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. 2026 Oct 21. Online ahead of print. doi: 10.1039/d6ra06899e

Hierarchical Ni-rGO nanocomposite derived from e-waste graphite for trace-level electrochemical sensing of metronidazole

Abdullah Ibn Masud Mubin a,c, Md Humayun Kabir b,✉, Md Qamrul Ehsan c, Othai Saha a, Sourav Datta a, Md Sanwar Hossain a, Md Yeasin Pabel a, Muhammad Shahriar Bashar d, Sabina Yasmin a,✉
PMCID: PMC13684875  PMID: 42909315

Abstract

Metronidazole (MTZ) accumulation in aquatic systems presents severe environmental and public health risks, requiring sensitive trace-level monitoring techniques. Here, we report the development of an electrochemical sensor based on a reduced graphene oxide (rGO) nanocomposite decorated with nickel-containing nanophases (Ni-rGO). The graphene oxide support was synthesized from graphite sustainably recovered from discarded lithium-ion batteries prior to integration via a one-pot, hydrazine-free hydrothermal method. Morphological and structural analyses (SEM, TEM, XRD, FTIR) reveal a hierarchical architecture where highly dispersed, ultra-small (1.5–3 nm) nickel-containing domains coexist with larger crystalline domains anchored to a porous rGO network. This dual-scale structure likely prevents graphene restacking, resulting in a 41.2% increase in electrochemically active surface area and a 43.35% reduction in interfacial charge-transfer resistance compared to unmodified glassy carbon electrodes. Voltammetric studies demonstrate that MTZ reduction at the Ni-rGO interface proceeds via a diffusion-dominated, proton-coupled electron transfer mechanism at physiological pH (7.4). Under optimal conditions, the sensor exhibits a wide linear range of 2–200 µM, a low detection limit of 122.6 nM, and a high sensitivity of 5111.9 µA mM−1 cm−2. The sensor exhibited high fabrication reproducibility (3.12% relative standard deviation across five independent electrodes), operational repeatability (95.4% signal retention over 10 continuous cycles), and storage stability (91.9% retention after 7 days). The analytical response remained unaffected by 10-fold excesses of common inorganic ions, endogenous biological species (dopamine, ascorbic acid, uric acid), and the structural analog imidazole, as well as 5-fold excesses of commonly used antibiotics, confirming the sensor's strict specificity toward the target analyte. Recovery rates of 87–108% across tap water and human urine matrices confirm the practical analytical utility of the Ni-rGO sensor for real-world MTZ monitoring in both environmental and clinical contexts.

A sustainable Ni-rGO electrochemical sensor derived from e-waste graphite enables sensitive and selective trace-level detection of metronidazole in environmental and biological samples.graphic file with name d6ra06899e-ga.webp

1. Introduction

The occurrence of pharmaceutical residues in aquatic environments has become an increasing environmental concern, particularly due to their contribution to antimicrobial resistance and long-term ecological disruption.1–5 Antibiotics at trace levels can still exert biological activity, selectively influencing microbial populations and accelerating the emergence of resistant strains.6–10 This has made the development of reliable trace-level monitoring strategies an important analytical and environmental priority.11

Among commonly detected antibiotics, metronidazole (MTZ), a nitroimidazole derivative, is widely used to treat anaerobic bacterial and protozoal infections in both human medicine and aquaculture.12 Its high aqueous solubility, recalcitrant nature, incomplete metabolic degradation, systemic excretion, and continuous pharmaceutical discharge have led to its widespread detection in wastewater effluents, groundwater, surface water, and drinking water, often bypassing conventional sewage treatment processes.13–15 The nitro functional group present in its structure is particularly concerning, as reductive activation in biological systems can generate reactive intermediates capable of inducing DNA damage, posing mutagenic risks to aquatic biota and potential carcinogenic risks to humans.16 Sensitive trace-level detection of MTZ is therefore essential for both public health protection and aquatic ecosystem preservation.17

Conventional analytical methods for MTZ determination include high-performance liquid chromatography (HPLC),18 supercritical fluid chromatography (SFC),19 capillary electrophoresis (CE),20 gas chromatography (GC),21 and UV-Vis spectrophotometry.22 Although well established in terms of accuracy and selectivity, these techniques are often limited by high operational costs, complex sample preparation, and dependence on centralized laboratory infrastructure, restricting their suitability for rapid or field-based monitoring.23 Electrochemical sensing platforms offer a compelling alternative, providing advantages such as simplicity, rapid response, low cost, and ease of miniaturization. The performance of such sensors is fundamentally governed by the properties of the electrode material and the kinetics of electron transfer at the electrode–electrolyte interface.24–28

A variety of electrode architectures have been reported for the electrochemical detection of metronidazole, with performance strongly governed by material composition and surface engineering. Pristine carbon electrodes exhibit poor sensitivity, with detection limits in the micromolar range (∼10−6 M), attributed to limited electrocatalytic activity.29 In contrast, noble metal- and graphene-based nanocomposites, such as Ag/Co-rGO and Au nanoparticle-modified electrodes, demonstrate markedly enhanced performance, achieving low nanomolar detection limits (74 nM and 97 nM, respectively),30,31 driven by improved conductivity and synergistic catalytic effects. Carbon-based nanocomposites and polymer-modified systems, including CB NPs/PGA and MWCNT-based electrodes, generally show intermediate performance (≈170–250 nM),32,33 where increased surface area is partially limited by diffusion and adsorption effects.

