Petal-like graphene–Ag composites with highly exposed active edge sites were designed and constructed for electrochemical determination of metronidazole

Abstract

Petal-like graphene–Ag (p-GR–Ag) composites with highly exposed active edge sites were designed and constructed in this work. Petal-like graphene (p-GR) was prepared using a HCl assisted hydrothermal method, which was made of basal planes and highly reactive edge planes to provide more active sites. Then the p-GR can be intentionally utilized as nucleation sites for subsequent Ag nanoparticles (NPs) deposition via modified silver mirror reaction. The composites were characterized by X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and electrochemical methods. The combination of zero-dimensional (0D) Ag NPs on a two-dimensional (2D) graphene (GR) support that came into being three-dimensional (3D) structure created a sensor for electrochemical detection of metronidazole. The designed sensor exhibited well bimodal linear behaviour in the metronidazole concentration range between 0.05 to 10 μM and 10 to 4500 μM with a detection limit of 28 nM (S/N = 3). The mechanism and the heterogeneous electron transfer kinetics constant of the metronidazole reduction were discussed in the light of the rotating disk electrode (RDE) experiments. Moreover, validation of the applicability of the prepared sensor was carried out by detecting metronidazole in human urine and local lake water samples.

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1. Introduction

The rational design and construction of materials with special structures at the nanoscale is paramount in developing advanced nanomaterials.^1,2 Novel properties may emerge when the structure is changed.³ Graphene (GR), a two-dimensional (2D) sheet of carbon arranged in a hexagonal lattice, has fascinated the scientific community in recent years due to its remarkable electronic conductivity, superior mechanical properties, and large surface area.^4,5 The unique structure and outstanding properties render GR highly promising for a diverse range of applications especially electrochemical sensors.^6–8 However, 2D GR is easy aggregation during synthesis process, resulting in poor electrical transport and low surface area.⁹ Furthermore, active sites for electrochemical catalysis are easily sandwiched between GR sheets or deeply hidden inside the aggregated GR.¹⁰ Thus, its electrocatalytic activity is limited. Previous studies based on GR electrochemical sensors have focused on enlarging the surface area of GR, improving the dispersion of GR, and combining GR with metallic NPs to enhance the catalytic activity. For example, Kung's group fabricated GR foam supported PtRu bimetallic nanocatalysts, which exhibited a remarkable performance to electrochemical oxidation of H₂O₂.¹¹ Li and co-workers utilized self-assembly techniques for GR immobilization, and the electrochemical sensor was prepared for detection of sildenafil.¹² Salamon et al. synthesized magnetite nanorods anchored over GR sheets for electrochemical determination of dopamine.¹³ But the planarity of conventional GR-based electrochemical sensors limits the active sites for electrochemical reactions. In view of this, we aimed to construct GR with highly exposed active sites structure so as to improve the electrochemical performance.

Learning from nature is generally considered a short cut to realize structural construction and functional integration.¹⁴ In nature, some plants' petals can suspend a water droplet and keep it in a spherical shape, which is called “rose petal effect”.¹⁵ This phenomenon has aroused the researchers' interest in fabricating the petal-like materials.^16–18 Previous studies have reported that the petal-like materials contained a large amount of open edge planes with high surface activity.¹⁹ Therefore, we designed and constructed petal-like GR (p-GR) with highly exposed active sites. To date, p-GR used as electrochemical sensor has rarely been reported.^20,21

On the other hand, coupling p-GR with metallic NPs is an effective strategy to improve the properties of p-GR and further broaden the application p-GR in the design of sensors. We utilized p-GR with highly exposed active sites to anchor metallic NPs, which can effectively increase the catalytic activity. Ag NPs is an important component of numerous catalysts,²² which is widely applied in electrical and chemical industries due to its highest electrical and thermal conductivities among all the metals, favorable microenvironment, and good biocompatibility.²³ Indeed, electrochemical sensors based on Ag NPs are conducive to the cathodic reactions of compounds.^24,25 The catalytic activity of metallic NPs is related with their size, distribution, and the support materials.²⁶ In order to maximize the catalytic activity of Ag NPs, control the size and morphology of Ag NPs rationally is necessary. Among various methods of preparing Ag NPs, interfacial reaction, based on the chemical reactions that happen at the interface of two different phases, is a special method.^27,28 Because the gas/liquid interface reaction can control the progress of the reduction reaction in a mild way by adjusting the reaction temperature, the reaction time, the gas pressures, and so on, the gas/liquid interface reaction can be used for the synthesis of Ag NPs with controlled size and morphology effectively.²⁹

