spectrometer (Bruker Tensor 27, Germany). Raman scattering was performed with a JY-1058 Raman spectrometer using a 520 nm laser source. UV-visible spectral analysis was carried out using a JoscoV-650 spectrophotometer. The surface area and pore size distribution of a nanocomposite was determined using a micromeritics ASAP 2020 surface area analyzer. The X-ray photoelectron spectroscopy (XPS) was carried out using a Kratos Axis Ultra-DLD X-ray photoelectron spectroscope (Manchester, U.K).

2.5. Fabrication of modified glassy carbon electrode using m-HAp/ECG nanocomposite

The m-HAp nanocomposite on ECG was dispersed in ethanol under sonication for 3 h and the resultant solution is hereafter represented as the m-HAp/ECG nanocomposite. Moreover, the surface of the GCE was carefully mirror polished with alumina slurry followed by washing with double distilled water and ultrasonicated in ethanol containing water for 20 min. Afterwards, ca. 8 μL of dispersed m-HAp/ECG (with optimal concentration) was drop cast on the GCE and dried in vacuum at 30 °C. The modified electrode is represented as m-HAp/ECG modified GCE and it can be exploited for electrochemical detection at room temperature under an inert atmosphere.

3. Results and discussion

3.1. Structural and morphological characterization of m-HAp/ECG

The schematic illustration for the formation of ECG sheets by high energy ball milling of natural graphite flakes in the presence of aspartic acid is shown in Fig. 1(a). Herein, the planetary ball mill with the speed of 300 rpm can be used to exfoliate the ECG sheets through interaction with aspartic acid under solid condition. The reactive carbon species (carboxylic ions in aspartic acid) and high speed rotation of the ball mill produces the cleavage of graphitic C–C bonds at the broken edges. Sufficient kinetic energy is generated during the high energy milling and it compensates the weak forces of van der Waals attraction between two adjacent π–π stacked graphite layers for bond cleavages in the C–C aromatic graphite structure.

Fig. 1 (a) Schematic illustration for the synthesis of ECG sheets by high energy ball milling in the presence of aspartic acid. FESEM images of (b) as-received natural graphite flakes, (c) ECG sheets, (d) exfoliated ECG sheets and (e) TEM images of ECG sheets.

The carbon species in aspartic acid react with the selective edges of graphite forming chemically functionalized graphite sheets during the milling process. The aspartic acid could effectively exfoliate graphite with edge-carboxylated species (–COO⁻) and it prevents the re-stacking of graphene layers. The obtained product was suspended in different solvents, such as water, ethanol, N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP), under ultra-sonication to remove any residual unreacted graphite. These ECG sheet dispersed solutions were kept for one week for the thorough settling of the unreacted graphite. The precipitate was removed and the resulting supernatant solutions were kept for 10 days at room temperature to confirm the dispersibility of ECG sheets. The ESI Fig. 1a† shows the dispersion of ECG with different solvents of water, ethanol, DMF and NMP for the exfoliating agent of aspartic acid. The as obtained ECG sheets indicate a good stability in DMF and NMP with the concentration of 1 mg ml⁻¹.

The possible stable highly concentrated dispersion of ECG sheets is due to the electrostatic stabilization and it is further confirmed by zeta potential analysis. The ESI Fig. 1(b)† shows the zeta potential of −40.7 mV corresponds to the ECG sheets in water. This reveals that the strong repulsive force arises due to the negatively charged carboxylic groups (–COO⁻) and it was present in the edges of graphene sheets. The morphology of as-received natural graphite flakes shows the larger grain sizes in the range from 80 to 100 μm with flake-like morphology, as shown in Fig. 1(b). After 40 h of ball milling, in the presence of aspartic acid, the reduction in the size range of 2–5 μm, as seen in Fig. 1(c), is due to mechanochemical cleavage of graphite. These ECG sheets were suspended in 10 ml of deionized water under ultrasonic dispersion for 30 min for FESEM and TEM analysis. Fig. 1(d and e) shows the FESEM and TEM images of exfoliated ECG sheets and it reveals that the graphene sheets are homogeneously dispersed (2–5 μm) and highly transparent under the electron beam. Fig. 3(a–d) show the surface morphology and microstructure of the m-HAp/ECG nanocomposite. Fig. 2(a and b) show the agglomeration free individual graphene sheets with uniformly distributed m-HAp nanoparticles on the surface as well as the edges of the ECG sheets. The high magnification FESEM images in Fig. 2(c and d) show mHAp nanoparticles uniformly dispersed on ECG sheets with an average diameter of 50–100 nm.