Despite these improvements, considerable variability remains across similar material systems, and certain transition-metal layered double hydroxide electrodes, such as Ni/Fe-LDH, still exhibit relatively higher detection limits (∼58 µM),34 reflecting intrinsic conductivity and charge-transfer limitations. Overall, balancing analytical sensitivity with fabrication simplicity, material sustainability, and avoidance of noble metal or multi-component precursors remains a key challenge in metronidazole sensor development.

Reduced graphene oxide (rGO) has been widely explored as an electrode modifier due to its excellent electrical conductivity and large electroactive surface area. However, the tendency of graphene sheets to restack via π–π interactions reduces accessible active sites and limits mass transport to the electrode surface.23,35 Incorporating nickel, a non-precious and earth–abundant transition metal, offers a cost-effective and sustainable alternative to noble metal or multi-component systems, while facilitating electrocatalytic reduction of the nitro group (–NO2) in MTZ and acting as a physical spacer that suppresses rGO restacking.36,37 The resulting Ni-rGO hybrid architecture in this work forms a three-dimensional porous conductive network that enhances electroactive surface area and facilitates analyte diffusion, yielding a strong and stable cathodic response.

Nickel-containing materials have previously been investigated for MTZ sensing. Darbandi et al. reported NiO and NiO–Ni nanoparticle-modified GCEs for the electrochemical determination of MTZ.37 In that study, the sensing electrodes were based on nickel oxide/nickel nanoparticles without a graphene support. Our previous work has also established the use of battery-derived rGO in electrochemical sensors, including rGO/ZnO for bisphenol A detection and rGO/CuO for glucose sensing.23,38 More recently, we reported an Ag/Co/rGO electrode for MTZ detection.30 The Ag/Co/rGO system is bimetallic, whereas the present electrode uses Ni as the sole metal component. Thus, the present Ni-rGO differs from the NiO/Ni nanoparticle-based MTZ sensor by incorporating rGO as the conductive support and from our previous system by employing a monometallic Ni-based composition. To the best of our knowledge, Ni-rGO has not previously been reported for electrochemical MTZ determination.

Conventional synthesis of Ni-rGO composites often involves multistep protocols requiring stringent reaction conditions and hazardous reducing agents such as hydrazine, raising both safety and environmental concerns.39,40 Uncontrolled nucleation in such approaches frequently results in metal particle aggregation, non-uniform dispersion, and reduced catalytic efficiency.41

To overcome these limitations, we report a facile one-pot hydrothermal synthesis using graphene oxide derived from graphite recovered from spent lithium-ion batteries. This sustainable approach enables simultaneous reduction of graphene oxide and in situ deposition of nickel-containing nanophases, producing a uniformly dispersed nanocomposite anchored across a porous rGO network while also valorizing electronic waste.42

The synthesized composite was characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The electrocatalytic performance toward MTZ reduction was evaluated through pH optimization, scan rate analysis, and charge-transfer studies. The selectivity of the sensor was assessed against common interfering species, including dopamine, ascorbic acid, uric acid, and common inorganic ions, in 10-fold excess. Additional interference studies were performed using the commonly used antibiotics – cefixime, tetracycline, cefuroxime, and doxycycline at 5-fold excess. Under optimized conditions, a detection limit of 122.6 nM was achieved with a wide linear dynamic range and high sensitivity. The sensor demonstrated good stability and reproducibility over repeated measurements. Finally, practical applicability was confirmed through successful MTZ detection in tap water and human urine samples, highlighting its potential for environmental monitoring applications.

2. Experimental

2.1. Reagents and chemicals

All reagents were of analytical grade and used without further purification. Metronidazole (MTZ, ≥99%, source: WXBF2223 V) was purchased from Sigma-Aldrich (USA) and used as the target analyte. Graphene oxide (GO) synthesis reagents included concentrated sulfuric acid (H2SO4, 98%, CAS: 7664-93-9) and potassium permanganate (KMnO4, ≥99%, CAS: 7722-64-7), procured from Scharlau (Spain); phosphoric acid (H3PO4, 85%, CAS: 7664-38-2) was obtained from Janssen Chimica (Belgium). Hydrogen peroxide (H2O2, 30%, CAS: 7722-84-1) and hydrochloric acid (HCl, 37%, CAS: 7647-01-0) were supplied by Sigma-Aldrich (USA). For Ni-rGO synthesis, nickel(ii) nitrate hexahydrate (Ni(NO3)2·6H2O, ≥98%, CAS: 13478-00-7) served as the nickel precursor, while sodium borohydride (NaBH4, ≥98%, CAS: 16940-66-2) was used as the reducing agent. Sodium hydroxide (NaOH, ≥98%, CAS: 1310-73-2) was used for pH adjustment.

Phosphate buffer solution (PBS, 0.1 M) was prepared from sodium phosphate monobasic dihydrate (≥98%, CAS: 13472-35-0) and used as the supporting electrolyte. All solutions were prepared using ultrapure deionized water (18.2 MΩ cm). Glassy carbon electrodes were polished using alumina slurry (0.05 µm) prior to modification.