Antibiotics make a contribution for human health and the development of the society, but antibiotics abuse phenomenon has caused attention recently. Metronidazole is an oral drug that belongs to a class of antibiotics known as nitroimidazole.³⁰ It is widely used for the treatment of amoebiasis and giardiasis caused by anaerobic protozoan.³¹ Quantitative detection of metronidazole is difficult and crucial for the bioanalysis of biological fluids and drug formulation because of the need to selectively remove the interferents such as proteins. Metronidazole contains a nitro group, which is reducible at the electrode surface.³² Therefore, it can be detected by electrochemical method.^33,34 However, the detection of metronidazole with low detection limit and high selectivity in a biological environment is still a major target of electroanalytical research.

Herein, we report a new strategy to construct petal-like graphene–Ag (p-GR–Ag) composites with highly exposed active edge sites as an efficient sensing material. The p-GR was first prepared by a HCl assisted hydrothermal method, and the p-GR can be intentionally utilized as nucleation sites for subsequent Ag NPs deposition via modified silver mirror reaction. The composites possessed three features. First, the interconnected p-GR formed the open and porous structure, providing large surface area and efficient channels for electron transport. Second, the unique petal structure favored the deposition of Ag NPs. Third, the 3D p-GR–Ag composites consisted of basal planes and highly reactive edge planes to provide more active sites, which could interact with metronidazole not only on the basal planes but also edge planes. The electrocatalytic and sensing performances of 3D p-GR–Ag based sensor were investigated in detail.

2. Experimental

2.1 Materials

Metronidazole was purchased from Sigma-Aldrich (USA). Natural graphite was obtained from Qingdao Hengrui Industrial, China. Silver nitrate (AgNO₃) was obtained from Tianjin Damao Chemical Factory. Hydrochloric acid (HCl) and formaldehyde (CH₂O) were purchased from Beijing Chemical Factory. All reagents were of analytical grade and used as received. The 0.2 M phosphate buffer solution (PBS) was made from potassium dihydrogen phosphate (KH₂PO₄) and disodium hydrogen phosphate (Na₂HPO₄). Aqueous solutions were prepared with redistilled water. Human urine samples were provided by a healthy volunteer.

2.2 Characterization

Scanning electron microscopy (SEM) measurements were performed on Hitachi S-4800 (Japan) to examine the morphologies of p-GR–Ag composites. The samples were cast on cleaning silicon wafers. The Raman spectra were obtained using Renishaw inVia Raman spectrometer with a 633 nm excitation source (England). The X-ray diffraction (XRD) images were recorded on Rigaku Miniflex 600 XRD diffractometer (Japan) with a CuKα source, having a wavelength of 0.154 nm. The diffractometer was scanned in the 2θ range of 5–90° and the scanning rate used was 1.2° min⁻¹. The Fourier transform infrared (FTIR) spectra were recorded on FTIR-8700 spectrometer (Japan). Powder samples were mixed with KBr and then press into pellets for FTIR measurements. Ultraviolet-visible (UV-vis) absorption spectra of the synthesized materials were recorded by a Shimadzu UV-vis spectrophotometer (Japan, UV-1700) in a range of 200–600 nm. X-ray photoelectron spectroscopy (XPS) analysis was measured on an ESCALAB-MKII 250 photoelectron spectrometer using Al as the exciting source.

Electrochemical studies were also carried out to assess the performance of the p-GR–Ag composites as catalysts and the current amplifier for the reduction of metronidazole. Cyclic voltammetry (CV), chronocoulometry, differential pulse voltammetry (DPV), and linear sweep voltammetry (LSV) measurements were performed using a CHI 660A electrochemical workstation (Shanghai Chenhua). A conventional three-electrode system was employed with a bare or modified glassy carbon electrode (GCE) with a diameter of 3 mm as working electrode, a platinum wire as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode. Rotating disk electrode (RDE) experiments were performed on a BAS RDE-2 electrode. All potentials reported in this paper were referenced to the SCE.