Fig. 2 (a–d) FESEM images of m-HAp/ECG nanocomposite with different magnifications.

A schematic representation for the synthesis and possible nucleation growth of the m-HAp/ECG nanocomposites is shown in Fig. 3. The edge-carboxylated grapheme sheets are prepared by high energy ball milling in the presence of aspartic acid. The aspartic acid reacts with the selective edges of graphite during the milling and forms carboxylic (–COO⁻) functionalized graphite sheets.

Fig. 3 Schematic representation for the new strategy for the formation mechanism of m-HAp nanoparticles grown on two dimensional ECG sheets.

A number of oxygen containing carbonyl and carboxyl groups was present at the edges, and epoxy and hydroxyl groups were located at the basal plane of the graphene sheets. These oxygen containing groups were possibly involved in the anchoring of m-HAp nanoparticles on the negatively charged ECG sheets. This edge-carboxylated graphite was further exfoliated under ultrasonication for 3 h to make highly stable, block colour suspension in water and the supernatant was collected for further experimental process.

In brief, a 30 mM concentration of CaCl₂·2H₂O was added to the supernatant of the ECG solution. The calcium ion (Ca²⁺) in the solution selectively bonded with epoxy, hydroxyl and carboxyl group through electrostatic interactions. This Ca²⁺ ion easily diffuses and is preferred for the crystallization and growth of HAp nuclei on the edges and basal planes of the ECG sheets. Furthermore, these Ca²⁺ ions react with phosphate (PO₄³⁻) ions via electrovalent bonds by adjusting the pH. Finally, the HAp nuclei were formed on the basal plans and edges of the ECG sheets. Moreover, during the nucleation and growth of HAp, a certain molar concentration of Fe²⁺and Fe³⁺ (1 : 2) was added for the preparation of m-HAp nanoparticles on the basal planes and the edges of the ECG sheets, which were confirmed through FESEM images. Fig. 4(a and b) shows the morphology of the m-HAp on ECG sheets, and it was grown on the edges of graphene sheets due to the presence of –COO⁻ ions in the graphene sheets. Fig. 4(c and d) shows the uniform dispersion of m-HAp nanocomposite on the edges of the graphene sheets with an average diameter of 80–100 nm. The energy dispersive X-ray (EDX) analysis and elemental mapping, as shown in ESI Fig. 2,† confirms the uniform distribution of carbon (C), oxygen (O), calcium (Ca), phosphorus (P) and iron (Fe) in the m-HAp/ECG sheets. It confirms that the P, Ca, Fe and O are well dispersed in the m-HAp nanorods and that the carbon was well dispersed in the ECG sheets. X-ray powder diffraction (XRD) was used to analyze the crystallinity and phases of the prepared m-HAp/ECG sheets.

Fig. 4 (a) Schematic illustration for the m-HAp nanoparticles grown on edges of two dimensional ECG sheets and (b–d) FESEM images of m-HAp on ECG sheets with different magnifications.

Fig. 5(a–c) shows the XRD patterns for the pure graphite, ball milled edge-carboxylated graphene and mHAp/ECG sheets. The XRD pattern for the m-HAp/ECG nanocomposite in Fig. 5(c) shows the dual phases of pure HAp (JCPDS #. 09-0432) and Fe₃O₄ (JCPDS #. 89-0688). Fig. 5(a) shows a strong diffraction peak at 26.45° for the (002) plane of natural graphite flakes with the d-spacing of 0.34 nm. In contrast, the ball milled graphite in the presence of aspartic acid shows the weak intense peak at 25.35° for the (002) plane in Fig. 5(b) confirming that the high degree of carboxyl groups are functionalized in the edges of graphite. It also confirms the mechanochemical cleavage of graphitic C–C bonds and also edge-selectively functionalized graphite.

Fig. 5 XRD patterns for (a) as-received natural graphite flakes, (b) ball milled edge-carboxylated graphite and (c) m-HAp/ECG nanocomposite.