2.2. Instrumentation

Material characterization was performed using structural, spectroscopic, morphological, and electrochemical techniques. X-ray diffraction (XRD) analysis was carried out using an ARL EQUINOX 1000 X-ray diffractometer (Thermo Scientific™, USA) with Cu Kα radiation (λ = 1.5406 Å) operated at 40 kV and 60 mA for phase identification and crystallinity evaluation. Fourier transform infrared (FTIR) spectroscopy (Shimadzu IRAffinity-1, Japan) was used to examine functional group evolution and chemical changes during synthesis in the mid-infrared region. Transmission electron microscopy (TEM) (Thermo Fisher Scientific Talos F200X, 200 kV) was employed to investigate nanoscale morphology, lattice features, and particle size distribution. Surface morphology and elemental composition were analyzed using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM–EDS) (Carl Zeiss EVO18, Germany) at an accelerating voltage of 20 kV. Electrochemical experiments were performed using a CHI660 electrochemical workstation in a standard three-electrode configuration. A glassy carbon electrode (GCE, 3 mm diameter) served as the working electrode, a saturated Ag/AgCl/KCl electrode (CHI111) was used as the reference electrode, and a platinum wire (BAS Inc., 23 cm) acted as the counter electrode.

2.3. Synthesis of graphene oxide (GO)

Graphene oxide was synthesized from graphite powder salvaged from discarded lithium-ion batteries – e-waste that would otherwise have gone to landfill. The extraction procedure for pulling graphite powder out of these batteries is described in our prior work.38 In brief: the graphite was mechanically cleaned, rinsed with deionized water, then stirred in aqua regia to strip out metallic impurities. This purified powder was the precursor for graphene oxide (GO), synthesized using an improved Hummers' method. 1 g of graphite powder went into a 9 : 1 mixture of concentrated sulfuric acid (120 mL) and phosphoric acid (14 mL), stirred continuously. KMnO4 (6 g) was then added gradually; adding it all at once risks a runaway exotherm, and the mixture was held at 50 °C for 6 hours under stirring. Once it cooled to room temperature, the flask was moved into an ice bath. 400 mL of DI water went in next, followed by 3 mL of 30% H2O2 to quench any KMnO4 that hadn't reacted. The reaction is aggressively exothermic; the ice bath kept the temperature pinned near 0 °C throughout this step. The resulting suspension was centrifuged at 4500 rpm and washed in sequence: water first, then 30% HCl to pull out residual metal ions, then a final round of deionized water and ethanol to clear away whatever contaminants remained. Vacuum drying at 45 °C for 48 hours yielded the final GO product.

2.4. Synthesis of the Ni-rGO nanocomposite

50 mg of graphene oxide (GO) was added to 50 mL of deionized (DI) water and ultrasonicated for 1 hour to obtain a homogeneous suspension. 50 mg of nickel nitrate hexahydrate was added to 10 mL of deionized water and ultrasonicated for 30 minutes. The nickel solution was then added dropwise under continuous stirring, and the mixture was ultrasonicated for 1 hour. 50 mg of sodium borohydride was dissolved in 10 mL of DI water, and this freshly prepared solution was added to the GO–Ni mixture dropwise under continuous stirring. The reaction mixture was then ultrasonicated for 30 minutes and placed in a hydrothermal autoclave for 4 hours at 170 °C.30 After turning off the oven, the hydrothermal pot was left inside the oven for 18 hours. The composite was then centrifuged and washed at 4500 rpm for 10 minutes each time. After that, it was washed with ethanol using the same process. The composite was collected in a Petri dish and dried in a vacuum oven at 60 °C overnight. Finally, it was stored in an airtight container (Scheme 1).

Scheme 1. Schematic illustration of the synthesis process of Ni-rGO nanocomposite from waste Li-ion batteries and its application in electrochemical detection.

Scheme 1

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2.5. Fabrication of the Ni-rGO modified GCE

The Ni-rGO ink was prepared by dispersing 2 mg of Ni-rGO composite in 2 mL of DI water, followed by ultrasonication for 1 hour. The GCE was polished thoroughly with 0.05 µm alumina slurry on a microcloth and then ultrasonicated in deionized water for 10 minutes to remove any residual particles. 6 µL of the ink was drop-cast onto the cleaned GCE surface. It was dried at room temperature, forming a catalyst film with a mass loading of 0.085 mg cm−2. All electrochemical measurements were performed using this Ni-rGO/GCE-modified electrode as the working electrode in a three-electrode system. Argon gas was purged for 5 minutes before each experiment to remove any oxygen and ensure stable and reproducible electrochemical response.

3. Results and discussion

3.1. Morphological and structural characterization

The morphological characteristics of the Ni-rGO nanocomposite were systematically examined using SEM and TEM to establish a structure–property relationship with its electrochemical behaviour. The SEM images (Fig. 1a and b) clearly display a highly crumpled and folded sheet-like morphology, forming an interconnected and loosely stacked porous framework. While rGO sheets inherently tend to restack irreversibly through π–π interactions,35 the incorporation of nickel based nanoparticles within this architecture serves as a physical spacer, suppressing this tendency as evidenced by the loosely stacked and porous framework observed here. As a result, a significant fraction of the graphene surface remains electrochemically accessible, which is critical for facilitating interfacial processes.

Fig. 1. (a and b) SEM images of Ni-rGO nanocomposite; (c and d) TEM images of Ni-rGO nanocomposite; (e) particle size distribution histogram; (f) EDS spectrum of Ni-rGO nanocomposite.

Fig. 1

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At high magnification, the wrinkled topology and overlapping sheet edges become more evident, suggesting the formation of a three-dimensional conductive network.43 Such a configuration is expected to provide multiple electron transport pathways while simultaneously allowing electrolyte penetration into the interior structure. This combination is particularly beneficial in electrochemical systems where both charge transport and mass accessibility play important roles.