2.3 Preparation of p-GR–Ag composites

Scheme 1a illuminated the preparation process of p-GR–Ag composites. First, graphene oxide (GO) was synthesized from natural graphite powder by the modified Hummers method.³⁵ 5.0 mg GO powder is fully dispersed in 10.0 mL redistilled water with ultrasonication for 1 h to form a homogenous GO aqueous dispersion. Then a certain amount of concentrated HCl was added to the GO dispersion with stirring for several minutes. Next the resulting suspension was transferred into a Teflon-lined stainless autoclave (20.0 mL), sealed and maintained at 180 °C for 12 h. After cooling to the room temperature, the obtained p-GR was filtered, washed with excess distilled water, and dried at 50 °C for overnight.

Scheme 1 (a) Scheme for the preparation process of p-GR–Ag composites; (b) schematic display of the electrocatalytic reduction of metronidazole on the p-GR–Ag/GCE.

The p-GR–Ag composites were further prepared according to the previous report with a slight modification.²⁹ Briefly, 5.0 mL 1.0 mg mL⁻¹ p-GR suspension was added to Ag[(NH₃)₂]⁺ solution, and the mixture was kept on ultrasonication for 20 min. After that, the mixture and another beaker with 10.0 mL CH₂O solution were put in a closed container. The reaction was kept stirring for 24 h under room temperature. The result composites were collected by filtration, washed with distilled water for several times, and dried at 50 °C in air for 10 h. For comparison, the GR–Ag composites were prepared using the same procedure, in which GO replaced p-GR.

2.4 Preparation of p-GR–Ag modified GCE

Prior to modification, the GCE was polished with 0.3 and 0.05 μm Al₂O₃ powder on the polishing cloth. Then it was washed successively with 1 : 1 nitric acid, ethanol, and water in an ultrasonic bath and dried under a stream of nitrogen. The p-GR–Ag/GCE was prepared by casting 10 μL of p-GR–Ag suspension (0.5 mg mL⁻¹) on the GCE surface and dried on air. For comparison, the GR–Ag/GCE and the p-GR/GCE were also prepared using the same method. 0.2 M PBS was used as buffer solution, and all electrochemical experiments were performed under a nitrogen atmosphere at room temperature. Scheme 1b illuminated the p-GR–Ag composites' electrocatalysis toward metronidazole reduction.

3. Results and discussion

3.1 Characterization of p-GR–Ag composites

Fig. 1 showed typical SEM images of the GR (Fig. 1a), the GR–Ag composites (Fig. 1b), the p-GR (Fig. 1c and d), and the p-GR–Ag composites (Fig. 1e and f). As shown in Fig. 1a, the GR had only a few wrinkles and exhibited a smooth featureless surface. In the GR–Ag samples, Ag NPs were only deposited on the outer surface of GR, and the average diameter was 56 nm. In contrast, it can be seen from the low-magnification SEM (Fig. 1c) that GR with petal structure were distributed continuously and randomly interlaced together, giving rise to 3D open and porous structure. As the electrode material, the 3D p-GR with open and porous structure makes optimal use of the surface readily accessible to liquid electrolyte and provides efficient channels for electron transport. A further enlarged view in Fig. 1d showed that p-GR consisted of edge planes and basal planes. This structure supplied more active edge sites, which allowed p-GR to interact with analyte not only on the basal planes but also on the edge planes. Thus, excellent electrochemical properties for p-GR can be anticipated. The SEM images of the p-GR–Ag composites with different magnifications were observed in Fig. 1e and f. It can be seen that a homogeneous distribution of discrete, spherical Ag NPs was apparent on the petals with negligible NPs agglomeration. The average size of Ag NPs in the p-GR–Ag composites was 40 nm, which was smaller than that in the GR–Ag composites. This should be mainly attributed to the porous structure of p-GR together with highly exposed surface active sites, which strongly absorb Ag[(NH₃)₂]⁺ to grow small Ag NPs via reduction by CH₂O.

