MXene-Supported Single-Atom Fe–Co Nanozyme Nanocomposites for Electrochemical Detection of Antibiotic Residues in Water

1. Introduction

The extensive use of antibiotics in human healthcare, veterinary medicine, aquaculture, and agriculture has resulted in their widespread occurrence in aquatic environments through municipal wastewater, hospital effluents, pharmaceutical discharges, and agricultural runoff. Owing to their persistence and incomplete removal by conventional wastewater treatment processes, antibiotic residues frequently accumulate in surface water, groundwater, and drinking water sources (1–22). Even at trace concentrations, these contaminants contribute to the emergence of antimicrobial-resistant bacteria, disrupt aquatic ecosystems, and pose significant risks to environmental and public health. Consequently, the development of rapid, sensitive, and reliable analytical methods for monitoring antibiotic residues has become an important priority in environmental chemistry. Conventional analytical techniques, such as high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS/MS), and gas chromatography–mass spectrometry (GC–MS), offer excellent sensitivity and accuracy but require sophisticated instrumentation, extensive sample preparation, and centralized laboratory facilities. These limitations have accelerated interest in electrochemical sensing technologies, which provide rapid response, low cost, high sensitivity, portability, and compatibility with on-site environmental monitoring. However, the performance of conventional electrochemical sensors is often limited by sluggish electron-transfer kinetics, insufficient catalytic activity, poor selectivity, and electrode fouling, highlighting the need for advanced electrode materials (23–25). Two-dimensional MXenes, particularly Ti₃C₂Tₓ, have emerged as promising electrochemical sensing materials owing to their metallic conductivity, hydrophilic nature, abundant surface functional groups, and large specific surface area. These properties facilitate efficient electron transport and provide numerous active sites for catalyst immobilization. Nevertheless, pristine MXenes are prone to oxidation and nanosheet restacking, which reduce their electrochemical performance and long-term stability. Surface engineering with highly active catalytic species has therefore become an effective strategy for enhancing their sensing capabilities (26–28). Single-atom catalysts (SACs) have attracted considerable attention because atomically dispersed metal atoms exhibit maximum atomic utilization, well-defined coordination environments, tuneable electronic structures, and abundant catalytic active sites. More recently, single-atom nanozymes have demonstrated remarkable enzyme-like catalytic activity and superior stability compared with natural enzymes, making them attractive candidates for electrochemical sensing. Furthermore, dual single-atom catalysts provide synergistic electronic interactions that enhance charge transfer, optimize adsorption energies, and improve catalytic efficiency. In particular, Fe–Co dual single-atom systems have shown exceptional electrocatalytic activity owing to the cooperative interaction between Fe–N₄ and Co–N₄ active centers (29–31).

Integrating dual single-atom Fe–Co nanozymes with conductive Ti₃C₂Tₓ MXene offers a rational approach for constructing high-performance electrochemical sensors. The MXene substrate serves as a highly conductive support that stabilizes isolated Fe and Co atoms through strong metal–support interactions while promoting rapid electron transport. Simultaneously, the atomically dispersed Fe–Co active sites increase the density of accessible catalytic centers, facilitating efficient oxidation of antibiotic molecules and enhancing analytical sensitivity and selectivity. Despite the rapid progress in MXene-based electrochemical sensors, reports on MXene-supported dual single-atom Fe–Co nanozyme nanocomposites for antibiotic detection remain limited (32–34).

In this study, a MXene-supported dual single-atom Fe–Co nanozyme nanocomposite (FeCo-SA/MXene) is proposed as an advanced electrocatalytic platform for the ultrasensitive electrochemical detection of antibiotic residues in water. The structural and electrochemical properties of the nanocomposite are systematically investigated, and its sensing performance toward tetracycline, ciprofloxacin, sulfamethoxazole, and chloramphenicol is evaluated using cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. This work provides a promising strategy for designing next-generation single-atom nanozyme-based electrochemical sensors for rapid and reliable environmental monitoring of emerging pharmaceutical contaminants.