Further structural insight is obtained from TEM analysis (Fig. 1c and d), where the rGO sheets appear as thin, semi-transparent layers with characteristic corrugation. The presence of nickel is confirmed by the appearance of darker contrast regions distributed across the graphene surface. These nanoscale features are reasonably well-dispersed and do not exhibit significant agglomeration, indicating that the synthesis approach enables effective immobilization of Ni species onto the rGO support. The absence of large clusters suggests that a substantial fraction of the metal phase remains exposed and available for interfacial interaction. Statistical analysis of particle size, based on measurements of the nanoparticles from TEM images, reveals a relatively narrow distribution primarily within the ∼1.5–3 nm range (Fig. 1e).

The elemental composition was evaluated using EDS (Fig. 1f), which confirms the presence of carbon, oxygen, and nickel with weight percentages of 48.99%, 26.93%, and 24.09%, respectively. The relatively high oxygen content indicates the partial retention of oxygen-containing functional groups within the rGO framework. These oxygen-containing functional groups serve as preferential nucleation and anchoring sites during metal deposition, contributing to the uniform dispersion of Ni nanoparticles observed across the rGO surface. The detected Ni content further verifies the successful incorporation of the metallic component within the composite matrix.

The structural features observed from microscopic analysis are consistent with the electrochemical characteristics discussed in subsequent sections. The Ni-rGO modified electrode exhibits a pronounced decrease in charge-transfer resistance along with an increase in electrochemically active surface area compared to the bare electrode. The combination of a porous graphene scaffold and dispersed Ni nanoparticles, therefore, provides a conductive and accessible interface that supports efficient charge-transfer processes. This structural configuration is considered to play a key role in enhancing the electrochemical response toward metronidazole reduction.

The crystalline structure and surface chemical evolution of the synthesized materials were investigated using X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, providing complementary evidence for the successful formation of the Ni-rGO nanocomposite.

The XRD pattern of the Ni-rGO composite shows a residual reflection near 10°, corresponding to the (002) plane of partially reduced graphene oxide (rGO)30,43 (Fig. 2a). The broadened and attenuated profile of this peak relative to the sharp, intense signature typically observed for pristine GO indicates successful exfoliation and the suppression of extensive graphitic restacking during the hydrothermal treatment. The absence of a distinct diffraction peak at ∼24–26°, typically associated with graphitic (002) stacking,44 further confirms incomplete restoration of graphitic order and supports the formation of partially reduced graphene oxide. Additional distinct reflections emerge at ∼34° and ∼60°, which are indexed to the (101) plane of Ni(OH)2 and the (220) plane of NiO, respectively.45,46 The absence of extraneous diffraction peaks confirms the phase purity of the deposited nickel species, while the broad underlying baseline signal originates from the amorphous carbon scaffold. The sharp, high-intensity nature of the Ni(OH)2 peak reveals a high degree of bulk crystallinity.46 While TEM imaging highlights a localized surface population of highly dispersed 1.5–3 nm nanoparticles, the bulk XRD data confirms the concurrent growth of larger crystalline domains. This points to a mixed-phase or hierarchical morphology generated during the one-pot synthesis. This structural duality is expected to be beneficial for electrochemical performance. The larger crystalline Ni(OH)2 and NiO domains act as physical intercalants that inhibit rGO restacking, while the coexisting 1.5–3 nm nanophase supplies the high density of accessible active sites required for efficient electrocatalysis.

Fig. 2. (a) XRD pattern of Ni-rGO nanocomposite; (b) FTIR spectra of Ni-rGO, rGO, and GO.

Fig. 2

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FTIR spectroscopy provides further evidence for the chemical changes accompanying the reduction of GO and the incorporation of Ni-based species (Fig. 2b). The GO spectrum shows the characteristic broad O–H stretching band in the ∼3200–3500 cm−1 region, together with a C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching feature in the ∼1700–1750 cm−1 region and C–O-related vibrations in the fingerprint region.47–49 After reduction, the intensity of the oxygen-containing functional-group bands decreases in the rGO spectrum, while the C Created by potrace 1.16, written by Peter Selinger 2001-2019 C skeletal vibration around ∼1580–1600 cm−1 remains evident, consistent with partial restoration of the conjugated carbon framework.47,50 In the Ni-rGO spectrum, the features below ∼800 cm−1 are consistent with Ni–O vibrations, supporting the presence of Ni–O-containing species in the composite.43 The remaining oxygen-containing groups may provide sites for interaction and anchoring of the Ni-based species on the rGO surface.

Collectively, the XRD and FTIR results support a partially restored graphitic structure decorated with nanoscale Ni-based phases with residual oxygen functionalities providing potential sites for interaction and anchoring of the Ni-based species. This hybrid architecture is consistent with the enhanced charge-transfer characteristics and increased electrochemical activity observed in subsequent electrochemical analyses.

3.2. Electrochemical characterization of the modified interface

Cyclic voltammetry analysis of 300 µM MTZ in 0.1 M argon-saturated phosphate buffer solution (PBS) shows different electrocatalytic behaviours among bare GCE, rGO/GCE, and Ni-rGO/GCE (Fig. 3a). The bare GCE showed a peak current of −27.03 µA at −0.619 V, and upon modification with rGO, it showed a peak current of −53.49 µA at −0.508 V, demonstrating a 97.9% increase relative to the bare GCE. The Ni-rGO/GCE demonstrated superior electrocatalytic performance, exhibiting a peak current of −68.79 µA at −0.561 V. This represents a total increase of 154.5% compared to the bare GCE and a 28.6% increase over rGO/GCE.