Fig. 1 (a) The SEM image of GR; (b) the SEM image of GR–Ag composites; (c) low-magnification SEM image of p-GR; (d) high-magnification SEM image of p-GR; (e) low-magnification SEM image of p-GR–Ag composites; (f) high-magnification SEM image of p-GR–Ag composites.

Fig. 2a compared the Raman spectra of GO, GR–Ag composites, and p-GR–Ag composites. All samples exhibited two prominent graphite-related bands, which correspond to the well-known D (∼1330 cm⁻¹) and G band (∼1600 cm⁻¹), assigned to the A_1g breathing mode of sp³ disordered graphitic carbon and E_2g mode of sp² ordered hexagonal carbon, respectively.³⁶ It is well documented that the D/G intensity ratio stands for a relative disorder in carbonaceous structures.³⁷ The D/G intensity ratio of GO, GR–Ag composites, and p-GR–Ag composites were 1.00, 1.14, and 1.19, respectively, demonstrating a good reduction degree of p-GR–Ag composites, and that the formation of p-GR–Ag composites did not destroy the intrinsic structural property of GR.

Fig. 2 (a) Raman spectroscopy of GO, GR–Ag composites, and p-GR–Ag composites; (b) FTIR spectroscopy of GO and p-GR–Ag composites; (c) XRD patterns of GO, GR–Ag composites, and p-GR–Ag composites; (d) UV-vis absorption spectra of GO and p-GR–Ag composites.

The GO and p-GR–Ag composites samples were further analyzed by means of FTIR spectroscopy (Fig. 2b). As expected, the peak at 3420, 1724, 1620, 1363, and 1116 cm⁻¹ were shown in the spectrum of GO, which were assigned to the –OH stretching vibrations, C=O stretching of COOH groups, C=C vibrations, epoxy C–O stretching vibrations, and alkoxy C–O vibrations, respectively, as previously reported.³⁸ Regarding the p-GR–Ag composites, intensity of the peaks characterizing the vibrations of the functional groups on GO became indistinguishable, indicating that most O-containing groups were removed by the reduction through hydrothermal method.

The structure and composition of GO, GR–Ag composites, and p-GR–Ag composites were characterized by XRD (Fig. 2c). An intense and sharp diffraction peak for GO appeared at 2θ = 10.9°, which was attributed to the (001) lattice plane. After the hydrothermal process, the peak of GO at 10.9° was disappeared and new peak at 24.4° corresponding to GR were observed in both the GR–Ag and p-GR–Ag samples,³⁹ suggesting that both of them were effectively reduced. In addition, the peaks at 38.2°, 44.3°, 64.5°, 77.4°, and 82.8° were assigned to the Ag (111) (200) (220) (311) (222) faces, respectively, indicating the face-centered cubic nature of the Ag NPs.⁷ However, the peaks of Ag NPs in the p-GR–Ag composites were more intense than those in the GR–Ag composites, this was because highly crystalline Ag NPs were deposited on the p-GR. Therefore, compared to traditional GR sheets, using p-GR as a catalytic support not only retained the unique property of GR itself, but also could supply more surface active sites as favorable nucleation sites for the highly crystalline Ag NPs deposition.

The UV-vis absorption spectra of GO and p-GR–Ag composites were shown in Fig. 2d. The two peaks at 230 nm and 303 nm were observed in the UV-vis spectrum of GO, due to the π–π* transitions of aromatic C=C bonds and the n–π* transitions of C=O bonds.⁴⁰ As for p-GR–Ag composites, the absorption peak of GO at 230 nm red-shifted to 258 nm, and the shoulder peak of 303 nm disappeared, indicating the partial restoration of electronic conjugation in the aromatic carbon structure of p-GR–Ag composites. Moreover, a clear broad absorption peak was observed at 399 nm, which was assigned to the surface plasmon resonance (SPR) absorption band of Ag NPs,⁴¹ confirming the formation of Ag NPs.