The FTIR spectra for pure graphite and ball milled ECG samples are shown in ESI Fig. 3(a–c).† The absorption peak at 3443 cm⁻¹ is due to the stretching vibration of O–H and the peak at 1637 cm⁻¹ is attributed to C=C stretching vibration. The FTIR spectrum for the ball milled sample in ESI Fig. 3(b)† shows a peak at 1739 cm⁻¹, attributed to the carboxylic group (C=O). The characteristic absorption peaks of methylene groups (CH₂) are observed for the ECG sheets and the m-HAp/ECG nanocomposite at 2853 and 2926 cm⁻¹, as shown in ESI Fig. 3(b and c).† The intense peaks at 565 and 1039 cm⁻¹ are due to bending vibrations and asymmetric stretching vibrations of the O–P–O and P–O in the PO₄³⁻ groups. The additional peak at 600 cm⁻¹ is attributed to the lattice absorption of iron oxide (Fe–O). Therefore, FTIR results confirm that the carboxylic group could be introduced at the broken edges of graphite during the ball milling and the m-HAp nanoparticles are successfully grown on edges of ECG sheets. The Raman spectra in Fig. 6 completes the structural analysis of pure graphite, ball milled edge-carboxylated graphene and the m-HAp/ECG nanocomposite. Fig. 6(a) shows the Raman spectrum for the pure graphite with two prominent peaks at 1580 and 2970 cm⁻¹ corresponding to the well documented G and 2D bands. The weak D band at 1346 cm⁻¹ was associated with the vibration of A_1g symmetry of sp³ carbon atoms and correlated to a defect ordered structural peak. It is well known that the G and 2D band corresponds to the first-order scattering of the E_2g phonon of carbon sp² atoms and second-order double resonant process with opposite momentum in the highest optical branch near the K points in the Brillouin zone (Bz) of graphene. The calculated intensity ratio of D to G (I_D/I_G) is approximately 0.02 for the pure natural graphite flakes. In contrast, the Raman spectrum for the ball milled edge-carboxylated graphene in Fig. 6(b) shows a strong D band at 1331 cm⁻¹. The intensity ratio of the D and G bands (I_D/I_G) is 0.5, suggesting significant edge functionalization in the presence of aspartic acid. Fig. 6(c) shows the Raman spectrum for the m-HAp/ECG nanocomposite.

Fig. 6 Raman spectra for (a) pure natural graphite flakes, (b) ball milled edge-carboxylated graphene sheets and (c) m-HAp/ECG nanocomposites.

The right inert in Fig. 6(c) shows the magnetite phase of the composite and the peak at 669 cm⁻¹ can be ascribed to the magnetite A_1g active mode and less intense peaks at 540 and 309 cm⁻¹ correspond to the Raman active modes of T_2g and E_g for magnetite. In addition, the characteristic band at 960 cm⁻¹ is assigned to the symmetric stretching (ν₁) of the phosphate group (PO₄³⁻). The optical absorption of the ECG sheets and m-HAp/ECG nanocomposites are studied by UV-vis spectroscopy with the initial concentration of 0.1 mg ml⁻¹ in water. Two prominent absorption peaks were observed at 230 and 300 nm for the ECG sheets, as shown in ESI Fig. 4(a).† The peak at 230 nm is due to the π–π* plasmon peak for the nanoscale sp² C=C aromatic transition bonds. Furthermore, the broader peak at 300 nm was attributed to the n–π* transition peak of sp³ C=O bonds. It suggests the edges of the graphite were highly carboxylated during the planetary milling with the presence of aspartic acid. The characteristic peak of ECG is red shifted from 230 to 255 nm may be due to the hydrothermal reduction reaction between ECG sheets and mHAp, as shown in ESI Fig. 4(b).† The phase and elemental composition of the as-prepared m-HAp/ECG nanocomposite was further analysed using XPS. Fig. 7 shows the wide scan XPS spectrum for the m-HAp/ECG nanocomposite, which exhibits photoelectron lines at binding energies of 347.07, 133.13, 284, 530 and 711 eV corresponding to Ca 2p, P 2p, C 1s, O 1s and Fe 2p. The right inset high-resolution C 1s XPS spectrum of the mHAp/ECG nanocomposite shows a sharp intense peak at 284.35 eV corresponding to the sp² aromatic structure of C–C bonds in a conjugated honeycomb lattice. The intense peak at 286.29 eV is attributed to the C–O bonding due to the harsh oxidation of graphene sheets by the mechanochemical synthesis in the presence of aspartic acid. The high-resolution Fe 2p XPS spectrum is placed in left corner of Fig. 7 and the binding energies at 711.2 and 724.8 eV are associated to Fe 2p_3/2 and Fe 2p_1/2, respectively. This result clearly confirms that the Fe₃O₄ and HAp phases are well dispersed on the mechanochemically synthesized ECG sheets through the hydrothermal process.