Fig. 3. (a) Comparison of Ni-rGO/GCE, rGO/GCE and GCE for sensing 300 µM MTZ in 0.1 M PBS; (b) Nyquist plots of Ni-rGO/GCE, rGO/GCE and GCE in 5 mM [Fe(CN)6]3−/4−; (c) CVs of different electrodes in 5 mM [Fe(CN)6]3−/4−; (d) CVs of Ni-rGO/GCE in 5 mM [Fe(CN)6]3−/4− at different scan rates.

Fig. 3

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The enhanced MTZ response of Ni-rGO/GCE can be associated with the combined contribution of the rGO framework and the Ni-containing nanostructures. Incorporation of the Ni-containing phases can reduce the restacking of rGO sheets and facilitate the formation of a more open nanostructured interface. This is consistent with the higher ECSA obtained for Ni-rGO/GCE compared with rGO/GCE and bare GCE. The resulting increase in electrochemically accessible surface area may provide more accessible sites for the interfacial reduction of the nitro group and thereby contribute to the enhanced MTZ response.

To evaluate the interfacial charge transfer efficiency of the bare GCE and the modified electrodes, EIS measurements were conducted in 5 mM [Fe(CN)6]3−/4− containing 0.1 M KCl supporting electrolyte. Nyquist plots were recorded at an AC amplitude of 5 mV over the frequency range of 105 Hz to 0.1 Hz (Fig. 3b). The diameter of the semicircle region at high frequencies corresponds to the charge-transfer resistance (Rct). The EIS data were fitted employing the Randles equivalent circuit model, which includes the solution resistance (Rs), charge transfer resistance (Rct), double-layer capacitance (Cdl), and Warburg impedance (W). The bare GCE showed an Rct value of 128.2 Ω, and after being modified with rGO, it showed an Rct value of 144.5 Ω. The observed increase in Rct upon rGO modification can be attributed to the negatively charged oxygen-containing functional groups on the rGO surface, which electrostatically repel the anionic [Fe(CN)6]3−/4− redox probe, hindering interfacial electron transfer.

Whereas the Ni-rGO/GCE achieved a significantly lower Rct of 72.62 Ω, which is 43.35% lower than that of the bare GCE and 49.74% lower than that of rGO/GCE. This large drop in the Rct value indicates the success of the Ni-based nanostructures as structural spacers, effectively decoupling the rGO sheets and thus preventing restacking and preserving a 3D porous architecture. This significant enhancement in interfacial charge-transfer efficiency at the Ni-rGO/GCE interface contributes to the improved electrochemical performance toward MTZ detection.

Fig. 3c shows 5 mM [Fe(CN)6]3−/4− in 0.1 M KCl under argon-saturated conditions was used to perform cyclic voltammetry to evaluate the electrochemical behaviour of bare GCE, rGO/GCE and Ni-rGO/GCE. These measurements were conducted to study how the surface modification affects the redox behaviour of the electrodes. The CVs explored a potential range of −0.3 V to +0.5 V at a scan rate of 100 mV s−1. The oxidation peak currents of bare GCE, rGO/GCE, and Ni-rGO were 111.2 µA, 108.1 µA, and 118.2 µA, respectively. The highest oxidation current observed for Ni-rGO is consistent with the improved charge transfer characteristics indicated by EIS analysis, confirming enhanced electrochemical activity upon Ni incorporation. The corresponding reduction peak currents for bare GCE, rGO/GCE, and Ni-rGO/GCE were −99.85 µA, −102.6 µA, and −96.09 µA, respectively. The slightly higher cathodic peak current observed for rGO/GCE may be attributed to increased surface adsorption and accumulation of the redox species on the rGO surface, which can enhance local concentration effects. Ni-rGO/GCE, however, exhibited a larger peak-to-peak separation than bare GCE and rGO/GCE, indicating a quasi-reversible electron-transfer process for the [Fe(CN)6]3−/4− redox couple at the modified interface. The increase in ΔEp with increasing scan rate further supports the quasi-reversible nature of the redox process. The CV results are consistent with the EIS findings and confirm that Ni-rGO modification enhances the electrochemical response of the electrode system.

In order to determine the ECSA value of GCE, rGO/GCE, and Ni-rGO/GCE, 5 mM [Fe(CN)6]3−/4− redox probe in 0.1 M KCl was used. Cyclic voltammograms recorded at different scan rates (30–100 mV s−1) showed a proportional increase in peak current with respect to the square root of scan rate (ν1/2), consistent with a diffusion-controlled electron transfer process (Fig. 3d and S1). The Randles–Ševčík relationship51,52 was applied:ip = 2.69 × 105n3/2AD1/2Cν1/2where the number of electrons transferred, n = 1 for the [Fe(CN)6]3−/4− couple, A is the electroactive surface area, the diffusion coefficient, D was taken as 7.6 × 10−6 cm2 s−1, and C = 5 × 10−6 mol cm−3. The observed slopes from the ipversus ν1/2 were 8.92866, 10.29226, and 12.60835 µA (mV s−1)−1/2 for bare GCE, rGO/GCE, and Ni-rGO/GCE, respectively. The corresponding ECSA values were 0.07615, 0.08778, and 0.1075 cm2, respectively. Thus, rGO/GCE exhibited a 15.3% higher ECSA than bare GCE, whereas Ni-rGO/GCE exhibited 41.2% and 22.5% higher ECSA than bare GCE and rGO/GCE, respectively. The increase in ECSA after rGO and Ni-rGO modification indicates a greater electrochemically accessible surface at the modified interfaces. The relatively larger ECSA of Ni-rGO/GCE compared with rGO/GCE is consistent with the higher MTZ response observed at the Ni-rGO/GCE. The geometric surface area of the 3 mm diameter GCE was 0.0707 cm2.