XPS study was carried out to further determine the surface composition of GO and p-GR–Ag. Fig. 3a showed the wide scan survey of XPS spectra of GO and p-GR–Ag composites. As expected, the bands centered at 284.8 and 531.0 eV were associated with C 1s and O 1s, respectively.⁴² In addition, there were two new peaks associated with Ag 3d in p-GR–Ag composites, suggesting the presence of Ag element in p-GR–Ag composites. As shown in Fig. 3b and c, the C 1s spectra of GO and p-GR–Ag composites consisted of four components including C–C (284.8 eV), C–O (286.7 eV), C=O (287.7 eV), and O–C=O (288.6 eV).⁴³ It was worth mentioning that the peaks assigned to C–C bond became predominant, while the peak for C–O bond was decreased dramatically in the C 1s spectrum of p-GR–Ag composites, demonstrating the GO had been deoxygenated. Moreover, there were two peaks emerged at 368.8 and 374.8 eV in Fig. 3d, corresponding to the Ag 3d_5/2 and Ag 3d_3/2 binding energy, respectively, which further supported the conclusion that p-GR–Ag composites had been synthesized successfully.⁴⁴

Fig. 3 (a) Wide scan survey of XPS spectra of GO and p-GR–Ag composites; (b) XPS C 1s spectra of GO; (c) XPS C 1s spectra of p-GR–Ag composites; (d) XPS Ag 3d spectra of p-GR–Ag composites.

The electrochemical active surface areas (A) of p-GR–Ag/GCE and GR–Ag/GCE were detected by chronocoulometry using 1 mM K₃[Fe(CN)₆] and 0.1 M KCl solution as model probe (ESI Fig. S1†), based on Anson equation.⁴⁵

Q(t) = 2nFAC_oD^1/2t^1/2/π^1/2 + Q_dl + Q_ads

where n is electron transfer number, C_o is bulk concentration, D is the diffusion coefficient, Q_dl is double layer charge, Q_ads is faradic charge, F, t, and π have usual values. By measuring the slope of the Anson plots, A was estimated to be 0.229 cm² and 0.179 cm² for p-GR–Ag/GCE and GR–Ag/GCE, respectively. The results indicated p-GR–Ag can enlarge electrochemical active surface area, arising from the creation of edge planes together with interconnected 3D open and porous structure.

3.2 Electrocatalytic activity of metronidazole on the p-GR–Ag/GCE

The cyclic voltammograms of metronidazole (300 μM) at the bare GCE (A), the p-GR/GCE (B), the GR–Ag/GCE (C), and the p-GR–Ag/GCE (D) in PBS (pH 6.0) buffer solution were shown in Fig. 4. The reduction peak current (I_pc) and peak potential (E_pc) of metronidazole at the bare GCE were 3.5 μA and −0.826 V, respectively, and no oxidation peak was visible in the reverse scan, indicating an irreversible reduction process. In contrast, at the p-GR/GCE, the I_pc increased by a factor of 3.1, and the E_pc was 0.270 V higher compared to the bare GCE. This phenomenon meant the p-GR had obvious electrocatalytic activity for metronidazole reduction. Moreover, it was observed that the I_pc of metronidazole was further improved with E_pc shifting to −0.469 V at the p-GR–Ag/GCE, which confirmed that the p-GR–Ag composites had superior electrocatalytic ability due to the addition of Ag NPs. It was noteworthy that the p-GR–Ag/GCE had 2-fold enhancement of the I_pc compared with the GR–Ag/GCE, and the detailed data were shown in Fig. S2.† The above results demonstrated the p-GR–Ag composites served as both the electrocatalyst and the current amplifier in this sensing system.

Fig. 4 Cyclic voltammograms of 300 μM metronidazole in PBS (pH 6.0) buffer solution at the GCE (A), the p-GR/GCE (B), GR–Ag/GCE (C), and p-GR–Ag/GCE (D). Scan rate: 50 mV s⁻¹.

The excellent sensing performance can be ascribed to the following major factors: (1) Ag NPs owned high electrical conductivity and excellent catalytic properties. (2) The interconnected p-GR formed the open and porous structure, which made optimal use of the surface readily accessible to liquid electrolyte and provided efficient channels for electron transport. (3) The 3D p-GR–Ag composites consisted of basal planes and highly reactive edge planes to provide more active sites, which could interact with metronidazole not only on the basal planes but also edge planes to amplify electrochemical signal of metronidazole.

