Enrofloxacin (ENR) is a quinolinemonocarboxylic acid and a third-generation fluoroquinolone. This antibiotic is extensively used in livestock and aquaculture as an antibacterial agent, showing high activity against both Gram-negative and Gram-positive bacteria . ENR, like other fluoroquinolones, is used to treat susceptible bacteria responsible for infections of the skin and soft tissue. The overuse of ENR causes severe adverse effects, including skeletal, reproductive, immune, and digestive disorders; moreover, ENR cannot be completely absorbed by most animals, it easily enters the environment through the excrement of organisms, also in the form of metabolites . Nowadays, the concentration of ENR in products, especially animal products intended for human consumption is limited in the USA, the EU, and China ; the FDA also bans the use of ENR in both poultry and livestock . Therefore, the determination of ENR residues is an urgent requirement. Various analytical techniques and methods have been developed to detect ENR, including microbiological methods, such as enzyme-linked immunosorbent assay (ELISA) , chromatography-mass spectrometry (LC-MS), chromatography (HPLC, UPLC), , flow immunochromatographic assays , and fluorescence measurements . However, these methods have high requirements regarding instruments, chemicals, time consumption, and preparation of the samples. Electrochemical methods to detect ENR have attracted great attention because of benefits including high accuracy, simplicity, and environmental friendliness . Electrochemical methods exhibited high efficacy in the detection of ENR with quick response, low cost, and simple handling; hence, these methods are promising to quantify ENR .

Noble metals and metal-organic frameworks (MOFs) are the most typical electrode materials to detect antibiotics . MOFs emerged as the outstanding material for electrochemical sensor applications because of their high loading quantity and surface area, defined structures, and chemical stability . Since the first report by Yaghi and his group in 1994 , MOFs have attracted great attention. The first publication related to a fluorescent sensor was presented by You et al. in 2002 . MOFs are crystalline hybrid materials with network structures formed by the self-assembly of metal ions or metal clusters and organic ligands, which give them ultrahigh porosity and enormous internal surface area. However, using MOFs for electrochemical sensors has some limitations associated with the low conductivity of MOFs. Therefore, the coupling with conducting materials, such as carbon-based materials, metal nanoparticles, and polymers, has been performed to enhance the electron charge transfer of MOFs ; single MOFs combined with carbon-based materials have been extensively studied . A sensitive and simplified electrochemical sensor using a Cu3(BTC)2-modified carbon paste electrode for detecting 2,4-dichlorophenol was reported by Dong and his group . Owing to the large specific surface area, high absorption capacity, and an acceptable efficacy of the electron transfer, Cu3(BTC)2 exhibited a good sensitivity to 2,4-dichlorophenol in the range from 0.04 to 1.00 μM with a limit of detection (LOD) of 9 nM in differential pulse voltammetry measurements. Moreover, the combination of metal oxides and MOFs showed better electrochemical detection ability than pristine MOFs. For example, Wang et al. developed a MOF/TiO2 composite to quantify chlorogenic acid in a range from 0.01 to 1.00 μM with a low LOD of 7 nM . Utilizing carbon-based materials can provide not only enhanced electron transfer but also catalytic functions for the MOFs. A sensor for simultaneously detecting hydroquinone and catechol in water using a Cu-MOF–graphene composite was developed by Li and colleagues. Measurement ranges beginning at 1.0 × 10−6 and 1.0 × 10−3 M were achieved, with LODs of 5.9 × 10−7 M and 3.3 × 10−7 M for hydroquinone and catechol, respectively . In addition, the coupling of MOFs with conducting polymers was investigated to modulate their electrical properties. Conducting poly(3,4-ethylenedioxythiophene) nanotubes were coated with porphyrin-based MOFs to detect dopamine in the range of 2 × 10−6 to 270 × 10−6 M with a LOD of 0.04 × 10−6 M . The combination of two types of MOFs has been rarely investigated for the detection of antibiotics.

To our current knowledge, no reports have been made on the use of electrodes modified with a mixture of MOFs for the detection of enrofloxacin. In this study, a sensor was developed using a mixture of CuBTC and FeBTC to modify carbon paste electrodes for the determination of enrofloxacin. The characterization and properties of the fabricated electrode, including molecular structure, morphology, and electrochemical characteristics, were thoroughly investigated using various analytical techniques. The synergistic effects of CuBTC and FeBTC are considered promising for enhancing the performance of enrofloxacin sensors in aqueous solutions.

Figure 1: (a) XRD pattern and (b) N2 adsorption/desorption isotherms of the (Cu)(Fe)BTC sample.

Figure 2: Full-scan (a) and high-resolution C 1s (b), O 1s (c), Fe 2p (d), and Cu 2p (e) XPS spectra of the (Cu)(Fe)BTC sample.

Figure 3: TEM image of (Cu)(Fe)BTC sample.

Figure 4: SEM images of (Cu)(Fe)BTC@CPE (a) and EDX maps of (Cu)(Fe)BTC@CPE (b).

Figure 5: Nyquist plots of the modified electrodes in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3−/4−.

Figure 6: (a) Cyclic voltammograms of (Cu)(Fe)BTC@CPE and CPE electrodes in a 200 µM ENR solution in PBS at scan rate of 0.1 V/s The green line shows a voltammogram measured in ENR-free solution. (b) Square wave voltammograms of the studied electrodes in 1 μM ENR solution at an accumulation time of 90 s. The green line shows a voltammogram measured in ENR-free solution.

Figure 7: The impact of electrode composition the peak current of 1 µM enrofloxacin.

Figure 8: Square wave voltammograms of 1 µM ENR in different electrolytes.

