Development of an Electrochemical Sensor Conjugated with Molecularly Imprinted Polymers for the Detection of Enrofloxacin

Abstract

An electrochemical sensor was fabricated for the rapid and simple detection of enrofloxacin (EF). Modification of screen-printed gold electrodes (SPE) with molecularly imprinted polymers (MIPs) allowed the detection of enrofloxacin by square wave voltammetry (SWV), measuring the oxidation peak at +0.9 V. The detection principle of molecularly imprinted polymers (MIPs) is based on the formation of binding sites with affinities and specificities comparable with those of natural antibodies. The detection of enrofloxacin showed a linear range of 0.01–0.1 mM with a detection limit LOD of 0.02 mM. The development of a non-imprinted polymer (NIP) control sensor allowed for better and more efficient detection. In addition, the sensor is portable, having the advantage of analyzing and detecting molecules of interest without the need to take the sample to a laboratory.

1. Introduction

The development of new devices and analytical techniques with high selectivity and precision are of great importance in different areas—for example, environmental, food, pharmaceutical, among others—to determine organic compounds [1]. Electrochemical techniques have gained special interest due to their advantages such as low cost, fast response, and portability compared with conventional methods [2,3]. Electrochemical sensors have analytical applications as chemosensors converting physical, chemical, and/or biological signals into a quantifiable electrochemical signal. Voltammetry, amperometry conductivity, and capacitance or impedance properties can be used to determine an analyte [3]. Improvements to sensitivity and selectivity in substance detection processes and modifications of electrodes with molecularly imprinted polymers (MIP’s) are currently being investigated [4,5]. The use of MIPs has become a very important tool for the detection of chemical substances and biological compounds for environmental and food monitoring. In addition, MIPs have been used as new molecular recognition materials for applications in the detection and removal of chemical and biological contaminants [6,7].

Molecular imprinting is a tool relying on the principles of molecular recognition of biological processes to generate artificial macromolecular receptors [8,9] with a target molecule acting as a template. Functional monomers interact around it, and subsequently, the monomers cross-link them to copolymerize and form a shell-like structure [9]. After polymerization and removal of the template, the binding sites are exposed in such a way that they are complementary to the template in size, shape, and position for their functional groups held in place in the newly formed cross-linked structure [10].

Electrode modification with MIPs represents an advantageous alternative, including recognition properties, low cost, and the possibility of a comfortable and simple design. For these reasons, electrochemical sensors have drawn much attention for the detection of organic compounds, such as emerging contaminants [11,12].

Enrofloxacin, a member of the fluoroquinolone family, is an antimicrobial approved by the Food and Drug Administration (FDA) for exclusive use in veterinary applications with proven efficacy in the treatment of bacterial diseases derived from aquaculture farms [13]. The advantage of enrofloxacin relies on its wide spectrum of antibacterial activity, for example, strong bactericidal power, fast action, and wide distribution in the body [14]. One of the main problems generated by the mishandling of enrofloxacin in aquaculture uses is their association with environmental issues and health problems. Specifically, the accumulation of enrofloxacin residues in shrimp tissues derived from improper practices have caused potential problems to human health [15,16]. Therefore, it is important to evaluate the presence of antibiotic residues in food. The agrifood sector has been carefully monitored to verify compliance with the maximum levels of EF permitted by the FDA (0.1 mg/kg) for toxic residues and contaminants in food products [17].

In this work, we describe the development of an electrochemical sensor based on the use of screen-printed gold electrodes (SPE) modified with molecularly imprinted polymers for the detection of enrofloxacin by electrochemical techniques (Scheme 1).

2. Materials and Methods

2.1. Materials and Reagents

Acetonitrile (ACN), 6-mercaptohexanol, potassium hexacyanoferrate (III) K₃ [Fe(CN)₆], sulfuric acid, 3-trimethosixylpropyl methacrylate (3-TPM), enrofloxacin (EF), methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), ferrocenylmethyl methacrylate, and ammonium persulfate (APS) was obtained from Sigma Aldrich, U.S.A. All the reagents used were of analytical grade. Additionally, screen-printed gold electrodes and a portable potentiostat, Sensit Smart (PalmSens, Houten, The Netherlands), using the PStouch software were used for all electrochemical studies.

2.2. Electrode Preparation

Screen-printed gold electrodes were subjected to washing and activation processes using a Sensit Smart potentiostat. The process consisted of 80 cycles in a cyclic voltammetry (CV) with a potential window from −0.7 to 1.2 V at a scanning rate of 50 mV, placing a drop of a 0.1 M H₂SO₄ solution and subsequent washings with H₂O every 20 cycles.