3.3 Optimal detection conditions

It is evident from Fig. 4 that p-GR–Ag composites can significantly improve the electrochemical response of metronidazole. Therefore, the effect of the immobilization amount of p-GR–Ag composites on the reduction peak current of 300 μM metronidazole was investigated (Fig. S3†). Fig. S3† showed a plot of the reduction peak current for metronidazole versus the volume of the dropped suspension. Clearly, it can be observed that 10 μL of the suspension was employed as the optimal amount and showed the best current response to metronidazole. However, when the suspension was less or more than 10 μL, the response to metronidazole was lower. This can be explained by the fact that with increasing the amount of p-GR–Ag composites, the active edge sites also increase, and more metronidazole molecules can be catalyzed by p-GR–Ag composites. As a result, the reduction peak current increased gradually. However, as the volume of the suspension increased, the formed film become too thick, which was unfavorable not only for the electron transfer but also for the diffusion of metronidazole to the modified electrode, leading to a lower response. So the optimum dosage of p-GR–Ag composites was 10 μL.

The influence of scan rate on the electrochemical response of 300 μM metronidazole in 0.2 M PBS buffer solution at the p-GR–Ag/GCE was investigated by CV (Fig. S4a†). It was observed that the E_pc shifted negatively with the increasing of the scan rate, confirming the irreversible nature of the reduction reaction. In addition, the I_pc varied linearly as the square root of the scan rate (ν^1/2) range from 10 to 410 mV s⁻¹ with the linear equation of I_pc (μA) = −1.758 ν^1/2 (mV s⁻¹)^1/2 − 4.072 (R = 0.9959) (Fig. S4b†), signifying a diffusion-controlled process.

The effect of pH on the I_pc and E_pc of 300 μM metronidazole at the p-GR–Ag/GCE were recorded by DPV over the pH range from 4.0 to 9.0, and the results were shown in Fig. 5. It was found that the peak potentials shifted negatively with the increase of solution pH, indicating that protons were involved in the electroreduction. The linear equations between E_pc and pH were E_pc = −0.04994 pH − 0.1280 (R = 0.9928). According to the Nernst equation, the proportion of electrons to protons involved in the reactions was calculated as approximately 1 : 1 based on the slope of −0.059 V pH⁻¹, which was consistent with the findings of previous studies.^46,47 The maximum I_pc was obtained at pH 6.0. Therefore, PBS with pH 6.0 was used as the supporting electrolyte in all determinations, which was close to physiological conditions.

Fig. 5 (a) Differential pulse voltammograms of 300 μM metronidazole at the p-GR–Ag/GCE in PBS with different pH. Pulse amplitude = 50 mV; pulse width = 50 ms; (b) the histograms of the cathodic peak potential of 300 μM metronidazole at the p-GR–Ag/GCE in PBS with different pH. Inset: the plot of peak potential versus pH values; (c) the histograms of the cathodic peak current of 300 μM metronidazole at the p-GR–Ag/GCE in PBS with different pH.

3.4 Determination of metronidazole by LSV

Using the optimal experiment conditions established above, the quantitative detection of metronidazole was further studied by LSV. The I_pc of metronidazole at various concentrations at the p-GR–Ag/GCE were recorded in PBS (pH 6.0) solutions at the scan rate of 50 mV s⁻¹ (Fig. 6). The I_pc was proportional to the concentration in the ranges of 0.05–10 μM (Fig. 6a) and 10–4500 μM (Fig. 6b). The corresponding linear regression equations were I_pc (μA) = −0.8679 − 0.1693C (μM) (R = 0.9962) and I_pc (μA) = −2.300 − 0.03997C (μM) (R = 0.9989), respectively. The detection limit was estimated as 28 nM (S/N = 3). The calibration plot was found to be bimodal with linear concentration range, which potentially resulted from the kinetically-controlled electrochemical process that related with the concentration of metronidazole. On the surface of p-GR–Ag/GCE, a large number of active sites existed. At low concentration, metronidazole was easy to be catalyzed and generated a sensitive current response. With the increase of concentration, the active sites were gradually buried. Therefore, the sensitivity of metronidazole decreased slowly with the increasing concentration of metronidazole. The linear response range was wider, and the detection limit was lower than previous reports.^31,46,48,49 In order to make a realistic comparison with previous reports, the characteristics of different electrochemical methods for metronidazole determination were summarized in Table 1. These results indicate that the p-GR–Ag is a preferable sensing platform for the determination of metronidazole.