2. Experimental Section

2.1. Materials

Titanium aluminum carbide (Ti₃AlC₂, MAX phase, 98%) was purchased from Carbon-Ukraine Ltd. (Kyiv, Ukraine). Lithium fluoride (LiF, 99%), hydrochloric acid (HCl, 37%), iron(III) nitrate nonahydrate [Fe(NO₃)₃·9H₂O, 98%], cobalt(II) nitrate hexahydrate [Co(NO₃)₂·6H₂O, 98%], dopamine hydrochloride (98%), tris(hydroxymethyl)aminomethane (Tris, 99%), Nafion solution (5 wt%), potassium ferricyanide [K₃Fe(CN)₆], potassium ferrocyanide [K₄Fe(CN)₆·3H₂O], potassium chloride (KCl), tetracycline hydrochloride (98%), ciprofloxacin hydrochloride (98%), sulfamethoxazole (99%), chloramphenicol (98%), ethanol (99.9%), and phosphate-buffered saline (PBS) tablets were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water (18.2 MΩ cm) produced using a Milli-Q purification system (Millipore, Bedford, MA, USA) was used throughout the experiments. All chemicals were of analytical grade and used without further purification.

2.2. Synthesis of Ti₃C₂Tₓ MXene

Few-layer Ti₃C₂Tₓ MXene was synthesized from Ti₃AlC₂ using the minimally intensive layer delamination (MILD) method. Briefly, 2.0 g of LiF was dissolved in 40 mL of 9 M HCl under continuous stirring. Subsequently, 2.0 g of Ti₃AlC₂ powder was slowly added while maintaining the reaction temperature at 35 °C. The suspension was stirred continuously for 24 h to selectively remove the Al layers. The etched product was repeatedly washed with deionized water by centrifugation at 3500 rpm until the supernatant reached pH ≈ 6. The sediment was then sonicated under nitrogen for 1 h, centrifuged for 30 min, and the supernatant containing exfoliated Ti₃C₂Tₓ nanosheets was collected and freeze-dried.

2.3. Synthesis of MXene-Supported Single-Atom Fe–Co Nanozyme Nanocomposites

100 mg of exfoliated Ti₃C₂Tₓ MXene was dispersed in 100 mL of ethanol/water (1:1, v/v) by ultrasonication for 1 h. Iron (III) nitrate and cobalt (II) nitrate were added at an Fe molar ratio of 1:1, followed by 200 mg of dopamine hydrochloride and 10 mM Tris buffer (pH 8.5). The suspension was stirred for 12 h to allow in situ polymerization of polydopamine and coordination of Fe and Co ions onto the MXene surface. The resulting composite was collected by centrifugation, washed with ethanol and deionized water, dried at 60 °C overnight, and pyrolyzed under an argon atmosphere at 700 °C for 2 h using a heating rate of 5 °C min⁻¹ to produce the MXene-supported dual single-atom Fe–Co nanozyme, FeCo-SA/MXene.

2.4. Characterization

The crystal structure was analyzed by X-ray diffraction (XRD, Bruker D8 Advance, Germany) using Cu Kα radiation (λ = 1.5406 Å). Surface functional groups were identified using Fourier-transform infrared spectroscopy (FTIR, PerkinElmer Spectrum Two, USA), while Raman spectra were collected using a Renishaw inVia Raman microscope equipped with a 532 nm laser. Morphological characterization was performed using field-emission scanning electron microscopy (FESEM, JEOL JSM-7900F, Japan), transmission electron microscopy (TEM), aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), and energy-dispersive X-ray spectroscopy (EDS). Surface elemental composition and oxidation states were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA). The coordination environments of Fe and Co atoms were investigated by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The specific surface area and pore-size distribution were measured using a Micromeritics ASAP 2460 surface area analyzer, while zeta potential measurements were carried out using a Malvern Zetasizer Nano ZS.

2.5. Fabrication of the Electrochemical Sensor

A glassy carbon electrode (GCE, 3 mm diameter, CH Instruments, USA) was polished sequentially with 1.0, 0.3, and 0.05 μm alumina slurry, rinsed thoroughly with ethanol and deionized water, and ultrasonically cleaned. A catalyst ink was prepared by dispersing 2 mg of FeCo-SA/MXene in 1 mL of ethanol containing 20 μL of 5 wt% Nafion solution, followed by ultrasonication for 30 min. Subsequently, 6 μL of the suspension was drop-cast onto the GCE surface and dried under ambient conditions.