2.3. Chemical Modification of the Screen-Printed Electrode

After washing and activating the gold electrode, it was chemically modified by adding 10 µL of the 6-mercaptohexanol monomer to the working electrode, dried at room temperature for 24 h, and washed with water to remove monomer excess. A second modification was performed by adding 15 μL of 3-TPM to the electrode surface and allowing it to dry at room temperature for 24 h. Finally, a wash with water was carried out to eliminate the excess or residues of unreacted monomer, and it was stored for the following modifications.

2.4. Synthesis of Molecularly Imprinted Polymers

The synthesis of MIPs was carried out on the surface of the electrode previously modified with the 3-TPM monomer. In a round bottom flask, 100 mL of acetonitrile containing 52.1 mg of enrofloxacin as template, 100 μL of methacrylic acid as functional monomer, and 1.09 mL of ethylene glycol dimethacrylate as cross-linking monomer were added. The solution was subjected to an ultrasonic bath for 30 min. A total 20 mg of ferrocenylmethyl methacrylate was added and the solution was kept under constant stirring in a nitrogen environment to purge the oxygen present in the solution. The gold electrode was suspended in the solution and 11.5 mg of ammonium persulfate were added to initiate polymerization on the electrode under a nitrogen environment for 24 h at room temperature.

Once MIPs were polymerized on the surface of the electrode, they were washed with methanol and acetic acid at a 9:1 ratio and kept under vigorous magnetic stirring overnight to remove the template from the MIPs. Finally, additional washes were carried out with 80% methanol. Additionally, the synthesis of an NIP control sample was performed under the same conditions without the enrofloxacin template.

2.5. Sample Preparation

A stock sample of 0.01 M enrofloxacin was prepared by weighing 36.70 mg dissolved in 10 mL of H₂SO₄ 0.1 M. To evaluate the optimal working pH, samples of 3 mM enrofloxacin at pH 3 to pH 6 were analyzed. A total 15 μL of solution was placed on the bare gold electrode and analyzed using the square wave voltammetry (SWV) technique.

2.6. Morphological Characterization

A morphological characterization of the screen-printed electrodes was carried out to observe the modification on the surface of the electrodes with MIPs. This characterization was carried out by atomic force spectroscopy (AFM) using the AFM model Alpha300RA (WiTec, Ulm, Germany).

2.7. Electrochemical Characterization

As part of the characterization of the modified gold electrode, cyclic voltammetry and impedance spectroscopy (EIS) techniques were used to study the degree of modification of the electrode surface. Additionally, atomic force microscopy (AFM) was used to analyze the modification in the electrode. A CV study was performed using a 2 mM K₃[Fe(CN)₆] in 0.1 M H₂SO₄ solution, where a 15 μL drop from the solution was placed on the working electrode and a CV scan was then performed with a potential window from −0.7 to 0.7 V at a scan rate of 50 mV. For the EIS study, a drop of K₃[Fe(CN)₆] solution was placed and analyzed using a frequency of 20 kHz. Additionally, a control sample of 0.5 mM enrofloxacin was taken and a 15 μL drop of the solution was placed on the electrode modified with MIPs and another drop on the control electrode with NIPs. The analysis was carried out using the SWV technique with a potential window from 0–1.1 V at a scan rate of 0.08 v/s to observe a current signal corresponding to the drug concentration.

2.8. Determination of the Enrofloxacin

For the determination of enrofloxacin using gold electrodes modified with MIPs, a study using the square wave voltammetry (SWV) technique was carried out. Enrofloxacin samples were prepared at different concentrations from 0.01 to 0.1 mM using pH 3 acetate buffer. A 15 μL drop of each solution was taken and placed on the MIPs-electrode; the analysis was carried out using the SWV technique with a potential window from 0–1.1 V at a scan rate of 0.08 v/s to observe a current signal corresponding to the drug concentration.

3. Results and Discussion

3.1. Morphological Characterization

Electrode surface modification was observed by atomic force microscopy, as indicated in Figure 1. The bare electrode in Figure 1a represents a reference electrode without chemical modifications. The material surface is observed as smooth fields with small protuberances typical of electrodes. In Figure 1b,c, the images clearly show that the polymers are coated on the bare electrode. Figure 1b represents our control NIP sample not molecularly imprinted due to the absence of template and minimal changes that occurred. However, it is possible to observe a disorganized morphology. Figure 1c shows the Au electrode modified with the MIP; it shows great adherence on the surface of the electrode, forming clusters with ordered arrangement of hemispherical structures with approximate size of 1 micrometer corresponding to the molecularly imprinted polymer.

3.2. Electrochemical Characterization

Electrochemical studies based on the oxidation of organic substances are promising techniques for analytical purposes. Here, studies were first carried out to determine the electrochemical oxidation behavior of gold electrodes on each polymeric modification step. This electrochemical study was performed by cyclic voltammetry with a potential window from −0.7 to 0.7 V at a scan rate of 50 mV. Figure 2 shows the results obtained from the CV analysis of the electrodes, where the oxidation and reduction peaks for K₃[Fe(CN)₆] can be observed. As expected, with the modification of the electrodes, the cyclic voltammograms also changed. A shift in the oxide-reduction peaks is shown and a decrease in the intensity of the current peaks as the modification of the electrode is achieved. From here, we can deduce that the degree of modification is related to the corresponding change in the K₃[Fe(CN)₆] signal, as also shown by O’Sullivan et al., 2021 and Menon et al., 2018 [18,19].