3.3. Electrochemical behaviour, kinetics and mechanism study

A clear understanding of electrochemical sensing kinetics depends on careful analysis of interfacial charge transfer processes.51 In particular, the electron stoichiometry involved in the rate-determining step plays a decisive role in controlling the reduction behaviour of metronidazole. Several cyclic voltammograms sensing 300 µM metronidazole in 0.1 M phosphate buffer solution were performed at different scan rates ranging from 20 mV s−1 to 100 mV s−1 (Fig. 4a).

Fig. 4. (a) CVs of 300 µM metronidazole in 0.1 M PBS at different scan rates (20–100 mVs−1) using Ni-rGO/GCE; (b) peak current versus the square root of scan rate from the CVs; (c) peak current versus scan rate; (d) log–log relationship between peak current and scan rate; (e) peak potential versus the logarithm of scan rate, (f) possible reaction of metronidazole at Ni-rGO/GCE in 0.1 M PBS.

Fig. 4

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The peak current exhibited a linear dependence on both square root of scan rate (ν1/2) and scan rate (ν) (Fig. 4b and c), described by:ip = −8.35376ν1/2 + 16.31282 (R2 = 0.99) and ip = −0.57268ν − 12.34011 (R2 = 0.99)respectively. These relationships suggest a combination of both diffusion and adsorption processes.51 To further study the kinetic behaviour, log(ip) versus log(ν) as plotted (Fig. 4d), which shows:log|ip| = 0.67771 log(ν) + 0.47347 (R2 = 0.99)

The slope value of 0.67771 lies between the theoretical limits for ideal diffusion control (0.5) and ideal adsorption control (1.0),53 indicating a diffusion-dominated mixed control mechanism with a measurable contribution from surface-associated process.

The dependence of the cathodic peak potential on the scan rate was further investigated to enhance understanding of the interfacial electron-transfer kinetics (Fig. 4e). Variation of scan rate led to a systematic shift in peak potential, with the relationship between peak potential (Ep) and logarithm of scan rate remaining linear:Ep = −0.06789 log(ν) − 0.42412 (R2 = 0.99)

The negative slope indicates that the peak potential shifts to more negative values with increasing scan rate, implying that a greater overpotential is required to drive the reduction at faster sweep rates.53 This behaviour reflects kinetic polarisation arising from finite electron-transfer rates at the electrode–electrolyte interface.

This response is characteristic of a quasi-irreversible system,53 in which increasing the scan rate moves the system further from equilibrium because of limited charge-transfer kinetics. The trend points to interfacial kinetic control governing the reduction of metronidazole under the present conditions.

For gaining deeper mechanistic insights into the rate-controlling processes at the interface of Ni-rGO/GCE, we performed Tafel analysis based on the cyclic voltammetric (CV) data acquired at 100 mV s−1 scan rate in the presence of 300 µM metronidazole. The charge transfer coefficient (α), which serves as a critical indicator of the reaction pathway's symmetry and the efficiency of potential utilisation, was derived from the slope (Δ) of the following equation:513.3.

In this formulation, n denotes the number of electrons participating in the rate-determining step (RDS), where α = charge transfer coefficient, universal gas constant R = 8.314 J mol−1 K−1, Faraday constant F = 96 485 C mol−1, and absolute temperature T = 298 K. Considering the stepwise reduction pathway of the nitro functional group, the initial single-electron transfer was treated as the kinetically limiting step; therefore, n = 1 was employed for the calculation of α. The Tafel plot exhibited a well-defined linear relationship, yielding a slope of 9.97696, corresponding to Δ = 0.10023 V per decade. Substitution into the above equation resulted in a charge-transfer coefficient of α = 0.59. This value reflects efficient interfacial electron transfer at the modified electrode surface,53 indicating that the applied potential is effectively utilised to drive the electrochemical reduction of metronidazole, yielding a sustainable faradaic response. The observed kinetic response aligns with the electrochemical impedance spectroscopy (EIS) data of Ni-rGO/GCE, which exhibits a charge-transfer resistance of 72.62 Ω – representing a 43.35% reduction compared to the bare GCE, and demonstrates a 41.2% increase in electrochemically active surface area (ECSA) relative to the unmodified surface.

The electrochemical response of 300 µM MTZ in 0.1 M PBS across various pH ranging from 5.0 to 9.0 was studied through cyclic voltammograms at a scan rate of 100 mV s−1. From Fig. 5b we can see the cathodic peak potential (Ep) shifts to more negative values when the pH is increased. It indicates the reduction of the nitro group (–NO2) is proton-dependent and that proton availability directly influences the redox energetics at the electrode interface. A plot of Ep against pH gave a regression equation of Ep = −0.04069(pH) – 0.2582, with R2 = 0.98. The slope magnitude of 40.69 mV pH−1 confirms a proton-coupled electron transfer (PCET) process.51 The deviation from the theoretical Nernstian response of 59 mV pH−1 suggests it does not follow a strict 1 : 1 stoichiometric coupling between proton transfer and electron transfer, potentially reflecting a modified PCET pathway influenced by interfacial reaction dynamics.

Fig. 5. (a) CVs of 300 µM MTZ at different pH levels (5.0–9.0) using Ni-rGO/GCE; (b) linear dependence of Ep with pH (left) and variation of ip with pH (right).

Fig. 5

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The plot illustrating peak current versus pH indicates that the maximum peak current value was observed at pH 7.4; consequently, this pH was selected for subsequent experiments. The alignment of this pH with the physiological conditions of the human body enhances the sensor's applicability, enabling it to operate effectively on real biological samples without necessitating pH adjustments. Such compatibility is crucial for the practical deployment of biosensors and supports their potential use in clinical and diagnostic applications.