Fig. 6 Linear sweep voltammograms of metronidazole with different concentrations at the p-GR–Ag/GCE in PBS (pH 6.0). Scan rate: 50 mV s⁻¹. (a) From top to bottom, 0, 0.05, 0.1, 1, 5, and 10 μM; (b) from top to bottom, 10, 50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 2500, 3000, 3500, and 4500 μM. Inset: the plots of peak current of metronidazole versus its concentrations.

Table 1 Performance comparison of the fabricated electrode for metronidazole detection with other electrodes

Electrode	Linear range (μM)	LOD (μM)	Method	Ref.
a CPE: carbon paste electrode.b SWASV: square wave anodic stripping voltammetry.
3D gold nanotube/CPE^a	0.1–200	0.063	SWASV^b	30
Copper–poly(cysteine)/GCE	0.5–400	0.37	LSV	31
Pretreated gold electrode	0.5–17, and 20–800	0.15	LSV	32
Graphene/ionic liquid/GCE	0.1–25	0.047	DPV	47
Poly(chromotrope 2B)/GCE	10–400	0.33	DPV	49
Cysteic acid/PDDA–graphene/GCE	0.01–1.0, and 70–800	0.0023	LSV	50
p-GR–Ag/GCE	0.05–10, and 10–4500	0.028	LSV	This work

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3.5 Rotating disk electrode investigation

Rotating disk electrode (RDE) technique is an effective method to obtain kinetic information of electrode reactions.⁵⁰ The RDE experiments of 300 μM metronidazole at p-GR–Ag/GCE were performed at various rotation rates from 200 to 4200 rpm and at scan rate of 5 mV s⁻¹ (Fig. 7). The linearity of the Levich plots at lower rotation rates indicated that the limiting current is mass-transport controlled. The Levich plots deviated from linearity at higher rotation rates, suggesting that a kinetic limitation is involved in the electron-transfer reaction. Under these conditions the RDE data were analyzed using the Koutecky–Levich equation.⁵¹

I⁻¹ = I_Lev⁻¹ + I_K⁻¹

in which I_Lev is the Levich current, I_K is the kinetic current. It can be further defined by

I_Lev = 0.62nFAD^2/3υ^−1/6ω^1/2C_o

I_K = nFAC_ok

where n is electron transfer number, F (96 485.3 C mol⁻¹) is the Faraday constant, A is the electrochemical active surface area, D (3.97 × 10⁻⁶ cm² s⁻¹) is the diffusion coefficient,³¹ υ (0.01 cm² s⁻¹) is the kinematic viscosity, ω (rad s⁻¹) is the rotation rate, C_o is bulk concentration, and k (cm s⁻¹) is the heterogeneous electron transfer rate constant. Based on the slopes of the curves of I⁻¹ versus ω^−1/2 at different electrode potentials (−0.80 V, −0.85 V, −0.90 V, −0.95 V, and −1.0 V), the average number of electron transfer was calculated to be 3.86 ≈ 4. Thus, the reduction of metronidazole at p-GR–Ag/GCE was probably to be a four-electron and four-proton electrode process, which was consistent with the previous reports.⁵⁰ This mechanism is attributed to the four-electron reduction of nitro functional group to hydroxylamine. According to the intercept of the curves, the average k value in the metronidazole concentration range was calculated to be 3.42 × 10⁻² cm s⁻¹. The kinetic parameter may be valuable to scientists who search metronidazole reduction catalysts.

Fig. 7 RDE polarization curves of 300 μM metronidazole at the p-GR–Ag/GCE in PBS (pH 6.0). Potential scan rate: 5 mV s⁻¹. The electrode rotation rates from top to bottom are 200, 600, 1000, 1400, 1800, 2200, 2600, 3000, 3400, 3800, and 4200 rpm, respectively. The insets are Levich plots (a) and Koutecky–Levich (b) at different electrode potentials.