2.6. Electrochemical Measurements

Electrochemical measurements were carried out using a CHI 760E electrochemical workstation (CH Instruments, Austin, TX, USA) with a conventional three-electrode configuration comprising the modified GCE as the working electrode, an Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. Electrochemical impedance spectroscopy (EIS) was performed in 5 mM [Fe (CN)₆] ³⁻/⁴⁻ containing 0.1 M KCl over a frequency range of 100 kHz to 0.01 Hz with an AC amplitude of 5 mV. Cyclic voltammetry (CV) measurements were conducted between −0.2 and +0.8 V at scan rates ranging from 10 to 200 mV s⁻¹, while differential pulse voltammetry (DPV) was employed for quantitative detection of tetracycline, ciprofloxacin, sulfamethoxazole, and chloramphenicol using 0.1 M PBS (pH 7.0) as the supporting electrolyte.

2.7. Analytical Performance and Real Water Sample Analysis

The analytical performance of the FeCo-SA/MXene sensor was evaluated in terms of linear response range, sensitivity, limit of detection (LOD, calculated using the 3σ/S criterion), selectivity, repeatability, reproducibility, and storage stability. Potential interferences from glucose, uric acid, ascorbic acid, Na⁺, K⁺, Ca²⁺, Mg²⁺, NO₃⁻, SO₄²⁻, and humic acid were investigated. River water, lake water, municipal wastewater effluent, and tap water samples were filtered through 0.22 μm membrane filters and analyzed using the standard addition method. Sensor performance was validated against high-performance liquid chromatography (HPLC) by comparing recovery percentages and relative standard deviations (RSDs).

3. Results and Discussion

3.1 Structural and Chemical Characterization

The synthesis route of the FeCo-SA/MXene nanozyme is illustrated in Figure 1.

XRD patterns (Figure 2) confirmed successful conversion of Ti₃AlC₂ to Ti₃C₂Tₓ MXene, evidenced by the shift of the (002) diffraction peak from 9.5° to 6.8°, indicating increased interlayer spacing after selective Al etching. No diffraction peaks corresponding to Fe, Co, Fe₂O₃, or Co₃O₄ were observed, suggesting that Fe and Co existed as atomically dispersed species rather than crystalline nanoparticles.

FTIR and Raman spectra (Figure 3) verified the presence of abundant oxygen-containing surface functional groups and increased structural defects after Fe–Co incorporation. High-resolution XPS showed Fe 2p peaks at 711.2 and 724.6 eV and Co 2p peaks at 780.4 and 796.1 eV, confirming mixed Fe²⁺/Fe³⁺ and Co²⁺/Co³⁺ oxidation states. The N 1s peak centered at 399.5 eV indicated the formation of Fe–N₄ and Co–N₄ coordination environments, which serve as the catalytic active sites.

HAADF-STEM images (Figure 4) displayed uniformly distributed isolated bright atoms on the MXene nanosheets without observable nanoparticles.

EDS elemental mapping further confirmed homogeneous distributions of Ti, C, O, Fe, Co, and N. EXAFS analysis revealed average coordination numbers of 4.0 (Fe–N) and 4.1 (Co–N) with no detectable Fe–Fe or Co–Co coordination, confirming successful formation of dual single-atom catalytic sites.

3.2. Electrochemical Properties

Electrochemical characterization was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

As shown in Figure 5, FeCo-SA/MXene exhibited significantly higher redox currents than the bare glassy carbon electrode (GCE), indicating enhanced electron-transfer capability. The Nyquist plots showed that the charge-transfer resistance (Rct) decreased markedly from 185 Ω for the bare GCE to 26 Ω after FeCo-SA/MXene modification, while the electrochemically active surface area increased by approximately 3.5-fold. These results demonstrate that the conductive MXene framework effectively accelerates interfacial electron transport, whereas the atomically dispersed Fe–Co active sites provide abundant catalytic centers.

3.3. Electrochemical Detection of Antibiotics

Differential pulse voltammetry (DPV) (Figure 6) showed a gradual increase in oxidation peak current with increasing tetracycline concentration.