Fig. 7 XPS wide scan spectrum of mHAp/ECG nanocomposite where the left and right inset figures are high resolution spectra of Fe 2p and C 1s, respectively.

The surface area and pore size distribution of the m-HAp/ECG nanocomposite was further determined from nitrogen physisorption (adsorption–desorption) measurements. The ESI Fig. 5† shows the adsorption–desorption isotherm and corresponding pore size distribution. The isotherm shows a type IV loop at a relative pressure between 0 and 1. The specific surface area and pore size distribution for the m-HAP/ECG nanocomposite using Barrett–Joyner–Halenda (BJH) calculations are 130 m² g⁻¹ and 5–20 nm, respectively. The magnetic properties of the nanocomposites have been investigated at room temperature using a vibrating sample magnetometer (VSM) with an applied magnetic field of ±2 KOe. The ESI Fig. 6† shows the magnetic hysteresis loop for the mHAp/ECG nanocomposite. The obtained saturation magnetization (M_s) and coercivity (H_c) are 11.12 emu g⁻¹ and 0.12 Oe, respectively.

To analyze the electrochemical sensing mechanism, resistance, diffusion coefficient, and charge transfer resistance at the electrode/electrolyte interfaces, the electrochemical impedance spectroscopy (EIS) study is performed. As we know, the diameter of the semicircle is a direct representation of the charge transfer resistance (R_ct). Fig. 8 depicts the Nyquist plots of pure graphene (a), pure HAp (b), m-HAp/ECG (c), m-HAp (d) and bare (e) modified GCE, studied in 5 mM Fe(CN)₆^3−/4− and 0.1 M KCl was used as the supporting electrolyte. Fig. 8 shows that the pure graphene and bare GCE has higher R_ct when compared with the other three modified electrodes, and the R_ct values of other modified GECs have effectively decreased. Hence, the m-HAp/ECG modified GCE exhibits the lowest R_ct value compared to the other modified electrodes, which suggests the m-HAp/ECG could be used as an optimized electrode for electrochemical analysis based on the obtained lower R_ct value.

Fig. 8 EIS plots of 5.0 mM [Fe(CN)6]^3−/4− in 0.1 M KCl recorded at different electrodes of pure graphene (a), pure Hap (b), m-HAp/ECG (c), m-HAp (d) and bare (e) modified GCE.

3.2. Electrochemical detection of 4-nitrophenol by modified GCE with m-HAp/ECG nanocomposite

The electrocatalytic performance of the m-HAp/ECG nanocomposite was studied by modifying the GCE with this nanocomposite using cyclic voltammograms (CV). Fig. 9 shows the cyclic voltammogram curves of 200 μM 4-NP in 0.05 M acetate buffer solution with a pH of 5.0 at various electrodes such as the bare GCE, m-HAp and m-HAp/ECG nanocomposite. There was no signal observed at the modified GCE without 4-NP. The addition of 4-NP in bare GCE shows not much difference in the CV curve with a weak peak current at E_pc (cathodic peak potential). Furthermore, a noticeable signal was observed at 0.77 V for the pure HAp, pure graphene, and m-HAp nanocomposite modified GCE. The well-defined redox peak and significant cathodic peak potential (E_pc) and anodic peak potential (E_pa) were observed at −0.77 and 0.21 V for the mHAp/ECG nanocomposite modified GCE. Fascinatingly, m-HAp/ECG-modified GCE exhibits a remarkably sharp peak at 0.76 V with enhanced peak current, as shown in ESI Fig. 7.† This concludes that the electrochemical detection of 4-NP is a reversible two electron transfer reduction–reduction process.^31–33 Subsequently, the molecular structure and the electron transfer mechanism can be explained in Scheme 1.

Fig. 9 (a) CV curve in the absence of 4-NP in the electrolyte. CV curves of (b) bare GCE, (c) pure HAp, (d) pure graphene (e) m-HAp and (f) m-HAp/ECG nanocomposite modified GCE in the presence of 200 μM 4-NP in 0.05 M acetate buffer solution with the pH of 5.0 at 50 mV s⁻¹.

Scheme 1 Molecular structures and electrochemical detection mechanism of 4-nitrophenol.