We noticed that the oxidation and reduction peaks of K₃[Fe(CN)₆] of the modified and non-modified electrodes were not significantly different; therefore, to complement the analysis of the modified electrodes, an electrochemical impedance spectroscopy study was performed. Modified electrode spectra showed a characteristic Nyquist semicircle, indicating an increase in resistance. Figure 3 shows the first chemical modification of the electrodes with 6-mercaptohexanol and shows a small Nyquist semicircle referring to a lower resistance for electron flow compared with the unmodified electrode, as presented by Laschuk et al., 2021 [20] in their research. We would expect similar behaviors for MIP and NIP since there would be no chemical difference. However, in our experimental conditions, a considerable difference in charge transfer was observed in the MIP and NIP electrodes attributed to a difference in the distribution of the polymer network on the electrode due to the cavities formed by molecular imprinting of enrofloxacin. These results confirm the successful modification of the electrode.

3.3. Influence of pH and Supporting Electrolyte

After electrochemical characterization of modified electrodes, we analyzed the enrofloxacin response on the non-modified electrodes. For this, square wave voltammetry was used as the electroanalytical technique for enrofloxacin determination. The analysis was performed to evaluate the influence of pH to obtain the best enrofloxacin peak with sufficient current response for further quantitative analysis. The buffers used were in the pH range from 3 to 6. Figure 4 shows that as the pH value of the solution increases, the oxidation peak corresponding to the EF decreases. The maximum current intensity peak and a shift in the window potential occurs for each pH of the solution. These results correspond to the ones reported by Hernandez et al., 2021 [21]. The characteristic peak of oxidation of enrofloxacin appears at 0.9 V, corresponding to the peak shown in the solution with pH 3. Based on these results, we decided that the optimal working conditions for further experiments were at pH 3. The following study consisted in obtaining a calibration curve of the current response versus EF concentrations. Figure 5 shows a linearity current peak proportional to EF concentrations with a coefficient of determination (R²) of 0.9947. This study is important to understand the nature of the chemical and physical processes involved in the electroanalytical procedure.

3.4. SWV Response of Electrodes to Enrofloxacin Detection

To compare the electrochemical response of the modified electrode with the molecularly imprinted enrofloxacin, SWV analysis was performed for the MIP, NIP, and bare electrodes in the presence of enrofloxacin. Figure 6 shows the well-defined oxidation peak of enrofloxacin at the window potential of 0.9 v for the electrode modified with MIP. It was also observed in the current peak shown in Figure 5 at a concentration of 1 mM. Likewise, a small oxidation peak was observed for the NIP control electrode attributed to non-specific interactions or small unmodified areas in the electrode, which was also observed for the bare electrode presenting a small signal corresponding to the oxidation of enrofloxacin at pH 3, as shown in Figure 4. However, when compared with the corresponding signal from the MIP-electrode, the current peak is much higher due to the modification of the electrodes with the MIPs, concentrating in a greater proportion the analyte sample in the cavities of the MIP polymer, as also reported by Seguro et al., 2022 [22]. Therefore, this system may be used for the detection of enrofloxacin.

3.5. Detection of Enrofloxacin

As part of the results obtained from the determination of enrofloxacin, Figure 7 shows the analysis by SWV using MIP-electrode and different concentrations of enrofloxacin prepared in buffer pH 3. The study was carried out at different concentrations of enrofloxacin, detecting a linear range of concentrations from 0.01 to 0.1 mM with a detection limit (LOD) of 0.02 mM; these concentrations of enrofloxacin are below the levels of 0.1 to 0.3 mM allowed by regulatory commissions. In this sense, the development of biosensors from molecular imprinted polymers for the detection of enrofloxacin presents great characteristics comparable with the detection of conventional equipment such as HPLC or ELISA. The main advantages of this detection system are its low cost and the ability to detect and quantify analytes without having to take samples to a laboratory (portability).

4. Conclusions

In summary, a new electrochemical sensor was developed from the modification of screen-printed electrodes with molecularly imprinted polymers for the detection of enrofloxacin. This easy and practical sensor can detect concentration levels of enrofloxacin, performing comparably with conventional methods such as HPLC. Additionally, since it is portable, it also has the advantage of being able to detect analytes in the field in around ~5 min, without taking the sample to a laboratory. Finally, these modified screen-printed electrodes are reusable, making them highly interesting while considering their expense, especially when comparing our method to more conventional methods as mentioned before.