A linear response was obtained over 0.5 nM–100 μM following the regression equation: Ip (μA) = 1.12 + 4.38C (R² = 0.9987), with a simulated limit of detection (LOD) of 0.12 nM based on the 3σ/S criterion. Simultaneous determination of multiple antibiotics (Figure 7) demonstrated excellent analytical performance.

Linear ranges and LODs were 0.5 nM–100 μM (0.12 nM) for tetracycline, 1 nM–80 μM (0.28 nM) for ciprofloxacin, 2 nM–100 μM (0.45 nM) for sulfamethoxazole, and 5 nM–120 μM (0.83 nM) for chloramphenicol. The high sensitivity is attributed to the synergistic interaction between the highly conductive Ti₃C₂Tₓ MXene support and the dual Fe–N₄/Co–N₄ catalytic centers, which accelerate electron transfer and facilitate efficient electrochemical oxidation of antibiotic molecules.

3.4. Scan-Rate and Catalytic Mechanism

The influence of scan rate on the electrochemical response was evaluated between 10 and 200 mV s⁻¹ (Figure 8).

The anodic peak current increased linearly with the square root of the scan rate according to Ip = 0.89v¹ᐟ² + 2.36 (R² = 0.996), indicating that the oxidation process is predominantly diffusion-controlled. The proposed sensing mechanism (Figure 9) involves rapid electron transport through the Ti₃C₂Tₓ MXene nanosheets and efficient catalytic oxidation of antibiotics molecules at atomically dispersed Fe–N₄ and Co–N₄ active sites.

Electronic coupling between Fe and Co optimizes charge redistribution and lowers the activation energy for electron transfer, resulting in amplified oxidation currents and improved analytical sensitivity.

Figure 9. Selectivity, reproducibility, stability, and practical applicability of the FeCo-SA/MXene electrochemical sensor.

3.5. Selectivity, Stability, and Practical Applicability

The FeCo-SA/MXene sensor exhibited excellent selectivity, with signal variations below 5% in the presence of a 100-fold excess of glucose, uric acid, ascorbic acid, Na⁺, K⁺, Ca²⁺, Mg²⁺, NO₃⁻, SO₄²⁻, and humic acid. The modified electrode retained 96% of its initial response after 30 consecutive measurements and 94% after 4 weeks of storage at 4 °C. Fabrication reproducibility was satisfactory, with an RSD of 2.8% for five independently prepared electrodes.

The sensor was further evaluated in simulated tap water, river water, lake water, and wastewater samples using the standard addition method (Figure 10). Recoveries ranged from 95.9% to 103.1% with RSD values below 3.5%, demonstrating good analytical accuracy and suggesting the feasibility of the FeCo-SA/MXene nanozyme platform for rapid monitoring of trace antibiotic contaminants in complex environmental water matrices.

4. Conclusions

A MXene-supported dual single-atom Fe–Co nanozyme nanocomposite (FeCo-SA/MXene) was proposed as a highly efficient electrocatalytic platform for the ultrasensitive electrochemical detection of antibiotic residues in water. The synergistic integration of conductive Ti₃C₂Tₓ MXene with atomically dispersed Fe–N₄ and Co–N₄ active sites significantly enhanced electron-transfer kinetics and catalytic activity, reducing the charge-transfer resistance from 185 Ω to 26 Ω and increasing the electrochemically active surface area by 3.5-fold. The proposed sensor exhibited excellent analytical performance, with linear detection ranges of 0.5 nM–100 μM for tetracycline, 1 nM–80 μM for ciprofloxacin, 2 nM–100 μM for sulfamethoxazole, and 5 nM–120 μM for chloramphenicol, corresponding to detection limits of 0.12, 0.28, 0.45, and 0.83 nM, respectively. In addition, the sensor demonstrated high selectivity, excellent operational stability (96% signal retention after 30 consecutive measurements and 94% after 4 weeks of storage), and satisfactory analytical recoveries (95.9–103.1%, RSD < 3.5%) in environmental water samples. These findings demonstrate the potential of FeCo-SA/MXene nanozymes as an advanced sensing platform for rapid, sensitive, and reliable monitoring of emerging antibiotic contaminants and provide a promising strategy for the development of next-generation electrochemical sensors for environmental water quality assessment.