Accordingly, redox peaks occur at peak potentials of −0.77 and 0.25 V due to 4e⁻/4H⁺ reduction of the nitro group of the hydroxylamine species. This suggests the 4-NP molecules could be adsorbed by the various modified electrodes and are reduced at −0.77 V. The CV curves clearly confirms that the m-HAp/ECG nanocomposite modified GCE exhibits the best electrochemical detection of 4-NP. The obtained cathode peak current is also higher than the already reported values for the sensing of 4-NP with modified electrodes.^24–32 This could be due to increase in the specific surface area and adsorption sites that increases the current response for the sensing of 4-NP in m-HAp/ECG nanocomposites. The influence of scan rate on the electrochemical activity of the m-HAp/ECG nanocomposite modified GCE was also studied by measuring the CV with different scan rates of 50 to 500 mV s⁻¹. Fig. 10 shows the CV curves with different scan rates of the m-HAp/ECG modified GCE for the detection of 4-NP. It shows that the reduction peak current increases with the increase of scan rate in the range from 50 to 500 mV s⁻¹. Moreover, a linear increase occurs between the redox peak current and the square root of the scan rate as expected for a typical diffusion-controlled process. For better understanding, we provide a correlation plot for E₀ vs. peak currents in ESI Fig. 7.† These results confirm that the observed CV responses are due to the detection of 4-NP molecules diffused on the surface of the m-HAp/ECG nanocomposite modified GCE.

Fig. 10 CV curves of m-HAp/ECG-modified GCE in the presence of 200 μM 4-NP in 0.05 M acetate buffer solution with pH 5.0 recorded at different scan rates from 50 to 500 mV s⁻¹. Inset: correlations between peak currents and square root of scan rate.

The inset in Fig. 10 shows the plot between reduction peak current and the square root of scan rate, which may be expressed by a linear regression equation as E_pa (V) = 1.3089x + 3.293, R² = 0.9968. All these results clearly confirm that the process is a diffusion controlled reversible process.^31–35 Fig. 11(a and b) shows the influence of accumulation potential and time on the electrochemical activity of the m-HAp/ECG nanocomposite modified GCE explored by DPV measurements. The peak current increases with the accumulation potential in the range from 0 to −0.4 V. This indicates the amount of adoptive accumulation of 4-NP on the surface of the m-HAp/ECG nanocomposites modified GCE. The adoptive accumulation of 4-NP reaches surface saturation at −0.6 V by further increasing the accumulation potential, as shown in Fig. 11(a). Fig. 11(b) shows the effect of accumulation time on the detection of 4-NP and a maximum peak current was observed at the accumulated time of 4 min. Therefore, the accumulation potential of −0.4 V and time of 4 min are chosen to achieve the optimal conditions for the detection of 4-NP.

Fig. 11 Variations of peak current with (a) accumulation potential and (b) time during the detection of 200 μM of 4-NP.

The analytical performance of the 4-NP sensor was compared by studying the electrocatalytic performance by using both CV and DPV. Fig. 12 shows the cyclic voltammogram curves for the detection of 4-NP with different concentrations of 4-NP in the range from 30–1455 μM in acetate buffer solution with pH 5. The peak current is increasing with the increasing concentration of 4-NP. The inset in Fig. 12 corresponds to the electrochemical analytical curve, which shows the linear dependence of peak current (I_pc) with the various 4-NP concentrations. Furthermore, the potential shift of the peak currents is not affected much by increasing 4-NP concentration, as shown in ESI Fig. 9.† The detection limit (LOD) can be calculated using the following equation.

Fig. 12 CV curves of m-HAp/ECG modified GCE under various 4-NP concentrations of 30–1455 μM at 50 mV s⁻¹. Inset; cathodic reduction peak current (I_pc) vs. 4-NP concentration.

The LOD can be calculated from the standard deviation of the mean value for ten voltammograms of the blank (S_b) and the slope of the straight line of the electrochemical analytical curve. The attributed analytical parameters of 4-NP are more comparable with the various modified electrodes summarized in Table 1. Differential pulse voltammetry (DPV) studies can be employed to obtain the sensitivity of the fabricated 4-NP sensor. Fig. 13 shows the DPV responses of the 4-NP reduction at different concentrations with the optimal experimental conditions. Fig. 13 shows the DPV response of the m-HAp/ECG modified GCE for the detection of 4-NP with different concentrations of 0.2–994 μM in the presence of 0.05 M acetate buffer solution with pH 5.