3.4. Analytical performance and selectivity

The analytical performance of the proposed Ni-rGO electrochemical sensor for sensing metronidazole was evaluated by taking linear sweep voltammograms of different concentrations of metronidazole in the range of 2 µM to 200 µM (Fig. 6a). The sensor response was further examined at MTZ concentrations above 200 µM. However, the response deviated from the linear relationship observed in the 2–200 µM range. Therefore, we considered 2–200 µM to be the linear range for quantitative determination of MTZ. The LSVs were taken in 0.1 M PBS of pH 7.4 at a sweep rate of 50 mV s−1. It detected metronidazole at a low concentration of 2 µM and the peak current increased with increasing concentration (Fig. 6b). A linear relationship was observed between the peak current and concentration, and a calibration curve was obtained for MTZ concentrations of 2 µM to 200 µM with a linear regression equation ofip = −0.3613[MTZ] − 21.95874 (R2 = 0.99)

Fig. 6. (a) LSV plots of metronidazole at different concentrations (2–200 µM) using Ni-rGO/GCE in 0.1 M PBS; (b) peak current versus concentration from the LSVs; (c) operational repeatability test of Ni-rGO/GCE with 300 µM metronidazole in 0.1 M PBS; (d) selectivity test of metronidazole with chronoamperometry.

Fig. 6

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The limit of detection (LOD) and limit of quantification (LOQ) were determined using the standard IUPAC approach:30,543.4.3.4.here, σ represents the standard deviation of baseline current obtained from 10 blank solution LSVs in 0.1 M PBS of pH 7.4 in absence of MTZ analyte. The value of σ was found to be 0.01476 µA. The S here represents the slope from the peak current versus MTZ concentration calibration curve, which is 0.3613 µA µM−1. Using the above equations, the LOD and LOQ were found to be 122.6 nM and 408.5 nM. The Ni-rGO sensor showed an excellent sensitivity of 5111.9 µA mM−1 cm−2, confirming a high electrochemical response toward MTZ at the electrode surface.

The detection limit of the Ni-rGO sensor is not the lowest among reported MTZ sensors (Table 1), but it balances performance and sustainability. It uses nickel as the only metal, contrasting noble-metal or bimetallic systems, and the rGO support is from graphite recovered from discarded Li-ion batteries. Made via a one-pot, hydrazine-free hydrothermal process, it has a detection limit of 122.6 nM, a linear range of 2–200 µM, good reproducibility, stability, and was tested in tap water and urine. These features make it appealing for sustainable use and simple fabrication alongside good sensitivity.

Table 1. Performance comparison of reported electrochemical MTZ sensors and present Ni-rGO system.

Modified electrode Method Linear range (µM) LOD (nM) Reference
Au-NP@PDDA/GH LSV 0.4–656.4 97 31
Hi Q Sil C18 column HPLC 1908 55
CB NPs/PGA DPV 0.5–10 170 32
MWCNTs-COOH CPE LSV 5–5000 250 33
AgNPs@CPE CV 1–1000 206 56
Poly(chromotrope 2B)/GCE DPV 10–400 330 57
Cd-MOF@MWCNT (1 : 1) DPV 0.4–500 250 58
Ni/Fe-LDH/GCE CV 5–1610 58 000 34
Activated GCE CV 2–600 1100 29
µAl2O3@CPE DPV 0.5–1000 253 59
Carbon fiber/MDE LSV 1 – 22 500 60
Ni-rGO LSV 2–200 122.6 This work
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The operational repeatability of the modified Ni-rGO sensor was evaluated by monitoring the cathodic peak current across 10 consecutive analyses (Fig. 6c). Following a strong initial faradaic response, the electrode was subjected to continuous analytical cycles, exhibiting only a marginal decline in current. This yielded an exceptional signal retention of 95.4% by the tenth cycle. The absence of significant signal attenuation indicates that the porous Ni-rGO architecture effectively resists surface passivation and fouling from MTZ reduction intermediates. The active catalytic sites remain highly stable and accessible across repeated sweeps, confirming the platform's structural integrity and its reliability for successive sample screening without necessitating tedious surface regeneration protocols.

The selectivity profile of the fabricated electrochemical sensor toward metronidazole was assessed via amperometric (i–t) measurements, as depicted in Fig. 6d. Upon successive injections of MTZ, a prompt and continuous increase in the cathodic current was recorded. The anti-interference capacity was subsequently evaluated by introducing common inorganic ions and potential organic co-interferents into the analytical matrix.

The addition of representative inorganic salts (NaNO3, KCl, NH4Cl) induced negligible deviation in the steady-state baseline current. Similarly, the introduction of common electroactive species, specifically dopamine (DA), uric acid (UA), and ascorbic acid (AA), resulted in no measurable signal perturbation. Notably, the injection of imidazole (IMZ), which is a structural analog belonging to the same fundamental molecular family as metronidazole, elicited no faradaic response. All of these interferents were introduced at 10-fold excess relative to MTZ. This observation confirms the sensor's strict specificity toward the reduction of the nitro group unique to MTZ, effectively bypassing false-positive signals from homologous ring structures.

Subsequent injections of MTZ continued to generate distinct, step-like current responses. This verifies that the presence of a high concentration of mixed interferents does not competitively inhibit the electrocatalytic active sites, ensuring reliable MTZ quantification in complex matrices.