3.6 Selectivity, stability and repeatability of the p-GR–Ag/GCE

For demonstrating the fabrication reproducibility of p-GR–Ag/GCE, a series of repeated determinations of 200 μM metronidazole with five different modified electrodes prepared under the same conditions were performed (Fig. S5†). The relative standard deviation (RSD) of the peak current is 2.49%. In order to further examine the stability of the electrode, eight consecutive measurements were carried out for 300 μM metronidazole at the same modified electrode and the RSD was 4.26% (Fig. S6†). After the modified electrode was kept for ten days at room temperature, the peak currents remained 90.2% of their initial values (Fig. S7†). The obtained operational stability of fabricated sensors was attributed to the structural stability and robustness of the prepared p-GR–Ag composites given via the p-GR matrix.

The possible interferents of some ions were also investigated (Table S1†). The results showed that 100-fold concentration of K⁺, Na⁺, Li⁺, Ca²⁺, Zn²⁺, Cl⁻, NO₃⁻, ClO₄⁻, SO₄²⁻, HPO₄²⁻, H₂PO₄⁻, 10-fold concentration of glucose, fructose, maltose, phenacetin, uric acid, and ascorbic acid had no effect on determination of metronidazole (signal change below 5%). Moreover, the 1 time concentration of 5-nitroimidazoles, including tinidazole and ornidazole can not interfere with the signals of metronidazole.

3.7 Analytical applications

The practical analytical utility of the modified electrode was evaluated by the determination of metronidazole in human urine and local lake water (South-Lake, Changchun, China). The samples were diluted 60-times with PBS solution (pH 6.0) and determined by LSV technique. The modified electrode had no response when metronidazole was not added in the samples. After the samples were spiked with a certain amount of metronidazole, well-defined reduction peak was observed on the modified electrode. The standard addition method was used for calculating the metronidazole concentrations. The obtained results were summarized in Table 2. The recoveries of the sample were 97.14–102.1% and 98.20–101.6% for urine and lake water, respectively. The RSD was below 3.7%. Therefore, the sensing platform based on p-GR–Ag is reliable for the determination of metronidazole in practical samples.

Table 2 Determination of metronidazole in practical samples

Sample	Added (μM)	Found^a (μM)	RSD (%)	Recovery (%)
a Average of three determinations.
Lake water	50.00	50.81	2.4	101.6
Lake water	150.0	147.3	3.1	98.20
Urine	50.00	48.57	3.7	97.14
Urine	150.0	153.2	2.8	102.1

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4. Conclusions

In summary, we report a facile and cost-effective method for the fabrication of p-GR–Ag composites with highly exposed active edge sites. The p-GR was prepared using a HCl assisted hydrothermal method, followed by Ag NPs decoration via modified silver mirror reaction. The p-GR–Ag composites possessed several features superior to traditional GR–Ag composites. Firstly, the p-GR–Ag composites was made of basal planes and highly reactive edge planes to expose more catalytically active edge sites, avoiding the planarity of conventional GR–Ag composites, which limited the active sites for electrochemical and catalytic reactions. Secondly, the p-GR possessed large surface area and supplied more active sites as favorable nucleation sites for the highly crystalline Ag NPs deposition, enabling improved performance for electrochemical and catalytic activity. Finally, the p-GR–Ag composites with 3D open and porous structure made optimal use of the surface readily accessible to liquid electrolyte and provided efficient channels for electron transport. These features presented a distinct opportunity to create a sensor for electrochemical detection of metronidazole, and the experimental results have proved the superior catalytic performance and signal amplification of the p-GR–Ag composites in metronidazole reduction and detection. Wide linear range, low detection limit, and stable response suggested that p-GR–Ag composites were a promising sensor for the detection of metronidazole. More importantly, it can be anticipate that the proposed strategy may provide a convenient and cheap approach in constructing p-GR with outstanding performance.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21375045), Natural Science Foundation of Jilin Province (No. 20130101118JC), and Project 2015036 Supported by Graduate Innovation Fund of Jilin University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01334a

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