Table 1 Comparison of analytical parameters for the detection of 4-NP with various modified electrodes

Electrode substrate	Detection limit (μM)	Concentration range (μM)	Sensitivity (μA μM^−1 cm^−2)	Reference
a ECG – edge-carboxylated graphene.b GCE – glassy carbon electrode.c AC – activated carbon.d Nf – Nafion.e SPCE – screen printed carbon electrode.f Au-gold nanoparticles.g HA-NP – hydroxyapatite nano powder.
mHAp/ECG^a/GCE^b	0.27	0.2–994	0.587 ± 0.002	Present work
AC^c	0.16	1–500	5.810 ± 0.24	26
Graphene/Nf^d/SPCE^e	0.6	10–620	—	36
Au NPs^f/GCE	8.0	10–1000	—	37
Ag particles/GCE	0.5	1.5–140	0.043	38
Nano-Cu2O/GCE	0.5	1–400	—	39
HA-NP^g/GCE	0.6	1–300	—	40

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Fig. 13 DPV curves of m-HAp/ECG modified GCE for various 4-NP concentrations of 0.2–994 μM. Inset-cathodic reduction peak current (I_pc) vs. 4-NP concentration.

Moreover, there is no saturation response observed for the concentration of 994 μM and the sensitivity calculated from the slope of the calibration plot is shown in the inset of Fig. 13. The calculated sensitivity is 0.587342 (±0.002) μA μM⁻¹ cm². The calculated lower detection limit (LOD) is 0.27 μM acc. to the formula of LOD = 3s_b/S (where s_b is the standard deviation of the blank signal, and S is the sensitivity). The calculated detection limit of 0.27 μM is lower than those required for environmental controls by the United States (US) EPA for the example of drinking water (0.43 μM). In addition, corresponding DPV curves at anodic peak current (I_pa) were also observed with the various concentrations between 23 and 472 μM. Fig. 13 shows, the catalytic activity of the 4-NP increases with increasing concentration of 4-NP and a linear regression values and correlation coefficient as I_pa (V) = 0.0068x + 0.2278 and R² = 0.9791 respectively. The detection limit was 2.5 μM observed from the slope of the calibration plot, as shown in the inset in Fig. 13. These results indicate that the catalytic performance of cathodic reduction is higher than the anodic oxidation. Moreover, the cathodic reduction is more favorable for the detection of 4-NP to avoid the interference arising from foreign species (particularly ascorbic acid) in real time applications.³² Evidently, the m-HAp/ECG modified GCE exhibits considerably higher sensitivity and a lower detection limit of 4-NP with higher concentrations compared to other reports (Table 1).

3.3. Stability, reliability and real sample analysis

The stability of the reported 4-NP sensor was examined using DPV measurements with the m-HAp/ECG modified GCE by measuring the cathodic peak current periodically. The sensor retains 91.36% of its initial reduction peak current response after 20 days at room temperature, which reveals its good storage stability. In addition, a separate DPV measurement also carried out for the 3 different m-HAp/ECG modified GCEs prepared under the same conditions, which shows an acceptable reproducibility with relative standard deviation (RSD) of 3.2%. Moreover, the repeatability for 5 successive measurements with the RSD of 2.16% for the determination of 200 μM 4-NP suggests a good repeatability of the sensor.

3.4. Real sample analysis

The m-HAp/ECG modified GCE was further explored to detect the 4-NP in tap and rain water for the real sample analysis. DPV was adopted to estimate the accuracy of the detection of 4-NP from these real samples. The obtained results are given in Table S1† and it confirms the good electrochemical responses of both tap and rain water. The obtained recovery values ranges from 97.4% and 105.9%, which reveals the appreciable practicality of the 4-NP sensor.

4. Conclusion

In summary, we have established a simple possible approach for the mass production of ECG sheets with the assistance of aspartic acid by an eco-friendly ball milling process under solid conditions. The obtained ECG sheets were highly dispersible in various solvents such as water, ethanol, DMF and NMP. The ECG sheets show a good stability of dispersion in water with a zeta potential of −40 mV. Furthermore, the m-HAp nanoparticles were uniformly grown on the edges and basal planes of ECG sheets using a hydrothermal process with an average diameter of 80–100 nm. A novel 4-nitrophenol detection with superior electrocatalytic performance was obtained by modifying the GCE with the prepared m-HAp/ECG nanocomposite. The electrocatalytic performance of the 4-NP sensor was evaluated by cyclic voltammogram (CV) and differential pulsed voltammetry (DPV). The fabricated sensor exhibits an excellent electrocatalytic performance with a low detection limit and high sensitivity of 0.27 μM and 0.587 μA μM⁻¹ cm⁻², respectively.

Acknowledgements

The authors would like to acknowledge the University Grant Commission (UGC), Government of India, and New Delhi for the financial support and DST – PURSE, Government of India, for FESEM facility.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16035e

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