To further evaluate the selectivity of the sensor toward MTZ, additional interference experiments were performed using representative commonly used antibiotics, including cefixime, tetracycline, cefuroxime, and doxycycline. As shown in Fig. S2, sequential addition of these antibiotics at fivefold excess relative to MTZ produced no discernible perturbation of the current response. Subsequent additions of 100 µM MTZ continued to produce clear responses, indicating that the presence of these antibiotics did not substantially affect the electrochemical response toward MTZ.

3.5. Real-sample application, reproducibility and stability

Real sample analysis was conducted using urine and tap water (Fig. 7a). The urine sample was collected in a sterile container, then filtered and centrifuged at 6000 rpm for 10 minutes to eliminate suspended protein and particles. Then it was diluted 10-fold using 0.1 M PBS of pH 7.4 to reduce matrix effect. Informed consent was obtained from the donor prior to the collection of the sample. No personally identifiable information was collected. Known amounts of MTZ were introduced to it, and peak current versus concentration calibration curve was constructed using a standard addition approach. Recovery experiments were performed by spiking the diluted urine samples with known concentrations of MTZ and determining the recovered amounts from the calibration curve. The recovery percentage was calculated as the ratio of detected concentration to the added concentration. Tap water was filtered and used as it is for the recovery analysis, as the above process. Recovery rate was good in both urine sample and tap water in the range of (87–108%). It is well within the acceptable range for sensors (80–120%). The recovery experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). Therefore, the sensor can be used for analysis of real-life samples. The data is summarized in the following table (Table 2).

Fig. 7. (a) Peak current versus concentration calibration curves of metronidazole in urine and tap water at Ni-rGO/GCE; (b and c) reproducibility test of Ni-rGO for metronidazole sensing with five different electrodes; (d) stability test of Ni-rGO/GCE for metronidazole sensing.

Fig. 7

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Table 2. Determination and recovery of metronidazole in tap water and human urine matrices using the modified Ni-rGO electrode.

Sample Concentration of MTZ (µM) Recovery (%)
Added Found
Urine 0 ND —
50 50.997 ± 0.16 102
100 107.572 ± 0.24 108
150 140.105 ± 0.22 93
Tap water 0 ND —
75 71.236 ± 0.14 95
100 102.115 ± 0.16 102
125 109.263 ± 0.30 87
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The reproducibility of the sensor was evaluated by preparing five independent electrodes under identical experimental conditions (Fig. 7b and c). The electrocatalytic responses of 300 µM metronidazole in 0.1 M PBS were investigated by cyclic voltammetry at a scan rate of 100 mV s−1. For each electrode, three consecutive measurements (n = 3) were recorded to ensure signal stability. The responses of the five independently prepared electrodes were highly consistent with an average peak current (ip) of −65.71 µA. The relative standard deviation (% RSD) of the peak current was 3.12%, indicating a good fabrication reproducibility of the modified electrode.

We evaluated the storage stability of the modified electrode over a 7 day period. Between measurements, the electrode was kept under dry ambient conditions. Cyclic voltammetry runs were recorded for 300 µM metronidazole in 0.1 M PBS on days 1, 2, 3, 5, and 7 at a scan rate of 100 mV s−1. As illustrated in Fig. 7d, the cathodic peak current (ip) stayed highly stable throughout the week. It started from an initial value of −70.75 µA on day 1, with the sensor retaining 92.3% of its initial response by day 5 and 91.9% by day 7. This slight variation in current reflects the excellent electrochemical stability of the modified electrode over one week under normal storage conditions.

4. Conclusion

In summary, a hierarchical Ni-rGO nanocomposite was successfully synthesized using graphene oxide derived from e-waste graphite. The subsequent one-pot, hydrazine-free hydrothermal route established a sustainable and highly sensitive electrochemical platform for metronidazole quantification. Structural characterization confirmed a mixed-phase morphology, wherein larger crystalline domains function as physical intercalants to suppress rGO restacking, while a highly dispersed 1.5–3 nm nanophase provides the requisite active site density. This structural duality directly enhanced interfacial kinetics, yielding a 41.2% increase in electrochemically active surface area and a 43.35% reduction in charge-transfer resistance relative to the unmodified electrode. Consequently, the sensor delivered robust analytical performance for MTZ reduction, characterized by a broad linear range (2–200 µM), high sensitivity (5111.9 µA mM−1 cm−2), and a low detection limit of 122.6 nM. The practical reliability of the sensor is supported by a 3.12% relative standard deviation across independent electrodes and a robust anti-interference capacity against common inorganic salts, concentrated endogenous species (DA, AA, UA), commonly used antibiotics, and the homologous interferent imidazole. The platform maintained structural integrity under stress, retaining 95.4% of its initial signal across continuous analytical cycles and 91.9% following a seven-day storage period. Finally, quantitative recoveries of 87–108% in tap water and human urine confirm the reliability of the sensor in complex matrices. The proposed Ni-rGO architecture presents a reproducible, scalable, and highly practical methodology for trace-level MTZ screening in both environmental and clinical applications.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

RA-OLF-D6RA06899E-s001

Acknowledgments

The authors are grateful to the Bangladesh Council of Scientific and Industrial Research (BCSIR) for facilities and financial support through Special R&D ref. no. 39.02.0000.011.14.180.2024/1107, dated: 13/01/2025.

Data availability

Data will be available upon request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra06899e.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-OLF-D6RA06899E-s001
RA-OLF-D6RA06899E-s001.pdf (331KB, pdf)

Data Availability Statement

Data will be available upon request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra06899e.

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