Figure 9: (a) SW-AdSV measurements of (Cu)(Fe)BTC@CPE in 1 µM ENR/PBS solution at different pH values from 6 to 9. (b) Plot of peak current vs pH. (c) Plot of peak potential vs pH.

Figure 10: The relationship between ENR peak currents and accumulation time at different concentrations of ENR of 0.02, 0.2, and 2 µM. Inset: rescaled plot for 0.02 µM ENR.

For a concentration of 0.2 µM, the peak current increased gradually and reached a steady state at tacc exceeding 300 s. This suggests that, at this intermediate concentration, the electrode surface reaches saturation after 300 s of accumulation, beyond which the peak current stabilizes. At the highest concentration tested (2 µM), the peak current experienced a significant increase with increasing tacc up to 120 s, followed by a gradual rise, until it reached equilibrium at tacc from 300 to 600 s. This behaviour indicates a rapid initial adsorption at high ENR concentration, with the surface reaching almost saturation relatively quickly, and then a slower adsorption to full saturation. These observations highlight the importance of optimizing tacc for different ENR concentrations to achieve the best analytical signal. Longer accumulation times are beneficial at lower concentrations, while higher concentrations require shorter times to reach equilibrium.

Figure 11: SW-AdSV measurements of successive measurements obtained in 0.20 µM ENR solution using electrodes (a) without renewal of the electrode surface and (b) with renewal of the electrode surface.

Figure 12: SW-AdSV measurements of ENR and calibration curves for (a) 0.005–0.100 µM (tacc of 600 s), (b) 0.1–1.0 µM (tacc of 90 s), and (c) 1–13 µM (tacc of 0 s).

From the calibration curve at the lowest concentration range from 0.005 to 0.100 µM, a detection limit for ENR of 3 nM was calculated through the formula LOD = 3σ/b, where σ is the standard deviation of blank signals, and b is the slope of the regression line. The LOD is quite comparable to those in previous studies . Additionally, the sensitivity of the sensor was calculated to be 56.14 µA/µM.

Figure 13: Influence of (a) inorganic ions and (b) organic compounds on the ENR signal.

Table 1: Analysis of ENR in spiked samples conducted with the (Cu)(Fe)BTC@CPE sensor.

Enrofloxacin (ENR, C19H22FN3O3, 98%) was purchased from Thermo Fisher Scientific. Graphite powder and paraffin oil were supplied by Thermo Scientific Acros. Trimesic acid (H3BTC, 98%), CuCl2·2H2O (≥99%), FeCl3·6H2O (98%), N,N-dimethylformamide (DMF, 98%) and C2H5OH (96%), H3PO4 (85%), KOH (≥85.0%), KH2PO4 (≥99.5%), K2HPO4 (≥98.0%), KCl (>99.5%), and HCl (37%) were bought from Fisher and Sigma-Aldrich. All chemicals and reagents were of analytical grade and used without further purification. K3[Fe(CN)6] (≥99.5%) and K4[Fe(CN)6] (≥99.5%) were purchased from China. Phosphate-buffered saline (PBS) was prepared by mixing 0.2 M KH2PO4 and 0.2 M K2HPO4 in double-distilled water. The pH of the solution used for the optimization of electrolyte solution was changed from 6.0 to 9.0 by controlling the amount of these phosphate salts. Enrofloxacin stock solution was prepared by dissolving ENR powder in double-distilled water. The ENR solutions with desired concentrations were prepared daily by diluting the ENR stock solution in with PBS solution.

The electrochemical properties of the developed electrode and enrofloxacin were evaluated through cyclic voltammetry by using a custom-made potentiogalvanostat (supplied by Laboratory of Informatics in Chemistry, Institute of Chemistry, Vietnam Academy of Science and Technology). Electrochemical impedance spectroscopy (EIS) was performed by using an Autolab PGSTAT 302 in a frequency range of 100 kHz to 0.01 Hz. A three-electrode electrochemical setup was used including a platinum wire counter electrode, an Ag/AgCl/saturated KCl reference electrode, and a carbon paste electrode (CPE) or MOF-modified electrodes as working electrodes.

Physical and chemical properties of MOFs were characterized by using field-emission scanning electron microscopy (FESEM, Hitachi S-4800, Japan), energy-dispersive X-ray spectroscopy (EDX, Horiba 7593-H, England), and X-ray photoelectron spectroscopy (XPS, Thermo VG Multilab 2000).

CuBTC and FeBTC were synthesized using the solvothermal method as described in our previous reports . Specifically, 12 mmol of trimesic acid was fully dissolved in 120 mL of N,N-dimethylformamide (DMF) (solution 1), and 12 mmol of the chloride salt of the corresponding metal was dissolved in 40 mL of water (solution 2). Solution 1 was then slowly added to solution 2 under vigorous stirring for 30 min before being transferred to a Teflon bottle. The mixture was heated at 150 °C for 12 h and stirred continuously to facilitate the MOF formation process. Finally, the synthesized powder was separated from the solution using a centrifuge at 6000 rpm, washed several times with DMF and ethanol, and dried to obtain CuBTC and FeBTC MOFs.

Table 2: Components of the studied electrodes.

Enrofloxacin was electrochemically detected by using square wave adsorptive stripping voltammetry (SW-AdSV) in PBS. Briefly, the modified electrode was immersed in the analytical solution for different accumulation times (0, 90, and 600 s) under stirring for enrofloxacin adsorption on electrode surface. Then, the SW-AdSV response of enrofloxacin was recorded in the potential window of 0.6–1.1 V.

Spiked samples of tap water and lake water were used to evaluate the performance of the modified electrode. After spiking known concentrations of enrofloxacin into the water samples, the added concentration was determined using the standard addition method. All experimental work was performed at room temperature (25 ± 1 °C).